Cancer Associated Viruses

Current Cancer Research Series Editor Wafik El-Deiry For further volumes: http://www.springer.com/series/7892 Erle S...

Author: Erle S. Robertson

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Current Cancer Research

Series Editor Wafik El-Deiry

For further volumes: http://www.springer.com/series/7892

Erle S. Robertson Editor

Cancer Associated Viruses

Editor Erle S. Robertson Professor of Microbiology Director of the Tumor Virology Training Program Perelman School of Medicine University of Pennsylvania Philadelphia, PA, USA [email protected]

ISBN 978-1-4419-9999-3 e-ISBN 978-1-4614-0016-5 DOI 10.1007/978-1-4614-0016-5 Springer New York Dordrecht Heidelberg London Library of Congress Control Number: 2011940811 © Springer Science+Business Media, LLC 2012 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)

In Memoriam

Baruch S. Blumberg, M.D., D.Phil.

Baruch Samuel (Barry) Blumberg died suddenly on April 5, 2011 shortly after giving a presentation on “citizen science” at the NASA Lunar Science Institute in Ames, California. Barry’s talk concerned making spacecraft data available to the public so that ordinary people could contribute to its interpretation. That final talk reflected his deep belief that anyone who was willing to invest time and thought could have ideas that would lead to new understandings of research information. This volume is about viruses and cancer, but Barry was neither a virologist nor an oncologist. However, he contributed fundamentally to both. Barry began his research career as a medical student at Columbia University when he took an elective in v

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Tropical Medicine in Suriname. There, he observed that filariasis was rampant, but that only some of the many infected people showed signs of disease. Suriname had many different ethnic groups and some of the diversity in responses was associated with ethnicity. That led him to wonder for the rest of his life why humans living in the same environment responded so differently to infectious agents. After an internship and assistant residency in medicine at Bellevue Hospital in New York City and a fellowship in rheumatology, Barry went to Oxford University to study biochemistry with Alexander Ogston. He was in the lab at the same time as Oliver Smithies who also went on to win a Nobel Prize. In England, he was exposed to the history of scientific discovery and began formulating his scientific ideas. It was there that he found his scientific inspiration in the lives and work of the nineteenth-century naturalists Charles Darwin and Alfred Russell Wallace. He began to apply the principles of evolution to his research. After receiving a doctorate, Barry went to an obscure unit called Geographic Medicine and Genetics of the National Institute of Arthritis and Metabolic Disease (NIAMD) in Bethesda, Maryland. He chose this hidden corner because he thought it would allow him to pursue his own ideas and travel wherever he pleased. He began a lifelong pattern of collecting blood samples everywhere he went, making observations about the people from whom they were drawn and often collecting samples of vegetation from their environments. It was at the NIH that Barry honed his interest in genetic polymorphisms in human blood, inherited variants of proteins or blood groups, which he believed were likely to be associated with human diseases. As a believer in the central importance of natural selection, he thought all such variants had to be important. Otherwise, they would not have persisted in human populations. In 1964, the Director of the Institute for Cancer Research (ICR, precursor to the Fox Chase Cancer Center) recruited Barry to become the head of a new Division of Clinical Research. The lure was that Barry was promised he could do whatever he wanted as long as his research ultimately had consequences for disease in humans. Barry was intrigued and immediately began assembling a small group of physicians to staff this new enterprise. He had complete faith that his approach: identifying variants in human blood and then finding out what they meant, would be much more informative than starting with a disease and trying to identify its causes. It was this approach that first resulted in identifying an antigen on a lipoprotein in serum, and subsequently to a different antigen, the “Australia antigen.” Barry focused the efforts of his new division on understanding the biological significance of Australia antigen. In a series of studies of diseases associated with the antigen, the group found that Australia antigen was closely associated with one form of viral hepatitis, later called hepatitis B. At the same time, they observed that Australia antigen was a particle similar in appearance to a virus. That was enough information for Barry to begin to develop a unique vaccine, one that was prepared from antigenic particles in human blood. The patent for the vaccine was submitted in 1969 and granted in 1971. By 1975, before the vaccine had even been tried in humans, Barry predicted in print that the vaccine would not only prevent infection

In Memoriam

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with the hepatitis B virus but that it would also prevent liver cancer. Therefore, it would be the first cancer vaccine. In 1976, Barry was awarded the Nobel Prize in Physiology or Medicine for “discoveries concerning new mechanisms for the origin and dissemination of infectious diseases.” Very few people are privileged to win a Nobel Prize. Barry made his success a celebration for everyone in his group. He took as many of his colleagues and staff in the division to the ceremony as he was permitted, and least 15 and their spouses. In 1989, he returned to Oxford to become the Master of Balliol College serving until 1994. Balliol was founded in 1263 and Barry was its first American Master. From 1999 to 2002, he was director of the NASA Astrobiology Institute. He continued his affiliation with NASA and in 2008 became a Senior Scientist at the NASA Lunar Science Institute. In 2005, he became the President of the American Philosophical Society, founded by Benjamin Franklin, and the oldest learned society in the Americas. Barry was always a happy person. He celebrated his own life by living it to the fullest. He cycled, hiked, ran, rock-climbed, canoed, and kayaked until the end of his life. Barry had a long and happy marriage. He was always proud of the accomplishments of his wife, Jean, his four children and nine grandchildren. He left a legacy of accomplishments that saved an enormous number of lives and prevented hundreds of millions of people from becoming ill with the hepatitis B virus. On every continent, his many friends and colleagues mourn his loss. Fox Chase Cancer Center Philadelphia, PA, USA

W. Thomas London

Preface

For almost 30 years, there has been no comprehensive text covering the many viral agents and their contributions to cancers or cell proliferation. The goal was to provide a relatively up-to-date tome, which would be a wonderful resource for the many investigators in the field of viral oncology. The chapters are meant to be a thorough review of the literature, which covers specific viruses as well as provide some synthesis of what we now know about viruses, cell proliferation and the genes that target specific cellular pathways. The previous works are outdated, and as this book is put to press, we would still have additional works to be published that will certainly be missed. We have tried to be as comprehensive as possible within the guidelines of the text without sacrificing the science and we have allowed authors much flexibility that would only be fitting if one takes on a job to complete a chapter that is as comprehensive and current as we have attempted in this book. The primary goal here was to provide the most comprehensive version of chapters covering the majority of viruses and cancers, which will be a major resource for all trainees in the field of viral oncology from undergraduates and graduate students to post-doctoral fellows in basic science and translational or clinical studies, as well as investigators related to viral oncology. This approach was certainly limited as we will, without a doubt, be missing some of the detail and intricate nuances of each viral system. That said, we certainly tried to encourage a general theme throughout and so the experienced readers in the field may find some aspects of it less inviting. Nevertheless, I think that overall, we have attained a level of scientific sophistication for each of the chapters that it would be worth the time of experts in the field to read and it would be a solid contribution enjoyed by all including novices, as well as the experts. Thus, I take full responsibility for any omission that may have occurred, unintentionally. I also want to say that each contributor has done a fantastic job in completing his or her chapter, and that one would have to give them all a tremendous thank-you for their efforts in making this project happen even with the burdensome task of meeting deadlines and responding to my many emails in nudging them along. The book begins with a chapter that introduces the father of viral oncology Professor Peyton Rous with some of his many interesting findings and his training, ix

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as well as his international interactions with many scientists across the world. This year would be the 100th anniversary of the initial discovery of the Rous Sarcoma Virus. This is followed by a chapter contributed by Baruch Bloomberg, one of the many Nobel laureates whose contributions to the field has made a huge impact in saving lives throughout the world. He was passionate about pushing for the development of the hepatitis B vaccine and in doing so, led to the vaccination of millions throughout the world who would have been infected and would have a higher probability of developing hepatocellular carcinoma. He presents a historical perspective on viruses and cancer. The chapters by Drs. Jae Jung, Blossom Damania and Robin Weiss present a broad outline of how viruses can contribute or drive the oncogenic process and the potential for cancer transmission. Dr. Alwine wrote the introductory chapter for the DNA tumor viruses and suggests that while some large DNA viruses may not be able to directly transform a cell, they can certainly alter signaling and metabolism in ways that can certainly drive the transformation and possible immortalization of the infected cells. The chapter by Dr. Bala Chandran brings together the many contributions by the large DNA herpesviruses and their ability to induce the oncogenic process. Further on this theme, we also cover specific chapters on viruses in herpesviridae with my group looking at the Lymphocryptoviruses; Dr. Schultz on the Rhadinoviruses; Dr. Rose on the contribution of the retroperitoneal fibromatosis herpesvirus to retroperitoneal fibromatosis, a Kaposi’s sarcoma-like disease in macaques with simian AIDS; Dr. Wong exploring the viruses in nonhuman primates; Dr. Speck presenting the murine herpesvirus model of tumorigenesis; and Drs. Parcells and Morgan who describe the Marek’s disease virus and its contribution to T-cell lymphomas in chickens. These chapters provide an in-depth analysis of these viral agents and their similarities and differences in driving the oncogenic process. We have had a great deal of success in bringing in a number of talented investigators looking at the small DNA tumor viruses, in particular the Polyoma and Papilloma viruses as well as the Adenoviruses. Dr. Gjoerup did a fantastic job in describing the many facets of the Polyomas and their contribution to cancer and this was followed by chapters from Drs. Butel, Hirsch, Khalili and Becker who provided a thorough review of the SV40 as a model system, the BK virus, the JC virus and the new Merkel cell Polyoma virus, respectively. I should also mention at this stage, that more recently in July 2010, there was a report of another virus, which belongs to the Polyoma virus family now called Trichodysplasia Spinulosa-associated Polyoma virus (TSV), which was identified in a rare skin disease called Trichodysplasia Spinulosa, exclusively seen in immunocompromised patients. It is yet to be seen if this is a ubiquitous virus in the population, which becomes opportunistic in these group of patients. A review of the Papilloma viruses covering the HPV and BPV systems was completed by Drs Jianxin You and Suzannne Wells. Adenoviruses, another group of oncogenic DNA viruses, were also included, although to date there has been no direct association with Adenoviruses and human cancers. However, there has been a wealth of information over more than 30 years showing that Adenoviruses are fully capable of meeting the major criteria for driving the oncogenic process using in vitro studies and also inducing tumors in an animal model.

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The Hepadnaviruses have also been addressed in the compendium, where we take a closer look at the contributions of the hepatitis viruses B and C. Professor Tim Block has done a marvelous job of reviewing the many general attributes of the hepatitis viruses and the cancers they are associated with from a virological to a more molecular perspective. This is followed by chapters on hepatitis B virus by Dr. Mason from the Fox Chase Cancer Center, where Barry Bloomberg spent a great many of his years as a scientist working on the hepatitis virus. Dr. Mason did a fantastic job in getting us up to date on the causes of chronic liver disease, cirrhosis and many years later HBV-induced hepatocellular carcinoma, which takes at times as long as 40 years. Dr. Bret Lindenbach explores the contributions of hepatitis C virus to the development of hepatocellular carcinoma and summarizes the clinical and molecular virology links between the HCV virus and HCC. Dr. Kathleen Boris-Lawrie finds an interesting angle to explore further the role of HIV-1 as a risk factor for the development of malignancies in AIDS patients by describing why Kaposi’s sarcoma, non-Hodgkin’s lymphoma and cervical carcinomas can function as prognostic indicators of AIDS and begins to suggest the relationship of coinfection and how this may contribute to the oncogenic process. We also cover the HTLV-1 and HTLV-2 where their contributions to cell proliferation were well described. Dr. Chou-Zen Giam focuses on the role of HTLV-1 in causing adult T-cell leukemia paying attention to the role of two viral proteins Tax and HBZ in viral replication and leukemogenesis. Dr. Patrick Green focuses on the biology and pathogenesis of HTLV-2 and further dissects the various cellular processes utilized by the virus in contributing to cell proliferation. The chapter on avian and murine retroviruses was skillfully put together by Drs. Karen Beemon and Naomi Rosenberg. This chapter provides information on viral oncogenes and the cooperation between these viral oncogenes as a major step in the development of cancer. They also describe the potential role of these viruses as vector systems. Dr. Leslie Parent goes into further detail in contributing the chapter on the Rous Sarcoma Virus which takes the reader from the provirus concept and how the integrated provirus, which certainly has transforming activities, led to the identification of the a cellular gene highly homologous to the viral transforming gene. Dr. Susan Ross thoroughly presents the mouse mammary tumor viruses (MMTV), which can cause breast cancer in mice by causing insertional activation of mutation of cellular oncogenes and provides an extremely useful model for understanding human breast cancer. Another very interesting virus is the Jaagsiekte Sheep retrovirus, which is associated with lung cancer. This virus causes ovine pulmonary adenocarcinoma which is derived from the secretory lung epithelial cells. Dr. Hung Fan describes here how the pathology of the virus is directly linked to the envelope protein (Env) and that JSRV is mostly pathogenic in the lung as the viral LTR is transcriptionally active only in differentiated airway epithelial cells. Drs. Renne and Swaminathan now contributed a chapter on the small RNAs and their role in viral-mediated cancers. The final chapter was a wonderful contribution by Dr. Charles Wood, who explores the role of immunodeficiency and opportunistic infections and their cooperation in driving the oncogenic process.

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Finally, I wanted to personally thank all the authors for their patience and their hard work in getting their contributions to me as timely as can be expected. Even the folks who were somewhat tardy in their delivery have made it worthwhile, and I can say overall that I am personally happy with the final product. I hope this project provides a renewed vigor to our community of scientists to explore the contributions of viruses to cancer. In my many discussions with investigators in the cancer field it is still amazing and a bit puzzling to me that a great many are still hesitant to acknowledge that viruses or infectious agents on a whole have much to do with cancer, even though it is well known that about 20% of all known cancers are associated with infectious agents. I hope this renewed thrust will minimize these concerns and provide new support for the many investigators who have spent their entire lives working towards understanding how viruses contribute to the oncogenic process. Happy reading. Cheers Philadelphia, PA, USA

Erle S. Robertson

Contents

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Peyton Rous: A Centennial Tribute to the Founding Father of Cancer Virology...................................................................... Volker Wunderlich and Peter Kunze

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Viruses and Cancer: A Historical Perspective – HBV and Prevention of a Cancer .................................................................... Baruch S. Blumberg

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3

Virus-Mediated Cell Proliferation ......................................................... Sun-Hwa Lee, Stacy Lee, and Jae Ung Jung

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4

Viral-Encoded Genes and Cancer ......................................................... Blossom Damania

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5

Oncogenic Viruses and Cancer Transmission ...................................... 101 Robin A. Weiss

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DNA Viruses and Cancer: Taking a Broader Look ............................. 119 James C. Alwine

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Herpesviruses and Cancer ..................................................................... 133 David Everly, Neelam Sharma-Walia, Sathish Sadagopan, and Bala Chandran

8

Lymphocryptoviruses: EBV and Its Role in Human Cancer ............. 169 Santosh Kumar Upadhyay, Hem Chandra Jha, Abhik Saha, and Erle S. Robertson

9

Nonhuman Primate Gamma-herpesviruses and Their Role in Cancer .................................................................................................. 201 Ryan D. Estep and Scott W. Wong

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Rhadinoviruses: KSHV and Associated Malignancies ........................ 215 Susann Santag and Thomas F. Schulz

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Contents

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Retroperitoneal Fibromatosis Herpesvirus and Kaposi’s Sarcoma-Like Tumors in Macaques ...................................................... 251 Laura K. DeMaster and Timothy M. Rose

12

Murine Gammaherpesvirus-Associated Tumorigenesis...................... 267 Kathleen S. Gray and Samuel H. Speck

13

Marek’s Disease Virus-Induced T-Cell Lymphomas ........................... 307 Mark S. Parcells, Joan Burnside, and Robin W. Morgan

14

Polyomaviruses and Cancer ................................................................... 337 Ole Gjoerup

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Polyomavirus SV40: Model Infectious Agent of Cancer ..................... 377 Janet S. Butel

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BK Polyomavirus and Transformation ................................................. 419 Tina Dalianis and Hans H. Hirsch

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Polyomavirus JC and Human Cancer: Possible Role of Stem Cells in Pathogenesis ................................................................. 433 Kamel Khalili, Martyn K. White, Jennifer Gordon, and Barbara Krynska

18

Merkel Cell Polyomavirus ...................................................................... 449 David Schrama and Jürgen C. Becker

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Human Papillomaviruses and Cancer................................................... 463 Jianxin You and Susanne Wells

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Tumorigenesis by Adenovirus Type 12 E1A ......................................... 489 Hancheng Guan and Robert P. Ricciardi

21

Overview of Hepatitis Viruses and Cancer ........................................... 509 Timothy M. Block, Jinhong Chang, and Ju-Tao Guo

22

Hepadnaviruses and Hepatocellular Carcinoma ................................. 531 William S. Mason

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Hepatitis C Virus and Hepatocellular Carcinoma ............................... 571 Brett Lindenbach

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Human and Animal Retroviruses: HIV-1 Infection Is a Risk Factor for Malignancy ............................................................ 585 Amy M. Hayes and Kathleen Boris-Lawrie

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HTLV-1 and Oncogenesis ....................................................................... 613 Chou-Zen Giam

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Human T-Cell Leukemia Virus Type 2 (HTLV-2) Biology and Pathogenesis ..................................................................................... 647 Rami Doueiri and Patrick L. Green

Contents

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Mechanisms of Oncogenesis by Avian and Murine Retroviruses ............................................................................................. 677 Karen Beemon and Naomi Rosenberg

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Rous Sarcoma Virus: Contributions of a Chicken Virus to Tumor Biology, Human Cancer Therapeutics, and Retrovirology.................................................................................... 705 Leslie J. Parent

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Mouse Mammary Tumor Virus and Cancer ........................................ 739 Susan R. Ross

30

Jaagsiekte Sheep Retrovirus and Lung Cancer ................................... 755 Chassidy Johnson and Hung Fan

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Small RNAs and Their Role in Herpesvirus-Mediated Cancers ........ 793 Sankar Swaminathan and Rolf Renne

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Viral Malignancies in HIV-Associated Immune Deficiency ................ 819 Pankaj Kumar, Veenu Minhas, and Charles Wood

Index ................................................................................................................. 853

Contributors

James C. Alwine Department of Cancer Biology, Abramson Family Cancer Research Institute, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA [email protected] Jürgen C. Becker Director, Division of General Dermatology, Department of Dermatology, Medical University of Graz, Auenbruggerplatz 8, A-8036, Graz, Austria [email protected] Karen Beemon Department of Biology, Johns Hopkins University, Baltimore, MD 21218, USA [email protected] Timothy M. Block Department of Microbiology and Immunology, Drexel University College of Medicine, Pennsylvania Biotechnology Center, Doylestown, PA, USA Hepatitis B Foundation, Pennsylvania Biotechnology Center, Doylestown, PA, USA [email protected] Baruch S. Blumberg Fox Chase Cancer Center, Philadelphia, PA 19111, USA [email protected] Kathleen Boris-Lawrie Department of Veterinary Biosciences, Center for Retrovirus Research, Center for RNA Biology, Comprehensive Cancer Center, Ohio State University, Columbus, OH 43210, USA [email protected] Joan Burnside Department of Animal and Food Sciences, University of Delaware, Newark, DE 19716, USA

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Contributors

Janet S. Butel Department of Molecular Virology and Microbiology, Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, TX 77030, USA [email protected] Bala Chandran H.M. Bligh Cancer Research Laboratories, Department of Microbiology and Immunology, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064, USA [email protected] Jinhong Chang Department of Microbiology and Immunology, Drexel University College of Medicine, Pennsylvania Biotechnology Center, Doylestown, PA, USA [email protected] Tina Dalianis Department of Oncology-Pathology, Karolinska Institutet, Cancer Center Karolinska R8:01, Karolinska University Hospital, 171 76, Stockholm, Sweden [email protected] Blossom Damania Department of Microbiology and Immunology and Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA [email protected] Laura K. DeMaster Department of Global Health, University of Washington, Seattle Children’s Research Institute, Seattle, WA, USA Rami Doueiri Department of Veterinary Biosciences, The Ohio State University, Columbus, OH 43210, USA Ryan D. Estep Vaccine and Gene Therapy Institute, Oregon Health and Science University, Beaverton, OR, USA David Everly H.M. Bligh Cancer Research Laboratories, Department of Microbiology and Immunology, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA Hung Fan Department of Molecular Biology and Biochemistry, Cancer Research Institute, University of California, Irvine, CA 9269, USA [email protected] Chou-Zen Giam Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814, USA [email protected] Ole Gjoerup Cancer Virology Program, University of Pittsburgh Cancer Institute, Research Pavilion Suite 1.8, 5117 Centre Avenue, Pittsburgh, PA, 15213, USA [email protected]

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Jennifer Gordon Department of Neuroscience and Center for Neurovirology, Temple University School of Medicine, 3500 N. Broad Street, Philadelphia, PA 19140, USA Kathleen S. Gray Department of Microbiology & Immunology, Emory Vaccine Center, Emory University School of Medicine, Atlanta, GA 30322, USA Patrick L. Green Center for Retrovirus Research, The Ohio State University, Columbus, OH 43210, USA Department of Veterinary Biosciences, The Ohio State University, Columbus, OH 43210, USA Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University, Columbus, OH 43210, USA Comprehensive Cancer Center and Solove Research Institute, The Ohio State University, Columbus, OH 43210, USA [email protected] Hancheng Guan Department of Microbiology, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA Ju-Tao Guo Department of Microbiology and Immunology, Drexel University College of Medicine, Pennsylvania Biotechnology Center, Doylestown, PA, USA Amy M. Hayes Department of Veterinary Biosciences, Center for Retrovirus Research, Center for RNA Biology, Comprehensive Cancer Center, Ohio State University, Columbus, OH 43210, USA Hans H. Hirsch Department of Biomedicine, Clinical and Transplantation Virology, Institute for Medical Microbiology, University of Basel, Basel, Switzerland Infectious Disease and Hospital Epidemiology, University Hospital Basel, Basel, Switzerland [email protected] Hem Chandra Jha Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA Abramson Comprehensive Cancer Center, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA Chassidy Johnson Department of Molecular Biology and Biochemistry, Cancer Research Institute, University of California, Irvine, CA 9269, USA Jae Ung Jung Department of Molecular Microbiology & Immunology, University of Southern California, School of Medicine, 2011 Zonal Avenue, HMR401, Los Angeles, CA 90033, USA [email protected]

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Contributors

Kamel Khalili Department of Neuroscience and Center for Neurovirology, Temple University School of Medicine, 3500 N. Broad Street, Philadelphia, PA 19140, USA [email protected] Barbara Krynska Center of Neural Repair and Rehabilitation and Shriners Hospitals Pediatric Research Center, Temple University School of Medicine, 3500 N. Broad Street, Philadelphia, PA 19140, USA Department of Neurology, Temple University School of Medicine, 3500 N. Broad Street, Philadelphia, PA 19140, USA Pankaj Kumar Nebraska Center for Virology and the School of Biological Sciences, Morrison Center, University of Nebraska-Lincoln, Lincoln, NE 68583, USA Peter Kunze Institute of Pathology “Georg Schmorl”, 01067, Dresden, Germany [email protected] Sun-Hwa Lee Department of Molecular Microbiology & Immunology, University of Southern California, School of Medicine, 2011 Zonal Avenue, HMR401, Los Angeles, CA 90033, USA [email protected] Stacy Lee Department of Molecular Microbiology & Immunology, University of Southern California, School of Medicine, 2011 Zonal Avenue, HMR401, Los Angeles, CA 90033, USA Brett Lindenbach Section of Microbial Pathogenesis, Yale University School of Medicine, 354C Boyer Center for Molecular Medicine, New Haven, CT 06536-0812, USA [email protected] William S. Mason Fox Chase Cancer Center, Philadelphia, PA 19111, USA [email protected] Veenu Minhas Nebraska Center for Virology and the School of Biological Sciences, Morrison Center, University of Nebraska-Lincoln, Lincoln, NE 68583, USA Robin W. Morgan Department of Animal and Food Sciences, University of Delaware, Newark, DE 19716, USA Mark S. Parcells Department of Animal and Food Sciences, University of Delaware, Newark, DE 19716, USA [email protected]

Contributors

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Leslie J. Parent Department of Medicine, Penn State College of Medicine, Hershey, PA 17033, USA Department of Microbiology and Immunology, Penn State College of Medicine, Hershey, PA 17033, USA [email protected] Rolf Renne Department of Molecular Genetics and Microbiology, UF Shands Cancer Center, University of Florida, Gainesville, FL 32610-3633, USA [email protected] Robert P. Ricciardi Department of Microbiology, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA Abramson Cancer Center, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA [email protected] Erle S. Robertson Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA Abramson Comprehensive Cancer Center, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA [email protected] Timothy M. Rose Department of Pediatrics, University of Washington, Seattle Children’s Research Institute, Seattle, WA, USA [email protected] Naomi Rosenberg Sackler School of Graduate Biomedical Sciences, Tufts University School of Medicine, Boston, MA 02111, USA [email protected] Susan R. Ross Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA Abramson Cancer Center, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA [email protected] Sathish Sadagopan H.M. Bligh Cancer Research Laboratories, Department of Microbiology and Immunology, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA Abhik Saha Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA Abramson Comprehensive Cancer Center, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA

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Contributors

Susann Santag Institute of Virology, Hannover Medical School, Hannover, Germany David Schrama Director Division of General Dermatology, Department of Dermatology, Medical University of Graz, Auenbruggerplatz 8, A-8036, Graz, Austria Thomas F. Schulz Institute of Virology, Hannover Medical School, Hannover, Germany [email protected] Neelam Sharma-Walia H.M. Bligh Cancer Research Laboratories, Department of Microbiology and Immunology Facilities, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA Samuel H. Speck Department of Microbiology & Immunology, Emory Vaccine Center, Emory University School of Medicine, Atlanta, GA 30322, USA [email protected] Sankar Swaminathan Division of Infectious Diseases, Department of Medicine, University of Utah School of Medicine, Salt Lake City, UT 84132, USA [email protected] Santosh Kumar Upadhyay Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA Abramson Comprehensive Cancer Center, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA Robin A. Weiss Division of Infection & Immunity, University College London, London, UK [email protected] Susanne Wells Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA Division of Hematology/Oncology, Cincinnati Children’s Hospital, Cincinnati, OH, USA [email protected] Martyn K. White Department of Neuroscience and Center for Neurovirology, Temple University School of Medicine, 3500 N. Broad Street, Philadelphia, PA 19140, USA

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Scott W. Wong Vaccine and Gene Therapy Institute, Oregon Health & Science University, 505 NW 185th AvenueBeaverton, OR 97006, USA Division of Pathobiology and Immunology, Oregon National Primate Research Center, Beaverton, OR, USA Department of Molecular Microbiology and Immunology, Oregon Health & Science University, Portland, OR, USA [email protected] Charles Wood Nebraska Center for Virology and the School of Biological Sciences, Morrison Center, University of Nebraska-Lincoln, Lincoln, NE 68583, USA [email protected] Volker Wunderlich Max Delbrück Center for Molecular Medicine, 13125, Berlin, Germany [email protected] Jianxin You Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA [email protected]

Chapter 1

Peyton Rous: A Centennial Tribute to the Founding Father of Cancer Virology Volker Wunderlich and Peter Kunze

Introduction On December 10, 1966, the American pathologist and cancer researcher Francis Peyton Rous (1879–1970) (Fig. 1.1), professor emeritus at the Rockefeller Institute for Medical Research, New York, was awarded the Nobel Prize for Physiology or Medicine “for his discovery of tumor-inducing viruses.” He received the award in Stockholm from the hands of the Swedish King Gustav VI Adolf (1882–1973). Fifty-five years after his discovery (Rous 1911a, b) and forty years after his first nomination by Karl Landsteiner (1868–1943) (Nomination Database 1901–1951), one of the great scientists of the twentieth century was awarded this long-deserved honor. His work launched a new era of medicine (Vogt 1996). Amazingly, however, up to now, science historians have not written a biography of Rous. Just a few months before the Nobel ceremony, Rous had received the prestigious Paul Ehrlich and Ludwig Darmstaedter Prize (Germany’s supreme medical accolade) in St. Paul’s Church in Frankfurt am Main on March 14, 1966. There, he began his award acceptance speech with the words: The joy that moves me on this festive occasion is particularly great because it brings to mind some personal memories. When I studied at The Johns Hopkins School of Medicine at the beginning of this century, all young physicians looked to Germany as a model. The dean of the school, Professor William Welch, an eminent pathologist, had many years earlier worked with Paul Ehrlich in Breslau (today Wroclaw) [both under the supervision of the pathologist Julius Cohnheim (1839–1884)], and upon his return to the United States had informed the American medical community of the major progress made in Germany during this time. (Rous 1966: 20) [German in original]

V. Wunderlich (*) Max Delbrück Center for Molecular Medicine, 13125 Berlin, Germany e-mail: [email protected] P. Kunze Institute of Pathology “Georg Schmorl”, 01067 Dresden, Germany E.S. Robertson (ed.), Cancer Associated Viruses, Current Cancer Research, DOI 10.1007/978-1-4614-0016-5_1, © Springer Science+Business Media, LLC 2012

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Fig. 1.1 Photograph of Peyton Rous, 1959 in Israel, courtesy of Dr. Inge Graffi, Berlin. Photo credit: Weizmann Institute of Science, Rehovot, Israel

In fact, during those years, German science (and in a broader sense European science) was a world leader in many fields within and outside of medicine, not only in the inner circle of Paul Ehrlich. As a result, it was extremely attractive for young scientists from other countries to come to Germany to work here temporarily. For a future career in the USA, proof of European experience could be quite helpful, not unlike as it is today with many appointments of professors in Germany, where a previous work stay in the USA is regarded a conditio sine qua non. If the young Rous, while still a student, dreamed of a sojourn in Germany, this dream was soon to become reality. In the official Nobel biography, which is based on Rous’s own autobiographical notes, an additional personal recollection of the laureate is recorded: [At The Johns Hopkins Medical School] he graduated in 1905 [Doctor of Medicine] and became an intern in its hospital. Then, finding himself unfit to be a “real doctor,” he turned to medical research instead, and for this purpose became an instructor in pathology at the University of Michigan on a beggarly salary. His work in the laboratory turned out to be mainly that of a technician because the University had small funds only, but with noble generosity Professor Alfred [sic] Warthin, head of the Department, came to his rescue, actually offering to “teach summer school” in his stead, and give Peyton the sum thus earned, if he would study German hard and use the money to go for the summer to a certain hospital in Dresden where morbid anatomy was taught. Dresden in 1907! Exquisite city in an exquisite land, with no hint of war in the air! (Anonymous 1966)

Rous, who in 1966 was well along in years, could still remember Dresden, although he seemed to have forgotten the names of the host institution (the specific hospital) and his Dresden mentor. Thus, these details remained largely unclear and

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were mentioned only very briefly in the Biographical Memoirs (Andrewes 1971; Dulbecco 1976) dedicated to Rous. However, his sojourn in Dresden was the only one abroad before Rous began his exceptionally successful career as independent research scientist at the Rockefeller Institute, and apparently – even in retrospect – the time spent in Dresden was very important to him. In this paper, we report on new research inquiries concerning Warthin, Schmorl, and Rous. But first, we shall briefly present some of the pathologists who influenced Rous before his stay in Dresden. In subsequent sections, we shall briefly present several aspects of Rous’ work in the years immediately following his Dresden stay that were crucial for tumor virology.

A Fresh Age of Medical Endeavor in America: William Henry Welch Welch and Warthin influenced the career of the young Rous in different ways. He studied at the School of Medicine at The Johns Hopkins University, America’s first research university, which opened in 1892. Apparently, William Henry Welch (1850–1934) (Fig. 1.2), the founding dean and professor of pathology at the medical school and at that time academic teacher, was able to spark Rous’ interest in experimental medicine in general and in pathology in particular. In Baltimore, Welch established the first pathology teaching laboratory in the USA. In general, Welch dedicated himself to an extraordinary degree to the modernization of American medicine in teaching and research [“a fresh age of medical endeavor came in – an

Fig. 1.2 Photograph of William H. Welch. Photo credit: Courtesy of the Rockefeller Archive Center

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age in which experiment largely took over from observation,” Rous wrote, in retrospect (Rous 1948: 611)], whereby the experience gained during his research stays in Europe helped him (1875–1878, 1884) (MacCallum 1936; Flexner and Flexner 1941; Flexner 1943; Brieger 1970). As first president of the Board of Scientific Directors at the Rockefeller Institute for Medical Research in New York (1901–1933), Welch took a keen interest in Rous’ subsequent rise to become a distinguished scientist. Just a few years after his death, Rous had the honor of presenting a William Henry Welch Lecture (Rous 1941). On another occasion, Rous noted: “It is not too much to say that modern scientific medicine reached America through William Henry Welch” (Rous 1949: 411). The Journal of Experimental Medicine was launched in 1896 by Welch as founding editor of a new type of medical publication, which was then developed into a highly prestigious journal by Rous during his extremely long tenure (1922–1970, until 1945 together with Simon Flexner).

Aldred Scott Warthin: A Consummate Pathologist As a young, freshly graduated medical doctor, Rous came to Aldred Scott Warthin (1866–1931) (Fig. 1.3) at the University of Michigan with the aim of doing experimental work and obtaining training in pathology. He could not have made a better choice. Warthin was initially trained as church musician before turning to the study of medicine. He received his MD in 1891 and his PhD in 1893. He worked temporarily in the Department of Internal Medicine at the University of Michigan before he

Fig. 1.3 Photograph of Aldred S. Warthin. Photo credit: A.S. Warthin Papers, Bentley Historical Library, University of Michigan

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started his academic career there as pathologist. Following various positions from 1896 on (among them as instructor, a position Rous later held under him), he was appointed professor and director of the Pathological Laboratories at the University of Michigan in Ann Arbor in 1903. Between 1893 and 1900, Warthin regularly traveled to Europe during the summer months in order to work at the institutes of pathology in Vienna, Freiburg, and Dresden and at the same time to pursue his multifaceted interests (music, art history, collection of old books, particularly of medical incunabula). While doing so, he in no way neglected his extensive pathological research. A number of eponyms are today associated with the name Warthin: Warthin’s sign (exaggeration of pulmonary sounds in acute pericarditis), Warthin’s tumor (benign salivary gland tumor with lymphoid tissue covered by epithelium), and Warthin–Finkeldey giant cells (multinucleated giant cells seen in the lymphoid tissues of patients with measles). Moreover, he translated Ernst Ziegler’s (1849–1905) Lehrbuch der allgemeinen und speziellen pathologischen Anatomie und Pathogenese [Text-Book of Pathological Anatomy and Pathogenesis] (two volumes; Jena, 1881–1882) from German into English. Warthin was, as one would say today, very well-networked and held important posts in numerous medical societies. In person and manner, Warthin was a model of virile fastidiousness. […] Warthin’s approach to pathology was based upon a familiarity with and keen interest in internal medicine. He had a full appreciation of the biological significance of pathology, but to him, study in this field represented a particular opportunity for advancement of medicine as a science. (Anonymous 1932: 134–35) [And Rous later noted]: Warthin was bright-eyed and fresh-colored, quick and strong. He was drastic yet kind, earnest yet cheerful, and most sensitive to beauty. He loved music, gardens, books, and friends. (Rous 1936: 494)

Warthin’s research on the familial incidence of cancer had a particularly lasting impact. In 1895, he initiated one of the most thoroughly documented and longest family histories ever recorded (Warthin 1913, 1925). Recently, a new update of this family, originally referred to as Warthin’s family G and subsequently described as Lynch syndrome family, has been published (Douglas et al. 2005). “He [Warthin] can properly be called the father of cancer genetics,” Henry T. Lynch affirmed, 90 years after the beginning of this study (Lynch 1985). In his later years, Rous drew attention to some forgotten, yet at their time very far-sighted, works of his teacher. In 1904 and again in 1906 Professor Aldred Warthin of the University of Michigan, a consummate pathologist whose abilities I came to know through serving under him as instructor, reported facts making plain that human leukemia is a neoplastic disease; and in 1907 he published a study showing that this held true of a leukemia he came upon in a chicken [Warthin 1907. At the end of this paper he stated: “The problem of leukemia, then, becomes identical with that of malignant neoplasms in general.” Not bad for 1907]. No causative agent was then perceptible within the neoplastic tissue, but in 1908 two Dutch [sic, correct would be Danish] workers, Ellermann and Bang, reported on a virus as causing a chicken leukemia [Ellermann and Bang 1908]. Soon after, they procured another agent from a leukemia of a differing sort, and by means of these agents they transferred the two diseases in fowl after fowl. Their findings were wholly convincing yet were written off because leukemia was not generally realized then to be a neoplastic disease. Indeed, this did not come about until the 1930s. Warthin’s papers had been completely overlooked. He and the [Danish] workers were more than 20 years ahead of their time. (Rous 1967a: 844)

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In the summer of 1898, Warthin was a guest of Schmorl at Dresden Friedrichstadt Municipal Hospital. During these months, he performed a number of dissections, the protocols of which are still preserved today in the journals of the institute (Fig. 1.4). Warthin must have enjoyed his stay so much that he – several years later – recommended his protégé Rous to go to Dresden, too, and work with Schmorl. From his own experience, he knew that for this purpose a sufficient command of the German language was required. As mentioned before, he proposed to Rous to finance his trip and stay in Dresden. Rous gladly accepted this suggestion and remained grateful to his mentor throughout his life. One can assume that Warthin also saw the famous Dresden Dance of Death while he was in Dresden, an over 12-m long sandstone relief with 27 figures dating from the year 1534 (Dresdner Totentanz). He must have pointed this out to Rous too. In any case, Rous titled his later lecture in Oxford, named after the British humanist and physician Thomas Linacre (1460–1524), “The Modern Dance of Death” with a picture of “The Physician” from Holbein’s “Dance of Death” from 1523 to 1526 on the cover of the printed version (Rous 1929). Warthin had been engaged with this subject for many years and published an in-depth and famous study in 1931 which traces the dance-of-death motif through six centuries (Warthin 1931).

Georg Schmorl: A Scientist with Most Infectious Enthusiasm The Pathological–Anatomical Institute of the Dresden Friedrichstadt Municipal Hospital had existed since 1849 and since 1894 was situated in a spacious new building (the present Institute of Pathology “Georg Schmorl”) (Kunze 1999: 22–25, 70–79) (Fig. 1.5). It was the domain of well-known pathologists, among them were Albert von Zenker (1825–1898), Felix Victor Birch-Hirschfeld (1842–1899), and Adolf Neelsen (1854–1894). Since 1894, Christian Georg Schmorl (1861–1932) (Fig. 1.6) had been head of the institute, which was already then very attractive, and helped give the place its special aura. Again and again, physicians from many countries came to Dresden to work with Schmorl. Their number reached hundreds, which is why it was not possible to mention the names of later prominent guest researchers in Schmorl’s obituaries. For instance, the British pathologist Hubert Maitland Turnbull (1875–1955) was a guest researcher at the institute from 1905 to 1906, and after him a long series of British scientists. Under the influence of Schmorl, Turnbull later performed a great service to British pathology (Russell 2004). Work in morbid anatomy at Dresden under the inspiring guidance of Professor Georg Schmorl revolutionized Turnbull’s ideas about his future. Schmorl was noted for his work on bone pathology, and Turnbull had gone to Dresden primarily to become versed in this as a prelude to entering orthopedic surgery. In the early years of this century morbid anatomy was regarded, at least in this country, as a subject which had nothing more to offer: it had been sucked dry by Virchow and his school, and bacteriology was in the ascendant. But at Dresden the lively teaching and work of Schmorl gave the lie to all this; so much so that Turnbull resolved to give his future to morbid anatomy and to redeem its status in England. (Russell 1956)

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Fig. 1.4 Excerpts from autopsy reports written by Warthin 1898 in Dresden. Reproduction with the permission of the Archive of the Institute of Pathology “Georg Schmorl,” Dresden Friedrichstadt Municipal Hospital, Germany

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Fig. 1.5 View of the Pathologic–Anatomical Institute, Dresden Friedrichstadt Municipal Hospital, in the year 1907, seen from Friedrichstrasse. The institute’s auditorium is located on the left side under the cupola. On the right, St. Matthew’s Church can be seen, which was constructed according to the plans of Daniel Pöppelmann in 1732. Contemporary postcard. Reproduction with the permission of the archive of the Institute of Pathology “Georg Schmorl,” Dresden Friedrichstadt Municipal Hospital, Germany

Fig. 1.6 Portrait of Christian Georg Schmorl. Oil painting (118 × 85 cm) by Robert Sterl (1867–1932), an important representative of German Impressionism, 1921. Reproduction with the permission of the Dresden Friedrichstadt Municipal Hospital, photo: Martin Würker

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The high standard of British pathology that is based on the schools of Schmorl and Turnbull (Storey 2008) certainly was a prerequisite for the remarkable performance that was achieved in this country in the field of chemical carcinogenesis since the 1920s (Lawley 1994). Like the famous German poet Gotthold Ephraim Lessing (1729–1781), Schmorl attended the renowned Fürstenschule (the Prince’s school) of St. Afra in Meißen. Later, he always emphasized the enduring influence of his schooling. After graduating, he studied mathematics and natural sciences for several semesters, before deciding to study medicine. After earning his doctorate in medicine in 1887, he became the assistant of Birch-Hirschfeld in Leipzig. There, he qualified to become lecturer in pathology in 1892. After Neelsen’s early death, he took on the position as prosector and director of the Pathological Institute of the Dresden Friedrichstadt Municipal Hospital in 1894, a position he held until 1932 (from 1903 on, as professor) (Turnbull 1932; Geipel 1934; Junghanns 1983; Scholz 2007). Schmorl was particularly interested in microscope technology. He was the author of the standard textbook “Pathological Histological Examination Methods” (1897), which within 37 years had seen 16 editions and throughout Germany became an indispensable reference as “Der kleine Schmorl” (the little Schmorl) for generations of young pathologists. Moreover, he had an affinity for photography, microphotography, and X-ray photography which he had developed for use in pathology and in which he achieved true mastery; preferably, he wanted to perform all the necessary work with his own hands. Noteworthy are his pioneering works on bone pathology, especially the work in which he for the first time describes “Nodulus intraspongiosus Schmorl.” Through his unique collection of pathological bone specimens (the “Georg Schmorl” Pathology Collection), he gained an international reputation. “Schmorl was possessed of tireless energy, a most infectious enthusiasm, great diligence, an astonishing memory and a lively imagination. To search with knife or microscope was obviously a joy to him” (Turnbull 1932: 982).

Peyton Rous in Dresden Whether Rous only stayed a summer (Anonymous 1966), a few months (Huggins 1970), or a whole year (Andrewes 1971; Dulbecco 1976) in Dresden cannot be concluded from the preserved documents. What can be verified is that he performed 23 dissections (Fig. 1.7) between the beginning of July until the end of August 1907 (Wunderlich and Kunze 2008). As the protocols show, he was proficient in German. Furthermore, a photograph is preserved showing Rous and other physicians together with Schmorl (Fig. 1.8) – further evidence for Rous’ stay in Dresden. The picture deserves a place in a future biography of Rous to be written by historians of science. Schmorl, the passionate photographer, was probably responsible for the carefully staged arrangement of people and objects in the photograph.

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Fig. 1.7 Excerpts from autopsy reports written by Rous 1907 in Dresden. Reproduction with the permission of the Archive of the Institute of Pathology “Georg Schmorl,” Dresden Friedrichstadt Municipal Hospital, Germany

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Fig. 1.8 Christian Georg Schmorl (fourth from left, seated) surrounded by visiting researchers (among them, the almost 28-year-old Peyton Rous, third from left) and some assistants. Pathological– Anatomical Institute of Dresden Friedrichstadt Municipal Hospital, summer 1907. Gustav Molineus (1880–1954), later professor of medicine in Düsseldorf, is standing to the left of Rous. Second from the right is Curt Oehme (1883–1963), later professor of internal medicine in Heidelberg. Details regarding the other people depicted in the photograph are not preserved. The photograph is clearly posed, as can be seen in the symmetrical arrangement of the people, among other things. The pathologists’ most important equipment of that time (microscope, hand microtome, staining solutions, and solvents) round off the picture. Reproduction with the permission of the Archive of the Institute of Pathology “Georg Schmorl,” Dresden Friedrichstadt Municipal Hospital, Germany

In view of the severe destruction through the devastating air raids in Dresden in February 1945 and the worst flood of the century in August 2002, which did not exclude Dresden Friedrichstadt Municipal Hospital, it is a stroke of good fortune that these documents could survive the past 100 years. “No hint of war in the air,” is how Rous in 1966 melancholically remembered the undamaged Dresden of 1907 (Anonymous 1966). On June 15, 1907, an article by Rous was published in the Journal of Infectious Diseases in which he suggested an improvement of Schmorl’s celloidin-plate method (Rous 1907). This enabled the simultaneous staining of many paraffin sections with additional dyes, making the overall staining procedure more efficient.

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This work, which apparently already originated in Ann Arbor, must have pleased Schmorl, the methodologist in quest of perfection. In a way, it was the admission ticket for Rous. From reports of other Schmorl students (Turnbull 1932; Geipel 1934; Junghanns 1983), we know a great deal about the work flow in the Schmorl Institute. Every coworker had to prepare his microscopic specimens himself, cut and stain them as well as obtain the necessary tools (staining solutions, microscope slides, and glass covers) himself. Although very many autopsies had to be performed, Schmorl found the time to discuss the results in the autopsy room every day and to give important information to the other autopsists. On Saturday mornings, the always well-attended demonstrations took place in the auditorium, which physicians from other institutions were allowed to attend as well. “Schmorl’s teaching was of the nature of personal coaching, and his pupils, who were all graduates, had the inestimable advantage of learning his method of working. […] He gave without stint from his store of knowledge, whether published or not, to any who wanted to learn” (Turnbull 1932: 984). Supposedly, Rous learned quite a lot about how to present results as well. Many of his later papers were “illustrated with excellent photographs and microphotographs […]. Descriptions were detailed, for Rous liked to have his observations fully documented, and the accounts were often full of vivid imagery” (Andrewes 1971: 648). Like Schmorl, he “photographed with meticulous attention to detail” (ibid: 652). Besides pathology, bacteriology was another key area in the Schmorl institute. A bacteriological research center was opened in 1897 in the Dresden Municipal Hospital, subordinate to the Pathological Institute and thus to Schmorl. By 1907, the number of examinations had increased to almost 7,000 per year. Among them were cases of important infectious diseases, such as diphtheria, typhoid fever, cholera, infection of wounds, anthrax, tetanus, influenza, pneumonia, tuberculosis, syphilis, and rabies. Therefore, Rous must have learned many new things about infectious diseases. This may have been unexpected for him, since for a long time his teacher Welch had not considered the etiological role of bacteria as having any particular significance (Temkin 1950) and Warthin had concentrated solely on research on syphilis and tuberculosis (Anonymous 1932; Rous 1936). It was also not clear at that time if the still young field of bacteriology was to be considered part of pathology or rather of hygiene. In any case, the broadening of Rous’ horizon very soon had consequences for his own research. Even in 1923, Rous commented on his own “inborn lack of aptitude for bacteriology” (Rous to Gye, quoted from Becsei-Kilborn 2010: 141). As Warthin had intended, the stay in Dresden superbly rounded off Rous’ training. Here, he experienced methodological perfection using state-of-the-art technology. After Welch and Warthin, Schmorl was another research personality who had a profound impact on his further career. Following the extraordinary achievements of bacteriology at the end of the nineteenth century and the associated perception that many diseases had a monocausal etiology, around 1900, many hopes were also placed on finding a rapid solution to the problem of cancer. In the following years, it was up to Rous to work out the first evidence of a multicausal explanation of carcinogenesis in order to later come to the confident realization that under natural conditions malignant tumors gradually develop in a multifactorial process (Rous 1967b).

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Simon Flexner and Medical Discovery During his studies, Rous was infected with tuberculosis bacteria while performing an autopsy, developed lymph-node TB, and was forced to interrupt his studies for a year. After his stay in Dresden, he was diagnosed with pulmonary tuberculosis, which forced him to pause again, but fortunately he recovered quickly. Afterward, it was Warthin, once again, who gave him a crucial bit of information: Dr. Warthin told Peyton Rous that the Rockefeller Institute for Medical Research [in New York] was casting a wide net of grants for beginners, and he asked him if Peyton would like him to apply for one that would free Peyton for experimental work. That grant enabled Rous to find out enough about lymphocytes to be deemed worth publishing in the Journal of Experimental Medicine [Rous published in 1908 three papers on lymphocytes in this journal], edited by Simon Flexner, who was also the director of the Institute; and after another few months Flexner asked Rous to take over the laboratory for cancer research [at the Department of Bacteriology and Pathology] which Flexner was quitting to learn more about poliomyelitis, then crippling many American children. (Anonymous 1966)

The Rockefeller Institute for Medical Research (from 1964 on, Rockefeller University) was founded in 1901 as a private institute by John D. Rockefeller (1839–1937) and opened in 1904. It was the first American institute that exclusively engaged in biomedical research. At first, research on infectious diseases and bacteriology was the focus of interest; however, the problem definition was very broad and comprised many branches of basic biological research (Corner 1964; Hollingsworth 2004). The founding director was the pathologist Simon Flexner (1863–1946) (Fig. 1.9), who held this position from 1901 to 1935 with great success. In his view,

Fig. 1.9 Photograph of Simon Flexner. Courtesy of the Rockefeller Archive Center

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the institute was “an attempt to add knowledge by discovery and to apply that knowledge to the prevention and alleviation of disease” (Rous 1949: 416). Flexner had studied pharmacy and medicine at the University of Louisville, and earned his PhD in 1889. As 27-year-old, he came to William Welch at The Johns Hopkins University in Baltimore and there experienced the stimulating founding phase of the School of Medicine. Welch became his teacher, promoter, and later his fatherly friend; Flexner soon became Welch’s closest colleague. The focus of Flexner’s research was on experimental pathology, bacteriology, and immunology. Because of his expertise in these areas, he was repeatedly appointed head of commissions to combat epidemics at home and abroad. In the years 1893 and 1909, he spent some time in Europe in order to work with Friedrich Daniel von Recklinghausen (1833–1910) in Strasbourg and later with Emil Fischer (1852–1919) and Ernst Leopold Salkowski (1844–1923) in Berlin, among others. Prior to becoming director of the Rockefeller Institute, he had been professor for Pathological Anatomy at The Johns Hopkins University since 1899 and since 1900 Head of the Department of Pathology at the University of Pennsylvania (Corner 1972). As researcher, Flexner became known especially for his work on epidemic cerebrospinal meningitis. At the Rockefeller Institute, he developed a serum treatment (Flexner’s serum), which proved successful first with monkeys and later, during an epidemic, with humans. A treatment with serum from immunized horses was introduced simultaneously and independently of Flexner by the German physician Georg Jochmann (1874–1915). Flexner’s research on poliomyelitis was of particular importance for Rous and the work that he had already begun at the Rockefeller Institute. At that time, outbreaks of polio were a major problem, not only in America. From 1908 to 1909, the team led by Flexner identified a filterable virus (!) as responsible agent of the disease [the RNA virus which was discovered independently in Vienna by Karl Landsteiner (1868–1943) and Erwin Popper (1879–1955) is today assigned to the Picornaviridae family]. Flexner and colleagues also identified the transmission path of the virus. They showed that it enters the body through the nose, attacking the olfactory nerve. They could also experimentally infect monkeys with poliomyelitis by administering the virus in the nasopharynx (Flexner and Lewis 1909). For his work on serum treatment of epidemic cerebrospinal meningitis and on transmission of poliomyelitis to monkeys, Flexner was nominated ten times for a Nobel Prize in Physiology or Medicine (Nomination Database 1901–1951), but did not get the Prize. However, contemporaries and historians regard Flexner’s greatest achievement to lie in the organization of medical research and especially in the unprecedented success of the Rockefeller Institute (Corner 1964; Hollingsworth 2004). “Perhaps no man save Welch has done so much for American medicine” (Rous [commenting on Flexner] 1948: 613). Although many other high-ranking researchers had worked at the Rockefeller Institute for a long time and could have been considered to pay tribute to Flexner in obituaries, this task fell on Peyton Rous (Rous 1948, 1949). He did it in a loving way. “He [Flexner] had proved that the experimental method can meet human needs if it be given its head, wide and free; and he had shown that discoverers can be discovered” (Rous 1948: 613).

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At the beginning, Flexner had also been head of a laboratory for cancer research at the Rockefeller Institute. Together with James W. Jobling (1876–1961), he discovered a transplantable tumor, a rat adenocarcinoma most likely of prostatic origin (Flexner–Jobling carcinoma). This particular tumor has served for many decades as a unique test material in cancer research (Triolo 1964: 12; Corner 1964: 59), for instance in the famous experiments of Otto Warburg on the energy metabolism of cancer cells. Because of urgent work in other areas, particularly in the research of poliomyelitis, Flexner gave up his position as head of the laboratory, after he had found an appropriate successor in the then 30-year-old Rous. However, a number of obstacles had to be overcome before Rous could take over. Rous later described that at this time youngsters were warned off from the Institute with the slurring phrase “Rockefeller money” (Rous 1949: 418). Since cancer research was considered a futile field among scientists, even Welch had implored him: “Whatever you do, don’t commit yourself to the cancer problem” (Andrewes 1971: 644). Luckily, Rous did not follow this advice. Presumably, it was again Warthin who, besides Flexner, encouraged Rous despite all the prophecies of doom to turn toward cancer research. Toward the end of his life, Rous was able to acknowledge that “[Cancer is] the most intellectually worthwhile of all diseases” (Rous to Heagensen 1957, quoted in Becsei-Kilborn 2010: 117).

A Discovery Greeted with Skepticism At the Rockefeller Institute, Rous soon had the luck of the diligent: still in 1909, a poultry farmer consulted him who had noticed a big tumor in the chest of one of his chickens (a Plymouth Rock breed). Worrying that this could be a threat for other chickens of his flock, the breeder consulted different pathologists without success. Only Rous realized that this was a spindle cell sarcoma. This malignant tumor served Rous in the following years as a model for basic research studies; it was, therefore, later termed the Rous sarcoma (classic chicken sarcoma). To begin with, Rous succeeded for the first time in the transmission of a chicken tumor by means of injection into healthy animals – however, only when the recipients were genetically compatible animals (Rous 1910). If a homogenate of such tumors was passed through a filter that was not permeable to cells or bacteria, this filtrate in turn generated spindle cell sarcomas in healthy chicks after inoculation, which Rous as an experienced pathologist could clearly identify (Rous 1911a, b). Although he initially did not use the term “virus” to explain his results (he spoke cautiously only of a “filterable agent”), the chosen experimental setup did not allow for any other interpretation: for the first time, it was shown that a “real” tumor was caused by an infectious agent, probably a virus. Leukemias which showed similar results, however less convincing, were reported by Vilhelm Ellermann (1871–1924) and Oluf Bang (1881–1937) (Ellermann and Bang 1908). However, at that time, leukemias were not considered to belong to the neoplastic diseases. In the 1890s, viruses as novel biological entities were recognized for their characteristic of passing through

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bacteria-proof filters while preserving their biological activity. In 1911, Rous published his findings. He soon succeeded in developing a detection method for the agent on the chorioallantoic membrane of the chicks. Shortly thereafter, he proved a virus etiology for two further distinct chicken sarcomas. Today, the term Rous sarcoma virus (RSV) not only includes Rous’ original isolates, but numerous other chicken viruses that have been isolated independently from each other and that induce sarcomas through a genetic mechanism similar to the original isolate. RSV was the first known tumor virus and the first representative of the retroviruses. The initial euphoria about Rous’ discovery was soon followed by disillusionment. Apparently, viruses were not generally responsible for the induction of tumors. Also, the virus theory could hardly be brought into conformity with contemporary beliefs about the causal genesis of cancer. Furthermore, with the outbreak of World War I, Rous had to devote himself to other subjects (among them, with great success, the conservation of blood). Recent historical research substantiates that Rous’ discovery triggered a longlasting scientific discussion (van Helvoort 1999, 2004; Becsei-Kilborn 2010). However, a detailed description of this discussion would go beyond the framework of this article. Rous found himself facing considerable skepticism, even resistance. Even James B. Murphy (1884–1950), temporarily his close colleague when carrying out these experiments and who later gained increasing influence in the US cancer research community, did not believe in the involvement of viruses, but interpreted the agent as a “transmissible mutagen.” Later, he thought to have proved it to be a ferment. Rous’ sharpest critic was James Ewing (1866–1943), who was very conscious of his power as pathologist and director of research at the Memorial Hospital for Cancer and Allied Diseases in New York City. Ewing largely rejected experimental pathology for the research of cancer etiology and believed the origin of cancer to be within the cell itself. Frequent points of criticism by other scientists about Rous’ experiments were: (1) when preparing the filtrate, a few tumor cells could have passed through the filter; (2) the effect might have been caused not by an infectious agent, but by products synthesized by tumor cells; (3) it was doubted that the induced sarcomas were real tumors, and it was suggested that they should rather be seen as “granulomas”; (4) since the agent could not infect most cells – with the exception of certain chicken cells – the high specificity of its effect remained enigmatic; and (5) whether tumor development could be caused by external factors at all or if tumors develop in an endogenous way was not decided at that time. However, the belief favoring purely endogenous reasons prevailed at that time. Although Rous could dispel some objections, he did not succeed in convincing his critics. All in all, the results were for a long time considered an oddity of chickens that were of no relevance to the situation in humans. But there were also advocates for Rous, in America most notably Flexner and the highly respected Leo Loeb (1869–1959). In Japan, Akira Fujinami (1870–1934) found a very similar agent in chicken sarcomas that is known today as Fujinami virus (Fujinami and Inamoto 1914). From the mid 1920s on, much-discussed studies by the British scientists Willam E. Gye (1884–1952) and Christopher H. Andrewes (1896–1988) kept Rous’ experiments from being forgotten. Rous himself did not take up work in this area until 1933, after Richard E. Shope (1901–1966) had

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succeeded with a cell-free transmission of papillomas of cottontail rabbits (Shope 1933). Shope left the alleged virus tumor to his colleague Rous for further experiments. The model gave Rous the opportunity to study many characteristics of the natural development of tumors. Under special conditions, real carcinoma developed from the papilloma. Rous found that tumors develop gradually. A phase of tumor initiation is followed by phases of promotion and progression up to the fully developed metastasizing tumor (Rous and Kidd 1941). This may cause a synergistic effect of viruses and chemical carcinogens. The terms “latent or dormant tumor cells” and “cancer as a multifactorial disease” were also introduced by Rous on the basis of these experiments (see Rous 1967b). The real breakthrough of the virus theory of cancer came in the 1950s. In 1951 and 1954, respectively, Ludwik Gross (1904–1999) in New York and Arnold Graffi (1910–2006) in Berlin were able to prove that viruses caused lymphatic (Gross 1951) and myeloid leukemia (Graffi et al. 1954) in mice. Numerous other isolations of DNA- or RNA-containing oncogenic viruses were made in a variety of animal species. Many of these became outstanding models of the emerging molecular biology. The golden age of tumor virology had begun. Rous had always believed in the viral nature of his agent. In a letter to his British colleague Stephen L. Baker (1888–1978), he confessed in 1930: “My own belief has always been that the agents causing these tumors are viruses.” But at that time, he had to continue carefully “[…] though the statement is confidential to you” (quoted from Becsei-Kilborn 2010: 132).

The Great Good Fortune of Rous I will always consider it good fortune that just when I finished my apprenticeship in physics, chemistry and medicine, the Kaiser Wilhelm Institute for Biology was founded in Berlin, and that upon its founding Emil Fischer, then Vice President of the Kaiser Wilhelm Society, forthwith appointed me as scientific member [upon the recommendation of Theodor Boveri (1862–1915)]. […] I have no doubt that my scientific achievements can mainly be attributed to the unusual degree of freedom and independence that I […] have received. [German in original]

This is how Otto Warburg (1883–1970) commented in retrospect on his career, according to his student and biographer Sir Hans Krebs (1900–1981) (Krebs 1978: 351; Krebs 1981). Warburg and Rous, who were almost the same age, were probably the most important cancer researchers of the early twentieth century (Fujimura 1996a, b). Like Warburg, Rous had completed a long period of apprenticeship served with Welch, Warthin, and Schmorl when he joined the new Rockefeller Institute, in which he then (from 1920, as member) was to experience an impressive career. Rous, too, always regarded these circumstances as good fortune: “Environment is everything for a scientific man,” he wrote (Rous 1949: 412). Both scientists benefited from developments that took place in these years. On the one hand, the character of cancer research changed: “Cancer research became an experimental science at the turn of the century, along with most of the biological

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sciences” (Fujimura 1996b: 247). On the other hand, novel institutions were founded for nonuniversity research: at first, the Rockefeller Institute for Medical Research in New York which later served as a model for the foundation of the German Kaiser Wilhelm Society with its various institutes. Therewith, a culture of excellence was institutionalized (Hollingsworth 2004) that was to lead to great achievements in the cases of Rous and Warburg: “Discovery [is] the most satisfying of human experiences” (Rous 1949: 423). However, the priorities which Flexner set from the beginning for the Rockefeller Institute were also a stroke of good fortune for Rous. Until the end of Flexner’s term of office, the role of pathology was sacrosanct: “Pathology is far more important for us than physiology and pharmacology, and the background of medicine than general science. Our pathologists are all moving on; pathology is the fundamental branch of medicine” (Flexner 1993, quoted from Corner 1964: 187). This was in the spirit of Rous, who told Jacob Furth (1896–1979) in 1942: “Experimental pathology has always been to me one of the most exciting of human activities” (quoted from Becsei-Kilborn 2010: 116). As mentioned before, Flexner was also a major proponent of virus research. At precisely the time when Rous began his research on chicken sarcoma, the studies of Flexner’s team reached a milestone with the evidence of a virus etiology of poliomyelitis. Rous’ decisive proposition – a cell-free transmission of chicken sarcoma – probably arose out of discussions within the Rockefeller Institute. Fortunately, Rous could count on Flexner’s continuing support during the yearlong controversies on the significance of his discovery. Rous himself was very confident early on. The identification of the chicken sarcoma had already been a considerable achievement in 1909 (very little research had been done on poultry tumors at that time), and he was now able to fend off his critics because of his versatility and expertise as a pathologist. He was aware that the nature of the tumors induced by cell-free transmission was a decisive argument. Evidence of their malignancy was in every respect unambiguous: cell-free filtrates of the same tumor induced reproducible, histologically identical neoplasms, each independently obtained agent induced tumors of various types, and all induced tumors had the ability for invasive growth and metastasizing, i.e., they were genuine malignant neoplasms. At the latest during these experiments, it became clear that Peyton Rous had been very fortunate in the choice of his teachers. He kept them in honorable memory throughout his life. In his obituaries for Simon Flexner, he also remembered William H. Welch, who had imparted a vision of an experimental pathology to the young Rous and who let him take part in the vibrant ambiance of scientific discovery (Rous 1948, 1949). Rous was indebted to Aldred S. Warthin for a thorough education as pathologist and for key professional guidance, such as providing the contact to Schmorl and Flexner, as well as encouraging the shift to cancer research. He commemorated him in a beautifully written obituary (Rous 1936) and as late as 1967 alluded to long-forgotten work of his teacher (Rous 1967a). In Dresden, he encountered European science and culture and was full of praise even 60 years later (Anonymous 1966). He studied the advances in bacteriology in Germany under Georg Schmorl. In the working method of Schmorl, he saw methodological perfection

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combined with the use of modern technical (especially photographic) equipment. Thus, he was well-prepared to seize the opportunities that were offered to him at the Rockefeller Institute. Among his teachers and promoters, it was Simon Flexner, who discovered the discoverer in Rous, whom he had known the longest and probably revered the most. He commemorated him in obituaries that are well worth reading even today (Rous 1948, 1949). Flexner was the guarantor of freedom of research at the Rockefeller Institute, and this proved to be of crucial importance for the seminal discovery of tumor-inducing viruses.

Epilogue Although Peyton Rous is first and foremost known as the “father of the tumor virus” (van Epps 2005; Moore and Chang 2010), his name is also associated with other important achievements. At the Rockefeller Institute, his well-rounded education bore rich fruit. During World War I, the preservation of living blood cells was an urgent medical need. Together with Joseph Richard Turner, Jr. (1889–?), Rous developed a method which enabled long-term storage of blood without clotting. “In a mixture of 3 parts of human blood […], 2 parts of isotonic citrate solution […] and 5 parts of isotonic dextrose solution […], the cells remain intact for about 4 weeks” (Rous and Turner 1916). This method enabled Oswald H. Robertson (1886–1966), the pioneer of transfusion medicine, to set up the world’s first blood bank behind the front line in Belgium in 1917. Rous cooperated closely with Robertson, as a number of joint publications demonstrate. The “Rous–Turner solution” is still in use for human blood transfusions, and in the 90 years since its first use it has saved countless lives. Rous was particularly proud of this achievement – and rightly so. A cell and tissue culture method which has been a laboratory standard up to the present day can also be traced back to Rous’ research. To obtain a suspension of individual living cells from the fixed tissue, he performed a digestion with trypsin (Rous and Jones 1916). The optimal trypsin concentration had to be determined separately for each kind of tissue. Once again, with this method, Rous proved his talent for finding solutions that could be carried out easily. In conjunction with his work on blood conservation, Rous also focused on the functions of the liver, gall bladder, and kidney as well as on the permeability of small vessels. The Rous test for hemosiderin in urine to detect hemosiderosis (Rous 1918) was one of the results of his research at that time. Any appraisal of Rous would be incomplete if it neglected to draw attention to Rous’ great merits as long-time editor (from 1922 to 1970) of the renowned Journal of Experimental Medicine. He invested a lot of time and energy in this task. According to contemporary witnesses, he continued working well into old age and was equally meticulous in revising manuscripts as he was in conducting experiments in the laboratory. Both activities – along with his modesty – contributed to Rous’ extraordinary reputation. As a young man, Cornelius Rhoads (1898–1959) experienced Rous as he revised his manuscript, and Rhoads described this later

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quite humorously: “Dr. Rous, gravely and patiently, reviewed my efforts with me, demolished my conclusions, refuted my claims and made clear the proper use of my native tongue. He then rebuilt on the ruins such a clear picture of the problem, and the procedure required to solve it, that my conceit was converted almost imperceptibly to inspiration, my enthusiasm to resolution. As I left the generous, patient, and kindly man, I was no longer the same individual” (Rhoads 1959). Time and again during the twentieth century, the RSV as a model for fundamental studies proved to be a serendipitous choice, and its scope extended far beyond oncology. After Rous, several scientists received the Nobel Prize in Medicine for studies in which this virus played a decisive role. In 1974, one of the prize winners was Albert Claude (1899–1983), who pioneered the fractionation of cells by differential centrifugation. This discovery was the prerequisite for making images of the structure of the RSV, among others, using an electron microscope – the first time for a tumor virus (Claude et al. 1947). Howard M. Temin (1934–1994) and David Baltimore (born 1938) discovered reverse transcriptase and were honored for their work in 1975. Their research was conducted with Rous sarcoma and Rauscher murine leukemia viruses (Temin and Mizutani 1970; Baltimore 1970). J. Michael Bishop (born 1936) and Harold E. Varmus (born 1939) received the Nobel Prize in 1989 for the discovery of cellular oncogenes. They were able to identify the cellular origin of retroviral oncogenes, among others, first for the src gene, the viral homolog of which is responsible for the transforming activity of the RSV (Stehelin et al. 1976). For their discovery of human immunodeficiency virus (Barré-Sinoussi et al. 1983), today besides the RSV the most well-known retrovirus, Françoise Barré-Sinoussi (born 1947) and Luc Montagnier (born 1932) were awarded the Nobel Prize in 2008 along with Harald zur Hausen. Other equally important discoveries in the field can also be mentioned, further substantiating George Klein’s appraisal: “Few fields in modern biology and certainly no other field in cancer research can be traced back to the work of one man in the same way that the foundation in the field of viral oncology can be traced back to the work of Peyton Rous in 1911” (Klein 1980). Acknowledgments We thank Carol Oberschmidt (Berlin) for translation and Professor Manfred F. Rajewsky (Essen) for critical reading of the manuscript.

References Andrewes CH (1971) Francis Peyton Rous 1879–1970. Biographical Memoirs of Fellows of The Royal Society 17:643–62 (with bibliography) Anonymous (1932) Aldred Scott Warthin. J Pathol Bacteriol 35:133–5 Anonymous (1966) Peyton Rous. Biography. The Nobel prize in physiology or medicine 1966. http://nobelprize.org/nobel_prizes/medicine/laureates/1966/rous-bio.html. Accessed 20 Jan 2011 Baltimore D (1970) RNA-dependent DNA-polymerase in virions of RNA tumour viruses. Nature 226:1209–11 Barré-Sinoussi F, Chermann JC, Rey F, Nugeyre MT, Chamaret S, Gruest J, Dauguet C, Axler-Blin C, Vézinet-Brun F, Rouzioux C, Rozenbaum W, Montagnier L (1983) Isolation of a

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T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS). Science 220:868–71 Becsei-Kilborn E (2010) Scientific discovery and scientific reputation: the reception of Peyton Rous’ discovery of the chicken sarcoma virus. J Hist Biol 43:111–57 Brieger GH (1970) Welch, William Henry. Dictionary of Scientific Biography 14:248–50 Claude A, Porter KR, Pickels EG (1947) Electron microscope study of chicken tumor cells. Cancer Res 7:421–30 Corner GW (1964) A history of the Rockefeller Institute, 1901–1953. Origins and growth. New York, Rockefeller Institute Press Corner GW (1972) Flexner, Simon. Dictionary of Scientific Biography 5:39–41 Douglas JA, Gruber SB, Meister KA et al (2005) History and molecular genetics of Lynch syndrome in family G: a century later. J Am Med Assoc 294:2195–202 Dresdner Toten Tanz (the Dresden dance of death) [in German]. http://www.derevo.org/common/ de/actions/projects/totentanz/tt1.html. Accessed 20 Jan 2011 Dulbecco R (1976) Francis Peyton Rous. October 5, 1879–February 16, 1970. Biogr Mem Members Natl Acad Sci 48:275–306 (with bibliography) Ellermann V, Bang O (1908) Experimentelle Leukämie bei Hühnern. Zentralbl Bakteriol 46:595–609 [in German] Flexner S (1943) William Henry Welch 1850–1934. Biogr Mem Natl Acad Sci 22:215–31 Flexner S, Flexner JT (1941) William Henry Welch and the heroic age of American medicine. New York, Viking Press [Reprinted 1966] Flexner S, Lewis PA (1909) The nature of the virus of epidemic poliomyelitis. J Am Med Assoc 53:2095 Fujimura JH (1996a) Standardizing practices: a socio-history of experimental systems in classical and virological cancer research, ca. 1920–1978. Hist Philos Life Sci 18:3–54 Fujimura JH (1996b) Crafting science. A sociohistory of the quest for the genetics of cancer. Harvard University Press, Cambridge, MA; London, UK Fujinami A, Inamoto K (1914) Über Geschwülste bei japanischen Haushühnern insbesondere über einen transplantablen Tumor. Z Krebsforsch 14:94–119 [in German] Geipel P (1934) Georg Schmorl. Verh Dtsch Pathol Ges 27:326–39, (with bibliography) [in German] Graffi A, Bielka H, Fey F et al (1954) Gehäuftes Auftreten von Leukämien nach Injektion von Sarkom-Filtraten. Naturwissenschaften 41:503–4 [in German] Gross L (1951) “Spontaneous” leukemia developing in C3H mice following inoculation, in infancy, with Ak-leukemic extracts, or Ak-embryos. Proc Soc Exp Biol Med 76:27–32 van Helvoort T (1999) A century of research into the cause of cancer: is the new oncogene paradigm revolutionary? Hist Philos Life Sci 21:293–330 van Helvoort T (2004) The start of a cancer research tradition: Peyton Rous, James Ewing, and viruses as a cause of cancer. In: Stapleton DH (ed) Creating a tradition of biomedical research: contributions to the history of The Rockefeller University. The Rockefeller University Press, New York, pp 191–209 Hollingsworth JR (2004) Institutionalizing excellence in biomedical research: the case of The Rockefeller University. In: Stapleton DH (ed) Creating a tradition of biomedical research: contributions to the history of The Rockefeller University. The Rockefeller University Press, New York, pp 17–64 Huggins CB (1970) Peyton Rous and his voyages of discovery. J Exp Med 150:734–5 Junghanns H (1983) Georg Schmorl, der Forscher und Lehrer (2.5.1861–14.8.1932). Medizinhist J 18:324–37 [in German] Klein G (1980) Introduction. In: Klein G (ed) Viral oncology. Raven Press, New York Krebs H (1978) Otto Warburg: Biochemiker, Zellphysiologe, Mediziner. Naturwiss Rundsch 31:349–56 [in German] Krebs H (1981) Otto Warburg: cell physiologist, biochemist, and eccentric. Oxford University Press, Oxford, UK Kunze P (1999) Vom Adelspalais zum Städtischen Klinikum. Geschichte des Krankenhauses Dresden-Friedrichstadt. Krankenhaus Dresden-Friedrichstadt, Dresden [in German]

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Lawley PD (1994) Historical origins of current concepts of carcinogenesis. Adv Cancer Res 65:17–111 Lynch HT (1985) Classics in oncology. Aldred Scott Warthin, M.D., Ph.D. (1866–1931). CA: Cancer J Clin 35:345–7 MacCallum WG (1936) Welch, William Henry. Dictionary of American Biography 19:621–4 Moore PS, Chang Y (2010) Why do viruses cause cancer? Highlights of the first century of human tumour virology. Nat Rev Cancer 10:878–89 Nomination Database for the Nobel Prize in Physiology or Medicine, 1901–1951. http://nobelprize.org/nobel_prizes/medicine/nomination/database.html. Accessed 20 Jan 2011 Pathologie-Sammlung “Georg Schmorl”. Technische Universität Dresden [in German] http://publicus.culture.hu-berlin.de/sammlungen/detail.php?dsn=766&view=2. Accessed 20 Jan 2011 Rhoads CP (1959) Citation and presentation of the Academy Medal to F. Peyton Rous, M.D. Bull N Y Acad Med 35:216–9 Rous FP (1907) A method for the simultaneous passage of many paraffin sections through the more difficult stains. J Infect Dis 4:382–4 Rous FP (1910) A transmissible avian neoplasm (sarcoma of the common fowl). J Exp Med 12:696–705 [Reprinted: (1979); ibid 150:729–753] Rous P (1911a) Transmission of a malignant new growth by means of a cell-free filtrate. J Am Med Assoc 56:198 [Reprinted: (1983) ibid 250:1445–1449 and http://caonline.amcancersoc.org/ cgi/reprint/22/1/23. Accessed 20 Jan 2011] Rous P (1911b) A sarcoma of the fowl transmissible by an agent separable from the tumor cells. J Exp Med 13:397–411 [Reprinted at http://www.euchromatin.com/Rous11b.htm. Accessed 20 Jan 2011] Rous P (1918) Urinary siderosis. Hemosiderin granules in the urine as an aid in the diagnosis of pernicious anemia, hemochromatosis, and other diseases causing siderosis of the kidney. J Exp Med 28:645–58 Rous P (1929) The modern dance of death. Cambridge University Press, Cambridge, UK Rous P (1936) Warthin, Aldred Scott. Dictionary of American Biography 19:493–4 Rous P (1941) The William Henry Welch lecture. I. The conditions determining cancer. II. The known causes of cancer. J Mt Sinai Hosp 8:184–7 Rous P (1948) Simon Flexner and medical discovery. Science 107:611–3 Rous P (1949) Simon Flexner. 1863–1946. Obituary Notices of Fellows of The Royal Society 6, No. 18:408–445 (with bibliography) Rous P. (1966) The dualism of the discoverer. In: Heymann G (ed) Festschrift anläßlich der Verleihung des Paul-Ehrlich-und Ludwig-Darmstaedter-Preises 1966 an Prof. Dr. Peyton Rous. Gustav Fischer, Stuttgart, pp 20–30 [text in German, the English version is unpublished]. Andrewes (1971) and Dulbecco (1976) did not include this paper in the list of all works by Rous. Rous P (1967a) Symposium on RNA viruses and neoplasia: comment. Proc Natl Acad Sci USA 58:843–5, Andrewes (1971) and Dulbecco (1976) did not include this paper in the list of all works by Rous Rous P (1967b) The challenge to man of the neoplastic cell. Nobel Prize lecture. Cancer Res 27:1919–24 Rous P, Jones FS (1916) A method for obtaining suspensions of living cells from the fixed tissues, and for the plating out of individual cells. J Exp Med 23:549–55 Rous P, Turner JR (1916) The preservation of living red blood cells in vitro I. Methods of preservation. J Exp Med 23:219–37 Rous P, Kidd JG (1941) Conditional neoplasms and subthreshold neoplastic states. A study of the tar tumors of rabbits. J Exp Med 73:365–90 Russell DS (1956) Hubert Maitland Turnbull. J Clin Pathol 9:78–9 Russell DS (2004) Turnbull, Hubert Maitland (1875–1955). Oxford Dictionary of National Biography 55:590–1 Scholz A (2007) Schmorl, Christian Georg. Neue Dsch Biogr 23:263–4 [in German]

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Shope RE (1933) Infectious papillomatosis of rabbits. J Exp Med 58:607–24 Stehelin D, Varmus HE, Bishop JM, Vogt PK (1976) DNA related to the transforming gene(s) of avian sarcoma viruses is present in normal avian DNA. Nature 260:170–3 Storey GO (2008) Hubert Maitland Turnbull (1875–1955): pathologist at The London Hospital. J Med Biogr 16:227–31 Temin HM, Mizutani S (1970) RNA-dependent DNA-polymerase in virions of Rous sarcoma virus. Nature 226:1211–3 Temkin O (1950) The European background of the young Dr Welch. Bull Hist Med 24:308–18 Triolo VA (1964) Nineteenth century foundation of cancer research. Origins of experimental research. Cancer Res 24:4–27 Turnbull HM (1932) Christian Georg Schmorl. J Pathol Bacteriol 35:982–5 Van Epps HL (2005) Peyton Rous: father of the tumor virus. J Exp Med 201:320 Vogt PK (1996) Peyton Rous: homage and appraisal. FASEB J 10:1559–62 Warthin AS (1907) Leukemia of the common fowl. J Infect Dis 4:369–81 Warthin AS (1913) Heredity with reference to carcinoma. Arch Intern Med 12:546–55 Warthin AS (1925) The further study of a cancer family. J Cancer Res 9:279–86 Warthin AS (1931) The physician of the dance of death. A historical study of the evolution of the dance of death mythus in art. Hoeber, New York Wunderlich V, Kunze P (2008) Vor 101 Jahren: Peyton Rous, ein zukünftiger Nobelpreisträger, im Krankenhaus Dresden-Friedrichstadt. Ärzteblatt Sachsen 19:375–81 [in German]

Chapter 2

Viruses and Cancer: A Historical Perspective – HBV and Prevention of a Cancer Baruch S. Blumberg

Introduction Viruses are thought to be the causative agent of about 15–20% of cancers, including some of the world’s most common cancers. As is the case for most diseases, cancers have multiple causes or factors that contribute to etiology, pathogenesis, prognosis, and response to treatment. In addition to the cancers with identified viral causes, there are probably additional cancer disease entities in which viruses contribute to pathogenesis and in which prevention of infection may significantly alter the outcome. In recent years, there have been reports, primarily in the general press, on the disaffection with the progress of cancer control and treatment. These often refer to the so-called War on Cancer (although that term was not the official designation) initiated on December 23, 1971 and, as perceived by the public, its failure to have a dramatic effect on control and treatment. There have been improvements in treatments for many cancers; for several childhood and usually relatively rare adult cancers, therapies have been impressive. But for many cancers, treatment has resulted in only limited increased survival and the treatments themselves may diminish the quality of life. The search for better treatments is a major research program. That War on Cancer campaign started with many goal-directed research programs. Viral-caused cancer was one of the goal-directed projects based on the models from virus/cancer relations in experimental, domestic, and wild animals. However, human cancers did not appear to closely follow the animal models, and by the mid-1970s support for virus-caused cancers in humans had diminished. But, as in many research areas, interest appears to wax and wane over the years. As evidenced by this book and others on the same topic, the increase in the number of research groups studying cancer and infectious agents, interest is growing once again.

B.S. Blumberg (*) Fox Chase Cancer Center, Philadelphia, PA 19111, USA e-mail: [email protected] E.S. Robertson (ed.), Cancer Associated Viruses, Current Cancer Research, DOI 10.1007/978-1-4614-0016-5_2, © Springer Science+Business Media, LLC 2012

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Prevention has had a major effect on decreasing the load of cancer in human populations. Primary prevention, for example smoking cessation programs, has resulted in major decreases in the incidence of cancer of the lung and other cancer as well as noncancer diseases. Dietary measures have probably decreased cancer of the colon and other organs. Cancer of the stomach has decreased dramatically, presumably as a consequence of changes in the environment, in diet, or some other factors that are not clearly identified. Secondary prevention, i.e., early detection and treatment, appears to have decreased the cancer load for cancers of the breast, colon, and others. The research on cancer-causing viruses (and other infectious agents) promises to facilitate even greater advances in prevention and treatment. The hepatitis B vaccine was invented in 1969 two years after the discovery and identification of hepatitis B virus (HBV). Product development began in the mid-1970s and it was approved for use in the early 1980s. It is now one of the most commonly used vaccines worldwide. The hepatitis B vaccination campaigns and other control measures have dramatically reduced the incidence of infections, the HBV carrier state, and acute and chronic liver disease. HBV, along with hepatitis C virus (HCV), is an etiological agent for over 80% of all primary cancers of the liver (hepatocellular carcinoma, HCC) and it is expected that in time there will be dramatic drops in the incidence of the cancer. Several studies have already shown significant decreases in the HCC incidence in countries with early and effective vaccination programs and a high incidence of the cancer. The CDC in its Hepatits B Vaccine Fact Sheet stated (Hepatitis B Vaccine: Fact Sheet First Anti-cancer Vaccine (2006) http:// www.cdc.gov/hepatitis, May 17, 2006): Hepatitis B vaccine prevents hepatitis B disease and its serious consequences like hepatocellular carcinoma (liver cancer). Therefore, this is the first anti-cancer vaccine.

The WHO has noted that, second to smoking cessation, HBV vaccination is the major cancer interventional prevention program. In 2007, about 25 years after the approval of HBV vaccine, a second cancer prevention vaccine was accepted by the FDA. Vaccines for several strains of papilloma virus have been shown to effectively prevent cancer of the cervix, other cancers, and common diseases of the reproductive system. Its use is now spreading widely as the appropriate populations for vaccination are being identified. As a consequence of these practical advances and a growing understanding of the viral-caused cancer process at the molecular level, we are now in a period of increased interest in cancer virology, as witnessed by this book and others on the subject, and the organization of new research groups. In this paper, I briefly review some of the established cancer virus relations and then discuss the research, public health, and clinical processes that led to the HBV vaccine and its consequences. The HBV story can serve as an example that can help to advance similar mass prevention programs. Most cancer therapies are dependent on either removing cancer by surgery or by the destruction of existing cancer cell with radiation, chemotherapy, or by altering the host immune system to reject cancer cells. For viral-induced cancers, these harsh methods may not be necessary. The cancer could be averted or delayed by the

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antiviral treatment of those already infected before symptoms appear and, possibly, also after clinical disease develops. This should be less toxic than many current therapies. The effectiveness of antiviral therapy would also be another direct demonstration of the etiological role of a virus. This is an exciting time for virus/cancer studies as the effectiveness of vaccines for at least two common cancers inspires a search for additional viral connections and applications.

Viral Origins of Human Cancers There are several excellent reviews of this subject, with an emphasis on molecular virology, for example Boccardo and Villa (2007) and DeVita et al. (2008). As Boccardo and Villa have noted, the strongest direct evidence for a meaningful etiologic relation is the prevention of the cancer by vaccination. This has been shown for HBV and HCC in large national studies (see, for example, Liang et al. 2009), and for papilloma virus [human papilloma virus (HPV)] and cancer of the cervix in field trials. As noted above, another direct demonstration of etiologic relation would be the prevention and/or treatment of a cancer with an antiviral. There are many other accepted virus/cancer etiologic relations. In many cases, they are believed to be indirect which appears to mean that they do not conform to molecular biological models of carcinogenesis. From a practical point of view, the question of a direct or secondary cause is not as important as an understanding of process that allows practical prevention or treatment. An important feature of cancer prevention by vaccination is the question of instituting universal vaccination to protect the relatively small portion of the population who develop cancer. However, vaccination is further justified if the program also protects against more common diseases. This is true for HBV vaccine, where protection is also offered against acute and chronic hepatitis, cirrhosis, and liver failure and probably some forms of kidney diseases, polyarteritis nodosa, and others. The same could be said for HCV if a vaccine is found for it. The HPV vaccine in addition to protecting against cancer of the cervix and other cancers protects against several common sexually transmitted and other diseases, including condyloma acuminate, warts, and recurrent respiratory papillomatosis. Vaccines are not currently widely used for other cancer-related viruses, but there is hope that they will be; other preventive methods against infectious agents can currently be used. The Epstein–Barr virus (EBV) is etiologically related to several cancers, some of them common in particular locations. They include nasopharyngeal carcinoma, Burkitt’s Lymphoma – one of the first virus cancer relations suspected on epidemiologic grounds – Hodgkin’s Disease, and several others. Intensive research on inventing a vaccine and determining its optimal use is in progress. Human T-cell leukemia virus (HTLV-1) is etiologically associated with adult T-cell leukemia as well as other noncancer diseases. These include HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), uveitis, and infective dermatitis.

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There are probably other causes of death associated with chronic HTLV-1 infection. In a historic prospective study in Zaire (now Democratic Republic of the Congo) where HTLV-1 is common (Lechat et al. 1997), after about 20 years, individuals who were HTLV-1 carriers in 1969 had a significantly lower survival compared to noncarriers. These studies confirmed a similar report from Japan. If further supported, this could justify the widespread use of an HTLV-1 vaccine when and if one is produced, in that vaccination could prevent many diseases in addition to the relatively rare leukemia and other identified associated diseases. HTLV-II has been isolated from a patient with hairy cell leukemia and there may be an etiologic relation of the virus with this disease. The EBV is associated with several cancers that are common in particular geographic regions. Denis Burkitt, a physician practicing and doing epidemiologic studies in equatorial Africa, described an unusual lymphoma, soon named after him. He showed that its distribution followed that of malaria and hypothesized that it had an infectious origin associated with mosquito transmission. Epstein and Barr, then, isolated a virus from the cells of a Burkitt’s lymphoma case and the virus was found to be extremely common in African cases. This was not true of sporadic cases found away from the tropics, suggesting multiple etiologies operating in different environment. EBV is also associated with nasopharyngeal carcinoma that is extremely common in China and elsewhere, and Hodgkin’s lymphoma. Human immunodeficiency virus types I and II (HIV-I, HIV-II) are associated with several cancers. These include other virus-related cancers: Kaposi’s sarcoma [associated with human herpes virus 8 (HHV-8) or Kaposi’s sarcoma herpes virus (KSHV)], Hodgkin’s lymphoma (associated with EBV), and cervical carcinoma (associated with HPV). The immune deficiency that characterizes AIDS may increase the susceptibility to these cancers, but there also appears to be an interaction of the genome of HIV in the cancer pathogenic process. These findings illustrate that the pathogenesis of viral-caused cancers may involve multiple viruses or other factors in pathogenesis. Humans have many more bacterial cells in their body than their “own” cells and even more viruses – and one or more of these may interact with the virus associated with the specific cancer. This may appear to make the problem incomprehensively complex, but the history of medicine has shown that effective measure can be found even though the entire process is not fully understood.

Discovery of the Hepatitis B Virus The discovery of the HBV did not start as a directed search for the virus but as a project in basic clinical research. It was a circuitous and convoluted process whose outcome would have been difficult to predict at its onset. (This and following sections are adapted in part from Blumberg 2002, 2010.) A striking feature of medicine is the great variation in host response to diseasecausing agents. Starting in 1957, we set out to find inherited polymorphic and acquired variation in the blood proteins that could be related to disease susceptibility

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(Blumberg 1961). This is analogous, at the phenotype (proteome) level, to the contemporary search for the relation between genomic polymorphic variation and disease using SNIPS and other databases. It was an interesting process as it required worldwide collections in different disease environments to compare the distribution of the polymorphic alleles and try to understand their relation to disease. Protein variation was determined using the recently introduced method of electrophoresis in gel. In 1961, another technique was deployed. Many serum proteins are polymorphic; hence, patients who had multiple transfusions would likely to be exposed to proteins they had not inherited. If some of the polymorphic proteins were antigenic, the patients might develop antibodies against them and their blood could be used as a “reagent” to discover and study antigenic protein polymorphisms. Using double diffusion in agar gel, we identified a complex, inherited antigenic system of the serum low-density lipoproteins (the “Ag System,” Allison and Blumberg 1961). Using these and other anti-lipoprotein antibodies identified by us (and to a greater measure by others), associations were found with cardiovascular disease, Alzheimer’s, and diabetes. We continued to test the sera of transfused patients against a panel of sera with the expectation that we would find additional antigen-antibody systems of interest. In 1963, a precipitin band was detected between the sera of a transfused hemophilia patient from New York and, among others, the sera of Australians (Blumberg et al. 1965). Much of the subsequent research was done on these sera; the reactant protein was called “Australia antigen” (Au). The next problem was to learn what it was. Au was rare in normal Americans but common in leukemia patients; this generated the hypothesis that people at high risk of leukemia might also have high frequencies of Au. Since patients with Down’s syndrome (DS, chromosome 21 trisomy associated with mental retardation) are at high risk for an unusual form of leukemia, we predicted that they would have a high frequency of Au; this was confirmed in a series of studies in large institutions for the mentally retarded (Blumberg et al. 1967) (Fig. 2.1). In the meanwhile, there were a series of observations that raised the possibility that Au was associated with hepatitis. It was found in transfused people, occasional patients with hepatitis, and in institutionalized patients (i.e., leprosy, mental retardation), where infectious spread would be expected. But, the most convincing observation was in a DS patient. He did not have Au when first seen but did so on subsequent testing; the appearance of Au coincided with the onset of hepatitis. We, then, formally tested the hypothesis that Au was associated with hepatitis by comparing the prevalence of Au in patients with and without clinical hepatitis; Au was much more common in the patients. The next series of tests were designed to determine if Au was a hepatitis virus or part of it. This was confirmed by clinical, electron microscope, transmission and other studies. The initial observations were soon confirmed by Okochi and Murakami (1968), Vierucci et al. (1968) and Prince (1968). Prince associated Au with the HBV that had been postulated by Krugman and other pioneers in the hepatitis field before our discovery of HBV. The identification of one virus facilitated the identification of others (HAV, HDV, HCV, HEV, etc.) in several other laboratories that greatly increased the probabilities of control and treatment of viral hepatitis (Fig. 2.2).

Fig. 2.1 Scan. ppt 112K. The first published image of the precipitin reaction in agar gel between “Australia antigen” (the surface antigen of HBV) in the top well, and the antibody against it in the bottom well. The top well contains serum form a patient with leukemia who is a carrier of HBV. The bottom well contains serum from a patent with hemophilia who has received many blood transfusions, some from blood donors who were HBV carriers, and developed antibodies against the surface antigen (Australia antigen)

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Fig. 2.2 Blumberg Surinam. ppt (299K). Surinam, South America, September 1950. The photograph was taken in a Djuka community near the mining town of Moengo on the Cottica River (Kotikaliba) during the course of an infectious disease survey and clinical care project. It was during these surveys that striking differences in response to infection were noted and reported

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There were immediate applications of the discovery of HBV. The “Au test,” as it was called, became widely used for the diagnosis of acute and chronic hepatitis, a further aid to control and treatment. It was a major step forward in viral diagnosis; a virus could be diagnosed within a few hours by direct detection. Previously, viral diagnosis often depended on comparing the titers of specific antibodies early in infection to titers during convalescence (Merigan 1997, personal communication). In 1969, we suggested testing blood donors to detect asymptomatic carriers of HBV (Blumberg et al. 1969). It was soon adapted at Philadelphia General Hospital (Senior et al. 1974), and later, after some controversy, at many other places. Soon, posttransfusion hepatitis due to HBV appeared to be under control. Subsequently, the discovery of HCV further reduced the frequency of posttransfusion hepatitis to the extent that it is no longer a major medical and surgical problem.

Invention of the Vaccine One of the major problems in vaccine invention is the identification of the specific antibody or cellular immune reaction that provides protection. The failure to do so has slowed the development of vaccines against HIV, HCV, tuberculosis, malaria, and other pathogens. The identification of a protective antibody for HBV was possible soon after the research began. By 1968, we recognized that antibodies (anti-HBs) against the surface antigen (HBsAg) of HBV were probably protective. We had rarely seen individuals who had both HBsAg (indicating infection) and anti-HBs in their blood; this is consistent with protection. Further, Okochi in Tokyo (Okochi et al. 1970) had shown that patients, who had anti-HBs before they were transfused with donor blood containing HBV, were much less likely to develop hepatitis than those who did not have anti-HBs. Later, in a multiyear study in a renal dialysis unit where HBV infection was endemic, Lustbader demonstrated a striking level of anti-HBs’ protection (Lustbader et al. 1976). A peculiar feature of HBV recognized after EM visualization of particles of the virus (Bayer et al. 1968; Dane et al. 1970) was that, in addition to the whole virus particles, there were very large numbers of spherical and rod-shaped particles in the peripheral blood of carriers and other infected individuals that contained only HBsAg. In some carriers, this amounted to more than 1% of their total serum protein. The vaccine was prepared from these particles. In 1969, the Institute for Cancer Research filed a patent in the USA and foreign countries for the process for extracting surface antigen particles free from whole virus as the source of the vaccine and for the product (Blumberg and Millman 1972; Blumberg 1972). To quote from the summary of the patent application: “… a vaccine against viral hepatitis is derived from blood containing Australia antigen, having particles resembling viruses which are substantially free of nucleic acid, of a size range of 180–210 A, substantially free of infectious particles. The vaccine where required is attenuated or altered. The preferred procedure of removing impurities including infectious components involves centrifugation, enzyme digestion, column

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gel filtration, differential density centrifugation in a solution of sucrose, dialysis, differential centrifugation in a solution of cesium chloride and dialysis.” At the time, this was a unique method for producing a vaccine. None had previously been produced from the blood of viral carriers and none has since then. Studies by Krugman and his colleagues increased the interest in the Blumberg/ Millman vaccine (Krugman et al. 1971). They inoculated children with a preparation of HBV-positive serum which had been boiled for 1 min. Subsequently, they evaluated whether these children had been protected against hepatitis by injecting them with untreated serum containing the virus. The heated serum provided substantial (but not complete) protection against HBV infection. This experiment had the effect of impressing potential manufacturers that the vaccine was feasible. In 1971, we received an assurance of interest from Merck Pharmaceuticals (Hilleman 1971, personal communication). By 1975, a sufficient amount of work had been done in laboratories in the USA and elsewhere to encourage us to recommend production and field testing of the vaccine; a licensing agreement was concluded with Merck. The noted vaccine expert Maurice Hilleman was given responsibility for the project and an experimental vaccine was produced and tested in animals (Buynak et al. 1976).

Vaccine Field Trials By the early 1980s, a series of vaccine field trials were completed, primarily by Szmuness and colleagues (Szmuness et al. 1980, 1981). The Szmuness trial has been described as “one of the best organized and executed trials of any human vaccine” and “a milestone in preventive medicine” (London and Blumberg 1985). It was primarily on the basis of this trial that the vaccine was approved by the FDA; it is described in some detail (summarized from London and Blumberg 1985). The first problem was to choose a population with a sufficiently high risk of infection to make a vaccine trial feasible. Szmuness believed that the trial should be carried out in a population which stood to benefit from an effective vaccine (Szmuness et al. 1980). Among the populations at high risk considered for the trial were residents of institutions for the mentally retarded (in whom we had earlier reported a high HBsAg frequency), patients undergoing maintenance hemodialysis, members of the medical staff of hemodialysis centers, American Indians, and homosexual men. By the late 1970s, very few new residents were being admitted to state institutions for the retarded, and the rate of new infections in long-term residents was quite low. Quarantine procedures had been instituted in Philadelphia (Snydman et al. 1976) and subsequently elsewhere; they had greatly reduced the incidence of hepatitis B infections. By 1975, Szmuness ascertained that the risk of infection among homosexual men in New York City was high and that they were cooperative, intelligent, and well-educated (Szmuness et al. 1975). The prevalence of hepatitis B markers was 68% among more than 10,000 men surveyed, and the annual incidence of infection was projected to be 19.2% (Szmuness et al. 1978), later estimated at 30% (Szmuness

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1980, personal communication). The study included 1,083 male subjects admitted between November 1978 and October 1979, of whom 549 received vaccine and 534 a placebo. The first two inoculations were given 1 month apart, and the third was given 6 months after the first. Follow-up was carried out for at least 6 months after the last dose of vaccine but for 12 months for most participants. Ninety-three percent of the subjects received all three inoculations, an outstanding compliance rate. The results were convincing. First, there was no difference in the frequency of severity of side effects between the vaccine and placebo groups. Second, the antibody response in the vaccinated groups was excellent; 77% of the vaccines had significant levels of anti-HBs within 2 months of the first inoculation, and 96% had antibody after the third dose, while only 2–5% of placebo recipients without evidence of active HBV infection developed anti-HBs. Third, there was a clear difference in the number of HBV infectious events between the vaccine and placebo recipients. Of 122 such events, 93 (76.2%) occurred in the placebo recipients and 29 (23.8%) in the vaccines (p < 0.0001). Fifty-two of the subjects had an event classified as “hepatitis B” (alanine aminotransferase levels ³90 IU plus the appearance of HBsAg in their serum). Only seven of these most serious events occurred in vaccinated men, and all but one of these occurred prior to the third dose of vaccine. There were 73 HBV events in the placebo group and 14 in the vaccines (p < 0.0001), and only 4 of the 14 events occurred after the third dose of vaccine. The efficacy ratio (incidence in placebo recipients over those in vaccines) reached 14.0 for the period from 5 to 18 months after vaccination. HBV infections which occurred in vaccine recipients of the full immunization schedule only happened in those who had not produced anti-HBs antibody. Finally, an unforeseen but clinically and biologically important result was that those vaccinated subjects who did not produce anti-HBs were not more likely to develop persistent infections than placebo recipients who became infected. Thus, Szmuness and his colleagues (Szmuness 1980, personal communication) were able to conclude that “this placebo-controlled, randomized, double-blind clinical trial, conducted in 1,083 subjects who had an unusually high risk of hepatitis B virus infection, proves the efficacy of the vaccine ….” Subsequent trials supported this conclusion (Maupas et al. 1981; Francis et al. 1982; Chan et al. 2004; Desmyter et al. 1983; Benahamon et al. 1984). The Szmuness trial was not only effective, but also efficient. Just over a 1,000 people were involved. Compare this to the more than one million children involved in the testing of polio vaccine and the thousands that have been involved in the so-far unsuccessful trials of an HIV vaccine. Millions of doses of the plasma-derived vaccine have been used. Reports of side effects have on occasion led to suspension of the vaccine programs, but they were subsequently reinstated (Marshall 1998). The effectiveness of HBsAg derived from plasma as a protection-inducing antibody validated its manufacture by recombinant methods, and recombinant vaccine is now the major source of the vaccine (McAleer et al. 1984). It was the first vaccine produced by the recombinant method and for many years the only one; it has helped to make the vaccine available worldwide as the cost of manufacture and distribution decreases.

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Global Vaccination Programs National vaccination programs began soon after the vaccine was available (see, for example, Gatcheva et al. 1995; Bonnanni 1995; Ginsberg and Shouval 1992; De la Torre and Esteban 1995). In February 1990, we convened The International Conference on Prospects for Eradication of Hepatitis B Virus that that included reports on the extent of the HBV endemic, the national resources available for vaccination programs, and the possibilities for control and, possibly, eradication (Blumberg 1990). In 1992, the WHO placed HBV vaccine on the Extended Program on Vaccination setting a target of 1997 for integration into national programs. By April of 2005, 158 of the 192 members of the World Health Organization had national vaccination programs (Kim et al. 2007). HBV is one of the most widely used vaccines worldwide. The results are impressive. Newborn and childhood vaccination was started in Taiwan in 1984 with excellent national participation and long-term reporting of the results (Chan et al. 2004). In 1999, 15 years later, the carrier prevalence had dropped from 9.8 to 0.7% (p < 0.001). The prevalence had also dropped significantly (but not as much) in children in a similar cohort who had not been vaccinated. There have been similar findings elsewhere (see, for example, Da Villa et al. 1992, 1995). This implies a type of “herd immunity” that could hasten the overall effect of the program and accelerate control and, possibly, eradication. There was also a striking drop in deadly fulminant hepatitis in young children (Kao et al. 2001). From 1975 to 1984, the average mortality from fulminant hepatitis was 5.36/100,000 infants; from 1985 to 1998 – after the vaccination program had started – it was 1.71/100,000. There have been reports from elsewhere of striking drops in the prevalence of HBV carriers and decrease in the incidence of clinical hepatitis. Several HBV carrier surveys before and after vaccination programs have been summarized (Blumberg 2004). In a regional study in the Peoples Republic of China, the prevalence of carriers decreased from 16.0 to 1.4%. The before and after percentages in other countries are similar: The Gambia 10.0 and 0.6%; Japan 2.7 and 0.9%; Saudi Arabia, 6.7 and 0.3%; Catalonia (Spain), 9.3 and 0.9%. In the USA, the rate of new HBV infections has declined significantly since 1991. It dropped from a peak of more than 70,000 cases in 1984 to less than 20,000 in 2006. The decline has been greatest among children born since 1991, when routine vaccination of children was recommended by the CDC (CDC Web site 2006). In Alaska, following an intensive vaccination campaign among Native Americans, the incidence dropped from 215 cases/100,000 before the vaccination programs to 7–14 cases/100,000 in 1993 after the program was in place. In 1995, no cases were reported (McMahon et al. 1996). A national hepatitis serological survey was conducted in China in 1992 (Liang et al. 2009). The authors estimated that there were 120 million HBV carriers, that 20 million patients suffered from chronic effects of HBV including chronic liver disease (cirrhosis, liver failure, etc.) and HCC, and that about 300,000 die annually from the consequences of late-stage HBV infection. Liver cancer and cirrhosis are

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both on the list of the ten most common causes of fatal diseases and HBV is the major cause of both of these. About one-third of all the HBV carriers in the world are in Peoples Republic of China. In 2009, a report was published on the effects of the vaccine program based on a national epidemiological study involving 160 counties, 369 township and village clusters, and 81,775 persons (Liang et al. 2009). The authors concluded that China had achieved the goal of reducing HBsAg prevalence to less than 1% in the vaccinated population (mostly children under 5 years) and 16–20 million HBV carriers had been prevented as a result of the infant vaccination program. Furthermore, in the vaccine-impacted population, i.e., young children, 2.8–3.5 million future deaths have been prevented. There are about 375–400 million carriers worldwide. A tentative projection to the whole world population can be attempted using the Chinese data. In many places worldwide, the vaccination programs began earlier and the compliance rates were somewhat higher. The Chinese estimates were based on the results in the childhood vaccinated population. In several studies, it has been shown that in countries with successful childhood vaccination program there is also a significant drop in HBV carrier incidence in the unvaccinated population, presumably as a consequence of herd immunity (see above). Taken these factors into account, about 15–25 million future deaths have been prevented worldwide. Plans for the continuation of the successful campaign in China are being discussed, including the possibility of eradication (So 2006).

HBV: The First Cancer Vaccine Perhaps, the most conceptually important outcome of the vaccination program is the decrease of HCC, primary cancer of the liver. HCC is one of the most deadly and common cancers worldwide. It is the third most common cause of death from cancer in males and the seventh most common in females. Most HCCs are caused by infection with HBV or HCV; HBV is said to account for 65–75% of the cases worldwide. In Taiwan, the yearly incidence of HCC in the vaccine-impacted population (age 6–14 years) declined from 0.7/100,000 (1981–1986) before vaccination programs were fully implemented to 0.36/100,000 between 1990 and 1994 after the vaccination program was in place (Huang and Lin 2000; Chan et al. 2004). A follow-up report found that the prevention of HCC has continued from “childhood to early adulthood” (Chang et al. 2009). The major cause of continued disease was inadequate vaccination and highly infective mothers. This is another indication for the treatment of HBV carrier mothers, particularly those with titers of HBV DNA. HBV vaccine is the first preventive cancer vaccine with a demonstrated impact on the world’s cancer load. In 2007, about 25 years after the introduction of the first, the second cancer prevention vaccine was launched. It provided effective protection against strains of papilloma virus preventing cancer of the cervix and other cancers (Chen and Berek 2007). There are also continuing studies on the

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possibility of vaccine protection against EB virus and the prevention of nasopharyngeal and other cancers (Hepeng 2008).

Secondary Antiviral Prevention: Prevention by Delay National vaccination programs are the most effective means of controlling HCC. But what of those already infected? There are about 400 million carriers of HBV and some 300 million carriers of HCV at risk for chronic liver disease and HCC. Antiviral treatment may delay or abort the risk of these diseases. We termed this “prevention by delay” (Blumberg and London 1981) as the antiviral treatment can prolong the symptom-free period until the carrier dies of other causes. Treatment of HBV carriers with antivirals (see, for example, Liaw et al. 2004) or by other means can greatly decrease the risk of HCC; this is discussed elsewhere in this book. A further advantage of treatment is that decreasing titers of virus decreases the infectivity of carriers and patients and, therefore, the risk of transmission to those who have not been vaccinated or were inadequately vaccinated. In particular, it could reduce the transmission of HBV from mothers to their children at the time of birth and soon afterward. This could hasten the control of HBV and increase the possibility of eradication. Fortunately, prevention of HCC, and probably of other viral-caused cancers, has the double arm of primary prevention and secondary prevention to aid in control.

The Genetics of Hepatitis B Virus The initial research on hepatitis B began as a study in the inheritance of susceptibility to HBV chronic infection (Blumberg et al. 1966), and more recent population and genomic studies have added rich detail (for review, see, Blumberg 2006a, b). There are multiple loci at which one allele increases susceptibility to chronic infection and an alternate allele increases the probability of developing protective antibody. An added interesting aspect of these observations is that these same alleles may affect susceptibility to other infectious agents. For example; the DRB1*1302 allele at the MHC Class II locus (chromosome 6) is related to susceptibility to HBV chronic infection, response to falciparum malaria, and response to papilloma virus. The VDR locus (chromosome 12) is related to responses to HBV, Mycobacterium tuberculosis, and Mycobacterium leprae; there are many other examples. We have categorized the organisms with affinities to the same genetic locus as Microorganism Gene Affinity Clusters (MIGAC). In some cases, an allele that confers an advantage to the host for one member of the cluster may be disadvantageous for another. These allelic variations constitute genetic polymorphisms and as such both advantages and disadvantages may be expected [for example, carriers of HBV bind larger quantities of iron than uninfected people. This may be an advantage in regions with low dietary

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iron intake (Sutnick et al. 1974; Felton et al. 1979)]. It is interesting to conjecture how members of the cluster are affected if one of the infectious agents in the cluster is greatly decreased as a result of a successful vaccination program.

Conjectures on the Future of Research on Viruses and Cancer 1. The Editor has asked me to comment on the possible directions for future research, including my preferences, in respect to cancer and viruses or, more broadly, infectious agents. Preventive measures have made an important contribution to the decrease of the load of human cancer and, obviously, research should be encouraged and increased. The apparent success of the HBV vaccine program in decreasing the incidence of one of the most common and deadly cancers was an encouragement to seek others. As already noted, it required about 25 years for the introduction of the second cancer prevention vaccine, against the papilloma virus, an indication of the lack of funding for this theater of research in the past. 2. There are multiple factors in the pathogenesis of cancers. Independent of what is considered the “real” cause, often expected to be some gene selection, introduction, or alteration, the most important “cause” may be distant from genetic effects but amenable to interventional change. For example, HBV and HCV may not be directly involved in genetic change consistent with theoretical expectations, but prevention or treatment of infection can considerably decrease risk. Control of other contributors to advancing pathogenesis, for example the removal of aflatoxins and/or other environmental enhancers of cancer, decreasing iron and iron storage levels (Weinberg 1984; Hann et al. 1989) controlling excessive alcohol intake, and probably many others, can contribute significantly to decreasing the risk of HCC and other untoward consequences of HBV infection (Blumberg 2002). 3. Viruses that are designated as the “main” cause of a cancer interact with other infectious agents in the host. As noted above, there are interactions of the genome of HIV with other cancer-associated virus. Humans are infected normally with vast numbers of bacteria and virus. It is unlikely that they would not interact with cancer-related viruses. Also, hosts are often coinfected if viruses have similar transmission routes. HBV infection is associated with infection with HCV, HIV, HTLV-1, and probably other blood-borne infectious agents. The host can respond differently to each of the following: acute disease, carrier state, development of chronic infection, production of protective and other antibodies, integration into the host genome, or genomes of the other organisms. The study of the interactions of viruses with each other and with the genome of the host can provide insight into the process of carcinogenesis, therapy, and prevention. 4. In their review, Boccardo and Villa (2007) cite many examples in which the same or similar viruses may cause several cancers (i.e., EBV and Burkitt’s Lymphoma, nasopharyngeal carcinoma, Hodgkin’s disease, etc.; HPV and genital carcinoma,

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carcinoma of the oropharynx, etc.). It would be fruitful to continue this search. There are several studies showing an association of HBV with cancer of the pancreas (see, for example, Iloeje et al. 2010). Duck HBV, a hepadnavirus (HBV-related viruses), localizes in the pancreas and HBV, although primarily localized to the liver, and is also found in the pancreas in humans (Coyne et al. 1970). These findings suggest that HBV has a role in cancer of the pancreas or that another virus that is cross antigenic with HBV may be involved. It is informative to determine if the HBV vaccination campaign has had an effect of the incidence of cancer of the pancreas. The first disease associations reported for HBV were with leukemias, but there has been little follow-up on these leads (Blumberg et al. 1967). HBV has recently been associated with lymphoma (Engels et al. 2010), and further studies on this connection are warranted. 5. Most cancer viruses are associated with more noncancer disease; in many cases, they are more common than the cancer. The association (noted above) of chronic HTLV-1 infection with decreased survival is an example of the potential public health importance of chronic infections with viral-caused cancers. The recent studies of zur Hausen and his colleagues (zur Hausen 2010) on the Torque Teno Virus (TTV) that is related to cancers, MS, and other disease characterized by inflammation is an important example of how this research is proceeding. 6. Prevention against and treatment of infectious agents has been one of the most successful accomplishments of scientific medicine and this can be extended to cancers caused or influenced by infectious agents. A future direction for cancer centers and research institutes, then, could or should be to seek viruses in patients with various cancers and, perhaps more importantly, those who are at the risk of cancer. A strategic approach would be to start with the null hypothesis that all cancers include one or more microorganisms (including viruses) in their etiological roster, and then use available methods (and methods to be developed) for detecting past or present infection. This could include infection in prior generations of the host that remain in the host’s genome. 7. Another fruitful area for research would be to continue the search for polymorphic genetic variation that increases the risk for and/or alters the pathogenesis and treatment of cancer. There have been important advances in this field since the early days of searching for polymorphic variants in serum proteins and elsewhere that influence susceptibility to disease and affect response to drugs (see, for example, Blumberg 1961). The use of whole genome sequences has facilitated the identification of many sites, but it has also made it more complicated and difficult to apply. As data accumulates, it is likely that general laws will emerge and the use of these variations to protect the most susceptible will increase. Identifying the genetic components of susceptibility makes it easier to find environmental agents in that a subpopulation can be identified in which the environmental influences are strong. Further investigation of “Magac” clusters (see above) can provide leads on disease connections and operations of many genes on the same outcome.

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8. The discovery and development of new and the improvement of current antivirals effective against specific or many viruses should be a major research program. The intensive research on treatments for HIV and AIDS has given strong support to pharmaceutical companies seeking useful and profitable products, and this can be extended to HBV and HCV. (There are worldwide about 700 million carriers of HBV and HCV.) It was recognized early in the HBV research programs that complete removal of the virus from its host may be difficult or impossible as the virus is often integrated into the host genome. However, decreased titers can considerably reduce the risk for the development of chronic liver disease and HCC. There are now several antivirals that are effective in doing this and others are in the process of development. In many cases, carriers of cancer-related viruses require long-term treatment using relatively low doses as reduction of titer rather than total elimination is the goal. This could also result in decreased side effects. Long-term treatments are attractive to pharmaceutical companies and could encourage research and development of the antivirals. The availability of effective and safe antivirals encourages the detection of carriers, of whom currently only a small percentage has been identified.

Conclusions Viruses cause many of the cancers that afflict humans and these same viruses often cause other noncancer diseases that may be more common than the cancers. This provides an additional justification for national and universal vaccination programs. Known cancer viruses are often implicated in the etiology or pathogenesis of other cancers and it is important to find these connections. If this is common, then the existing cancer vaccine programs and those added in the future may lead to decreases in the incidence of other cancers not now recognized as virally caused. There are now two vaccination programs – HBV and HPV – that are in place and appear to be successful; it is expected that others will be added in time as vaccines or other preventive measures are found for the existing virus-related cancers. It is likely that viruses have a role in the pathogenesis of many other cancers than those already identified and an important direction for cancer research would be to find these and develop countermeasures, including vaccination. There is also the possibility of developing new and better antvirals to treat those chronically infected with known cancer viruses. As experience with secondary prevention of cancer with antivirals increases, treatment may be a useful method to determine if a cancer is caused by a virus. The HBV vaccine cancer prevention program has been in place for nearly 30 years. It has served as a model for future programs and has been described in some detail in this chapter. HBV was discovered in a nongoal-directed project, as is often the case in the solution of medical and biological problems. The original vaccine was derived from a portion of the virus – the surface antigen particles that

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exist in large quantities in the blood of carriers of HBV; this was a unique method of making a vaccine that did not require cell culture. HBV vaccine was subsequently made by the recombinant method, the first vaccine so produced. It is now one of the most commonly used vaccines in the world. (However, there are still regions that are inadequately served.) The reduction in the prevalence of HBV carriers and the incidence of acute and chronic liver disease and HCC has been profound. Rigorous control and even eradication may be possible. We are in a period of increased awareness of the virus–cancer connection and the major effect it has and can have on the control of several and perhaps many cancers. Strong support for continued research and application moves these programs forward.

References Allison AC, Blumberg BS (1961) An isoprecipitation reaction distinguishing human serum protein types. Lancet 1:634–637 Bayer ME, Blumberg BS, Werner B (1968) Particles associated with Australia antigen in the sera of patients with leukemia, Down’s syndrome and hepatitis. Nature 218:1057–1059 Benahamon E, Courouce AM, Jungurs P et al (1984) Hepatitis B vaccine: randomized trial of immunogenicity in hemodialysis patients. Clin Nephrol 3:102–103 Blumberg BS, Millman I (1972) Vaccine against viral hepatitis and process. US Patent Office no. 3,636,191 Blumberg BS (1961) Inherited susceptibility to disease: Its relation to environment. Arch Environ Health 3:612–636 Blumberg BS (1990) Proceedings of International Conference on Prospects for Eradication of Hepatitis B Virus. In Vaccine 8 Introduction S5 Conclusion S139 Blumberg BS (1972) Viral hepatitis, Au antigen, and hope for a vaccine. Gastroenterology (Med. World News) 14–18 Blumberg BS, Alter HJ, Visnich S (1965) A “new” antigen in leukemia sera. JAMA 191:541–546 Blumberg BS, Gerstley BJS, Hungerford DA et al (1967) A serum antigen (Australia antigen) in Down’s syndrome leukemia and hepatitis. Ann Int Med 66:924–931 Blumberg BS, Melartin L, Guinto RA et al (1966) Family studies of a human serum isoantigen system (Australia antigen). Am J Human Genet 18:594–608 Blumberg BS, London WT, Sutnick AI (1969) Relation of Australia antigen to virus of hepatitis. Bull Path 10:164 Blumberg BS (2002) Hepatitis B. Princeton University Press, Princeton NJ, The Hunt for a Killer Virus Blumberg BS (2006a) Hepatitis B virus: conjectures on human interactions and the origin of life. In: Seckbach J (ed) Life as We Know It. Springer, New York, pp 213–235 Blumberg BS (2006b) The curiosities of hepatitis B virus: prevention, sex ratio, and demography. Proc Am Thorac Soc 3:14–20 Blumberg BS (2004) The impact of hepatitis B vaccine worldwide. In: Vierucci Alberto (ed) The Proceedings of the Società Italiana de Allergolgia ed Immunologia Pediatrica, 57–63 Firenza, 18–20 EDITEAM, Cento, (Florence) Blumberg BS (2010) Hepatitis B. In: Artenstein AW (ed) Vaccines: a biography. Springer, New York, pp 301–315 Blumberg BS, London WT (1981) Hepatitis B virus and the prevention of primary hepatocellular carcinoma. Editorial. N Engl J Med 304:782–784

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Boccardo e, Villa LL (2007) Viral origins of human cancers. Curr. Med. Chem. 14(24): 2526–2539 Bonnanni P (1995) Implementation in Italy of a universal vaccination program against Hepatitis B. Vaccine 13:68–71 Buynak EB, Roehm RR, Tytell AA et al (1976) Development and chimpanzee testing of a vaccine against human Hepatitis B. Proc Soc Exp Biol Med 151:694–700 Chan C-Y, Lee S-D, Lo K-J (2004) Legend of Hepatitis B vaccination: the Taiwan experience. J Gastrolenterol and Hepatol 19:121–126 Chang MH, You SL, Chen CJ, Liu CJ, Lee CM, Lin SM, Chu HC, Wu TC, Yang SS, Kuo HS, Chen DS, the Taiwan Hepatoma Study Group (2009) Decreased incidence of hepatocellular carcinoma in Hepatitis B vaccinees: a 20-year follow-up study. J Natl Cancer Inst 101(19):1348–1355 Chen JK, Berek JS (2007) Impact of the human papilloma vaccine on cervical cancer. J Clin Oncology 25:2975–2982 Coyne (Zavatone) V, Millman I, Cerda J, Gerstley BJS, London WT, Sutnick AI, Blumberg BS (1970) The localization of Australia antigen by immunofluorescence. J Exp Med 131:307–320 Da Villa G, Piazza M, Iorio R et al (1992) A pilot model of vaccination against hepatitis B virus suitable for mass vaccination campaigns in hyperendemic areas. J Med Virol 36:274–278 Da Villa G, Picciottoc L, Elia S et al (1995) Hepatitis B vaccination: universal vaccination of newborn babies and children at 12 years of age versus high risk groups. A comparison in the field. Vaccine 13:1240–1243 Dane DS, Cameron CH, Briggs M (1970) Virus-like particles in serum of patients with Australia antigen-associated hepatitis. Lancet 1:695 Devita VT, Chu E (2008) A history of cancer chemotherapy. Cancer Res. 68(21):8643–8653. De la Torre J, Esteban R (1995) Implementing universal vaccination programs: Spain. Vaccine 13:72–74 Desmyter J, Colaert J, DeGroote G et al (1983) Efficacy of heat-inactivated hepatitis B vaccine in hemodialysis patients and staff: double blind placebo-controlled trial. Lancet 2:1323–1328 Engels EA, Cho ER, Jee SH (2010) Hepatitis B virus infection and risk of non-Hodgkin lymphoma in South Korea: a cohort study. Lancet Oncol 11:827–834 Felton C, Lustbader ED, Merten C et al (1979) Serum iron levels and response to hepatitis B virus. Proc Natl Acad Sci USA 76:2438–2441 Francis DP, Hadler SC, Thompson SE (1982) The prevention of hepatitis B with vaccine: report of the centers for disease control multi-center efficacy trial among homosexual men. Ann Intern Med 97:362–366 Gatcheva N, Vladimirova N, Kourtchatova A (1995) Implementing universal vaccination programs: Bulgaria. Vaccine 13:82–83 Ginsberg GM, Shouval D (1992) Cost-benefit analysis of a nationwide neonatal inoculation program against hepatitis B in an area of intermediate endemicity. J Epidemiol Community Health 46:597–594 Hann HL, Kim CY, London WT, Blumberg BS (1989) Increased serum ferritin in chronic liver disease: A risk factor for primary hepatocellular carcinoma. Int J Cancer 43:376–379 Hepatitis B Vaccine: Fact Sheet First Anti-cancer Vaccine (2006) http://www.cdc.gov/hepatitis May 17, 2006 Hepeng J (2008) A controversial bid to thwart the “Cantonese Cancer”. Science 321:1154–1155 Huang K-Y, Lin S-R (2000) Nationwide vaccination: a success story in Taiwan. Vaccine 18:S35–S38 Kao JH, Hsy HM, Shau WY et al (2001) Universal hepatitis B vaccination and the decreased mortality from fulminant hepatitis in infants in Taiwan. J Pediatr 139:349–352 Kim S-Y, Salomon JA, Goldie SJ (2007) Economic evaluation of hepatitis B vaccination in lowincome countries: using cost-effectiveness affordability curves. Bull World Health Organ 85:821–900

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Krugman S, Giles JP, Hammond J (1971) Viral hepatitis, type B (MS-2 strain): studies on active immunization. JAMA 217:41–45 Lechat MF, Shrager DI, Declercq E, Bertrand F, Blattner WA, Blumberg BS (1997) Decreased survival of HTLV-1 carriers in leprosy patients from the Democratic Republic of the Congo: a historical prospective study. J Acquir Immune Defic Syndr Hum Retrovirol 15:387–390 Liang X, Bi S, Yang W, Wang L, Cui G, Cui F, Zhang Y, Liu J, Gong X, Chen Y, Wang F, Zheng H, Wang F, Guo J, Jia Z, Ma J, Wang H, Luo H, Li L, Jin S et al (2009) Epidemiological serosurvey of Hepatitis B in China. Declining HBV prevalence due to Hepatitis B vaccination. Vaccine 27:6550–6557 Iloeje UH, Yang HI, Jen CL, Su J, Wang LY, You SL, Lu SN, Chen CJ (2010) Risk of pancreatic cancer in chronic hepatitis B virus infection: data from the REVEAL-HBV cohort study. Liver Int 30:423–429 Liaw YF, Sung JJ, Chow WC, Farrell G, Lee CZ, Yuen H, Tanwandee T, Tao QM, Shue K, Keene ON, Dixon JS, Gray DF, Sabbat J, Cirrhosis Asian Lamivudine Multicentre Study Group (2004) Lamivudine for patients with chronic hepatitis B and advanced liver disease. N Engl J Med 351:1521–1531 London WT, Blumberg BS (1985) Comments on the role of epidemiology in the investigation of hepatitis B virus. Epidemiol Rev 7:59–79 Lustbader ED, London WT, Blumberg BS (1976) Study design for a hepatitis B vaccine trial. Proc Natl Acad Sci USA 73:955–959 McAleer WJ, Buynak EB, Maigetter RZ et al (1984) Human hepatitis B vaccine from recombinant yeast. Nature 307:178–180 Marshall E (1998) A shadow falls on hepatitis B vaccination effort. Science 281:630–631 Maupas P, Chiron VP, Barn F et al (1981) Efficacy of hepatitis B vaccine in prevention of early HBsAg carrier in children: controlled trial in an endemic area (Senegal). Lancet 1:289–292 McMahon B, Mandsager R, Wainwright K, et al. (1996) The Alaska native hepatitis B control program. Proc. IX Triennial International Symposium on Viral Hepatitis and Liver Disease, Rome, Italy, abstract 74 Okochi DJ, Murakami S (1968) Observations on Australia antigen in Japanese. Vox Sang 15:374–385 Okochi DJ, Murakami S, Nonomiya K et al (1970) Australia antigen, transfusion and hepatitis. Vox Sang 18:289–300 Prince AM (1968) An antigen detected in the blood during the incubation period of serum hepatitis. Proc Natl Acad Sci USA 60:814–821 Senior JR, Sutnick AI, Goeser E et al (1974) Reduction of post-transfusion hepatitis by exclusion of Australia antigen from donor blood in an urban public hospital. Amer J Med Sci 267:171–177 Snydman DR, Bryan JA, London WT et al (1976) Transmission of hepatitis B associated with hemodialysis: role of malfunction (blood leaks) in dialysis machines. J Infect Dis 134:562–570 So S (2006) A comprehensive national strategy to eliminate hepatitis B in China should include an expanded national immunization program to provide free catch-up vaccination for every child and adolescent in addition to universal newborn vaccination. Opening keynote address, China Hepatitis Prevention and Control Conference, Nov 16–18, 2006, Chengdu, China Sutnick AI, Blumberg BS, Lustbader ED (1974) Elevated serum iron levels and persistent Australia antigen (HBsAg). Ann Intern Med 81:855–856 Szmuness W, Harley EJ, Ikram H et al (1978) Socio-demographic aspects of the epidemiology of hepatitis B. In: Vyas G, Cohen SN, Schmid R (eds) Viral hepatitis. Franklin Institute Press, Philadelphia, pp 297–320 Szmuness W, Much MI, Prince AM et al (1975) On the role of sexual behavior in the spread of hepatitis B infection. Ann Intern Med 83:489–495 Szmuness W, Stevens CE, Harley EJ et al (1980) Hepatitis B vaccine: demonstration of efficacy in a controlled clinical trial in a high-risk population in the United States. N Engl J Med 303:833–841

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Szmuness W, Stevens CE, Harley EJ et al (1981) A controlled clinical trial of the efficacy of the hepatitis B vaccine (heptavax B); a final report. Hepatology 8:119–121 Vierucci A, Bianchini AM, Morgese G et al (1968) L’antigene Austrlia. I Rapporte con l’epatite infettiva e da siero. Una ricerca in pazienta pediatrici. Pediatria Int 18:3–11 Weinberg ED (1984) Iron withholding: a defense against infection and neoplasia. Physiol Rev 64:65–102 Zur Hausen H (2010) In New virus behind cancer, MS? – The scientist – Magazine of the Life Sciences http://www.the Scientist.com/news/display/57623/#ixzz0x1SJXy4g.

Chapter 3

Virus-Mediated Cell Proliferation Sun-Hwa Lee, Stacy Lee, and Jae Ung Jung

Introduction It is estimated that 15–20% of all human cancers are linked to human tumor viruses, including hepatitis B virus (HBV), hepatitis C virus (HCV), human papillomavirus (HPV), human T-cell lymphotropic virus (HTLV), Epstein–Barr virus (EBV), and Kaposi’s sarcoma-associated herpesvirus/human herpesvirus type 8 (KSHV/HHV-8). HTLV is an RNA tumor virus associated with adult T-cell leukemia, whereas HBV, HPV, EBV, and KSHV are DNA tumor viruses associated with liver cancer (HBV), cervical and other anogenital cancers (HPV), Burkitt’s lymphoma and nasopharyngeal carcinoma (EBV), and Kaposi’s sarcoma (KS) (KSHV) (Dayaram and Marriott 2008). Simply defined, cell proliferation is the increase in cell numbers as a result of cell growth and division. For normal cells, entry into an active proliferative state from a quiescent state (G0) depends on the presence of exogenous, mitogenic, growth stimulatory signals, such as diffusible/soluble growth factors, components of the extracellular matrix (ECM), and cell-to-cell adhesion/interaction molecules, since the binding of these stimulatory signals to their receptors induces a variety of intracellular signaling transduction pathways involved in cellular proliferation (Hanahan and Weinberg 2000). Tumor cells, however, depend less on exogenous, growth stimulatory signals in the initiation of proliferation. Thus, the ability to proliferate in the absence of external growth factors is suggested to be one of the hallmarks of tumor cells and is generally achieved by either overexpression of growth receptors and/or ligands, mutations in receptors, or downstream signaling molecules whose activities are independent of ligand binding or defects in specific components of the cell cycle machinery (Hanahan and Weinberg 2000).

S.-H. Lee (*) • S. Lee • J.U. Jung Department of Molecular Microbiology and Immunology, University of Southern California, School of Medicine, 2011 Zonal Avenue, HMR401, Los Angeles, CA 90033, USA e-mail: [email protected] E.S. Robertson (ed.), Cancer Associated Viruses, Current Cancer Research, DOI 10.1007/978-1-4614-0016-5_3, © Springer Science+Business Media, LLC 2012

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Likewise, tumor viruses appear to have evolved numerous strategies that promote the proliferation of infected host cells in the absence of external growth stimulatory signals for their replication and survival, thereby ultimately contributing to the transformation of infected host cells. To this end, several viral proteins encoded by tumor viruses have been shown to dysregulate normal cellular processes, such as cell cycle progression, apoptosis, immune surveillance, and antiviral responses, allowing viral replication and survival. Many excellent reviews on the impact of tumorigenic viruses on cellular processes, including cell cycle progression, have been published (Damania 2007; Dayaram and Marriott 2008; Gatza et al. 2005; Helt and Galloway 2003; Hoppe-Seyler and Butz 1995; McLaughlin-Drubin and Munger 2008; Sears and Nevins 2002). This review focuses on the biochemical and molecular strategies used by oncogenic HHVs, including EBV and KSHV, to enhance cell proliferation.

Oncogenic Human Herpesviruses The Herpesviridae family comprises large, double-stranded DNA viruses with a genome size of 100–200 kb. Members of this family are classified as three subfamilies based on their genomic organization and biological characteristics: alpha (a), beta (b), and gamma (g). Eight HHVs are known so far. Members of the a-HHV include herpes simplex viruses (HSV) 1 and 2 (HHV-1 and HHV-2) and varicellar zoster virus (VZV; HHV-3). Members of the b-HHV include cytomegalovirus (CMV; HHV-5), HHV-6 variants A and B, and HHV-7. Members of g-hepesviruses are further classified as g1-herpesviruses (lymphocryptoviruses) and g2-herpesviruses (rhadinoviruses). EBV (HHV-4) and KSHV (HHV-8) belong to g1- and g2HHV, respectively (Damania 2007). Among the members of the HHV family, only EBV and KSHV have been implicated in a variety of human cancers. Association of both EBV and KSHV with a number of human cancers derives from two distinct features of their life cycles, latency and lytic cycle. In a lytic cycle, viruses replicate extensively and express virtually all viral genes, ultimately leading to the production of progeny viruses and the death of infected host cells (Ganem 2007). This lytic infection probably occurs either during primary infection or periodically in certain physiologic conditions, causing viral spread between cells and hosts (Kalt et al. 2009). In latency, on the other hand, the viral genome is maintained as a circular episome in the nuclei of infected host cells. Only a handful of viral genes are expressed and progeny viruses are not produced. In addition to these two life cycles, both EBV and KSHV have other common features: (1) both can infect B lymphocytes, (2) their latency is associated with human cancers, and (3) it is difficult to model their lytic replication cycles in vitro (Hume and Kalejta 2009). EBV (HHV-4). EBV is the first human virus to be directly implicated in carcinogenesis and over 90% of the global adult population is infected with EBV. It is usually asymptomatic, but a proportion of EBV-infected individuals develop infectious mononucleosis (IM), a disease characterized by lymphadenopathy and fatigue, later in life. During acute infection, EBV primarily infects and replicates in the stratified

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squamous epithelium of the oropharynx. This is followed by latent infection of B lymphocytes. While lytic infection is known to be associated with oral hairy leukoplakia, a proliferative disorder in immunocompromised patients, most EBVassociated malignancies are caused by latent infection (Kutok and Wang 2006). EBV-associated malignancies include Burkitt’s lymphoma (BL), Hodgkin’s lymphoma (HL), nasopharyngeal carcinoma (NPC), T and natural killer (NK) cells lymphoma, and posttransplant lymphoma (Kutok and Wang 2006). BL is a malignancy principally found in children, especially those who live in regions of Africa with a high incidence of malaria, although it also occurs more sporadically in other areas of the world. BL tumor cells are highly linked with EBV as almost all African BL patients are EBV positive. NPC is a malignancy of the squamous epithelium situated in the nasopharynx. EBV-associated NPC frequently occurs in Southern China, Northern Africa, and among Eskimo populations and is thought to arise from clonal expansion of latently infected cells. HL is the most common EBV-associated malignancy occurring in the Western world (about 30–90% of all HL patients are EBV positive). EBV is also highly present in immunoblastic lymphomas in HIVinfected individuals (about 70%) as well as in immunosuppressed posttransplant patients (100%) (Damania et al. 2000). EBV can infect primary human B lymphocytes in vitro, converting them into continuously growing, semiactivated, immortalized, and transformed lymphoblastoid cell lines (LCLs). Within LCLs, EBV is latent. Among the >85 genes encoded by EBV, only 11 viral proteins are expressed in LCLs: six nuclear antigens (EBNAs 1, 2, 3A, 3B, 3C, and 5), three latent membrane proteins (LMPs 1, 2A, and 2B), and two EBV-encoded small nonpolyadenylated (noncoding) RNAs (EBERs 1 and 2). Among these, EBNA-2, -3A, -3C and LMP1 are required for the in vitro immortalization of B lymphocytes by EBV (Rickinson and Kieff 2007). KSHV (HHV-8). KSHV is the second HHV implicated in human malignancies. KSHV is uncommon in the general population (less than 7%, but some geographical areas have infection rates as high as 60%). KSHV is primarily transmitted through saliva, although other transmission routes, including vertical, sexual, blood, and transplantrelated transmission, have also been reported (Pica and Volpi 2007). KSHV can infect many different types of cells in vitro, including B cells, epithelial cells, endothelial cells, and cells of the monocyte/macrophage lineage (Brinkmann and Schulz 2006). In addition, KSHV has been shown to immortalize primary human endothelial cells to have long-term proliferation and survival and to establish latency in B cells and endothelial cells in vivo (Brinkmann and Schulz 2006; Damania et al. 2000). KSHV is the etiologic cause of Kaposi’s sarcoma, an endothelial neoplasm. Globally, KS is the fourth most common cancer caused by infection, after gastric cancer (Helicobacter pylori), cervical cancer (human papillomavirus), and liver cancer (hepatitis cirrhosis). KS remains a major cause of cancer-related deaths among immune-suppressed and organ-transplant patients (Kalt et al. 2009). KSHV infection is also highly associated with two rare atypical B-cell lymphoproliferative diseases: primary effusion lymphomas (PEL)/body cavity B-cell lymphomas (BCBL) and multicentric Castleman’s disease (MCD). These are principally or exclusively of B-cell origin. MCD is a polyclonal B-cell lymphoproliferative

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disease with dissemination to multiple lymph nodes and other lymphoid tissues. In AIDS patients, MCD is invariably associated with KSHV infection, whereas approximately half the cases of HIV-negative individuals are KSHV associated. PEL is a monoclonal B-cell lymphoproliferative disease marked by rapid proliferation of cells in the pleural, peritoneal, and pericardial cavities. It usually occurs in AIDS or other immunosuppressed individuals and is often associated with KSHV and EBV coinfection. Most of what we know about KSHV biology has been obtained from studying viral gene expression in cultured PEL cells, which grow readily in culture, stably maintain latent KSHV genome, and express latent transcripts and proteins in virtually all cultured cells (Ganem 2006). KSHV proteins expressed during latency are believed to contribute to the development of KSHV-associated diseases. Five major latency-associated viral proteins have been identified in latently infected lymphoma cells: the ORF73-71 locus encoding latency-associated nuclear antigen (LANA-1, ORF73), viral cyclin (v-Cyclin, ORF72), viral FLICE inhibitory protein (vFLIP, ORF71, K13), viral interferon regulatory factor (vIRF) 3 (LANA-2, K10.5), and vIL-6 (K2) (Ganem 2007). All, except for LANA-2 which is exclusively expressed in PEL and MCD, are also expressed in all KSHV-infected cells and have been shown to affect cellular proliferation and survival (Ganem 2006).

Viral Strategies for Self-Proliferation Oncogenic HHVs, including EBV and KSHV, are equipped with strategies that promote the proliferation of infected host cells for their survival and replication. Both lytic and latent EBV and KSHV viral proteins demonstrate the ability to activate growth signaling by functioning as (1) growth factor receptors, (2) growth factor receptor ligands, (3) signal transduction molecules, (4) cell cycle regulators, or (5) transcription factors. Interestingly, some viral proteins are of cellular origin.

Viral Proteins Mimicking Growth Receptor A common feature shared by EBV and KSHV is the presence of membrane-associated viral proteins located at the left and right ends of the coding regions of their viral genomes. These viral proteins are LMP1 (EBV), LMP2A (EBV), K1 (KSHV), and K15 (KSHV). Unlike LMP1 and LMP2A, both K1 and K15 are mainly expressed during the lytic replication cycle (Brinkmann and Schulz 2006). There is no sequence homology among the proteins, although there is a limited structural similarity (Fig. 3.1). Yet, they all function as constitutively active receptors capable of inducing an array of cellular signaling pathways in a ligand-independent manner. In addition, they all have distinct oncogenic/transforming potentials. Thus, it has been suggested that these viral proteins act by mimicking normal growth signals required for the

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EBV LMP1

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Lyn

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I T A M

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Vav

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TRADD

COOH

MAPK

PI3K/Akt

MAPK

NH2

JAK

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2+

Ca

Fig. 3.1 Membrane-associated viral proteins encoded by both ends of EBV and KSHV genomes

proliferation and survival of cells, which ultimately contributes to the transformation of infected cells (Brinkmann and Schulz 2006; Damania et al. 2000; Schulz 2006). This section briefly discusses EBV and KSHV viral proteins which enhance selfproliferation by acting as growth factor receptors and their ligands. EBV LMP1. LMP1, the first open reading frame (ORF) of EBV, is expressed in all EBV-related malignancies. It is essential for the immortalization of primary B lymphocytes to LCLs in vitro (Rickinson and Kieff 2007). It is an integral membrane protein containing a short cytoplasmic N-terminus (23 aa), six transmembrane (TM)-spanning domains, and a long cytoplasmic C-terminus (200 aa). A substantial amount of experimental evidence suggests that the six TM domains and the two cytoplasmic C-terminal domains of LMP1, termed transformation effector sites (TESs) 1 and 2 or C-terminal NF-kB-activating regions (CTARs)1 and 2, are critical for the conversion of primary B lymphocytes to LCLs by LMP1 (Fig. 3.1, Soni et al. 2007). It has been shown that LMP1 markedly mimics an important B-cell activation receptor CD40, which induces the activation of NF-kB, a key transcription factor involved in the regulation of cell growth, antiapoptosis, and expression of numerous cytokines upon CD40–ligand interaction (Glenn et al. 1999; Hatzivassiliou et al. 1998; Kilger et al. 1998). Like CD40, LMP1 when expressed in B cells recruits tumor necrosis factor (TNF) receptor-associated factors (TRAFs) and the TNF receptor-associated death domain (TRADD) through its CTAR1 and CTAR2, respectively (Devergne et al. 1998; Eliopoulos et al. 1996; Eliopoulos and Young 1998; Izumi et al. 1997). Unlike CD40, however, LMP1 transduces signals in the absence of extracellular ligands or cross-linking by self-oligomerizing through its N-terminal and the TM domains on the plasma membrane. This self-oligomerization mimics the effects of receptor/ligand interactions, giving rise to the constitutive activation of a number of downstream signaling pathways (Clausse et al. 1997;

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Eliopoulos and Young 1998; Floettmann et al. 1996; Hatzivassiliou et al. 1998; Huen et al. 1995; Izumi et al. 1997; Izumi and Kieff 1997; Laherty et al. 1992). Thus, by mimicking the B-cell activation receptor CD40, LMP1 contributes to the progression of EBV-associated malignancies. More detailed molecular mechanisms by which LMP1 activates NF-kB signaling are discussed in later in this review. In addition to NF-kB signaling, activation of both MAPK and JAK/STAT is also implicated to be the function of LMP1 (Eliopoulos et al. 1999; Eliopoulos and Young 1998; Gires et al. 1999; Huen et al. 1995). The MAPK family, a group of serine/threonine kinases activated in response to extracellular environment signals such as growth factor, cytokines, and stress signals, is involved in a variety of key events, including proliferation, differentiation, apoptosis, and migration. The MAPK family consists of three parallel pathways, namely ERK, JNK, and p38. In epithelial cells, LMP1-mediated activation of JNK and p38 depends more on its CTAR2 domain (Eliopoulos et al. 1999; Eliopoulos and Young 1998). In lymphocytes, however, both CTAR1 and CTAR2 are necessary for the activation of JNK and p38 (Soni et al. 2007). LMP1-mediated activation of JNK requires TRAF6, TAK1, and TAB1, but not TRAF2, TRADD, IRAK4, MyD88, and RIP, indicating that LMP1 selectively utilizes cellular signaling molecules involved in TNFR or IL-1/TLR receptors that maximize growth and survival signals without inducing apoptosis (Soni et al. 2007). Meanwhile, JAK/STAT signaling mediated by LMP1 integrates with the AP-1 transcription factor pathway. The region between CTAR1 and CTAR2 contains proline-rich sequences and is involved in the interaction with members of the JAK family. Thus, this motif of LMP1 is believed to play a role in JAK3-mediated activation of STAT3 (Gires et al. 1999). Taken together, LMP1 appears to mediate constitutive activation of cellular signaling pathways important for controlling EBV-infected cell survival and proliferation by mimicking activated receptors. EBV LMP2A. LMP2A is another membrane protein expressed in EBV latently infected B cells. LMP2A contains 12 TM domains linked by loops, a long cytoplasmic N-terminal (119 aa), and a short C-terminal domain (27 aa). LMP2A aggregates in the plasma membrane. The N-terminal cytoplasmic domain of LMP2A contains three tyrosine-based SH2 binding motifs, two of which form a functional immunoreceptor tyrosine-based activation motif (ITAM) (Fig. 3.1, Fruehling and Longnecker 1997). ITAM motifs [(D/E)x7(D/E)x2YxxL/I/Vx6–8YxxL/I/V, where x is any amino acid] are composed of a stretch of negatively charged amino acids followed by two SH2 binding motifs (YxxL/I/V). Found in the cytoplasmic domains of T- or B-cell receptors, ITAM motifs are tyrosine phosphorylated by Src family protein tyrosine kinases (SF-PTKs) upon ligand engagement. Signaling molecules containing SH2 domains are then subsequently recruited, leading to the induction of an array of intracellular signaling pathways. In the case of LMP2A, its ITAM is tyrosine phosphorylated and is required for LMP2A association with the SH2 domain of the nonreceptor tyrosine kinases, such as Lyn, Fyn, Syk, and Csk (Beaufils et al. 1993; Burkhardt et al. 1992; Longnecker et al. 1991; Scholle et al. 1999), to induce intracellular calcium mobilization and cytokine production.

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LMP2A interaction with Lyn and Syk mimics BCR signaling, inducing the activation of the PI3K/Akt survival pathway in the absence of BCR signals (Caldwell et al. 1998). The PI3K/Akt signaling pathway plays an important role in mediating transformation, antiapoptotic effects, invasion, and adhesion. Akt is a serine/threonine kinase, which phosphorylates and regulates the activities of cell cycle regulatory proteins, such as GSK-3b and cyclin D. LMP2A is also involved in the activation of MAPK signaling in various EBV-infected cell lines in vitro (Chen et al. 2002; Panousis and Rowe 1997). LMP2A binds to and is phosphorylated by ERK, leading to the activation of MAPK signaling in B-cell lines (Panousis and Rowe 1997). LMP2A activates JNK, but not p38, in NPC cell lines (Chen et al. 2002). This in vitro observation is supported by a recent study, in which LMP2A expression in transgenic mice induced the constitutive activation of the ERK/MAPK and PI3K/Akt pathways, resulting in cell proliferation and survival (Anderson and Longnecker 2008). KSHV K1. KSHV K1 is the first ORF located at a position equivalent to that of the LMP1 of EBV (Lagunoff et al. 1999; Lee et al. 1998b; Zong et al. 1999). K1 is a 46-kDa transmembrane glycoprotein consisting of an N-terminal extracellular domain, a TM domain, and a short cytoplasmic C-terminal domain (Fig. 3.1). Its N-terminal extracellular domain contains several N-glycosylation sites and displays a high degree of genetic variability between different KSHV isolates. A survey of isolates led to the identification of five major subtypes of K1 (A–E), each containing several distinct variants (Brinkmann and Schulz 2006). In contrast, its C-terminal cytoplasmic (38 aa) is relatively well-conserved and contains a functional ITAM similar to the one found in LMP2A (Lee et al. 1998a). The K1 ITAM motif is phosphorylated and recruits Lyn, Syk, PLCg2, the p85 subunit of PI3K, Vav1, SHP1 and SHP2, RAS-GAP, and growth factor receptor-bound protein 2 (Lagunoff et al. 1999; Lee et al. 2005). Interaction of K1 with these cellular proteins results in the activation of several transcription factors, including AP-1, NF-AT, and Akt-driven forkhead box containing proteins, all of which are involved in preventing apoptosis (Tomlinson and Damania 2004). In addition, K1 interaction with Lyn leads to the activation of NF-kB in B cells (Prakash et al. 2005). Similar to EBV LMP1 and LMP2A, K1-mediated signaling occurs constitutively, independent of ligand binding by self-oligomerization through its extracellular domain (Lagunoff et al. 1999). In addition, K1 induces VEGF and matrix metalloproteinase-9 (MMP-9) in endothelial cells (Greene et al. 2007), suggesting that K1 may contribute to angiogenesis and cell division. KSHV K15. The K15 gene of KSHV is located at a position equivalent to that of EBV LMP2A (Choi et al. 2000; Glenn et al. 1999; Poole et al. 1999). Sequence analysis of the K15 gene from different KSHV isolates revealed that the K15 gene is highly variable, showing as much as 60–70% divergence at the amino acid level (Poole et al. 1999). K15 is a transmembrane protein consisting of 4–12 TM domains and a short cytoplasmic C-terminal domain (amino acid 335–489) (Fig. 3.1). Like LMP2A, the cytoplasmic domain of K15 contains multiple signaling motifs, which have been shown to be highly conserved in most isolates (Poole et al. 1999): a putative SH3 binding motif (P386PPLPP), a potential SH2 binding motif (Y481EEVL), a

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tyrosine-based signaling motif (Y431ASIL), and a putative TRAF binding motif (A473TQPTDD) (Glenn et al. 1999; Poole et al. 1999). The cytoplasmic domain of K15 binds to TRAF1, TRAF2, and TRAF3 in vitro (Glenn et al. 1999). The tyrosine residue within the Y481EEVL motif of K15 is constitutively tyrosine phosphorylated by cellular SF-PTKs, including Src, Hck, Lck, Fyn, and Yes (Burkhardt et al. 1992; Choi et al. 2000; Rajcani and Kudelova 2003). Once phosphorylated, K15 activates the ERK2 and JNK pathways, which together lead to AP-1 activation (Brinkmann et al. 2003). TRAF2 interaction with the region containing Y481EEVL is involved in K15mediated activation of the MAPK pathway. Tyrosine phosphorylation within the Y481EEVL motif is required for TRAF2 binding and NF-kB activation (Brinkmann et al. 2003), indicating the importance of the Y481 residue of K15 in its activation of diverse signaling pathways. It has been reported that K15 is expressed in KSHVlatently infected PEL and MCD cells (Sharp et al. 2002), leading to the suggestion that the interaction between K15 and SF-PTKs plays a role in the maintenance of latency (Cho et al. 2008; Pietrek et al. 2010). Taken together, these findings suggest that K15 seems to function similarly to LMP1 (e.g., the recruitment of TRAFs and the activation of NF-kB and JNK) as well as to LMP2A (e.g., the recruitment of SF-PTKs).

Modulation of Chemokine/Cytokine System The mammalian chemokine system is composed of chemokines and chemokine receptors. Members of the chemokine superfamily currently consist of at least 46 members that are structurally related small proteins around 8–10 kDa in size. Four subfamilies have been identified so far based on the relative position of their cysteine (C) residues, which form conserved disulfide bonds: CCL, CXCL, XCL, and CX3L. The majority are either CCL chemokines (with no intervening amino acids between two cysteine residues) or CXCL (with a single intervening amino acid (X) between two cysteine residues) (Mantovani et al. 2010). The main function of chemokines is to attract different cells. For example, the CCL family members are known to attract a variety of cells from immune system, whereas the CXCL family members mainly attract neutrophils and lymphocytes. However, chemokines also regulate other biological activities, such as cell proliferation and differentiation, survival, angiogenesis, and senescence (Mantovani et al. 2010). Thus, deregulation of chemokine expression has been implicated in tumor growth, angiogenesis, and metastasis (Mantovani et al. 2010). Biological effects of chemokines are mainly mediated by G protein-coupled receptors (GPCRs), a diverse family of membrane receptors containing seven TM domains. While the extracellular domains of GPCRs engage with a variety of ligands, its cytoplasmic domain couples to heterotrimeric G proteins made up of an a subunit and a bg heterodimer. Following ligand binding, its TM domains undergo a series of conformational changes, catalyzing GDP to GTP exchange on a Ga subunit and subsequently generating a free GTP-bound Ga subunit and a free Gbg heterodimer to activate downstream signaling effector proteins, including PLC and

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adenylate cyclase (AC), which function as secondary messenger molecules. Although mutant forms of GPCR or heterotrimeric G proteins are not frequently found in human cancer, malignant cells often subvert the normal physiological functions of GPCRs to promote autonomous proliferation, evade immune responses, increase blood supply, and invade surrounding tissues and disseminate to other organs (Dorsam and Gutkind 2007). Several members of the HHV family, including EBV, KSHV, HHV-6, -7, and hCMV-1, encode mimicries of cellular chemokines and chemokine receptors, indicating that these viruses might have evolved to hijack host chemokine system for their propagation and replicative advantage (Rosenkilde et al. 2008). KSHV Viral Chemokines. KSHV expresses at least three chemokine homologs of cellular macrophage-inhibitory proteins (MIPs), which are members of the CCL chemokine family (Moore et al. 1996). For this reason, they were previously named vMIPI (vCCL1), vMIPII (vCCL2), and vMIPIII (vCCL3) and are encoded by ORFs K6, K4, and K4.1, respectively. These KSHV-encoded chemokines act on cellular chemokine receptors expressed on Th2 cells, such as CCR8 (vCCL1 and vCCL2), CCR3 (vCCL2), and CCR4 (vCCL3), from which their immunomodulatory functions are derived (Boshoff et al. 1997; Dairaghi et al. 1999; Endres et al. 1999; Stine et al. 2000). Interestingly, vCCLs, unlike their cellular homologs, have been shown to promote angiogenesis in certain experimental systems (Boshoff et al. 1997; Simonart et al. 2001; Stine et al. 2000). Since angiogenesis is a key feature of KS, vCCLs were speculated to be possible angiogenic factors of KS. For instance, vCCL1 activates the induction of a potent angiogenic factor, VEGF, and its receptor KDR (Flt-1) in vCCL1-expressing cells. Thus, it has been suggested that increased signaling mediated by upregulated VEGF and its receptor by vCCL1 may enhance new blood vessel formation and proliferation of tumor cells within the microenvironment of angiogenic KS lesions (Liu et al. 2001). Since KS lesions are also characterized by the presence of infiltrating leukocytes and high levels of inflammatory cytokines, the chemotactic properties of vCCLs may further promote infiltration of monocytes/macrophages to KS lesions to facilitate the production of inflammatory cytokines and proangiogenic factors (Direkze and Laman 2004). KSHV vGPCR. The KSHV vGPCR, encoded by ORF74, is homologous to the human chemokine receptors CXCR1 and CXCR2, which are the receptors for the angiogenic chemokines IL-8 (also known as CXCL8) and growth-related oncogene a (Gro-a, also known as CXCL1). Similar to cellular GPCRs, KSHV vGPCR is a seven TM protein with conserved glycosylation sites in its N-terminal and cysteine residues in its extracellular loops (Fig. 3.2a, Arvanitakis et al. 1997; Bais et al. 1998). Unlike cellular GPCRs, however, vGPCR is ligand-independent and constitutively active due to the presence of several structural changes, including a mutation (Asp142Val) within its highly conserved DRY (Asp-Arg-Tyr) motif (Rosenkilde et al. 2008). Moreover, compared to other viral and cellular chemokine receptors, vGPCR shows a higher degree of promiscuity, capable of binding not only cellular CXCL1 and CXCL8 but also vCCL2, indicating that vGPCR can induce signaling in a ligand-dependent fashion as well (Geras-Raaka et al. 1998; Gershengorn et al. 1998).

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a

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Fig. 3.2 Signaling mediated by viral chemokine receptors

Thus, both ligand-dependent and -independent activation of vGPCR may contribute to tumorigenesis and/or viral replication uniquely in the microenvironment of KSHV-associated tumors. The broad signaling capability of vGPCR also encompasses signaling through several G-protein a subunit families (Gas, Gaq, and Gai) (Shepard et al. 2001; Smit et al. 2002) as well as Rac-1, a member of the Rho family of monomeric G-proteins (Montaner et al. 2004). Signaling pathways constitutively activated by KSHV vGPCR include MAPK, PLC, PI3K, and Akt. Expression of KSHV vGPCR in human umbilical vascular endothelial cells (HUVECs) results in the induction of PI3K and Akt activity, which in turn plays a central role in promoting cell survival (Bais et al. 2003; Montaner et al. 2001; Sodhi et al. 2004). Akt activity is tightly regulated by PI3K. When growth factor receptors bind to ligands, the catalytic subunit (p110) of PI3K is activated via recruitment of the regulatory subunit (p85) of PI3K or via Ras activation, both of which lead to the production of PIP3. Akt in the cytoplasm then binds to PIP3 and subsequently translocates to the plasma membrane, where its kinase activity is then fully induced by PI3K-dependent kinases (PDKs). The tumor-suppressor protein PTEN antagonizes PI3K by dephosphorylating PIP3, reducing Akt translocation to the cellular membrane, thereby downregulating Akt activity. It has been reported that treatment of an Akt inhibitor prevents the proliferation of vGPCR-expressing endothelial cells in vitro and inhibits the tumorigenic potential of vGPCR in mice (Sodhi et al. 2004). This suggests that the activation of Akt is a significant factor in the development of vGPCR-induced sarcoma and perhaps KS. Recent studies have further demonstrated that vGPCR-mediated Akt activity can activate the mTOR pathway, which also plays a central role in cell proliferation, metabolism, and angiogenesis (Sodhi et al. 2006). Constitutive activation of Akt by vGPCR-mediated PI3K

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activation results in phosphorylation and subsequent degradation of the tuberous sclerosis complex (TSC), a negative regulator of mTOR (Sodhi et al. 2006). As a result, mTOR activity is induced, giving rise to the activation of ribosomal p70 S6 kinase and 4EBP1, the key regulators of the translational machinery, and the promotion of cell proliferation. Treatment of rapamycin, a pharmacological inhibitor of mTOR, showed a dramatic suppression of vGPCR-expressing cell proliferation in vitro as well as tumor growth in vivo, indicating that vGPCR-mediated activation of the PI3k/Akt/mTOR cascade may contribute to vGPCR-mediated sarcomagenesis (Montaner 2007). Similar to vCCLs, vGPCR has also been reported to have angiogenic functions. Endothelial cells ectopically expressing vGPCR have constitutively active VEGF receptors and increased proliferation (Bais et al. 2003). In addition to VEGF, vGPCR also upregulates angiogenic chemokines IL-8 and Gro-a in a manner similar to human CXCR2, a chemokine receptor associated with angiogenesis in a number of tumors (Montaner et al. 2004). Another signaling pathway induced by vGPCR is the activation of p38 and ERK and the subsequent phosphorylation of hypoxia-induced factor 1a (HIF-1a). This, then, acts on the VEGF promoter, resulting in the induction of VEGF expression and secretion (Sodhi et al. 2000). vGPCR can also activate such transcription factors as NF-AT, AP-1, and NF-kB and promote the expression of a number of autocrine and paracrine proinflammatory cytokines and growth factors, such as IL-1b, IL-6, GM-CSF, TNFa, IL-8, and MIP-1, as well as adhesion molecules, such as VCAM-1, ICAM-1, and E-selectin (Cannon et al. 2003; Couty et al. 2001; Montaner et al. 2001; Pati et al. 2001, 2003; Schwarz and Murphy 2001; Shepard et al. 2001; Smit et al. 2002). As discussed above, vGPCR appears to exert broad effects on cell proliferation, angiogenesis, and inflammation for a mere single viral gene product. Thus, KSHV vGPCR, although expressed during the lytic phase of its viral life cycle, clearly stands out as one of the key factors involved in the proliferation of KSHV-associated tumor cells. EBV BILF1. EBV encodes a GPCR homolog called BILF1, which is expressed in the lytic phase of the viral replication cycle. Although it displays a limited homology to chemokine receptors, sequence analysis revealed that BILF1 has several features belonging to GPCRs. It contains seven TM domains, conserved cysteine residues in its N-terminus and in its extracellular loops, seven N-glycosylation sites, and four intracellular phosphorylation sites (Fig. 3.2b, Paulsen et al. 2005). BILF1 is known to constitutively activate signaling pathways involved in proliferation (NF-kB) through Gai (Paulsen et al. 2005). Similar to KSHV vGPCR, the DRY motif in BILF1 is replaced with an EKT (Glu-Lys-Thr) motif (Paulsen et al. 2005). Alternative DRY motifs found in KSHV vGPCR have been previously shown to be associated with constitutive activity as well as transforming activity (Rosenkilde et al. 2008). Unsurprisingly, a recent study demonstrated that BILF1, similar to KSHV vGPCR, has both of these abilities in vitro and in vivo (Lyngaa et al. 2010). This study shows that the EKT motif plays a key role in the constitutive activation of BILF1 and that constitutive signaling through Gai is associated with BILF1-mediated cell transformation, VEGF secretion, and tumor formation (Lyngaa et al. 2010). Although these

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studies imply that like KSHV vGPCR BILF1 may play an important role during EBV infection by mimicking a functional GPCR, additional roles of BILF1 in immune evasion by targeting PKR and MHC class I (Garcia et al. 2006; Zuo et al. 2009) suggest that BILF1 may function distinctly from KSHV vGPCR. Moreover, it still remains unknown whether chemokines and other ligands bind to BILF1. KSHV vIL-6 (ORF K2). Cellular interleukin (IL)-6 is a multifunctional cytokine involved in the regulation of immune responses, inflammation, oncogenesis, and angiogenesis (Nishimoto and Kishimoto 2006). Signaling induced by IL-6 requires the IL-6 receptor (IL-6R) complex composed of gp80 (the a subunit) and gp130 (the b subunit). gp130 is ubiquitously expressed and homodimerizes to transduce intracellular signaling, whereas gp80 binds to IL-6 but is limited in its availability. IL-6/IL-6R signaling induces signaling pathways, such as the Ras/MAPK and JAK/ STAT pathways, which are in turn involved in the activation of transcription of IL-6 responsive genes (c-Fos and c-Jun). Many B-cell tumor cell lines depend on IL-6 signaling for growth (Nishimoto and Kishimoto 2006). KSHV encodes a homolog of cellular IL-6, vIL-6 encoded in (ORF) K2 (Neipel et al. 1997). It has 24.6% amino acid sequence homology to cellular IL-6. Although expressed abundantly during lytic replication, various levels of vIL-6 expression have been detected in latently infected cells (MCD>PEL>>KS; <5% of cells in PEL and in about 5–20% of KSHV-infected lymphoid cells in extracavity PEL and MCD, but not in KS) (Du et al. 2007). Unlike cellular IL-6, however, vIL-6 is selectively glycosylated and signals through only gp130 to activate IL-6 responsive genes and promote B-cell survival (Chatterjee et al. 2002; Hoischen et al. 2000; Martin and Gutkind 2009; Molden et al. 1997; Mullberg et al. 2000). Thus, vIL-6 bypasses the normal cellular checkpoint of gp80 coupling with gp130 for IL-6 signaling (Molden et al. 1997). Similar to cellular IL-6, vIL-6 has multifunctional roles, targeting various cellular processes, such as cell proliferation, oncogenesis, and immune responses (Nicholas 2005). Interestingly, it has been reported that vIL-6 can functionally replace cellular IL-6 in many B-cell tumor cell lines, which are dependent on IL-6 for their growth. This indicates the substantial functional homology between viral and cellular IL-6 (Burger et al. 1998; Moore et al. 1996). In addition, it has been shown that vIL-6 activates all of the known cellular IL-6-induced signaling pathways, including the Ras/MAPK and JAK/STAT signaling cascades. Activation of the JAK/STAT pathway by vIL-6 results in the induction of VEGF (Molden et al. 1997), underscoring the likely importance of the angiogenic property of vIL-6 in the development of KSHV-associated malignancies. In addition, several lines of evidence suggest that both cellular and viral IL-6 are important in the proliferation of KSHV-associated B-cell lymphomas: (1) PEL-derived cell lines depend on vIL-6 and IL-10 for their growth and the induction of cellular IL-6 expression (Jones et al. 1999); (2) vIL-6 can induce the secretion of cellular IL-6 (Mori et al. 2000); (3) in addition to vIL-6, LANA-1, vFLIP, and vGPCR can also induce cellular IL-6 expression, most likely by activating AP-1 and NF-kB (An et al. 2002; Du et al. 2007; Montaner et al. 2004; Polson et al. 2002); and (4) high serum levels of IL-6 are detected in patients with PEL and MCD (Ganem 2007). Thus, both cellular IL-6 and vIL-6 signaling may

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contribute to PEL cell proliferation and to the angiogenesis present in patients with KSHV-associated lymphoproliferative diseases.

Modulation of Cell Cycle Machinery Normal tissues maintain cellular quiescence and tissue homeostasis by employing multiple antiproliferative signals (Hanahan and Weinberg 2000). Similar to growth stimulatory signals, growth inhibitory signals, including both soluble and immortalized inhibitors embedded in the ECM and on the surface of nearby cells, are received by TM receptors and transduced into intracellular antiproliferative signaling circuits, which eventually block proliferation. Depending on the incoming signal, cells in the G1 phase of the cell cycle decide whether to initiate proliferation or to prevent proliferation either by forcing themselves out of the active proliferative cycle into a quiescent (G0) state or by inducing permanent (irreversible) entry into a postmitotic and differentiated state (Dayaram and Marriott 2008). Much of the antiproliferative signals induced in normal cells upon receiving growth inhibitory signals are associated with cell cycle block, in which tumor-suppressor genes, such as p53, and members of the retinoblastoma (Rb) family (Rb, p107 and p130) play critical roles at the molecular level. Rb and p53 mutations are observed at high frequencies in a wide variety of clinically important cancers (Hanahan and Weinberg 2000). Most DNA tumor viruses encode viral oncoproteins, which can disrupt the function of the p53 and Rb family members at least in part by directly interacting and inactivating them, which presumably allow infected cells to escape from p53- or Rb-mediated cell cycle arrest (Hume and Kalejta 2009; O’Shea 2005). Both EBV and KSHV appear to encode proteins capable of targeting multiple cellular components of the cell cycle machinery, including Rb and p53, to promote cell proliferation. Thus, this section briefly introduces the cellular antiproliferative pathways mediated by Rb and p53 prior to the discussion of how viral proteins encoded by EBV and KSHV modulate cellular antiproliferative signals to ultimately contribute to the proliferation of tumor cells. Rb-mediated Cell Cycle Arrest. Progression of normal cell cycle transition from G1 through S, and G2 into M, occurs in a sequential and coordinated fashion. For example, the sequential activation of cyclin and cyclin-dependent kinase (Cdk) complexes is a central event in cell cycle transitions: cyclin D and Cdk4, Cdk6 and Cdk2 in G1, cyclin E and Cdk2 in late G1, cyclin A and Cdk2 in S and G2 phase, and cyclins B and A and Cdk1 in M phase (cyclin B is synthesized in the G2 and M phase). Activated Cdks then accelerate cell cycle progression (Hume and Kalejta 2009). One of the main substrates of Cdks is the Rb protein, which is known to contain multiple putative Cdk phosphorylation sites. In the absence of growth stimulatory factors, Rb, constitutively expressed during the G1 phase, exists in a hypophosphorylated form and forms a complex with the E2F transcriptional activator, resulting in the repression of E2F-mediated transcription of cellular genes required for a G1 to S phase transition (Rb-mediated G1 cell cycle arrest) (Fig. 3.3, Hume and

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Fig. 3.3 Rb- and p53-mediated antiproliferative pathways

Kalejta 2009). In the presence of mitogenic growth factors, however, D-type cyclins (cyclins D1, D2, and D3) are synthesized throughout the G1 phase and activate Cdk4/6 by forming a complex with either Cdk4 or Cdk6. Active cyclin D-Cdk4/6 complexes then phosphorylate Rb, and hyperphosphorylated Rb is unable to interact with E2F. The released E2F is then able to stimulate the transcription of cellular genes essential for subsequent cell cycle progression into the S phase. Moreover, cell cycle is intricately regulated by a sophisticated interplay of positive and negative regulatory signals. In the absence of stimulatory growth signals, negative regulatory signals prevent uncontrolled cellular proliferation by maintaining Cdks in an inactive state. This is achieved by two distinct classes of Cdk inhibitors (CKIs): the Cip/Kip family includes p21Cip1, p27Kip1, and p57Kip2 and inhibits G1 cyclin–Cdk and cyclin B–Cdk1 complexes, and the other class is the INK4 family including p16INK4a, p15INK4b, p18INK4c, and p19INK4d, which specifically inhibits G1 cyclin–Cdk complexes (cyclin D/Cdk4 or cyclin D/Cdk6). p16INK4a is an inhibitor of cyclin D-dependent kinases, such as Cdk4 and Cdk6, and blocks Rb function, whereas p19INK4d blocks the murine double minute 2 (Mdm2)-mediated destruction of p53, thereby inducing the expression of the Cdk inhibitor p21Cip1 (Vermeulen et al. 2003). p53-mediated Cell Cycle Arrest. Cellular DNA is continuously exposed to a variety of endogenous and exogenous genotoxic influences. Thus, cells must have an efficient surveillance system to monitor the integrity of their DNA and eliminate acquired DNA damage. Upon detecting genomic injuries in the G1 phase, the DNA damage checkpoint response is induced by activating the DNA damage sensors

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ATM and ATR; protein kinases capable of phosphorylating a large number of protein substrates involved in cell cycle control and DNA repair, including the tumor-suppressor protein p53; and checkpoint kinases (Chk) 1 and 2. Phosphorylation of p53 by activated ATM/ATR increases its stability and its function as a transcriptional activator. Activation of p53 by DNA damage promotes the transcription of p21, an inhibitor of G1 cyclin–Cdk, which consequently causes G1 cell cycle arrest (p53-mediated G1 cell cycle arrest) (Fig. 3.3). Since genes induced by activated p53 are involved in apoptosis, induction of p53 also leads to apoptosis (p53-mediated apoptosis) (Fig. 3.3). Under normal conditions, a low level of p53 is maintained by Mdm2, an E3 ubiquitin ligase that exerts its negative effect on p53 in two ways. First, the N-terminus of Mdm2 interacts with the transactivation domain of p53, thus inhibiting the transcriptional activity of p53. Second, the C-terminal ubiquitin ligase domain of Mdm2 ubiquitinates p53, resulting in p53 degradation through the ubiquitin–proteasome pathway. Additionally, Mdm2 and p53 are components of an autoregulatory negative feedback loop, wherein p53 induces Mdm2 expression, while Mdm2 represses p53 activity (Fig. 3.3). This feedback loop confers the tight regulation necessary for proper p53 function (Lee et al. 2009). Thus, the subversion of cell cycle components involved in the Rb pathway as well as the p53 pathway could render tumor cells insensitive to a number of antiproliferative signals. EBV LMP1. LMP1 has been reported to modestly induce cyclin D2 expression, perhaps indirectly through the induction of c-Myc and AP-1 transcription factors, and to maintain Rb in a hyperphosphorylated state in B cells treated with TGF-b (Arvanitakis et al. 1995; Dirmeier et al. 2005; Hume and Kalejta 2009). In addition, LMP1 downregulates the expression of CKI p16INK4a and inhibits Ras-mediated induction of p16 and p21 (Yang et al. 2000). Recent studies have demonstrated that LMP1 decreases p27 transcription by inducing the binding of E2F4 and p130, a member of the Rb family, to the predicted E2F site within the p27 promoter. Furthermore, it increases the levels of Cdk2 and Rb phosphorylation by inducing the PI3K–Akt signaling pathway (Everly et al. 2009; Mainou et al. 2005, 2007; Mainou and Raab-Traub 2006). Thus, by inducing cyclin D and downregulating both p16 and p27, LMP1 may play a pivotal role in EBV-associated lymphoproliferative diseases. EBV EBNA-1. EBV EBNA-1 is the only EBV protein consistently expressed in all proliferating infected cells. It is a multifunctional sequence-specific DNA binding phosphoprotein required for the replication and maintenance of the EBV genome (Rickinson and Kieff 2007). The best known function of EBNA-1 is to maintain latent episomal DNA as an episomal plasmid, which must be distributed into daughter cells during division (Thompson and Kurzrock 2004). Four functional modules have been characterized (Rickinson and Kieff 2007): (a) an arginine-rich N-terminal spanning 89 amino acids; (b) a region encompassing amino acids 90–327 of GGA repeats; (c) amino acids 328–386 which are also arginine rich and contain a nuclear localization signal at amino acids 379–386, and (d) the C-terminal comprising amino acids 451–608 that allows sequence-specific DNA binding and dimerization of EBNA-1 molecules (Rajcani and Kudelova 2003). The two arginine-rich regions mediate homotypic interactions between DNA-bound EBNA-1 molecules and are

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involved in transcriptional activation, plasmid maintenance, and DNA replication (Mackey and Sugden 1999). Recently, it has been reported that EBNA-1 increases cell survival and proliferation by targeting HAUSP, a deubiquitinase that plays a key role in the regulation of p53 and Mdm2. Overexpression of HAUSP stabilizes p53, resulting in p53-mediated growth repression and apoptosis, whereas decreased HAUSP levels destabilize p53. HAUSP also stabilizes Mdm2, a negative regulator of p53, as described above. Thus, HAUSP plays multiple roles in regulating the p53–Mdm2 pathway. The HAUSP N-terminal TRAF domain (amino acids 53–208) binds not only to the C-terminal regulatory region of p53 (amino acids 357–382), but also EBNA-1 (amino acids 395–450) (Holowaty et al. 2003). However, EBNA-1 binds HAUSP much stronger (tenfold) than the p53 regulatory region. The EBNA-1 395–450 peptide also displaces the p53 peptide from the p53–HAUSP complex. EBNA-1 binds HAUSP in EBV-infected cells and EBNA-1 interferes with the UV-induced stabilization of p53 when expressed at levels similar to those in EBVinfected cells. Taken together, several lines of evidence indicate that EBNA-1 can indirectly destabilize p53 by binding to HAUSP, which could be important for initial cell immortalization by EBV, continued proliferation and survival of latently infected cells, and/or malignant transformation. EBV EBNA-3C. The EBNA-3 gene family comprises three genes placed tandemly on the EBV genome. The products of EBNA-3 genes are EBNA-3A, -3B, and -3C. These proteins are nuclear phosphoproteins and function mainly as transcriptional regulators. Among these, EBNA-3A and -3C are crucial for B-cell transformation in vitro, whereas 3B is dispensable (Rickinson and Kieff 2007). In addition to acting as a transcriptional regulator, a number of studies indicate that EBNA-3C can also regulate the cell cycle by interacting with and modulating several components of the cell cycle (Kashuba et al. 2003, 2008; Knight and Robertson 2004; Knight et al. 2004, 2005a, b; Parker et al. 1996, 2000; Saha et al. 2009; Yi et al. 2009). Earlier studies have shown that EBNA-3C directly interacts with Rb in vitro (Parker et al. 1996, 2000). This interaction conferred rodent fibroblasts resistance to the effects of p16INK4a. Subsequently, it was reported that EBNA-3C is required for LCLs to proliferate continuously and to maintain low levels of both p16INK4a protein and mRNA. Although this study suggests that EBNA-3C may repress p16 expression, evidence of EBNA-3C interaction with Rb in virus-infected cells was not provided (Parker et al. 2000). Another mechanism by which EBNA-3C may modulate the Rb pathway is by forming a complex with cyclin A, an activator of S-phase cell cycle (Knight and Robertson 2004; Knight et al. 2004). EBNA-3C was shown to associate with cyclin A in vitro as well as in LCLs. Interestingly, it was also shown that the expression of ENBA-3C induces cyclin A/Cdk2 kinase activity and disrupts cyclin A interaction with p27Kip1, a potent inhibitor of cyclin A/Cdk2 (Knight and Robertson 2004; Knight et al. 2004). Recent studies further explore the potential roles of EBNA-3C as a modulator of cell cycle by regulating both Rb and p53 (Kashuba et al. 2008; Knight et al. 2005a, b; Saha et al. 2009; Yi et al. 2009). It has been reported that EBNA-3C recruits the SCFSkp2 E3 ubiquitin ligase complex, known to promote the polyubiquitination and degradation of p27, E2F, and c-Myc, to facilitate the degradation of p27Kip1 and Rb

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(Knight et al. 2005a, b). One study demonstrated that EBNA-3C can bind to and translocate MRS18-2, a mitochondrial protein capable of binding to both hypo- and hyperphosphorylated forms of Rb, into the nucleus, where MRS18-2 competes with E2F for binding to Rb, thereby allowing free E2F to induce S-phase entry (Kashuba et al. 2008). In addition to Rb, EBNA-3C has been shown to directly interact with p53 in vitro (Saha et al. 2009; Yi et al. 2009). Interaction of EBNA-3C and p53 substantially reduces luciferase activity under the control of a p53-responsive promoter and blocks p53-mediated apoptosis (Yi et al. 2009). Moreover, EBNA-3C was shown to form a ternary complex with Mdm2 and p53, although EBNA-3C interaction with Mdm2 was reduced in the presence of p53. EBNA-3C binding to Mdm2 results in the stabilization of Mdm2, promoting p53 polyubiquitination and subsequent p53 degradation in various cell lines (Saha et al. 2009). EBV ENBA-5 (EBNA-LP). EBNA-5 is a nuclear phosphoprotein expressed early during EBV infection of B cells. Early studies have suggested that EBNA-5 may dysregulate cell cycle progression by binding to both Rb and p53 (Hoppe-Seyler and Butz 1995; Szekely et al. 1993). EBNA-5 was reported to bind to Rb in GST pull-down experiments despite lacking either an LxCxE motif or hydrophobic patch (Szekely et al. 1993). However, the biological significance of Rb binding remains unclear since ENBA-5 was unable to counteract the repressive effects of Rb or p107 on a reporter construct under the control of a Gal4–E2F–1 fusion protein (Inman and Farrell 1995). Another mechanism by which EBNA-5 may dysregulate cell cycle progression is by cooperating with ENBA-2. EBNA-5 was shown to interact with EBNA-2 to drive resting B cells that were stimulated with the EBV gp340 envelope protein into the G1 phase of the cell cycle by upregulating cyclin D2 expression (Sinclair et al. 1994). Moreover, recent studies have hypothesized that EBNA-5 binds p14ARF and Mdm2, two proteins involved in p53 regulation (Kashuba et al. 2003, 2010), leading to the inhibition of the transactivating function of p53. KSHV vCyclin. KSHV vCyclin encoded in ORF72 is a homolog of cellular cyclin D2, displaying around 53% sequence similarity to cyclin D2 (Li et al. 1997). Although vCyclin weakly binds to Cdk2, Cdk3, and Cdk4, it strongly forms a complex with Cdk6 (Chang et al. 1996; Godden-Kent et al. 1997; Li et al. 1997). Tight interaction of vCyclin with Cdk6 results in the robust activation of Cdk6, leading to enhanced phosphorylation of Rb (Fig. 3.3, Li et al. 1997). Although vCyclin is structurally and functionally similar to type D cyclins, it has several unique features. First, unlike cellular type D cyclins, vCyclin exhibits promiscuous substrate specificity, targeting substrates of type A and E cyclins coupled to Cdk2 kinases. These include targets of the replication machinery, such as histone H1, p27, Cdc25a (targets of the cyclin E/Cdk2 complex), ORC1, and Cdc6 (targets of the cyclinA/Cdk2 complex) (Ellis et al. 1999; Laman et al. 2001; Mann et al. 1999), indicating that vCyclin/ Cdk6 mimics the combined activities of G1- and S-phase cyclin/Cdk complexes (Verschuren et al. 2004). Second, unlike the cellular cyclin D/Cdk6 complex, which normally requires Cdk6 phosphorylation through the activation of a Cdk-activating kinase (CAK), vCyclin is less dependent on host kinases since the vCyclin/ Cdk6 complex does not require phosphorylation by CAK (Child and Mann 2001;

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Kaldis et al. 2001). Third, the vCyclin/Cdk complex can escape from CKI inhibition by targeting p27 and p21, potent inhibitors of cyclin D, A, and E and weak inhibitors of cyclin B-associated kinases. vCyclin modulates p27 levels by inducing p27 degradation, which normally occurs in the presence of A/E type cyclins, but not D type cyclins (Ellis et al. 1999; Mann et al. 1999; Rajcani and Kudelova 2003). Recently, it has been demonstrated that vCyclin stably associates with p27 in PEL cells, and that the vCyclin/Cdk6 complex phosphorylates p27 on serine 10, leading to its sequestration in the cytoplasm and thereby inactivating its antiproliferative function (Sarek et al. 2006). This may allow PEL cells to proliferate despite their high levels of p27. Similarly, the vCyclin/Cdk6 complex phosphorylates p21 on serine 130, bypassing p21-mediated G1 arrest (Jarviluoma and Ojala 2006). Thus, the vCyclin/Cdk6 protein complex is resistant to many cell cycle checkpoints, including Rb and CKI, contributing to enhanced rates of cell proliferation. KSHV LANA-1. KSHV LANA-1 (ORF73) is a major latency-associated nuclear protein expressed in PEL, MCD, and KS. Thus, it is the most widely used marker for KSHV latency. There is no known mammalian homolog. It is composed of three subdomains: (a) a central region containing variable numbers of highly acidic repeats; (b) a more basic C-terminal region involved in DNA binding and oligomerization; and (c) an N-terminal region implicated in chromatin attachment and corepressor recruitment (Ganem 2006). LANA-1 is a multifunctional protein. The best-characterized function of LANA-1 is its involvement in the establishment and maintenance of the latent viral episome in the nucleus by tethering the viral episome to host chromatin during mitosis to ensure segregation of viral genomes to daughter cells (functionally similar to the EBV EBNA-1 protein) (Ballestas et al. 1999; Cotter and Robertson 1999). It has been reported that KSHV LANA-1 targets both the Rb pathway and p53 pathway (Friborg et al. 1999; Radkov et al. 2000). The finding that LANA-1 directly binds Rb through a highly acidic region in vitro and in PEL cells, transactivating E2F-dependent promoters and the cyclin E promoter (Radkov et al. 2000) indicates that LANA-1 alleviates Rb-mediated cell cycle arrest. LANA-1 blocks p53-mediated apoptosis by binding to p53 and inhibiting the transcriptional activity of p53 in PEL cells (Friborg et al. 1999). This results in the prolonged life span of primary endothelial cells overexpressing LANA-1 (Watanabe et al. 2003), suggesting a potential role of LANA-1 in promoting cell survival. KSHV vIRFs. The KSHV genome encodes four potential homologs of cellular IRFs and they are thus named vIRF 1 (K9), 2 (K11), 3 (K10.5; LANA-2), and 4 (K10). KSHV vIRFs are numbered based on the order of their discovery and characterization, not on their homology to any particular cellular IRF. For example, vIRF3 is not the viral homolog of cellular IRF3; it is more closely related to IRF4 (Ganem 2006). Most of vIRFs are lytic proteins, but vIRF3 is a latent gene primarily expressed in latently infected B cells, not in KS spindle cells (Ganem 2006). In addition to the downregulation of IFN-mediated host innate immune responses, KSHV vIRFs share one more common feature, which is the targeting of p53-mediated cell growth control (Lee et al. 2009). vIRF1 interacts with p53 to suppress its acetylation, inhibiting

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the transcriptional activation of p53 and efficiently preventing p53-mediated apoptosis (Nakamura et al. 2001; Seo et al. 2001). Moreover, vIRF1 interacts with ATM to block its activity, thereby reducing p53 phosphorylation at its serine 15 residue and increasing p53 ubiquitination (Shin et al. 2006). In conjunction with vIRF1, the B cell-specific, latently expressed vIRF3 also interacts with p53, thereby inhibiting p53-mediated transcription and apoptosis (Rivas et al. 2001). Silencing it by siRNA/ shRNA in PEL cell lines results in increased apoptosis and caspase3/7 activity, suggesting that this protein contributes to the survival of PEL cells (Carbone and Gloghini 2008). Recently, it was shown that vIRF4 interacts with Mdm2, leading to a reduction of p53 via proteosomal degradation, thereby effectively suppressing p53mediated apoptosis and establishing favorable circumstances for viral replication (Lee et al. 2009). Collectively, it is clear that the downregulation of p53-mediated cell growth control is a common characteristic of the vIRFs (Lee et al. 2009).

Subversion of Cellular Signaling Pathways Associated with Cell Proliferation Constitutive Activation of NF-kB Signaling Pathway. One of the unique mechanisms that both EBV and KSHV utilize to promote lymphoma induction is the constitutive activation of the NF-kB pathway, which regulates the expression of a large number of genes involved in cell proliferation (de Oliveira et al. 2010). NF-kB, a transcription factor, is composed of homo- and heterodimers of five subunits, including c-Rel, NF-kB1 (p50), NF-kB2 (p52), p65 (RelA), and RelB. NF-kB1 and NF-kB2 are produced as p105 and p100 precursors, respectively. In nonstimulated cells, NF-kB is sequestered in the cytoplasm by forming a complex with inhibitory kB (IkB). Various biological stimuli, including TNFa, interleukin-1, the lipopolysaccharide (LPS), viral infection, and ligands for antigen receptors, however, lead to the phosphorylation and subsequent ubiquitination of IkB for proteosomal degradation. NF-kB is then released and translocated to the nucleus to induce the transcription of target genes. Two protein kinases, IkB kinase a (IKKa) and IKKb, mediate the phosphorylation of IkB and represent a convergence point for signal transduction pathways leading to NF-kB activation. Most of the IKKa and IKKb molecules in the cell are in IKK complexes that also contain a regulatory molecule, IKKg/Nemo. The interaction of IKKg/Nemo with IKKa and IKKb is critical for the assembly of the IKK complex and for increasing IKK activity (Hacker and Karin 2006). Two distinct NF-kB pathways, termed canonical (classical) and noncanonical (alternative), have been defined. IKKb and IKKg/Nemo play a major role in IkB phosphorylation in canonical NF-kB activation upon TNFa, IL-1, and LPS stimulation. Noncanonical NF-kB activation involves proteasome-mediated processing of p100 into p52 and this requires IKKa and NF-kB-inducing kinase (NIK), a member of the MAP3K superfamily responsible for the phosphorylation and activation of IKKa, to respond to a subset of TNFa family members, such as B-cell activating factor (BAFF), CD40 ligand, and lymphotoxin (LT)a–LTb heterodimers (Hacker and Karin 2006).

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Fig. 3.4 Activation of NF-kB signaling by EBV and KSHV

Several viral proteins expressed during lytic and latent infection of EBV and KSHV have been shown to modulate NF-kB signaling pathways in favor of viral replication and survival (Fig. 3.4, de Oliveira et al. 2010). Among them, EBV LMP1 and KSHV vFLIP, expressed during latency, are largely responsible for the constitutive activation of NF-kB in infected cells (Fig. 3.4, de Oliveira et al. 2010). In addition, these two viral proteins can similarly activate both canonical and noncanonical NF-kB (de Oliveira et al. 2010). EBV LMP1. LMP1 activates NF-kB via its CTAR1 and CTAR2 regions by recruiting TRAFs and TRADD, respectively. The TRAF family consists of seven members, all of which contain a RING finger domain associated with E3 ligase activity and several zinc finger motifs at their N-terminus with the exception of TRAF1. The C-terminal domain of all TRAF proteins have a novel TRAF domain composed of a coiled-coil domain (TRAF-N) and a conserved TRAF-C domain at the C-terminus, which is responsible for homo- and heterodimerization of the TRAF proteins as well as for their direct and indirect interactions with cognate surface receptors. TRAF proteins interact with members of the TNFR superfamily, which directly or indirectly recruits specific TRAFs to their intracellular domain to mediate various downstream signaling pathways involved in survival, proliferation, differentiation, activation, and migration. Both TNFR2 and CD40 directly recruit TRAFs via a consensus TRAF-binding motif (PxQxT, where x is any amino acid) in their intracellular domain. Members of TNFR superfamily that contain a death domain (DD) in their intracellular domain, such as TNFR1, first recruit a

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DD-containing adaptor protein, TRADD, via a DD–DD homotypic interaction. TRADD then recruits TRAF2 and RIP for survival signaling, and FADD and caspase-8 for the induction of apoptosis (Soni et al. 2007). LMP1 CTAR1 has a PxQxT TRAF binding motif and is therefore able to recruit DD-containing domain proteins, such as TRADD and RIP1. LMP1 CTAR1 can activate the noncanonical NF-kB pathway in fibroblast cell lines and epithelial and B cells by recruiting TRAFs (1, 2, 3, and 5), whereas LMP1 CTAR2 has a YYD motif and can activate the canonical NF-kB pathway by directly interacting with RIP and TRADD, followed by the recruitment of TRAF6. Although TRAF6 does not bind to LMP1 directly, it is essential for LMP1-mediated NF-kB activation in MEFs (Soni et al. 2007). Thus, LMP1 can activate NF-kB signaling by acting as a constitutively active TNFR, producing effects similar to those induced by B-cell activation receptor CD40 or a combination of TNFR1 and TNFR2 (Figs. 3.1 and 3.4a, Rickinson and Kieff 2007). KSHV K13 (vFLIP). The KSHV K13, encoded in ORF71 of the viral genome, is a homolog of cellular FLIP [FLICE (FADD-like IL-1b converting enzyme)-inhibitory protein], thus named vFLIP. Similar to cellular FLIPs (cFLIPs) and several other vFLIPs encoded by herpesvirus saimiri (HVS), equine herpesvirus, and Molluscum contagiosum poxvirus, KSHV vFLIP contains two tandem death-effector domains (DEDs) and can block Fas-mediated apoptosis (Belanger et al. 2001; Bertin et al. 1997; Hu et al. 1997; Thome et al. 1997). Among the known vFLIPs, only KSHV vFLIP shares functional homology to cFLIPs, with the ability to activate the NF-kB pathway but in a constitutively active manner (Chaudhary et al. 1999). This may indirectly contribute to antiapoptotic function of vFLIP by enhancing the transcription of a number of antiapoptotic proteins. In PEL cells, vFLIP interacts with components of the IKK complex and RIP, a protein kinase that plays an essential role in TNFR1-mediated NF-kB activation (Liu et al. 2002). Yeast two-hybrid screening identified IKKg/Nemo as a binding partner of vFLIP (Fig. 3.4b, Field et al. 2003). In addition, KSHV vFLIP associates with Hsp90, an important component of IKK complexes (Field et al. 2003). Inhibition of Hsp90 in PEL cells results in a significant reduction of cell death and IKK activity induced by vFLIP, suggesting that the activity of the vFLIP–IKK complex depends on Hsp90 (Field et al. 2003). Despite its weak detection in PEL cells and endothelial cells carrying KSHV, vFLIP is the major factor promoting tumor cell survival. In PEL cells, siRNA-mediated knockdown of vFLIP expression significantly reduced NF-kB activity by approximately 80% and increased the sensitivity of PEL cells to external apoptotic signals, subsequently enhancing apoptotic levels, suggesting that vFLIP is essential for PEL cell survival (Guasparri et al. 2004). KSHV vFLIP also constitutively activates the noncanonical NF-kB pathway by upregulating the expression of NF-kB precursor protein p100 and enhancing its processing to p52 subunit in MEF and 293T cells stably expressing KSHV vFLIP (Matta and Chaudhary 2004). Knockdown of KSHV vFLIP by siRNA in PEL cells leads to a marked reduction of p100 processing and a significant decrease in cell proliferation (Matta and Chaudhary 2004), indicating that activation of the noncanonical NF-kB pathway induced by KSHV vFLIP is also required for the growth

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and proliferation of PEL cells. One of the unique features of KS is the involvement of cytokines and growth factors in the pathogenesis of this disease. The NF-kB pathway is well-known for its role in the transcriptional activation of several cytokines and chemokines. In fact, vFLIP mediates the induction of IL-6 and IL-8 expression through its activation of NF-kB (An et al. 2002; Sun et al. 2006). Both KSHV vIL-6 and KSHV vGPCR are proposed to contribute to the production of cytokines involved in KS development as discussed above. Unlike KSHV vFLIP, however, none of these genes are major latency-associated genes. Thus, vFLIP expressed in latently infected cells is a likely candidate for viral genes responsible for the promotion of tumor cell proliferation. Modulation of Notch Signaling Pathway. Notch signaling is involved in diverse cellular processes, including cell fate decision, differentiation, and proliferation. Deregulation of Notch signaling has been implicated in a number of human malignancies (Bolos et al. 2007; Leong and Karsan 2006). In mammals, the Notch receptor family consists of four members, Notch1, 2, 3, and 4, and the Notch ligand family consists of five members, Jagged-1 and −2 and Delta-like-1, -3, and −4. Both Notch receptors and Notch ligands are type I single-pass TM proteins. Notch receptors have a large extracellular domain and a cytoplasmic domain. The extracellular domain of Notch receptors consists of 29 and 36 EGF-like repeats involved in ligand binding, followed by three cysteine-rich LIN12/Notch repeats (LNRs) involved in the prevention of receptor activation in the absence of ligands. The Notch intracellular domain (NotchIC or NICD) consists of a RAM23 domain, six ankyrin/cdc10 repeats involved in protein–protein interactions, two nuclear localization signals (NLSs), a transcriptional activation domain (TAD), and a PEST sequence involved in degradation of Notch. Two consecutive proteolytic cleavages of these receptors occur upon ligand binding: TNF-a converting enzyme (TACE)mediated cleavage on the extracellular side near the TM domain, followed by g-secretase complex-mediated intramembrane cleavage within the TM domain. As a result, NotchIC is released and translocated into the nucleus to induce target gene transcription. In the absence of nuclear NotchIC, the transcription-repressor protein C protein-binding factor 1 (CBF1, also known as RBP-Jk or CSL) binds to CBF1 binding sites of Notch target gene promoters and represses transcription of Notch target genes (Fig. 3.5a). When Notch signaling is activated, NotchIC enters the nucleus, binds CBF1, and recruits mastermind-like (MAML) transcription activator. MAML then recruits the histone acetyltransferase p300/CBP, resulting in the activation of Notch target gene transcription (Fig. 3.5b). Activated Notch signaling has been linked to the development of hematologic malignancies and solid tumors in which a number of cellular signaling pathways are modulated to result in increased cell proliferation and inhibition of apoptosis (Leong and Karsan 2006). Both EBV and KSHV exploit components of the Notch signaling pathways to promote proliferation and viral gene expression (Hayward 2004; Hayward et al. 2006). EBV and Notch Signaling. EBV EBNA-2, one of the first viral proteins expressed after EBV infection in vitro, is essential for the proliferation of EBV-infected cells. It is a transcriptional activator that upregulates almost all viral genes and many cellular

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genes by interacting with other transcription factors, not by directly binding to DNA (Hayward 2004; Hayward et al. 2006). Similar to NotchIC, EBNA-2 interacts with the repression domain of CBF1/RBP-Jk, which is the same binding domain of NotchIC, resulting in the activation of CBF1/RBP-Jk-responsive reporter gene promoters, EBV latency promoters (with the exception of LMP1), and affecting the expression of the same cellular genes targeted by NotchIC, including c-Myc (Fig. 3.5c, Hayward 2004). Interaction of EBNA-2 with CBF1/RBP-Jk is thought to be necessary for the proliferation of EBV-infected lymphoblastoid cells since blockage of EBNA-2–CBF1/RBP-Jk interaction with a cell-permeable EBNA-2TAT peptide caused growth arrest and concurrent induction of p21 (Farrell et al. 2004). Unlike Notch receptor, however, EBNA-2 functions as a constitutively active Notch receptor because it activates target genes in the absence of ligands (Kohlhof et al. 2009). In addition, unlike NotchIC, which is mainly regulated by processes, such as differential expression of Notch ligands, receptor recycling, and protein

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turnover, the activity of EBNA-2 is either negatively or positively regulated at the level of the CBF1/RBP-Jk complex by other viral proteins; the EBNA-3 family competitively binds CBF1/RBP-Jk, resulting in the loss of CBF1/RBP-Jk binding to its target promoters and subsequent downregulation of EBNA-2-mediated transcriptional activation, while EBNA-5 (EBAN-LP) acts as a coactivator of EBNA-2mediated transcriptional activation (Hayward 2004). Nonetheless, Notch1-IC, only if expressed at extremely high levels or in combination with LMP1, has been shown to rescue proliferation of immortalized B cells to some extent in the absence of EBNA-2 (Gordadze et al. 2001; Hofelmayr et al. 2001). A more recent genomewide expression analysis utilizing EBV-infected B cells, however, has shown that neither Notch1-IC nor Notch2-IC can functionally replace EBNA-2 function and that Notch1 is more potent in regulating genes associated with differentiation/ development while EBNA-2 is more potent in inducing viral and cellular genes involved in proliferation, survival, and chemotaxis (Kohlhof et al. 2009). Thus, this study suggests that Notch1 and EBNA-2 have profoundly different effects on cellular processes. KSHV and Notch Signaling. Unlike EBV, subversion of Notch signaling pathway by KSHV appears to be critical for a productive lytic infection rather than latency. The expression of KSHV replication and transcription activator (RTA) protein is necessary and sufficient for KSHV reactivation from latency. KSHV RTA activates target gene expression not only by directly binding to recognition sequences in the KSHV genome, but also by indirectly interacting with cellular DNA-binding proteins on responsive promoters. One of the cellular DNA-binding proteins targeted by RTA is the CBF1/RBP-Jk protein, notable because RTA target genes are less responsive in fibroblasts derived from RBP-Jk null mice (Liang and Ganem 2003). RTA also binds the same repression domain of CBF1/RBP-Jk targeted by NotchIC and EBV EBNA-2, resulting in the activation of both cellular (CD21 and CD23) and viral (vIL-6 and vGPCR) gene transcription (Fig. 3.5d, Chang et al. 2005; Hayward 2004; Hayward et al. 2006; Liang et al. 2002; Liang and Ganem 2003). In tetracycline-inducible BCBL-1 PEL cells, NotchIC was capable of inducing the expression of 24 KSHV genes, including the vIL-6, but did not induce the full spectrum of viral lytic gene expression and replication (Chang et al. 2005), indicating that the transcriptional responses to NotchIC overlap with but differ from those of KSHV RTA. Although KSHV latent genes expressed in KSHV-associated tumor cells play significant roles in the development of KSHV-associated diseases, lytic KSHV genes expressed from a small percentage of cells also contribute to KSHV-associated pathogenesis. Two lytic genes, such as vIL-6 and vGPCR, are now recognized to have NotchIC-responsive promoters (Liang and Ganem 2003). Thus, the induction of vIL-6 and vGPCR by NotchIC may contribute to the proliferation of KSHVinfected tumor cells. Modulation of Wingless-Type (Wnt) Signaling Pathway. The Wnt-mediated signaling pathway is a highly conserved pathway between species and has been implicated in embryonic development, cell polarity and adhesion, apoptosis, and tumorigenesis (Akiyama 2000; Polakis 2000). It is activated when Wnts, secreted glycoproteins,

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a

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bind and activate the Frizzled (Fz) receptors, seven-pass TM receptors with an extracellular N-terminal cysteine-rich domain, on target cells in a paracrine manner. Fzs then cooperate with LRP5/6 coreceptor, a single-pass TM protein, to activate b-catenin, a key transcriptional activator of the Wnt signaling pathway involved in the transcription of more than 30 different genes in the nucleus by forming a complex with the T-cell factor (Tcf)/lymphoid-enhancer factor (Lef) family of transcription factors. In the absence of Wnt signaling, the cytosolic pool of b-catenin is held in a destruction complex comprising casein kinase 1 (CK1), glycogen synthase kinase 3-b (GSK-3b), axin, and adenomatous polyposis coli (APC) (Fig. 3.6a).

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Both axin and APC function as scaffold proteins, binding both GSK-3b and b-catenin. GSK-3b, a serine/threonine kinase, phosphorylates both axin and APC, strengthening the formation of the complex. CK1 and GSK-3b sequentially phosphorylate N-terminal Ser/Thr residues of b-catenin. Phosphorylated b-catenin is then recognized by b-TrCP, a component of an E3 ubiquitin ligase complex, resulting in the rapid degradation of b-catenin by the 26S proteasome. In the presence of Wnt, the activated receptor Fz binds dishevelled (Dvl) proteins, which recruit the destruction complex through its interaction with axin. Coreceptors LPR5/6 are then phosphorylated by GSK-3b and CK1g and subsequently recruit axin, which leads to dissociation of destruction complex and stabilization of b-catenin in the cytoplasm. Stabilized b-catenin then enters the nucleus, where it associates with Tcf/Lef transcription factors to activate many target genes involved in cell cycle progression, cell growth, and cell proliferation. Notable b-catenin target genes include c-Myc, c-Jun, and cyclin D1. Both c-Myc and Cyclin D1 can promote G1 to S phase cell cycle transition (Fig. 3.6b, Clevers 2006; Karim et al. 2004). Dysregulation of Wnt signaling pathway and its components has been implicated in different stages of tumorigenesis ranging from initiation, proliferation, and progression to the accumulation of mutations (Karim et al. 2004). Both EBV and KSHV are capable of constitutively activating Wnt signaling pathways by directly or indirectly inactivating GSK-3b, resulting in the enhanced proliferation of virus-infected cells. EBV and Wnt Signaling. EBV LMP2A expression in epithelial cells activates the PI3K/Akt pathway, resulting in the inactivation of GSK-3b, increased cytoplasmic accumulation of b-catenin, enhanced nuclear translocation of b-catenin, and subsequently upregulation of a reporter responsive to Tcf (Morrison et al. 2003). EBV infection in B cells constitutively induces the accumulation of b-catenin in the cytoplasm and the translocation of phosphorylated and inactivated GSK-3b into the nucleus. Unlike LMP2A-mediated regulation of b-catenin in epithelial cells, however, there was no nuclear localization of b-catenin in B cells nor increased transcription of b-catenin-dependent target genes (Everly et al. 2004). EBV LMP1 has been shown to upregulate b-catenin in B-lymphoma cells by increasing its stability through the inhibition of Siah-1, an E3 ubiquitin ligase that binds APC and promotes b-catenin degradation in a GSK-3b-independent fashion (Jang et al. 2005). Thus, EBV proteins expressed during latency target GSK-3b, an inhibitor of Wnt signaling, resulting in the constitutive activation of the Wnt signaling pathway. KSHV and Wnt Signaling. It has been reported that KSHV LANA-1 dysregulates the Wnt pathway by binding and sequestrating GSK-3b in the nucleus, which leads to b-catenin stabilization, nuclear entry, and subsequent activation of cyclin D1, a protein involved in cell proliferation (An et al. 2005; Fujimuro and Hayward 2003; Fujimuro et al. 2005, 2003; Hayward et al. 2006). Consistent with this, transient LANA-1 expression stimulates S-phase entry (Fujimuro and Hayward 2003). These studies indicate that tumor cells expressing LANA-1 are more prone to survive and proliferate in the absence of Fz receptor ligands.

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Summary Although the pathogenesis of EBV and KSHV-induced malignancies is still not completely understood, similar to the accumulative mutational process of cancer induction, these viruses affect a variety of host cell processes, including cell cycle progression, DNA damage response, and numerous intracellular signaling pathways to promote cellular proliferation, and ultimately facilitate the development of malignancies. This review summarizes the recent progress made in our understanding of the biochemical and molecular mechanisms, whereby EBV and KSHV, two oncogenic, large DNA HHV, induce cell proliferation, with a particular focus on the contribution of viral proteins to this process. Both EBV and KSHV can induce uncontrolled cell proliferation, resulting in lymphomas, Hodgkin’s diseases, and Kaposi’s sarcoma. Overall tumor cell number is determined by the rate of not only cell proliferation (birth), but also apoptosis (death). Although this review does not discuss EBV- or KSHV-mediated antiapoptosis mechanisms, most of viral proteins discussed in this review, in fact, have demonstrated the ability to inhibit apoptosis as well. Moreover, both EBV and KSHV are equipped with many different strategies to evade cellular apoptosis machineries and immune surveillance systems, suggesting that the promotion of cellular proliferation may represent one of the several mechanisms employed by these viruses for enhancing their survival and replication and to contribute to tumorigenesis. Thus, understanding the functional interplay between viral proteins with cellular proteins involved in cellular proliferation may provide a basis for the development of future therapeutic strategies to tackle virus-mediated tumorigenesis. Acknowledgment This work was partly supported by the U.S. Public Health Service grants CA082057, CA31363, CA115284, Hasting Foundation, and Fletcher Jones Foundation.

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Chapter 4

Viral-Encoded Genes and Cancer Blossom Damania

Tumor Viruses and Cancer Cancer is a multistage process. Most cancers do not arise from mutation of a single gene, but rather from cumulative accumulation of multiple gene mutations. Mutations that contribute to tumorigenesis generally occur in one of three types of genes – a proto-oncogene; a tumor suppressor gene; or genes involved in DNA replication and repair, chromosome segregation, or cytokinesis. The definition of a proto-oncogene is a wild-type gene, which when mutated can give rise to an oncogene. Proto-oncogenes include genes that encode for proteins involved in cell growth, cell survival, or cell signaling. The accumulation of multiple genetic insults in a single cell leads to the loss of cell cycle checkpoints and the loss of programmed cell death (or apoptosis), which consequently results in continual cell proliferation. In contrast, tumor suppressors are proteins that normally function to inhibit cell growth and enhance cell death pathways. When mutated, tumor suppressors lose the ability to block cell growth or activate apoptosis; thereby, contributing to the proliferative phenotype of a cancer cell. The combination of oncogenes and tumor suppressor mutations occurring in a multistage process leads to transformation. Several studies have supported this multistage mutational model of cancer. Both breast and colorectal tumor models reveal many gene mutations (Wood et al. 2007). Generally speaking, the road that a normal cell traverses to become a cancer cell includes (1) hyperplasia or an abnormal increase in cell number; (2) dysplasia, which results in disorganized tissue architecture; (3) benign tumor, which is a hyperproliferative tissue mass that is not invasive; (4) invasive tumor, where the tumor begins to penetrate into adjacent tissues; and (5) metastasis, where the

B. Damania (*) Department of Microbiology and Immunology, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA e-mail: [email protected] E.S. Robertson (ed.), Cancer Associated Viruses, Current Cancer Research, DOI 10.1007/978-1-4614-0016-5_4, © Springer Science+Business Media, LLC 2012

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tumor cells move to distal sites. Each of these stages is driven by genetic mutation, with the end result being a combination of gene mutations that give rise to cancer. Thus, each type of cancer that arises has a specific “genetic signature” comprised of a particular combination of genetic mutations (Nowell 2002). Although the above descriptors seem primarily related to cell growth, the prevention of cell death, a contributing factor to the development of cancer, is as important as cell proliferation. Apoptosis or programmed cell death is a tightly orchestrated process in which cells die in response to particular stimuli. Apoptosis is an important cellular defense mechanism against transformation. The p53 tumor suppressor is one of the major regulators of apoptosis in the cell. Thus, cellular gene mutations that prevent a damage-induced apoptotic response in the cell contribute to the tumorigenesis process. Oncogenic viruses are comprised of both RNA and DNA tumor viruses. These tumor viruses are associated with cancer in their natural hosts or in non-native hosts. Viruses have contributed to, and revolutionized, the field of cancer biology. The study of tumor viruses has been the key to understanding the genetic basis of transformation. In addition, the study of these viruses has also contributed to our understanding of virus-induced cancers. The study of tumor viruses initiated the concept of “oncogenes” and “tumor suppressors” in modern cancer biology.

Retroviruses and the Concept of Oncogenes Retroviruses are RNA viruses that undergo reverse transcription as part of their lifecycle. Following infection, the viral RNA genome is transcribed by the reverse transcriptase enzyme that is packaged in the virion. This produces a double-stranded DNA copy of the RNA genome with the help of the viral integrase can be integrated into the host chromosomal DNA. The avian sarcoma virus, Rous sarcoma virus (RSV), was the first known tumor virus and also a retrovirus. The study of RSV led to the discovery of oncogenes (Vogt 1997). RSV was shown to be capable of transforming cells in culture assays (Temin and Rubin 1958). The RSV-encoded gene named src or viral src (v-src) was essential for the transformation of this virus. Additionally, src was also found to be present in normal cells. This discovery was seminal in linking the concept of cancer development to a transforming gene like src. It also revealed that normal cells contained genes that were homologous to v-src and led to the concept of protooncogenes that were present in normal cells. Since the discovery of RSV and src, many other oncogenic retroviruses of chickens and mice, which contained transforming genes, were identified. These included the Abelson murine leukemia virus (Ab-MLV) containing the abl oncogene, the Harvey murine sarcoma virus (Ha-MSV) containing the ras oncogene, the MC29 avian myelocytomatosis virus containing the myc oncogene and the Moloney

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Table 4.1 A subset of retroviruses encoding for viral oncogenes that are homologous to cellular proto-oncogenes Virus Viral oncogene Viral oncoprotein Rous sarcoma virus src Tyrosine kinase Abelson murine leukemia virus abl Tyrosine kinase Moloney murine sarcoma virus mos Serine–threonine kinase Murine sarcoma virus 3611 raf Serine–threonine kinase Hardy–Zuckerman-4 feline sarcoma kit Receptor tyrosine kinase virus Avian erythroblastosis virus erbB Epidermal growth factor receptor Harvey murine sarcoma virus H-ras GDP/GTP binding Kirsten murine sarcoma virus K-ras GDP/GTP binding FBJ osteosarcoma virus fos Transcription factor Avian sarcoma virus-17 jun Transcription factor MC29 myelocytoma virus myc Transcription factor Reticuloendotheliosis virus rel Transcription factor

murine sarcoma virus (Mo-MSV) containing the mos oncogene (Rosenberg and Jolicoeur 1997). Similar to the situation with src, all the oncogenes carried by these tumor retroviruses were subsequently shown to be homologous to genes present in the normal cells. The cellular proto-oncogenes were of many different types including kinases, G proteins, growth factors, and growth factor receptors, all of which were involved in signal transduction and cell proliferation (Table 4.1). This led to the concept of molecular piracy by the virus. However, the viral gene and the cellular counterpart are not usually identical. Retroviral oncogenes are generally mutated and expressed constitutively from the control of the viral long terminal repeat (LTR), which is thought to lead to transformation of the host cell or animal. Moreover, certain retroviruses contain two oncogenes, e.g., an isolate of avian erythroblastosis virus encodes for v-erbA and v-erbB oncogenes. These observations gave rise to the idea that oncogenes can synergize with each other since the isolate containing two oncogenes was more transforming than an isolate containing the v-erbB oncogene (Rosenberg and Jolicoeur 1997). The retroviruses described earlier are oncogenic retroviruses. However, a second class of non-oncogenic retroviruses exist that do not contain viral oncogenes, e.g., avian leukosis virus (ALV). Rather, these viruses induce transformation because their viral DNA genome is integrated upstream of a cellular proto-oncogene resulting in dysregulated expression of the cellular gene. This allows the cellular protooncogene to be expressed constitutively and at very high levels (Kung et al. 1991). Such expression of a growth regulatory proto-oncogene allows for cellular transformation by giving the cell a proliferative and survival advantage over normal cells. This initiates the process of tumorigenesis and additional mutations acquired after this event may give rise to bonafide tumors.

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Small DNA Tumor Viruses and the Concept of Tumor Suppressors Small DNA tumor viruses include the adenoviruses, polyomaviruses, and papillomavirus families. Unlike retroviruses, these DNA viruses do not encode homologs of cellular proto-oncogenes, but contain unique viral oncogenes that are important for cell transformation (Cole 1996; Howley 1996; Shenk 1996). Studies of these small tumor DNA viruses led to the identification of “tumor suppressor” genes. Due to their small genome size, these DNA tumor viruses are dependent on the host cell machinery to replicate their viral genomes. Viral proteins stimulate quiescent cells to enter the cell cycle to replicate both the cellular and the viral genomes. Simian virus 40 (SV40) was the first small DNA tumor virus to be studied extensively due to its presence as a contaminant in the Sabin polio vaccine. Polyomaviruses are small DNA tumor viruses that are widely prevalent throughout the animal kingdom. SV40, the prototype polyomavirus, encodes for a protein named large T antigen (T ag), which is required for viral DNA replication and for pushing the cell into the cell cycle. Homologs of large T antigen are found in all human polyomaviruses, including JCV, BKV, and Merkel cell polyomavirus (MCV) (White et al. 2009). SV40 large T is a potent transforming protein in many different cell and animal model systems (Butel and Lednicky 1999). SV40 large T antigen was found to coimmunoprecipitate with p53, which was later coined a tumor suppressor gene due to its ability to inhibit cell proliferation (Lane and Crawford 1979; Linzer and Levine 1979). Hence, the p53 protein was discovered due to its interaction with SV40 large T antigen. Adenoviruses are a different small DNA tumor virus family that encode for two different viral oncoproteins, E1A and E1B (Shenk 1996). E1B was also found to bind p53 while a different tumor suppressor protein, Rb, was identified as a cell protein that interacted with the E1A oncoprotein in adenovirus-transformed cells (Whyte et al. 1988). SV40 Tag was later found to interact with Rb as well. The papillomaviruses are a third group of small DNA tumor viruses, which encode their own unique transforming proteins, E6 and E7. Key to E6’s and E7’s ability to immortalize or transform cells is their ability to bind p53 and Rb, respectively (Howley 1996).

Common Properties Shared by the Small DNA Tumor Viruses Small DNA tumor viruses share the common property of inducing DNA synthesis in resting cells. This is an essential function of small DNA tumor viruses since they need to activate the DNA replication machinery of the host cell to replicate their own viral genomes. The binding of different viral oncoproteins with the cellular tumor suppressor proteins, Rb and p53, is key for allowing the small DNA tumor viruses to induce cell proliferation, and as a result, transform these cells.

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Large T E1B E6

Rb p53

Rb

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Large T E1A E7

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Fig. 4.1 Common targets of small DNA tumor viruses. (a) p53 negatively regulates cell cycle progression and positively regulates the induction of apoptosis in response to aberrant proliferation signals, DNA damage or cellular stress. Binding of p53 by the small DNA tumor viral proteins, Large T, E6 and E1B allows cells to survive and escape p53 checkpoints in the face of DNA damage stress signals. This allows for cell cycle progression and replication of the viral genome. (b) E2F is a transcription factor that under normal conditions is bound to Rb and upon phosphorylation by cyclin-dependent kinases is released from Rb enabling its binding to cellular promoters of genes involved in cell cycle progression. Small DNA tumor viral proteins, large T, E1A and E7 dissociate Rb from E2F

Each small DNA tumor virus encodes unique proteins that bind and inactivate the function of these tumor suppressor proteins, which are common targets of these viruses. Figure 4.1 depicts how binding p53 and Rb by proteins encoded by the small DNA tumor viruses may lead to cell cycle progression and cellular transformation. SV40 large T antigen, HPV E6, and adenovirus E1B-55K bind p53 and prevent its DNA binding activity, induce its degradation, or inhibits its transactivation, respectively; thereby, inactivating p53 function. Large T antigen, HPV E7 and adenovirus E1B all possess a common LXCXE motif, which bind Rb, release E2F, and initiate cell cycle progression (Butel 2000). Although SV40 encodes for one protein (large T antigen) that inactivates both Rb and p53, the adenoviral proteins E1A and E1B-55K as well as the HPV proteins E6 and E7 show functional cooperativity in inactivating the function of both p53 and RB. This synergistic inactivation of the tumor suppressor genes results in transformation. Since the discovery of these critical proteins, the cancer biology field has also determined the transforming potential of viral oncogenes in combination with cellular oncogenes. For example, transfection of fibroblasts by a cellular ras oncogene did not induce transformation by itself. However, the fibroblasts did get transformed if a second oncogene such as a viral gene (large Tag) or cellular gene (myc) was introduced together with the cellular ras gene (Land et al. 1983). Just as oncogenes cooperate to induce transformation, it has also been shown that expression of an oncogene together with the loss of a tumor suppressor gene or expression of a mutant tumor suppressor gene can cooperate to induce tumorigenesis (Zambetti et al. 1992).

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Viruses Associated with Human Cancer Several types of neoplastic disease in humans are linked to viral infection. Viruses from diverse families have been etiologically linked to human malignancy (Table 4.2). Two RNA viruses hepatitis C virus (HCV) and human T-cell leukemia virus 1 and 2 (HTLV-I, 2) are linked to human cancer. HTLV-1, 2 is associated with adult T-cell leukemia (ATL) (Yoshida 1999) and HCV is the etiological agent of hepatocellular carcinoma (HCC) (zur Hausen 1999). Among the small DNA tumor viruses, the aforementioned human papillomavirus is linked to cervical cancer, a subset of head and neck cancers, skin cancers in patients with epidermodysplasia verruciformis (EV), and anogenital cancers (zur Hausen 1996). Additionally, MCV polyomavirus has been linked to cases of Merkel cell carcinoma (Feng et al. 2008). There is also a question as to whether SV40 and the human polyomaviruses, JCV and BKV, are linked to different types of brain tumors (Pagano et al. 2004). Another virus, HBV, is associated with HCC in addition to HCV (Robinson 1999). Two DNA herpesviruses are also linked with human malignancies. EBV is the etiological agent of nasopharyngeal carcinoma (NPC), African Burkitt’s lymphoma, posttransplant lymphomas (PTLD), Hodgkin’s disease, and some gastric cancers (Damania 2004; Pagano et al. 2004). Kaposi’s sarcoma-associated herpesvirus (KSHV) is linked to Kaposi sarcoma (KS) and two lymphoproliferative diseases, primary effusion lymphoma and multicentric Castleman’s disease (Damania 2004; Pagano et al. 2004). In this chapter, we discuss the details of a subset of genes encoded by these oncogenic viruses that appear to play a contributing role in the development of human cancer. These genes modulate diverse cellular pathways including transcription, cell cycle progression, signal transduction, and apoptosis (Table 4.3).

Table 4.2 Viruses associated with human malignancies Virus family Virus Type of human cancer Retroviridae HTLV-1, 2 T-cell leukemia Flaviviridae HCV Hepatocellular carcinoma Hepadnaviridae HBV Hepatocellular carcinoma Polyomaviridae MCV Merkel cell carcinoma JCV, BKV, SV40 Brain and other tumors? Papillomaviridae HPV Cervical cancer Herpesviridae EBV Burkitt’s lymphoma, Hodgkin’s lymphoma, nasopharyngeal carcinoma, post-transplant lymphoproliferative disease, gastric cancer KSHV Kaposi’s sarcoma, primary effusion lymphoma, multicentric Castleman’s disease

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Table 4.3 Viral oncogenes encoded by human tumor viruses Virus Oncogene(s) Mode of action HTLV-1, 2 Tax Transactivator of viral and cellular genes HCV Core proteins, NS3, Inhibition of apoptosis, induction of oxidative NS4B and NS5A stress, activation of signal transduction proteins pathways, immune evasion HBV HBx Transactivator of viral and cellular genes MCV Large T antigen Inactivation of tumor suppressor proteins, promotion of cell cycle progression JCV, BKV, SV40 Large T antigen Brain tumors? HPV E6, E7 Inactivation of tumor suppressor proteins; induction of genomic instability EBV LMP1 Activation of NF-kB and signal transduction pathways LMP2A Signal transduction; Inhibition of B cell differentiation EBNA-1 Inhibition of apoptosis, promotion of tumorigenesis, viral latency EBNA-2 Transactivator of viral and cellular genes EBNA-3A, B, C Inactivation and degradation of p53 and Rb; promotes transformation and metastasis KSHV K1 Activation of signal transduction pathways; angiogenesis; promotes transformation LANA Inactivation of tumor suppressors, viral latency vFLIP Inhibition of apoptosis vGPCR Activation of signal transduction pathways; angiogenesis; promotes transformation Kaposin Activation of signal transduction pathways; promotes transformation vIL-6 Activation of signal transduction pathways; cell proliferation vIRF-1 Evasion of host immune responses, promotes transformation

HTLV-1-Encoded Oncogenes Unlike the aforementioned murine and avian retroviruses, HTLV-1 does not contain a viral oncogene derived from a cellular proto-oncogene. It encodes a unique protein, Tax, which is a transcription factor that is essential for cellular transformation. Tax has been shown to potently activate the NF-kB pathway and turn on expression of NF-kB responsive genes such as the IL-2 receptor and several NF-kB responsive cytokines involved in T-cell proliferation. Tax upregulates the expression of Bcl-XL; thereby, conferring resistance of Tax expressing cells to apoptosis (Matsuoka and Jeang 2007). Furthermore, Tax interacts with chromatin remodeling proteins including SWI/SNF and HDAC-1 and inhibits DNA repair mechanisms, which leads to genomic instability and transformation (Matsuoka and Jeang 2007).

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HCV-Encoded Oncogenes Transgenic mice that contain the entire HCV genome or just the HCV core protein have been shown to develop liver steatosis and hepatocellular carcinogenesis (Lerat et al. 2002; Moriya et al. 1998). It is thought that the HCV core protein is a major factor in the induction of oxidative stress leading to the induction of transformation. Additionally, the HCV NS5A protein can sequester p53 in the cytoplasm, activate STAT3 signaling, and inhibit TNF alpha-mediated apoptosis (Levrero 2006). HCV core, NS3, NS4B, and NS5A proteins, have been shown to transform cells in vitro and alter oncogenic signaling pathways (Levrero 2006). HCV NS3/4A also inhibits innate immune responses through cleavage of the mitochondrial antiviral signaling (MAVS) protein (Li et al. 2005).

HPV-Encoded Oncogenes Almost 100% of cervical cancers contains HPV genomic DNA. Additional cancers associated with HPV include vulvar, vaginal, penile, oropharyngeal, and some skin cancers. Of the more than hundred HPV strains known thus far the “high-risk” viruses HPV type 16 and 18 are associated with cervical and anal cancer. HPV strains 31 and 45 are also found in these cancers and together with 16 and 18 account for 80% of them. Low-risk viruses (strains 6 and 11) are associated with benign lesions such as condyloma accuminata. The E6 and E7 genes encode for the major oncoproteins of HPV. In general, E6 and E7 proteins of high-risk strains of HPV are more efficient at inactivating tumor suppressor proteins than those of the low risk types. In high grade cervical dysplasias, HPV 16 and 18 E6 and E7 oncoproteins are consistently over-expressed as a consequence of deletion of the E2 repressor gene upon integration of the HPV genome into the host chromosome. HPV E6 induces the ubiquitination-mediated degradation of p53 through the ubiquitin ligase, E6-AP, which prolongs survival of the infected cell (Scheffner et al. 1993). HPV E7 induces the proteosomal degradation of Rb and related proteins so that cell growth is dysregulated (Dyson et al. 1989). E7 also induces genomic instability (Duensing et al. 2000).

HBV-Encoded Oncogenes The HBx protein encoded by HBV is considered to be a viral oncogene since transgenic mice expressing HBV X develop hepatocarcinogenesis (Zeichner et al. 1991). The HBx protein is a transcription factor that transactivates cellular growth factors, cytokines, and proto-oncogenes. HBx binds to p53, thereby inactivating it. HBx also activates several cell signaling molecules including src kinase through activation of Pyk-2 kinase and induces cytosolic calcium (Bouchard et al. 2001).

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EBV-Encoded Oncogenes EBV encodes for many different viral proteins that control various cellular pathways. The primary EBV oncogene, LMP1, is essential for EBV transformation of B cells and can transform rodent fibroblasts (Rickinson and Kieff 2001). LMP1 interacts with TNF receptor-associated factors (TRAFs) (Mosialos et al. 1995). LMP1 is a potent activator of the NF-kB signaling pathway in both lymphocytes and epithelial cells (Miller et al. 1998). Transgenic mice expressing LMP1 under the control of the immunoglobulin promoter have an increased frequency of B-cell lymphomas (Uchida et al. 1999). LMP1 activates the expression of cellular genes involved in cell signaling and proliferation including Bcl-2, ICAM-1, epidermal growth factor receptor (EGFR), matrix metalloproteinase 9, vascular endothelial growth factor (VEGF), and hypoxia-inducible factor HIF-1alpha (Pagano et al. 2004). Additionally, LMP1 activates the phosphatidylinositol 3-kinase (PI3K) and mitogen activated kinase pathways (MAPK) (Dawson et al. 2003; Mainou et al. 2005; Mainou and Raab-Traub 2006). Although LMP2A is dispensable for EBV-immortalization of B lymphocytes (Longnecker et al. 1993), it induces a hyperproliferative response and prevents differentiation in epithelial cells and is likely to contribute to epithelial transformation (Scholle et al. 2000). The cytoplasmic domain of LMP2A contains several tyrosine-based SH2 binding motifs including a functional ITAM (Fruehling and Longnecker 1997). In keratinocytes, LMP2A activates both the PI3K and betacatenin signaling pathways (Morrison et al. 2003). Additionally, LMP2A transgenic mice do not progress to full B cell development, causing immunoglobulin-negative B cells to home to peripheral lymphoid organs (Caldwell et al. 1998). It is therefore thought that LMP2A mediates survival of immature EBV-infected B cells in the absence of functional BCR signaling. EBV nuclear antigen 2 (EBNA-2) is a promiscuous transcriptional activator, of both viral and cellular genes (Grossman et al. 1994; Kaiser et al. 1999; Wang et al. 1991, 2000). Deletion of the EBNA-2 gene from wild-type EBV renders the mutant virus incapable of immortalizing B lymphocytes (Cohen et al. 1989; Hammerschmidt and Sugden 1989; Rabson et al. 1982). It is believed that the transactivation ability of EBNA-2 contributes to the transforming mechanism of EBV. The genes encoding EBNA 3A, 3B, and 3C lie in a tandem array in the viral genome. EBNA 3A and 3C are essential for B-cell transformation (Sample and Parker 1994; Tomkinson et al. 1993), while 3B has been shown to be dispensable (Tomkinson and Kieff 1992). All three EBNA-3 proteins can prevent EBNA-2 transactivation function by hindering its ability to bind RBP-Jk (Le Roux et al. 1994; Marshall and Sample 1995; Robertson et al. 1996). EBNA 3C has been shown to cooperate with the proto-oncogene, Ras, to immortalize and transform rodent fibroblasts (Parker et al. 1996) and promote metastasis (Kaul et al. 2007). It can directly interact with the retinoblastoma (Rb) tumor suppressor protein, rendering it inactive, promoting its degradation, and thereby contributing to tumorigenesis (Knight et al. 2005; Parker et al. 1996). EBNA 3C also induces ubiquitin-mediated

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degradation of p53 through stabilization of Mdm2 (Saha et al. 2009). In sum, EBNA 3C promotes cellular proliferation by overcoming cell cycle control checkpoints. It also cooperates with EBNA-2 and 3A to modulate cellular gene expression in EBVinfected lymphocytes. EBV EBNA-1 might also contribute to EBV transformation since an EBNA-1deleted virus showed greatly reduced ability to immortalize B cells (Humme et al. 2003). Additionally, EBNA-1 transgenic mice develop B-cell lymphomas (Wilson et al. 1996) and expression of EBNA-1 can enhance the tumorigenicity of EBVnegative NPC epithelial cells (Sheu et al. 1996). This property of EBNA-1 may be linked to its ability to induce Bcl-xL (Tsimbouri et al. 2002). Similar to EBNA 3C, EBNA-1 can also promote metastasis (Kaul et al. 2007).

KSHV-Encoded Oncogenes Similar to EBV, KSHV encodes for a number of different viral genes, some of which have been shown to be transforming in different model systems. Located in a similar position as EBV LMP1, the first open reading frame of KSHV encodes for K1, a 46-kDa type I transmembrane protein (Lagunoff and Ganem 1997). K1 is expressed at low levels during latency and is highly upregulated during early lytic replication (Chandriani and Ganem 2010; Wang et al. 2006). Its expression has been detected in KS, PEL, and MCD (Jenner et al. 2001; Lagunoff and Ganem 1997; Lee et al. 2003; Wang et al. 2006). The K1 protein possesses an immunoreceptor tyrosine-based activation motif (ITAM) that is important for lymphocyte activation signaling (Lee et al. 1998) and has been shown to transform Rat-1 rodent fibroblasts by inducing morphological changes and foci formation (Lee et al. 1998). Transgenic mice expressing the K1 gene develop tumors with features resembling spindle-cell sarcomatoid tumors and malignant plasmablastic lymphomas (Prakash et al. 2002). K1 activates PI3K (p85 subunit), Akt, Vav, and Syk kinases in B cells (Lee et al. 1998; Tomlinson and Damania 2004). In addition, K1 can prevent death receptor-mediated apoptosis of B lymphocytes by inhibiting the induction of FasL expression and activating the PI3K/Akt pathway (Tomlinson and Damania 2004), and requires heat shock proteins (Hsp) 40 and 90 for its antiapoptotic function (Wen and Damania 2010). In epithelial and endothelial cells, K1 induces the expression and secretion of angiogenic factors, including VEGF and matrix metalloproteinase-9 (Wang et al. 2004) and activates the PI3K/Akt/mTOR pathway (Wang et al. 2006). Furthermore, K1 can immortalize and extend the life span of primary endothelial cells (Wang et al. 2006). Cumulatively, these data suggest a paracrine model in which K1-mediated secretion of cytokines is involved in the development of KSHV-associated diseases (Wang et al. 2004). Thus K1 appears to be important in KSHV-associated tumorigenesis and angiogenesis. Another transforming protein of KSHV is the viral G-protein-coupled receptor encoded by Orf74 (Arvanitakis et al. 1997; Guo et al. 1997). vGPCR can transform murine NIH3T3 cells (Bais et al. 1998) and a subset of vGPCR transgenic mice developed

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KS-like angioproliferative lesions with surface markers and cytokine profiles resembling those of KS (Guo et al. 2003; Montaner et al. 2003; Yang et al. 2000). The expression of vGPCR can be found in a small fraction of KS, PEL, and MCD samples (Sodhi et al. 2000). Like K1, vGPCR can immortalize endothelial cells (Bais et al. 2003; Montaner et al. 2001). In contrast to its cellular homologs, vGPCR is constitutively active (Rosenkilde et al. 1999). Several signaling pathways including the mitogen-activated protein kinases (MAPKs) (Sodhi et al. 2000), PLC (Bais et al. 2003), PI3K (Bais et al. 2003), and Akt (Montaner et al. 2001) pathways have been shown to be activated by vGPCR. In addition to its role in the establishment and maintenance of KSHV latency (reviewed in Dittmer 2008; Rainbow et al. 1997; Verma et al. 2007), the latencyassociated nuclear antigen (LANA) may also be involved in oncogenesis due to its ability to bind p53 and inhibit p53-driven transactivation and apoptosis (Friborg et al. 1999; Si and Robertson 2006). LANA can also inactivate the tumor suppressor retinoblastoma (Rb) gene, allowing the E2F transcription factor to induce G1/S cell cycle progression (Radkov et al. 2000). LANA also interacts with the tumor suppressor, Sel10, and suppresses ubiquitination and degradation of intracellular Notch (Lan et al. 2007). LANA induces expression of human telomerase reverse transcriptase (hTERT) and survivin (Lu et al. 2009; Verma et al. 2004; Watanabe et al. 2003). Finally, transgenic mice expressing LANA under the endogenous LANA promoter developed splenic follicular hyperplasia with increased germinal centers as well as lymphomas (Fakhari et al. 2006). Another latent gene product, viral Fas-associated death-domain like IL-1 beta-convertase enzyme (FLICE) inhibitory protein (vFLIP) is encoded by Orf71 (Dittmer et al. 1998; Fakhari and Dittmer 2002; Jenner et al. 2001). Similar to cellular FLIPs, vFLIP may inhibit death receptor signaling by preventing the association between caspase-8 and Fas-associated death domain (FADD) (Belanger et al. 2001; Djerbi et al. 1999). vFLIP is also a strong activator of the NF-kB pathway (Chaudhary et al. 1999; Field et al. 2003; Guasparri et al. 2004, 2006; Liu et al. 2002). The enhanced NF-kB signaling appears to contribute to the transforming potential of vFLIP in Rat-1 fibroblast assays and tumors in nude mice (An et al. 2003). Kaposin A, B, and C are three proteins encoded by the K12 gene of KSHV. The smallest isoform, Kaposin A, demonstrates oncogenic potential in different assays (Kliche et al. 2001; Muralidhar et al. 1998). Viral interleukin-6 encoded by OrfK2 is a homolog of cellular IL-6 (Neipel et al. 1997) and is detected in KS, PEL, and MCD samples (Parravicini et al. 2000; Staskus et al. 1999). Similar to cellular IL-6, vIL-6 activates MAPK and the Janus tyrosine kinases signal transducers and activators of transcription (JAK/STAT) (Molden et al. 1997). This results in increased VEGF expression and signaling in an autocrine/paracrine fashion (Liu et al. 2001). However, in contrast to cellular IL-6, vIL-6 signaling occurs through gp130 and does not require the gp80 subunit (IL-6Ra) (Molden et al. 1997). Additionally, vIL-6 also transforms murine fibroblasts and when these cells were injected into immunocompromised mice, they formed highly vascularized tumors (Aoki et al. 1999). Another viral homolog, the viral interferon regulatory factor 1 (vIRF-1) suppresses both type I and type II interferon responses (Burysek et al. 1999; Gao et al. 1997; Li et al. 1998).

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In addition to suppressing the host antiviral response, vIRF-1 is a potential oncogene since NIH3T3 cells stably expressing vIRF-1 are transformed and form tumors in nude mice (Gao et al. 1997).

Common Properties Shared by the Large DNA Tumor Viruses A common property shared by the large DNA tumor viruses EBV and KSHV is that they are both members of the gammaherpesvirus family and induce tumors in their hosts. Additionally, both EBV and KSHV contain a distinct ORF at the left end of their respective genomes (LMP1 and K1, respectively) that have characteristic transforming potential. Although LMP1 and K1 do not share sequence similarity, the ability to transform cells and activate critical signaling pathways to augment cell survival and proliferation is conserved. Additionally, these viruses also encode for similarly functioning proteins at the right end of the viral genome, EBV LMP2A and KSHV K15. These two proteins interact with kinases associated with the B-cell receptor (BCR) and alter signaling pathways in the cell (Beaufils et al. 1993; Brinkmann et al. 2003; Brinkmann and Schulz 2006; Burkhardt et al. 1992; Glenn et al. 1999; Longnecker et al. 1991; Scholle et al. 1999). Cross-linking of the B-cell antigen receptor (BCR) triggers a signal transduction cascade that leads to the activation of B cells. Both EBV LMP2A and KSHV K15 antagonize BCR-induced signaling events (Beaufils et al. 1993; Burkhardt et al. 1992; Glenn et al. 1999; Longnecker et al. 1991; Scholle et al. 1999). It is thought that this helps maintain viral latency in B lymphocytes. The viral proteins encoded close to the terminal repeats of EBV (LMP1 and LMP2A) and KSHV (K1 and K15) also exhibit the highest sequence divergence among individual viral isolates (Nicholas et al. 1997, 1998; Palefsky et al. 1996; Zong et al. 1999, 2002). This is likelya result of their close proximity to the terminal repeats of the viral genome, which is a region of high mutagenicity.

Summary There is a lot to be learned about cancer biology by studying the common and distinct mechanisms by which tumor viruses modulate cell pathways. As described above, all tumor viruses encode proteins that interact intimately with host cell pathways to mediate cell proliferation and cell survival. Most of these viruses target common cellular proteins and cell signaling pathways that appear to be essential for the survival of all tumor viruses in the host cell. These include activation of multiple oncogenes and inhibition of tumors suppressors, evasion of the host immune system, immune evasion, and the induction of cell proliferation and cell survival programs.

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Acknowledgments BD is a Leukemia & Lymphoma Society Scholar and a Burroughs Welcome Fund Investigator in Infectious Disease. Her work is supported by grants CA096500, DE18281, and HL083469 from the NIH.

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Chapter 5

Oncogenic Viruses and Cancer Transmission Robin A. Weiss

Infection and Human Cancer Physicians are able to reassure patients and those who care for them that cancer is “not catching” in the sense that you do not develop cancer as a result of close contact with someone who has the disease. Veterinarians might be more cautious, as there is a malignancy of dogs and another of the Tasmanian devil that are transmitted directly through contact, as described at the end of this chapter. However, while human cancer is not directly contagious, there is compelling evidence that nearly 20% of the global burden of human cancer has an infectious etiology amounting to almost 2 million new cases each year (Parkin 2006). Approximately 5.5% of tumors are attributable to bacteria, such as Helicobacter pylori (Polk and Peek 2010) causing stomach cancer and mucosal-associated lymphoid tumors (MALTs), and to helminths associated with cancers of the urinary bladder and the gall bladder. The remaining infective cancers are due to virus infection representing some 13.5% of all human cancer. This is good news for cancer prevention because it opens the path to cancer control through screening and immunization. The notion that some types of cancer might be transmissible was first promulgated in 1842 by Domenico Rigoni-Stern (Scotto and Bailar 1969), who was a surgeon with epidemiological and statistical interests in Verona in Northern Italy. He observed that nuns in the city’s convents tended to develop breast cancer more frequently than other women, but suffered less cancer of the uterine cervix. He suggested that the constriction of tight corsets might promote breast tumors but that cervical cancer was associated with sexual activity. With modern eyes, we can say that the higher incidence of breast cancer had an endocrine explanation and the lower rate of cervical cancer owed its benefit from less exposure to human papillomaviruses (HPVs).

R.A. Weiss (*) Division of Infection and Immunity, University College London, London, UK e-mail: [email protected] E.S. Robertson (ed.), Cancer Associated Viruses, Current Cancer Research, DOI 10.1007/978-1-4614-0016-5_5, © Springer Science+Business Media, LLC 2012

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Nulliparous women in general develop breast cancer more frequently than those who have children; moreover, nuns were usually recruited from relatively wealthy families, and there is a positive correlation between body mass index and breast cancer. On the other hand, most of the women who joined religious orders (other than widows) would not have encountered infection by a sexually transmitted virus. Rigoni-Stern conducted his analysis before the germ theory of transmissible diseases was accepted (although he was an active promoter of vaccination) and before the elaboration of the cell theory of cancer, yet his observations remain pertinent today. The infectious agents known to be associated with human cancers have been identified during the last 50 years (Table 5.1). A vintage year for discovery was 1983, when HPV-16, HIV-1, and H. pylori were first reported (Weiss 2008). Where there was clear epidemiological evidence of a transmissible agent causing the cancer, the discovery of the virus often resulted from a specific search for it, e.g., cervical carcinoma, Burkitt’s lymphoma, and Kaposi’s sarcoma. On the other hand, some cancers were not identified as having an infectious etiology until well after the agent had been characterized, e.g., hepatocellular carcinoma (HCC), nasopharyngeal carcinoma, and stomach cancer. Virus discovery was usually followed by meticulous epidemiological analyses using serological or molecular markers and established the links between virus infection and malignant disease. For example, it took more than 12 years following the discovery of hepatitis B virus (HBV) by Baruch Blumberg (Chap. 2) as a cause of serum hepatitis and cirrhosis for R. Palmer Beasley to demonstrate unequivocally through a study of a cohort of 22,707 Taiwanese men that, contrary to the prevailing view, HBV was also a major cause of liver cancer (Beasley et al. 1981). The discovery of human T-cell leukemia virus type 1 (HTLV-1) in the USA (Poiesz et al. 1980) in a case of cutaneous CD4-positive lymphoma was soon followed by its link to adult T-cell leukemia (ATL) in Japan (Miyoshi et al. 1981; Hinuma et al. 1981), and later to tropical spastic paraparesis in the West Indies (Gessain et al. 1985). The identification of the Caribbean islands as an endemic region of HTLV-1 transmission (Blattner et al. 1982) followed on from an observation in London (Catovsky et al. 1982) that immigrants from Jamaica presented with a type of leukemia indistinguishable from Japanese ATL and were seropositive for HTLV-1. The public health importance of different oncogenic viruses varies greatly, ranging from major global causes of mortality, such as liver and cervical cancer, to relative rarities, like ATL and Merkel cell skin cancer (Table 5.1). Given that the Merkel cell polyomavirus (PyV) was discovered as recently as 2008 (Feng et al. 2008; Becker, Chap. 18), further cancer –associated viruses may be revealed. That seems possible for rare cancers which are increased in immunodeficiency, for example conjunctival squamous carcinoma in African AIDS (Waddell et al. 2010), although a sensitive subtractive transcriptome analysis of this tumor did not reveal any viral sequences (Feng et al. 2007). Further investigation of Epstein–Barr virus (EBV)-negative AIDS lymphomas might also lead to the discovery of a new agent. Acute lymphocytic leukemia (ALL) in children is thought to be linked to delayed infection among children living in relative isolation (Kinlen et al. 1990; Greaves 2006). However, a specific oncogenic virus is not necessarily involved as several

1967

1979 1980 1983

1983

1989

1994

HBV

HPV-5 HTLV-1 HIV

HPV-16, -18, etc.

HCV

KSHV

Skin Ca ATL KS Lymphoma Cervical Ca Other anogenital Ca HCC

Tumor BL NPC HCC

490,000 65,000 195,000

<1,000 3,340

Estimated annual new cancer cases globally 6,700 78,000 340,000

~3.0 ~3.0 ~5.0

<0.1 0.5 ~20

Carriers who develop cancer (%) <0.1 <1.0 ~15

Hepatitis Cirrhosis None

Genital warts

Nonmalignant disease Infectious mononucleosis Hepatitis Cirrhosis Wart TSP/HAM AIDS

Blood

Blood Sexual Contact Milk, blood Sexual Blood Sexual

Transmission route Saliva

KS 66,000 <1.0 Saliva PEL, CD <0.1 Merkel PyV 2008 Skin Ca ~1,000 <0.1 None Contact EBV Epstein–Barr virus, HBV hepatitis B virus, HPV human papillomavirus, HTLV human T-cell leukemia virus, HIV human immunodeficiency virus, KSHV Kaposi’s sarcoma herpesvirus, PyV polyomavirus, BL Burkitt’s lymphoma, NPC nasopharyngeal carcinoma, HCC haptocellular carcinoma, Ca cancer, ATL adult T-cell leukemia, KS Kaposi’s sarcoma, PEL primary effusion lymphoma, CD Castleman’s disease, TSP/HM tropical spastic paraparesis/HTLV-associated myelopathy, AIDS acquired immune deficiency syndrome

First reported 1964

Virus EBV

Table 5.1 Viruses causing cancer in humans

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kinds of infection promoting lymphocyte activation could be triggers for the appearance of ALL if infection is delayed (Greaves 2006). The infection hypothesis is consistent with the higher incidence of children’s ALL in more affluent social classes, as was seen for paralytic poliomyelitis in the 1950s, because poorer children tended to acquire asymptomatic poliovirus infection and subsequent immunity early in life. It represents a situation analogous to the hygiene hypothesis for the increasing prevalence of allergies (Rook 2007). Certain common human viruses can transform cells in culture and can induce tumors in experimentally inoculated animals, yet are not known to cause tumors in their natural host. Thus, the human polyomaviruses, BK, and JC (Hirsch, Chap. 16; Khalili, Chap. 17) and human adenovirus types 2 and 12 (Ricciardi, Chap. 20) can transform rodent cells in culture, and are potently oncogenic in newborn rats and hamsters. However, there is no firm epidemiological evidence to suggest that these commonplace human infections are linked to any human malignancy. One possible explanation for this paradox is that human cells are permissive for lytic replication of these viruses which would kill the host cell, whereas rodent cells permit the expression of only the early, transforming genes, such as polyoma T antigens and adenovirus E1A and E1B. But these viruses also spawn defective variants during the multiple rounds of replication in the human body, so cell transformation by viruses lacking late, lytic viral genes might be expected to occur. One could argue that the human immune system would nonetheless control or eliminate the development of viral tumors, yet even in immunocompromised people (Wood, Chap. 32), tumors caused by polyomaviruses or adenoviruses have not been documented. Perhaps this question merits reinvestigation. Conversely, Zur Hausen (2001) has suggested that zoonotic animal viruses that can induce cell proliferation might be tumorigenic in humans if human cells were nonpermissive for lytic replication.

Contribution of Tumor Viruses of Animals to Molecular Biology Veterinary malignant diseases were first shown to be transmissible in the nineteenth century which we now know are caused by oncogenic viruses. Enzootic bovine leukosis is caused by a deltaretrovirus related to the HTLV and Jaagsiekte in sheep, caused by the betaretrovirus, JRSV (Fan, Chap. 30). The first cell-free filtrate – hence a virus – shown to cause malignancy was the erythroleukemia virus of chickens reported by Ellerman and Bang in 1908 (Coffin et al. 1997). And 100 years ago, Peyton Rous described his eponymous sarcoma virus (Rous 1911; Parent, Chap. 28). In 1932, Richard Shope discovered the first oncogenic papillomaviruses in rabbits (Tooze 1980) while in 1936, Bittner’s milk factor (mouse mammary tumor virus (MMTV)) was the first oncogenic virus to be defined in laboratory mice (Weiss et al. 1984). It is curious that in some animal species the proportion of tumors with a viral etiology is higher than that of humans (e.g., cats and chickens), whereas other well-studied species do not appear to harbor any oncogenic viruses (e.g., dogs and horses), although cross-species infection of horses by bovine papillomavirus

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type 1 induces the benign tumor, equine sarcoid. Cancer in wild animals is more difficult to study (McAloose and Newton 2009) and therefore few oncogenic viruses of wild species are known. One that has recently emerged is the retrovirus-causing acute leukemia in koalas (Tarlinton et al. 2006). In addition to their importance as pathogens, tumor viruses have played a pivotal role in gaining insight into basic molecular and cell biology and into mechanisms of nonviral oncogenesis. This benefit to our understanding resulted from the intensity of studies of animal tumor viruses as models from the 1960s onward (Tooze 1980; Weiss et al. 1984; Coffin et al. 1997). Thus, splicing of eukaryotic mRNA transcripts was first discovered in polyomaviruses and adenoviruses. The first nuclear location signal in a protein was identified in SV40 large T antigen, and origins of DNA replication and control in mammalian systems were first analyzed with these DNA tumor viruses. The tumor-suppressor gene, p53, was first identified as a protein to which large T antigen bound and inhibited or sequested its function (Howley and Livingston 2009). RNA tumor viruses led to the identification of oncogenes before cellular oncogenes were discovered, reverse transcriptase which allowed the synthesis of cDNA, and retroviral vectors which became standard tools in gene transfer and gene therapy.

Control and Prevention of Viral Cancers We live in an age of optimism that we can prevent millions of potential cancers through screening and immunization with tumor virus vaccines. Cervical cancer screening by cytologists essentially entails the recognition of abnormal, HPV-infected cells. In Japan, the screening of blood donations for HTLV-1 has led to a drastic diminution of horizontal virus transmission, and antenatal screening helps to prevent the next generation acquiring HTLV-1 vertically via mothers’ milk, which is the main route of transmission. The first really successful anticancer vaccine was the Marek’s disease virus (MDV) vaccine against this alpha herpes virus causing T-cell lymphomas in the domestic fowl (Parcells and Morgan, Chap. 13). However, as this vaccine protects against tumor development rather than virus infection, it has led to increased MDV virulence later. Immunization against HBV (Blumberg, Chap. 2; Mason, Chap. 22) prevents HBV infection and hence hepatitis. It was initially prepared by purification of noninfectious 22-nm particles present at high titer in the plasma of viremic patients, but then the subunit vaccine was produced which represents the first vaccine of any kind to be developed through recombinant DNA technology. There have now been sufficient years of follow-up to discern a fall in hepatocellular cancer incidence in immunized populations. The recent development of two commercially available, efficacious vaccines against genital HPV transmission promises to greatly reduce cervical cancer (Roden and Wu 2006). The delivery and coverage of HBV and HPV immunization programs in resourcepoor countries, where these viruses are most prevalent, presents a challenge in

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public health economics and disease prevention. Protective vaccines against EBV and HTLV-1 could be developed and produced if they were deemed to be of public health benefit, but to date they have not been regarded as a major market opportunity for vaccine companies. HCV and HIV vaccines would be of obvious and immediate benefit, but alas, we still await safe and efficacious vaccines for these viruses.

Transmission Dynamics of Oncogenic Viruses DNA Tumor Viruses Many DNA viruses with oncogenic potential are acquired early in life, but the tumors, if any, tend to present much later. For example, the major route of transmission of EBV and Kaposi’s sarcoma-associated herpesvirus (KSHV or HHV-8) is from mother to child via saliva (Boshoff and Weiss 2006; Schulz, Chap. 10), but Kaposi’s sarcoma occurs mainly in elderly men, although KSHV-associated lymphoma may occur in children and young adults. Evidence from seroconversion also suggests that polyomavirus infections, such as BK and JC, occur in childhood. A recent study indicates that Merkel cell polyomavirus and novel human polyomaviruses are chronically shed from the skin and may, therefore, be transmitted by contact (Schowalter et al. 2010). HPVs infect the dividing, basal cells of epithelia but its late proteins for viral maturation are only expressed in differentiated squamous cells. Thus, carcinoma in situ and eventually invasive cancer develop in cells that do not achieve sufficient differentiation to complete lytic viral replication. HPV infections of the skin (e.g., HPV-1, HPV-5) are similarly shed in squames, and some genital HPV types are vertically transmitted, as HPV-11 and HPV-16 have been found in pediatric laryngeal warts. Transmission probably occurs when the baby passes through the dilated cervix. Of course, the main route of transmission of these genital HPV strains is sexual (You and Wells, Chap. 19).

Oncogenic Retroviruses The transmission dynamics and pathogenesis of retroviruses vary according to the type of tumor virus and its mode of transmission. Let us take the classical example of avian leukosis viruses in chickens (Weiss et al. 1984). Horizontal transmission between birds (except to the newly hatched) usually results in a transient viremia followed by an immune response that controls the virus load in the tumor-target organ (the bursa of Fabricius producing B cells) so that leukemia rarely ensues. However, if the same virus infects the chick embryo through secretion into egg albumen in the hen’s oviduct, then chronic viremia without an immune response often results – many weeks after hatching – in B-cell leukemia. The mechanism of

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oncogenesis is through the integration of the proviral long terminal repeat (LTR) adjacent to a cellular oncogene (Beemon and Rosenberg, Chap. 27), so the higher the rate of replication, the greater the chance of integration proximal to an oncogene. A similar mechanism applies to MMTV acquired through suckling colostrums immediately after birth, except that lymphocytes provide an intermediate cellular reservoir until the offsprings’ mammary glands become infected upon maturity and pregnancy (Ross, Chap. 29). On the other hand, Jaagsiekte virus of sheep (Fan, Chap. 30) has a strict cell tropism for type II pneumocytes which require lungto-lung transmission via aerosols. Murine and feline leukemia viruses present a rather more complex series of events leading to cancer (Beemon and Rosenberg, Chap. 27). An ecotropic virus is the primary agent, replicating from cell to cell and increasing in titer. It has an endogenous, Mendelian origin in mice, such as the AKR strain, but is horizontally, exogenously acquired among cats (FeLV-A). However, a recombinant virus between these transmissible, ecotropic viruses and endogenous retroviral envelopes with xenotropic or polytropic properties eventually emerges and it is this recombinant genome that has oncogenic properties. Thus, the oncogenic agent itself is generated anew in each infected host, and the transmissible virus is a precursor. Retroviruses have also been transmitted across species barriers, sometimes between large taxa, to infect new hosts. For instance, the gibbon ape leukemia virus and the koala virus are both closely related to gammaretroviruses of rodents in Southeast Asia. Whether retroviruses and polyomaviruses of animal origin are circulating in humans is discussed later. Retroviruses which bear oncogenes of cellular origin appear to be severely restricted in transmission. Most are defective, requiring a “helper” leukemia virus to complement missing or defective replicative genes (Beemon and Rosenberg, Chap. 27; Parent, Chap. 28). Despite the immense interest in these viruses for unraveling molecular mechanisms of oncogenesis, I am not aware of any cases of oncogenebearing viruses which are naturally transmitted between hosts, with the possible exception of the cyclin-bearing fish retroviruses (Kurth and Bannert 2010). Each one of the sarcoma viruses or acutely transforming leukemia viruses of chickens, mice, cats, and primates appears to be transmissible only by experimental inoculation. We can regard them as one-off, dead-end transductions whose tumors came to the notice of pathologists like Peyton Rous, but which play no role in the natural history of virus transmission.

Iatrogenic Infections Hepatitis viruses are transmitted parenterally (Lindenbach, Chap. 23; Mason, Chap. 22). HBV is often naturally acquired early in life, perinatally in Asia and during infancy in Africa. But iatrogenic (medically induced) transmissions also play a significant role in tumor virus transmission. Where nonsterile injection equipment was used, the dynamics and prevalence of infection by HBV, HCV, and HIV may

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have been greatly increased in Africa during the twentieth century, as argued by Drucker et al. 2001. The campaign to eradicate bilharzia (Schistosomiasis) in Egypt from the 1950s to 1980s by using an intravenous drug, tatar emetic, greatly exacerbated the spread of HCV because syringes and needles were not replaced for each individual when entire villages were treated (Frank et al. 2000; Strickland 2006). A related veterinary example was the spread of bovine leukemia virus (BLV) infection when whole herds were dehorned using common equipment, although injection for brucellosis vaccination did not markedly increase the risk of BLV transmission (Lassauzet et al. 1990). Regarding zoonotic infection by tumor viruses, millions of humans were exposed to SV40 virus because it was a containment of poliovirus vaccine propagated in primary kidney cultures derived from rhesus macaques (Shah and Nathanson 1976). Whether or not SV40 has taken off as a circulating virus in the human population remains controversial (Butel, Chap. 15, and Sect. 4.7). The discovery of SV40 in rhesus kidney cultures (Sweet and Hilleman 1960) soon led to the replacement by African green monkey kidneys as the preferred substrate for the propagation of polio vaccines. It was not known at the time that African green monkeys frequently harbor a strain of simian immunodeficiency virus (SIVagm), but fortunately SIVagm does not transfer to humans as it is sensitive to human host cell restriction factors. A claim that HIV-1 came into humans as a result of a batch of polio vaccine being prepared in chimpanzee kidney cultures (Hooper 2001) has not been upheld for two major reasons: first, a vial from the incriminated lot of polio vaccine was analyzed and found to have been prepared in rhesus cells and was not contaminated by SIVcpz (Berry et al. 2001); second, estimates of the time since the most recent common ancestor of HIV-1 Group M (the pandemic group) reliably show that HIV started to circulate in humans in Cameroon and the Congo several decades before the polio vaccine trials were conducted there (Sharp et al. 2001). These tales of contamination and of “near misses” emphasize how important it remains to ensure that biologicals and the injecting equipment used to deliver them are free from contaminating viruses. Novel therapies, such as xenotransplantation, need to consider the potential hazards of virus transmission (Weiss 1998).

Transmission and Virulence Cancer is a multistep process of progressive somatic mutations, and oncogenic viruses may push the infected cell past one or more of those steps, but not all of them. Cancer-associated viruses are persistent infections in the hosts in which the cancer develops. Indeed, the virus is usually present in the cancer cell clone itself: EBV genomes are found in the nasopharyngeal carcinoma and lymphoma cells with which it is associated, HTLV-1 in ATL cells, HBV in HCC cells (often integrated into chromosomal DNA), but HCV-linked liver tumors do not invariably contain the virus. However, HIV is a cancer-associated virus without infecting the tumor cells. It acts indirectly by allowing opportunistic oncogenic viruses to exert their effect via

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immune deficiency (Wood, Chap. 32), although some investigators have proposed an additional role for the Tat protein in oncogenesis (Aoki and Tosato 2007). One of the reasons that certain DNA viruses are potentially oncogenic is that they need to kick-start the cell-replicative machinery – the S phase of the cell division cycle – in order to replicate themselves. The small DNA tumor viruses, such as HPV, polyomaviruses, and adenovirses, do not encode all the enzymes for DNA replication in the viral genome, and require cellular enzymes to achieve replication. Oncogenic herpesviruses may be similar in this regard, but pox viruses, which replicate in the cytoplasm, are less dependent upon the cell cycle. This reasoning may explain the convergent evolution of disparate DNA tumor viruses to target the inactivation of host tumor suppressors, such as retinoblastoma protein and p53 (Jung, Chap. 3). Oncogenic retroviruses in general need to infect dividing cells. An interesting exception is JSRV (Fan, Chap. 30) which infects terminally differentiated type II pneumocytes, and its envelope glycoprotein serves as an “oncogene” which returns these cells to a dividing state. It is not clear how early after an infection an incipient precancerous cell lineage occurs. The tumors tend to present late in the life span of the host. Moreover, tumors develop in only a minority of infected individuals, perhaps up to 20% in HBV infection, around 1% in HTLV-1 and in genital HPV infection, and <0.01% in EBV and cutaneous HPV infection (Table 5.1). Thus, the rare malignant state of an infected cell is not directly related to virus transmission; rather, it should be regarded as collateral damage, an occasional “side effect” of infections that require the host cells to be in a proliferative state. As oncogenic viruses are necessary if not sufficient for the tumors to develop, immunization against infection can prevent the oncogenic process commencing. Marek’s disease (MD) (Parcells and Morgan, Chap. 13) is an interesting exception in that the vaccines protect mainly against tumorigenesis rather than against infection. MD vaccines protect with great efficacy against the development of the disease but do not prevent MDV transmission. Nonetheless, the MD vaccines dampen down virus transmission and, therefore, elicit selective pressure on the virus to circumvent this restriction. Over the 40 years since the first MDV vaccine was introduced, virus escape has resulted in the emergence of progressively more virulent MDV strains as novel, more potent vaccines were developed (Gimeno 2008). MD represents the best example for tumor viruses of a positive correlation between virulence and onward transmission. A corollary of this phenomenon is that vaccines which provide sterilizing immunity against primary infection (as for HBV and genital HPV) are less likely to exert selective pressure for the evolution of virulence.

Multifactorial Facets of Viral Cancers As oncogenic viruses only cause cancer in a minority of infected individuals, many cofactors influence the likelihood of malignancies developing. Further somatic mutations in the cell lineage leading to cancer are necessary, and several

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Mendelian-inherited predispositions both to viral infection and cancer emergence affect cancer incidence. These genetic predispositions are best understood for retroviral oncogenesis in mice, but they are also well-recognized in humans. In addition, environmental agents, including other infections, also play a role in certain types of viral cancer. Three human examples of the multifactorial elements in human cancer illustrate the complexities of viral oncogenesis.

Hepatitis B Virus and Hepatocellular Carcinoma HCC is the third most common cause of cancer incidence (Parkin 2006), but the substantial proportion of the burden due to HBV should be preventable by vaccination (Goldstein et al. 2005). Liver mycotoxins in the diet, such as aflatoxin B, act synergistically with HBV infection to increase the incidence of HCC three- to sixfold over than seen for exposure to chemical or virus alone (Kirk et al. 2006; Wild and Montesano 2009). Aflatoxin B is a product of Aspergillus mold species which grow on maize and groundnuts, especially in tropical humid conditions. Aflatoxin B is often present in the diet in regions, such as China and West Africa, where HBV infection is also prevalent. It is a genotoxic agent making mutagenic adducts to DNA and it induces mutations in key genes, such as the tumor suppressor p53. Aflatoxin B becomes concentrated and metabolized in the liver which is its main site of mutagenic action. Regenerating hepatocellular cells in HBV infection, therefore, become targets for the chemical carcinogen in the diet. It is not yet known precisely to what extent dietary aflatoxin B interacts with HCV (Wild and Montesano 2009). Because primary HCV infection usually occurs at a later age than HBV, the synergy for HCC may be lower, although aflatoxin B can act as an initiating carcinogen. Clearly, it is of health benefit to reduce the levels of mycotoxins in the diet through dry storage and other means, as well as controlling hepatitis infections.

Epstein–Barr Virus-Associated Tumors EBV infects the vast majority of people in all populations, and is linked to several types of lymphoma, nasopharyngeal carcinoma, and gastric cancer (Robertson, Chap. 8), yet few infected individuals develop EBV-associated diseases. The virus is usually acquired via saliva in infancy – to no ill effect unless the child inherits the rare genetic disorder, Duncan’s syndrome. Delaying infection until adolescence (and probably receiving a higher infecting dose through kissing) is likely to result in infectious mononucleosis, a polyclonal lymphoproliferative but nonmalignant disease. As discussed earlier, it is not uncommon to see an increase with age in the virulence of primary infections, e.g., polio virus and varicella-zoster virus, as maternal antibodies partially protect the infant from disease.

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EBV-linked tumors require cofactors in addition to EBV. Burkitt’s lymphoma (BL) in children only occurs in areas of endemic malaria infections, and the chronic activation of B cells due to malaria may allow EBV-infected premalignant clones to expand. Moreover, in every case of BL, including the EBV-negative adult cases, the lymphoma cells have chromosome translocations linking one of the highly active promoters of immunoglobulin genes to c-myc on chromosome 8. Thus, of the oncogenic virus, malaria infection and somatic rearrangement of chromosomes act in combination to promote the malignancy. A further cofactor might be local inflammation because BL in children frequently presents in the upper or lower jaw during the time of secondary tooth development. Undifferentiated nasopharyngeal carcinoma (NPC) causes considerably more mortality than BL (Table 5.1), although the tumor has been studied less intensively. Like BL, NPC prevalence tends to be geographically restricted and it occurs most commonly among people in Southern China and in people of Chinese origin. It is thought that predisposing host genetic factors must be determinants of tumor development. Certain major histocompatibility complex genes are associated with risk of NPC (Hildesheim et al. 2002), although the association does not appear to be strong enough to wholly explain the geographic clustering. There is also a theory that an environmental factor, such as nitrosamines in the diet, may cause somatic mutations that trigger NPC several decades later (Zheng et al. 1994). Other tumors which are sometimes associated with EBV infection, such as Hodgkin’s lymphoma, gastric cancer, and immunoblastic large cell lymphoma (LCL), have other risk factors, e.g., immune deficiency for LCL.

Epidermodysplasia Verruciformis Epidermodysplasia verruciformis (EV) is a very rare genetic disorder affecting DNA repair and immune functions in the skin. This leads to multiple warts developing all over the body, and these warts are initiated by papillomaviruses of the HPV-5 group (Kremsdorf et al. 1982). If the skin of EV patients is exposed to sunlight or other sources of ultraviolet radiation, malignant skin tumors appear. Thus, two distinct environmental factors – HPV and sunlight – generate a malignancy which is vastly exacerbated in patients carrying defective EV alleles. Skin tumors of the type seen in EV patients are rarely seen in people not carrying EV-defective genes. But HPV-5-related viruses are thought to be near-universal infections because all EV-affected individuals develop warts if not malignancies. Moreover, using polymerase chain reactions (PCRs), the viruses can be detected in normal skin and are expressed in psoriasis (Orth et al. 2001). However, other HPV types are more commonly associated with squamous skin cancer and can be frequently detected in normal skin and hair follicles (Plasmeijer et al, 2010). Like EBV, usually nonpathogenic, ubiquitous cutaneous HPV strains elicit a lethal malignant disease in rare individuals and occur more frequently in immunocompromised individuals.

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Distinguishing Tumor Viruses from “Rumor” Viruses Distinguishing genuine infection from artifact is not as easy as it might at first sight appear. Voisset et al. (2008) reviewed this topic in detail concerning the claims of retroviruses in humans, and similar problems arise with certain DNA tumor viruses. Our interest was aroused when we imagined that we had discovered a novel human retrovirus linked to rheumatoid arthritis (Griffiths et al. 1997) but later found that it was actually an endogenous retroviral genome of rabbits (Griffiths et al. 2002). The virus could be found at extremely low virus load usually requiring nested PCR for detection. The error only became evident when we managed to clone host DNA sequences at the integration sites flanking the LTRs of the virus, and a BLAST search showed that they were rabbit sequences. Strangely, the “false-positive” detection of this retrovirus was also detected by independent groups in Europe and the USA (Voisset et al. 2008), and occurred significantly more frequently in pathological tissue samples than in controls. The only reason we could suggest for this discrepancy was that the pathology samples might have been opened more often than the control blood samples and hence have had more opportunity for contamination by extraneous DNA molecules in the laboratory. There is a long history of reports of DNA sequences and antigens related to animal tumor viruses in human tumors (Voisset et al. 2008). The presence or not of MMTV in human breast cancer biopsies remains controversial, but definitive evidence, to my mind, has never quite materialized. PCR detection of MMTV sequences is genuine enough (Pogo et al. 2010) but could well result from contamination like we found with the rabbit virus. Antigens cross-reacting with antibodies to MMTV envelope glycoproteins gp52 are not quite convincing, and the in situ hybridization of MMTV on human chromosomes only lights up one chromatid of a mitotic pair (Liu et al. 2001), again leading one to doubt the interpretation that this represents reliable evidence of MMTV infection in human breast cancer. A virus sequence similar to xenotropic mouse leukemia virus is called xenotropic murine-related retrovirus (XMRV) (Singh, this volume). XMRV was detected in stromal cells of a subset of human prostate cancer biopsies in patients with a defective RNase L gene, which affects the interferon response pathway (Urisman et al. 2006). Such a defect might predispose people to zoonotic infection with a mouse retrovirus or aid its transmission from human to human. But the link to RNase L alleles has not been upheld in another study which detected XMRV in the prostatic epithelium rather than in the stroma (Schlaberg et al. 2009). Another report (Hohn et al. 2009) failed to detect XMRV in prostate cancer. XMRV has also been reported in a majority of patients with chronic fatigue syndrome (CFS) (Lombardi et al. 2009) but could not be confirmed in other cohorts with similar symptoms (Erlwein et al. 2010; Groom et al. 2010; Switzer et al. 2010). XMRV has been isolated in infectious form as well as by PCR (Lombardi et al. 2009) from prostate tumor cell lines and CFS biopsies. However, it should be born in mind that any human tumor that has been grown as a xenograft in mice or has been propagated in culture in the same facility as cell lines producing xenotropic

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murine retroviruses is at risk of contamination. Many murine monoclonal antibody preparations contain xenotropic MLV (Weiss 1980), and therefore the viral genes are present in most research laboratories. XMRV has a signature in the gag sequence that distinguishes it from classical MLV-X from NZB mice, but different mouse strains harbor many variants of this retrovirus. Thus, the link between XMRV and prostate cancer or CFS and whether the virus is a genuine human transmissible agent remains to my mind unresolved. The current XMRV controversy is reminiscent of the SV40 issue in the mid 1990s (Brown and Lewis 1998). As already mentioned, this polyomavirus of rhesus macaques did contaminate certain early batches of polio vaccine to which millions of people were exposed (Shah and Nathanson 1976) and hence it might have established a foothold in the human population. Thus, when Carbone et al. (1994) reported the presence of SV40-like DNA in human mesothelioma biopsies, many investigators turned their attention to it. Indeed, Bergsagel et al. (1992) had previously detected SV40-related sequences in pediatric central nervous system tumors, such as ependymoma and choroid plexus tumors. Assuming that these tumors contained SV40 rather than a human polyomavirus, such as JC on BK, the data suggest that SV40 has now become a transmissible human virus (rather like HIV-1) because these cancer cases occurred in children born one or two generations after polio vaccines were cleaned up. In the years since this controversy was actively debated, the evidence does not appear to have hardened for a role of SV40 in human tumors (Shah 2007). So I veer toward the skeptical view, whereas Butel (Chap. 15) has a more positive opinion. An impediment to sorting tumor virus from rumor virus is that investigators tend to become drawn into taking entrenched positions. Those who obtain positive results may declare that those who cannot confirm such data do not know how to do PCR detection diligently! On the other hand, the doubters like me point out that the laboratories with positive results are often those that work on that type of virus in any case, and therefore would be more prone to contamination by virus or PCR. As detection techniques become increasingly sophisticated, we must be careful to match sensitivity with specificity, including the purity of tissue sources.

Transmissible Cancer Cells Around the time that HIV-1 was discovered, I read a paper by Hayes et al. (1983) postulating that Kaposi’s sarcoma (KS) in AIDS may have a similar mode of transmission to canine-transmissible venereal tumor (CTVT) of dogs, namely, that the sexually transmissible agent could be the tumor cell itself. This hypothesis was disproved for KS when the Kaposi’s sarcoma-associated herpesvirus was discovered (Chang et al. 1994; Schulz, Chap. 10), but the question whether it remained true for the canine tumor intrigued me. CTVT was first experimentally transplanted in 1876. It was extensively used in cancer research before inbred rodents became available, and it was cited (as Sticker’s

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sarcoma) by Rous (1911). The cellular theory of CTVT transmission was based on its ease of experimental transmission and on the presence of marker chromosomes in tumors arising in different dogs (Murgia et al. 2006; Murchison 2008). Further suggestive evidence for cellular transmission came from the detection in all CTVT biopsies of a unique LINE-1 retrotransposon insertion site (Amariglio et al. 1991). Many veterinarians seemed to accept the cellular theory of CTVT transmission, but when I discussed the hypothesis with oncologists and immunologists, it was usually greeted with disbelief. We, therefore, investigated the phenomenon employing modern forensic DNA markers in order to test whether CTVT differed genetically from the dogs bearing the tumors. Indeed, CTVT specimens collected from five continents differed from the host dogs, but belonged to a single clonal lineage of cancer cells. Thus, a canine somatic cell had evolved to become a transmissible oncogenic parasite (Murgia et al. 2006). A second example of a parasitic cancer has been found in the marsupial Tasmanian devil known as Tasmanian devil facial disease (TDFD) (Pearse and Swift 2006; Murchison 2008; Murchison et al. 2010). While CTVT is mainly spread through sexual intercourse, TDFD is transmitted through biting and fighting. TDFD was first seen in 1996 and has recently emerged in a population which is partially inbred and shows restricted MHC polymorphism (Jones et al. 2004). CTVT probably originated in a gray wolf some thousands of years ago and can spread to all dog breeds and mongrels (Murgia et al. 2006; Rebbeck et al. 2009). These two tumors in animals represent transmissible cancers that are not caused by viruses. The question arises whether similar tumors might be found in humans. To date, only iatrogenic cases are known, when immunosuppressed transplant recipients have been unwittingly transplanted with apparently healthy tissue or organs that contained occult cancer cells (Barozzi et al. 2003; MacKie et al. 2003). The world of transmissible tumor viruses and tumor cells is fascinating.

References Amariglio EN, Hakim I, Brok-Simoni F, Grossman Z, Katzir N, Harmelin A, Ramot B, Rechavi G (1991) Identity of rearranged LINE/c-myc junction sequences specific for the canine transmissible venereal tumor. Proc Natl Acad Sci USA 88:8136–8139 Aoki Y, Tosato G (2007) Interactions between HIV-1 tat and KHSV. Curr Top Microbiol Immunol 312:309–326 Barozzi P, Luppi M, Facchetti F, Mecucci C, Alu M, Sarid R, Rasini V, Ravazzini L, Rossi E, Festa S, Crescenzi B, Wolf DG, Schulz TF, Torelli G (2003) Post-transplant Kaposi sarcoma originates from the seeding of donor-derived progenitors. Nat Med 9:554–561 Beasley RP, Hwang LY, Lin CC, Chien CS (1981) Hepatocellular carcinoma and hepatitis b virus. A prospective study of 22,707 men in Taiwan. Lancet 2:1129–1133 Bergsagel DJ, Finegold MJ, Butel JS, Kupsky WJ, Garcea RL (1992) DNA sequences similar to those of simian virus 40 in ependymomas and choroid plexus tumors of childhood. N Engl J Med 326:988–993 Berry N, Davis C, Jenkins A, Wood D, Minor P, Schild G, Bottiger M, Holmes H, Almond N (2001) Vaccine safety: analysis of oral polio vaccine CHAT stocks. Nature 410:1046–1047

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Hinuma Y, Nagata K, Hanaoka M, Nakai M, Matsumoto T, Kinoshita KI, Shirakawa S, Miyoshi I (1981) Adult T-cell leukemia: antigen in an ATL cell line and detection of antibodies to the antigen in human sera. Proc Natl Acad Sci USA 78:6476–6480 Hohn O, Krause H, Barbarotto P, Niederstadt L, Beimforde N, Denner J, Miller K, Kurth R, Bannert N (2009) Lack of evidence for xenotropic murine leukemia virus-related virus (XMRV) in German prostate cancer patients. Retrovirology 6:92 Hooper E (2001) Experimental oral polio vaccines and acquired immune deficiency syndrome. Philos Trans R Soc 356:803–814 Howley PM, Livingston DM (2009) Small DNA tumor viruses: large contributors to biomedical sciences. Virology 384:256–259 Jones ME, Paetkau D, Geffen E, Moritz C (2004) Genetic diversity and population structure of Tasmanian devils, the largest marsupial carnivore. Mol Ecol 13:2197–2209 Kinlen LJ, Clarke K, Hudson C (1990) Evidence from population mixing in British New Towns 1946–85 of an infective basis for childhood leukaemia. Lancet 336:577–582 Kirk GD, Bah E, Montesano R (2006) Molecular epidemiology of human liver cancer: insights into etiology, pathogenesis and prevention from The Gambia, West Africa. Carcinogenesis 27:2070–2082 Kremsdorf D, Jablonska S, Favre M, Orth G (1982) Biochemical characterization of two types of human papillomaviruses associated with epidermodysplasia verruciformis. J Virol 43: 436–447 Kurth R, Bannert N (2010) Retroviruses: Molecular Biology, Genomics and Pathogenesis. Caister Academic Press, Norfolk Lassauzet ML, Thurmond MC, Johnson WO, Stevens F, Picanso JP (1990) Effect of brucellosis vaccination and dehorning on transmission of bovine leukemia virus in heifers on a California dairy. Can J Vet Res 54:184–189 Liu B, Wang Y, Melana SM, Pelisson I, Najfeld V, Holland JF, Pogo BG (2001) Identification of a proviral structure in human breast cancer. Cancer Res 61:1754–1759 Lombardi VC, Ruscetti FW, Das Gupta J, Pfost MA, Hagen KS, Peterson DL, Ruscetti SK, Bagni RK, Petrow-Sadowski C, Gold B, Dean M, Silverman RH, Mikovits JA (2009) Detection of an infectious retrovirus, XMRV, in blood cells of patients with chronic fatigue syndrome. Science 326:585–589 MacKie RM, Reid R, Junor B (2003) Fatal melanoma transferred in a donated kidney 16 years after melanoma surgery. N Engl J Med 348:567–568 McAloose D, Newton AL (2009) Wildlife cancer: a conservation perspective. Nat Rev Cancer 9:517–526 Miyoshi I, Kubonishi I, Yoshimoto S, Akagi T, Ohtsuki Y, Shiraishi Y, Nagata K, Hinuma Y (1981) Type C virus particles in a cord T-cell line derived by co-cultivating normal human cord leukocytes and human leukaemic T cells. Nature 294:770–771 Murchison EP (2008) Clonally transmissible cancers in dogs and Tasmanian devils. Oncogene 27(Suppl 2):S19–S30 Murchison EP, Tovar C, Hsu A, Bender HS, Kheradpour P, Rebbeck CA, Obendorf D, Conlan C, Bahlo M, Blizzard CA, Pyecroft S, Kreiss A, Kellis M, Stark A, Harkins TT, Marshall Graves JA, Woods GM, Hannon GJ, Papenfuss AT (2010) The Tasmanian devil transcriptome reveals Schwann cell origins of a clonally transmissible cancer. Science 327:84–87 Murgia C, Pritchard JK, Kim SY, Fassati A, Weiss RA (2006) Clonal origin and evolution of a transmissible cancer. Cell 126:477–487 Orth G, Favre M, Majewski S, Jablonska S (2001) Epidermodysplasia verruciformis defines a subset of cutaneous human papillomaviruses. J Virol 75:4952–4953 Parkin DM (2006) The global health burden of infection-associated cancers in the year 2002. Int J Cancer 118:3030–3044 Pearse AM, Swift K (2006) Allograft theory: transmission of devil facial-tumour disease. Nature 439:549 Plasmeijer EI, Neale RE, Buettner PG, de Koning MN, Ter Schegget J, Quint WG, Green AC, Feltkamp MC (2010) Betapapillomavirus infection profiles in tissue sets from cutaneous squamous cell-carcinoma patients. Int J Cancer 126:2614–2621

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Pogo BG, Holland JF, Levine PH (2010) Human mammary tumor virus in inflammatory breast cancer. Cancer 116(11 Suppl):2741–2744 Poiesz BJ, Ruscetti FW, Gazdar AF, Bunn PA, Minna JD, Gallo RC (1980) Detection and isolation of type C retrovirus particles from fresh and cultured lymphocytes of a patient with cutaneous T-cell lymphoma. Proc Natl Acad Sci USA 77:7415–7419 Polk DB, Peek RM (2010) Helicobacter pylori: gastric cancer and beyond. Nat Rev Cancer 10:403–414 Rebbeck CA, Thomas R, Breen M, Leroi AM, Burt A (2009) Origins and evolution of a transmissible cancer. Evolution 63:2340–2349 Roden R, Wu T-C (2006) How will HPV vaccines affect cervical cancer? Nat Rev Cancer 6:753–763 Rook GA (2007) The hygiene hypothesis and the increasing prevalence of chronic inflammatory disorders. Trans R Soc Trop Med Hyg 101:1072–1074 Rous P (1911) A sarcoma of the fowl transmissible by an agent separable from the tumor cell. J Exp Med 14:397–417 Schlaberg R, Choe DJ, Brown KR, Thaker HM, Singh IR (2009) XMRV is present in malignant prostatic epithelium and is associated with prostate cancer, especially high-grade tumors. Proc Natl Acad Sci USA 106:16351–16356 Schowalter RM, Pastrana DV, Pumphrey KA, Moyer AL, Buck CB (2010) Merkel cell polyomavirus and two previously unknown polyomaviruses are chronically shed from human skin. Cell Host Microbe 7:509–515 Scotto J, Bailar JC (1969) Rigoni-Stern and medical statistics: a nineteenth-century approach to cancer research. J Hist Med Allied Sci 24:65–75 Shah KV (2007) SV40 and human cancer: a review of recent data. Int J Cancer 120:215–223 Shah K, Nathanson N (1976) Human exposure to SV40: review and comment. Am J Epidemiol 103:1–12 Sharp PM, Bailes E, Chaudhuri RR, Rodenburg CM, Santiago MO, Hahn BH (2001) The origins of acquired immune deficiency syndrome viruses: where and when? Philos Trans R Soc 356:867–876 Strickland GT (2006) Liver disease in Egypt: hepatitis C superseded schistosomiasis as a result of iatrogenic and biological factors. Hepatology 43:915–922 Sweet BH, Hilleman MR (1960) The vacuolating virus, SV40. Proc Soc Exp Biol Med 105:420–427 Switzer WM, Jia H, Hohn O, Zheng H, Tang S, Shankar A, Béannert N, Simmons G, Hendry RM, Falkenberg VR, Reeves WC, Heneine W (2010) Absence of evidence of xenotropic murine leukemia virus-related virus infection in persons with chronic fatigue syndrome and healthy controls in the United States. Retrovirology 7:57 Tooze J (ed) (1980) Tumor viruses. Cold Spring Harbor Laboratory Press, New York Tarlinton RE, Meers J, Young PR (2006) Retroviral invasion of the koala genome. Nature 442: 79–81 Urisman A, Molinaro RJ, Fischer N, Plummer SJ, Casey G, Klein EA, Malathi K, Magi-Galluzzi C, Tubbs RR, Ganem D, Silverman RH, DeRisi JL (2006) Identification of a novel gammaretrovirus in prostate tumors of patients homozygous for r462q RNAsel variant. PLoS Pathog 2:e25 Voisset C, Weiss RA, Griffiths DJ (2008) Human RNA “rumor” viruses: the search for novel human retroviruses in chronic disease. Microbiol Mol Biol Rev 72:157–196 Waddell K, Kwehangana J, Johnston WT, Lucas S, Newton R (2010) A case-control study of ocular surface squamous neoplasia (OSSN) in Uganda. Int J Cancer 127:427–432 Weiss RA (1980) Retroviruses produced by hybridomas. N Engl J Med 307:1587 Weiss RA (1998) Transgenic pigs and virus adaptation. Nature 391:327–328 Weiss RA (2008) On viruses, discovery, and recognition. Cell 135:983–986 Weiss RA, Teich NM, Varmus HE, Coffin J (eds) (1984) RNA tumor viruses. Cold Spring Harbor Laboratory Press, New York Wild CP, Montesano R (2009) A model of interaction: aflatoxins and hepatitis viruses in liver cancer aetiology and prevention. Cancer Lett 286:22–28

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Chapter 6

DNA Viruses and Cancer: Taking a Broader Look James C. Alwine

Introduction Increasingly evidence suggests that a variety of infectious agents, including bacteria, parasites, and viruses, contribute to oncogenesis. At present, more than 20% of worldwide cancers are believed to be caused or associated with infection by these agents (zur Hausen 2009). Mechanistically, these agents may contribute to oncogenesis directly or indirectly. For example, many of the viruses encode oncoproteins, which can directly transform cells by affecting the function of major cellular growth control proteins such as p53 and the retinoblastoma proteins (Rb) (O’Shea 2005); or transform using modified host gene, which have integrated into the viral genomes (Chen and Barker 1985). Other viruses, as well as the bacteria and parasites, may function more indirectly through immunosuppression, chronic inflammation, suppression of apoptosis, and induction of genetic instability (zur Hausen 2009). It is highly likely that we have only begun to discover the extent to which infectious agents contribute to oncogenesis especially via an indirect route. In this regard, it is worthwhile to take a broader look at the potential means by which those DNA viruses not known to frankly transform cells may mediate more subtle means of manipulating cells toward oncogenesis (oncopotentiation), or tumor cells toward more serious malignancy (oncomodulation) (Michaelis et al. 2009). The successful replication of mammalian DNA viruses such as polyomaviruses, adenoviruses, and herpesviruses requires viral adaptation of the host cell to establish an environment that can accommodate the increased demands for nutrients, energy, and macromolecular synthesis that accompany viral infection. In order to do this DNA viruses must control key cellular processes, for example, signaling pathways, stress responses signaling, and metabolism, that affect broad aspects of cellular

J.C. Alwine (*) Department of Cancer Biology, Abramson Family Cancer Research Institute, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA e-mail: [email protected] E.S. Robertson (ed.), Cancer Associated Viruses, Current Cancer Research, DOI 10.1007/978-1-4614-0016-5_6, © Springer Science+Business Media, LLC 2012

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Fig. 6.1 The phosphotidylinositol-3¢ kinase (PI3K)–Akt–tuberous sclerosis complex (TSC)– mammalian target of rapamycin (mTOR) signaling pathway. The diagram shows the control of mTOR complex 1 (mTORC1) in the phosphorylation of p70S6 kinase (S6K) and the eIF4Ebinding protein (4E-BP), which control cap-dependent translation by controlling the formation of the eIF4F translation initiation complex

synthesis, growth and survival. Thus we examine how DNA viral-mediated alterations in these processes might contribute to the progression toward transformation when the virus infects preoncogenic cells; or how these viral effects could increase malignancy if the virus infected a tumor cell. We consider these effects for DNA viruses in general, but also focus more specifically on the b-herpesvirus, human cytomegalovirus (HCMV). HCMV is not believed to be capable of frankly transforming human cells; although, there are reports in the literature suggesting a hit and run transformation mechanism (Shen et al. 1997). However, the recent development of more sensitive methods for the detection of HCMV has implicated the virus in the development of several cancers, including glioblastoma (Cobbs et al. 2008; Mitchell et al. 2008), colorectal cancer (Harkins et al. 2002), and prostate cancer (Samanta et al. 2003). The role of HCMV in these cancers has been proposed to by oncomodulation (reviewed in Michaelis et al. 2009; Soderberg-Naucler 2008). It is proposed that by this process, HCMV in tumor cells increases their malignancy by deregulating vital cellular processes (Bentz and Yurochko 2008; Castillo and Kowalik 2004; Lukac and Alwine 1999; Sanchez and Spector 2008; Streblow et al. 2008; Yu and Alwine 2002; Zhu et al. 1995). In the following section, we will examine how HCMV and other DNA viruses can alter the phosphotidylinositol-3¢ kinase (PI3K)–Akt–tuberous sclerosis complex (TSC)– mammalian target of rapamycin (mTOR) pathway (Fig. 6.1) in ways that mimic effects seen in oncogenesis. We will also discuss viral alterations of cellular metabolism that also mimic what happens in many tumor cells. The data suggest that while some DNA viruses, like HCMV, may not frankly transform cells, they can certainly alter signaling and metabolism in ways that could potentiate transformation or increase the malignancy of a cancer cell.

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The PI3K–Akt–TSC–mTOR Signaling Pathway A good example of a major cellular signaling pathway targeted by many DNA viruses is the PI3K–Akt–TSC–mTOR pathway (Fig. 6.1). As discussed below, mammalian DNA viruses interact with one or a few components of this pathway at some point in their life cycle. This is done in order to activate or inhibit the growth, metabolic, antiapoptotic, and translational functions the pathway controls (Cooray 2004; Datta et al. 1999). The significance of this is that each component of the PI3K–Akt–TSC–mTOR pathway is known to be an oncoprotein. Thus the DNA viruses are altering the pathway in a manner that could contribute to transformation or the progression of oncogenesis. Akt (also called PKB) is the cellular homolog of the oncoprotein encoded by the AKT8 retrovirus (Bellacosa et al. 1991). Akt can be activated by several mechanisms (Datta et al. 1999; Plas and Thompson 2005; Sarbassov et al. 2005b), the best understood involves PI3K in response to insulin and other tropic factors. Akt activation depends on activation of PI3K to phosphorylate PIP2 (phosphotidylinositol (PI)-3,4-bisphosphate), creating PIP3 (PI-3,4,5-triphosphate) at the plasma membrane. For example, insulin binding to the insulin receptor leads to the activation of PI3K and converts PIP2 to PIP3. PIP3 binds both Akt and PDK1 (phosphoinositidedependent protein kinase-1), recruiting both to the plasma membrane allowing PDK1 to phosphorylate Akt on threonine 308 (T308). Activated Akt affects multiple cellular targets, which increase metabolism, growth, synthetic processes, and proliferation while suppressing apoptosis (Cass et al. 1999; Datta et al. 1999; Hill et al. 1999; Summers et al. 1998; Ueki et al. 1998). Since these processes benefit viral replication, it is not surprising that many DNA viruses have developed means to activate the Akt pathway (Cooray 2004). However, these viruses can affect more aspects of the pathway than just Akt. An important downstream effect of Akt is the activation of mTOR kinase (also known as RAFT1 or FRAP), which exists in two complexes, mTOR complex 1 (mTORC1) and complex 2 (mTORC2) (Fig. 6.1). The major difference between the two is the substrate specificity factors, raptor in mTORC1 and rictor in mTORC2 (Kim et al. 2002; Sarbassov et al. 2004). Functionally, the two complexes have very different substrates in uninfected cells. The substrates and functions of mTORC2 are less well characterized than mTORC1; however, the data suggests that mTORC2 functions in (1) organizing actin cytoskeleton (Jacinto et al. 2004; Sarbassov et al. 2004), (2) regulating cell growth and proliferation, (3) promoting the activation of the serum glucocorticoid-induced protein kinase (SGK), and (4) promoting the phosphorylation of proline-directed serine or threonine sites in the turn motif of Akt and protein kinase C isoforms (reviewed in Alessi et al. 2009). A primary function of mTORC1 is to control cap-dependent translation (Mamane et al. 2006; Reiling and Sabatini 2006; Sarbassov et al. 2005a). When mTORC1 is active, it phosphorylates p70S6 kinase (S6K) and the eukaryotic initiation factor 4E (eIF4E) binding protein (4E-BP1) (Fig. 6.1). Phosphorylation of S6K activates it, promoting the formation of translation initiation complexes (Mamane et al. 2006); this includes the phosphorylation

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of ribosomal protein S6. The phosphorylation of 4E-BP1 is a major point of control in cap-dependent translation and regulates the function of the eIF4F translationinitiation complex. eIF4F binds to the 5¢-cap of an mRNA, which is the first step in the initiation of cap-dependent translation. The eIF4F complex (Fig. 6.1) contains eIF4E, which is the subunit that actually binds to the 5¢-cap, eIF4G, eIF4A, and the Mnk1 kinase. To form the eIF4F complex eIF4E must bind to eIF4G, the scaffolding protein of the eIF4F complex. However, eIF4G binding and cap-dependent translation, can be inhibited by 4E-BP1 binding to eIF4E, which displaces eIF4G and inhibits eIF4F complex formation. mTORC1 regulates the binding of 4E-BP1 to eIF4E by controlling 4E-BP1 phosphorylation. Under positive growth conditions, mTORC1 is active and maintains 4E-BP1 in its hyperphosphorylated state where it is incapable of binding eIF4E. This allows eIF4E to remain active in the eIF4F complex and promote cap-dependent translation. However, under negative growth conditions, for example, during stress or inhibition of mTOR kinase activity, mTORC1 is inactive; 4E-BP1 becomes hypophosphorylated, binds efficiently to eIF4E, displaces eIF4G and inhibits cap-dependent translation (reviewed in Buchkovich et al. 2008b). The signaling between Akt and mTORC1 involves the TSC, which comprises TSC1 and TSC2 (also known as hamartin and tuberin, respectively) (Jozwiak 2006; Krymskaya 2003), and Rheb-GTP (Astrinidis and Henske 2005; Long et al. 2005a, b). Rheb-GTP function is mediated by the TSC, which stimulates the intrinsic GTPase activity of Rheb, converting it from Rheb-GTP to Rheb-GDP, which cannot activate mTOR kinase. Akt phosphorylation of the TSC inactivates the TSC, allowing Rheb-GTP levels to remain high and maintain mTOR kinase activity in mTORC1 (Long et al. 2005a). A number of cellular stress responses activate the TCS to inhibit mTORC1 and cap-dependent translation. Viral infections activate many of these stress responses and many viruses have developed means to circumvent them to maintain cellular functions like translation. As discussed below, a number of viruses circumvent the effects of stress responses by targeting the TSC for inhibition or degradation, thus maintaining mTORC1 activity. Hence, viruses that want to maintain mTORC1 activity and cap-dependent translation will want to either (1) activate Akt to maintain the TSC in an inactive state, (2) directly block TSC function, or (3) in some other way maintain mTORC1 activity.

The Effects of Mammalian DNA Viruses on the Activation of PI3K–Akt–TSC–mTOR Pathway and the Maintenance of Cap-Dependent Translation Nearly every family of mammalian DNA viruses can be shown to affect some aspect of the PI3K-Akt-TSC-mTOR pathway. This has been previously reviewed in Buchkovich et al. (2008b). In the following discussion, we will consider the past and more present data regarding the polyoma, papilloma, adeno, and herpes viruses, i.e., the mammalian DNA viruses, which replicate in the nucleus and have

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double-stranded DNA genomes. Thus we will not discuss the single-stranded DNA parvoviruses and the cytoplasmic-replicating Pox viruses. The polyoma viruses, for example, mouse polyoma (Py) virus and simian virus 40 (SV40) and the papilloma viruses contain covalently closed circular double stranded (ds) DNA genomes of 5–8,000 base pairs encoding 5–8 temporally expressed early and late proteins. Differential splicing of the early gene transcript of both viruses produces mRNAs for several early proteins called large and small tumor (T) antigens (LT and ST, respectively) for both Py and SV40, and middle T antigen (MT), which is specific to polyoma (Ichaso and Dilworth 2001). The plasma membrane-bound PyMT is a potent oncogene that alters the activities of important signaling proteins (Gottlieb and Villarreal 2001; Utermark et al. 2007), including PI3K, which is important for transformation mediated by PyMT (Utermark et al. 2007). PI3K activation by PyMT leads to the phosphorylation of a number of cellular targets, including Akt. Presumably, the activation of Akt leads to the activation of mTORC1. SV40 large T (SVLT) transforms mouse and human cells primarily by binding and inactivating p53 and the Rb family proteins (Simmons 2000). Additionally, it has been reported that SVLT transformation requires insulin receptor substrate-1 (IRS-1) (Rundell and Parakati 2001), which is key in the activation of PI3K by insulin. SV40 small t (SVSt) binds to protein phosphatase 2A (PP2A), a major phosphatase controlling many cellular functions. This interaction is required for SV40-mediated transformation of human cells (Rundell and Parakati 2001). Akt and mTOR are activated early during SV40 infection. Both SVLT (Yu and Alwine 2002; Yu et al. 2005) and SVSt (Yuan et al. 2002) have been shown to activate Akt; however, the mechanisms involved remain unclear. Activation by SVLT can be inhibited by PI3K inhibitors, indicating that SVLT activates PI3K. The interaction between SVSt and PP2A is required for the SVSt-mediated activation of Akt (Yuan et al. 2002), thus inhibition of PP2A appears to be needed to maintain Akt activity. The papillomaviruses cause warts and several members of this virus family cause cancer, including the high-risk human papillomaviruses (HPVs), which cause cervical cancer (Mirzamani et al. 2006; Schiffman et al. 2007). HPVs express several early proteins, two of which, E6 and E7, are required for maintenance of HPVrelated oncogenesis through interactions with p53 and the retinoblastoma protein (Rb), respectively. However, it has been shown that HPV E7 can interact with PP2A, interfering with its interaction with Akt and, similar to SVSt, inhibiting Akt dephosphorylation (Pim et al. 2005). It has also been reported that the HPV 16 E5 protein may be required for activation of PI3K and Akt (Kim et al. 2006). As the papillomavirus infection progresses, the need to activate Akt specifically to maintain mTOR activity can be circumvented by the E6 protein, which binds to tuberin in the TSC; this directs tuberin to proteasome-mediated degradation (Lu et al. 2004). As a result, mTORC1 remains active. The adenoviruses possess linear dsDNA genomes of 30–38 kb encoding 30–40 proteins. Early adenovirus proteins are responsible for preparing the cell to execute viral replication, adenovirus proteins, E1A and E1B-55K, target Rb and p53, respectively (Helt and Galloway 2003; Querido et al. 1997, 2001). It has been suggested that E1A mediates increased phosphorylation of 4E-BP and S6K (de Groot et al. 1995;

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Gingras and Sonenburg 1997); other studies suggest that the early adenoviral proteins E4-ORF1 and E4-ORF4 are involved in the activation of PI3K, resulting in the activation of Akt and mTORC1 (O’Shea et al. 2005). The human herpesviruses (HHV) include herpes simplex virus (HSV) 1 and 2, varicella zoster virus (VZV), HCMV, Epstein-Barr virus (EBV), HHV-6, HHV-7, and Kaposi’s sarcoma herpesvirus (KSHV, also known as HHV-8). These are large DNA viruses with linear dsDNA genomes up to 230 kb, capable of encoding up to 200 proteins, as is the case with HCMV, the largest HHV. The host ranges and the rates of the replicative cycles of the various HHVs differ greatly. For example, HSV-1 has a broad host range and replicates very rapidly. Conversely, HCMV is human specific and has a very slow replicative cycle, thus it must keep host cells relatively functional for an extended period despite the stress of the infection on the cells. Data suggest that HCMV does this very well by affecting many cellular signaling pathways in a way that inhibits stress signals or circumvents their inhibitory effects (Alwine 2008; Buchkovich et al. 2008a, b; Child et al. 2004; Chuluunbaatar et al. 2010; Hakki et al. 2006; Isler et al. 2005a, b; Kudchodkar et al. 2004, 2006, 2007; Mohr 2006; Moorman et al. 2008; Walsh et al. 2005; Xuan et al. 2009). HCMV infection can be sustained under stress conditions that would kill uninfected cells (Buchkovich et al. 2008a; Isler et al. 2005a, b). Regarding the effects of HHVs on the PI3K–Akt–TSC–mTOR pathway, much is known about HCMV and HSV. HCMV infection activates Akt through stimulation of T308 phosphorylation via activation of PI3K (Johnson et al. 2001; Yu and Alwine 2002) and stimulation of S473 phosphorylation via activation of mTORC2 (Kudchodkar et al. 2006). However, the need for AKT activation during lytic infection for both HCMV and HSV infections has come under question by the observations that Akt becomes highly hypophosphorylated after 24 hpi in HCMV infected cells (Kudchodkar et al. 2006) suggesting that it is only needed during the early stage of infection. Additionally, the HCMV early protein UL38 binds to and inactivates the TSC to eliminate TSC-mediated inhibition of mTOR (Moorman et al. 2008), thus the activity of Akt as an inhibitor of the TSC is moot. HSV appears to eliminate the need for Akt altogether, recent data has shown that it encodes an Akt surrogate (HSV Us3), which allows mTORC1 to be constitutively active (Chuluunbaatar et al. 2010). HCMV infection also appears to targets the mTOR complexes and functionally alters them to maintain 4E-BP phosphorylation and cap-dependent translation (Kudchodkar et al. 2006). Additionally, it can act downstream of mTOR by inducing the activity of the Mnk1 kinase to phosphorylate eIF4E in the eIF4F complex (Walsh et al. 2005) (Fig. 6.1). Figure 6.2 summarizes this discussion showing all of the intersections between DNA viruses and the PI3K–Akt–TSC–mTOR pathway. We see events that activate PI3K, Akt, mTORC1, and inhibit the TSC. Each one of these activation or inhibition events is known to be associated with oncogenesis (Averous and Proud 2006; Engelman 2009; Henske 2004; Lu et al. 2010; Zhou and Huang 2010). Thus the PI3K–Akt–TSC–mTOR pathway provides a vivid example of how DNA viruses can affect cellular signaling in way that can be oncopotentiating and oncomodulatory.

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Fig. 6.2 The points in the PI3K–Akt–TSC–mTOR signaling pathway that are activated or inhibited by polyoma, adeno, and herpes viruses. See text for details

Viruses and the Warburg Effect: Viral Effects on Glucose and Glutamine Metabolism The effect of viruses on metabolism has recently begun to be examined in detail. The findings, bases primarily on studies of HCMV, suggest that some DNA viruses can alter metabolism in a manner similar to that seen in many tumor cells, thus suggesting another way viruses may promote the progression of oncogenesis. In 1924, Otto Warburg observed that cancer cells metabolize glucose differently than normal cells (Warburg et al. 1924). Like normal cell, cancer cells use glycolysis to convert glucose to pyruvate (Fig. 6.3); however, normal cells and cancer cells differ in their utilization of pyruvate. Pyruvate enters the tricarboxylic acid (TCA) cycle in normal cells where it is catabolized to CO2 and promotes oxidative phosphorylation. In cancer cells, pyruvate is converted to lactate (Fig. 6.3), and does not enter the TCA cycle, even if sufficient oxygen is present to support mitochondrial oxidative phosphorylation (Vander Heiden et al. 2009). Such utilization of glucose is classically known as the Warburg effect and it has several important implications. Specifically, when pyruvate does not enter the TCA cycle only two ATP molecules are produced per molecule of glucose; however, by completing the TCA cycle and oxidative phosphorylation, an additional 36 ATP molecules are produced per molecule of glucose. While this appears inefficient, the shift from the complete catabolism of glucose in cancer cells, and other rapidly proliferating cells, results in the utilization of glucose carbon in biosynthetic pathways for the production of macromolecules needed to construct progeny cells (Vander Heiden et al. 2009, 2010). Another implication of this utilization of glucose is that glucose-derived carbon does not enter the TCA cycle; this not only limits ATP production, but also limits the production of TCA cycle intermediates, which are important biosynthetic precursors.

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Fig. 6.3 The metabolism of glucose and glutamine in normal, tumor, and HCMV-infected cells. See text for details

Depriving the TCA cycle of glucose-derived carbon must be compensated and recent studies show that in cancer cells exogenous glutamine is used as a carbon source (DeBerardinis et al. 2007, 2008; Wise et al. 2008). Glutamine is converted to a-ketoglutarate via glutaminase (Glnase) and glutamate dehydrogenase (GDH) (Fig. 6.3). Thus, in a process called anaplerosis glutamine provides TCA cycle intermediates to maintain the cycle (Fig. 6.3) and NADH for oxidative phosphorylation (DeBerardinis et al. 2007, 2008). Most normal, quiescent cells use only a small amount of consumed glutamine in this way. Thus, both glucose and glutamine metabolism are dramatically altered in tumor cells (DeBerardinis et al. 2008; Newsholme et al. 1985; Wise et al. 2008). Interestingly, HCMV-infected cells appear to induce metabolic changes similar to those observed in tumor cells. Recent studies have shown that glucose metabolism is dramatically altered in HCMV-infected cells (Munger et al. 2006, 2008). In these studies, it was shown that glucose carbon does not complete the TCA cycle. Instead, much of the glucose derived pyruvate into the TCA cycle as far as citrate, which is formed via the conversion of pyruvate to acetyl CoA (AcCoA) and the subsequent reaction of AcCoA with oxaloacetate (OAA) (Fig. 6.3). Citrate in the mitochondrion can be shuttling from the mitochondria to the cytoplasm where it is converted back to AcCoA and OAA by ATP-citrate lyase (ACL). This provides AcCoA in the cytoplasm for use in synthetic processes such as fatty acid synthesis (Fig. 6.3) (Munger et al. 2008). The utilization of glucose carbon for fatty acid synthesis is essential for the success of the HCMV infection (Munger et al. 2008). This is presumably due to the large amounts of membranes required during infection (Buchkovich et al. 2010; Das et al. 2007; Homman-Loudiyi et al. 2003; Sanchez et al. 2000; Seo and Britt 2006).

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The diversion of citrate from the TCA cycle can be considered a modified Warburg effect since glucose-derived carbon is being removed from the TCA cycle for synthetic purposes. HCMV-infected cells, like tumor cells, must find other ways to maintain the TCA cycle and produce ATP. Several recent studies have shown that HCMV induces a program for the anaplerotic use of glutamine. Metabolomic data charting metabolic flux suggested that glutamine utilization in the TCA cycle was increased in HCMV-infected cells (Munger et al. 2006, 2008). The critical nature of glutamine for the success of HCMV infection was demonstrated by direct measurement of glutamine metabolism in infected cells (Chambers et al. 2010). These data showed that glutamine was necessary for ATP production in infected human fibroblasts, but not in uninfected cells. HCMV-induced glutamine utilization was also indicated by increased glutamine uptake and increased ammonia production resulting from glutaminolysis. Correspondingly, there were HCMV-induced increases in the levels and enzymatic activities of glutaminase and GDH, the two enzymes that must be activated to convert glutamine to a-ketoglutarate for entry into the TCA cycle (Fig. 6.3). That glutamine is used to maintain the TCA cycle in infected cells was confirmed by the observation that TCA cycle intermediates such as a-ketoglutarate (Fig. 6.3) could rescue both ATP production and viral growth under conditions of glutamine deprivation (Chambers et al. 2010). Hence, HCMV-infected cells, like many tumor cells, activate a program, whereby, glutamine utilization increases specifically to maintain the TCA cycle allowing glucose to be used biosynthetically. There remains much to be determined about the effects of HCMV and other viruses on metabolism and how this relates to pathogenesis. However, it is important to understand since it may provide new insights into viral therapy and new drug targets for viral diseases. However, there is more to consider. As described above, the alterations in cellular metabolism during HCMV infection are comparable to that seen in many tumor cells. As we noted in the case of viral alterations in cell signaling what HCMV is doing could potentiate oncogenesis by setting up a metabolic condition that favors the needs of the pre-cancerous or cancerous cell. Indeed, the viral-mediated alterations in cellular signaling, stress responses, and metabolism could result in not only oncopotentiation and oncomodulation, but also unexpected pathogenesis, potentially implicating HCMV and other viruses as agents or subtle cofactor in many maladies.

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Chapter 7

Herpesviruses and Cancer David Everly, Neelam Sharma-Walia, Sathish Sadagopan, and Bala Chandran

Introduction Over 200 herpesviruses that are known to infect humans and a spectrum of animal species including oysters are classified under Herpesviridae (order: Herpesvirales) due to common characteristics such as large double-stranded, linear DNA genomes encoding 100–200 genes encased within an icosahedral capsid, which is itself wrapped in the tegument protein layer containing both viral proteins and viral mRNAs and a lipid bilayer envelope bearing many viral glycoproteins. Most importantly, they establish lifelong latent infection in the infected host and periodically reactivate to reinfect. It is obvious that for the successful persistence in the host these viruses must have evolved to utilize several avenues such as evading the host intrinsic, innate, and adaptive immune responses, and modulation of apoptosis, transcription, host cell metabolism, transport, and cell cycle. Though transformation leading to cancer and death of the host is not an advantage for the maintenance of species, events such as reduction in immune and other surveillance mechanism can lead to tumor formation by some of the members of herpesviruses. The notion that herpesviruses play roles as the etiological agents of human and animal malignancies came from the initial electron-micrographic discovery of Epstein-Barr virus (g-1 herpesvirus) particles in Burkitt’s lymphoma (BL) cells in 1966 by Epstein, Achong, and Barr (Epstein et al. 1966a, b). This was further substantiated by the identification of Marek’s disease virus (MDV), an a-herpesvirus, as the causative agent of Marek’s disease (MD). Subsequent studies discovered that Kaposi’s sarcoma-associated herpesvirus (KSHV), a g-2 herpesvirus, is etiologically associated with Kaposi’s sarcoma (KS).

B. Chandran (*) • D. Everly • N. Sharma-Walia • S. Sadagopan H.M. Bligh Cancer Research Laboratories, Department of Microbiology and Immunology, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064, USA e-mail: [email protected] E.S. Robertson (ed.), Cancer Associated Viruses, Current Cancer Research, DOI 10.1007/978-1-4614-0016-5_7, © Springer Science+Business Media, LLC 2012

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EBV is a g-1 herpesvirus and the type species of the genus Lymphocryptovirus (LCV). Thirty different LCV, 26 of them novel, were detected in primates by a panherpesvirus PCR assay. Nineteen LCV from chimpanzees, bonobos, gorillas, and other Old-World primates were closely related to EBV. Seven LCV originating from New-World primates were related to callitrichine herpesvirus 3 (CalHV-3), the first recognized New-World LCV. Another LCV from gorillas and three LCV from orangutans and gibbons were only distantly related to EBV and CalHV-3, which were tentatively assigned to a novel genogroup of Old-World primate LCV. Another transforming, EBV-related virus from spontaneous B cell lymphomas of common marmosets (Callithrix jacchus) has been isolated. KSHV is classified as a member of the rhadinovirus subgroup of gammaherpesviruses. Rhadinoviruses have been found in many species, including cattle, mice, and both Old-World and New-World primates such as herpesvirus saimiri (HVS), Rhesus rhadinovirus (RRV), mouse herpesvirus 68 (MHV-68), alcelaphine herpesvirus 1 (AHV1), bovine herpesvirus 4 (BHV-4), equine herpesvirus 2 (EHV2), and ovine herpesvirus 2 (OvHV-2). Viruses that infect New-World monkeys include HVS, infecting the squirrel monkey, and herpesvirus ateles (HVA), infecting the spider monkey. The macaques of Asia are the only Old-World primate species documented thus far to harbor rhadinoviruses. RRV is widespread among rhesus macaques (Macaca mulatta). Its genome organization is very similar to that of KSHV, with homologues of several potentially pathogenic KSHV genes. Another rhadinovirus sequences have been identified in macaques suffering from retroperitoneal fibromatosis (RF), a mesenchymal neoplasm with vascular components. Both the rhesus macaque and the pigtailed macaque (Macaca nemestrina) harbor RF herpesviruses (RFHVMm and RFHVMn). RF herpesviruses are more closely related to KSHV than KSHV is to RRV. Individual excellent chapters of this book discuss separately the role of LCV, RRV, RFHV, MHV-68, and MDV in cancer. The focus of this chapter is primarily on the general biology of the two human oncogenic herpesviruses: EBV and KSHV.

EBV Biology After the initial identification of EBV in a BL biopsy, seroepidemiological studies demonstrating increased antibody titers to EBV antigens solidified the connection between the virus and BL (Burkitt 1958; Epstein et al. 1964; Levy and Henle 1966). Subsequently, serological analyses of EBV antigens made the connection to infectious mononucleosis and undifferentiated nasopharyngeal carcinoma (NPC) (Klein et al. 1968; Henle et al. 1970; zur Hausen et al. 1970). Since then, EBV has been associated with an increasing number of human malignancies. Although EBV is an extremely successful pathogen that has infected greater than 95% of the individuals by adulthood worldwide, only a subset of infected individuals develops malignancies containing EBV. Understanding the virus’s contribution to malignant transformation has revealed a complex relationship between the virus with host factors, genetic events, environmental influences, and other pathogens. The contribution of EBV to

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Gastric and Nasopharyngeal Carcinoma Latent infection of mucosal epithelial cells in the presence of other mutations (Latency II)

Hodgkin’s disease EBV infection in the presence of other mutations (Latency II)

EBV

EBV EBV

EBV EBV T EBV EBV

Germinal Center B

EBV EBV

EBV

EBV EBV

Immunosuppressed lymphomas Proliferation of latently infected Bcells (Latency III)

Memory B-cell

EBV

EBV EBV

EBV EBV

EBV EBV EBV

Burkitt’s lymphoma C-myc translocation in the presence of EBV (Latency I)

Fig. 7.1 Model of EBV life cycle and cancer contribution. EBV normal infectious cycle (solid arrows) is initiated when the virus infects and replicated in the oral epithelial cells leading to the latent infection of B-cells. Infected B-cells can enter lytic cycle leading to the infection of epithelial cells and possible transmission of the virus or can remain latent and go through the germinal center reaction. Eventually the latently infected cell becomes a memory B-cell. EBV can also contribute to the oncogenic process (dotted arrows). In the presence of immunosuppression EBV can drive the proliferation of latently infected cells (immunosuppressed lymphomas). Alternatively, germinal center B-cells latently infected with EBV that undergo mutation, e.g. c-myc translocation (Burkitt’s lymphoma) or other mutations (Hodgkin’s disease), can lead to lymphoma development. Latent infection of epithelial cells in the presence of mutations and/or environmental or genetic predispositions can lead to carcinoma (gastric or nasopharyngeal carcinoma). Viral gene expression profile is indicated

cancer is intimately related to the natural life cycle of the virus (Fig. 7.1). Understanding the mechanisms of cancer development have yielded insights into the viral life cycle, and conversely, understanding the viral life cycle has led to insights into the processes of malignant transformation. EBV primarily infects two cell types: B-lymphocytes and epithelial cells. Like all herpesviruses, infection with EBV can result in either lytic or latent infection. Infection of epithelial cells primarily results in lytic infection. Lytic infection results in production of infectious viral particles and the death of the infected cell. Infection of B-lymphocytes normally results in latent infection. In latent infection, there is limited viral gene expression and the virus seeks to maintain the viral DNA within a cellular reservoir, i.e., memory B-cells for EBV. The malignancies of EBV are associated with the latent cycle of the virus. The viral proteins that ensure the survival of the latently infected cell and maintenance of viral DNA are hypothesized to play roles in the development of the malignancies associated with EBV (Table 7.1).

Latency 3 Latency 3 Latency 2, 3

Latency 2, 3

Latency 2, 3 Latency 1, 2, 3 Variable

EBNA-LP BHRF1 LMP1

LMP2A

LMP2B EBERs miRNAs

Augment EBNA2 activity Bcl2 homolog TRAF-binding; NF-kB, PI3K, MAPK, and JNK signaling Syk and Lyn-binding; PI3K signaling Unknown PKR-binding Unknown targets

Table 7.1 EBV proteins with potential involvement in oncogenesis Gene Expression Function EBNA1 Latency 1, 2, 3 Episomal origin binding EBNA2 Latency 3 RBP-Jk binding; transcription activator EBNA3A Latency 3 RBP-Jk and CtBP binding EBNA3B Latency 3 RBP-Jk binding EBNA3C Latency 3 RBP-Jk and CtBP binding, Deubiquitination

Regulate LMP2A function Inhibit innate immunity Cellular and viral gene expression

B-cell receptor mimic

Unknown Increased tumorigenicity Unknown

BIM and p16INK4A downregulation Unknown BIM and p16INK4A downregulation, Rb and p27KIP1 regulation, Mdm2 regulation Unknown Antiapoptotic Growth, antiapoptotic, and increased motility and invasion Cell growth and survival

Transcriptional and epigenetic regulation Unknown Transcriptional and epigenetic regulation

Transcriptional regulator Antiapoptotic CD40 mimic, antiapoptotic

Possible cancer contribution Genomic instability Myc upregulation

Proposed latency function Episome replication and partitioning LMP and cellular gene transcription

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During latent infection, a variety of latent proteins and RNAs can be expressed depending upon promoter usage. EBV nuclear antigens (EBNAs) are encoded from several alternatively spliced primary transcripts. The transcripts are named for the BamH I library fragment from which the transcript arises. EBNA transcripts arise from promoters in the W, C, and Q fragments named Wp, Cp, and Qp, respectively. Wp and Cp transcripts are alternatively spliced to encode EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, and EBNA-LP, while Qp transcripts express only EBNA1. One of the viral Bcl2 homologues, BHRF1, can also be expressed from Wp and Cp. The latent membrane proteins (LMPs), LMP1, LMP2A, and LMP2B, are expressed from individual promoters near the terminal repeats of the genome. EBV also expresses noncoding RNAs. EBER1 and EBER2 are abundant nonpolyadenylated of 167 and 173 base pairs, respectively. A number of viral microRNAs (miRNAs) are also expressed during viral latency from the BamH I A and BHRF1 RNAs. Increased antibody titers to EBV latent proteins and in some cases EBV lytic proteins are associated with the development of EBV-associated malignancies. The correlation between the increased EBV-specific antibodies and the development of malignancies suggested a role of the virus in contributing to cancer. At least three different, increasingly restrictive latent gene expression patterns have been observed, termed latency III, latency II, and latency I. Alternatively, these patterns have been described as the growth program, default program, and latency program, respectively. The later nomenclature accounts for what is hypothesized to occur during B-cell infection in a healthy individual. Following initial infection of a naïve B-cell, all of the latent proteins are expressed in the growth program to induce the proliferation of the latently infected cell. As the latently infected cells move through the germinal center reaction from centroblasts to centrocytes and in the face of increased immune selection, only the LMPs and EBNA1, the default program, are expressed. Finally, as the infected cell is differentiated into a memory B-cell, only EBNA1 of the latency program is expressed. It is hypothesized that if the latently infected memory B-cell stops dividing altogether, no EBV proteins may be expressed, also called “latency 0”. These gene expression profiles approximate the gene expression profiles observed in different EBV-associated cancers. However, there can be significant variability in the EBV genes expressed in specific cancers that are not accounted for by the latency nomenclature. The different EBV latent proteins that play roles in the establishment of latency undoubtedly play roles in cancer development under specific circumstances.

EBV and Burkitt’s Lymphoma Although the BL was the first cancer to be associated with EBV, understanding the contribution of EBV to the development of the tumor has been a longstanding mystery. BL is endemic to equatorial Africa and is nearly always associated with EBV; by contrast, spontaneous BL is also observed but it is rarely associated with EBV. The primary oncogenic event of BL is a reciprocal chromosomal translocation

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between the cellular oncogene, c-myc, and the genes of the immunoglobulin loci, 75–85% t(8;14)(q24;q32) Ig heavy-chain (m), 5% t(2;8)(p12;q24) Ig light-chain (k), and 10% t(8;22)(q24;q11) Ig light-chain (l) (Dalla-Favera et al. 1982; Lenoir et al. 1982; Magrath 1990). This translocation results in the uncontrolled expression of c-myc, which is both necessary and sufficient to induce the proliferation of the tumor cells (Lombardi et al. 1987; Polack et al. 1996). However, overexpression of c-myc can also activate p53-dependent antioncogenic pathways (Cherney et al. 1997). The Em-myc mouse model, which mimics the translocation, demonstrates that genetic or epigenetic changes in the ARF/MDM2/p53 pathways are required for tumor development (Eischen et al. 1999). Two nonmutually exclusive hypotheses for the contribution of EBV to BL are (1) EBV may increase the chance of the c-myc translocation or (2) EBV may antagonize the cellular antioncogenic defenses. Interestingly, EBV-associated BL development is also associated with infections of the malarial parasite Plasmodium falciparum (Kafuko and Burkitt 1970) or HIV infection in the context of AIDS. The association with malaria and AIDS is suggestive of the contribution of immunosuppression and thus higher levels of virus to the development of BL. P. falciparum can induce reactivation of lytic EBV replication in EBV-infected BL cell lines and infected primary B-cells that may increase the pool of latently infected B-cells in which the c-myc translocation might occur (Chene et al. 2007). The contribution of the viral proteins and RNAs of EBV to the development of BL has been difficult to decipher. Paradoxically, the transforming proteins of EBV are not expressed in BL, reinforcing the fact that the primary oncogenic event of BL is the overexpression of c-myc. The presence of EBV within BL cell lines increases their resistance to cytotoxic agents and leads to increased tumorigenicity. Most BL express only EBNA1 and the EBERs. EBNA1, which is the expressed in almost all EBVpositive BL, is required for the maintenance of the viral episome within proliferating cells. Expression of EBNA1 in EBV-negative BL and expression of dominant-negative versions of EBNA1 in EBV-positive BL suggest that EBNA1 may also increase cell survival and inhibit p53-dependent apoptosis (Kennedy et al. 2003; Saridakis et al. 2005). A role for EBNA1 in inducing genetic instability has also been proposed (Gruhne et al. 2009). The expression of the EBERs has been reported to increase the tumorigenicity of BL cell lines (Komano et al. 1999; Ruf et al. 2000, 2005). Examining the viral genomes within BL has yielded interesting clues as to how the virus might contribute to the survival of the tumor cells. A series of deletions have been identified in up to fifteen percent of BL samples examined (Kelly et al. 2002, 2005). Although the exact coordinates of the deletion vary, nearly all remove the EBNA2 coding sequences. Such deletions produce viruses are defective in transformation and establishment of latency, suggesting that the selection for these deletions in BL is specific for the development of cancer rather than selection for increased fitness of the resulting viruses. Expression of RNAs capable of encoding BHRF1 and EBNA3 proteins from the Wp promoter has been detected in the deleted BL (Kelly et al. 2006). EBNA2-deleted episomes have increased expression of one of the viral Bcl2 homologues, BHRF1, that was previously only observed to be expressed during lytic replication. BHRF1, which has been found to be expressed

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during latency as well as during lytic replication, is a potent inhibitor of apoptosis (Kelly et al. 2009). This could be a critical inhibitor of myc-induced apoptosis following the chromosomal translocation. Both EBNA3A and 3C have been reported to downregulate the proapoptotic protein Bim, and deletion of EBNA3A and 3C results in increased sensitivity to cytotoxic agents (Anderton et al. 2008). EBNA3C also regulates MDM2/p53 and Rb function (Knight et al. 2005; Saha et al. 2009; Yi et al. 2009). Expression or roles of EBV miRNAs in BL development have not been established. In summary, it is clear that the primary event of BL development is the c-myc translocation. EBV may play a role in compensating for the translocation by inhibiting apoptosis. A number of functions of the viral proteins which may help in inhibiting apoptosis have been identified. EBV may also increase directly or indirectly increase the probability of translocation. The discrete roles of these EBV proteins as well as the interaction between EBV and other infectious agents will continue to be elucidated to determine the pathogenesis of EBV in BL.

EBV and Nasopharyngeal Carcinoma EBV was associated with NPC shortly after BL and is the cancer that is most highly associated with EBV. Like BL, NPC has a specific geographical distribution suggestive of specific genetic and/or environmental influences to the development of cancer. NPC is endemic to regions of China and South-East Asia. Incidence is particularly high in Cantonese males (Yu and Yuan 2002). Computation approaches have suggested that EBV latent proteins may be poorly recognized by the MHC haplotypes of the affected ethnicities (Edwards et al. 2004). In addition, the environmental cofactor of nitrosamines consumed in salted fish at an early age is thought to be an important contributing factor to the development of cancer (Huang et al. 1978; Yu et al. 1986). Increased IgA antibodies to the EBV viral capsid antigen (VCA) are useful in the diagnosis of the disease (Henle and Henle 1976). Unlike most of the other cancers associated with EBV, NPC results from latent infection of epithelial cells rather than B-lymphocytes. Although infection of epithelial cells by EBV was initially controversial, lytic infection of the epithelial cells, particularly in the oral mucosa, is now assumed to be important for the amplification of the virus in primary infection to establish a sufficient latent B-cell reservoir. Inefficient in vitro epithelial infection models have made it difficult to understand the factors that influence how or why latent infection is induced in epithelial cells. Genetic and epigenetic analyses have clearly defined several events that are required for the development of cancer. Loss or silencing of genes on chromosomes 3p, 9p, and 14q are responsible for increased susceptibility presumably through the loss of tumor suppressors p16IN4A, p14ARF, RASSF1A, and most recently a novel tumor suppressor, Mirror-Image POLydactyly 1 (MIPOL1) (Lo and Huang 2002; Cheung et al. 2009). Current models of NPC development suggest that following loss of

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these tumor suppressors EBV latently infects the epithelial cells and induces precancerous lesions. Following subsequent genetic and epigenetic events, the precancerous lesions develop into invasive and metastatic NPC (Raab-Traub 2002). Several EBV genes are expressed in NPC, which is the prototypical latency II profile. EBNA1, EBERs, LMP1, and LMP2A are consistently detected in EBVpositive NPC, although the LMPs are detected less often, especially at the protein level (Tsao et al. 2002a). LMP1 is assumed to drive the proliferation and induce some of the transformed phenotypes of the tumor cells. Expression of LMP1 in epithelial cells and EBV-negative NPC cell lines induces increased motility and invasion (Tsao et al. 2002b; Shair et al. 2008). LMP1-specific therapies are being developed and tested. Interestingly, the EBV miRNAs arising from the BART RNAs are highly expressed in NPC, although the function of these miRNAs and their role in NPC pathogenesis are unknown currently (Cai et al. 2006).

EBV and Hodgkin’s Disease Like BL and NPC, an infectious agent was suspected as a contributor to Hodgkin’s Disease (HD) and increased antibodies to EBV-specific antigens initially solidified this connection (MacMahon 1966; Levine et al. 1971). In situ hybridization established the presence of EBV DNA and EBER RNA in the tumors as well as the fact that the HD tumors were clonal for the EBV episome (Anagnostopoulos et al. 1989; Wu et al. 1990; Weiss et al. 1991). This suggests that the tumor arises from the expansion of a single transformed clone. Increased risk of HD is the only EBVassociated cancer correlated with a history of infectious mononucleosis (Gutensohn and Cole 1980). Association between EBV and HD varies by ethnic group, sex, and age (Armstrong et al. 1998). Generally, HD in children and older adults is associated with EBV, while HD in young adults is not associated as often with EBV. Although HD is only slightly increased in AIDS, the majority of them contain EBV (Uccini et al. 1990). HD has a latency II EBV gene expression pattern (Deacon et al. 1993). The malignant cells of HD, Hodgkin Reed–Sternberg cells, express LMP2A and/or the viral oncoprotein LMP1 in the absence of EBNA2 (Weiss et al. 1987, 1989; Pallesen et al. 1991; Murray et al. 1992). There is some correlation between the pathways and proteins activated by LMP1 in vitro and the EBV-positive HD, suggesting that LMP1 signaling is active in the tumor cells (Herbst et al. 1996; Durkop et al. 1999; Murray et al. 2001; Hinz et al. 2002). However, EBV-negative HD often has similar profiles, suggesting that the tumors may arise by alteration of the cellular environment by either cellular or viral methods (Knecht et al. 1999). In particular, NF-kB and possibly Akt appear to be activated and critical to the growth of both EBVpositive and EBV-negative tumors (Bargou et al. 1997; Morrison et al. 2004). Comparison of EBV-positive and -negative tumors has highlighted the importance of deregulation of specific pathways critical to the development of HD whether by viral or other means.

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EBV and Lymphomas in Immunosuppression A number of EBV-associated malignancies occur in the context immunosuppression. Lymphoproliferations that occur during immunosuppression as a result of therapies after organ transplantation are known as posttransplant lymphoproliferative disorders (PTLD) (Gulley et al. 1993; Capello et al. 2005; Gottschalk et al. 2005; Taylor et al. 2005). Similar tumors are observed in inherited immunodeficiency and in the context of AIDS (Ambinder 2001). The majority of the lymphomas in the context of immunosuppression has gene expression of Latency III/growth program and is analogous to cell lines formed as the result of immortalization of B-cells in vitro by infection with EBV. PTLD will often regress following removal of immunosuppression but aggressive non-Hodgkin’s lymphoma, which does not resolve, also can result. Like latency III, generally all of the proteins of EBV are expressed in immunosuppressed lymphomas. The latent membrane proteins, LMP1, LMP2A, and LMP2B, and EBV nuclear antigens, EBNA1, EBNA2, EBNA3A-C, and EBNA-LP, are all expressed and thought to drive the proliferation of the tumor cells in the absence of immune surveillance. The noncoding RNAs, EBERs and miRNAs, are also expressed, but their role in tumor growth and development has not been determined. As the viral proteins are hypothesized to induce the growth of these tumors, they likely would be the most sensitive to therapies that specifically target viral proteins.

EBV and Gastric Carcinoma Gastric carcinoma is the second most common carcinoma worldwide and 10% of the cancers contain EBV making the new cases of EBV-gastric cancer nearly 90,000 worldwide (Takada 2000). Like HD and NPC, gene expression in gastric carcinoma is latency II. EBNA1 is the only EBNA detected in gastric carcinoma. The EBERs and EBV lytic protein BZLF1 is also detected in the tumors. Detection of the LMPs in gastric carcinoma is variable and LMP2A is most often detected while LMP1 expression has also been reported (Imai et al. 1994; Ryan et al. 2009). As with BL and HD, there may be other factors that influence the association of EBV with the tumor.

EBV and Other Cancers EBV is also associated with other cancers. It is associated with some T-cell and NK-cell lymphomas (Jones et al. 1988; Kikuta et al. 1988; Harabuchi et al. 1990; Pallesen et al. 1993; Nagata et al. 2001; Lan et al. 2008). EBV is also associated with leiomyosarcoma (McClain et al. 1995). There have been reports of EBV in breast cancer and hepatocellular carcinoma (Bonnet et al. 1999;

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Sugawara et al. 1999). However, some of these associations are controversial and the necessary immunohistochemical and gene expression profiles have not been rigorously demonstrated.

EBV Animal Models Because of the clinical importance of EBV and the importance of developing EBVtargeted therapies, a number of EBV animal models have been developed. Nonhuman primates have yielded limited effectiveness. Old-World monkeys are not infected by EBV possibly due to presence of related cross-reacting antibodies from endogenous herpesviruses as detailed above. Several species of New-World monkeys can be infected with EBV; however, they cannot be infected via the natural, oropharyngeal route and fail to develop most EBV-related pathologies (Niedobitek et al. 1994). Although EBV does not infect mouse cells, a number of mouse models have been developed that mimic aspects of EBV disease. Tumor xenografts can be grown in nude or SCID mice. SCID mice injected with lymphocytes from seropositive donors can develop lymphoproliferative diseases similar to PTLD (Mosier et al. 1988). A number of knock-in mouse models have been developed in which the expression of latent viral proteins is targeted to specific cell types, including LMP1 (epithelial targeted (Wilson et al. 1990; Curran et al. 2001), LMP1 (B-cell targeted (Kulwichit et al. 1998; Uchida et al. 1999), LMP2A (Caldwell et al. 2000), and EBNA1 (Kang et al. 2005). Recently, humanized mice in which immunocompromised or immunodeficient mice, typically nude, SCID, or NOG mice, are populated with human immune stem cells and then used to study human adaptive and innate immune responses to EBV infection (Melkus et al. 2006). Each model system has offered unique insights into EBV biology and/or the pathogenesis of EBV-related malignancies but also fails to fully explain the complex interconnection between EBV and its host leading cancer development. Understanding the contribution of EBV to the development of human cancer helps scientists understand the nature of cancer and the pathogenesis of the disease. It also offers insights into the viral life cycle and how the virus uses the host cell to its advantage. The presence of EBV within the tumor also offers a unique pharmacological target in which the virus and thus the tumor cells may be subject of a therapy that does not affect normal cells and tissues.

KSHV or Human Herpesvirus-8 (HHV-8) and Kaposi’s Sarcoma KS was recognized as early as 1872 by the Hungarian dermatologist Moritz Kaposi as a rare “multiple idiopathic pigmented hemangiosarcoma” seen as purple blotches on the skin of extremities with some visceral involvement. This form

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of KS known as classical KS occurs most commonly in Eastern Europe and North America among older men of Italian or Jewish ancestry. However, in the 1950s and 1960s, another aggressive form known as endemic KS was recognized among younger individuals in central Africa and KS was reported to represent 9% of all neoplasms in Uganda (Tornesello et al. 2010 ; Buonaguro et al. 2003). Clinically, the African aggressive forms have been described as nodular, florid, infiltrative and lymphoadenopathic. The lymphoadenopathic form is usually seen in African children and young adults. The disease manifests mainly with the involvement of lymph nodes and is rapidly fatal. During 1970s, KS was also detected in the USA and in Europe before the AIDS epidemic era in patients receiving immunosuppressive therapy for renal transplantation and other medical conditions (Tornesello et al. 2010; Buonaguro et al. 2003). In transplant recipients, KS is reported to be the second most common tumor after lymphoma to arise and occurs more often (10 vs. 3% of all transplant-associated neoplasms) in patients who receive cyclosporin as part of their immunosuppressive regimen. Several studies support the idea that KS is a disorder of growth factor production (Boshoff et al. 1995; Boshoff and Weiss 1997, 1998, 2002). Even though HIV protein-tat involvement in KS is hypothesized to induce cytokines, it was recognized that this may not be the sole inciting event in KS etiology, since KS also occurs in the absence of HIV infection. Although KS is also manifested in a small number of HIV-positive transfusion patients and intravenous drug users, it is very rare in hemophiliacs, which indicated that the KS agent is cell-associated or that is inactivated during factor VIII purification. AIDS-KS is almost exclusively confined to homosexual men with AIDS, bisexual men and their contacts. Gay and bisexual men are about 20 times more likely than other HIV-positive patients to develop KS. This has suggested that a sexually transmitted cofactor, inefficiently transmitted by blood or blood products may be required for KS development (Chang and Moore 1996; Chang et al. 1996; Gaidano et al. 1996; Moore et al. 1996a, b). The occurrence of KS lesions in homosexual men without HIV infection supports this hypothesis. Using “representational difference analysis (RDA)”, Chang et al. (1994) reported the identification of a novel herpesvirus DNA sequences in AIDS associated KS. This unique DNA is closely related to HVS as well as to EBV. Currently, this new herpesvirus is called KSHV or HHV-8. KSHV DNA was present in more than 95% of AIDS-KS tissues, >15% of the non-KS tissue samples from AIDS patients (Chang et al. 1994). In KS patients, KSHV DNA was detected only in the skin near the KS tissue and in some adjoining lymph nodes, but not in other tissues. KSHV DNA was also detected in all the non-AIDS classical KS tissues from southern Europe and Africa (Chang and Moore 1996; Chang et al. 1996; Gaidano et al. 1996; Moore et al. 1996a, b). In addition, PBMC of AIDS patients obtained prior to the development of KS lesions was positive for KSHV DNA and was negative in matched HIV positive individuals without KS (Chang and Moore 1996; Chang et al. 1996; Gaidano et al. 1996; Moore et al. 1996a, b). These studies strongly suggested an association of KSHV with KS.

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Cells of KS Lesions KS lesions are characterized by proliferating spindle shaped endothelial cells, neovascular structures, inflammatory cells, and an abundance of inflammatory cytokines (ICs), growth factors (GFs), angiogenic factors (AFs), and invasive factors. Whether KS is a true malignancy or a well-characterized consequence of infection with unique hyperplasia is still a debatable subject. The discrepancy between KS and other tumors is more evident in the histopathology of the tumor. Most human tumors are derived from the clonal outgrowth of a single cell type. By contrast, KS lesions show a variety of cell types. Advanced lesions include a predominance of spindle-shaped cells, thought to be derived from cells in the endothelial or mesenchymal lineage (Wang et al. 2004). All KS lesions display a proliferation of aberrant, slit-like neovascular spaces as well as variable quantities of infiltrating inflammatory cells, including plasma cells, T cells, and monocyte/macrophages (Boshoff and Weiss 1998; Blauvelt 2001; Wang et al. 2004). KS tumors lack many of the features of classical tumor cells as they are often oligo- or polyclonal, lack a fully transformed phenotype in vitro, and do not form tumors upon implantation in nude mice (Boshoff and Weiss 1998). In vitro cultures of KS cells generally produce IL-6, granulocyte-macrophage colony-stimulating factor (GMCF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PGF), and basic fibroblast growth factor (BFGF) (Samaniego et al. 1998). The cells cultured from KS tumors almost always have normal diploid karyotype, grow for a limited number of passages in culture, and contain a mixture of different cell types. The dominant cell types are fibroblasts and endothelial cells, both of which have a spindle shape (Samaniego et al. 1998). These spindle cells are dependent upon exogenous growth factors and are not fully transformed by most laboratory criteria (Samaniego et al. 1998). They generally fail to grow in soft agar and do not induce tumors upon transplantation in immunodeficient mice. KSHV was found in peripheral blood CD19 positive B cells (Sin et al. 2010; Dittmer et al. 1999). Using in situ PCR hybridization, KSHV DNA was demonstrated in the flat endothelial cells lining vascular spaces of KS lesions as well as in typical KS spindle cells of all forms of KS (Chang et al. 1994). KSHV DNA does not persist in the cultures of KS tissues and the KS cell lines are not positive for KSHV DNA and the reason for this is not clear. KSHV encodes ~86 ORFs of which at least 22 are potentially immunomodulatory and antiapoptotic (Boshoff et al. 1995) and the ORFs are named based on their homology to HVS. In KS lesion endothelial cells, KSHV is in a latent form with about 10–20 copies of the viral episome per cell and lytic replication is observed in a low percentage of infiltrating inflammatory monocytes. Only a small proportion (<10%) of spindle-shaped endothelial cells are positive for KSHV in early KS lesions. In advanced KS lesions, KSHV is latently expressed in >90% of the tumor (spindle) cells, indicating that paracrine mechanisms are probably important in the initiation and progression of KS. In nodular (advanced) lesions, all the spindle cells contain latent KSHV (Boshoff et al. 1995), suggesting that KSHV latent proteins

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provide a growth advantage to the infected cells. KS spindle cells are referred to as the driving force of KS histogenesis (Ensoli et al. 1992).

KSHV and Primary Effusion Lymphoma PEL, a rare type of B-cell lymphoma detected in HIV+ and HIV− patients, is also called as body-cavity-based lymphoma (BCBL). PEL are presented as malignant lymphomatous effusions in the pleural, pericardial, or peritoneal cavities, usually without significant tumor mass or lymphadenopathy/lymph nodal involvement and these lymphomas occur predominantly in HIV-positive individuals with immunosuppression (Boshoff et al. 1998). KSHV viral DNA was first detected in patients during a survey of 193 lymphomas with and with out AIDS. KSHV sequences were detected in eight non-Hodgkin’s lymphomas (NHLs) in HIV infected individuals, all of which were EBV positive (AIDS-BCBLs) (Cesarman et al. 1995). These lymphomas appear to represent a distinct group of B cell NHLs, without c-myc rearrangement and with striking characteristics such as absence of most lineage-associated antigens that are distinct from the vast majority of AIDS-related lymphomas (Karcher and Alkan 1997). Only two of the eight patients with BCBLs had KS indicating that KSHV-associated BCBLs can occur independently of KS in patients with AIDS. BCBL cell lines containing only KSHV were isolated ,which suggests that KSHV may be involved in this NHL independently of EBV. Unlike KS, PEL has characteristics of a typical malignancy, with the B cell being of clonal origin. Circular KSHV episome is seen in every cell in the tumor. These cells grow well in culture and have been an ideal system to study KSHV replication in vitro. PEL has poor prognosis and median survival of approximately six months (Carbone and Gloghini 2007), and there are no specific treatments targeting PEL. PEL is characterized by the expression of KSHV latency proteins LANA-1 (ORF73), vCyclin (ORF72), v-FLIP (ORF71), Kaposins (ORFK12), LANA-2, K1 and vIL-6 as well as >18 miRNAs with 1–5% of cells spontaneously entering lytic cycle (Chandriani and Ganem 2010 ; Jenner et al. 2003; Klein et al. 2003; Carbone et al. 2009).

KSHV and Multicentric Castleman’s Disease Multicentric Castleman’s Disease (MCD) is an atypical lymphoproliferative disorder (ALPD) defined using clinical and pathologic criteria. MCD’s association with KS was observed during the clinical course of HIV infection. Soulier et al. (1995) reported the presence of KSHV-like DNA sequence in MCD. This is polyclonal involving multiple lymphoid organs. KSHV-associated MCDs can occur independently of HIV infection and KS. KSHV-associated MCD is the highly indolent systemic variant of Castleman’s disease (CD) and is also designated as plasmablastic variant of MCD (Dupin et al. 1999). It is a lymphoproliferation accompanied by

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weight loss and fever and is more commonly diagnosed in HIV-infected patients and has very poor prognosis (Chadburn et al. 1997; Dupin et al. 1999). Unlike KSHV-positive PEL cells, the plasmablasts in MCD are KSHV-positive and EBV negative.

KSHV Biology In vivo KSHV infects variety of target cells including human B cells (Chandran 2010; Mesri et al. 1996; Akula et al. 2001), macrophages, endothelial cells, keratinocytes and epithelial cells. Several B-cell lines have been established from PEL tumors bearing either KSHV alone (BCBL-1 and BC-3) or KSHV and EBV (BC-1, JSC-1, HBL-6). KSHV has also been shown to infect a variety of cell types in vitro including human B, endothelial, epithelial, and fibroblast cells as well as variety of animal cells such as owl money kidney cells, baby hamster kidney fibroblast cells, Chinese hamster ovary cells (CHO) cells and mouse fibroblasts. Infection of human primary B cells by EBV results in the establishment of latent infection, transformation, and B-lymphoblastoid cell lines. By contrast, primary nonstimulated B cells are poorly infected with KSHV and infection does not lead to immortalization. Instead, a lytic replication is reported in activated B cells. In vitro KSHV infection of adherent target cells does not result in a productive lytic cycle (Naranatt et al. 2004). Instead, KSHV infection of human endothelial cells (human microvascular dermal (HMVEC-d), human umbilical vein endothelial cells (HUVEC), human foreskin fibroblasts (HFF), human endothelial cells immortalized by telomerase (TIME), and human embryonic kidney epithelial cells (HEK 293) results in the expression of latency-associated genes and thus providing a reasonable model for studying latency in vitro (Naranatt et al. 2004). Lytic replication could be induced from these cells by chemicals or by the lytic switch KSHV-ORF 50 (RTA- replication and transcription activator). In vitro, the switch from latency to lytic can be achieved by treating latently infected cells with phorbolesters or sodium butyrate or by constitutively expressing RTA. The stimuli for in vivo lytic activation are not well understood and there are evidences for cytokine mediated lytic cycle activation.

KSHV Latent Proteins and Oncogenesis Like EBV, KSHV’s latency program is directly linked to oncogenesis due to its antiapoptotic role and by increasing cell survival (Table 7.2). The lytic cycle was presumed not to contribute to oncogenesis as the cells that enter lytic cycle die. One of the major lytic gene vGPCR coded by ORF74 was demonstrated to have oncogenic properties (Table 7.2). However, in situ hybridization studies have shown only 1–3% of latently infected spindle cells express lytic genes, thus supporting the importance of latent gene expression in tumor development.

Latency

Latency

Latency and Lytic replication

ORF71 (vFLIP/K13)

ORF72 (v-Cyclin)

ORFK12 (Kaposin)

Reported to be present in most spindle cells of all stages of KS and in PEL KSHV-transforming gene Multiple transcripts of various lengths being produced from K12 locus resulting in Kaposin A, B, and C forms

Structurally similar to cellular D-type cyclins and forms an active kinase complex with cellular CDK6 kinase Stimulate the expansion of KSHV-infected cells

Blocks KSHV lytic replication

Table 7.2 KSHV proteins with potential involvement in oncogenesis Gene Expression Proposed functions in KSHV life cycle ORF73 Latency Tethering viral episome to the host chromosome (LANA-1) KSHV episome replication/segregation Associates with a host cell DNA polymerase and RNA helicase

(continued)

Possible cancer contribution Inactivates pRb and p53 proteins Interacts with GSK3-ß, promotes nuclear ß-catenin accumulation Activates aberrant c-Myc stabilization Cooperates with H-Ras to transform fibroblasts Complexes with P53 in PEL cells Stabilizes intracellular activated Notch protein Inhibits TGF-ß signaling Associates with a number of cell cycle proteins and DNA methyltransferases (Dnmts) Transcriptional coactivator of c-Jun Drives cell transformation in vitro Can inhibit the extrinsic apoptosis pathway by preventing the activation of caspases Involved in constitutive NF-kB activation through the IkB kinase (IKK) complex Contributes to PEL cell survival Extends lifespan of spindle cells Provides the inflammatory phenotype to KS lesions. Cooperates with Myc to promote lymphoma Suppresses autophagy Suppresses CXCR4 by upregulating miR-146a Regulates the transcription of S-phase genes Stimulates quiescent fibroblast cells to enter S-phase Can phosphorylate CDK2 substrates such as ORC1, CDC6, p27Kip1, histone H1, Bcl-2 and p53 Activates p38/MK2 pathway and to stabilize various cytokine mRNA containing AREs Kaposin A can lead to cell transformation in vitro

Lytic

Latency

K1

v-BCL2

v-GPCR (ORF74)

Lytic

ORF-K2 (v-IL6) Lytic/latent

Latency

miRNAs

Table 7.2 (continued) Gene Expression

Expressed in KS lesions and in cell lines derived from PEL Plays an antiapoptotic role in virus infected cells Expressed in only a few cells of KS lesions, but in a greater number of cells in PEL and MCD lesions Early lytic gene vIL-6 play important roles in KSHV replication and pathogenesis Characterized as viral oncogene Likely to play a critical role in both the initiation and promotion of KS tumor development Can activate the promoter of the lytic cycle switch ORF50

18 mature miRNAs have been detected so far in latently KSHV-infected cells miR-K3 can suppress both viral lytic replication and gene expression miR-132 regulates antiviral innate immunity Contribute to viral-induced reprogramming by silencing the cellular transcription factor MAF Deletion of a 14 miRNA cluster from the KSHV genome significantly enhances viral lytic replication as a result of reduced NF-κB activity K1 is expressed at very low levels in KSHV-infected cell lines Induced during lytic replication

Proposed functions in KSHV life cycle

Possible cancer contribution

Powerful signaling capacity Possesses a cytosolic ITAM motif and mimics signaling through the B-cell receptor (BCR) Activates tyrosine kinases Lyn and Syk, as well as PLC-g2 and vav Akt activation Angiogenesis and inflammation Important regulator of cell death Interacts with cell survival proteins Enhances cell transformation Mediates signaling through the gp130 signal transducer Can stimulate the growth of KSHV-infected lymphoma cells Promotes hematopoiesis Acts as an angiogenic factor through the induction of VEGF Creates tumor friendly milieu through paracrine mechanisms Secretion of chemokines and growth factors Activates mitogen- and stress-activated kinases, and induces transcription via multiple transcription factors including AP-1, CREB, NFAT and NFκB Causes cellular transformation in vitro and leads to KS-like tumors in transgenic mouse models Mediates angiogenesis, and inflammation

Inhibit p21 and attenuates cell cycle arrest miR-K12-11 upregulates xCT expression and increases cell permissiveness for KSHV infection and protects infected cells from death induced by reactive nitrogen species (RNS) Induce IL-6 and IL-10 secretion by macrophages and monocytes

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LANA-1 (ORF73). KSHV LANA-1 expressed during latency in KS, MCD, and PEL cells is a large nuclear phosphoprotein with no known mammalian homolog (Rainbow et al. 1997). It binds the KSHV terminal-repeat (TR) regions and tethers the viral episome to the host mitotic chromosome through interactions with cellular chromosome-associated proteins (Cotter and Robertson 1999). LANA-1 has similarities to EBNA-1, which include the presence of central repeat region of variable length composed of acidic amino acids, essential roles in viral episome maintenance and segregation. LANA-1 has been shown to perform multiple functions which contribute to KSHV-associated neoplasia-like inactivation of pRb and p53 proteins (Fujimuro et al. 2003), upregulation of ß-catenin, and stabilization of its expression by sequestering its inhibitor, glycogen synthase kinase 3-ß. LANA has been shown to physically interact with cellular proteins, including p53, pRB, RING3, histone H1, ATF4/ CREB2, and members of the mSin3 corepressor complex. LANA interacts with GSK3-ß, leading to the accumulation of cytoplasmatic and nuclear ß-catenin (Fujimuro et al. 2003), activation of the Tcf/Lef transcription factors and aberrant c-Myc stabilization (Bubman et al. 2007). LANA-1 can cooperate with H-Ras to transform fibroblasts (Radkov et al. 2000). LANA-1 is also referred to as the guardian of KSHV latency, as it modulates several cell-cycle pathways of infected cells. vFLIP (K13/ORF71). v-FLIP is a homologue of the cellular protein FLIP and the vFLIP gene is expressed in late stage KS lesions and PEL cells from a polycistronic mRNA encompassing the latency locus containing an internal ribosome entry site (IRES) located within the v-cyclin ORF (Thome et al. 1997; Boshoff et al. 1998; Talbot et al. 1999). KSHV FLIP protein is an important component of KSHV pathogenesis. vFLIP protein has the ability to drive cell transformation in vitro (Sun et al. 2003) and can inhibit the extrinsic apoptosis pathway by preventing the activation of caspases (Djerbi et al. 1999). Its ectopic expression can lead to constitutive NF-kB activation through the IkB kinase (IKK) complex (Chaudhary et al. 1999; Keller et al. 2000) and can contribute to survival of PEL cells (Godfrey et al. 2005; Guasparri et al. 2006). v-FLIP contains death-effector domains which interact with the adapter protein FADD, inhibiting the recruitment and subsequent activation of the protease FLICE by the CD95 (Fas) death receptor and protects cells from Fas, TNFR-1, and TNF-related apoptosis-inducing ligand receptor (TRIAL-R)-mediated apoptosis. v-FLIP expression in B-cells leads to development of tumors in mice by preventing death receptorinduced apoptosis triggered by CTL immunosurveillance. Expression of v-FLIP in spindle cells has been shown to extend their lifespan (Chugh et al. 2005) and provides the inflammatory phenotype to KS lesions. Finally, activation of NF-kB by v-FLIP has been shown to oppose lytic reactivation that thereby stabilizes KSHV latency. vCyclin (ORF72). KSHV-encoded v-cyclin is a human cyclin D homolog (closely related to cyclin D2). v-cyclin forms a ternary complex with CDK6 and INK4. Distinct from cellular cyclins, v-cyclin protein has longer half-life. It activates the cyclin-dependent kinases (CDKs) CDK4 and CDK6, thus phosphorylates pRb, releases the repression on E2F and that subsequently upregulates the transcription of S-phase genes, whose products modulate the progression of cells from G1- to S-phase (Kang and Lieberman 2009). Furthermore, unlike cellular D cyclin/CDK6 complexes, KSHV-cyclin/CDK6 activity is resistant to inhibition by the CDK inhibitors

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(CKI) p16, p21, and p27 (Swanton et al. 1997). v-Cyclin/CDK6 expression can also phosphorylate histone H1, the CDK2 substrates (p27KIP1, Id-2, cdc25a, E2F-4), replication proteins (orc1 and cdc6), and thus allows cell to overcome cell cycle arrest. Ectopic expression of v-cyclin in quiescent fibroblasts prevents CDK inhibitors (CKIs; p16INK4a, p21CIP1 and p27KIP1)-imposed G1 arrest and stimulates entry into S-phase (Sharp and Boshoff 2000). Interestingly, v-Cyclin has also been reported to trigger apoptosis in cells with elevated levels of CDK6 probably by phosphorylating and inactivating the cellular antiapoptotic factor Bcl-2. Kaposin. Kaposin or K12-containing mRNAs have been reported in most spindle cells of all stages of KS and in PEL cells during latency and lytic replication (Staskus et al. 1997; Sturzl et al. 1997). Translational regulation of the K12 transcript is very complex and yields a minimum of three protein species (Kaposin A, Kaposin B, and Kaposin C), of which Kaposin B is the most abundant in BCBL-1 cell lines and is encoded by the sequences upstream of K12, but not by K12 itself. Kaposin B is a scaffolding protein and has been shown to perform immunomodulatory functions such as increasing the expression of certain cytokines by stabilizing their mRNAs (McCormick and Ganem 2005) via p38 MAPK pathway and inhibiting the decay of AU-rich elements (AREs) in 39-untranslated regions of mRNAs. Recently, kaposin locus was shown to code for KSHV micro-RNAs (Cai et al. 2005; Pfeffer et al. 2005; Samols et al. 2005, 2007), which may be involved in the regulation of viral and cellular immune responses. Kaposin C is a chimera of direct-repeat sequences DR1, DR2, and K12. Kaposin A is the predicted product of K12, and has been reported to be involved in cell transformation (Muralidhar et al. 1998, 2000; Tomkowicz et al. 2005). Kaposin A is a 60-aa transmembrane protein whose overexpression in fibroblasts can lead to their transformation in vitro, suggesting that the molecule can stimulate signaling pathways linked to growth deregulation (Muralidhar et al. 1998). The mechanism of Kaposin-mediated transformation is not clear but has been linked to its ability to bind cytohesin-1 (Moss and Vaughan 2002), an exchange factor for ADP ribosylation factor (ARF) family GTPases, key regulators of vesicular trafficking and of the dynamics of the actin cytoskeleton (Moss and Vaughan 2002). KSHV miRNA. miRNAs are small noncoding RNAs of ~22 nucleotides expressed by virtually all multicellular organisms and are known to regulate gene expression either at posttranscriptional level by binding to 3’UTRs of target and/or repression of their translation. miRNAs have been shown to regulate cellular differentiation, proliferation, apoptosis, metabolism, and immune responses. The KSHV miRNAs are transcribed during latency and their expression has been confirmed in KS but their functions are not yet fully characterized. The kaposin transcription unit of KSHV encodes 12 pre-miRNAs (Cai et al. 2005; Pfeffer et al. 2005; Samols et al. 2005; Ziegelbauer et al. 2006, 2009; Ganem and Ziegelbauer 2008) and these pre-miRNAs can engender 18 mature miRNAs (Umbach and Cullen 2010). Some of these miRNAs appear to function as modulators of the latent-lytic switch (Bellare and Ganem 2009; Ziegelbauer et al. 2009). KSHV miRNA (miRK12-5) has been identified to be capable of suppressing ORF50 mRNA probably by decreasing histone H3 K9 methylation or increasing

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histone H3 acetylation, and a striking loss of DNA methylation throughout the viral and cellular genome (Ablashi et al. 2002). KSHV miRNA cluster as a genetic element activates CpG methylation through an indirect mechanism involving modulation of DNA methyltransferase (DNMT) gene transcription regulation that helps maintain the latent state of the viral chromosome (Ablashi et al. 2002). Recent study by Lei et al. (2010) showed that the deletion of a 14 miRNA cluster from the KSHV genome significantly enhanced KSHV lytic replication as a result of reduced NF-kB activity. Expression of miR-K1 has been indicated to efficiently rescue NF-kB activity and inhibit viral lytic replication, whereas inhibition of miR-K1 in KSHV-infected PEL cells displayed the opposite effect. Recent reports identified few potential KSHV miRNA targets as thrombospondin (THBS; a major regulator of cell adhesion, migration, and angiogenesis), BRCA1associated C-terminal helicase (BACH1; DNA repair protein supporting BRCA1 damage response), leucine zipper transcription factor MAF (musculoaponeurotic fibrosarcoma oncogene homolog), and BCLAF1 (Gottwein and Cullen 2010; Samols et al. 2007; Skalsky et al. 2007; Ziegelbauer et al. 2009), which primarily control viral reactivation, apoptosis, cell survival, immune responses (Umbach and Cullen 2010), cellular differentiation, and transcriptional reprogramming of host cells. The KSHV miR-K12-11 seed region shares complete homology to that of hsa-miR-155, a multitasking immune-specific miRNA controlling B cell maturation, lymphomagenesis, transformation and cancer (Qin et al. 2010; Gottwein et al. 2007; Samols et al. 2007; Zhao et al. 2009). High levels of the precursor miRNA mir-24 recently emerged as a biomarker in patient derived KS samples (O’Hara et al. 2009). Cellular mRNAs encoding the cellular cyclin-dependent kinase inhibitor p21, a key inducer of cell cycle arrest has been shown to be direct target for KSHV miR-K1. Ectopically, expression of KSHV miR-K1 specifically inhibits the expression of endogenous p21 in KSHV-negative cells and attenuates the cell cycle arrest that normally occurs upon p53 activation suggesting that KSHV miR-K1 is likely to contribute to the oncogenic potential of KSHV.

Cytokines and Angiogenic Factors in KS A marked characteristic of KS is the infiltration of inflammatory cells accompanied by inflammatory cytokines, angiogenic factors, growth factors, and chemokines, making it an angioproliferative lesion. The importance of some of these cytokines and angiogenic factor in establishment of latency and ultimately in tumor progression is discussed here (Fig. 7.2). KSHV induced proinflammatory cytokines. A variety of ICs are produced in lesions from all forms of KS. These include, IL-1b, IL-6, IL-8, Cox-2, TNF, IFNg. IL-1 produced and released by endothelial cells in a KS lesion induces autocrine growth of the endothelial cells. Studies have also shown that their growth effects are mediated by induction of bFGF production which appears to be the final mediator of KS cell growth (Ensoli et al. 1994; Samaniego et al. 1995, 1997; Ensoli and Sturzl 1998;

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Late phase of signal pathway activation Secretion of cytokines and growth factors

PI3K

Early phase of signal pathway activation Activation of NF-kB, ERK1/2

p38 MAPK

AKT

NF-kB, ERK1/2

Stabilization of Cytokine mRNA

Cell Proliferation /anti apoptosis Induction and regulation of host genes

Concurrent persistent latent and transient lytic gene expression

Tube formation

K12 Anti-inflammatory Chemokines cytokines Growth and Pro-inflammatory angiogenicfactors cytokines

Maintenance of latency LANA, vFLIP, vCyclin, K12, mRNA

Autocrine/Paracrine action

Cell migration

Dissolution of basement membrane

Plasmin generation

Fig. 7.2 Model depicting the early and late phases of KSHV infection of HMVEC-d cells, transcription factor regulation, establishment and maintenance of infection, and cytokine secretion. Virus binding and entry lead to signal pathway induction, such as FAK, Src, PI 3-K, AKT, PKC-ζ, MAPK-ERK1/2, and NF-κB signal molecules which coincides with viral-DNA entry into the infected-cell nuclei, concurrent transient expression of limited viral lytic genes, and persistent latent gene expression. Expression of several host genes including cytokines, growth factors, transcription factors etc., is initiated by AP-1, ERK1/2 and NF-κB. Released host factors act in autocrine and paracrine fashions on the infected, as well as neighboring, cells. The autocrine action of these factors, along with viral gene expression, probably contributes to the late phase of signal pathway activation including sustained NF-κB activation and phosphorylation of p38 MAPK, ERK1/2, and AKT required for the maintenance of latency

Faris et al. 1998). IL-1b increases the expression of adhesion factors on endothelial cells to enable transmigration of leukocytes. IL-1b dependent NF-kB activation mediates PGE2 release via the expression of COX2 and PGE2 synthase (Catley et al. 2003). KSHV mediated COX2 expression is critical for PGE2 release, which in turn is responsible for the maintenance of latency (George Paul et al. 2010; Sharma-Walia et al. 2010), IL-1b could be a feed back loop factor responsible for COX-2 induction via NF-kB. Latency protein vFLIP is known to induce COX2 expression (Matta et al. 2007) and recent reports have shown KSHV induced COX2 to be a key factor in latency inflammation, angiogenesis and cell survival (Sharma-Walia et al. 2010). IL-6 is an autocrine growth factor known to be over expressed in KS tumors, PEL, MCD (Miles et al. 1990; Yang et al. 1994) and is implicated in the growth and

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survival of PEL cells (Asou et al. 1998). IL-6 is known to be regulated both by AP-1 and NF-kB (An et al. 2003; Xie et al. 2005). LANA-1 and vFLIP are known to induce IL-6 secretion via the activation of AP-1 or NF-kB (An et al. 2003, 2004). IL-8 is another chemokine expressed in KS lesions, by cultures endothelial cells and by IC activated endothelial cells. IL-8 is a chemoattractant for neutrophils and stimulates angiogenesis and tumor growth (Koch et al. 1992; Sparmann and BarSagi 2004). In addition, IL-8 has prominent role in endothelial cell migration, it might also contribute to the angiogenesis found in the KS lesion. Recent studies have shown that IL-8 plays a pivotal role in pathogenesis of KS and vFLIP mediates this IL-8 production (Sun et al. 2006; Wilson et al. 2008). IFNg mediates several immunomodulatory functions including recruitment of leukocytes to the site of infection, increased inflammation and the regulation of Th-2 response. IFNg mediates an antiviral activity by decreasing the NF-kB activation in CMV infected cells (Sedger et al. 1999), whereas KSHV appears to induce IFNg for its benefit. The administration of IFN and IL-2 has resulted in KS progression or onset (Monini et al. 1999). IFNg induces the endothelial cells to acquire the same feature of KS cells, which includes the KS spindle morphology and the specific cell marker expression (Fiorelli et al. 1998). KSHV induced growth factors. Leptin is circulating pleotropic hormone involved in the regulatory process of immunity, inflammation hematopoiesis and angiogenesis. Administration of leptin in vivo and in vitro leads to the stimulation of proliferation and angiogenesis of endothelial cells. Leptin is known to stimulate VEGF secretion in endothelial cells (Misztal-Dethloff et al. 2004; Naranatt et al. 2004). Leptininduced IkBa degradation in astrocytes potentiates the release of IFNg, TNFa and IL-1b (Raso et al. 2002). It is also known to stimulate the release of IL-6, TNFa, and prostaglandins via NF-kB and ERK1/2 pathway. Hence, it is evident that one cytokine induced by KSHV could initiate the secretion of various other cytokine and growth factors, leading to the activation of signaling pathway responsible for the establishment of infection and tumor progression. PDGF is one of the numerous growth factors that regulate cell proliferation and survival. PDGF plays a role in embryonic development, cell proliferation, cell migration and angiogenesis. PDGF-b is a potent paracrine-acting mitogen for cultured endothelial spindle cells that is expressed in vivo by subpopulation of cells that are intermingled with the spindle cells. Endothelial cells express PDGF b-receptor suggesting that PDGF-b may activate the proliferation of KS spindle cells by paracrine mechanism. PDGF has also been reported as an autocrine growth factor in KS mRNA for angiogenic factor receptors such as VEGF receptor (VEGFR 1 and 2), the angiopoietin receptors Tie1 and Tie2, and the PDGFR b were reported to be upregulated by vGPCR induction (Jensen et al. 2005). GMCSF is a pleotropic cytokine that induces the differentiation and proliferation of granulocyte and macrophage precursor cells. GMCSF is produced by spindle endothelial cells and by inflammatory cells in a KS lesion. GMCSF is known to influence the proliferation and migration of endothelial cells and enhance the production of IL-8 and MIP-1a. GMCSF can be also stimulated via NF-kB pathway by IGF and PDGF, which were also upregulated upon KSHV infection. GMCSF was thought to

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contribute to differentiation of monocytes into macrophages and dendritic cells that are found in KS lesions (Clark and Kamen 1987). Hence, a controlled interplay is seen between these cytokines and is probably regulated by KSHV for its own benefit. KSHV induced angiogenic factors. Angiogenesis, which is the development of new blood vessels from preexisting vasculature, is fundamental to tumor growth. Such neovascularization may be stimulated by factors released from tumor cells, tumorassociated inflammatory cells, or the extra cellular matrix (Loffek et al. 2006). These factors include VEGF, angiogenin, basic fibroblast growth factor (bFGF) family members, TGF-b, and E-cadherin. Among angiogenic factors, VEGF-A is the bestcharacterized positive regulator, with its distinct specificity for vascular endothelial cells. VEGF-A is a proangiogenic molecule and has been reported to play a crucial role in the development of KS and PEL, and may potentially be involved in multicentric Castleman’s disease. VEGF-A stimulates endothelial cell proliferation, migration, differentiation, tube formation, increased vascular permeability, and maintenance of vascular integrity. VEGF-A has been identified in KS lesions, and VEGF-C has gained attention because of the presence of its receptor VEGFR-3 in KS lesions and its ability to induce lymphangiogenesis. Coexpression of VEGF-A and bFGF has been shown in AIDS-KS and classic KS lesions, and the production of these factors is believed to be induced synergistically by inflammatory cytokines. Angiogenin can affect vascular endothelium directly and can facilitate the role of other angiogenic growth factors, which are elevated significantly during disease progression in patients with carcinoma. Angiogenin a multifunctional protein was observed to be upregulated in KS lesions (Sadagopan et al. 2009) and PEL tissues. Angiogenin translocates into the nucleus in endothelial cells and helps in rRNA transcription. The increased secretion of angiogenin could regulate a KS tumor, which is predominated by endothelial cells. Angiogenin was found to be upregulated only in KSHV-associated lymphomas and not in EBV-associated lymphomas signifying its role in KSHV mediated pathogenesis. Angiopoietin-2 (ang-2) mRNA was observed in KS lesions by insitu hybridization, and its levels are upregulated in KS as determined by gene expression microarray analysis (Wang et al. 2004; Bureau et al. 2006). Ang2 levels are increased in the plasma of individuals with KS, its expression is known to increase with increasing number of lesion and was observed to reduced upon antiretroviral therapy when KS resolves. SDF-1 (Stromal derived factor-1) is a chemokine produced by stromal cells as well as a variety of cells. SDF-1 promotes hematopoietic cell movement and cancer cell metastasis. It acts as a critical regulator of cell recruitment from the blood stream to specific tissues by promoting transendothelial migration through chemokine gradients across the endothelium. VEGF is known to promote constitutive SDF-1 expression. Once present on the surface of endothelial cell, SDF-1 supports the lymphocyte arrest and promotes the recruitment of KSHV infected cells to the skin (Yao et al. 2003). Leptin is known to induce VEGF and VEGF induces SDF-1 secretion. Hence, there exists a loop between these cytokines that is magnificently modulated by KSHV probably for its advantage.

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Cell cultures composed of characteristic spindle-shaped tumor cells have been established from KS lesion explants by the addition of cytokines such as TNFa, TNFb, IFNg, IL-1, IL-6, GMCSF, and oncostatin M, highlighting the role of these paracrine factors in KS lesion cell survival. It is believed that KSHV tumorigenesis and disease progression are predominantly driven by both paracrine and autocrine mechanisms, where KSHV infection could induce an angiogenic growth factor and MMPs-rich microenvironment and a strong cytokine network. These events via their synergistic actions and communications could support continued proliferation and migration of KSHV latently infected cells (Sharma-Walia et al. 2010; Dupin et al. 1999; Qian et al. 2007). There seems to be a tight network existing between several cytokines during KSHV pathogenesis, which is orchestrated by the latency proteins including LANA, vFLIP, and Kaposin.

KSHV Animal Models The experimental barrier to working with KSHV has been the lack of suitable animal model. KSHV goes into latency upon infection and establishment of latency is a complex phenomenon, which is yet to be fully understood. There are multiple cytokines playing important roles both during latent and lytic phase of infection, the exact combination of cytokines in the milieu is not known. Hence, it is difficult to develop an animal model where the virus upon infection goes into latency and with a known trigger switches to lytic cycle. Lytic cycle activation could be induced by addition of agents such as phorbol esters and is limited only to in vitro cell culture model and cannot be extrapolated in vivo animal model systems. Xenograft models of KSHV lymphoma have been able to mimic tumors seen in humans. Initial studies on the establishment of PEL-like tumors was done using KSHV+, EBV- cell lines BCP-1 and KSHV+, EBV+ cell lines HBL-6, PEL cells in nonobese diabetic/severe combined immunodeficiency disease Nod/SCID mice (Boshoff et al. 1998). In this study, all mice injected intraperitoneally with either BCP-1 or HBL-6 developed lymphomatous effusion similar to PEL, while all mice injected subcutaneously with HBL-6 formed solid tumor, but only 1 of 4 injected with BCP-1 formed solid tumor, thus providing evidence about the importance of the route of injection in the development of a suitable animal model. These studies opened up a new area of investigation on xenograft model, and lead to the understanding of the involvement of tumor microenvironment (Staudt et al. 2004) and host signal molecules (Keller et al. 2006) in the development of lymphomas by other investigators. Dittmer et al. 1999 (Dittmer et al. 1999) used SCID-hu Thy/Liv mice reconstituted with the liver and thymus of human fetuses to study viral transcription as well as the susceptibility of the mice to infection with BCBL-1 derived KSHV. In addition, Parsons et al. (2006) investigated the immune response to KSHV by implanting NOD/SCID mice with functional human hematopoetic tissue grafts. Furthermore, they have shown that NOD/SCID mice infected with purified KSHV provide a system for demonstrating latent and lytic replication.

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Besides these lymphoma animal models, KS animal models were also developed. Yang et al. (2000) reported that transgenic mice expressing the KSHV- encoded chemokine receptor (viral G protein-coupled receptor) vGPCR within hematopoietic cells develop angioproliferative lesions in multiple organs that morphologically resemble KS lesions. Grisotto et al. (2006) used doxycycline inducible vGPCR expressing system and demonstrated that vGPCR expressing cells accumulated in areas where angioproliferation was observed. KSHV vGPCR expression was sufficient to induce angioproliferative tumors that resembled human KS (Montaner et al. 2003). Guo et al. (2004) produced ORF74 transgenic mice and showed that these mice develop tumors resembling KS on the tail or legs. The tumors were reported to be highly vascularised with characteristic CD31+ve spindle shaped cells expressing VEGF-C. ORF74 expressing SV-40 T antigen immortalized murine endothelial cells when injected into nude mice produced KS-like tumors (Montaner et al. 2006). Mutlu et al. (2007) transfected KSHV bacterial artificial chromosome (KSHVBAC36) into mouse bone marrow endothelial-lineage cells, generated mECK36 that formed KS-like tumors. In nude mice, mECK36 cells formed vascular spindle cell sarcomas that displayed KSHV and host transcriptomes reminiscent of KS. They further demonstrated that siRNA suppression of KSHV vGPCR inhibited angiogenicity and tumorigenicity. In a recent study, common marmoset, a New-World primate, injected intravenously with KSHV seroconverted and maintained high antibody titers for more than one year. Infection of common marmoset with rKSHV.219 via the oral route developed a KS-like skin lesion with the characteristic spindle shaped cells along with small blood vessels (Chang et al. 2009). Although animal model studies have provided useful insight into the understanding of the tumors and had been instrumental in the identification of possible therapeutic targets, mechanisms of latency establishment and switch from latency to lytic cycle still remain unanswered. The plausible reasons for this could be that the immune response in human is not completely identical to mouse system and apart from the physical condition, emotional status and stress-related response in human might be a contributing factor. The viruses are more adapted than the human race; they tend to acclimatize themselves to peacefully coexist in the host, establishment of latency, tumorigenicity and cell death are mechanisms adopted by the virus for a successful infection. Counteracting million years of virus evolution need intense research and immense understanding of the virus.

Marek’s Disease Virus Marek’s Disease Virus (MDV) or Gallid herpesvirus 2 (GaHV-2) is a highly contagious herpesvirus whose infection affects predominantly chickens as well as other avian species such as turkeys, pheasants, quail, and game fowl worldwide. Mortality rates can be very high in susceptible birds. MD can develop in chickens as young as 3 week of age which is characterized by the T cell lymphoma infiltrating the nerves and organs resulting

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in enlarged nerves and tumors in nerve, organ, muscle and epithelial cells leading to paralysis of legs, wings, and neck, loss of weight and vision impairment. Following lytic infection, latency is established mainly in activated CD4+ T cells, which may be transformed with differing efficiencies, depending on the genotype of the infected chicken, and result in lymphoma formation. Irrespective of the transformation event, infection of feather follicle epithelial cells in the skin by migrating lymphocytes leads to the production of infectious particles that are shed into the environment, providing a continuous source of infectious virus. Though MDV is a highly cell-associated, it is readily transmitted usually via respiratory tract. The mechanism by which MDV transforms cells is not clearly understood. Nevertheless, vaccination developed in 1970 has been used successfully as the central strategy for the prevention and control of MD. Since vaccination prevented the clinical disease, MDV vaccine perhaps can be considered as the first cancer vaccine. The earlier vaccine contained the antigenically similar turkey herpesvirus, which is serotype 3 of MDV. While vaccination prevented clinical disease and reduced shedding of infective virus, it did not prevent infection. This resulted in the evolution of MDV with increased virulence and resistance to this vaccine. Current vaccines use a combination of vaccines consisting of HVT and gallid herpesvirus type 3 or an attenuated MDV strain. For additional information, please look up the chapter by Parcells and Morgan of this book, which deals exclusively on MDV and T cell lymphomas. The above summary will be a starting point to understand EBV ad KSHV role in cancers, and the readers will benefit from the various chapters of this book detailing the mechanism of oncogenesis by herpesviruses.

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Chapter 8

Lymphocryptoviruses: EBV and Its Role in Human Cancer Santosh Kumar Upadhyay, Hem Chandra Jha, Abhik Saha, and Erle S. Robertson

Lymphocryptoviruses: Introduction Lymphocryptovirus (LCV) or g1-herpesvirus is a genus of g-herpesvirinae that along with Rhadinovirus or g2-herpesviruses constitutes this subfamily of the herpesviridae family. The herpesviridae family is composed of three subfamilies: a-herpesvirinae, b-herpesvirinae, and g-herpesvirinae. Originally, these subfamilies were created based on their distinct biological properties such as cell tropism; however, the present classification depends on the genomic properties of each viral species (Roizmann et al. 1992; Lacoste et al. 2010). Initially, LCVs were thought to be restricted to human and Old-World primates, whereas Rhadinoviruses were considered to be inhabitants of new-world primates. In addition, geographical severance was considered to be the reason behind their evolutionary differences. However, this paradigm was challenged with the discovery of the human Rhadinovirus, Kaposi’s sarcoma-associated herpesvirus (KSHV) and a marmoset LCV Callitrichine herpesvirus 3 (Rivailler et al. 2002a; Lacoste et al. 2010). Regardless of their classifications, these viruses share a common structure, comprised of a 100–200-kb long linear double-stranded genome surrounded by an icosahedral capsid and subsequently by a lipid bilayer envelop derived from host’s cell membrane (Carville and Mansfield 2008). There are approximately 30 known members of LCVs. Examples include the following: Epstein–Barr virus (EBV) or human herpesvirus 4 (HHV4), rhesus monkey LCV, herpesvirus papio of baboons, and LCVs of African green monkeys, orangutan,

S.K. Upadhyay • H.C. Jha • A. Saha • E.S. Robertson (*) Department of Microbiology and Abramson Comprehensive Cancer Center, University of Pennsylvania School of Medicine, 201E Johnson pavilion, 3610 Hamilton walk, Philadelphia, PA 19104, USA Abramson Comprehensive Cancer Center, University of Pennsylvania School of Medicine, 201E Johnson pavilion, 3610 Hamilton walk, Philadelphia, PA 19104, USA e-mail: [email protected] E.S. Robertson (ed.), Cancer Associated Viruses, Current Cancer Research, DOI 10.1007/978-1-4614-0016-5_8, © Springer Science+Business Media, LLC 2012

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Table 8.1 Viral species of genus lymphocryptovirus (00.031.3.01.) Species name ICTVdB virus code Isolate(s)/strain(s) Alternate name(s) Callitrichine 00.031.3.01.009. Callitrichine Marmoset herpesvirus 3 herpesvirus 3 lymphocryptovirus (Ca1HV-3) Cercopithecine 00.031.3.01.002. Cercopithecine Baboon herpesvirus, herpesvirus 12 herpesvirus 12 Herpesvirus papio (CeHV-12) Cercopithecine 00.031.3.01.003. Cercopithecine African green monkey herpesvirus 14 herpesvirus 14 EBV-like virus (CeHV-14) Cercopithecine 00.031.3.01.004. Cercopithecine Rhesus EBV-like herpesvirus 15 herpesvirus 15 herpesvirus, rhesus (CeHV-15) lymphocryptovirus Human herpesvirus 4 00.031.3.01.005. Human herpesvirus 4 Epstein–Barr virus (HHV-4) Pongine herpesvirus 1 00.031.3.01.006. Pongine herpesvirus 1 Herpesvirus pan (PoHV-1) Pongine herpesvirus 2 00.031.3.01.007. Pongine herpesvirus 2 Orangutan herpesvirus (PoHV-2) Pongine herpesvirus 3 00.031.3.01.008. Pongine herpesvirus Gorilla herpesvirus 3(PoHV-3) Source: ICTV database (http://www.ictvdb.org/Ictv/fs_herpe.htm#Genus31)

and gorilla (Table 8.1). The common members of the Rhadinoviruses are Kaposi’s sarcoma-associated herpesvirus (KSHV) or human herpesvirus 8 (HHV8), herpesvirus saimiri (HVS), mouse herpesvirus 68 (MHV68), ovine herpesvirus 2 (OvHV-2), equine herpesvirus 2 (EHV-2), and rhesus monkey rhadinovirus (RRV) (Damania and Jung 2001; Ackermann 2006; Ehlers et al. 2010). The members of gammaherpesvirinae are lymphotropic. However, some are capable of undergoing lytic replication in fibroblasts, endothelial and epithelial cells (Damania and Jung 2001). Importantly in Old-World nonhuman primates, the natural hosts for LCVs, the infection remains persistent in the blood cells.

Lymphocryptoviruses and Cancer Similar to EBV, Old-World LCVs can also immortalize primary B-cells in vitro, and LCV infection has been found to be tightly associated with tumorigenesis in vivo (Rivailler et al. 2002a). Simian LCV infection of immunocompromised macaques at various National Primate Research Centers (NPRCs) has been found to be associated with a condition similar to oral hairy leukoplakia, which is frequently seen in HIV-infected patients and is associated with lytic EBV infection of epithelial cells on the tongue (Carville and Mansfield 2008). Non-Hodgkin lymphoma (NHL) is the second most commonly diagnosed malignancy in HIV-infected populations, and similarly, the incidence of NHL in cynomolgus (M. fascicularis) and rhesus

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macaques infected with SIV is 4–15% and 40%, respectively. M. nemestrina, the pigtailed macaque, housed at a NPRC developed a mycosis-fungoides-like lymphoma in association with LCV infection, with characteristic features of a T-cell lymphoma of the skin revealed on histological examination (Rivadeneira et al. 1999). Like other malignancies caused by EBV in the human host, nonhuman primates may also get posttransplant lymphoproliferative disorder (PTLD) on infection with their respective LCV, during organ transplantation. Out of 160 cases of cynomolgus macaque renal transplant 9 (5.6%) had evidence of PTLD at the time of necropsy, 28–103 days after transplantation (Schmidtko et al. 2002). Studies on EBV infection of tamarins and owl monkeys revealed that cross species transmission of LCVs from the natural to inadvertent host may also be associated with oncogenesis and the development of malignant lymphoma (Carville and Mansfield 2008). EBV, the prototypic member of the LCV genus, infects most of adult humans and usually persists asymptomatically for the life of the host. It is the most well studied and the only identified LCV known to infect humans (Moghaddam et al. 1997).

EBV: LCV Causing Cancer in Human LCVs naturally infecting Old-World primates are known to be biologically similar to EBV (Rivailler et al. 2002a). LCV infection is ubiquitous in adult Old-World nonhuman primates, and they harbor persistent LCV infection in their blood. OldWorld rhesus macaques have been used as animal model for EBV infection, as experimental infection of naive rhesus macaques with rhesus LCV reproduced acute and persistent infection similar to EBV infection in humans (Moghaddam et al. 1997). The genome sequence of rhesus LCV showed a high degree of amino-acid sequence homology (75%) with EBV providing the genetic validation for the similarities between EBV and rhesus LCV infection (Rivailler et al. 2002a, b). EBV infects more than 95% of the human population within the first decades of life and in the majority of cases infection is asymptomatic. However, later in life, a proportion of EBV-infected individuals can develop IM, a disease that is characterized by lymphadenopathy and fatigue, (Damania and Jung 2001). In contrast to most of the viruses identified as the cause of acute infections, EBV was discovered because of its isolation from cells obtained from tumor-bearing children with BL. The discovery of BL in late 1950s by Denis Burkitt was based on the climatic and geographical distribution of this lymphoma with an environmental or infectious etiology. However, a decade or more later in 1964, Anthony Epstein, Yvonne Barr, and Bert Achong identified virus particles in BL cells sent by Denis Burkitt by electron microscopy (Epstein et al. 1964; Kutok and Wang 2006). Subsequent studies revealed the association of EBV with a variety of other human tumors such as posttransplant B-cell lymphomas, HD, and NPC. Cells in these tumors contain characteristic multiple extrachromosomal copies of the circular viral genome (Young and Murray 2003). Association of this virus was also evidenced by its unique ability to efficiently transform resting B-cells in vitro into continuously growing lymphoblastoid cell lines (LCLs) (Young and Murray 2003).

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EBV Genome Among the herpesviruses, the EBV genome was the first that was cloned and subsequently sequenced (Baer et al. 1984). The genome of EBV is a linear doublestranded DNA of approximately 184 kb as packaged in the virion particle. It has a series of 500-bp terminal direct repeats (TRs) and internal repeat sequences (IRs) that divide its genome into short and long unique sequence domains (US and UL) which contains almost all the genome coding capacity. A BamHI fragment-cloned library was used for sequencing the entire EBV genome. Thus, any particular region of the EBV genome is referred with respect to its location within the BamH1-digested fragment of the genome, to a specific BamHI fragment, from A to Z, in descending order of fragment size (Young and Murray 2003). Upon EBV infection the linear termini of the viral genome are joined intracellularly to form circular episomal DNA (Kaschka-Dierich et al. 1976; Lindahl et al. 1976; Given et al. 1979). The process of genome circularization creates a signature number of TRs. In the dividing latently infected host cells, the number of TRs remains constant during episomal replication, and all the daughter cells derived from a single infected cell have been shown to have identical numbers of TRs in their EBV episomes (Kutok and Wang 2006). The TR number can also be used as a marker to determine the clonality of latently infected host cells (Kutok and Wang 2006). The EBV genome encodes nearly 100 proteins, and genes expressed during the lytic phase of infection have homologues in other human herpesviruses. However, the genes expressed during latent infection appear to be unique to EBV and are responsible for targeting a range of cellular pathways that lead to induction of EBV-associated cancers (Table 8.2).

Structure of EBV Similar to other herpesviruses, EBV has an outer envelope with glycoprotein spikes, a toroid-shaped DNA core in a nucleocapsid containing 162 capsomeres, and a protein tegument between the nucleocapsid and envelope (Epstein et al. 1965; Dolyniuk et al. 1976a, b; Kieff and Rickinson 2007). EBV capsids from purified enveloped virus are composed of EBV homologues of five previously purified herpesvirion proteins, the 18-kDa small capsid protein, the 30-kDa minor capsid protein, the 40-kDa minor capsid protein binding protein, the 155-kDa major capsid protein, and the 68-kDa portal protein (Kieff and Rickinson 2007). Among the common herpesvirus tegument proteins, the 350-kDa large tegument protein (BPLF1), the 140-kDa large tegument protein binding protein (BOLF1), the 15-kDa myristylated protein (BBLF1), the 32-kDa myristylated protein binding protein (BGLF2), the 58-kDa capsid-associated protein (BVRF1), the 58-kDa packaging protein (BGLF1), the 27-kDa palmitylated protein (BSRF1), and a 47-kDa protein kinase (BGLF4) are present in EBV. The gammaherpesvirus-specific proteins of the EBV tegument are the 140-kDa major tegument protein (BNRF1), the 19-kDa BLRF2, the 72-kDa BRRF2, the 54-kDa BDLF2, and the 42-kDa BKRF4 (Johannsen et al. 2004; Kieff

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Table 8.2 EBV lytic and latent genes and their functions EBV Gene Function Lytic genes BZLF1/BRLF1 Transcriptional activators of lytic gene expression BHRF1 Prevents apoptotic cell death in lytic EBV infection, Dispensable for B-cell growth transformation and for virus replication in culture BCRF1 Homologous to human IL-10; may downregulate the host immune response during viral replication BLLF1(gp350/220) Mediates adsorption between EBV and CD21 BKRF3,BBLF2/3, BMRF1, Replication factors BSLF1, and BBLF4 BALF2 Replication factor; single-strand DNA binding protein BALF5 Replication factor; core DNA polymerase Latent genes EBNA1 EBNA2

EBNA3A EBNA3B EBNA3C

EBNALP LMP1

LMP 2A/2B

EBER-1/2 BARTs

Tethers viral genome to the host chromosome, interacts with p53 (antiapoptotic function) Transactivator for many viral (LMP1 and LMP2A) and cellular (CD21, c-Myc,CD23) genes, essential for cellular transformation, interacts with RBP- Jk, and modulates Notch signaling. EBNA2 antagonist and transcriptional coactivator, modulates Notch signaling Interacts with RBP-Jk, modulates Notch signaling Essential for B-cell transformation by EBV, Ubiquitination of pRb and p27, modulates cell cycle through interaction with chk2, c-myc, Cyclin A/E/D1, p53 and Mdm2, chromatin remodeling by interaction with HDAC1 and 2, Interacts with RBP-Jk, modulates Notch signaling. Necessary for the efficient outgrowth of LCLs, transcriptional activation of viral promoters in association with EBNA2 Mimics the function of the B lymphocyte CD40 receptor and contributes to the EBV-induced transformation of primary B cells; Interacts with TRAFs LMP2A sequesters tyrosine kinases from the BCR, block EBV lytic cycle in B cells, LMP2B competes with LMP2A function Most abundant RNAs in latently infected cells, inhibit PKR and thus the antiviral effects of the interferons Different BARTs: interfere with the normal function(s) of RACK1 protein, modulate LMP1-induced NF-kB signaling, interact with RBP-Jk, modulate Notch signaling

and Rickinson 2007). Interestingly, a number of cellular proteins such as actin, HSP70, Cofilin, b-tubulin, enolase, and HSP90 are also contained within the EBV tegument (Kieff and Rickinson 2007). The lipid envelop contains a number of glycoproteins including gp350 (BLLF1), gH (BXLF2), gB-N, gB-C, and full-length gB (BALF4), gp42 (BZLF2), gM (BBRF3), gp78 (BILF2), gN (BLRF1), gp150 (BDLF3), and gL (BKRF2) (Johannsen et al. 2004; Kieff and Rickinson 2007).

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EBV-Encoded Proteins Implicated in Driving the Associated Pathologies During the interaction with its host, EBV goes through various phases of its infection cycle: (1) it infects B-lymphocytes and induces proliferation of infected cells; (2) it enters into a latent phase that drives the proliferative phase; and finally, (3) it can be reactivated to produce infectious progeny by inducing lytic cycle (Bornkamm and Hammerschmidt 2001). The transforming potential of the EBV genome is maintained within one third of the viral genome, and only a limited number of viral genes are expressed in EBV immortalized cells (Kempkes et al. 1995; Robertson and Kieff 1995). These include three latent membrane proteins (LMP1, LMP2A, and LMP2B), six nuclear antigens (EBNA1, 2, 3A, 3B, 3C, LP), and two short nonpolyadenylated RNAs (EBER1 and EBER2). BARF0 a reading frame reported to be expressed in NPCs (Hitt et al. 1989) is not required for the B-cell immortalization (Robertson et al. 1994; Bornkamm and Hammerschmidt 2001). Similarly, viral interleukin 10 (vIL10) earlier thought to have a B-cell growth factor activity, and BHRF1, a viral Bcl-2 homologue have been found to be nonessential for B-cell immortalization (Marchini et al. 1991; Bornkamm and Hammerschmidt 2001). EBV-encoded latency program is crucial for its stable persistence in host. By evading the host immune system, a program of EBV latency is established primarily in resting B-lymphocytes (Kumar et al. 2010). Depending on the spectrum of viral genes expressed, the latent

Fig. 8.1 Differential expression of EBV genes in the different latency programs. The expression pattern of the latent transcripts is a characteristic of the specific latency programs associated with EBV infection. The associated EBV malignancies that are typically linked to particular latency programs are shown in italics. Latency 0–III are shown and are represented by the inclusive oval shapes for the different transcripts. Latency IV has more recently been described with an EBNA2+ and LMP1- phenotype but EBNA1, EBERs, LMP2A, and EBNA3s have not been determined and are shown by a broken line including EBNA2 but may also have other transcripts. PEL primary effusion lymphoma, PTLD posttransplant lymphoproliferative disease

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program can be growth and proliferative type (latency III) or more restricted type (latency 0, I, II, or IV). Specific latency programs have been found to be associated with the different malignant pathologies associated with EBV infection (Kuppers 2003; Kumar et al. 2010) (Fig. 8.1).

LMP1 LMP1 is an integral membrane protein of 63 kDa that has three domains: an N-terminal cytoplasmic tail (amino acids 1–23), which tethers LMP1 to the plasma membrane and orientates the protein; six transmembrane-spanning domains loops, which are involved in self-aggregation and oligomerization (amino acids 24–186); and a C-terminal cytoplasmic domain (amino acids 187–386), which carries out most of the signaling activity for the molecule. This C-terminal domain is further divided into two subdomains, C-terminal activation regions 1 and 2 (CTAR1 and CTAR2) on the basis of their ability to activate the NF-kB pathway (Huen et al. 1995; Kutok and Wang 2006). LMP1 is one of the more carefully characterized molecules and is known for its transforming potential. It transforms rodent fibroblasts and is also essential for the immortalization of primary B-lymphocytes to LCLs (Wang et al. 1985, 1988; Baichwal and Sugden 1988). Expression of the LMP1 is also known to induce B-cell lymphoma in transgenic mice (Kulwichit et al. 1998). By mimicking the function of the B-lymphocyte CD40 receptor, LMP1 is known to contribute to EBV-induced transformation of primary B-cells (Gires et al. 1997; Hatzivassiliou et al. 1998; Kilger et al. 1998; Damania and Jung 2001). Similar to CD40 receptor the C-terminal domain of LMP1 is capable of interacting with many signal transducers such as TNF receptor-associated factors (TRAFs), TNF receptor-associated death domain (TRADD), and receptor-interacting protein (RIP) (Devergne et al. 1996; Eliopoulos et al. 1996; Izumi et al. 1997, 1999; Izumi and Kieff 1997; Sandberg et al. 1997; Devergne et al. 1998; Eliopoulos and Young 1998). In case of CD40 activation, the signal transduction usually takes place when CD40 is multimerized by binding with an extracellular ligand (Werneburg et al. 2001); however, LMP1 is capable of multimerization through its transmembrane domains, thereby generating a constitutively active signal that results in multiple downstream activities including the activation of NF-kB and JNK mediating activity and the induction of numerous cellular genes including bcl-2, bclx, mcl1, and A20 (Laherty et al. 1992; Hsu et al. 1995; Huen et al. 1995; Floettmann et al. 1996; Izumi et al. 1997; Izumi and Kieff 1997; Eliopoulos and Young 1998; Hatzivassiliou et al. 1998; Eliopoulos et al. 1999; Fries et al. 1999; Takeshita et al. 1999; Damania and Jung 2001), all critically important for EBV-induced immortalization of B cells. Recently, LMP1 has been discovered to signal the Janus kinase 3 (JAK3) and extracellular signal-regulated kinase 1/2 (ERK1/2) pathways upon the activation of STAT3 and STAT transactivation activity. LMP1-induced vascular endothelial growth factor (VEGF) expression via the JAK/STAT and mitogen-activated protein kinase (MAPK)/ERK signaling pathways is thought to be involved in the invasion of EBV-positive nasopharyngeal carcinoma (Wang et al. 2010).

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LMP 2A and 2B The LMP2 gene encodes two distinct integral membrane proteins, LMP2A and LMP2B (Laux et al. 1988). As the exons for LMP2 gene are located at both the ends of linear EBV genome, they could only be expressed from the circular EBV episome during latent infection (Longnecker et al. 1991). Both LMP2A and 2B have 12 transmembrane domains and a 27-amino-acid cytoplasmic C-terminus in common. However, LMP2A contains an additional 119 amino-acid cytoplasmic N-terminal domain (Longnecker and Kieff 1990). Both LMP2A and 2B oligomerize to form patches within the plasma membrane of latently infected B-lymphocytes where LMP2B has been observed to reduce the impact of LMP2 expression probably by imposing a dominant negative effect on LMP2 oligomers (Raab-Traub 2009). Although LMP2 has been found to be consistently expressed in most of the malignancies associated with EBV, none of the LMP2 proteins are essential for B-cell transformation in vitro (Longnecker 2000). Two of the tyrosine residues in LMP2A aminoterminal domain (Y74 and Y85) form an immunoreceptor tyrosine-based activation motif (ITAM) (Fruehling and Longnecker 1997), which mimic the ITAM present in the B-cell receptor (BCR). The ITAM present in BCR is known to play an important role in proliferation and differentiation of lymphocytes by activation of the src and syk protein tyrosine kinases (PTKs). Phosphorylated ITAMs of LMP2A compete with ITAM of BCR for binding with these PTKs and thereby negatively regulate the PTK activity (Fruehling and Longnecker 1997). Thus, LMP2A is implicated in blocking BCR-mediated calcium mobilization, tyrosine phosphorylation, and activation of the EBV lytic cycle in B-cells (Miller et al. 1995). In transgenic mice, LMP2A has been shown to drive the proliferation and survival of B-cells in the absence of BCR signaling (Caldwell et al. 1998). Inhibition of BCR signaling by sequestering of tyrosine kinases by LMP2A prevents unwanted antigentriggered activation of EBV-positive B-cells, thereby preventing their entry into the lytic cycle. However, LMP2A itself is able to stimulate these tyrosine kinases and thus provide the survival signal to the B-cells (Kuppers 2003). Therefore, LMP2A modifies B-cell development to favor the maintenance of EBV latency and also prevents inappropriate activation of the EBV lytic cycle, whereas LMP2B plays the modulatory role in LMP2A function (Longnecker 2000; Young and Murray 2003). LMP2A recruits Nedd4-like ubiquitin ligases through phosphotyrosine motifs, resulting in degradation of LMP2A through a ubiquitin-dependent mechanism (Ikeda et al. 2000) (Fig. 8.2). LMP2A also interacts with ERK1-MAPK, leading to self-phosphorylation at two serine residues (S15 and S102), which ultimately contributes to JUN activation (Chen et al. 2002; Young and Rickinson 2004). A recent study provides more conclusive evidence that LMP2B negatively regulates LMP2A function in preventing the switch from latent to lytic EBV replication (Rechsteiner et al. 2008). In EBV-harboring Akata cells, overexpression of LMP2B increased the magnitude of EBV switching from its latent to its lytic form upon BCR cross-linking, as indicated by more-enhanced upregulation and expression of EBV lytic genes and significantly increasing production of transforming EBV (Rechsteiner et al. 2008).

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Fig. 8.2 LMP1 and LMP2-mediated cell signaling in EBV infected B cells. LMP1 deregulates cell signaling through NF-kB, MAPKKK and PI3K by interaction with TRAF2 in mammalian cells. LMP2A cell signaling is primarily mediated through PI3K and AKT pathways by activation of the SRC family kinases, which leads to cell survival and motility

LMP2B also reduces the degree of BCR cross-linking required to induce lytic EBV infection and restored calcium mobilization upon BCR cross-linking, a signaling process inhibited by LMP2A (Rechsteiner et al. 2008).

EBNA1 EBNA1 is a DNA-binding nuclear phosphoprotein that is crucial for the maintenance of EBV episome and its replication. Short dyad symmetry (DS) Cis-acting elements at the origin of latent replication oriP have been identified as EBNA1 binding sites on

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the viral episome (Rickinson and Kieff 2001). EBNA1 increases the transcriptional activity from both Cp and the LMP1 promoters (Rickinson and Kieff 2001), while at the same time negatively regulating its own expression through the Qp promoter (Nonkwelo et al. 1996). Interestingly, EBNA1 is not presented by MHC class I molecules so that EBV-positive B-cells expressing this antigen are not recognized by cytotoxic T-cells and thus increases EBNA1 protein stability, which is possibly achieved by the internal Gly-Ala repeat domain (Levitskaya et al. 1995, 1997). A direct role for EBNA1 in oncogenesis has been suggested by studies performed on transgenic mice, where directing the EBNA1 expression in B-cells resulted in B-cell lymphomas (Wilson et al. 1996; Young and Murray 2003). EBNA1 along with another latent antigen EBNA3C has been recently identified to interact with a tumor metastasis suppressor, Nm23-H1. Using nude mice model, these EBV antigens have been evidently shown to promote the growth of transformed cells by rescuing Nm23-H1-mediated suppression (Kaul et al. 2007).

EBNA2 EBNA2 is one of the first EBV-encoded antigen to be expressed following primary B-cell infection in vitro and also indispensable for cellular transformation. Although EBNA2 does not bind to DNA directly, it can control many viral and cellular gene expression, such as that of LMP1, LMP2A, CD21, CD23, CD25, and C-FGR, via engaging various transcription regulators including RBP-Jk, SPI1/PU.1, TAF40, TFIIB, TFIIE, TFIIH, and RPA70 (Tong et al. 1995a, b, c; Damania and Jung 2001; Klein et al. 2010). Most of the EBNA2-responsive promoters possess a common DNA sequence (GTGGGAA) for binding to RBP-Jk, the master regulator of the Notch signaling pathway (Grossman et al. 1994). Notch genes encode cell surface receptors that regulate development of several cell types. In Drosophila melanogaster, mutations in Notch loci led to abnormalities in the notching of the wing, whereas in human these can lead to development of T-cell malignancy (Artavanis-Tsakonas et al. 1995). As the ligand (Jagged 1, 2 in humans) binds to the extracellular part of the Notch receptor, its intracellular domain is cleaved off and targeted to the nucleus to complex with RBP-Jk. This complex then transcriptionally activates genes including Hairy/Enhancer of split (HES) family, cyclin D1, and p21 (Aster et al. 2008; Borggrefe and Oswald 2009). Both EBNA2 and the cleaved intracellular region of Notch (RAMIC) transactivate the same set of viral and host gene promoters as they shown to compete for binding to RBP-Jk. This indicates that their interaction sites on RBP-Jk overlap at least partially (Sakai et al. 1998). Furthermore, interaction between EBNA2 and RBP-Jk (or CBF1) has been found to be essential for EBVmediated transformation, as a specific deletion of the RBP-Jk binding domain within the EBNA2 gene in the context of the whole virus renders the virus incapable of immortalizing B-lymphocytes in vitro (Cohen et al. 1991; Harada et al. 2001). As little or no RBP-Jk association with Notch1 has been observed in EBV-positive B-lymphocytes compared to the RBP-Jk associated with Notch1 in T-cell lines,

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EBNA2 is thought to compete for the available pool of RBP-Jk more effectively in B-cells, offering a potential explanation for the ability of EBV to efficiently infect and immortalize primary human B-cells in vitro (Callahan et al. 2000). EBNA2 also plays a role in chromatin remodeling in regulating both viral and host gene expressions by interacting with numerous modulatory proteins actively involved in this process. This includes the SNF5 subunit of the SWI/SNF chromatin remodeling complex (Wu et al. 1996), histone acetyltransferases (HATs) – P300/ CBP, PCAF (Wang et al. 2000), and the helicase DP103 (Grundhoff et al. 1999; Klein et al. 2010). Deletion mutation analysis of the EBNA2 protein showed that the Pro-rich aminoterminal and a domain within the divergent region mediate the interaction between EBNA2 and hSNF5/INI1 (Wu et al. 1996). EBNA2 engages the hSNF-SWI complex to generate an open chromatin conformation at the EBNA2responsive target genes, thereby facilitating the activity of the RBP-Jk/EBNA2/ RNA-polymerase II transcription complex (Wu et al. 1996). This hypothesis was strengthened by the additional observation where antibodies directed against components of the hSNF-SWI complex precipitated chromatin-associated DNA that contained a targeted EBNA2-responsive element in the context of both episomal and cellular chromatin. This enrichment could not be observed in the EBV-negative cells or when the EBNA2-responsive element was mutated (Wu et al. 2000).

EBNA3 Family of Proteins: 3A, 3B, and 3C The three members of the EBNA3 family, EBNA3A, -3B, and -3C, lie in a tandem array on the EBV genome. These proteins share an amino-terminal domain with a 20–25% homology, but different carboxy-terminal domains. They are hydrophilic nuclear proteins that contain heptad repeats of Leu, Ile/Val that can act as dimerization domains (Young and Murray 2003). Genetic studies have revealed that both EBNA3A and EBNA3C are essential for in vitro B-cell transformation, whereas EBNA3B is dispensable (Robertson 1997; Young and Murray 2003). Members of the EBNA3 family participate in transcriptional regulation of both cellular and viral genes. EBNA3C in particular transcriptionally activates cellular genes, such as CD21, CD40, Vimentin, and the viral encoded LMP1 (Allday and Farrell 1994; Silins and Sculley 1994). Moreover, EBNA3C also acts as a transcriptional repressor from the viral Cp promoter (Radkov et al. 1997). All EBNA3 proteins repress the EBNA2-mediated transactivation by associating with RBP-Jk and disrupting its binding to the cognate DNA sequence and also to EBNA2 (Robertson 1997; Young and Murray 2003). Thus, both cellular and viral promoters containing RBP-Jk cognate sequences are cooperatively regulated by EBNA2 and EBNA3 family proteins (Young and Murray 2003). EBNA3C plays a key role in deregulating the cell cycle and transformation of B-cells in vitro by interfering with the function(s) of a number of important cell-cycle regulators (Kumar et al. 2010). EBNA3C interacts with and enhances Cyclin A/CDK2 dependent kinase activity and efficiently rescues p27-mediated repression of Cyclin A/CDK2 kinase activity by decreasing

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the molecular association between the Cyclin A/CDK2 complex and p27 in EBV-transformed LCLs (Knight and Robertson 2004). EBNA3C also interacts specifically with metastatic suppressor protein Nm23-H1 and reverses its ability to suppress the migration of BL cells and breast carcinoma cells (Subramanian et al. 2001) and was also shown to regulate transcription of a number of genes involved in regulation of cell migration and, so, demonstrates a transcriptional component of this interaction (Kuppers et al. 2005; Choudhuri et al. 2006; Kaul et al. 2007, 2009). EBNA3C is also shown to rescue the pRb-induced flat cell phenotype and target pRb for proteasome-ubiquitin mediated degradation (Knight et al. 2005a). Further mechanistic studies revealed that EBNA3C specifically recruits the ubiquitin ligase, Skp1/Cul1/F-box complex (SCFSkp2), which adds ubiquitin moieties to many cellcycle regulators such as pRb and p27, marking them for subsequent degradation (Knight et al. 2005a, b). Additional studies also demonstrated that EBNA3C forms a ternary complex with the p53 tumor suppressor protein and its negative modulator Mdm2 (Saha et al. 2009; Yi et al. 2009). Mdm2 is a specific ubiquitin ligase that is known to negatively regulate the tumor suppressor activity of p53 by facilitating its ubiquitin mediated degradation. Interestingly, EBNA3C has been shown to suppress the p53’s function by stabilizing Mdm2 through deubiquitination (Saha et al. 2009; Yi et al. 2009). Furthermore, Knight et al. showed that EBNA3C manipulates the cellular deacetylase activity and thereby the chromatin structure through interactions with ProTa in association with deacetylases (HDAC1 and HDAC2) and corepressors (mSin3A and NCoR) (Knight et al. 2003). In a recent study, the vitamin D receptor (VDR) has been identified as a binding partner of EBNA3A, which subsequently activates VDR-dependent genes to protect LCLs against vitamin-D3-induced growth arrest and apoptosis (Yenamandra et al. 2010).

EBNALP EBNALP (EBV nuclear antigen leader protein) is encoded by a small ORF in the leader exons of the EBNA transcripts and is composed of repetitive units derived from repetitive nucleotide sequences in the EBV internal repeat (IR1) sequence (Bodescot et al. 1986). Therefore, EBNALP protein may vary in size depending on the number of BamHI W repeats contained by a particular EBV isolate (Young and Murray 2003). Although EBNALP does not seem to be absolutely required for B-cell transformation in vitro, mutant viruses for EBNALP have reduced efficiency for immortalization of B-cells and it is shown to be necessary for the efficient outgrowth of LCLs (Hammerschmidt and Sugden 1989; Mannick et al. 1991; Allan et al. 1992). EBNALP colocalizes with the promyelocytic leukemia (PML) protein, which is known to be involved in the sequesteration of transcription factors. It has been suggested that EBNALP disrupts PML function, which leads to the transcriptional activation of viral promoters in association with EBNA2 (Allan et al. 1992; Nitsche et al. 1997; Young and Murray 2003). Both EBNALP and EBNA2 have been found to form complexes with HDAC4 in an LCL background, where EBNALP coactivated

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transcription by relocalizing HDAC4 and HDAC5 from EBNA2 activated promoters to the cytoplasm (Portal et al. 2006). EBNALP was suggested to interact with both pRb and p53 (Jiang et al. 1991; Szekely et al. 1993); however, these interactions have not been shown to affect either pRb or p53 mediated signaling pathways (Young and Murray 2003). Recently, studies have shown that EBNALP may form a ternary complex with p53 and Mdm2, where Mdm2 serves as a bridge (Kashuba et al. 2010). Although EBNALP has been found to be neutralized by Mdm2 destabilizing the effect on p53, it can efficiently block p53-dependent gene activation in LCLs, thus providing a potential mechanism for the accumulation of p53 levels in LCLs without the induction of p53-mediated apoptosis (Kashuba et al. 2010).

EBERs EBV expresses two small nonpolyadenylated (noncoding) RNAs - EBER1 and EBER2 in almost all the forms of latency (Fig. 8.1). Although EBERs have not been found to be essential for the EBV-induced primary B-lymphocytes transformation (Young and Rickinson 2004), a potential role for them has been suggested, which relates to the maintenance of viral persistence (Nanbo et al. 2002). Through assembling with autoantigen La and ribosomal protein L22, EBERs are shown to bind and interfere with the function of the interferon inducible, double-stranded-RNAactivated protein kinase PKR (Takada and Nanbo 2001). As PKR has a role in mediating the antiviral effects of the interferons, it has been suggested that EBERmediated inhibition of PKR function could be important for viral persistence (Nanbo et al. 2002). Interestingly, expression of the EBERs in BL cell lines has been found to increase tumorigenicity, promote cell survival, and induce interleukin-10 (IL-10) expression (Ruf et al. 2000; Takada and Nanbo 2001; Young and Rickinson 2004). These studies suggest that EBV genes previously shown to be dispensable for the B-cell transformation may be involved by contributing in a critical way to the pathogenesis of a number of EBV-associated malignancies (Young and Murray 2003).

BARTs A group of highly spliced transcripts encoded by the BamHI A region of the EBV genome, which are abundantly expressed in EBV-associated malignancies, are commonly referred to as either BamHI A rightward transcripts (BARTs), or complementary-strand transcripts (CSTs) or BARF0 (Karran et al. 1992; Smith et al. 2000). The function of most of the BARTs is not conclusively known but their detection in B-cells from normal donors and in many EBV-associated tumors suggests that they are likely to have critical roles in viral persistence and in development of the associated pathologies. For example, the RPMS1 CST encodes a nuclear protein that binds to RBP-Jk and modulates EBNA2-mediated Notch signal transduction (Smith et al. 2000). Another cytoplasmic protein encoded by A73 CST

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interacts with RACK1 protein (Smith et al. 2000). Since RACK1 modulates signaling from protein kinase C and Src tyrosine kinases, this suggests a possible role for A73 in growth control (Smith et al. 2000). EBV-encoded BART miRNAs also target the 3¢ UTR of the LMP1 gene and negatively regulate LMP1 protein expression (Lo et al. 2007). These miRNAs have also been found to modulate LMP1-induced NF-kB signaling and alleviate the cisplatin sensitivity of NPC cells (Lo et al. 2007). Another important transcript generated from the BamHI A region is BARF1, which encodes a 31-kDa secreted protein expressed as a latent protein in EBV-associated NPCs and gastric carcinomas (GCs) (Decaussin et al. 2000; zur Hausen et al. 2000). BARF1 shares limited homology with the human colony-stimulating factor (CSF) 1 receptor and displays oncogenic activity when it is expressed in rodent fibroblasts and simian primary epithelial cells (Sheng et al. 2001).

Host Machinery Targeted by EBV to Induce Tumorigenesis Cell Signaling EBV is a highly immunogenic virus, as confirmed by the strong response induced at the time of primary contact, which successfully constrains the virus in a rigorously latent, immunologically silent status. After spreading through activation of the lytic cycle, EBV establishes a latent infection in the memory B-cells by negatively regulating the expression of the major immunogenic latent antigens (Merlo et al. 2010). A large body of evidence has clearly shown that EBV-encoded proteins can critically modulate the function of major players in different cell-signaling cascades in the development of associated cancers. The activation of a signaling cascade leading to the posttranscriptional modification and nuclear translocation of NF-kB and Rel-family transcription factors modifies the expression of numerous proteins involved in various phases of cancer development, including cell proliferation (e.g., cyclin D1 and E), survival (e.g., cFLIP and IAPs), inflammation and angiogenesis (e.g., IL-1, IL-6, VEGF, ICAM-1, and E-selectin), epithelial-mesenchymal transition (e.g., vimentin and cathepsin-B and -Z), invasion and metastasis (e.g., uPA, MMPs 2 and 9, and ICAM-1), and many others (Basseres and Baldwin 2006). EBV has developed sophisticated mechanisms to hijack the NF-kB signaling pathway to achieve a successful infection and promote survival signals. There is a critical interdependence between the host cell and virus to finetune NF-kB-mediated functions, both positively and negatively, which has allowed survival and efficient proliferation of these tumor viruses during evolution. During latent EBV infection, NF-kB constitutively activates its downstream targeted genes, which subsequently allows for viral persistence. The current understanding of the molecular controls suggests a plausible model whereby the inflammatory microenvironment is sensed by EBV through NF-kB activation in infected cells. Inflammation may have an adverse effect on the biology of the virus due to the immunogenic response against EBV-infected cells; however, it has also been reported that EBV

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subtly exploits NF-kB-mediated signaling pathways to elicit the viral latency program (de Oliveira et al. 2010). To date, a number of EBV-encoded antigens are well defined and are also shown to be associated with the deregulation of various cellular signaling pathways. Further studies are required to identify detailed mechanisms for EBV specific manipulation of host-signaling pathways in human. The EBV-encoded LMP1 is involved in several signaling pathways including NF-kB, AP-1, JAK/STAT, PI3K/AKT, and ERK-MAPK and has been shown to regulate their downstream effects (Dawson et al. 2003; Mainou et al. 2005; Zheng et al. 2007). LMP1 also activates the PI3K/ AKT/mTOR signaling pathway in B-lymphocytes (Lambert and Martinez 2007). The mTOR signaling pathway has been identified as a downstream component of the PI3K/AKT pathway in the LMP2A-transfected NPC cell lines (Moody et al. 2005). Furthermore, LMP1 can mimic CD40 signaling to induce B-cell activation and differentiation in vivo (Rastelli et al. 2008) by usurping its activities through interaction with important signaling components such as TRAF1, 2, 3, and 5 involved in various pathways including the NF-kB, JNK, p38/MAPK, PI3K/AKT, and JAK/ STAT signaling pathways (Uchida et al. 1999). NF-kB activity is repressed by expression of EBNA1 in numerous carcinoma cell lines suggesting that its activation is not due to clonal variation or cell line specificity. It has also been reported that a number of malignancies are associated with chronic activation of NF-kB (Valentine et al. 2010). In spite of the constitutive expression of several viral antigens during latent infection, many EBV-associated tumors are poor targets for the cellular immune response, suggesting that escape from immune surveillance is an early event in the development of oncogenesis (Dantuma and Masucci 2003).

Chromatin Remodeling A number of recent studies show that similar to many other tumor viruses, EBV has also evolved sophisticated strategies to manipulate cellular chromatin-modifying enzymes to precisely control viral as well as cellular genes expression. The EBV proteins known to critically regulate the chromatin remodeling mechanism include latent antigens EBNA2, EBNA3C, BGLF4 (Cotter and Robertson 2000; Knight et al. 2003; Lee et al. 2007) and the lytic cycle inducer Zta (Deng et al. 2003; Wei and Zhou 2010) (Fig. 8.3). EBNA2 activates the viral LMP1 promoter via interaction with many histone acetyltransferases (HATs), including p300, CBP, and PCAF, whereas the transcriptionally inactive point mutants of EBNA2 lack binding affinity to these HATs and are unable to activate LMP1 (Wang et al. 2000). EBNA2 has been suggested to utilize the intrinsic HAT activity for positively regulating viral genes transcription (Wang et al. 2000). In another study, a phosphorylated fraction of lymphocyte EBNA2 has been found to be associated with a component of the hSWI/SNF HAT complex, hSNF5/Ini1 (Wu et al. 1996) (Fig. 8.3). The SWI/SNF complex can activate or repress transcription of a subset of genes through alteration of the chromatin

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Fig. 8.3 EBV proteins influence the remodeling of the chromatin structure during infection and development of associated malignancies. This schematic shows the functions of various molecules of the chromatin remodeling machinery, which have altered functions due to their interaction with a number of EBV-encoded antigens. EBNA3C is the encoded transcription regulator that binds to HDACs, ProTa and p300; BGLF4, the kinase seen in the tegument, can be associated with condensin phosphorylation and TopoII activation; EBNA2 is the major EBV transcription factor also interacting with p300, CBP, PCAF, and the SWI/SNF complex; and Zta is the immediate early viral transcription activator in its interaction with HATs including CBP

structure by altering the effects on transcription imposed by nucleosomal packaging of DNA (Kwiatkowski et al. 2004). Chromatin immunoprecipitation (ChIP) assays showed that EBNA2 recruits this complex to the LMP2A regulatory segment (Wu et al. 2000). Moreover, the relocalization of hSWI/SNF complex found to be dependent on binding of RBP-Jk to its recognition sequence, as well as on EBNA2 expression (Wu et al. 2000; Hayward 2004). Additionally, EBNA2 also modulates the expression of the CD23 gene expression through recruitment of the hSWI/SNF complex to its regulatory region (Wu et al. 2000). EBNA3C also delicately controls chromatin structure by modulating cellular chromatin remodeling machineries (Radkov et al. 1999; Cotter and Robertson 2000; Knight et al. 2003). EBNA3C has been found to recruit histone deacetylase (HDAC1) enzyme activity (Radkov et al. 1999) (Fig. 8.3). Interestingly, as both EBNA3C and RBP-Jk share similar binding region to HDAC1, it has been suggested that through recruitment of HDAC activity, EBNA3C serves as a bridge between RBP-Jk and HDAC1 interaction, to repress the transcription activation from the viral Cp promoter (Radkov et al. 1999). EBNA3C has also been found to interact with prothymosin a (ProTa), a cellular protein known to interact with histones and likely to be involved in the chromatin remodeling machinery (Gomez-Marquez and Rodriguez 1998; Cotter and Robertson 2000). Moreover, EBNA3C also interacts with the cellular HAT p300 (Cotter and Robertson 2000).Typically, addition of acetyl groups to the core histone molecules results in disassociation of the compact chromatin structure,

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which renders the genetic regulatory sites accessible to the transcriptional machinery (Shiama 1997; Giles et al. 1998). As a result, the interactions of EBNA3C with ProTa and p300 suggest another important role for EBNA3C in modulating the chromatin structure for transcriptional activation. On the contrary, EBNA3C expression downmodulates EBNA2-mediated increased HAT activity in a dose–responsive manner (Cotter and Robertson 2000). In another study, EBNA3C was also shown to form a complex with ProTa, HDAC1, HDAC2 and the transcriptional corepressors mSin3A and NCoR (Knight et al. 2003) (Fig. 8.3). Thus, EBNA3C plays an extremely important role during EBV-mediated immortalization of infected cells by precisely balancing the overall transcriptional events. EBV lytic cycle activator Zta is a bZIP protein shown to also have a role in stimulating nucleosomal HAT activity of the CREB binding protein (CBP) (Fig. 8.3). Zta and CBP colocalize to viral immediate-early promoters and overexpression of Zta has been found to lead to a robust increase in H3 and H4 acetylation at various regions of the EBV genome (Deng et al. 2003). In a recent study, in EBV infected B-cells, the latency-associated viral antigens have been suggested to inhibit expression of the proapoptotic Bcl-2-family member Bim and enhance cell survival through epigenetic regulations (Paschos et al. 2009). However, the actual viral latent protein involved in this phenomenon is yet to be identified (Paschos et al. 2009). Along with controlling chromatin transcription levels, EBV antigens can also modulate the higher order chromatin structure formation. The EBV-encoded kinase BGLF4 has been shown to induce cellular DNA condensation through condensin phosphorylation and topoisomerase II (Topo II) activation (Lee et al. 2007). EBV reactivation leads to cellular chromatin condensation and interchromosomal space enlargement, and BGLF4 is considered to be responsible for this activity as its expression in various cell types caused a prophase-like individualized condensation pattern for chromatin (Lee et al. 2007). Overall, the reprogramming events that occur in infected B-cells facilitate EBV persistence and the development of EBV-mediated carcinomas.

Cell-Cycle Progression Cell-cycle progression is exclusively regulated by four major families of proteinsthe cyclins, the cyclin-dependent kinases (CDKs), retinoblastoma family of proteins (pRb and the related “pocket proteins”), and the cyclin-dependent kinase inhibitors (CDKIs) (Johnson and Walker 1999; Obaya and Sedivy 2002). In mammals, there are at least nine CDKs and 16 cyclins, and each CDK combines with a specific cyclin to generate an active holoenzyme (Ekholm and Reed 2000; Obaya and Sedivy 2002). The CDKs (CDK1, 2, 4, 6, and 7) are required for cell-cycle progression, and they are expressed throughout the cell cycle, but their catalytic activity requires binding to a cyclin, which acts as a positive regulatory subunit for determination of the target specificity of the kinase. Cyclins can be categorized by the cell-cycle phase in which they are expressed. The G1 cyclins (Cyclins D1, D2, D3, and E) are required for G1 to S phase transition, followed by the activation of an S-phase cyclin (Cyclin A) for the completion of S phase. Further, a mitotic cyclin (Cyclin B) facilitates

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Fig. 8.4 Deregulation of cell cycle phases by the EBV-encoded antigens. EBV-encoded latent antigens – EBNA2, EBNA3C, LMP1, and LMP2 – deregulate different phases of cell cycle by interacting with many important cell cycle regulators. These include the cyclins, cyclin inhibitors, and the tumor suppressors, which include p53, SKp2, and p16

the entry into mitosis (M) phase from G2 (Morgan 1997; Saha et al. 2010c). The levels of different cyclins oscillate throughout the cell cycle as a result of coordinated synthesis and ubiquitin-proteasome-mediated degradation, ensuring the correct temporal activation of each CDK and imposing directional irreversibility to the progression of the cell cycle (Fig. 8.4) (Morgan 1997). In the G1 phase, an active cyclin/CDK complex phosphorylates the pRb and related pocket proteins releasing the E2F family of transcriptional factors (E2F1-8) and subsequently transactivates an array of genes accountable for initiating DNA replication facilitating G1 to S phase transition (Saha et al. 2010c). In the absence of mitogenic signals, CDKs remain inactive, and are negatively controlled by a number of CDKIs including p15, p14ARF/p16, p21WAF1/CIP1, and p27KIP1, which ultimately prevent aberrant cell-cycle proliferation (Saha et al. 2010c). In normal cells, the integrity of DNA replication is protected. This relies on the p53 tumor suppressor protein along with other DNA damage response proteins to repair the damage. In response to various genotoxic stresses, p53 either blocks the cell cycle to allow repair or induces apoptosis if the cellular injury cannot be repaired

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(Saha et al. 2009, 2010c). For example, p53 enhances transcription of the specific CDKI, p21WAF1/CIP1, which in turn interacts with and inhibits CDK2 to arrest the cellcycle progression. Thus, p53 is one of the prime targets for oncogenic viruses and is the most frequently mutated gene in human cancers (Saha et al. 2010c). Earlier, it was shown that EBV fails to block p53-mediated apoptosis in LCLs induced by variety of genotoxic agents, suggesting that, unlike other small DNA tumor viruses, such as SV40 or HPVs, EBV does not compromise p53 function (Allday 2009). On the contrary, it has also been shown that EBV can disrupt G1 cell-cycle checkpoint by negatively acting downstream of p53-mediated pathway (Leao et al. 2007). However, more recently, it has been clearly shown that EBNA3C can directly bind to the p53 protein and repress its functions in part by blocking its transcriptional activity as well as facilitating its degradation through stabilization of its negative regulator, Mdm2 (Saha et al. 2009; Yi et al. 2009). In addition, EBNA3C has further been shown to negatively regulate p53-mediated functions by interacting with its regulatory proteins, the inhibitor of growth family proteins, ING4 and ING5, known to be frequently deregulated in different cancers (Saha et al. 2010a). To facilitate cell-cycle progression, EBNA3C also targets the CDKI p27KIP1 and pRb for ubiquitin-proteasome dependent degradation through the recruitment of the SCFSkp2 E3 ligase activity (Knight et al. 2005a, b). By disrupting p27KIP1 from Cyclin A/CDK2 complexes, EBNA3C enhances CDK activity (Knight and Robertson 2004). EBNA3C can also override p16INK4A-mediated suppression during EBVmediated in vitro transformation, consistent with EBNA3C targeting the checkpoint at the G1/S transition regulated by pRb (Saha et al. 2010c). As a result, similar to the adenovirus-encoded E1A and HPV-encoded E7 oncoproteins, EBNA3C can also cooperate with oncogenic mutant H-ras for immortalization and transformation of rat embryonic fibroblasts (REFs) (Saha et al. 2010c). EBV encodes another oncoprotein LMP1, which when expressed can repress transcription from the p16INK4A promoter. However, it did not have any significant effect on the p21WAF1/CIP1 promoter (O’Nions and Allday 2004). Furthermore, it is known that constitutive expression of the c-Myc oncoprotein in B-lymphocytes induces overall protein synthesis and cellcycle division. Additionally, c-Myc can also stimulate the expression of D-type cyclins and cyclin E and downregulate p21WAF1/CIP1 and p27KIP1 (O’Nions and Allday 2004; Saha et al. 2010c). The EBV-encoded latent antigen, EBNA2 directly activates c-Myc further increasing transcription of the cyclin D2 gene, whose enhanced expression is generally found in EBV-associated lymphomas (Saha et al. 2010c). Earlier reports have shown that in vitro EBV infection or its transforming antigen LMP1 upregulates only cyclin D2 but not cyclin D1 gene expression in primary B-lymphocytes as well as BL cells (Saha et al. 2010b). However, by contrast, Cyclin D1 protein level has been shown to be significantly expressed in a number of EBVpositive LCLs (Kim et al. 2002; Park et al. 2004) or SCID mice lymphomas (Murai et al. 2001). Surprisingly, these abovementioned studies did not directly set out to explore the contribution of Cyclin D1 in EBV-mediated B-cell oncogenesis. Nonetheless, more recent studies have shown that EBNA3C stabilizes as well as enhances the kinase activity of the Cyclin D1/CDK6 complex, as well as the nuclear localization of Cyclin D1 to bypass the G1 restriction point (Saha et al. 2010b).

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Importantly, this particular study provides evidence that shows that EBNA3C specifically targets Cyclin D1 activity, which subsequently nullifies pRb-mediated growth suppression (Saha et al. 2010b). This describes a fundamental mechanism by which EBV can deregulate the mammalian cell cycle in EBV-associated human cancers (Saha et al. 2010b).

Diagnosis of EBV Infections and Malignancies EBV is the causative agent of infectious mononucleosis and also contributes significantly to the pathologies of associated benign and malignant lesions. These include oral hairy leukoplakia, inflammatory pseudotumor, HD, NHL, NPC, and GC (Kumar et al. 2010). Molecular diagnosis of these diseases is very important for monitoring and treatment of patients affected by these diseases. In biopsy tissues, in situ hybridization for EBV-encoded RNA transcripts is considered the gold standard for identification of EBV-related histopathological lesions and is routinely used with LMP1 immunostaining to detect latent EBV infections. Except for the case of immunocompromised patients, high serological titers may serve as tumor marker for EBV-related malignancies (Gulley 2001). Because EBV elicits a rapid antibody response, testing acute-phase serum samples for the presence of EBV specific antibodies provides a means for detection of the early phase of EBV infection. Antibody tests for EBV can measure the presence and/or the concentration of at least six specific EBV antibodies. By evaluating the results of these different tests, the stage of EBV infection can be determined (http://www.cdc.gov/ncidod/diseases/ebv.htm). EBV viral load testing from blood samples by quantitative PCR is now a promising test for early diagnosis and monitoring patients with PTLD (Tsai et al. 2008). Another new and powerful approach of diagnosis of EBV-related pathologies is using gene expression profiling, which provides the added advantage of subclassifying EBV-related diseases and more comprehensive monitoring in response to therapy (Gulley 2001).

Treatment Options for EBV-Associated Malignancies Treatment options for EBV-mediated tumors may include manipulating the balance between outgrowing EBV-infected B cells and the EBV-cytotoxic T lymphocytes (CTL) response, or targeting the B cells with monoclonal antibodies, chemotherapy or radiation therapy.

Immunotherapy EBV-associated tumors express viral encoded antigens and are excellent antigenpresenting cells, expressing high levels of immune system costimulatory molecules (Heslop 2009). Therefore, one therapeutic option is to manipulate the immune

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system to target and eradicate these malignancies. Immunotherapy with EBV-specific CTLs has proved effective in PTLDs, which are highly immunogenic tumors expressing type III latency (Fig. 8.1). The malignant cells in HD and NPC express type II latency and hence a more restricted pattern of EBV antigens that suggests less immunogenicity (Straathof et al. 2003).

Use of Cytotoxic T Cells EBV-Specific T Cells EBV-specific CTLs could be generated from EBV transformed LCLs. In a study where donor-derived EBV-specific CTLs have been administered to more than 100 patients after hematopoietic stem cell transplantation (HSCT), the results suggest that this approach is highly effective as prophylaxis in high-risk patients with a history of PTLD or patients receiving selective T-cell depletion (Heslop et al. 1996; Rooney et al. 1998; Heslop 2009).

Unmanipulated Donor T Cells EBV-specific T-cell response can also be provided by infusing unmanipulated donor lymphocytes from EBV-seropositive HSCT cell donors. This approach has been shown to have a more than 70% response rate in HSCT patients with established PTLD (Heslop 2009). However, it has an associated risk of inducing severe or fatal Graft-versus-host disease (GVHD), as the frequency of alloreactive T cells in the cell product has been found to be more than a log higher than the frequency of virus-reactive T cells (Heslop et al. 1994; O’Reilly et al. 1997). In an approach to circumvent this problem, T cells have been transduced with the thymidine kinase (TK) suicide gene, which can be activated by infusion of ganciclovir if the recipient develops GVHD. This approach has shown promising results in early-phase trials (Heslop 2009).

Targeting B-Cells Antibody Therapy One strategy of prevention and treatment of EBV-associated tumors could be to eliminate EBV-infected B cells. In this approach, B cell-specific surface antigens present on the EBV transformed malignant cells are targeted with antibodies. The most commonly used antibody for this purpose is a chimeric murine or human

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monoclonal anti-CD20 antibody, Rituximab (Straathof et al. 2003). This antibody has been used as prophylaxis and treatment for PTLD after HSCT, with promising results showing initial response rates between 55% and 100% (Kuehnle et al. 2000; van Esser et al. 2002; Powell et al. 2004; Brunstein et al. 2006). However, as CD20 expression is not confined to the malignant cells, normal B-cells are also targeted resulting in marked immunosuppression. Fatal viral infections have also been reported after rituximab therapy (Suzan et al. 2001; Heslop 2009).

Antiviral Agents For the treatment of EBV infected patients, nucleoside analogs, which target the virus-specific enzyme, thymidine kinase (TK) expressed in lytically infected cells, are currently in use (Heslop 2009). However, as EBV tumors usually show a latent pattern of gene expression and thus lack the TK expression, it makes antiviral therapy alone ineffective as an antineoplastic therapy (Heslop 2009).

Radiation Therapy and Surgery When the virus associated tumor is confined to a single site, radiation and/or surgery can be effective. Surgery and radiation may also have a role in managing local complications because of malignancies, such as compression of vital organ structures (Heslop 2009).

Chemotherapy Chemotherapy with regimens used in lymphoma therapy, such as CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone), could be a therapeutic option for patients who do not respond to immune manipulation or rituximab (Heslop 2009). However, chemotherapy has the risk of putting the patient at increased risk of infection particularly when they have preexisting immune suppression (Heslop 2009).

Vaccination Against EBV The development of an EBV vaccine may be protective against primary infection and hence presumably reduce the burden of EBV-associated cancers. EBV neutralizing antibodies mainly targeting the major virus surface glycoprotein gp350/220 and

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several vaccine candidates based on gp350/220 have been successfully developed (http://www.who.int/vaccine_research/diseases/viral_cancers/en/index1.html). Live recombinant vaccinia virus vectors have also been used to express the gp350/220 antigen and have been found to confer protection in primates and elicit antibodies in EBV-negative Chinese infants (World health organization Web site: http://www. who.int/vaccine_research/diseases/viral_cancers/en/index1.html). Soluble recombinant gp350/220 produced in CHO cells was found to be safe in humans; however, they need strong adjuvants to elicit acceptable immunogenicity (codevelopment by MedImmune, GSK and Henogen) (World health organization Web site: http://www. who.int/vaccine_research/diseases/viral_cancers/en/index1.html). Phase II clinical trials of this candidate vaccine are under way. Clinical trials of an EBNA-3A peptide are also being conducted in Australia (World health organization Web site: http://www.who.int/vaccine_research/diseases/viral_cancers/en/index1.html)

Conclusion Capability of lifelong asymptomatic persistence in their host keeps LCVs among the most successful category of viruses. Deregulated immune system of host, however, makes the hibernating virus exert its pathogenic role, mainly contributing to induction of a range of cancers in the host. Its infectivity in the human host makes EBV one of the most well-studied LCV. Extensive molecular studies revealed that EBV-virulence is distributed among a number of proteins and RNA, encoded by its genome, with specific antigens contributing more to the critical functions than others. EBV proteins identified as essential for host-cell transformation have been thought to provide the scope or molecular blueprint for target based drug design to treat the EBV-induced pathologies. However, to date the therapies implicating the manipulation of the immune system have appeared to be more progressive and promising. Combinatorial therapy designed on the basis of the clinical condition of patient may be more useful in the majority of the cases, as it may have a higher potential for success.

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Klein G, Klein E, Kashuba E (2010) Interaction of Epstein–Barr virus (EBV) with human B-lymphocytes. Biochem Biophys Res Commun 396:67–73 Knight JS, Lan K, Subramanian C, Robertson ES (2003) Epstein–Barr virus nuclear antigen 3C recruits histone deacetylase activity and associates with the corepressors mSin3A and NCoR in human B-cell lines. J Virol 77:4261–4272 Knight JS, Robertson ES (2004) Epstein–Barr virus nuclear antigen 3C regulates cyclin A/p27 complexes and enhances cyclin A-dependent kinase activity. J Virol 78:1981–1991 Knight JS, Sharma N, Robertson ES (2005a) Epstein–Barr virus latent antigen 3C can mediate the degradation of the retinoblastoma protein through an SCF cellular ubiquitin ligase. Proc Natl Acad Sci USA 102:18562–18566 Knight JS, Sharma N, Robertson ES (2005b) SCFSkp2 complex targeted by Epstein–Barr virus essential nuclear antigen. Mol Cell Biol 25:1749–1763 Kuehnle I et al (2000) CD20 monoclonal antibody (rituximab) for therapy of Epstein–Barr virus lymphoma after hemopoietic stem-cell transplantation. Blood 95:1502–1505 Kulwichit W, Edwards RH, Davenport EM, Baskar JF, Godfrey V, Raab-Traub N (1998) Expression of the Epstein–Barr virus latent membrane protein 1 induces B cell lymphoma in transgenic mice. Proc Natl Acad Sci USA 95:11963–11968 Kumar P, Saha A, Robertson ES (2010) Epstein–Barr virus hijacks cell-cycle machinery. Microbe 5:251–256 Kuppers DA, Lan K, Knight JS, Robertson ES (2005) Regulation of matrix metalloproteinase 9 expression by Epstein–Barr virus nuclear antigen 3C and the suppressor of metastasis Nm23-H1. J Virol 79:9714–9724 Kuppers R (2003) B cells under influence: transformation of B cells by Epstein–Barr virus. Nat Rev Immunol 3:801–812 Kutok JL, Wang F (2006) Spectrum of Epstein–Barr virus-associated diseases. Annu Rev Pathol Mech Dis 1:375–404 Kwiatkowski B, Chen SY, Schubach WH (2004) CKII site in Epstein–Barr virus nuclear protein 2 controls binding to hSNF5/Ini1 and is important for growth transformation. J Virol 78:6067–6072 Lacoste V, Lavergne A, de Thoisy B, Pouliquen JF, Gessain A (2010) Genetic diversity and molecular evolution of human and non-human primate Gammaherpesvirinae. Infect Genet Evol 10:1–13 Laherty CD, Hu HM, Opipari AW, Wang F, Dixit VM (1992) The Epstein–Barr virus LMP1 gene product induces A20 zinc finger protein expression by activating nuclear factor kappa B. J Biol Chem 267:24157–24160 Lambert SL, Martinez OM (2007) Latent membrane protein 1 of EBV activates phosphatidylinositol 3-kinase to induce production of IL-10. J Immunol 179:8225–8234 Laux G, Perricaudet M, Farrell PJ (1988) A spliced Epstein–Barr virus gene expressed in immortalized lymphocytes is created by circularization of the linear viral genome. EMBO J 7:769–774 Leao M, Anderton E, Wade M, Meekings K, Allday MJ (2007) Epstein–Barr virus-induced resistance to drugs that activate the mitotic spindle assembly checkpoint in Burkitt’s lymphoma cells. J Virol 81:248–260 Lee CP et al (2007) Epstein–Barr virus BGLF4 kinase induces premature chromosome condensation through activation of condensin and topoisomerase II. J Virol 81:5166–5180 Levitskaya J et al (1995) Inhibition of antigen processing by the internal repeat region of the Epstein-Barr virus nuclear antigen-1. Nature 375:685–688 Levitskaya J, Sharipo A, Leonchiks A, Ciechanover A, Masucci MG (1997) Inhibition of ubiquitin/proteasome-dependent protein degradation by the Gly-Ala repeat domain of the Epstein– Barr virus nuclear antigen 1. Proc Natl Acad Sci USA 94:12616–12621 Lindahl T, Adams A, Bjursell G, Bornkamm GW, Kaschka-Dierich C, Jehn U (1976) Covalently closed circular duplex DNA of Epstein–Barr virus in a human lymphoid cell line. J Mol Biol 102:511–530 Lo AK et al (2007) Modulation of LMP1 protein expression by EBV-encoded microRNAs. Proc Natl Acad Sci USA 104:16164–16169

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Powell JL, Bunin NJ, Callahan C, Aplenc R, Griffin G, Grupp SA (2004) An unexpectedly high incidence of Epstein–Barr virus lymphoproliferative disease after CD34+ selected autologous peripheral blood stem cell transplant in neuroblastoma. Bone Marrow Transplant 33:651–657 Raab-Traub N (2009) Epstein–Barr virus transforming proteins: biologic properties and contribution to oncogenesis; DNA tumor viruses. Springer, Heidelberg Radkov SA, Bain M, Farrell PJ, West M, Rowe M, Allday MJ (1997) Epstein–Barr virus EBNA3C represses Cp, the major promoter for EBNA expression, but has no effect on the promoter of the cell gene CD21. J Virol 71:8552–8562 Radkov SA et al (1999) Epstein–Barr virus nuclear antigen 3C interacts with histone deacetylase to repress transcription. J Virol 73:5688–5697 Rastelli J, Hömig-Hölzel C, Seagal J, Müller W, Hermann AC, Rajewsky K, Zimber-Strobl U (2008) LMP1 signaling can replace CD40 signaling in B cells in vivo and has unique features of inducing class-switch recombination to IgG1. Blood 111:1448–1455 Rechsteiner MP et al (2008) Latent membrane protein 2B regulates susceptibility to induction of lytic Epstein–Barr virus infection. J Virol 82:1739–1747 Rickinson AB, Kieff E (2001) Epstein–Barr Virus. In: Knipe DM, Howley PM (eds) Fields Virology, 4th edn. Lippincott Williams and Wilkins, Philadelphia Rivadeneira ED et al (1999) A novel Epstein–Barr virus-like virus, HV(MNE), in a Macaca nemestrina with mycosis fungoides. Blood 94:2090–2101 Rivailler P, Cho YG, Wang F (2002a) Complete genomic sequence of an Epstein–Barr virusrelated herpesvirus naturally infecting a new world primate: a defining point in the evolution of oncogenic lymphocryptoviruses. J Virol 76:12055–12068 Rivailler P, Jiang H, Cho YG, Quink C, Wang F (2002b) Complete nucleotide sequence of the rhesus lymphocryptovirus: genetic validation for an Epstein–Barr virus animal model. J Virol 76:421–426 Robertson E, Kieff E (1995) Reducing the complexity of the transforming Epstein–Barr virus genome to 64 kilobase pairs. J Virol 69:983–993 Robertson ES (1997) The Epstein–Barr virus EBNA 3 protein family as regulators of transcription. Epstein–Barr Virus Rep 4:143–150 Robertson ES, Tomkinson B, Kieff E (1994) An Epstein–Barr virus with a 58-kilobase-pair deletion that includes BARF0 transforms B lymphocytes in vitro. J Virol 68:1449–1458 Roizmann B, Desrosiers RC, Fleckenstein B, Lopez C, Minson AC, Studdert MJ (1992) The family Herpesviridae: an update. The Herpesvirus Study Group of the International Committee on Taxonomy of Viruses. Arch Virol 123:425–449 Rooney CM et al (1998) Infusion of cytotoxic T cells for the prevention and treatment of Epstein– Barr virus-induced lymphoma in allogeneic transplant recipients. Blood 92:1549–1555 Ruf IK, Rhyne PW, Yang C, Cleveland JL, Sample JT (2000) Epstein–Barr virus small RNAs potentiate tumorigenicity of Burkitt lymphoma cells independently of an effect on apoptosis. J Virol 74:10223–10228 Saha A, Bamidele A, Murakami M, Robertson ES (2010a) EBNA3C attenuates the function of p53 through interaction with the inhibitor of growth family proteins, 4 and 5. J Virol 85:2079–2088 Saha A et al (2010b) Epstein–Barr virus nuclear antigen 3C facilitates G1-S transition by stabilizing and enhancing the function of cyclin D1. PLoS Pathog 7(2):e1001275 Saha A, Kaul R, Murakami M, Robertson ES (2010c) Tumor viruses and cancer biology: modulating signaling pathways for therapeutic intervention. Cancer Biol Ther 10:961–978 Saha A, Murakami M, Kumar P, Bajaj B, Sims K, Robertson ES (2009) Epstein–Barr virus nuclear antigen 3C augments Mdm2-mediated p53 ubiquitination and degradation by deubiquitinating Mdm2. J Virol 83:4652–4669 Sakai T et al (1998) Functional replacement of the intracellular region of the Notch1 receptor by Epstein–Barr virus nuclear antigen 2. J Virol 72:6034–6039 Sandberg M, Hammerschmidt W, Sugden B (1997) Characterization of LMP-1’s association with TRAF1, TRAF2, and TRAF3. J Virol 71:4649–4656 Schmidtko J et al (2002) Posttransplant lymphoproliferative disorder associated with an Epstein– Barr-related virus in cynomolgus monkeys. Transplantation 73:1431–1439

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Sheng W, Decaussin G, Sumner S, Ooka T (2001) N-terminal domain of BARF1 gene encoded by Epstein–Barr virus is essential for malignant transformation of rodent fibroblasts and activation of BCL-2. Oncogene 20:1176–1185 Shiama N (1997) The p300/CBP family: integrating signals with transcription factors and chromatin. Trends Cell Biol 7:230–236 Silins SL, Sculley TB (1994) Modulation of vimentin, the CD40 activation antigen and Burkitt’s lymphoma antigen (CD77) by the Epstein–Barr virus nuclear antigen EBNA-4. Virology 202:16–24 Smith PR et al (2000) Structure and coding content of CST (BART) family RNAs of Epstein–Barr virus. J Virol 74:3082–3092 Straathof KC, Bollard CM, Rooney CM, Heslop HE (2003) Immunotherapy for Epstein–Barr virus-associated cancers in children. Oncologist 8:83–98 Subramanian C, Cotter MA 2nd, Robertson ES (2001) Epstein–Barr virus nuclear protein EBNA-3C interacts with the human metastatic suppressor Nm23-H1: a molecular link to cancer metastasis. Nat Med 7:350–355 Suzan F, Ammor M, Ribrag V (2001) Fatal reactivation of cytomegalovirus infection after use of rituximab for a post-transplantation lymphoproliferative disorder. N Engl J Med 345:1000 Szekely L, Selivanova G, Magnusson KP, Klein G, Wiman KG (1993) EBNA-5, an Epstein–Barr virus-encoded nuclear antigen, binds to the retinoblastoma and p53 proteins. Proc Natl Acad Sci USA 90:5455–5459 Takada K, Nanbo A (2001) The role of EBERs in oncogenesis. Semin Cancer Biol 11:461–467 Takeshita H et al (1999) Matrix metalloproteinase 9 expression is induced by Epstein–Barr virus latent membrane protein 1 C-terminal activation regions 1 and 2. J Virol 73:5548–5555 Tong X, Drapkin R, Reinberg D, Kieff E (1995a) The 62- and 80-kDa subunits of transcription factor IIH mediate the interaction with Epstein–Barr virus nuclear protein 2. Proc Natl Acad Sci USA 92:3259–3263 Tong X, Drapkin R, Yalamanchili R, Mosialos G, Kieff E (1995b) The Epstein–Barr virus nuclear protein 2 acidic domain forms a complex with a novel cellular coactivator that can interact with TFIIE. Mol Cell Biol 15:4735–4744 Tong X, Wang F, Thut CJ, Kieff E (1995c) The Epstein–Barr virus nuclear protein 2 acidic domain can interact with TFIIB, TAF40, and RPA70 but not with TATA-binding protein. J Virol 69:585–588 Tsai DE et al (2008) EBV PCR in the diagnosis and monitoring of posttransplant lymphoproliferative disorder: results of a two-arm prospective trial. Am J Transplant 8:1016–1024 Uchida J et al (1999) Mimicry of CD40 signals by Epstein–Barr virus LMP1 in B lymphocyte responses. Science 286:300–303 Valentine R et al (2010) Epstein–Barr virus-encoded EBNA1 inhibits the canonical NF-kappaB pathway in carcinoma cells by inhibiting IKK phosphorylation. Mol Cancer 9:1 van Esser JW et al (2002) Prevention of Epstein–Barr virus-lymphoproliferative disease by molecular monitoring and preemptive rituximab in high-risk patients after allogeneic stem cell transplantation. Blood 99:4364–4369 Wang D, Liebowitz D, Kieff E (1985) An EBV membrane protein expressed in immortalized lymphocytes transforms established rodent cells. Cell 43:831–840 Wang D et al (1988) Epstein–Barr virus latent infection membrane protein alters the human B-lymphocyte phenotype: deletion of the amino terminus abolishes activity. J Virol 62:4173–4184 Wang L, Grossman SR, Kieff E (2000) Epstein–Barr virus nuclear protein 2 interacts with p300, CBP, and PCAF histone acetyltransferases in activation of the LMP1 promoter. Proc Natl Acad Sci USA 97:430–435 Wang Z et al (2010) STAT3 activation induced by Epstein–Barr virus latent membrane protein1 causes vascular endothelial growth factor expression and cellular invasiveness via JAK3 and ERK signaling. Eur J Cancer 46:2996–3006 Wei H, Zhou MM (2010) Viral-encoded enzymes that target host chromatin functions. Biochim Biophys Acta 1799:296–301

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Chapter 9

Nonhuman Primate Gamma-herpesviruses and Their Role in Cancer Ryan D. Estep and Scott W. Wong

Introduction The Gamma-herpesvirinae are a subfamily of lymphotropic herpesviruses that infect and replicate mainly in lymphoid cells and are capable of causing cellular transformation. Importantly, viruses belonging to this subfamily have been associated with oncogenesis in both humans and nonhuman primates, and have been linked to such human diseases as nasopharyngeal carcinoma, Burkitt’s lymphoma, Kaposi’s sarcoma, multicentric Castleman’s disease, and non-Hodgkin’s lymphoma. The contributions of gamma-herpesvirus infections to the development of other cancers are also a possibility, and thus, an intense focus has been placed on these viruses to better understand the mechanisms by which they induce oncogenesis. Overall, the gamma-herpesviriniae represent examples of fine tuned virus–host relationships, in which typical infection of natural host species is relatively harmless, and only under specific and much less frequent circumstances, may be capable of promoting the development of cancer. The gamma-herpesvirus subfamily can be further divided into the lymphocryptovirus (or g-1) genus and the rhadinovirus (or g-2) genus, based on genomic organization and sequence homology. In humans, the lymphocryptovirus genus is

R.D. Estep Vaccine and Gene Therapy Institute, Oregon Health & Science University, 505 NW 185th Avenue, Beaverton, OR 97006, USA S.W. Wong (*) Vaccine and Gene Therapy Institute, Oregon Health & Science University, 505 NW 185th Avenue, Beaverton, OR 97006, USA Division of Pathobiology and Immunology, Oregon National Primate Research Center, Beaverton, OR, USA Department of Molecular Microbiology and Immunology, Oregon Health & Science University, Portland, OR, USA e-mail: [email protected] E.S. Robertson (ed.), Cancer Associated Viruses, Current Cancer Research, DOI 10.1007/978-1-4614-0016-5_9, © Springer Science+Business Media, LLC 2012

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Fig. 9.1 Alignment of specific primate gamma-herpesviruses that have been sequenced. Conserved blocks of genes are indicated (black) along with genes that are unique (white) to the each gammaherpesvirus. The genomes of Epstein–Barr virus and rhesus lymphocryptovirus are represented as one, as they are nearly identical (Rivailler et al. 2002)

represented by Epstein–Barr virus (EBV), while the rhadinovirus genus is represented by Kaposi’s sarcoma-associated herpesvirus (KSHV). Lymphocryptoviruses with similarity to human EBV have been identified in many species of Old- and New-World monkeys (Dunkel et al. 1972; Kalter et al. 1972; Landon and Malan 1971; Levy et al. 1971; Naito et al. 1971), and similarly, rhadinoviruses have been identified in many primates (Damania and Desrosiers 2001; Greensill et al. 2000; Lacoste et al. 2000a, b; Melendez et al. 1968; Simas and Efstathiou 1998). Species specificity appears to preclude infection of nonhuman primates with human gammaherpesviruses (Renne et al. 2004; Wang et al. 2001), thus preventing the study of the mechanisms of pathogensis of these human viruses in primate model systems. However, due to the discovery of highly related primate counterparts of these human viruses, and their ability to promote the development of similar disease sequelae as their human counterparts, these primate viruses provide relevant model systems for understanding and deciphering the oncogenic mechanisms of these herpesviruses in vivo. Rhesus macaque lymphocryptovirus (LCV), Herpesvirus saimiri (HVS), and Rhesus macaque rhadinovirus (RRV) are discussed in the following sections as models of primate gamma-herpesviruses oncogenesis that are important for understanding mechanisms of primate herpesvirus oncogenesis. To illustrate the relatedness of the simian gamma-herpesviruses to the human, the genomes of the viruses are depicted in Fig. 9.1.

Herpesvirus Saimiri Herpesvirus saimiri (HVS, saimirine herpesvirus 2) is the prototypical rhadinovirus, the natural host of which is the squirrel monkey (Saimiri sciureus). The virus was originally isolated from a kidney cell culture of a healthy squirrel monkey (Melendez et al. 1968) and was also identified in peripheral blood cells of persistently infected

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squirrel monkeys by coculture with permissive cell lines such as owl monkey kidney (OMK) cell line (Desrosiers and Falk 1982). In the wild, most squirrel monkeys are naturally infected with HVS within the first two years of life and remain asymptomatically and persistently infected for the remainder of their lives, without the development of any overt disease (Melendez et al. 1968). Although infections of squirrel monkeys with HVS are generally asymptomatic, the virus can cause fatal T cell lymphomas in other New-World primates, including, marmosets, tamarins, and owl monkeys, as well as Old-World monkeys, including rhesus macaques and cynomolgus monkeys (Fickenscher and Fleckenstein 2001), suggesting that in its natural host HVS is not oncogenic, but when introduced into other nonhost species, the virus has the potential to produce cancer. HVS strains are classified into three subgroups (A, B, and C), based on their pathogenic qualities and genomic similarity. While most genes are well conserved amongst different HVS strains, there is extensive sequence divergence at the left end of the HVS L-DNA of the different subgroups. Subgroup C viruses possess the highest oncogenic properties, and are the only subgroup that is capable of immortalizing human, rabbit, and rhesus monkey lymphocytes, and causing fulminant lymphomas in both Old-World and New-World primates. The genomes of HVS strains A11 and C488 have been fully sequenced (Albrecht et al. 1992; Ensser et al. 2003), with both HVS genomes containing 76–77 open reading frames (ORFs). The genomes of all HVS strains possess homologues of several cellular genes, which are thought to have been pirated from host cell genes during evolution of the virus. These genes include those involved in various processes, such as regulation of cellular growth and proliferation (e.g., viral cyclin), nucleotide metabolism (e.g., viral dihydrofolate reductase [DHFR]), complement cascade activation (e.g., viral complement control protein homologue [CCPH]), apoptosis (e.g., viral Bcl-2), and cytokine signaling (e.g., viral IL-17 and vGPCR) (Fickenscher and Fleckenstein 2001). All of these genes could potentially contribute to the oncogenic potential of the virus, although evidence appears to suggest that they are not required for T cell transformation and pathogenicity, and are likely more important for persistence of the virus within its natural host species. However, a gene encoding a protein with limited homology to any cellular protein is located near the left end of the genome of all three HVS subtypes. This protein, termed the Saimiri transformation-associated protein (Stp), has been shown to play a critical role in direct transformation of T cells by HVS in vitro and in vivo (Duboise et al. 1998; Jung and Desrosiers 1991). Interestingly, although Stp of HVS subgroup A and C (StpA and StpC) are both transforming in vitro, with StpC displaying a higher transforming potential than StpA, Stp of HVS subtype B (StpB) does not appear capable of inducing transformation (Choi et al. 2000; Jung et al. 1991). These findings correlate closely with the oncogenic potential of the different HVS subtypes in vivo and indicate the importance of Stp to viral oncogenesis (Tsygankov 2005). StpC has been extensively studied and has been found to be a perinuclear membrane protein that is capable of interacting with and activating host cell signaling pathways, including the Ras-ERK pathway and NF-kB pathways (Jung and Desrosiers 1995; Lee et al. 1999; Tsygankov 2005). Thus, as a result of alteration of normal cellular signaling, expression of StpC may

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result in cellular transformation. In addition to Stp, Subgroup C strains also encode another transforming protein termed the tyrosine kinase-interacting protein (Tip), which has also been found to be important for viral transformation. The tip gene is located directly downstream from the stp gene and is transcribed as part of a single bicistronic mRNA that encodes both proteins. Tip was initially identified as a protein that interacted with a T cell-specific tyrosine kinase Lck (Biesinger et al. 1995; Fickenscher and Fleckenstein 2001). Indeed, Tip has been shown to activate Lck (Wiese et al. 1996) and, in addition, has been found to be capable of activating STATs, NF-AT, and the nuclear mRNA export protein Tap (Hartley and Cooper 2000; Isakov and Biesinger 2000; Lund et al. 1997; Yoon et al. 1997). Although Tip is also capable of inducing T cell lymphomas in transgenic mice (Wehner et al. 2001), the exact role that Tip may play in cellular transformation remains unclear (Tsygankov 2005). However, based on in vivo studies using Stp and Tip deleted forms of HVS, it is apparent that both proteins are essential for T cell transformation of subgroup C viruses in vivo and in vitro (Duboise et al. 1998). The fact that subgroup C viruses encode two highly oncogenic viral proteins may also partly explain why these viruses are more oncogenic in vivo than subgroup A and B viruses. Although there is no true counterpart to HVS that has been identified in humans, the value of this animal model is still extremely important. The mechanisms by which HVS is capable of existing within its host population with no apparent disease manifestation yet remains capable of inducing lymphoma in other nonhost species suggest a close coevolution of virus and host that allows viral persistence and spread with no overt symptoms in the host. Understanding the mechanisms by which both the virus and natural host maintain this close relationship, and what exactly prevents induction of viral-related disease, provides an important model to help understand species restriction in terms of oncogenic herpesviruses, and could provide insight into potential targets for treatment and prevention of herpesvirus-induced cancers.

Rhesus Macaque Lymphocryptovirus Several lymphocryptoviruses have been identified in both Old- and New-World monkeys based upon cross-reactivity with antibodies directed against human EBV proteins, due to the conservation of sequences between these viruses (Dunkel et al. 1972; Kalter et al. 1972; Landon and Malan 1971; Levy et al. 1971; Naito et al. 1971). Rhesus macaque LCV (RhLCV), or Cercopithecine herpesvirus 15, is emerging as a useful animal model for studying EBV infection and disease development in vivo using the rhesus macaque as a model (Carville et al, 2008). RhLCV, like EBV in humans, is spread through oral secretions, and is generally ubiquitous within captive rhesus macaque populations, with essentially all animals seroconverting by the time they reach 2 years of age (Carville and Mansfield 2008). Two distinct lineages of RhLCV (RhLCV1 and RhLCV2) have been identified (Cho et al. 1999), and the complete sequence of the RhLCV1 genome has recently been determined (Rivailler et al. 2002). The RhLVC genome is 171,096 nucleotides in length, with a GC content of 62%,

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Fig. 9.2 Lymphoma and retroperitoneal fibromatosis (RF) in RRV-infected rhesus macaques. (a) Hematoxylin and eosin (H&E) stain of lymphoma isolated at biopsy from the breast of animal 19185, original magnification ×1,000. (b) Combined in situ hybridization of lymphoma with RRV cosmid probe (purple) and immunohistochemistry with mouse anti-CD20 (brown) and anti-CD3+ (gray) demonstrates that RRV is present in CD20+ cells comprising the lymphoma, original magnification ×1,000. (c) Same as (b), except cosmid vector control replaces the RRV cosmid probe. (d) H&E stain of lymphoma isolated from liver of animal 19286 during necropsy, original magnification ×200. (e) Same as (b) except RRV cosmid probe detected by immunofluorescence (purple) with no evidence of CD20 staining. (f) Similar to (c), this is the control for panel (e). (g) H&E stain of RF attached to the stomach of animal 18483, original magnification ×400. (h) Combined in situ hybridization with RRV cosmid probe (purple) and immunohistochemistry with anti-CD20 (brown) and anti-CD3 (gray) shows that RRV is not present in the CD20- or CD3positive cells, original magnification ×1,000. Arrows point to RRV-positive cells that have spindlelike morphology. (i) Identical to (h), except vector control replaces the RRV cosmid, original magnification ×1,000. (This research was originally published in Blood. Orzechowska et al. (2008) Rhesus macaque rhadinovirus-associated non-Hodgkin’s lymphoma: animal model for KSHV associated malignancies. 112:4227–4234. © the American Society of Hematology)

which closely compares to the genome of EBV. Further, the overall nucleotide homology between the RhLCV and EBV genomes is 65%, and the genetic organization of these viruses is colinear (Fig. 9.2). RhLCV encodes eighty open reading frames (ORFs), all of which share some level of homology to genes in human EBV, with the average homology between EBV and RhLCV ORFs being 75.6% (Rivailler et al. 2002).

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Human EBV is generally spread via oral secretions, and initially infects and replicates in the oropharynx, with the subsequent development of a generally asymptomatic and persistent latent infection of peripheral blood B lymphocytes. In addition to the typical disease associated with primary infection, EBV is capable of inducing transformation of infected cells, and has been linked to the development of several human malignancies, including Burkitt’s lymphoma, Hodgkin’s lymphoma, and nasopharyngeal carcinoma (Fields et al. 2007). Previous attempts to infect nonhuman primates with human EBV have been generally unsuccessful and may largely be due to species-specific restrictions that prevent LCVs from immortalizing B cells other than those derived from their natural host species (Moghaddam et al. 1998; Wang et al. 2001). Thus, infection of nonhuman primates with LCVs specific to the host species in question has been examined as a more relevant experimental model system to better understand EBV infection in vivo. Recently, RhLCV has emerged as a feasible model, allowing for the study of LCV infection and pathogenesis in rhesus macaques. RhLCV infection of rhesus macaques has been shown to result in disease manifestations very similar to those associated with EBV infection in humans. For example, experimental oral inoculation of naïve rhesus macaques with RhLCV results in the development of lympadenopathy, lymphocytosis, and an increase in CD23+ B cells, all of which are symptoms of primary EBV infection and mononucleoisis in humans (Moghaddam et al. 1997). Further, RhLCV is also capable of infecting peripheral blood B cells, in which it establishes a persistent latent infection, with occasional reactivation of virus from these cells, followed by shedding of virus in oral secretions. Thus, primary infection of RhLCV mirrors very closely the symptoms of primary EBV infection in humans. In addition to disease associated with acute RhLCV infection, several RhLCV-associated malignancies have also been shown to occur in rhesus macaques coinfected with SIV, and are similar to EBV-associated cancers that frequently occur in AIDS patients. For example, B cell tumors can develop in SIV-infected rhesus macaques that display many similarities to some Non-Hodgkin’s lymphomas (NHLs) seen in AIDS patients that are associated with EBV infection (Carville and Mansfield 2008; Shibata et al. 1993). SIV-associated NHLs show a similar pattern of development to those seen in HIV-infected patients, and direct examination of these lymphomas for the presence of RhLCV sequences demonstrates a strong association of the virus with their development (Carville and Mansfield 2008; Habis et al. 1999, 2000). In addition to lymphoma, other manifestations such as squamous epithelial proliferative lesions have also been found to develop in SIV-infected rhesus monkeys and appear similar to oral hairy leukoplakia that is commonly associated with EBV infection in humans. Indeed, these lesions have been found to contain EBV-like sequences by immunohistochemistry and in situ hybridization, suggesting a causative role for RhLCV in this disease in rhesus macaques (Baskin et al. 1995). Also of importance, experimental studies involving inoculation of LCV-seronegative macaques with RhLCV-immortalized B cells indicate that RhLCV infection of immunnosuppressed animals results in the induction of lymphomagenesis (Rivailler et al. 2004). Taken together, these studies provide strong evidence that RhLCV infection and disease development in rhesus macaques

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closely parallels EBV infection in humans, and thus provides a novel system to allow for the analysis of the mechanisms of LCV pathogenesis in vivo. Several EBV-encoded genes have been implicated as major regulators of viral transformation and oncogenesis, namely, the latently expressed Epstein–Barr nuclear antigens (EBNAs) and latent membrane proteins (LMPs) (Farrell 1995). Although these genes are normally important for establishment of latency and persistence of virus within infected cells, under certain conditions expression of these genes can also result in cellular transformation, leading to aberrant cell growth and ultimately the development of viral-related cancers (Young and Murray 2003). Importantly, RhLCV possesses homologues of these EBV genes, suggesting that these two highly related viruses may have similar mechanisms of transformation in vivo (Wang et al. 2001). Indeed, in vitro studies of several RhLCV oncogenes indicate that they may function similarly, if not identically, to their EBV counterparts. For example, RhLCV LMP2A protein, which is 62% identical to EBV LMP2A, has been shown to be capable of activating cellular signaling pathways and inhibiting differentiation of epithelial cells, suggesting similar properties to its EBV counterpart and a possible role in pathogenesis (Siler and Raab-Traub 2008). RhLCV EBNA genes have also been examined and have been shown to share similar structural and functional similarities to the homologous EBV genes (Jiang et al. 2000; Peng et al. 2000). Further, analysis of tumors from RhLCV-infected rhesus macaques indicates that expression of RhLCV homologs of EBV transforming genes are associated with development of malignant lymphomas (Blaschke et al. 2001). Therefore, as is the case for EBV, expression of transforming viral genes during infection may result in the development of viral cancers in vivo. In addition to the known transforming genes in EBV, other viral factors conserved between EBV and RhLCV with potential roles in oncogenesis are likely yet to be identified. This is evidenced by the recent discovery of several noncoding viral microRNAs (miRNAs) in RhLCV that share significant homology to those found in EBV (Cai et al. 2006). miRNAs have been identified in numerous organisms and viruses and are capable of posttranscriptionally regulating gene expression via interactions with target mRNAs (Gottwein and Cullen 2008). Importantly, miRNAs have also been proposed to be involved in the development of cancer (McManus 2003), and given that viral miRNAs play roles in regulating the expression of both viral and cellular genes, it is plausible that in some cases, viral miRNAs such as those encoded by RhLCV may be involved in viral oncogenesis (Dykxhoorn 2007; Gottwein and Cullen 2008). Further analysis will be necessary to determine the exact role of RhLCV and EBV miRNAs in viral oncogenesis. Since RhLCV is capable of inducing disease manifestations in infected rhesus macaques similar to those seen in EBV-infected humans, and given the high genetic similarity of these viruses, RhLCV provides a highly relevant and functional animal model system that allows for in vivo analysis of the mechanisms of EBV-associated pathogenesis and oncogenesis. Further experimental analysis of RhLCV using this system may lead to a better understanding of the exact mechanisms of viral transformation, and allow for the determination of roles of individual viral genes in oncogenesis. Most importantly, the use of the RhLCV/rhesus macaque model will allow for

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the development and testing of potential vaccine strategies and therapeutics to prevent or treat LCV-induced malignancies.

Rhesus Macaque Rhadinovirus Rhesus macaque rhadinovirus (RRV) is a g-herpesvirus of rhesus macaques, which has been shown to be closely related to human herpesvirus 8 (HHV-8)/Kaposi’s sarcoma-associated herpesvirus (KSHV). KSHV is the causative agent of Kaposi’s sarcoma (KS), as well as B cell lymphoproliferative disorders (LPD) primary effusion lymphoma (PEL), multicentric Castleman’s disease (MCD), and some non-Hodgkin’s lymphomas (Cesarman et al. 1995; Chang et al. 1994; Oksenhendler et al. 2002; Soulier et al. 1995), diseases that are frequently associated with HIV infection. An RRV strain (RRV17577), was obtained from a simian immunodeficiency virus (SIV)-infected macaque that had developed B cell hyperplasia, and upon isolation and characterization of this herpesvirus isolate, the RRV17577 genome was found to be essentially colinear with KSHV, retaining many of the ORFs believed to be involved in the pathogenesis of KSHV (Searles et al. 1999) (Fig. 9.2). Specifically, 67 of 79 ORFs are similar to those in KSHV. The general structure of the RRV17577 genome is similar to other herpesviruses and consists of a long unique region (LUR) of ~131 kb in length, flanked on both ends by regions of terminal repeats. RRV is a natural pathogen of macaques, with greater than 90% of captive rhesus macaques having been found to be seropositive for RRV (Damania and Desrosiers 2001). RRV transmission between susceptible hosts is believed to occur via shedding in saliva, and similar to KSHV, RRV is capable of establishing a latent infection in B cells (Bergquam et al. 1999). KSHV infection of primates has thus far proven unsuccessful, and due to the high genetic similarity and pathogenic properties to KSHV, RRV infection of rhesus macaques has become a widely utilized animal model to study KSHV disease development in vivo, particularly in the setting of SIV induced immunodeficiency. Experimental inoculation of rhesus macaques with RRV17577 that have previously been infected with SIVmac239 promotes the development of B cell hyperplasia, persistent lymphadenopathy, splenomegaly, and hypergammaglobulinemia (Bergquam et al. 1999; Wong et al. 1999). The hyperplastic lymphoproliferative disorder (LPD) in RRV-infected macaques closely resembles the plasma cell variant of multicentric Castleman’s disease B cell hyperplasia seen in humans infected with KSHV. More recently, RRV infection has also been associated with lymphomagenesis and a mesenchymal cell proliferative lesion in SIVinfected rhesus macaques (Orzechowska et al. 2008). Thus, RRV infection of immune compromised rhesus macaques is an invaluable model for KSHV-associated disease development that is utilized to dissect the mechanisms of viral pathogenesis and to develop potential targets for vaccine development against KSHV. Numerous genes in KSHV have been implicated in oncogenesis, and a majority of these genes are conserved in the RRV genome (Searles et al. 1999; Wen and Damania 2010). Many of these viral oncogenes are homologues of cellular genes,

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from which they are believed to have been pirated during evolution of the virus, and are capable of affecting numerous processes, such as cellular proliferation (e.g., viral Cyclin) (Li et al. 1997), apoptosis (e.g., viral Bcl-2) (Sarid et al. 1997), and immunoregulation (e.g., viral interferon regulatory factors/vIRFs) (Gao et al. 1997). In general, the main function of these viral genes is likely not to induce transformation per se, but rather to help establish an environment that favors viral infection, replication, and persistence within an infected host. However, due to the fact that these genes dysregulate normal cellular function and immune control, their expression may also result in cellular transformation and the development of cancer. Examination of several RRV homologues of KSHV genes has indicated that these genes retain similar function to their KSHV counterparts and, thus, are likely to play similar roles in regard to oncogenesis in vivo. For example, the KSHV encoded vIL-6 molecule has been shown to be a functional homologue of cellular interleukin 6 (IL-6), and expression of this protein has been associated with KS, PEL, and MCD (Staskus et al. 1999). RRV also encodes a vIL-6 homologue, and studies indicate that RRV vIL-6 is capable of promoting B cell proliferation in vitro (Kaleeba et al. 1999) and that expression of this viral protein is associated with the development of lymphoproliferative and mesenchymal cell proliferative disorders in RRV-infected rhesus macaques (Orzechowska et al. 2008, 2009) (Fig. 9.3). Another RRV gene, ORF74, which encodes the viral G protein-coupled receptor (GPCR), shares sequence similarity to the KSHV vGPCR, a protein that has been suggested to play a major role in viral oncogenesis (Arvanitakis et al. 1997; Bais et al. 1998; Cannon 2007; Cesarman et al. 1996). The vGPCRs of KSHV and RRV are homologues of CXCR2, the cellular receptor for IL-8, and are thus capable of regulating cellular signaling pathways in cells in which they are expressed. Like the KSHV vGPCR, the RRV vGPCR has been shown to possess constitutive and ligand-dependent signaling abilities, promote cytokine secretion, and induce transformation of cells in which it is expressed (Estep et al. 2003). Thus, the RRV vGPCR is likely to contribute to the development of RRV-associated malignancies in a fashion similar to the vGPCR of KSHV. Other RRV genes with no apparent cellular counterparts are also likely to play a role in viral transformation. For example, the R1 protein of RRV, which shares similarity to KSHV K1, has been shown to be transmembrane signaling proteins with oncogenic potential in vitro and in vivo (Damania et al. 1999, 2000). In addition, RRV encodes several microRNAs miRNAs that may be involved in pathogenesis, with the majority sharing no apparent homology to those that were similarly identified in KSHV (Schafer et al. 2007; Umbach et al. 2010). Importantly, all of the RRV miRNAs that have been identified have been found to be expressed within B cell lymphoma and retroperitoneal fibromatosis tissues isolated from RRVinfected macaques, suggesting that the expression of these miRNAs may be involved in the development of RRV related cancers (Umbach et al. 2010). Further in vivo analysis of the roles of the numerous RRV-encoded oncogenes will be the focus of future research, particularly through the creation of recombinant forms of RRV with mutated or deleted forms of these genes, and the use of the rhesus macaque model of infection for the assessment of effects on disease development (Estep et al. 2007). By studying the function of these viral oncogenes in the context of an in vivo infection

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Fig. 9.3 Detection of vIL-6 in RRV-associated lymphomas, RF and MCD. Lymphoma from RM 19185 was stained with murine monoclonal antibodies specific for RRV vIL-6 and CD20 (a), lymphoma from animal 19286 was stained with murine monoclonal antibodies specific for RRV vIL-6 and goat anti-human IgM, and nuclei (c). (b) and (d) are isotype controls, original magnification ×630. Panel (a), vIL-6 (green) and CD20 (red); panel (b), isotype controls for vIL-6 and CD20; panel (c), vIL-6 (green) and IgM (red), and nuclei (blue); and panel (d), isotype controls for vIL-6 and IgM. RF from animal 18483 was stained for vIL-6 (green), vimentin (red), and nuclei (blue) (panel e), original magnification ×630. MCD lesion from animal 19455 was stained for vIL-6 (green), CD20 (red) and nuclei (blue) (panel f), original magnification ×630. (This research was originally published in Blood. Orzechowska et al. (2008) Rhesus macaque rhadinovirusassociated non-Hodgkin’s lymphoma: animal model for KSHV-associated malignancies. 112:4227–4234. © the American Society of Hematology)

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in a primate model that closely recapitulates KSHV infection of humans, the contributions of individual genes to viral pathogenesis can be examined, and this in turn will provide unique insight into the roles they and their KSHV counterparts may play in oncogenesis.

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Chapter 10

Rhadinoviruses: KSHV and Associated Malignancies Susann Santag and Thomas F. Schulz

Kaposi’s Sarcoma Herpesvirus Kaposi’s Sarcoma Herpesvirus (KSHV), also termed human Herpesvirus 8 (HHV8), was discovered by Y. Chang and P. Moore in a Kaposi’s Sarcoma tissue of an AIDS patient (Chang et al. 1994). KSHV is the only known Rhadinovirus capable of infecting humans. It is known to cause three human neoplastic diseases and may be involved in other clinical manifestations.

Origin and Evolution KSHV is today of low prevalence (0–5%) in Northern Europe (Preiser et al. 2001), most of North America (Baillargeon et al. 2001; Kouri et al. 2004; Engels et al. 2007) and most of Asia (de Sanjose et al. 2009), of intermediate prevalence (5–20%) in countries around the Mediterranean Sea (Whitby et al. 1998), in parts of South America (Mohanna et al. 2005) and in some ethnic populations [e.g. some South American Indian tribes (Biggar et al. 2000; Whitby et al. 2004; Mohanna et al. 2007), South Siberia (Cassar et al. 2010) or Xinjiang in China (Dilnur et al. 2001)] but of high prevalence (20–80%) in most countries of sub-Saharan Africa (Dedicoat and Newton 2003; Klaskala et al. 2005; Adjei et al. 2008). There is good evidence that KSHV has co-evolved with human populations. Thus, sequence variation, mainly in the variable K1 gene of KSHV, has been used to define five lineages (A–E) and additional subgroups (Hayward 1999). Of these, lineage B and subtype A5 are commonly found in sub-Saharan Africa, lineage D in

S. Santag • T.F. Schulz (*) Institute of Virology, Hannover Medical School, Hannover, Germany e-mail: [email protected] E.S. Robertson (ed.), Cancer Associated Viruses, Current Cancer Research, DOI 10.1007/978-1-4614-0016-5_10, © Springer Science+Business Media, LLC 2012

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old Australasian populations (Cassar et al. 2007) and lineage E in native South American populations (Biggar et al. 2000). This pattern suggests that KSHV spread across the globe with migrating human populations. Why KSHV should have become infrequent in many populations and geographic areas, while remaining highly prevalent in some, remains unexplained.

Transmission and Risk Factors Horizontal transmission via saliva seems to be a common route of infection (Vieira et al. 1997; Cattani et al. 1999; Dedicoat et al. 2004; Casper et al. 2006). In endemic areas, infection occurs frequently during childhood and increases with age (Mayama et al. 1998; Whitby et al. 2000; Cunha et al. 2005; Plancoulaine et al. 2000; MalopeKgokong et al. 2010). In addition, sexual transmission of KSHV contributes to its spread in endemic countries (Butler et al. 2009) and appears to be the main transmission route among individuals at increased risk of sexually transmitted disease both in endemic and non-endemic countries. Less important routes of infection include injection drug use and blood borne transmission (including transmission via transfusion) as well as organ transplantation (Atkinson et al. 2004; Hladik et al. 2006; Cannon et al. 2009; Vamvakas 2010).

KSHV Associated Diseases Table 10.1 summarises the disease manifestations that have been attributed to KSHV infection. For three neoplastic diseases, Kaposi’s Sarcoma (KS), primary effusion lymphoma (PEL) and the plasma cell variant of multicentric Castleman’s disease (MCD), a causative role of KSHV is accepted. A recent evaluation of the available epidemiological and molecular evidence by an IARC (International Agency for Research on Cancer)/WHO working group led to the classification of KSHV as a group I carcinogen (Bouvard et al. 2009). Reported manifestations of primary KSHV infections are based on relatively few case reports and small studies (see below). In addition, a long list of clinical conditions has been attributed to KSHV infection, but hardly any have been corroborated by a sufficient number of laboratories for such associations to have become generally accepted.

KSHV Primary Infection Several case reports suggest that KSHV primary infection in HIV-infected patients or organ transplant recipients can occasionally lead to fever, arthralgia, cervical lymphadenopathy, splenomegaly and pancytopenia, which spontaneously resolves within

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Table 10.1 Spectrum of KSHV-associated diseasesa Stage of infection/KSHV association Disease Primary infection Mostly asymptomatic; on rare occasions infectious mononucleosis-like illness, KS, MCD or rare cases of bone marrow failure in immunosuppressed patients (section “KSHV Primary Infection”) Accepted causative role/strong • KS (Newton et al. 2006) (all four types) (section association “Kaposi Sarcoma”) • Primary effusion lymphoma (PEL) (Cesarman et al. 1995) (section “Primary Effusion Lymphoma”) • Plasma cell variant of multicentric Castleman’s disease (MCD) (Soulier et al. 1995) (section “Multicentric Castleman’s Disease”) • Plasmablastic lymphoma arising from MCD (Dupin et al. 2000; Oksenhendler et al. 2002; Dargent et al. 2007; Ustun et al. 2009) Unconfirmed associationb • Multipe Myeloma (Rettig et al. 1997; Said et al. 1997); could not be confirmed by others (MacKenzie et al. 1997; Brander et al. 2002) • Mesenchymal tumours (1 case KSHV-positive out of 76) (Kazakov et al. 2002) • Hemophagocytic syndrome (Luppi et al. 2002); could not be confirmed by others (Mikala et al. 1999) • Other lymphomas (Lazzi et al. 1998; de Sanjose et al. 2004; Lazzi et al. 2006) • Large-cell centroblastic lymphomas (Hansen et al. 2000) • Autoimmune bullous dermatoses: • Pemphigus vulgaris and pemphigus folicaeus (Tye 1970; Memar et al. 1997) • Bullus phemigoid (Gaspari et al. 1997) • Idiopathic pulmonary arterial hypertension (IPAH) (Cool et al. 2003); could not be confirmed by others (Henke-Gendo et al. 2005; Katano et al. 2005) • Sarcoidosis (Di Alberti et al. 1997); could not be confirmed by others (Lebbe et al. 1999) • Kikuchi’s disease (Huh et al. 1998); could not be confirmed by others (George et al. 2003; Cho et al. 2007) • Prostate cancer (Hoffman et al. 2004); could not be confirmed (Jenkins et al. 2007) a KSHV association was based on PCR, IgG antibody responses, in situ hybridisation on tumour cells and immunohistochemistry on tissue sections b On the basis of KSHV detection by PCR or immunohistochemistry in some, but not most, studies

the course of several weeks (Oksenhendler et al. 1998; Luppi et al. 2000). In a study of HIV1-negative men, primary infection was associated with mild, non-specific signs and symptoms of diarrhoea, fatigue, localised rash and lymphadenopathy (Wang et al. 2001). Goudsmit and colleagues showed that within a Dutch MSM (men who have sex with men) cohort, KSHV seroconversion in HIV-infected patients

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follows a brief, low-grade viremia which was mostly asymptomatic (Goudsmit et al. 2000). In an endemic region (Egypt), Andreoni and colleagues observed that primary KSHV infection in immunocompetent children can be symptomatic and associated with a transient febrile skin rash (Andreoni et al. 2002). These results were confirmed by a study analysing primary g-herpesviral infection among Zambian children, showing a weak association between the history of rashes in children with primary KSHV infection (Minhas et al. 2010). These findings indicate that generally mild symptoms can occur during primary infection of immunocompetent individuals and that these can be more severe in immunocompromised patients.

Kaposi Sarcoma In 1872, the Hungarian dermatologist Moritz Kaposi published a description of an entity termed “idiopathic multiple pigmented sarcoma of the skin” (Kaposi 1872), which was afterwards renamed Kaposi’s sarcoma (KS). He described several cases of elderly patients with skin lesions, typically on the lower extremities. In his histological examination, Moritz Kaposi noted”… a picture of small cell carcinoma, with cells appearing in masses and clumps”. All patients described by Kaposi died of Kaposi’s sarcoma. For more than a century, this disease was known as a rare and low-grade malignancy in Europe and North America. By 1950, the total number of cases reported was approximately 600 (McCarthy and Pack 1950). Until 1980, KS had incidence rates of 0.02–0.06/100,000 in the USA, seen mostly in persons of European descent (Safai and Good 1981) and up to 1/100,000 (men) or 0.27/100,000 (women) in Italy, with higher incidence rates in Sicily (Geddes et al. 1994). In 1981, several groups observed an epidemic of KS in young, homosexually active men (Friedman-Kien 1981; Gottlieb et al. 1981; CDC 1981). The course of disease in these patients was often fulminant and malignant. Friedman-Kien noted several opportunistic infections and suspected this to be the tip of the iceberg (Friedman-Kien 1981). He was proved correct in the following years and AIDS-KS, also called epidemic KS, is until today one of the first manifestations of AIDS. It took again more than 10 years until KSHV, the infectious agent necessary to develop KS, was discovered in 1994. The epidemiological evidence for a causative role of KSHV in the pathogenicity of KS is extensive and convincing and supported by numerous cohort and case–control studies. KSHV DNA could be detected in most KS lesions, independently of HIV status (Dupin et al. 1995; Dictor et al. 1996) and within these lesions, KSHV could be localised to the spindle cells, the hallmark of the tumour (section “Clinical Features and Treatment”) (Boshoff et al. 1995). There is a strong correlation between KSHV seroprevalence and KS incidence. Worldwide, seroprevalence for KSHV is high in AIDS risk groups that also show an increased incidence of KS, and is low in groups in whom KS is rare (Whitby et al. 1995; Gao et al. 1996; Moore et al. 1996; Rezza et al. 1999). Furthermore, KSHV seroprevalence is high in organ transplant recipients who developed KS after transplantation

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(Parravicini et al. 1997; Regamey et al. 1998). Globally, KS is more common in regions with an intermediate or high prevalence of KSHV (e.g. Mediterranean region, Africa), whereas the incidence of KS is low in regions with lower prevalence of KSHV (e.g. the USA, Northern Europe) (Gao et al. 1996). Both case–control and prospective cohort studies strongly link infection with KSHV to KS (Chang et al. 1994; Melbye et al. 1998; Grulich et al. 1999; Sitas et al. 1999; Newton et al. 2003). The detection of KSHV DNA in peripheral blood often precedes the development of KS lesions and is associated with an increased risk of the subsequent appearance of KS lesions (Moore et al. 1996; Laney et al. 2007). In addition, several cohort studies on AIDS- or iatrogenic KS demonstrated that high antibody titres against KSHV prior to diagnosis are associated with an increased risk of KS (Regamey et al. 1998; Renwick et al. 1998; Frances et al. 1999; O’Brien et al. 1999; Newton et al. 2006). In general, 90–100% of patients with KS have high antibody titres to KSHV, independent of ethnicity or geographic localisation (Chatlynne and Ablashi 1999).

Different Forms of KS Clinically and epidemiologically, four variants of KS have been defined. They are histologically indistinguishable and represent variants of the same disease.

Classical KS Classical KS represents the original form of KS described by Moritz Kaposi, which occurs mainly in elderly men with Mediterranean or eastern European Jewish ancestry. Even though KS was first described as an aggressive tumour (Kaposi 1872), this form is today known to be a rare, mainly indolent and usually involves the skin of the lower and higher extremities as well as the trunk. Internal involvement does occur in approximately 10% of all cases. Classic KS is more common in men than in women with a male–female ratio between 15:1 and 3:1 (Antman and Chang 2000; Geddes et al. 1994) and occurs predominantly in elderly people with a mean age at diagnosis between 65 and 70 years (DiGiovanna and Safai 1981).

Endemic KS Endemic KS is more aggressive, may involve lymph nodes in addition to the skin and affects HIV-negative people and children (Slavin et al. 1970; Ziegler and Katongole-Mbidde 1996). It occurs mainly in certain parts of central and eastern Africa. The endemic form appeared before the HIV epidemic and was well documented in Uganda in the 1960s (Hutt and Burkitt 1965).

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Iatrogenic KS The incidence of KS is about 100- to 500-fold increased in patients receiving immunosuppressive treatment after solid organ transplantation in comparison to the general population (Zavos et al. 2004; Tessari et al. 2006; Grulich et al. 2007). As a result of the different KSHV prevalence rates, KS occurs in less than 1% of transplant recipients in the USA and Western Europe (Farge 1993) but increases up to 4% of transplant patients in Saudi Arabia (Qunibi et al. 1998).

Epidemic (AIDS-) KS Epidemic or AIDS-KS is the most common and most aggressive form of KS. It not only involves the skin and lymph nodes but often also disseminates to the inner organs, including lung, gastrointestinal tract, liver and spleen and is frequently fatal. AIDS-KS became the most common HIV-associated malignancy in the 1990s particularly affecting young homosexual men who had a 20-fold higher risk of KS development compared to other HIV transmission risk groups (Beral et al. 1990). Cohort studies revealed a high incidence of KS among homosexual men who were infected with both HIV and KSHV at baseline: the 10-year probability to develop KS was nearly 50% (Martin et al. 1998; Renwick et al. 1998). Even though adjusted incidence rates for AIDS-associated KS declined from 15.2 in years 1992–1996 to 4.9 during 1997–1999 after the introduction of effective anti-HIV therapy (Carrieri et al. 2003), this form of KS is still one of most common HIV-associated malignancies (Dal Maso et al. 2009). Owing to the widespread HIV epidemic in Africa, KS is currently the most common cancer in men in Africa (Dedicoat and Newton 2003).

KS and Its Co-factors Globally, HIV-infection is associated with an approximately 3,000-fold increase in risk for KS and is its most important co-factor (Grulich et al. 2007). The risk for an HIV- and KSHV-coinfected person to develop KS increases with immunosuppression (Mbulaiteye et al. 2003). HIV-infected homosexual men who subsequently acquire KSHV have an approximately 50% chance to develop AIDS-KS within 5–10 years (Renwick et al. 1998; O’Brien et al. 1999; Stein et al. 2008). HIV aggravates KSHV pathogenicity probably on several levels: Immune suppression is a potent risk factor, as indicated by the 200-fold increase of KS in transplant recipients relative to the general population (Vajdic et al. 2006; Grulich et al. 2007). However, given that in industrialised countries, KS is approximately 3,000-fold more common in HIV-infected patients than in the general population, other aspects of HIV infection may promote KS development. These range from inflammatory cytokines to the HIV transactivating protein Tat (Aoki and Tosato 2007), which was reported to activate lytic replication of KSHV (Zeng et al. 2007) and to accelerate

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the tumorigenic effects of the KSHV G-protein coupled receptor (vGPCR) (Pati et al. 2001; Guo et al. 2004). The importance of the immune system is also illustrated by the fact that KS can regress after reduction of immunosuppressive treatment in solid organ transplant recipients (Moray et al. 2004) and the improvement of KS in AIDS patients after the introduction of highly active antiretroviral therapy (HAART) (Dupont et al. 2000; Bower et al. 2009). In addition, mild immune suppression was suggested as a risk factor for classic KS (Brown et al. 2006b). Although the incidence of KS correlates broadly with the worldwide variation of KSHV prevalence (section “Kaposi Sarcoma”), several exceptions exist: Seroprevalence for KSHV is comparable in the African countries Uganda, Zambia, Gambia and Ivory Coast, but juvenile and endemic KS is found mainly in East Africa, around the great lakes (Gompels and Kasolo 1996; Dedicoat and Newton 2003). Furthermore, even though KSHV prevalence is high in Papua New Guinea, KS prevalence is low in this country (Rezza et al. 2001; Dedicoat et al. 2004). These observations point to a role of other still unknown factors involved in the aetiology of KS. These co-factors may involve host genetic polymorphisms, e.g. in VEGF or IL8-receptor genes (Brown et al. 2006a; Zanetti et al. 2010) or certain HLA types (Alkharsah et al. 2007). In addition, environmental factors, such as volcanic soil, durum wheat, blood-sucking insects and plant-derived chemicals, that can reactivate KSHV from latency in tissue culture, have been suggested as possible co-factors (Whitby et al. 2007; Ascoli et al. 2009; Pelser et al. 2009; Goedert et al. 2010).

Clinical Features and Treatment KS ranges from indolent to aggressive forms associated with significant mortality. In classical KS, typical cutaneous lesions consist of purple-blue or reddish-brown plaques and nodules, appearing initially on the hands and feet and progressing up the extremities over a period of years to decades. Extracutaneous spread can occur. More aggressive forms disseminate to lymph nodes and to the inner organs including the pulmonary and gastrointestinal tract, liver and spleen. KS is a vascular tumour with a complex histology. All KS lesions are composed of spindled-shaped tumour cells (the hallmark of the tumour), atypical endothelial cells and an inflammatory cellular infiltrate (Fig. 10.1). The spindle-shaped tumour cells express endothelial markers including CD31 and CD34 (Nickoloff 1991, 1993), but also markers of the lymphatic endothelium such as VEGFR3 (Dupin et al. 1999). However, some cells express markers for dendritic cells, macrophages or smooth muscle cells. These observations suggest that the cells are either derived from a pluripotent mesenchymal progenitor or KS lesions might represent a heterogeneous population of cells. KSHV can influence the differentiation pattern of vascular endothelial cells towards that of lymphatic endothelial cells (Wang et al. 2004a). In addition, there are some suggestions that KSHV can induce endothelial– mesenchymal transition (P. Ojala et al., pers. comm.).

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Fig. 10.1 Kaposi’s sarcoma. Histological section of a lymph node metastasis from an 45-year-old HIV-positive patient. (a) Staining with haematoxylin–eosin (×250 magnification) shows vascular slits and elongated spindle cells. (b) Staining with an antibody to KSHV LANA shows the presence of LANA in the nuclei of numerous spindle cells (brown staining, ×125 magnification). Image kindly provided by Dr. Buesche, Dept. Pathology, Hannover Medical School

Most cells are latently infected and express only a limited number of viral genes including the latency-associated nuclear antigen-1 (LANA), viral cyclin (vcyclin) and viral FLICE inhibitory protein (vFLIP) (section “Viral Proteins Involved in Tumorigenesis”); only in a small proportion of the cells does KSHV undergo lytic replication (Staskus et al. 1997). KS lesions evolve from early (inflammatory) patches to plaques that develop further to tumour nodules. All three kinds of lesions can be detected in one KS-patient in parallel. Treatment of AIDS-KS initially involves an efficient control of HIV replication by HAART (Dupont et al. 2000; Bower et al. 2009). For transplant KS a reduction in immune suppression can be attempted, but has to be balanced against the risk of transplant rejection (Moray et al. 2004). In general, local therapy including radiotherapy is applied for mild to moderate local KS whereas systemic chemotherapy has to be applied in case of aggressive progression including visceral involvement. Although the productive replication of KSHV responds to some anti-herpesviral drugs such as gancyclovir or cidofovir (Casper 2006), the efficacy of these drugs against established KS lesions is limited. HIV-infected patients treated with gancyclovir for other indication (usually Cytomegalovirus – CMV – disease) have a lower risk of developing AIDS-KS (Glesby et al. 1996), suggesting that a reduction of productive KSHV replication can prevent the progression to KS. More recent strategies to treat KS target cellular growth and angiogenic pathways, such as vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF) and matrix metalloproteinases (MMPs) that are dysregulated by the virus. Rapamycin, the inhibitor of mTOR, showed promising results in the treatment of iatrogenic KS (Stallone et al. 2005). Further experimental compounds include COL3, an inhibitor of MMPs (Dezube et al. 2006) and imatinib, a tyrosine kinase inhibitor of c-kit and PDGF (Koon et al. 2005). However, continued efforts will be essential to further optimise existing strategies and find new targets for future therapy.

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KS: A True Malignancy or a (Chronic) Inflammatory, Proliferative Disorder? KS lesions consist of spindle-shaped cells of endothelial origin, but infiltrated plasma cells, lymphocytes and other inflammatory cells are also present. Early KS lesions contain only a small number of spindle cells (less than 10%) and a large number of infiltrated immune cells. The number of spindle cells increases within the development of the lesion towards the later nodular stage, during which KSHV is present in more than 90% of spindle cells, but not in normal vascular endothelium (Dupin et al. 1999). Spindle cells explanted from KS tumours release several growth factors and cytokines such as IL-1, bFGF, VEGF, interleukin 8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) (Salahuddin et al. 1988; Ensoli et al. 1989), which may play a role in the aberrant angiogenesis seen in KS lesions. Injection of cultured KS cells into mice causes a strong angiogenic reaction of mouse cell origin, but no tumour growth (Salahuddin et al. 1988), suggesting that they do not represent classical transformed cells that are able to grow in immunodeficient mice. However, the interpretation of this observation is complicated by the fact that cultured KS cells lose KSHV after a few passages and the reported lack of tumorigenicity of cultured KS spindle cells could, therefore, conceivably be linked to the absence of KSHV. Clonality studies suggest that KS is an oligoclonal and multifocal disease (Judde et al. 2000): in one study nearly 80% of the KS lesions were oligoclonal, with evidence of clonal and oligoclonal KS lesions within the same patient (Duprez et al. 2007). Taken together, these observations lead to the widely held view that especially the early stages of KS are defined by a benign and inflammatory-driven, proliferative process rather than by true tumorigenesis. From this early, mainly polyclonal stage, the lesion might – depending on immunosuppression or other still unknown circumstances – proceed towards a true malignancy. Therefore, KS could be termed an inflammatorydriven tumorigenic process or a paracrine neoplasia (Mesri et al.). These issues are dealt with in more detail in other chapters of this book and also in recent reviews (Douglas et al. 2007; Mesri et al. 2010; Riva et al. 2010). A more detailed discussion of viral proteins thought to be involved in KS pathogenesis is provided in section “Mechanisms of Tumorigenesis in KSHV-Associated Diseases” of this chapter.

KSHV-Associated Lymphomas In addition to KS, KSHV is also associated with two B-cell-lymphoproliferate diseases, primary effusion lymphoma and multicentric Castleman’s disease.

Primary Effusion Lymphoma Shortly after the discovery of KSHV as causative agent of KS, E. Cesarman and colleagues identified KSHV DNA in a subgroup of AIDS-related non-Hodgkin

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lymphomas (NHL) (Cesarman et al. 1995). These malignancies were subsequently called “primary effusion lymphoma” (PEL) (Nador et al. 1996). PEL is a very rare subgroup of B-cell NHL, comprising 3% of AIDS-related lymphomas (Little et al. 2001). Owing to the rarity of the disease, only case reports and no systematic case–control or cohort studies are available. Epidemiological evidence for the role of KSHV in PEL is, therefore, limited to the detection of KSHV in all reported cases of PEL. In addition to KSHV, more than 90% of all AIDS-PEL cases show co-infection with EBV. However, only a restricted set of latent EBV genes is expressed (Horenstein et al. 1997) and the role of EBV in PEL is not fully understood. Gene expressing profiles of EBV-positive and EBV-negative PEL as well as other NHL have revealed the important role of KSHV in the oncogensis of PEL, as its presence selects for a very distinct cellular gene expression and a clearly different lymphoma type (Fan et al. 2005). Even though the majority of PEL cases is found in HIV-positive patients, several HIV-negative PEL patients have been described. These patients are likely to have an underlying immunosuppression (e.g. after solid organ transplantation – iatrogenic PEL). PEL has also been diagnosed in two elderly HIV-negative men in Italy, a country with intermediate prevalence for KSHV (Ascoli et al. 1999). Weakened immune functions in old age may, therefore, contribute to PEL development in geriatric patients. HIV-negative PEL share several features with AIDS-associated PEL at the morphologic and molecular level as well as the immunophenotype, but are characterised by onset at an older age and a less aggressive clinical course are reported (Ascoli et al. 1999).

Solid PEL Even though the majority of PEL cases have been reported as lymphomatous effusions in the absence of a solid tumour mass (section “Primary Effusion Lymphoma”), a few patients with KSHV-positive extracavitary solid tissue lymphomas have been observed (Dotti et al. 1999). These solid variants – which can occur before, after or independently of effusion lymphoma – often appear at extranodular sites and are similar to the effusion type with regard to morphology, immunophenotype and molecular characteristics. Therefore, these two variants have been grouped as a single clinicopathologic entity and the extracavitary form is classified as “solid”, whereas the effusion type is classified as “classic” PEL. The hallmark of both variants and their distinctive feature in comparison to other NHL is the presence of KSHV.

Clinical Features and Therapy PEL is clinically characterised by the lymphomatous growth in a liquid phase in body cavities such as pleural, pericardial or peritoneal spaces, mainly without identifiable associated extracavitary tumour mass. Morphologically, PEL cells are large

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in size, have a basophilic cytoplasm and round to irregular nuclei. The cells exhibit a range of appearances from immunoblastic to plasmablastic to anaplastic. Gene expression profiles revealed that PEL cells express a distinct pattern of genes, distinguishable from other NHL or normal B-cells and indicating a plasmablastic derivation with immunoblastic features (Jenner et al. 2003; Klein et al. 2003). PEL cells have a characteristic phenotype with expression of the lymphocyte marker CD45, the activation markers CD30, CD38, CD71 and epithelial membrane antigen (EMA), as well as markers associated with plasma cell differentiation such as CD138/Syndecan-1 and MUM1/IRF4 (Carbone et al. 2001). However, classical B-cell markers (CD19, CD20) or T-cell markers (CD2, CD3, CD5 and CD7) are not detectable in general (Nador et al. 1996), but may be expressed occasionally on tumour cells (Brimo et al. 2007). Immunoglobulin gene rearrangements define the B-cell origin of PEL cells (Nador et al. 1996). Rearranged immunoglobulin genes show high levels of somatic mutation in their heavy chain variable (VH) regions, suggesting a postgerminal B-cell origin (Matolcsy et al. 1998). Furthermore, gene expression profiles showed an expression pattern of PEL distinct but related to both immunoblasts and plasma cells (Jenner et al. 2003; Klein et al. 2003). In conclusion, it is likely that the lymphoma is derived from a transition state between antigen-selected germinal centre B-cells and terminally differentiated plasma cells. The prognosis of PEL is extremely poor with a median survival of less than 6 month. No widely used standards for therapy have been established and the disease is not curable. The use of the antiviral therapy HAART is associated with a better prognosis (Boulanger et al. 2005) and, therefore, recommended for HIV-positive PEL patients. Some case reports showed prolonged remission of PEL with antiretroviral therapy alone (Hocqueloux et al. 2001). These findings imply an important role of immune reconstitution in the control of this aggressive lymphoma (Chen et al. 2007). As for other NHL, a systemic combination therapy based on cyclosphamide, doxorubicin, vincristine and prednisone (CHOP) is frequently used. Novel approaches include antiviral components such as cidofovir (De Clercq 2003). Many antiviral drugs are most effective during viral lytic phase; therefore, their effect might be improved with the additional administration of drugs such as valproate, an inducer KSHV of lytic replication (Klass et al. 2005). Other options for treatment, such as reactivation of the p53 pathway using the murine double minute 2 (MDM2) inhibitor Nutlin 3a to induce apoptosis in PEL tumour cells (Sarek et al. 2007), are actively discussed at the moment.

The Role of KSHV in PEL Pathogenesis How KSHV promotes the development of PEL is still under intense investigation. As in KS, most PEL tumour cells are latently infected, but a small percentage of infected cells enters the productive replication cycle at any one time. During latency, the same viral proteins found in KS are also expressed in PEL. In addition, one of the KSHV interferon regulatory factor (IRF) homologues, vIRF-3 (also called

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LANA-2), is expressed during latency in B-cells and there is also substantial expression of the lytic protein vIL6, a homologue of human interleukin 6 (IL-6) (section “Viral Proteins Involved in Tumorigenesis”). Using RNAi, several viral proteins have been shown to be essential for the continuous proliferation and survival of PEL cell lines. While silencing LANA by RNAi did not induce apoptosis but reduced the copy number of the viral episome, presumably due to its role in episomal persistence (Godfrey et al. 2005), targeting vFLIP and vcyclin by RNAi caused efficient apoptosis in all PEL cell lines tested (Guasparri et al. 2004; Godfrey et al. 2005). Furthermore, knockdown of vIRF3 (LANA-2) resulted in repressed proliferation of PEL cells and activation of caspase-3 and -7 (Wies et al. 2008). PEL are devoid of gene rearrangements such as chromosomal translocations including bcl-6, bcl-2 and c-myc, which are associated with other aggressive B-cell NHLs (Cesarman et al. 1995; Nador et al. 1996). However, mutation in the 5¢ noncoding regions of BCL-6 are found in a fraction of PEL (Carbone et al. 2000).

Multicentric Castleman’s Disease Castleman’s disease (CD), also called angiofollicular lymph node hyperplasia, is a polyclonal lymphoproliferative disease. It was originally described by Benjamin Castleman in 1956 (Castleman et al. 1956). CD represents several clinical and pathological entities, it can affect a single lymph node (localised or unicentric CD) or can be generalised (multicentric form). Multicentric Castleman’s disease (MCD) presents with lymphadenopathy and frequently multi-organ involvement; it is associated with systemic manifestations such as inflammation and B-cell hyperreactivity and is more aggressive. MCD is less common than localised CD and occurs in the HIV-negative population in the sixth decade, but in HIV-positive patients also at younger age. Following the histopathology of CD, the disease can similarly be divided into two groups: the hyaline vascular type is more common and tends to be localised, whereas the plasma-cell variant is only rarely localised but often multicentric. Furthermore, mixed types exist, complicating an accurate classification of individual cases. MCD and KSHV The incidence of MCD increased with the emergence of the HIV epidemic. Several groups described the co-existence of KS and MCD in patients (Rywlin et al. 1983; Lachant et al. 1985) and shortly after the detection of KSHV in KS, J. Soulier and colleagues could demonstrate the presence of KSHV DNA in HIV-positive and HIV-negative MCD samples (Soulier et al. 1995). KSHV could be detected in 13% of patients with localised CD and 75% of patients with MCD (Bowne et al. 1999). Immunohistochemical studies revealed that KSHV is found in the plasmablasts within MCD lesions; these plasmablasts occur as isolated cells in the mantle zone of B-cell follicles and seem to be absent in KSHV-negative MCD (Dupin et al. 1999; Parravicini et al. 2000). KSHV-infected plasmablasts have germ line immunoglobulin

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genes but are lambda light chain restricted and appear to represent pre-germinal centre naïve B-cells that are polyclonal but monotypic (Du et al. 2001). These plasmablasts can aggregate and form microlymphomas or even plasmablastic lymphoma. HIV-positive patients with KSHV-dependent MCD have a 15-fold higher incidence of NHL, e.g. plasmablastic lymphoma, compared to the general HIV-positive population (Oksenhendler et al. 2002). The viral expression pattern in KSHV-associated MCD differs from KS and PEL, which show mainly expression of latent proteins. In the affected B-cells in KSHVassociated MCD, viral cytokines such as vIL-6 are expressed in addition to the viral latent proteins LANA, v-cyclin, vFLIP and vIRF3 (Parravicini et al. 2000).

The Role of IL-6 in MCD The characteristics of MCD are partly due to cytokine dysregulation, which includes increased levels of the pleiotropic cytokine IL-6 (Yoshizaki et al. 1989; van Kooten et al. 1993; Oksenhendler et al. 2000); IL-6 is known to be involved in B-cell stimulation, mediates B-cell differentiation and promotes the growth of B-cell associated diseases. Anti-IL-6 or anti-IL-6-receptor (IL-6R) antibodies have been shown to abrogate CD-related symptoms and led to involution of the affected lymph node(s) (Nishimoto et al. 2000). Furthermore, hIL-6 transgenic mice develop CD-like syndromes, which can be reversed with the application of antibodies against hIL-6R (Katsume et al. 2002). KSHV vIL-6 mimics many biological functions of its human homologue IL-6 (Viral proteins involved in Tumorigenesis) and might play an important role in the apparent B-cell proliferation in MCD (Aoki et al. 1999; Uldrick et al. 2010). It has been hypothesised that activation of hIL-6 pathways by vIL-6 might transform naïve B-cells into plasmablasts, leading to the lymphoproliferative diseases associated with KSHV, including MCD (Du et al. 2001).

Clinical Features and Treatment MCD has multiple manifestations including fever, weakness, severe fatigue and lymphadenopathy, followed by splenomegaly and hepatomegaly. MCD in HIV-positive patients is more aggressive with clinical features including severe constitutional symptoms, generalised lymphadenopathy and frequent bone marrow involvement. The diagnosis is mostly established by lymph-node biopsy. While surgical excision is often the first line therapy for local CD, treatment for MCD is not standardised and due to its comparative rarity, no randomised clinical trials have yet been performed. Corticosteroids are given to improve symptoms in case of acute aggravation. Several cases were reported to have had a partial or complete response following treatment with rituximab (Corbellino et al. 2001; Nicoli et al. 2009), an antibody against the B-cell surface antigen CD20, which is used as therapy for B-cell lymphomas and autoimmune diseases (Lim et al. 2010). Targeting hIL-6 or its receptor

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hIL-6R with specific antibodies (tocilizumab, siltuximab) also showed partial or complete response in several cases. Other strategies include the application of immunomodulatory agents (such as INF-a) or systemic chemotherapy, e.g. the combination therapy CHOP. In line with the role of KSHV in MCD development, treatment with the antiviral agent gancyclovir led to remission of KSHV- and HIVassociated MCD (Casper et al. 2004); however, the antiviral agent cidofovir was shown not to be effective against HIV-associated MCD (Berezne et al. 2004). HAART is used as first line therapy for HIV and it could recently be shown in a systematic review of the literature that life expectancy in HIV- associated MCD cases has improved in the HAART era; however, a direct role could not be established and the effect of HAART might be explained by the immune reconstitution in treated patients (Mylona et al. 2008).

Mechanisms of Tumorigenesis in KSHV-Associated Diseases General Mechanisms of KSHV-Mediated Tumorigenesis In the absence of HIV or iatrogenic immune suppression, KSHV-associated disorders (Table 10.2) are rare, even in populations with a high KSHV seroprevalence. Thus, it is probable that KSHV infection represents only one of several events involved in pathogenesis. In KS, PEL and also MCD, the majority of infected cells is latently infected; however, lytic replication takes place in a fraction of infected cells. In KS and PEL, this fraction generally makes up less than 5–10% but may reach 5–25% of the infected cells in MCD (Parravicini et al. 2000). Several KSHV proteins have been shown to have transforming and oncogenic properties in in vitro and in vivo assays (section “Viral Proteins Involved in Tumorigenesis”, Table 10.3). Latent proteins may directly promote the growth of infected cells, whereas viral proteins only expressed during the productive replication cycle are suspected to act mainly via autocrine or paracrine mechanisms. Several virus-encoded proteins share high homology with cellular partners (Table 10.3) and are thought to have been acquired from the host in the distant past during co-evolution of a KSHV progenitor within mammalian hosts. These viral homologues often mimic, or may go beyond, the effects of their cellular counterparts. Their expression often enables infected cells to manipulate their environment (Choi et al. 2001). Distinct expression patterns of KSHV proteins are detected in different KSHV associated diseases (Parravicini et al. 2000). It is unclear if these differences are due to cell type or tissue specific control of KSHV gene expression. The exact contribution of individual proteins to the development of KSHV-associated diseases is not well understood in spite of experimental evidence that some of them, in particular the proteins encoded by open reading frame (orf) K1, K9 (an interferon regulatory factor homologue), 74 (a chemokine receptor homologue with constitutive signalling activity) as well as Kaposin A and LANA may have transforming properties (Viral proteins involved in Tumorigenesis).

Endothelial cells

Spindle-shaped cells of endothelial origin, monocytes

Cellular origin

Infected cells

Table 10.2 Comparison of KSHV-associated diseases KS (all forms) Neoplasm Inflammation-driven neoplastic process/ paracrine neoplasia Clinical presentation Predominantly in immunodeficient patients but also in elderly immunocompetent individuals (classic KS), sometimes systemic symptoms, prognosis depends on extent of disease Sites Mainly skin, frequent involvement of oral cavity and viscera KSHV Present in all cases

B-cells, transition state between antigen-selected germinal centre B-cells and terminally differentiated plasma cells B-cells • Related to immuno-blasts and plama cells • “Null-phenotype”: often lack B-cell markers • Express B-cell activation markers (CD30, CD38) • Express plasma cell markers (CD138, MUM1)

Body cavities, extranodular sites for solid form Present in all cases (diagnostic criterion)

Predominantly in immunodeficient patients, systemic symptoms, poor prognosis

PEL (effusion and solid form) Malignant tumour

(continued)

B-cells • Plasmablasts • Express B-cell markers (such as CD20) • Lack activation markers • Monotypic IgMl expression

Present in most HIV-associated and ~50% of HIV-noninfected cases of the plasma cell variant of MCD B-cells, naïve, pre-plasma state

Lymph nodes, spleen

KSHV-associated MCD lymphoproliferative disease; can rarely develop into plasmablastic lymphoma Predominantly in immunodeficient patients, systemic symptoms, poor prognosis

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Mono- and oligo-clonal Not known

Most cells latently infected, mainly latent proteins expressed, a small percentage shows evidence of productive lytic replication

Absent

Expression of viral proteins

EBV co-infection

KS (all forms)

Clonality Mutations in infected cells

Table 10.2 (continued) PEL (effusion and solid form) Monoclonal No chromosomal abnormalities but mutation in the 5´-noncoding regions of BCL-6 Most cells latently infected, mainly latent proteins expressed, a small percentage shows evidence of productive lytic replication, vIRF3 (LANA-2) expressed Frequent

KSHV-associated MCD

Rare

Most cells latently infected, mainly latent proteins expressed, latent and lytic genes (in 5–25% of infected cells) expressed, vIRF3 (LANA-2) expressed

Polyclonal Not known

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Table 10.3 Selection of KSHV proteins and miRNAs involved in oncogenesis Protein (gene) Latent/lytic Cellular homologue Mode of action LANA (ORF 73) Latent None Inhibits p53 and pRB-E2F pathway, upregulates hTERT, interacts with Brd2/4, traps GSK-3b – thereby deregulation of Wnt-pathway; transforming activities (in presence of h-Ras) vcyclin (ORF 72) Latent D-type cyclin Constitutively activates CDK6, increases cellular proliferation, causes genomic instability, cytokinesis defects and polyploidy vFLIP/K13 (ORF 71) Latent FLIP Activates NFkB; antiapoptotic function, induces endothelial spindle cell formation Kaposin A (ORF K12) Latent None Interacts directly with cytohesin-1, transforming activities in rodent fibroblasts LANA2/vIRF3 (ORF Latent IRFs Expressed only in B-cells, K10.5) inhibits p53, inhibits apoptosis in PEL cells, antagonises IFN K1/VIP (ORF K1) Lytic None Induces NFkB, NFAT and bFGF, prevents death-receptor mediated apoptosis, activates PI3K, Akt, Vav and Syk kinases, induces MMP-9 and VEGF in endothelial cells, causes immortalisation of primary endothelial cells, vascular tumours in transgenic mice vGPCR (ORF 74) Lytic IL-8 receptor Activates MAPK and PI3K pathways, increases VEGF and VEGF receptor 2 (VEGFR-2) secretion, transforms murine NIH3T3 cells and immortalises human endothelial cells in tissue culture (see also text) vIL-6/K2 (ORF K2) Lytic IL-6 Induces human IL-6 secretion, triggers JAK/STAT-, MAPK-dependent pathways, autocrine/paracrine signalling, supports B-cell proliferation vBCL2 (ORF16) Lytic BCL2 Forms heterodimer with human bcl-2, inhibits bax-mediated apoptosis (continued)

232 Table 10.3 (continued) Protein (gene) Latent/lytic Viral miRNAs (miR-K1 Latent to miR-K12)

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Cellular homologue miR-K12-11: miR155

Mode of action Suppress lytic replication, influence cell differentiation and angiogenesis, downregulate several tumour suppressors, thereby involved in KSHV-mediated oncogenesis: • miR-K1: targets cyclindependent kinase p21 • miR-K3: targets NFIB • miR-K9*: targets KSHV ORF50 (RTA) • miR-K11: targets NFkB regulator IkBa • miRK12-3 and -7: target C/ EBPb isoform p20/LIP • miR-K12-7: targets the NK cell ligand MICB • miR-K12-10a: targets TWEAKR • miR-K12-11: targets e.g. BACH-1 and IkB kinase e (IkKe) • miR-K12-6 and -11: target transcription factor MAF Abbreviations: LANA latency associated nuclear antigen, pRB retinoblastoma protein, E2F E2 transcription factor, hTERT human telomerase reverse transcriptase, BRD2/4 Bromodomaincontaining protein 2/4, GSK-3b Glycogen synthase kinase-3 b, Ras rat sarcoma protein, CDK6 Cyclin-dependent kinase 6, FLIP FLICE inhibitory protein, NFkB nuclear factor “kappa-lightchain-enhancer” of activated B-cells, IFN interferon, VIP variable ITAM-containing protein, NFAT Nuclear factor of activated T-cells, bFGF basic fibroblast growth factor, PI3K phosphoinositide 3-kinase, MMP-9 Matrix metallo protease-9, VEGF vascular endothelial growth factor, GPCR G protein-coupled receptor, IL interleukin, MAPK Mitogen-activated protein kinase, VEGFR-2 VEGF-receptor 2, JAK Janus kinase, STAT signal transducer and activator of transcription, MAPK mitogen-activated protein kinase, BCL2 B-cell lymphoma 2, NFIB nuclear factor I/B, C/EBP b CCAAT-enhancer-binding protein b, MICB MHC class I polypeptide-related sequence B, TWEAKR tumour necrosis factor-like weak inducer of apoptosis receptor protein, BACH-1 BTB and CNC homology 1- basic leucine zipper transcription factor 1

It could be shown in different experimental systems that KSHV can induce features linked to tumorigenesis: KSHV infection of primary endothelial cells induces cell survival and angiogenesis without resulting in the transformation of endothelial cells (Wang and Damania 2008). Even though KSHV infection can also not transform mature B-cells in culture (Kliche et al. 1998), KSHV is required for survival of PEL cells in culture (Godfrey et al. 2005; Guasparri et al. 2004; Wies et al. 2008) and individual KSHV proteins possess transforming capacities in different experimental systems as well as in transgenic mice. KSHV targets several tumour suppressor proteins, such as p53 and pRB, thereby promoting cell proliferation and survival, which facilitate tumour development; this includes also inhibition of apoptosis and the interference with cell cycle regulation, caused by several latent and lytic proteins (see below).

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Genetic instability is another hallmark of tumorigenesis, which is found in KS and PEL. KSHV infection can induce chromosomal instability and the proteins LANA and v-cyclin might be partially responsible (Si and Robertson 2006; Verschuren et al. 2002; Koopal et al. 2007). Furthermore, DNA damage response (DDR), a critical mechanism for the maintenance of genomic integrity, is influenced by several KSHV proteins, which either activate DDR (Koopal et al. 2007) or prevent response after damage has already occurred (Shin et al. 2006). Another key aspect in neoplastic development is the stimulation of angiogenesis and the accompanying secretion of cytokines. Tumour angiogenesis is a complex process involving different pro-angiogenic factors, which are induced in KSHV infection, including VEGF, bFGF, IL-8, MMPs and cyclooxygenase-2 (COX-2) (Sodhi et al. 2000; Prakash et al. 2002; Brinkmann et al. 2007; Qian et al. 2007; Sharma-Walia et al. 2010).

Viral Proteins Involved in Tumorigenesis LANA The latency associated nuclear antigen-1 (LANA) is expressed during latency in KS, PEL and MCD cells. In addition to its role in episomal persistence and suppression of lytic replication (Verma et al. 2007), LANA contributes to tumorigenesis though interference with several pathways, e.g. p53-mediated transcriptional activation and apoptosis (Friborg et al. 1999), pRB/E2F- as well as GSK3b/b-cathenin mediated cell cycle regulation (Radkov et al. 2000; Fujimuro and Hayward 2003). Furthermore, LANA interacts with RING3/Brd2 (Viejo-Borbolla et al. 2005) and in cooperation with the cellular oncogene h-Ras is able to transform primary rat fibroblasts (Radkov et al. 2000). vcyclin Viral cyclin (vcyclin), expressed during latency in the majority of infected cells (Davis et al. 1997), is a homologue of cellular D-type cyclins, important regulators of the cell cycle. vcyclin associates with cellular cyclin dependent kinases (Cdk), preferentially with Cdk6 (Godden-Kent et al. 1997), and phosphorylates pRB in vitro and in vivo (Chang et al. 1996). The vcyclin-CDK6 complex has an extended set of substrates compared to the cellular cyclinD-CDK6 complex, including histone H1, p21(KIP1), cdc25A and BCL-2 (Godden-Kent et al. 1997; Mann et al. 1999; Ojala et al. 2000). Furthermore, the vcyclin-CDK6 complex is more resistant to the inhibitory effects of CDK inhibitors (CDKIs) such as p16(INK4), p21(CIP) and p27(KIP) (Swanton et al. 1997) and can inactivate p27(KIP1) through phosphorylation (Sarek et al. 2006). In addition to the cell cycle regulatory effects, expression of vcyclin induces replicative stress, which can lead to senescence and activation of DNA damage response (Koopal et al. 2007) and it was also shown to affect latency

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by phosphorylation of nuceleophosmin (Sarek et al. 2011). However, the physiological role of vcyclin during KSHV infection is still not understood.

vFLIP The viral FLICE (Fas-associated death-domain-like IL-1 b-converting enzyme) inhibitory protein (vFLIP) inhibits, similarly to cellular FLIP proteins, death receptormediated apoptosis, thereby providing a survival advantage to latently infected cells. Additionaly, vFLIP effectively suppress autophagy, thereby regulating cell death (Lee et al. 2009). vFLIP is a potent activator of the NFkB pathway. It binds the kB kinase-g (IKKg) (Liu et al. 2002), which leads to the induction of many cytokines (Sakakibara et al. 2009) and induces the spindle shape morphology of KSHV-infected endothelial cells (Grossmann et al. 2006).

miRNAs During latency, KSHV expresses 18 mature miRNAs, which originate from 12 viral pre-miRNAs (Cai et al. 2005; Pfeffer et al. 2005; Grundhoff et al. 2006). The viral miRNAs target host and viral mRNAs, are involved in the suppression of lytic replication and can influence cell differentiation, cell proliferation and survival as well as angiogenesis. miR-K12-11 is an orthologue of cellular miR155 and downregulates an extensive set of common targets, including the tumour suppressor BACH-1 (Gottwein et al. 2007; Skalsky et al. 2007) and the IkB kinase e (IKKe) (Liang et al. 2011), thereby regulating interferon (IFN) signalling and suppressing antiviral immunity. miR-K11 targets IKBa, an inhibitor of the NFkB pathway (Lei et al. 2010). Cytokine secretion in macrophages and monocytes is affected by miR-K12-3 and -7; these miRNAs have been shown to downregulate the negative regulator C/EBPb p20 (LIP) leading to induction of IL-6 and IL-10 (Qin et al. 2009). miR-K1 represses expression of p21, a cyclin dependent kinase with known tumour suppressor functions (Gottwein and Cullen 2010). Effects of further KSHV miRNAs are listed in Table 10.3. These varied effects of KSHV miRNAs point to a multifaceted involvement in KSHV biology.

vIL-6 This homologue of human IL-6 (Neipel et al. 1997) can be detected in KS, PEL and MCD samples (Parravicini et al. 2000). vIL-6 binds directly to the human gp130 receptor without the need of the hIL-6 receptor a-chain (hIL-6R), thereby activating the JAK/STAT pathway and further hIL-6 induced pathways (Osborne et al. 1999), leading also to increased VEGF expression. Owing to the changed requirements for receptor binding, vIL-6 can theoretically activate every cell in the body expressing the gp130 signalling chain, which is shared by many cytokine receptors. However, its

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binding affinity is lower compared to hIL-6 (Aoki et al. 2001) and vIL-6 expression in KSHV infected cells is limited. The expression of vIL-6 in a minority of infected cells suggests a paracrine model for vIL-6 (Liu et al. 2001). Furthermore, vIL-6 can activate hIL-6 (Mori et al. 2000). vIL-6 potentially contributes to KSHV-related disease progression by continued activation of IL-6-stimulated growth and anti-apoptotic pathways; its role especially in MCD is mentioned in section “Clinical Features and Treatment”. Further details on vIL-6 are reviewed elsewhere (Suthaus et al. 2010).

vGPCR This early lytic protein is a constitutively active member of the family of CXC chemokine G protein coupled receptors (GPCR) (Arvanitakis et al. 1997) and is a homologue of the human angiogenic IL-8 receptor (Cesarman et al. 1996). Several studies showed the oncogenic potential of this protein, which can activate both MAPK and PI3K pathways, leading to the activation of several cell-signalling networks (Sodhi et al. 2000; Montaner et al. 2001). vGPCR has been shown to transform murine NIH3T3 cells and immortalise human endothelial cells (Montaner et al. 2001; Bais et al. 2003). A transgenic mouse that enabled endothelial cell-specific infection revealed that vGPCR was the only viral gene analysed able to cause vascular tumours in a subset of mice (Montaner et al. 2003). However, while interpreting these effects obtained with overexpressed vGPCR, it has to be taken into account that in culture, KSHV cannot immortalise primary endothelial cells. VEGF and VEGF receptor 2 (VEGFR-2) expression is increased in vGPCR-induced tumours in vitro and in vivo (Sodhi et al. 2000; Guo et al. 2003). vGPCR is expressed only in the minority of productively infected cells in KS, PEL or MCD, leading to hypothesis that vGPCRmediated effects are driven by spontaneous productive (lytic) reactivation in the background of latency. These data implicate that vGPCR signals via autocrine or paracrine mechanisms, thereby influencing KSHV driven oncogenesis.

K1/VIP The lytic transmembrane signalling protein K1/VIP (variable immunoreceptor tyrosine-based activation motif (ITAM) containing protein) displays a high degree of variability between different KSHV isolates (Hayward 1999; Zong et al. 1999; Biggar et al. 2000) (section “Origin and Evolution”). Expression of the K1 gene in rodent fibroblasts led to morphologic changes and focus formation indicative of transformation (Lee et al. 1998). Furthermore, transgenic mice expressing the K1 gene under transcriptional control of the SV40 promoter developed tumours with sarcomatoid features and malignant plasmablastic lymphoma (Prakash et al. 2002). K1 expression leads to the activation of different signalling pathways including the PI3K/Akt pathway, thereby inactivating pro-apoptotic factors (Tomlinson and Damania 2004; Wang et al. 2006). K1 blocked Fas-mediated apoptosis was shown to increase cell survival (Wang et al. 2007). Furthermore, K1 expression leads to

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increased phosphorylation of syk and phospholipase Cg2, as well as increased activity of NFkB and NFAT (Lagunoff et al. 1999). In addition, K1 was reported to both augment and repress reactivation in B-cells (Lagunoff et al. 2001; Lee et al. 2002) and to downregulate the B-cell antigen receptor (BCR) surface expression by inhibition of its intracellular transport (Lee et al. 2000). The K1 protein induces expression of angiogenic and invasion factors including VEGF, suggesting a role of K1 in KSHV pathogenesis via paracrine mechanisms (Wang et al. 2004b).

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Chapter 11

Retroperitoneal Fibromatosis Herpesvirus and Kaposi’s Sarcoma-Like Tumors in Macaques Laura K. DeMaster and Timothy M. Rose

Kaposi’s Sarcoma Kaposi’s sarcoma (KS) is a vascular neoplasm that was first identified in elderly men in the Mediterranean region. Classical KS commonly presents as a relatively benign multifocal lesion on the skin of the extremities. A highly aggressive, epidemic form of KS emerged in association with HIV and the acquired immunodeficiency syndrome (AIDS) epidemic and was largely confined to men who have sex with men (Antman and Chang 2000). AIDS-associated KS was frequently fatal and involved lymph nodes, viscera, and mucosa, in addition to the skin. An endemic form of KS, found in children and young adults in Sub-Saharan Africa prior to the AIDS epidemic, has now reached epidemic proportions in association with AIDS. Women and children are frequently affected, with a high tumor burden, rapid disease progression, and limited life expectancy. Finally, organ-transplant recipients and patients receiving immunosuppressive therapy are at higher risk of developing classical KS. Although KS occurs with various morphological forms during the time course of disease, all forms present with characteristic elongated spindle-shaped tumor cells. Early KS lesions show scattered spindle cells in a background of neovascular tissue with obvious inflammatory infiltration. In advanced KS lesions, the spindle cells proliferate and dominate the lesion. Most spindle cells express endothelial markers CD31 and CD34 and the lymphatic endothelial markers VEGFR-3 and podoplanin,

L.K. DeMaster Department of Global Health, University of Washington, Seattle Children’s Research Institute, Seattle, WA, USA T.M. Rose (*) Department of Pediatrics, University of Washington, Seattle, WA, USA Seattle Children’s Research Institute, 1900 Ninth Ave, Seattle, WA 98101, USA e-mail: [email protected] E.S. Robertson (ed.), Cancer Associated Viruses, Current Cancer Research, DOI 10.1007/978-1-4614-0016-5_11, © Springer Science+Business Media, LLC 2012

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suggesting that spindle cells derive from the lymphatic, rather than vascular, endothelial lineage. However, KS spindle cells can also express markers for smooth muscle cells and macrophages suggesting possible pluripotency of the spindle cell precursor.

KSHV Is the Etiological Agent of All Forms of KS In 1994, KS-associated herpesvirus (KSHV) was first identified in KS lesions of HIVinfected individuals (Chang et al. 1994). Based on sequence analysis, KSHV was classified as a gammaherpesvirus within the rhadinovirus genus. Significant genomic colinearity was detected between KSHV and other tumorigenic gammaherpesviruses, including Epstein–Barr virus (EBV), a member of the lymphocryptovirus genus, and herpesvirus saimiri (HVS), the rhadinovirus prototype of New World primates. Both EBV and HVS are able to transform cells in vitro. While EBV is associated with Burkitt’s lymphoma, nasopharyngeal carcinoma, and hairy leukoplakia in its natural host, HSV induces lymphomas and leukemias in nonnatural host species of New World primates. A causal linkage between KSHV and KS is now widely accepted and was established through a combination of epidemiological and molecular studies. Populationbased studies show strong geographic localization for KS tumor incidence. Serologic assays developed against viral antigens revealed that KSHV distribution is consistent with KS disease epidemiology. In most of the world, KS is a rare disease, but classical and endemic KS have long been recognized in the Mediterranean and in regions of Sub-Saharan Africa. Correspondingly, KSHV seroprevalence is low in North America (2–5%), where the disease incidence is low, and high in the Mediterranean and regions in Sub-Saharan Africa (30–80%), where the disease incidence is high (Ablashi et al. 2002). High seroprevalence was also observed in populations known to be at increased risk of KS, such as men who have sex with men (Kedes et al. 1996; Moore and Chang 1998). Molecular evidence linking KSHV to KS disease initially focused on association of viral genetic material with KS tumor tissue. Putative KSHV DNA sequences were detected in KS tumor tissue by representational difference analysis (RDA) (Chang et al. 1994). These viral sequences were found in all KS tissue samples from HIV-infected patients but not in unaffected controls. Furthermore, KSHV DNA was found at higher levels in KS lesions than in nontumor tissues of KS patients, demonstrating a strong association of viral DNA with KS tumors. An estimation of the viral copy indicated that each KS tumor cell contained two viral genomes (Chang et al. 1994). The presence of KSHV genomic DNA in tumor cells has now been demonstrated for all epidemiological forms of KS (Boshoff et al. 1995; Moore and Chang 1995). Evidence of viral gene expression in tumor cells provided additional support for a causal role of KSHV. Although viral transcripts are consistently detected in KS tumor cells, initial findings revealed a highly restricted expression of KSHV latent genes, demonstrating that tumor cells were predominantly latently infected (Zhong et al. 1996; Staskus et al. 1997; Dittmer et al. 1998). One of the major gene products

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expressed during latent infection is open reading frame (ORF)73, which encodes the latency-associated nuclear antigen (LANA) (Kedes et al. 1997; Rainbow et al. 1997). KSHV LANA has a number of functions within infected cells, including maintenance of the viral genome and deregulation of cell cycle and tumor-suppressor pathways (Ballestas et al. 1999; Friborg et al. 1999; Radkov et al. 2000). Nearly all KS tumor cells express nuclear LANA protein, and LANA is now considered a histological hallmark of latent KSHV infection. While latency is the predominant program of infection in KS spindle cells, latent infection of KS tumor cells is unstable in vitro, as cultured cells rapidly lose viral genomes when passaged (Grundhoff and Ganem 2004). Pleural effusion lymphomas (PELs) have been identified in AIDS patients that are latently infected with KSHV and carry the viral episome at a high copy number (Cesarman et al. 1995). PEL cell lines that maintain KSHV genomes after continuous culture have been derived from the lymphomas. Treatment with sodium butyrate or phorbol esters induces lytic activation of the latent KSHV genomes resulting in viral replication and release of infectious virions. PEL cell-derived virus has proven to be an invaluable tool in KSHV research. Though many studies have demonstrated the importance of individual viral genes to KS tumor formation, an in-depth understanding of KSHV pathogenesis has been hindered by the lack of relevant animal models. Unfortunately, introduction of KSHV into heterologous animals, such as macaques, has failed to produce a useful animal model for KS (Renne et al. 2004).

Retroperitoneal Fibromatosis: A KS-like Disease of Macaques Retroperitoneal fibromatosis (RF) is a multifocal, fibroproliferative syndrome associated with retrovirus-induced simian immunodeficiency in macaques. RF was first recognized as a disease syndrome in 1976 (Giddens et al. 1979), and epidemic outbreaks of RF occurred at the Washington National Primate Research Center (WaNPRC) and other primate centers (King et al. 1983) in the 1980s, which resulted in a 1% overall mortality and 10% mortality in juvenile macaques (Tsai et al. 1985). RF disease was originally associated with a form of simian AIDS (SAIDS) caused by infection with simian D-type retrovirus-2 (SRV-2) (Tsai 1993). RF lesions are highly proliferative and arise primarily in the peritoneum, in the area of the ileocecal junction and adjacent mesenteric lymph nodes, and secondarily as KS-like lesions in the skin (Giddens et al. 1985; Tsai 1993). Similarly to KS, RF tumors have a characteristic spindle-shaped tumor cell (Tsai 1993). Lesions have a significant vascular component and are infiltrated by inflammatory immune cells. The morphological similarities between RF lesions in macaques and KS lesions in humans were noted early on, and RF, especially in its skin-associated form, was characterized as a KS-like lesion (Tsai 1993). Additional cases of RF continue to occur in captive macaque populations in association with natural infections of SRV-2 or experimental infections with SIV or SIV/HIV hybrid viruses (Shibata et al. 1997; Bielefeldt-Ohmann et al. 2005).

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Fig. 11.1 Phylogenetic analysis of partial DNA polymerase sequences of RFHVMn and RFHVMm amplified from RF tumor samples. RFHVMn and RFHVMm cluster with KSHV within the rhadinovirus genus of the subfamily of lymphotrophic gammaherpesviruses. Gammaherpesviruses: (Rhadinovirus genus) RFHVMn, RFHVMm, KSHV, equine herpesvirus 2 (EHV2), herpesvirus saimiri (HVS); (lymphocryptovirus genus) Epstein–Barr virus (EBV); alphaherpesviruses: herpes simplex virus 1 (HSV1), varicella zoster virus (VZV); betaherpesviruses: human herpesvirus 6 (HHV6), human cytomegalovirus (HCMV). Bootstrap analysis using 100 replicates is shown. Adapted from Rose et al. (1997), Copyright American Society for Microbiology, J Virol 71, 1997, 4138–4144

The epidemic nature of RF suggested that the disease might have an infectious etiology, and this hypothesis was supported by experimental transmission studies using inocula of RF tissue (Giddens et al. 1985). Due to the strong association between SAIDS RF and SRV-2 infection, it was initially hypothesized that the type D retrovirus SRV-2 was the causative agent of RF (Tsai et al. 1985). After the discovery of KSHV in KS tumor tissue, however, the morphological and histological similarities between RF and KS suggested the involvement of a herpesvirus in RF pathogenesis. Using consensus degenerate hybrid oligonucleotide primers (CODEHOP) (Rose et al. 1998) that were highly sensitive and broadly reactive with the different members of the herpesvirus family (VanDevanter et al. 1996), novel herpesvirus DNA sequences were identified in RF tissue of two species of SRV-2-infected macaques (Rose et al. 1997). Initial alignments revealed a 70% nucleotide identity with the DNA polymerase gene of KSHV. Subsequent studies with CODEHOP PCR primers targeting the conserved herpesvirus glycoprotein B gene identified nucleotide sequences in the RF tissue that displayed 75% identity with the glycoprotein B gene of KSHV (Bosch et al. 1998). Phylogenetic analyses demonstrated that the novel macaque virus sequences were highly related to each other and to KSHV and clustered with members of the lymphotropic rhadinovirus genus of herpesviruses (Fig. 11.1). This indicated that the amplified DNA sequences were derived from novel KSHV-like rhadinoviruses of macaques,

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which were designated as retroperitoneal fibromatosis-associated herpesvirus (RFHV) from pig-tailed macaques (Macaca nemestrina, RFHVMn) and rhesus macaques (M. mulatta, RFHVMm) (Rose et al. 1997).

Genomic Conservation Between KSHV and RFHV The complete genome sequence of KSHV was determined from cosmid and phage libraries prepared from KSHV-infected PEL cell lines, revealing the presence of more than 90 viral genes (Russo et al. 1996). The majority of these genes encoded structural and enzymatic proteins that were highly conserved with other herpesviruses, especially the gammaherpesviruses. However, several divergent regions dispersed within blocks of conserved sequences were identified. These divergent loci contained a large number of genes with homology to cellular regulatory genes (Russo et al. 1996). These viral homologs of cellular genes have been shown to function in the disruption of several cellular processes, including apoptosis, cell cycle, and antiviral immunity, and are thought to contribute to the unique biology of KSHV and function in the development of KS tumors (Nicholas et al. 1997). The complete sequence of the RFHV genome has not yet been determined. Similar to the situation with KSHV-infected KS tumor cells, culture of tumor cells derived from RF tumors results in the loss of the viral genome. Furthermore, no RFHV-infected lymphomas have been identified that would contain high numbers of the viral episome. Thus, sequence analysis of RFHV has relied upon the use of CODEHOP-based PCR to amplify regions of interest from DNA isolated from archived RF biopsies. The sequence of a 4.3-kb region of the genomes of RFHVMn and RFHVMm, including the DNA polymerase gene and flanking sequences, has been determined (Rose et al. 2003). This region corresponds to the divergent locus B of KSHV that contains a number of viral homologs of cellular genes known to play a role in the pathogenesis of KSHV. As shown in Fig. 11.2, the RFHV genomes are colinear with the KSHV genome, containing the glycoprotein B gene (ORF8), the DNA polymerase gene (ORF9), ORF10, ORF RF2 (the viral interleukin-6 (IL-6) homolog), ORF 02 (the viral dihydrofolate reductase (vDHFR) homolog), ORF RF3 (the K3 MIR1 homolog), and ORF70 (the viral thymidylate synthase (vTS) homolog). Both RFHVMn and RFHVMm genomes lack an ORF 11 homolog. Though the function of ORF 11 is not yet clear, sequence conservation suggests that it is evolutionarily related to ORF 10. Phylogenetic analysis of the sequences in divergent locus B revealed a consistent clustering with the corresponding gene from KSHV, substantiating the original close phylogenetic relationship between RFHV and KSHV determined using the DNA polymerase gene (Rose et al. 2003). Sequence analysis of the divergent locus B region of RFHV revealed the conservation of two genes directly implicated in the pathogenesis of KSHV. First, the RFHV genome contained a viral homolog of the cellular IL-6 cytokine that has strong sequence homology to the KSHV vIL-6. The KSHV vIL-6 induces STAT

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Fig. 11.2 Relative organization of ~7.7 kb of the RFHV genome between the glycoprotein B (gB) and viral thymidylate synthase (vTS) genes in comparison to other primate rhadinoviruses, including KSHV (human), RRV (rhesus macaque), HVS (South American squirrel monkey). Missing genes are indicated with a dashed line, and noncontiguous regions of the genome are indicated with a double-slanted line. Copyright American Society for Microbiology, J. Virol 77, 2003, 5084–5097

signaling through direct interaction with the gp130 signal transducer. This is believed to enhance the growth and survival of cells latently infected with KSHV, thus contributing to virus-induced neoplasia (Chen et al. 2009). The RFHV vIL-6 sequence is 35% conserved with KSHV vIL-6 and the primary hydrophobic sites of protein interaction with gp130 are maintained, suggesting that RFHV vIL-6 would display the same binding affinities and function as KSHV vIL-6 (Rose et al. 2003). The RFHV genome also contains a homolog of the KSHV modulator of immune recognition (MIR). MIR1 homologs exist in several herpesviruses and are thought to play a critical role in immune evasion and viral persistence (Stevenson et al. 2000; Lehner et al. 2005). Two MIR homologs, ORFK3 (MIR1) and ORFK5 (MIR2), have been identified in KSHV. Both MIR1 and MIR2 function as ubiquitin ligases to downregulate major histocompatibility class I (MHC-I) and other immune molecules on the surface of infected cells (Coscoy and Ganem 2000; Ishido et al. 2000). The MIR1 homolog, RF3, shares positional homology with K3, and considerable sequence similarity exists among the zinc finger domains of RF3, K3, and K5 as well as within C-terminal conserved regions (Rose et al. 2003). Like the KSHV MIR1, the RFHV MIR1 homolog is able to downregulate cell surface MHC-1, suggesting that it also plays an important role in virus-induced neoplasia (Harris et al. 2010). The RFHV ORF73 LANA homolog was targeted for sequence analysis due to the critical role of KSHV LANA in viral persistence and tumorigenesis. As shown in Fig. 11.3, RFHV LANA is similar in size and structurally analogous to KSHV

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Fig. 11.3 Comparison of rhadinovirus ORF73 LANA homologs. (a) RV1 lineage rhadinoviruses: KSHV (human; NP_572129) and RFHVMn (pig-tailed macaque, ABH07414). (b) RV2 lineage rhadinoviruses: RRV (rhesus macaque, AAD21406) and MneRV2 (pig-tailed macaque, ABH07415). (c) New World rhadinoviruses: HVS (squirrel monkey; NP_040275, AtHV3) (spider monkey; NP_048045). Murine rhadinovirus: MHV68 (vole; NP_044913). Adapted from Burnside et al. (2006). Reprinted from Virology 354, Burnside et al., 103–115, 2006, with permission from Elsevier

LANA (Burnside et al. 2006). It contains an N-terminal serine/proline-rich region, a large internal glutamic acid-rich repeat region, and a conserved C-terminal domain. KSHV LANA binds to both host chromatin and to the KSHV viral DNA, thus tethering the viral episome to chromatin. LANA, therefore, plays an important role in the maintenance of viral infection during latency (Ballestas and Kaye 2001). The amino acid sequences critical for KSHV LANA binding to chromatin have been identified (Wong et al. 2004). Analysis of the RFHV LANA sequence revealed the complete conservation of these residues, suggesting that RFHV LANA would serve a similar role in the maintenance of the RFHV genome during latency (Burnside et al. 2006). The large internal glutamic acid-rich repeat in RFHV LANA contained multiple repetitions of the consensus sequence “EEPEPEPE” (Burnside et al. 2006). A monoclonal antibody (mAB247) developed against an “EPEPEP” repeat in the procyclin protein of trypanosomes was found to react specifically with RFHV LANA expressed as a recombinant protein in COS-7 cells, which was targeted directly to the nuclei. RFHV LANA was also recognized by the anti-KSHV LANA LN53 monoclonal antibody which binds to “EQEQ” repeats in the large glutamic acid-rich repeat region in KSHV LANA, suggesting similarities in the structure of the repeats (Burnside et al. 2006).

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Macaques and Other Old World Primates Are Host to Two Distinct Lineages of KSHV-Like Rhadinoviruses Subsequent to the discovery of RFHV, it was found that pig-tailed and rhesus macaques are host to additional closely related rhadinoviruses with similarity to KSHV. A novel virus called rhesus rhadinovirus (RRV) was isolated by two different groups from healthy (Desrosiers et al. 1997) and SIV-infected (Wong et al. 1999) rhesus macaques. Both isolates of RRV have been sequenced, with only minimal nucleotide differences observed (Searles et al. 1999; Alexander et al. 2000). Limited sequence information available from a rhadinovirus isolated from a pig-tailed macaque designated M. nemestrina rhadinovirus 2 (MneRV2) revealed a close phylogenetic relationship with RRV (Auerbach et al. 2000; Schultz et al. 2000; Burnside et al. 2006; Bruce et al. 2009). Sequence analysis revealed that MneRV2 and RRV were distinct from RFHVMn and RFHVMm, even though they infected the same macaque species. Two distinct KSHV-like herpesviruses have also been identified in many Old World primate host species, including African green monkeys, mandrills, gorillas, and chimpanzees (Greensill et al. 2000; Strand et al. 2000; Lacoste et al. 2001). Phylogenetic analysis of partial DNA polymerase sequences revealed that these viruses cluster into two distinct lineages (Schultz et al. 2000;Greensill et al. 2000). RFHVMn and RFHVMm cluster with KSHV and other closely related Old World primate viruses within the RV1 rhadinovirus lineage while RRV and MneRV2 cluster with a distinct set of other Old World primate viruses within the RV2 rhadinovirus lineage. No RV2 lineage virus has been identified in humans to date. The complete sequences of the ORF59 DNA polymerase processivity factors from RV1 and RV2 rhadinoviruses from chimpanzees and three species of macaque were determined and compared to the KSHV ORF59 (Bruce et al. 2009). Sequence and phylogenetic analysis clearly demonstrated that each primate species was host to a distinct RV1 and RV2 rhadinovirus and that individual animals were often coinfected with both types of rhadinovirus (Fig. 11.4).

Genetic Similarities Between RV1 and RV2 Rhadinoviruses Due to their ability to replicate in primary rhesus fibroblast cell lines and produce significant quantities of infectious virus, the two isolated RRV variants have been completely sequenced (Searles et al. 1999; Alexander et al. 2000). The RRV genomes were highly homologous and colinear with the KSHV and RFHV genome sequences. Gene sequence comparisons revealed a strong conservation of nucleotide and amino acid sequences between homologous genes of RFHV and RRV. Nucleotide sequence identity averaged 66% over the more highly conserved herpesvirus genes, including the DNA polymerase and glycoprotein B genes. Significantly higher nucleotide sequence identity reaching 88% was detected in numerous regions of these genes. Amino acid identity between RFHV and RRV sequences ranged from 19% (vIL-6) to 69% (vTS), demonstrating a close evolutionary relationship.

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Fig. 11.4 RFHVMn and RFHVMm are the macaque homologs of KSHV and phylogenetically cluster with members of the RV1 lineage of Old World primate rhadinoviruses. Protein maximum likelihood analysis of the complete sequences of homologs of the ORF59 DNA polymerase processivity factor from the known macaque, chimpanzee, and human RV1 and RV2 Old World primate rhadinoviruses. The ORF59 homolog of the New World primate rhadinovirus, herpesvirus saimiri (NP_040261), was used as an out-group. Bootstrap values for 100 replicate samplings and the scale for substitutions per site are provided. From Bruce et al., Virol J 2009 6:205, BioMed Central

Homologs of nearly every KSHV gene were identified in the RRV genomes. However, as shown in Fig. 11.2, some divergence between the RV1 and RV2 rhadinoviruses was present. In the divergent locus B, the RRV genome contained vIL-6 and vTS homologs, but lacked a homolog of the MIR1 ORF K3. Since MIR1 plays an important role in immunoevasion, the lack of this gene suggests that RRV may utilize a different pathway for maintenance of infection during latency. Analysis of the RRV and MneRV2 ORF73 LANA homologs also showed significant differences with the RV1 LANA homologs of KSHV and RFHV. The RV2 LANA homologs lacked the conspicuous, large, internal, glutamic acid-rich repeat region found in KSHV and RFHV

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LANA (Fig. 11.3). Furthermore, critical residues in the chromatin-binding motif of KSHV were not conserved in the RV2 LANA homologs, suggesting a difference in the tethering function of LANA during latency (Burnside et al. 2006).

Prevalence of RFHV and RRV in Captive Macaque Populations Serological studies in different National Primate Research Centers have detected antibodies to rhadinovirus antigens in 93–98% of the macaque populations (Desrosiers et al. 1997; White et al. 2009). However, none of these studies distinguished whether the antibodies were cross-reactive to both RV1 and RV2 macaque rhadinovirus antigens. Therefore, the actual seroprevalence of RFHV or RRV in these colonies is unknown. A real-time quantitative PCR (qPCR) assay was developed that targets common gene sequences within the RV1 variants from pig-tailed (RFHVMn), rhesus (RFHVMm), and fascicularis macaques (RFHVMf), but does not react with RV2 variants (Bruce et al. 2006). Additional qPCR assays have been developed that target either RRV alone (White et al. 2009) or common gene sequences within the RV2 variants from pigtail (MneRV2), rhesus (RRV), and fascicularis (MfaRV2) macaques (Bruce et al. 2005). The latter study demonstrated the important lack of cross-reaction with RV1 variants. Using these assays, studies in the California National Primate Research Center (rhesus macaques) and Washington National Primate Research Center (pig-tailed macaques) determined the prevalence of RFHV DNA in 35–42% of the animals, RRV/MneRV2 DNA in 48–81% of the animals, and coinfections in 20–42% of the animals (White et al. 2009; Rose et al. unpublished data). These studies indicate that macaque RV1 and RV2 rhadinovirus infections are endemic in the National Primate Research Centers.

Evidence for an Etiological Association of RFHV with RF Although RFHV was originally detected in RF tumors from different macaque species, the high prevalence of the closely related RV2 rhadinoviruses in the same species of macaques highlights the need to carefully distinguish between the two viral lineages during an assessment of etiology. Initially, the lineage-specific qPCR assays, described above, were used to quantitate the levels of the RV1 and RV2 rhadinoviruses in a set of archived RF tumor samples at the Washington National Primate Research Center that had a corresponding nonaffected spleen sample for comparison (Bruce et al. 2006). Six RF cases were identified, all associated with an SAIDS-like syndrome. Five animals were naturally infected with SRV-2. One SRV-2 animal had been experimentally infected with an SIV/HIV hybrid virus. The other animal was SRV-2 negative, but had been experimentally infected with a pathogenic SIV. All six animals contained multifocal fibrous nodules on the skin or on visceral organs, diagnosed as RF. qPCR analysis revealed that all six animals

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Fig. 11.5 Viral load of macaque rhadinoviruses in RF tumor lesions. The levels of RV1 (RFHVMn/ RFHVMm) and RV2 (MneRV2/RRV) were determined in RF tumor lesions and nonaffected spleen samples from SAIDS–RF pigtail (RF-1–4, 6) and rhesus macaques (RF-5), respectively, using RV1and RV2-specific TaqMan qPCR assays. The results were normalized to cell number by comparison to a single-copy cellular gene. Viral load is expressed as genome equivalent copies per million cells. From Bruce et al. 2006, J. Gen. Virol. 87, 3529–3538, Society for General Microbiology

were coinfected with both RV1 and RV2 rhadinoviruses. However, the RV1 levels were on average 1,000-fold higher than the RV2 levels in the RF tumor samples (Fig. 11.5). Furthermore, the RV1 levels in RF tumor samples were 1,000-fold higher than the corresponding levels in spleen, showing a strong tumor association. The RV1 tumor load was determined to be 1.8 × 106 viral copies per million cells while the spleen load was 2.9 × 103 copies per million cells. In contrast, the RV2 tumor load (mean of 1.6 × 104 copies per million cells) was lower than the load in the spleen (mean of 2.4 × 104 copies per million cells) (Bruce et al. 2006), showing no tumor association.

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Recently, a case of RF was reported in a rhesus macaque that had been experimentally infected with SIV and RRV (Orzechowska et al. 2008). No RFHV was detected in the lesion by PCR; however, the sensitivity or specificity of the PCR assay was not indicated. This study detected RRV in higher amounts in tissue derived from the RF lesion than in other tissue; however, no quantitative analysis of the viral load was determined. A similar situation was seen in the RF2 animal (Fig. 11.5), where the RV2 viral load was higher in RF tissue than spleen. However, the viral load for the RF sample was only 2.1 × 104 copies per million cells, indicating that maximally only 1 in 50 cells in the lesion could be infected (Bruce et al. 2006). In Orzechowska et al., in situ hybridization of RRV cosmid clones representing 50% of the viral genome showed localization to cells within the RF lesion with a spindle morphology, suggesting that the tumor cells were infected with RRV (Orzechowska et al. 2008). However, no controls were included that demonstrate the specificity of this hybridization, an important issue given the close sequence similarity between the RV1 and RV2 rhadinovirus DNA. To determine whether the spindle tumor cells in RF lesions are infected by RFHV, immunohistochemical analysis of RF sections was performed using both the anti-EP repeat and anti-KSHV LANA LN53 monoclonal antibodies which react with recombinant RFHV LANA in Western blot and immunofluorescence analysis and are nonreactive with recombinant RRV or MneRV2 LANA (Burnside et al. 2006). Both monoclonals showed strong specific nuclear staining of spindle cells in the RF tumor lesions (Fig. 11.6). Essentially, every spindle cell in the tumor was positive for RFHV LANA demonstrating that the tumor cells were latently infected by RFHV. Coupled with the qPCR data, this indicated that each tumor cell contained approximately two viral episomes. This is similar to the latent infection of KS spindle cells by KSHV (Boshoff et al. 1996; Ablashi et al. 2002; Curreli et al. 2003) and is consistent with an etiological association of RFHV with RF. Immunohistochemical analysis of nontumor lymph nodes from a macaque with RF, using the LN53 anti-KSHV LANA antibody, revealed distinct staining of occasional small lymphocytes (Bielefeldt-Ohmann et al. 2005). This correlates with the qPCR data of Bruce et al., from the spleen samples of the RF animals, which suggested that only 1 in 300 spleen cells were infected (Bruce et al. 2006). Although no IHC staining was determined for the RV2 rhadinoviruses, RRV, or MneRV2, in the RF tumor lesions or spleen samples in either of the studies by Bielfeldt-Ohmann et al. or Bruce et al., the level of RV2 virus determined by qPCR was not consistent with an infection of the tumor cells and more likely was due to an inflammatory infiltrate containing latently infected lymphocytes. In the single RF case identified by Orzechowska et al., spindle cells within the RF lesion reacted with a monoclonal antibody raised against the RRV vIL-6 (Orzechowska et al. 2008). However, the abnormal nuclear localization of this reactivity and the lack of specificity controls for RFHV vIL-6 rendered these results inconclusive. Both Orzechowska et al. and Bielefeldt-Ohmann et al. detected strong vimentin staining in the cytoplasm of the RF tumor cells with no desmin staining suggesting that the RF lesions, in both cases, were proliferative mesenchymal lesions (Bielefeldt-Ohmann et al. 2005; Orzechowska et al. 2008).

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Fig. 11.6 Immunohistochemical localization of RFHV LANA in RF tumor cells. Paraffin-embedded RF tumor (a, b) and normal jejunum (c, d) tissues were deparaffinized, subjected to antigen retrieval, incubated with the LN53 anti-KSHV LANA monoclonal antibody (a, c) or the anti-EP monoclonal antibody (b, d), and counterstained with hematoxylin. Inserts show magnification of positive nuclei in spindle-shaped tumor cells. Reprinted from Virology, 354, Burnside et al. 103–115, 2006, with permission from Elsevier

Conclusion Different species of macaque are infected with RFHV, the macaque homolog of the human herpesvirus, KSHV. Although its complete sequence is not known, RFHV exhibits a close similarity to KSHV in gene sequence and content. Its major latency protein, LANA, is recognized by the KSHV LANA monoclonal antibody and is expressed in spindleoid tumor cells in RF lesions, a KS-like neoplasm in macaques. In all cases examined, RFHV LANA was expressed as a major nuclear antigen in essentially all of the spindle-shaped tumor cells in RF lesions, demonstrating that the tumor cells were latently infected with RFHV. This was corroborated by qPCR analysis which detected elevated levels of the viral genome in RF lesions, which were determined to be approximately two viral genomes per cell. These results parallel the association of KSHV with KS tumor cells. While much work remains to show a conclusive etiological association, the current data suggest that the macaque RV1 rhadinovirus, RFHV, plays a major role in the induction of RF tumors in the background of a retrovirus-induced immunosuppression. In all cases examined, RF

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tumors were detected in animals also infected with a closely related rhadinovirus belonging to the RV2 lineage of Old World primate rhadinoviruses, including RRV and MneRV2. The role of the RV1 rhadinovirus and its interactions with the coinfecting RV2 rhadinovirus remain to be delineated.

References Ablashi DV, Chatlynne LG et al (2002) Spectrum of Kaposi’s sarcoma-associated herpesvirus, or human herpesvirus 8, diseases. Clin Microbiol Rev 15(3):439–464 Alexander L, Denekamp L et al (2000) The primary sequence of rhesus monkey rhadinovirus isolate 26–95: sequence similarities to Kaposi’s sarcoma-associated herpesvirus and rhesus monkey rhadinovirus isolate 17577. J Virol 74(7):3388–3398 Antman K, Chang Y (2000) Kaposi’s sarcoma. N Engl J Med 342(14):1027–1038 Auerbach MR, Czajak SC et al (2000) Species specificity of macaque rhadinovirus glycoprotein B sequences. J Virol 74(1):584–590 Ballestas ME, Chatis PA et al (1999) Efficient persistence of extrachromosomal KSHV DNA mediated by latency-associated nuclear antigen. Science 284(5414):641–644 Ballestas ME, Kaye KM (2001) Kaposi’s sarcoma-associated herpesvirus latency-associated nuclear antigen 1 mediates episome persistence through cis-acting terminal repeat (TR) sequence and specifically binds TR DNA. J Virol 75(7):3250–3258 Bielefeldt-Ohmann H, Barouch DH et al (2005) Intestinal stromal tumors in a simian immunodeficiency virus-infected, simian retrovirus-2 negative rhesus macaque (Macaca mulatta). Vet Pathol 42(3):391–396 Bosch ML, Strand KB et al (1998) Gammaherpesvirus sequence comparisons. J Virol 72(10):8458–8459 Boshoff C, Schulz TF et al (1995) Kaposi’s sarcoma-associated herpesvirus infects endothelial and spindle cells. Nat Med 1(12):1274–1278 Boshoff C, Talbot S et al (1996) HHV8 and skin cancers in immunosuppressed patients. Lancet 348(9020):9138 Bruce AG, Bakke AM et al (2005) Development of a real-time QPCR assay for the detection of RV2 lineage-specific rhadinoviruses in macaques and baboons. Virol J 2:2 Bruce AG, Bakke AM et al (2006) High levels of retroperitoneal fibromatosis (RF)-associated herpesvirus in RF lesions in macaques are associated with ORF73 LANA expression in spindleoid tumour cells. J Gen Virol 87(Pt 12):3529–3538 Bruce AG, Bakke AM et al (2009) The ORF59 DNA polymerase processivity factor homologs of Old World primate RV2 rhadinoviruses are highly conserved nuclear antigens expressed in differentiated epithelium in infected macaques. Virol J 6:205 Burnside KL, Ryan JT et al (2006) RFHVMn ORF73 is structurally related to the KSHV ORF73 latency-associated nuclear antigen (LANA) and is expressed in retroperitoneal fibromatosis (RF) tumor cells. Virology 354(1):103–115 Cesarman E, Chang Y et al (1995) Kaposi’s sarcoma-associated herpesvirus-like DNA sequences in AIDS-related body-cavity-based lymphomas. N Engl J Med 332(18):1186–1191 Chang Y, Cesarman E et al (1994) Identification of herpesvirus-like DNA sequences in AIDSassociated Kaposi’s sarcoma. Science 266(5192):1865–1869 Chen D, Sandford G et al (2009) Intracellular signaling mechanisms and activities of human herpesvirus 8 interleukin-6. J Virol 83(2):722–733 Coscoy L, Ganem D (2000) Kaposi’s sarcoma-associated herpesvirus encodes two proteins that block cell surface display of MHC class I chains by enhancing their endocytosis. Proc Natl Acad Sci USA 97(14):8051–8056

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Curreli F, Robles MA et al (2003) Detection and quantitation of Kaposi’s sarcoma-associated herpesvirus (KSHV) by a single competitive-quantitative polymerase chain reaction. J Virol Methods 107(2):261–267 Desrosiers RC, Sasseville VG et al (1997) A herpesvirus of rhesus monkeys related to the human Kaposi’s sarcoma-associated herpesvirus. J Virol 71(12):9764–9769 Dittmer D, Lagunoff M et al (1998) A cluster of latently expressed genes in Kaposi’s sarcomaassociated herpesvirus. J Virol 72(10):8309–8315 Friborg J Jr, Kong W et al (1999) p53 inhibition by the LANA protein of KSHV protects against cell death. Nature 402(6764):889–894 Giddens WE Jr, Bielitski JT et al (1979) Idiopathic retroperitoneal fibromatosis: an enzootic disease in the pigtail monkey, Macaca nemestrina. Lab Invest 40:294 Giddens WE Jr, Tsai CC et al (1985) Retroperitoneal fibromatosis and acquired immunodeficiency syndrome in macaques. Pathologic observations and transmission studies. Am J Pathol 119(2):253–263 Greensill J, Sheldon JA et al (2000) Two distinct gamma-2 herpesviruses in African green monkeys: a second gamma-2 herpesvirus lineage among old world primates? J Virol 74(3):1572–1577 Grundhoff A, Ganem D (2004) Inefficient establishment of KSHV latency suggests an additional role for continued lytic replication in Kaposi sarcoma pathogenesis. J Clin Invest 113(1):124–136 Harris S, Lang SM et al (2010) Characterization of the rhesus fibromatosis herpesvirus MARCH family member rfK3. Virology 398(2):214–223 Ishido S, Wang C et al (2000) Downregulation of major histocompatibility complex class I molecules by Kaposi’s sarcoma-associated herpesvirus K3 and K5 proteins. J Virol 74(11):5300–5309 Kedes DH, Lagunoff M et al (1997) Identification of the gene encoding the major latency-associated nuclear antigen of the Kaposi’s sarcoma-associated herpesvirus. J Clin Invest 100(10):2606–2610 Kedes DH, Operskalski E et al (1996) The seroepidemiology of human herpesvirus 8 (Kaposi’s sarcoma-associated herpesvirus): distribution of infection in KS risk groups and evidence for sexual transmission. Nat Med 2(8):918–924 King NW, Hunt RD et al (1983) Histopathologic changes in macaques with an acquired immunodeficiency syndrome (AIDS). Am J Pathol 113(3):382–388 Lacoste V, Mauclere P et al (2001) A novel gamma 2-herpesvirus of the Rhadinovirus 2 lineage in chimpanzees. Genome Res 11(9):1511–1519 Lehner PJ, Hoer S et al (2005) Downregulation of cell surface receptors by the K3 family of viral and cellular ubiquitin E3 ligases. Immunol Rev 207:112–125 Moore PS, Chang Y (1995) Detection of herpesvirus-like DNA sequences in Kaposi’s sarcoma in patients with and without HIV infection. N Engl J Med 332(18):1181–1185 Moore PS, Chang Y (1998) Kaposi’s sarcoma (KS), KS-associated herpesvirus, and the criteria for causality in the age of molecular biology. Am J Epidemiol 147(3):217–221 Nicholas J, Ruvolo V et al (1997) A single 13-kilobase divergent locus in the Kaposi sarcomaassociated herpesvirus (human herpesvirus 8) genome contains nine open reading frames that are homologous to or related to cellular proteins. J Virol 71(3):1963–1974 Orzechowska BU, Powers MF et al (2008) Rhesus macaque rhadinovirus-associated non-Hodgkin lymphoma: animal model for KSHV-associated malignancies. Blood 112(10):4227–4234 Radkov SA, Kellam P et al (2000) The latent nuclear antigen of Kaposi sarcoma-associated herpesvirus targets the retinoblastoma-E2F pathway and with the oncogene Hras transforms primary rat cells. Nat Med 6(10):1121–1127 Rainbow L, Platt GM et al (1997) The 222- to 234-kilodalton latent nuclear protein (LNA) of Kaposi’s sarcoma-associated herpesvirus (human herpesvirus 8) is encoded by orf73 and is a component of the latency-associated nuclear antigen. J Virol 71(8):5915–5921 Renne R, Dittmer D et al (2004) Experimental transmission of Kaposi’s sarcoma-associated herpesvirus (KSHV/HHV-8) to SIV-positive and SIV-negative rhesus macaques. J Med Primatol 33(1):1–9 Rose TM, Ryan JT et al (2003) Analysis of 43 kilobases of divergent locus B of macaque retroperitoneal fibromatosis-associated herpesvirus reveals a close similarity in gene sequence and genome organization to Kaposi’s sarcoma-associated herpesvirus. J Virol 77(9):5084–5097

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Rose TM, Schultz ER et al (1998) Consensus-degenerate hybrid oligonucleotide primers for amplification of distantly related sequences. Nucleic Acids Res 26(7):1628–1635 Rose TM, Strand KB et al (1997) Identification of two homologs of the Kaposi’s sarcomaassociated herpesvirus (human herpesvirus 8) in retroperitoneal fibromatosis of different macaque species. J Virol 71(5):4138–4144 Russo JJ, Bohenzky RA et al (1996) Nucleotide sequence of the Kaposi sarcoma-associated herpesvirus (HHV8). Proc Natl Acad Sci USA 93(25):14862–14867 Schultz ER, Rankin GW Jr et al (2000) Characterization of two divergent lineages of macaque rhadinoviruses related to Kaposi’s sarcoma-associated herpesvirus. J Virol 74(10):4919–4928 Searles RP, Bergquam EP et al (1999) Sequence and genomic analysis of a Rhesus macaque rhadinovirus with similarity to Kaposi’s sarcoma-associated herpesvirus/human herpesvirus 8. J Virol 73(4):3040–3053 Shibata R, Maldarelli F et al (1997) Infection and pathogenicity of chimeric simian-human immunodeficiency viruses in macaques: determinants of high virus loads and CD4 cell killing. J Infect Dis 176(2):362–373 Staskus KA, Zhong W et al (1997) Kaposi’s sarcoma-associated herpesvirus gene expression in endothelial (spindle) tumor cells. J Virol 71(1):715–719 Stevenson PG, Efstathiou S et al (2000) Inhibition of MHC class I-restricted antigen presentation by gamma 2-herpesviruses. Proc Natl Acad Sci USA 97(15):8455–8460 Strand K, Harper E et al (2000) Two distinct lineages of macaque gamma herpesviruses related to the Kaposi’s sarcoma associated herpesvirus. J Clin Virol 16(3):253–269 Tsai CC (1993) Fibromatosis in macaques infected with type D retroviruses. In: Jones TC, Mohr U, Hunt RD (eds) Nonhuman primates, 1st edn. Springer-Verlag, Berlin Tsai CC, Giddens WE Jr et al (1985) Retroperitoneal fibromatosis and acquired immunodeficiency syndrome in macaques: epidemiologic studies. Lab Anim Sci 35(5):460–464 VanDevanter DR, Warrener P et al (1996) Detection and analysis of diverse herpesviral species by consensus primer PCR. J Clin Microbiol 34(7):1666–1671 White JA, Todd PA et al (2009) Prevalence of viremia and oral shedding of rhesus rhadinovirus and retroperitoneal fibromatosis herpesvirus in large age-structured breeding groups of rhesus macaques (Macaca mulatta). Comp Med 59(4):383–390 Wong SW, Bergquam EP et al (1999) Induction of B cell hyperplasia in simian immunodeficiency virus-infected rhesus macaques with the simian homologue of Kaposi’s sarcoma-associated herpesvirus. J Exp Med 190(6):827–840 Wong LY, Matchett GA et al (2004) Transcriptional activation by the Kaposi’s sarcoma-associated herpesvirus latency-associated nuclear antigen is facilitated by an N-terminal chromatinbinding motif. J Virol 78(18):10074–10085 Zhong W, Wang H et al (1996) Restricted expression of Kaposi sarcoma-associated herpesvirus (human herpesvirus 8) genes in Kaposi sarcoma. Proc Natl Acad Sci USA 93(13):6641–6646

Chapter 12

Murine Gammaherpesvirus-Associated Tumorigenesis Kathleen S. Gray and Samuel H. Speck

Introduction The gammaherpesviruses subfamily of herpesviruses is distinguished from their alpha and beta relatives by sequence homology, tropism for lymphocytes, and strong association with tumorigenesis in several host organisms. For the human gammaherpesvirses, Epstein–Barr virus (EBV) is linked to numerous malignancies, including Burkitt’s lymphoma and Hodgkin’s lymphoma, as well as nasopharyngeal and gastric carcinomas. Likewise, Kaposi Sarcoma-Associated herpesvirus (KSHV) is the etiologic agent of Kaposi’s sarcoma, a tumor of mixed lymphocyte and endothelial cell origin, and is associated with primary effusion lymphoma (PEL) and multicentric Castleman’s disease (MCD), two malignancies of lymphoid origin. Herpesvirus saimiri (HVS), a natural pathogen of nonhuman primates, is linked to lymphomagenesis in New World primates and induces fulminant T-cell lymphomas in rabbits. Although gammaherpesvirus-related tumors are relatively rare, their incidence is greatly increased in immunocompromised individuals. For example, prevention and treatment of posttransplant lymphoproliferative disorder (PTLD) or lymphoma is now an important aspect in the clinical management of transplant and AIDS patients. The association of gammaherpesviruses with human disease has made their study an area of active interest for nearly 6 decades. Until more recently, the breadth of studies was limited by the narrow host tropism of EBV and KSHV, making it difficult to examine major aspects of pathogenesis including primary infection and subsequent events leading to lymphomagenesis. The identification, initial characterization, and sequencing of murine gammaherpesvirus 68 (MHV68), a naturally

K.S. Gray • S.H. Speck (*) Department of Microbiology and Immunology, The Emory Vaccine Center, Emory University School of Medicine, Atlanta, GA 30322, USA e-mail: [email protected] E.S. Robertson (ed.), Cancer Associated Viruses, Current Cancer Research, DOI 10.1007/978-1-4614-0016-5_12, © Springer Science+Business Media, LLC 2012

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Fig. 12.1 Schematic depicting unique and conserved g2-herpesvirus genes. As shown, most of the genes are highly conserved among the rhadinoviruses (g2-herpesviruses) MHV68, KSHV, and HVS. The majority of these encode replication-associated proteins essential for virus replication and virion assembly. However, there are a number of genes that are not well conserved among the rhadinoviruses, and many of these have demonstrated roles in viral latency (note the presence of several “unique” genes within each annotated genome). Despite limited sequence homology, several of these genes encode a protein of orthologous function and are interspersed among regions of high sequence conservation. The genomes are not drawn to scale (genome size for MHV68, KSHV, and HVS is approximately 120, 138, and 113 kb, respectively) and compensatory adjustments were made for the additional copies of Orf75 at the right-end of the MHV68 genome. Essential, attenuated, and nonessential genes were determined by two independent transposon mutagenesis analyses of the MHV68 genmome (Gong et al. 2009; Song et al. 2005)

occurring rodent gammaherpesvirus, has provided the basis for a model system using MHV68 infection of laboratory mice to study key aspects of gammaherpesvirus pathogenesis. MHV68 shares considerable overall sequence homology with EBV, KSHV, and HVS and encodes homologs of several genes that exhibit transforming properties in human and nonhuman primate systems (Fig. 12.1). These include genes affecting cell cycle progression, survival, proliferation, and immune evasion as discussed below. In addition, MHV68 infection of laboratory mice, as well as wild rodents, shares several important immunological characteristics with EBV and KSHV infection, including the kinetics of the adaptive immune response, latent infection of B lymphocytes, and a requirement for an intact immune system to limit viral replication and associated pathologies. Although MHV68 is becoming increasingly well accepted as a model to study several aspects of gammaherpesvirus pathogenesis, some speculation exists in the field as to its usefulness in the further understanding of gammaherpesvirus-induced transformation. EBV has clear transforming potential, and is ubiquitously used to generate immortalized B cell lines in diverse areas of study. Likewise, KSHV also has transforming potential, and the association of EBV and KSHV with human malignancy is well-established.

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Despite the high level of sequence conservation among MHV68 and the human gammaherpesviruses, it has been argued that no direct evidence exists to corroborate the classification of MHV68 as a DNA tumor virus. However, as we discuss below, MHV68 encodes several proteins that alter the biology of infected cells with consequences highly reminiscent of transformed cells. The inability to efficiently infect primary mouse B cells with MHV68 in vitro has complicated efforts to demonstrate commonalities between the mouse and human gammaherpesviruses, and therefore, true MHV68 transforming potential has not been conclusively assessed. Importantly, however, MHV68 infection promotes lymphoproliferative disease in both immunocompetent mice as well as mice with specific immune defects (see Table 12.1). Initial studies aimed at evaluating MHV68-mediated tumorigenesis demonstrated that slightly more than 10% of infected mice of various genetic backgrounds developed malignant disease; around 10% of these mice had lymphoproliferative disease (LPD), and around 4% developed high-grade lymphomas (Sunil-Chandra et al. 1994). Characteristics of tumors in these studies mimic pathologies observed in human disease and provide further justification for the use of MHV68 to examine the contribution of gammaherpesviruses to lymphomagenesis. Subsequent experiments using cell lines derived from MHV68-positive lymphomas have also revealed fundamental similarities to human gammaherpesviruses with regard to tumorigenesis, latency, and reactivation (Husain et al. 1999; Moser et al. 2005; Robertson et al. 2001; Usherwood et al. 1996b; Wu et al. 2000). In addition to MHV68, five other viruses isolated from wild rodents (MHV60, MHV72, MHV76, MHV78, and MHV-Sumava) are highly genetically similar to MHV68 and have also been associated with the development of lymphomas in mice (Blaskovic et al. 1980; Hrabovska et al. 2010; Mistrikova et al. 2004; Mrmusova et al. 2002; Pappova et al. 2004) The following sections review several key aspects in progression to MHV68-associated lymphoproliferative disease and lymphoma. We discuss putative viral oncogenes, as well as oncogenic signaling pathways induced by MHV68 infection not yet ascribed to specific viral proteins. We also address the role of the immune system in controlling MHV68-associated disease, both directly (by way of cell-mediated cytotoxicity) and indirectly (by controlling inflammation-induced damage resulting from persistent lytic viral replication). Finally, we discuss the potential contributions of the MHV68 system to furthering the understanding and treatment of human malignancies associated with gammaherpesvirus infection.

MHV68 Encoded Proteins with Possible Direct Roles in Tumorigenesis Viral proteins have long acted as harbingers for cellular oncogene discovery. Many tumor-virus oncogenes function by altering and exploiting key aspects of cellular biology to favor self-propagation and promote survival of the host cell. As a consequence of viral gene expression and perhaps secondary mutagenic events, infected cells may come to exhibit several or all hallmarks of transformation: self-sufficiency in growth signals, insensitivity to antigrowth signals, evasion of apoptosis, limitless

Table 12.1 Characteristics of lymphoma or lymphomagenic disease associated with MHV68 infection Viral genome Time to disease Pathology Mouse strain Cell type/Site of pathology positive? (months) Incidence References Lymphoproliferative disease C57Bl/6, Balb/c CB/CCa/lung, K, L, spl, Yes (ISH) 5.5–28 5% (n = 220)c Sunil-Chandra et al. (1994) MLN, intestine B lymphocytesa/spl, lung Yes (ISH-vtRNA) 6–7 50% infected micee Tarakanova et al. (2005) Hyperplasia Balb/c-b2Md (Lymphoid or B10Br B lymphocytes/lung Yes (ISH) 21 0.5% (n = 220) Sunil-Chandra et al. (1994) follicular) C57Bl/6-IFNgR−/−f Lymphocytes/Lung n.d.g 2–12 35% (n = 20) Lee et al. (2009) Lymphoma Balb/c-b2M IB/CBa,b/spl, lung Yes (ISH-vtRNA) 11.6 (avg)j 29% infected miceh Tarakanova et al. (2005) & L metastases B220+ and CD3+ CBA, C57Bl/6, Yes (ISH) 5.5–28 4% (n = 220) Sunil-Chandra et al. (1994) lymphocytesa,b/spl, L, K, Balb/ce lung, MLN, pancreas, ovary, K, adrenal gland Pulmonary lymphoma C57Bl/6-IFNgR-/-f B220+/lungi, LN, heart, Yes (RT-PCR) <12k 45% (n = 20)l Lee et al. (2009) (pathology similar brain to LyG) Abbreviations: L Liver, MLN mesenteric lymph node, spl spleen, K kidney, LN lymph node, IB immunoblast, CB centroblast, LyG lymphomatous granulomatosis a plasmacytic markers present b evidence of clonality in tumors (determined by Ig transcript analysis) c Total incidence of LPD: 60% (mice treated with cyclosporine A) versus 20% (untreated mice) d disease severity affected by absence of v-cyclin, v-bcl2, and M1 genes e 50% infected mice (versus 0% mock-infected); 67% infected (versus 22% mock-infected) developed LH or lymphoma f Mice infected with both wild-type and v-cyclin-deficient virus g Lungs from infected mice at day 90 infected mice were positive for polIII, but not Rta, transcripts by ISH h 29% infected mice developed lymphoma (versus 6% mock infected) i Seven of nine B cell lymphomas presented in lung j Compare to 15.8 months (mean) time to disease for mock-infected mice k Forty-five percent of infected mice developed B cell lymphoma after 1 year l Developed frank tumors

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replicative potential, sustained angiogenesis, tissue invasion and metastasis, and genome instability. In the following section, we review putative MHV68 oncogenes encoding proteins that may facilitate the acquisition of transforming characteristics.

v-Cyclin General characteristics. Sequencing of the MHV68 genome revealed a viral ortholog to the cellular D-type cyclins. The presence of D-type cyclin orthologs in KSHV and HVS and modulation of cellular cyclin D expression by EBV indicate a common and important theme in gammaherpesvirus pathogenesis (Chang et al. 1996; Nicholas et al. 1992; Palmero et al. 1993; Sinclair et al. 1995). The overall genomic position of the gene encoding the gammaherpesvirus cyclin homolog is largely conserved, as are many key residues dictating interactions with host cyclin-dependent kinases (CDKs) (Virgin et al. 1997). Despite its location in a region associated with latent gene transcription, v-cyclin is not considered a latency-associated gene. Based on a study using conservative criteria to define latency MHV68 candidates, v-cyclin was excluded based on the abundant expression of v-cyclin transcripts during lytic replication (Virgin et al. 1999). V-cyclin transcripts were not detected in latently infected PECs or splenocytes in this study, and studies of lytic infection in fibroblasts had originally defined MHV68 v-cyclin as a lytic gene with leaky-late expression kinetics (van Dyk et al. 2000). However, subsequent analyses using latently infected animals and cells have suggested that v-cyclin is more likely a latency-associated gene with immediate-early kinetics during lytic replication or reactivation (Allen et al. 2007; Forrest and Speck 2008; Martinez-Guzman et al. 2003). The original classification of v-cyclin as a lytic gene is not surprising given the proposed role of the protein during MHV68 infection. Despite sharing 25 and 21% sequence identity with the KSHV and HVS cyclins, respectively, MHV68 v-cyclin has been shown to preferentially interact with CDK2 and cdc2, rather than CDK6, a characteristic that distinguishes it from the other gammaherpesvirus cyclins (Card et al. 2000; Upton et al. 2005). However, like k-cyclin, MHV68 v-cyclin complexes perpetuate phosphorylation of several targets in the G1 to S transition (discussed further below), which presumably facilitates cell-cycle progression and DNA replication in infected cells (Upton et al. 2005). Also, crystal structure analyses of MHV68 v-cyclin bound to cellular CDKs suggest that despite the sequence divergence among gammaherpesvirus D-type cyclins, interactions with respective CDK binding partners are mediated by key conserved residues (Card et al. 2000). MHV68 v-cyclin null mutants have profound reactivation defects as assessed by ex vivo analyses. While the virus establishes latency in splenocytes and peritoneal exudate cells (PECs) at a similar frequency to wild-type or a genetic marker-rescue virus in C57BL6 mice, it is impaired for virus reactivation in both cellular compartments (Hoge et al. 2000; van Dyk et al. 2000). The defect is more apparent in PECs, and B-cell-deficient mice infected with a cyclin-LacZ insertion virus exhibit an exaggerated reactivation defect (van Dyk et al. 2003). This reactivation defect most

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likely contributes to the deterioration in the frequency of infected cells by 6 months postinfection. These data suggest that, in addition to B lymphocytes, v-cyclin plays a critical role in launching MHV68 lytic replication/reactivation and maintaining latent infection in the non-B cell reservoir (e.g., macrophages). This supports a role for v-cyclin in promoting lytic viral gene transcription and cell-cycle progression in resting lymphocytes, perhaps simultaneously driving cellular proliferation and reactivation from latency. Interestingly, viral mutants containing only mutations in key residues important for CDK-interaction and activation display a significant decrease in reactivation from splenocytes, but do not appear compromised for reactivation in PECs (Upton and Speck 2006). This defect, however, may be explained by the replication defects seen in the lung of infected animals, which may then result in decreased establishment and reactivation in the spleen. This indicates that although the overall result of v-cyclin expression may be cell-cycle progression, the mechanism for overcoming growth barriers in infected cells varies according to cell type and ultimately requires both CDK-dependent and independent functions for a successful in vivo infection. The role of v-cyclin in permissive lytic infection is not fully elucidated, but also supports the possibility of cell-type dependent mechanisms. While high-dose intranasal inoculation of C57BL/6 mice with v-cyclin mutants produces lung titers similar to those with a marker-rescue virus, inoculation with a lower dose of virus reveals a requirement for an intact v-cyclin – including the amino acids essential for CDKbinding and activation (Upton and Speck 2006; van Dyk et al. 2000). This characteristic does not appear to be essential for all lytic replication, as v-cyclin is dispensable for lytic replication in vitro in NIH 3T12 fibroblasts and some lung epithelial cell lines, as well as for virus replication in the spleen following intraperitoneal infection (Hoge et al. 2000; Upton and Speck 2006; van Dyk et al. 2000). However, the in vitro analyses are complicated by the fact that cells used in these experiments were either actively cycling (in vivo), or serum-starved but capable of reentering the cell cycle (in vitro), thereby potentially masking the requirement for v-cyclin-CDK-interactions (via the action of host cyclin-CDK complexes). Thus, it appears that the MHV68 v-cyclin plays a specific role in driving cell-cycle progression and lytic replication in terminally differentiated cells in the lung in vivo. Evidence for v-cyclin as an oncogene. Much evidence exists to support the classification of gammaherpesvirus v-cyclins as oncogenes (Mittnacht and Boshoff 2000). In the case of MHV68, some of the most convincing data are those demonstrating T-cell lymphomagenesis in transgenic mice expressing v-cyclin under the control of the lck promoter (van Dyk et al. 1999). These studies support a proproliferative role for v-cyclin, as T lymphocytes exhibited a marked increase in BrdU-incorporation and the percentage of S/G2/M cells compared to total splenocytes. However, despite the increased proliferation of thymocytes in these animals, there was only a slight increase in total T cell number, a disconnect attributed to increased apoptosis of v-cyclin-expressing cells in the thymus. Although several factors may contribute to cell death in the transgenic animals, one possible conclusion is that v-cyclin is sufficient to support cell cycle progression and proliferation yet insufficient to inhibit apoptosis in the context of v-cyclin-driven thymocyte expansion.

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The effects of v-cyclin-CDK complexes on the activation status of key cell-cycle regulatory proteins support its classification as an oncogene. The tumor suppressors Rb, p21Cip1, p53, and p27Kip1, as well as the antiapoptotic protein bcl-2, have been shown to be phosphorylation targets of MHV68 v-cyclin in a host-CDK-dependent fashion (Card et al. 2000; Upton et al. 2005). This observation is in apparent contrast to those made in the context of MHV68 infection of endothelial cells. While infection of epithelial cells results in productive viral replication followed by cell lysis, infection of endothelial cells appears to support persistent infection wherein cells produce titers equivalent to epithelial cell cultures but retain viability for weeks following infection (Suarez and van Dyk 2008). This prolonged survival appears to be dependent on v-cyclin, as cells infected with a v-cyclin-null virus produce infectious virus but exhibit greatly reduced viability. This seems to argue for a prosurvival role for v-cyclin during productive replication in endothelial cells. It may, therefore, be that v-cyclin is not sufficient to rescue cells forced into cycling from apoptosis (as in the transgenic mouse system) but is, nonetheless, required for protection against cell death in a persistently infected cell. A particularly notable aspect of endothelial cell infection was the distinct downregulation of cellular adhesion molecules on infected cells, accompanied by uniform changes in cell size and morphology. These changes could be ameliorated by treatment with PAA, which blocks late viral gene expression and, therefore, were most likely a direct result of viral infection. Infected cells displayed markedly reduced expression of ICAM-1 and VCAM-1, two molecules involved in cellular adhesion. This complements the observation that a significant number of endothelial cells continued to survive and produce virus even after detaching from the tissue culture matrix. It was, therefore, concluded that MHV68 infection contributes to the acquisition of anchorage-independent growth characteristics via the down regulation of cellular adhesion molecules, a process with strong implications for both trafficking of infected cells as well as tumorigenesis. V-cyclin is not required for these changes in cell surface marker expression, so it, therefore seems as if its role in endothelial cell infection is primarily to promote sustained viability of cells undergoing active viral replication. Though not the same mechanism for tumorigenesis as cell-cycle promotion, this function of v-cyclin also has implications for the preferential survival and outgrowth of infected cells. v-cyclin and tumorigenesis. Although BALB/c b2m−/− mice are predisposed to developing tumors, MHV68 infection both increases the incidence and accelerates the development of lymphomas in these mice (Tarakanova et al. 2005). In particular, MHV68 exacerbated the development of what the authors termed atypical lymphoid hyperplasia (ALH) in BALB/c b2m−/− mice, a lesion of hyperproliferation characterized by MHV68-positive cells and high numbers of plasmacytic CD138+ cells (Tarakanova et al. 2005). ALH lesions preceded the development of B cell lymphoma and shared similarities with a subset of PTLD. ALH lesions were, therefore, used as a measure of MHV68-induced tumorigenesis. A subsequent study found that mice infected with a v-cyclin-null virus displayed significantly less splenic ALH than mice infected with wild-type virus, despite the presence of latent viral genomes (Tarakanova et al. 2008). Mice infected with the v-cyclin null virus did,

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however, exhibit plasmacytosis at a frequency similar to wild-type virus-infected mice. This observation highlights the complexity of events leading to MHV68mediated tumorigenesis. While individual viral oncogenes, such as v-cyclin, may contribute directly to cellular transformation and lymphomagenesis by promoting replication or cell survival, it is important to consider that MHV68 infection as a whole may also contribute to this process by promoting a proinflammatory environment, which leads to cell damage and the acquisition of transforming mutations.

v-bcl2 General characteristics. A key component of successful virus replication is the override of host-derived cell death signals in response to viral infection. To this end, many viruses have evolved specific mechanisms to allow survival of an infected cell by suppressing multiple cell death pathways, such as apoptosis, necrosis, or autophagy. Prosurvival genes are an especially important component of oncogenic virus infection for obvious reasons; pathogen-aided viability of a cell with compromised genomic integrity can have deleterious consequences for the host organism should this cell sustain further insult and acquire growthpromoting characteristics. However, given the hostile environment created by the likely induction of interferon production and the antiviral state during the initial stages of viral replication, it is essential that host-cell death signals be suppressed for infection to progress and latency to be established, particularly in B cells and macrophages. The apoptotic pathway has been long-studied as a mechanism of programmed host-cell death and is regulated by homo- and heterotypic interactions amongst a family of pro- or antiapoptotic proteins. Very simplistically, the proapoptotic proteins Bad, Bid, and Bax are antagonized by their association with antiapoptotic proteins Bcl-2 and BclXL. Cells respond to numerous insults, including viral infection, by undergoing programmed cell death. Premature cell death is likely deleterious to the long-term pattern of viral replication, latent persistence and reactivation. Therefore, many viruses have evolved proteins that inhibit apoptosis by blocking the actions of host proteins involved in cellular suicide. KSHV, EBV, HVS, and MHV68 all encode molecules with homology to a cellular protein, bcl-2, that prevents cell death elicited by mitochondrial insults. (Virgin et al. 1997). M11/v-Bcl2 and MHV68 v-cyclin are encoded on opposite strands in close proximity (Virgin et al. 1997). Despite the immediate juxtaposition of M11 and v-cyclin, the expression profiles of their transcripts are quite distinct with regard to acute replication. The original characterization of M11 transcription in B-cell deficient mice suggested that M11/v-Bcl2 is expressed at low levels during lytic replication, but it is easily detected in latently infected peritoneal cells and is, therefore designated a latency-associated gene (Virgin et al. 1999). Subsequent studies in wild-type animals indicated that M11 is actively transcribed during both acute lytic and persistent infection (Roy et al. 2000).

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The gammaherpesvirus Bcl-2 orthologs bear less than 25% sequence homology to their cellular counterparts and do not appear to contain distinct BH3 or BH4 domains (Tschopp et al. 1998). MHV68 M11, in particular, is also divergent from the other gammaherpesvirus proteins in that it contains only a BH1 domain and lacks an obvious BH2 domain consistently found in other v-Bcl2s. BH3 and BH4 domains are required for interaction of cellular Bcl-2 and Bcl-xl proteins to potentially block antiapoptotic function. Therefore, the lack of these domains may make the M11 protein refractory to regulation by proapoptotic host proteins. Earlier studies demonstrated that M11 localizes to the cytoplasm and that overexpression was sufficient to protect cells from TNF and Fas-induced apoptosis (Roy et al. 2000; Wang et al. 1999), but no comprehensive analysis of cellular interacting partners has been performed to verify M11’s involvement in the host apoptotic pathway. More recent work has demonstrated that despite the absence of domains required for association with BH3-domain containing proteins, M11 tightly associates with the proapoptotic Bim, Bak, Bid, Bmf, Puma, and Noxa (Ku et al. 2008). In addition, these and other studies revealed an additional prosurvival role for M11 in the inhibition of autophagy via inhibition of Beclin-1 (Xiaofei et al. 2009; Ku et al. 2008; Sinha et al. 2008). Beclin-1 is a proautophagic BH3-domain containing protein whose action is also inhibited by host Bcl-2. It was shown not only that M11 tightly associates with Beclin-1 by isothermal titration calorimetric analyses but also that this interaction was significantly stronger than Beclin-1 with host Bcl-2. It was subsequently shown that in addition to protection from apoptosis M11 could not only protect against rapamycin-induced autophagy but could also do so more efficiently than cellular Bcl-2 (Xiaofei et al. 2009). These analyses suggest that sequence divergence from host Bcl-2 proteins has not compromised the effectiveness of viral Bcl-2 homologs, but rather conferred upon them enhanced prosurvival functions capable of protection against multiple host-cell-induced death pathways. It would seem that survival of the host cell in the midst of death signals triggered by lytic viral infection would require the function of virus-encoded antiapoptotic/ autophagic proteins, yet studies using viruses lacking the Bcl2 homolog demonstrated that M11, like BHRF1 of EBV, is dispensable for acute replication in vitro. Similarly, M11-null MHV68 replicates equivalent to wild-type virus in the lung and other sites in vivo (de Lima et al. 2005; Gangappa et al. 2002b). However, while M11 is not required for the establishment of latent infection, it appears to contribute to the efficiency of virus reactivation, a characteristic shared with the v-cyclin-null virus as discussed above and made more interesting by the demonstration that both v-cyclin and M11 appear to be involved in persistent replication and the associated severity of inflammatory disease observed in IFNg−/− and IFNgR−/− mice (Gangappa et al. 2002b; Tarakanova et al. 2008). The fact that two viral proteins involved in forcing cell-cycle progression and survival would be dispensable for acute replication, yet important for reactivation, provides important clues about the pathogenesis of latent viral infection. It has been hypothesized that low-level, spontaneous reactivation in vivo is one of the mechanisms by which herpesviruses maintain long-term latency. Given the observations above, it seems that reactivation from a latently infected, perhaps resting, lymphocyte

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requires viral proteins to not only stimulate the cell cycle, but to preserve viability in the cell driven into a cycling state. How this compromises the functional and genetic integrity of the infected cell is not entirely clear, but may set the stage for transforming events to occur. As discussed below, the involvement of the host immune system in this process further highlights the complexity of these interactions. Evidence for v-bcl2 as an oncogene. As mentioned above, several studies have demonstrated the potent antiapoptotic activity of the M11 protein. When overexpressed, M11 can protect against Sindbis virus-induced apoptosis in BHK cells, as well as TNF-a and Fas-induced apoptosis in HeLa cells (Bellows et al. 2000; Roy et al. 2000; Wang et al. 1999). The cell death pathways triggered by intrinsic factors (such as virus infection) and extrinsic factors (such as TNF/Fas-ligation) have overlapping yet distinct pathways. It, therefore seems that the M11 can protect against both intrinsic and extrinsic insults that may otherwise result in cell death in the context of these studies. M11 likely supports survival during MHV68 infection and, given the data regarding M11-mediated protection from Fas-ligation, may also protect against CTL-mediated killing during an immune response. M11 may, therefore, enhance survival of infected cells that have sustained transforming mutations potentially facilitating tumorigenesis. Since autophagy also mediates tumor-suppressive functions in vivo, subversion of autophagy by M11 (Xiaofei et al. 2009; Ku et al. 2008; Sinha et al. 2008) via suppression of Beclin-1-mediated autophagosome formation may also result in viruspromoted survival of a cell that would otherwise be eliminated. Similar to v-cyclin-null MHV68, M11-null virus does not induce ALH as efficiently as M11 competent MHV68 following infection of BALB/c β2m−/− mice. Since the autophagic pathway is important in tumor suppression (reviewed in Bialik and Kimchi 2008; Gozuacik and Kimchi 2004), it may be that v-bcl2-mediated inhibition of this process contributes to the development of ALH in the absence of a CD8+ T cell response. In the coincident absence of an optimal immune response and antiautophagic or antiapoptotic v-bcl2, cell death may be induced by other extrinsic or intrinsic signals, thus limiting the amount of cellular proliferation and controlling lymphoid hyperplasia, limiting the initiating events of lymphomagenesis.

vGPCR General characteristics. A third conserved gammaherpesvirus protein is that encoded by MHV68 Orf74, giving rise to a product orthologous to host G-proteincoupled-receptors (GPCRs). These viral GPCRs (v-GPCR) share characteristics reminiscent of host receptor proteins and are either activated by similar ligands or signal through common pathways (Nicholas 2005) Like the KSHV vGPCR, the MHV68 GPCR bears significant sequence homology to host CXCR2 receptors, which binds several chemokines and has particularly high affinity for IL-8 (Virgin et al. 1997; Wakeling et al. 2001). The KSHV vGPCR is highly transforming, which is thought to be due in part to its ability to induce prosurvival and proangiogenic

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signaling in response to IL-8 and other ligands (Arvanitakis et al. 1997; Sodhi et al. 2000; Yang et al. 2000). Similarly, MHV68 vGPCR confers on NIH 3T3 fibroblasts the ability to form foci in culture and in soft agar, suggesting that the MHV68 protein may also be transforming (Wakeling et al. 2001). As a delayed-early gene, Orf74 is transcribed weakly and variably during lytic replication, but is relatively abundant in latently infected PECs, and it has, therefore been classified as a latencyassociated gene (Virgin et al. 1999). This classification would support a potential role in tumorigenesis, as the majority of gammaherpesvirus-associated malignancies are associated with latent infection. Indeed, Orf74 appears to be dispensable for lytic replication in vitro; however, Orf74 is required for augmented viral production in response to CXCR2 agonists (Lee et al. 2003; Verzijl et al. 2004). One of these studies also demonstrated the involvement of MEK and PI3K in Orf74-mediated effects (Lee et al. 2003). These data suggest that Orf74 plays a specific role in manipulating cell signaling in infected cells to exaggerate or induce a response to extracellular stimuli during latent infection. This has several potential outcomes, including prolonged survival, angiogenesis, chemotaxis, or viral reactivation from the infected cell. In support of this hypothesis, Orf74 mutant viruses display normal acute replication but are slightly compromised for reactivation. Separate studies have demonstrated a requirement for Orf74 in efficient reactivation from splenocytes (Lee et al. 2003; Moorman et al. 2003a). Evidence for vGPCR as an oncogene. Although the in vivo evidence for the MHV68 vGPCR is not as compelling as that for v-cyclin or v-bcl2 in terms of transforming capabilities, it is important to consider its potential, more subtle effects on MHV68 pathogenesis. The vGPCR of KSHV is thought to contribute more to the growth and migration of KSHV-positive tumors, facilitating survival of a transformed cell rather than directly inducing its transformation (Arvanitakis et al. 1997). The MHV68 vGPCR may have a limited role or, an as yet undefined function in the context of normal infection, yet confer tumor-promoting properties in a cell already compromised by a previous transforming event (a “promoter” rather than an “initiator”). That the viral protein bears resemblance to a host chemokine receptor, specifically one involved in inflammatory responses and angiogenesis, raises the possibility that the vGPCR is involved in the trafficking of infected cells during latency. The observation that infection with a vGPCR-deletion mutant results in an increased frequency of infected peritoneal cells may support this possibility (Moorman et al. 2003a). In the same infections, however, the increase in genome positivity did not correlate with an increase in reactivation frequency, suggesting at least two possible explanations (1) the vGPCR is involved in reactivation from latency, or (2) absence of the vGPCR results in altered trafficking of cells into the peritoneum that are genome-positive but do not efficiently reactivate at this stage of latency (reminiscent of splenocytes at day 42). Should the latter be true, it can be extrapolated that a viral protein capable of altering cell migration patterns, in the presence or absence of an inflammatory response, might contribute significantly to dissemination of MHV68-infected cells within the host. These changes may have effects on invasion and metastasis in the MHV68-mouse model system, or in natural infection, that have yet to be appreciated.

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mLANA General characteristics. MHV68 Orf73 encodes a protein with homology to the latency-associated nuclear antigen (LANA) of KSHV, which also shares limited homology with HVS LANA (Virgin et al. 1997). Although significantly variant by sequence from the Epstein–Barr nuclear antigen 1 (EBNA1), the gamma-2-herpesvirus (KSHV, HVS, MHV68) LANA proteins function similarly to EBNA1. The best studied of these functions is tethering of the viral genome to host chromatin during latency. During latency, the viral genome is maintained as a circular episome; during host cell division, EBNA1 and LANA are thought to be essential for the faithful segregation of the viral genome to host daughter cells, although this notion has not been directly tested in EBV or KSHV-infected cells. From studies using an MHV68 LANA (mLANA)-null virus, mLANA also appears to be critical in the establishment of MHV68 latency in the spleen following intranasal infection (Moorman et al. 2003b). This phenotype is complicated, however, by the observation that the mutant virus has a lytic replication defect in the lung, which may compromise the process by which cells traffic from the lung to the spleen where they seed the primary latency reservoir (Moorman et al. 2003b). Importantly, recent data demonstrate that the splenic establishment defect following intranasal infection can be partially ameliorated by infecting mice intraperitoneally (Paden et al. 2010). While mLANA-null genome-positive cells persist, the viral genome does not appear to be episomal in Orf73-null infected cells and reactivation is entirely compromised. These data emphasize that initial events taking place in the lung following intranasal infection are key to establishing splenic latency, inextricably linking early lytic infection and long-term latent infection with the host immune response. mLANA as an oncogene. In addition to its role in episomal maintenance, gamma-2herpesvirus LANA is also a potent transcriptional regulator of both host and viral gene transcription. A dual role for LANA in lytic and latent infection is evidenced by the kinetics of LANA transcription. Orf73 was originally classified as a latencyassociated gene based on the specific and ready detection of LANA protein and transcript in KSHV-associated malignancies (Courville et al. 2002; Si et al. 2005). However, Orf73 transcription has immediately early kinetics, with protein detected as early as 4 h postinfection and at low levels throughout lytic infection (Forrest et al. 2007). In studies with KSHV and HVS, LANA has been reported to directly interact with or regulate the activity of several host proteins with known roles in tumorigenesis, such as HIF1a, DNMT3A, p53, Rb, and beta-catenin (Borah et al. 2004; Cai et al. 2006; Forrest et al. 2007; Liu et al. 2007; Shamay et al. 2006; Verma et al. 2007). LANA’s involvement with these proteins could influence lytic replication from both the virus and host cell prospective. For example, KSHV LANA interacts with HIF1a to activate transcription of the primary lytic transactivator, Rta, under hypoxic conditions, yet acts as a transcriptional repressor of Rta during normoxia – interactions with a potentially strong impact on initial infection and reactivation (Cai et al. 2006). With respect to host proteins, KSHV LANA promotes Rb phosphorylation and inactivation of p53 to facilitate cell-cycle progression and override DNA damage signals during infection (Friborg et al. 1999).

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Recently, MHV68 LANA was shown to influence cell survival via these host pathways. A common cellular defense in response to viral infection is via activation of p53, followed by subsequent cell cycle arrest and induction of apoptosis. MHV68 induces DNA damage signals upon infection, including phosphorylation of gH2AX (Tarakanova et al. 2007) and, as discussed above, also encodes proteins to promote cell-cycle progression despite this arrested state. Subversion of p53 activity during this process is, therefore critical to productive viral replication, and mLANA has been shown to be a central mediator by coordinating viral gene expression and destabilizing p53. In these experiments, cells infected with an mLANA-null virus demonstrated increased p53 activation and reduced viability, while mLANA overexpression afforded protection from etoposide-induced cell death (Forrest et al. 2007). The implications of a p53-inactivating viral protein in oncogenic transformation are clear, as has been extensively demonstrated for a number of viral proteins (e.g., HPV E6 and SV40 large T-antigen). A more subtle contribution of mLANA to MHV68-induced tumorigenesis may be through its role as a transcriptional modulator. KSHV LANA expressed from its endogenous promoter in transgenic mice has been shown to increase the incidence of lymphoma (Fakhari et al. 2006), a result most likely linked to a combination of p53 subversion and regulation of host transcription. Although mLANA appears to be essential for reactivation, a separate role for mLANA in viral gene transcription during latency has yet to be clearly defined. It is possible that constitutive LANA expression during latency results in compromised p53 function and virus-controlled host and viral gene transcription, thereby contributing to tumorigenesis by increasing an infected cell’s vulnerability to secondary transforming mutations.

Prooncogenic Alterations in Cell Signaling Associated with MHV68 Infection Many cellular alterations induced upon viral infection have been ascribed to specific MHV68 proteins. However, there are signaling pathways affected by viral infection that are most likely influenced by not-yet-identified viral proteins that act either individually or in concert. This section highlights examples of host-cell signaling networks altered upon infection that are likely targeted by viral proteins. Moreover, these pathways are often altered in transformed cells, supporting the hypothesis that perturbations incurred during MHV68 infection may facilitate tumorigenesis.

NF-kB NF-kB is a key transcriptional regulator with diverse roles in numerous cellular processes. It has been extensively studied in the context of inflammation due to its role in regulating a myriad of functions including differentiation, metabolism, and

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the development and execution of immune responses. It has also been a subject of intense focus in tumorigenesis, particularly blood-based lymphomas and leukemias. NF-kB has potent prosurvival, proproliferative effects and is often constitutively active in tumors of this origin. It is widely believed that prolonged inflammation promotes tumorigenesis – thus, in addition to its direct association with survival and proliferation, constitutive NF-kB activation has also been implicated as a central factor linking inflammation and cancer. The NF-kB pathway is of central importance in normal lymphocyte biology and is the converging point for several extrinsic and intrinsic B cell signals. Several studies have demonstrated that gammaherpesviruses exploit NF-kB signaling to achieve and maintain latent infection, promoting activation and translocation of NF-kB complexes in infected cells (Brown et al. 2003; Krug et al. 2009; Krug et al. 2007; Sgarbanti et al. 2004; Thornburg et al. 2006; Yao et al. 1995). Indeed, studies have shown that NF-kB is dispensable for lytic replication, but plays a clear role in the establishment and, interestingly, the control of latent MHV68 infection (Krug et al. 2009). Gel-shift analyses demonstrated activated p50 during both latent (S11E lymphoma cells) and lytic infection. It, therefore, seems likely that NF-kB plays a role in both promoting cell survival and proliferation during lytic replication and reactivation, but also in controlling reactivation in a latently infected cell. This implies that NF-kB is constitutively active throughout the duration of MHV68 infection, acting both on viral and host genes at each stage of the viral life cycle. Several roles for NF-kB in inflammation-associated tumorigenesis have been proposed with regard to the development of Hodgkin’s lymphoma (for a review see Khan 2006). The effects of constitutive NF-kB activation on the state of host gene transcription during MHV68 infection are unknown, but it seems plausible that constitutive activation of NF-kB as an inflammatory mediator contributes to oncogenic transformation through a combination of environmental (cytokines, chemokines) and intrinsic (antiapoptotic/proproliferative signals) alterations in infected cells.

PI3K/Akt The PI3K/Akt signaling axis is implicated in numerous cancers such as prostate, thyroid, lung, and breast. It is also affected by MHV68 infection. As mentioned previously, PI3K signals are perpetuated by the v-GPCR. Further, inhibiting PI3K/ Akt activation during MHV68 infection results in increased lytic replication, presumably mediated by increased Rta activity (Peng et al. 2010). This implies a mechanism by which MHV68 infection stimulates PI3-K/Akt pathways to facilitate the establishment of latent infection by limiting lytic replication. This is similar to the proposed mechanism of NF-kB, perhaps because the PI3K pathway is upstream and feeds into the NF-kB pathway in some signaling pathways. Given potential involvement of PI3K/Akt signaling in tumorigenesis, MHV68-mediated activation of PI3K/ Akt may also play a role in the promotion of tumorigenesis in the context of MHV68 infection.

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Twist/Snail As mentioned above, MHV68 infection of endothelial cells results in downregulation of cellular adhesion molecules and the subsequent loss of anchoragedependent growth. The loss of adhesion molecules on tumor cells has been extensively studied in the epithelial-to-mesenchymal transition (EMT), the process by which tumor cells are thought to detach from the initial tumor site, enter the blood or lymphatic system, and metastasize to distant anatomical locations. Lymphocytes are circulating cells by nature, but their role, as well as the role of non-B cells, in virus trafficking and the dissemination of infection is not defined. The transcription factor Twist has been shown to be a positive regulator of EMT by contributing to the downregulation of several epithelial markers and acquisition of mesenchymal cell markers. Recent studies indicate a role for Twist in promoting cell-cycle progression via inactivation of pRb and p53. Indeed, high Twist expression may reflect a poor prognosis in phenotyping for certain cancers (Ansieau et al. 2010). A recent study using MHV68 in a mouse model for idiopathic pulmonary fibrosis demonstrated that MHV68 infection enhances Twist expression in lung epithelial cells (Pozharskaya et al. 2009). Increased Twist expression is accompanied by the expression of mesenchymal and epithelial markers on the same cell, such that infected cells exhibit a mixed phenotype. Analyses of Twist expression have not been performed in MHV68-infected lymphocytes, but it is possible that this pathway is involved in the migration of infected cells to and from sites of infection.

MHV68 Immunomodulation Gammaherpesviruses establish latent infection in immune cells. The immune response is, therefore key to shaping and promoting a successful latent infection in terms of (1) successful host immune evasion by the virus; and (2) the contribution of the affected cell during long-term infection. As a result, these viruses have evolved mechanisms to modulate the immune response in favor of establishing and maintaining latency in these specialized cell populations. Several viral proteins have been shown to promote and maintain latent infection by directly manipulating infected cells, while others indirectly affect surrounding cells to facilitate immune evasion and create an environment conducive to latency establishment. Many of these proteins are powerful modulators whose expression is sustained throughout chronic infection, which is perhaps why the majority of gammaherpesvirus-related tumors are associated with latent infection. In combination with immunosuppression, these proteins may create the idea environment for tumorigenesis. Below, we describe a few MHV68 proteins with known immunomodulatory functions and discuss their possible contribution to MHV68-mediated tumorigenesis.

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M2 General characteristics. MHV68, like the other characterized gamma-herpesviruses, harbors several genes that encode proteins with no known host or viral homologues (Virgin et al. 1997). However, despite the lack of obvious homologues in the other gammaherpesviruses, frequently these “novel” gammaherpesvirus gene products share common functions. One key example is the M2 protein of MHV68. Both KSHV and EBV encode proteins that manipulate host cytokine production, in particularly the IL-10 and IL-6 signaling pathways which have significant effects on B lymphocyte proliferation, differentiation, and survival. In humans, IL-10 influences B cell proliferation and differentiation, a link with implications for gammaherpesvirusrelated lymphomagenesis. The BCRF1 gene of EBV encodes an IL-10 homolog (vIL-10), while KSHV encodes an IL-6 homolog (vIL-6), both of which have been linked to increased proliferation of infected cells in vitro and in virus-positive tumor cells (Nicholas 2005). Although M2 is not a cytokine homolog, it induces the production of several cytokines pivotal to B cell physiology, most notably IL-10, and to a lesser extent, IL-6 (Siegel et al. 2008). In addition, M2 induces expression of both the high-affinity IL-2 receptor and IL-2 in primary murine B cells (Siegel et al. 2008) which may also play a role in driving B cell proliferation. How M2 manipulates cytokine signaling pathways remains unclear; however, M2 does contain several motifs capable of interacting with proteins involved in lymphocyte signal transduction (discussed below). M2 expression is primarily limited to latent infection, and it is, therefore considered a latency-associated gene (Virgin et al. 1999). The phenotype of an M2-null virus is complex and is dependent on both dose and route of infection. M2 is critical for the efficient establishment of and reactivation from latency following low-dose (100 pfu) intranasal infection (Herskowitz et al. 2005; Jacoby et al. 2002). Increasing the size of the intranasal dose (106 pfu) partially ameliorates the defect in the establishment of latency but the reactivation defect remains. Infection via the intraperitoneal route largely overcomes the establishment of latency defect, with low-dose intraperitoneal infection largely recapitulating the high-dose intranasal phenotype (Herskowitz et al. 2005). Interestingly, under infection conditions where the frequency of infected cells is largely similar in M2-null and marker rescue-infected animals, the profile of M2-null-infected cells is altered; while the majority of viral genome-positive cells in wild-type infection bear hallmarks of class-switched, germinal-center experienced B cells (sIgD-), mice infected with M2-null virus harbor significantly increased frequencies of viral genome-positive naïve B cells (sIgD+). This phenotype is resolved by 6 months postinfection, when the majority of viral genome-positive cells for both mutant and marker rescue virus are sIgD-. Immunomodulatory functions of M2 may promote lymphomagenesis. The significance of the M2-null phenotype in long-term latent infection increases given recent data demonstrating that M2 drives B-cell differentiation and class-switching (Liang et al. 2009; Siegel et al. 2008), a mechanism integral to the proposed model for

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EBV-driven B cell differentiation via viral proteins such as LMP1 and LMP2A. These studies collectively showed that M2 overexpression in naïve primary B cells or a pregerminal center B cell line resulted in partial loss of B-cell and acquisition of plasma-cell-surface phenotypic markers. M2-expressing cells also exhibited a blast-like phenotype and enhanced proliferation, further supporting the hypothesis that M2 expression in infected cells serves to promote the differentiation of naïve B cells in vivo. How M2 drives this transition is not completely understood. The M2 protein contains two tyrosines which, when phosphorylated, may provide targets for SH2domain containing proteins (Herskowitz et al. 2008; Madureira et al. 2005; Pires de Miranda et al. 2008; Rodrigues et al. 2006). It also has several PxxP motifs, which function as docking sites for SH3-domain containing proteins. SH2 and SH3 domains are critical components of lymphocyte signaling cascades, allowing protein–protein interactions that perpetuate signals from the cell surface to the nucleus to modulate transcription. Studies have reported the interaction of M2 with several proteins involved in signal transduction, such as Vav, Grb2, and Fyn, as well as Ras GTPase-activating protein 1, and Rho GTPase-activating protein 4 (Herskowitz et al. 2008; Madureira et al. 2005; Pires de Miranda et al. 2008; Rodrigues et al. 2006). As previously mentioned, constitutively active or deregulated signaling, for example that resulting from mutated proteins or gene fusions, has been implicated in innumerable cancers – there is, therefore, a possibility that M2 function may have implications for tumorigenesis in MHV68-infected animals. The relationship between M2-host protein interactions and M2-mediated effects has not been clearly established, but it is hypothesized that the greatly augmented IL-10 produced in M2-transduced cultures is a result of a sequence of signaling events initiated by M2 at the plasma membrane. IL-10 has pleiotropic effects on murine lymphocytes; it enhances B cell survival while suppressing proinflammatory Th1-type immune responses, including T cell and NK cell function. Mice infected with M2-null virus have decreased serum IL-10 and increased MHV68-specific CD8+ T cells (Siegel et al. 2008). We later discuss the potential role of CD8+ T and NK cells in tumor surveillance with regard to MHV68 infection. By simultaneously promoting B cell viability and suppressing anti-MHV68 immunity, one can imagine a scenario in which M2 function serves as the fulcrum in the balance between successful longterm latency and the advent of malignant disease in infected animals. Further studies delineating the direct effects of M2 in host cells will provide more clues as to how this balance is maintained in healthy animals. Overexpression of M2 in naïve B cells results in cells displaying a “preplasmablast” phenotype; they downregulate hallmark B lineage markers such as B220, MHC class II, and sIgD, while retaining CD19 and IgG expression and only modestly upregulating expression of Syndecan-1, a marker for differentiated plasma cells (Siegel et al. 2008). Yet M2 seems to be crucial for class-switching in infected primary B cells and induces IgM secretion in BCL-1 cells, accompanied by transcriptional changes associated with plasma cell differentiation – as well as acquisition of a large, granular, blast-like appearance (Liang et al. 2009). In other words,

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M2 seems to drive naïve cells to a “gray zone” of differentiation, too differentiated to be called a true B cell, but not yet a full-fledged plasma cell. Importantly, this partially differentiated phenotype has been observed in several human lymphomas, some of which are associated with gammaherpesvirus infection. Hodgkin Reed– Sternberg cells, the diagnostic marker for classical Hodgkin’s lymphoma, contain rearranged immunoglobulin loci and are, therefore, presumably derived from germinal center-experienced B lymphocytes. However, many HD tumors do not express CD19, CD20, or a functional BCR (Kuppers 2009; Schwering et al. 2003). Accumulating genetic evidence supports the B-cell origin of these cells – it has, therefore, been hypothesized that HRS cells are B cells that have undergone a “crippling” mutation, but are “rescued” from apoptosis by an alternative transforming event (e.g., bcl-2 translocation). These cells then survive the germinal center reaction and persist as genetically and phenotypically altered cells of B cell origin that function neither as a plasma cell nor a traditional differentiated memory B cell (Brauninger et al. 2003; Kanzler et al. 1996; Kuppers 1999). EBV-positive HRS cells exhibit a type II latency program and express LMP1 and LMP2A. LMP1, via CD40-like signal transduction, has been shown to generate cells with a post-GC, preplasma cell phenotype (Rastelli et al. 2008; Uchida et al. 1999; Vockerodt et al. 2008), while LMP2A has been shown to compete for binding of Src-family kinases in EBV-transformed cells (Fruehling et al. 1996; Fruehling and Longnecker 1997; Miller et al. 1995; Miller et al. 1994). The consequences of M2 overexpression in naïve B cells share remarkable similarities with these particular LMP1 and LMP2A-mediated effects, and support the possibility that M2 may serve a hybrid LMP1/LMP2A function in MHV68 infection. The possibility that M2 can also “rescue” cells from apoptotic cell death in vivo remains to be tested, but evidence shows that in addition to its role in driving proliferation and differentiation, as well as cytokine production, M2 also interacts with the DDB1/COP9/cullin repair complex and the ATM DNA damage signal transducer to protect cells from DNA-damage-induced apoptosis, such as that which is sustained by B cells during the germinal center reaction (Liang et al. 2006). The phenotype resulting from M2 overexpression in B cells is also reminiscent of that observed in several KSHVassociated malignancies, including primary effusion lymphomas and a plasmablastic variant of multicentric Castleman’s disease (MCD). Cells from these tumors share important characteristics with M2-expressing cells in that they retain several features to indicate a B cell origin such as immunoglobulin gene rearrangement, yet acquire several characteristics of plasma cells, such as CD38 cell surface expression and transcription of plasma-cell specific genes (Du et al. 2007, 2001; Dupin et al. 2000; Jenner et al. 2003). It, therefore seems that partial differentiation of B lymphocytes into plasma cells is an associated consequence of gammaherpes virus infection. The fact that M2, an MHV68 protein lacking any known cellular or viral homologs, can induce similar phenotypic changes in B cell lines and primary splenocytes is quite intriguing and may provide an opportunity to better understand mechanisms involved in the transformation in human lymphocytes.

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mK3 A critical aspect to successful herpesvirus infection is the ability to escape immune clearance by CD8+ cytotoxic T cells (CTL). Many viruses have achieved this by encoding proteins specifically designed to reduce MHC class I expression on infected cells, thereby hindering the cell’s ability to present viral antigen to activated CTLs. The mechanisms by which viral proteins downregulate MHC class I expression vary. Like KSHV, MHV68 encodes a zinc finger protein, MK3, which functions as a ubiquitin ligase to target newly synthesized MHC class I for degradation (Boname and Stevenson 2001; Lybarger et al. 2003). MK3 is localized to the ER membrane, where it binds to and ubiquitinates the cytoplasmic tails of nascent MHC class I molecules, leading to rapid proteosomal degradation (Boname and Stevenson 2001). However, this mechanism does not seem to be the sole process responsible for MHV68 immune evasion. First, infected B cells can also present to CD8+ T cells via the nonclassical class I glycoproteins Qa-1 and Qa-2, which are not susceptible to ubiquitination. Second, sufficient levels of IFNg, such as those present as a result of MHV68 infection, have been shown to overcome MHC class I virus-mediated degradation. Further studies have demonstrated that in addition to targeting classical MHC class I, MK3 also facilitates the degradation of TAP, a major component of the class I peptide loading complex (Boname et al. 2004; Boname et al. 2005). This mechanism inhibits both classical and nonclassical MHC class I presentation, as well as conferring increased resistance to the effects of IFNγ. The consequences of reduced MHC class I expression on infected cells are advantageous from the viral standpoint of CD8+ T cell immune evasion. However, cells lacking MHC class I are susceptible to clearance by host natural killer (NK) cells (“missing-self” hypothesis). Many viruses circumvent this by encoding inhibitory receptors that block NK cytolytic activity. Alternatively, while the synthesis of many types of MHC class I molecules is affected by the action of viral proteins, there is evidence from KSHV studies that some HLA subtypes are spared, thus still allowing for inhibitory receptor ligation on NK cells (Ishido et al. 2000; Means et al. 2002). It has been suggested that mK3 serves different roles during lytic and latent infection; the gene is transcribed in proliferating, latently infected germinal center B cells, as well as in lung tissue during lytic infection (Marques et al. 2003; Stevenson et al. 2002). Studies characterizing the pathogenesis of mK3 mutant viruses demonstrated reduced splenomegaly and infectious centers compared to wild-type virus (Stevenson et al. 2002). This reduction in latency establishment was attributed to an increased antigen-specific effector CD8+ T cell response, as depletion of CD8+ T cells resulted in the restoration of wild-type latency. Interestingly, mice infected with mutant mK3 virus exhibited a marked reduction in Vb4+ CD8+ T cells (discussed below), most likely a result of the decreased frequency of latently infected cells (Stevenson et al. 2002). These studies indicate that MHV68 K3 specifically inhibits the cytolytic activity of MHV68-specific CD8+ T cells, thus

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preventing them from clearing their infected target cells following infection. This may allow an infected cell to escape CTL tumor surveillance, and whether that cell is transformed by MHV68 or by a secondary mechanism, this escape may have implications for the development of MHV68-positive tumors.

M1 Another unique MHV68 protein is that encoded by the M1 ORF. Original sequence analysis noted that M1 shared sequence homology to a poxvirus serpin, but the absence of key residues required for serpin function make it unlikely that M1 is a functional serpin (Virgin et al. 1997). Subsequent experiments have demonstrated that M1, whose transcripts can be detected during lytic replication, encodes a secreted protein required for the massive clonal expansion of Vb4+ CD8+ T cells observed following the initial establishment of MHV68 latent infection (Evans et al. 2008). In C57Bl/6 mice infected with wild-type MHV68, CD8+ T cells bearing this isoform of the TCR can comprise nearly half of the entire CD8+ T cell population (Flano et al. 2004; Tripp et al. 1997). The frequency of these cells remains high throughout the lifetime of the infected host, and contrary to the functional exhaustion seen in other models of chronic viral infection, the MHV68-induced Vb4+ T cells retain the ability to produce inflammatory cytokines upon ex vivo stimulation and do not upregulate expression of the cell surface marker PD-1 (Evans et al. 2008). A similarly disproportionate expansion of CD8+ T cells bearing a particular Vb chain is also seen following infection with EBV in human peripheral blood. Studies using large deletion mutants, and subsequently an M1-specific null virus, have demonstrated that M1 is required to induce the Vb4+ expansion in vivo (Clambey et al. 2002, 2000; Evans et al. 2008). Notably, evidence also indicates that M1 is able to induce T cell activation in the absence of MHC class I, and it has, therefore, been proposed as a “superantigen-like” molecule, presumably capable of bridging the TCR and coreceptor molecules to induce activation and negating the need for presentation via cognate MHC (Evans et al. 2008). Infection of IFNγ −/− mice with wild-type MHV68 infection results in severe pathology, characterized by multiorgan fibrotic disease, arteritis in the great elastic vessels and hyper-reactivation of MHV68 from peritoneal exudate cells (PECs) (Ebrahimi et al. 2001; Tibbetts et al. 2002; Weck et al. 1997). Identification of M1’s causal role in the Vb4+ CD8+ T cell expansion was facilitated by observations made following infection of IFNγ −/− mice with an MHV68 virus containing a large deletion within the M1 open reading frame (Clambey et al. 2000; Evans et al. 2008). Infection with this mutant virus ameliorated disease, providing the first evidence that the viral component contributing to the extreme pathology in IFNγ −/− may be encoded by the M1 open reading frame. It was subsequently demonstrated that M1-null viruses not only failed to induce the Vb4+ CD8+ T cell expansion in C57Bl/6 or IFNγ −/− mice, but also did not induce fibrotic disease, thereby linking these “effector-memory-like” CD8+ T cells to the severe pathology observed in

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MHV68-infected immunocompromised mice (Evans et al. 2008). The resulting model from these infections implies a role for Vb4+ CD8+ T cells in controlling MHV68 reactivation via IFNg-mediated repression, with M1 playing a pivotal role in their expansion. IFNγ has been shown to suppress reactivation from macrophages, perhaps by inhibiting the Rta promoters (Goodwin et al. 2010; Steed et al. 2007; Steed et al. 2006), and it was further proposed that reactivation specifically from alveolar macrophages is a primary target for Vb4+ CD8+ T cell effector function. Implications of Vb4± CD8± T cells in tumorigenesis. IFNg is a potent proinflammatory cytokine with multiple functions, produced by multiple cell types. The frequency of IFNg-producing cells increases during the initial stages of MHV68 infection and these cells most likely play dual roles in controlling lytic replication and shaping the immune environment in the infected host (Stevenson and Doherty 1998). Although the overall frequency of effector CD8+ T cells wanes during later infection, the frequency and effector functions of the expanded Vb4+ population are maintained (Evans et al. 2008; Tripp et al. 1997). Countless studies have documented the effects of IFNg on various players in the global immune response, including T cells, macrophages, and NK cells. An expanded, functionally active population of IFNγproducing cells persisting throughout the duration of MHV68 infection most likely has implications not only for subsequent host immune responses to pathogens, but also for tumorigenesis. CD8+ T cells and NK cells involved in tumor surveillance may be affected by the Vb4+ population, and long-term maintenance of a proinflammatory environment by constitutive IFNγ production may recapitulate certain events now documented as contributors to inflammation-associated tumorigenesis, including constitutive activation of NF-kB, macrophage infiltration, and angiogenesis. In addition to IFNγ, the Vb4+ T cells also produce high levels of TNFa, which may also contribute to inflammation-mediated tumorigenesis (Evans et al. 2008).

Role of Immune System in Control of MHV68 Lymphomagenesis Viral infection typically induces a Th1-biased immune response, yet gammaherpesviruses encode proteins promoting Th2-polarization and the production of Th2-type cytokines. MHV68 infection results in a strong B cell and antibody response, but also induces nonspecific polyclonal and MHV68-specific CD4+ and CD8+ T cell expansions. As discussed above, a subset of CD8+ T cells is specifically induced by an MHV68 protein and actively involved in controlling reactivation. This suggests that a fine balance in the immune response exists throughout the course of the immune response to gammaherpesvirus infection. When this balance is perturbed – such as during immunosuppressive therapy, immune senescence, or genetic immunodeficiency – it may greatly increase the risk for developing gammaherpesvirusrelated disease. Below, we describe the general features of the MHV68 immune response as a model for gammaherpesvirus pathogenesis and discuss its potential role in limiting MHV68-related lymphomagenesis.

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Kinetics of MHV68 Immune Response Although the natural route of infection is not known, the immune response to MHV68 in inbred mice has been best characterized following intranasal infection. Studies using bioluminescent viruses have verified the previously proposed course of infection and localization of lytic virus during the early stages of infection. Following intranasal inoculation, some degree of lytic replication occurs in the nasal epithelium before the virus trafficks to the lung where the majority of lytic replication occurs. Viral particles are detectable in the lung by day 4, with lytic titers peaking by day 9 following infection. Based on the poor establishment of latency in the spleens of B cell-deficient mice following intranasal inoculation (Weck et al. 1999a), it is hypothesized that B cells are infected in the lung during this period of lytic replication and serve to traffic virus to the spleen to seed latent infection. Lytic virus is detectable in the spleen by day 9 and reaches peak titer by day 12 postinfection. It is possible that the secondary wave of acute replication observed in distal organs is initiated by reactivation of virus from latently infected B cells that have trafficked to those sites. Indeed, this mechanism may link the M2-associated reactivation defect with the failure to efficiently establish latency in the spleen following intranasal inoculation (see discussion of M2 null virus phenotype above). By day 16, the infection is largely latent, with the majority of viral load accounted for by infected B cells bearing markers of either active participation in or transit from the germinal center. MHV68 can establish latency in macrophages and dendritic cells, but the majority of long-term latent infection is maintained in the memory B cell population (CD19+sIgD-) (Flano et al. 2000; Willer and Speck 2003). By 3 months postinfection, a steady-state frequency of MHV68-infected cells has been reached and persists largely unchanged for the lifetime of the infected animal. How this latency reservoir is maintained is a subject of great interest. It has been hypothesized that while there may be long-lived cells that carry the viral genome and persist throughout the lifetime of the host, there is most likely an active cycle of reactivation and reinfection – a dynamic process referred to as “reseeding” the latency reservoir. Recent studies have demonstrated that during early infection, the majority of reactivation from splenocytes can be accounted for by plasma cells – nearly 100% of sorted CD138 + B220low cells reactivate upon explant into culture, while the plasmacell-depleted population contains very few reactivating cells (Liang et al. 2009). Previous studies with KSHV and EBV have indicated that plasma cell differentiation is one of several mechanisms to elicit virus reactivation from infected B cells (Bhende et al. 2007; Jenner et al. 2003; Laichalk and Thorley-Lawson 2005; Yu et al. 2007). It is, therefore, likely that throughout the lifetime of the infected host, low-level episodic reactivation and lytic replication occurs. How this changes the immune landscape is not yet well-defined, but evidence exists to suggest an ongoing effort on the part of the host to control chronic MHV68 infection (discussed further below). Also, as mentioned above, the Vb4+ CD8+ T cell population expands around the same time serum antibody is readily detected, and remains constant and functional long after initial infection – most likely serving as another safeguard to control long-term latency.

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Innate response. The importance of NK cells in controlling herpesvirus infections has been demonstrated both in humans with genetic NK cell disorders, as well as NK-deficient transgenic mouse models of herpesvirus infection. However, the role of NK cells in limiting MHV68 infection is less obvious – initial studies using IL15-deficient (which lack NK and NKT cells) and NK-cell depleted mice revealed no direct requirement for this cell population in controlling lytic replication in the lung or spleen (Usherwood et al. 2005). The authors did note an increase in the total number of NK cells in infected mice versus naïve controls, an observation also made in a subsequent study (Thomson et al. 2008). In addition to the expanded NK population, this second study also demonstrated that these cells were activated, IFNg-producing cells with MHV68-infected cytolytic activity. However, depletion studies carried out to 5 days postinfection failed to reveal a requirement for NK cells in controlling lytic replication in the lung, mesenteric lymph nodes, or spleen. The role of NK cells in persistent or latent infection has not been addressed, nor has the contribution of NKT cells. Both these cell types are capable of producing IFNg and, therefore, have the potential to contribute to both long-term control of MHV68 reactivation and pathology related to constitutively high levels of inflammatory cytokines. A recent study demonstrated that mice infected with latent MHV68 harbor a population of “armed” NK cells capable of rapid effector functions upon ex vivo stimulation, including cytotoxicity and IFNg production (White et al. 2010). These cells also appear to protect against a lethal MHV68 infection and, therefore, most likely contribute to the control of latent MHV68 infection, at least in part via production of IFNg. Although these innate cytolytic cell populations do not appear to be essential to control acute viral replication, they may in fact contribute to MHV68 latency and, therefore, impact MHV68-mediated tumorigenesis. More obvious is the requirement of an innate cytokine response for control of MHV68 lytic replication. While mice deficient in type II interferon responses (IFNg−/− or IFNgR−/−) display pathology associated with unresolved, persistent infection, interferon ab-receptor knockout mice (IFNabR−/−) succumb to MVH68 infection within 12 days following inoculation with an intermediate-level dose of virus (Barton et al. 2005). Stat-1 deficient mice, like IFNabg-receptor knockout mice, are even more susceptible, exhibiting 100% lethality even at doses as low as 10 pfu, suggesting a nonredundant role for type I and type II interferons and further supporting an undefined role for NK cells during acute infection. This study also revealed perturbations in reactivation and viral gene expression in latently infected IFNabR−/− mice and mice in which IFNa and IFNb had been depleted in vivo. This supports a role for type I interferons in regulating MHV68 latency and further emphasizes the importance of the intersecting of innate and adaptive immunity for the control of long-term infection.

Adaptive Immune Response Antibody response. B cells are an important aspect of MHV68 infection due to the intimate association of viral pathogenesis and B cell biology, but the humoral immune response is also involved in shaping the course of infection. As previously

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mentioned, B cells are primary MHV68 long-term latency reservoir, but latency is still established in B-cell-deficient mice (Weck et al. 1996). Subsequent studies demonstrated that macrophages and DCs can also support MHV68 latent infection (Weck et al. 1999b). Several lines of evidence have indicated that the phenotype of the infected cell influences the nature of MHV68 latency (Gray et al. 2009; Steed et al. 2007; Weck et al. 1999b). Not surprisingly then, latent infection in B-cell deficient mice is inherently different from wild-type latency; at six weeks postinfection, mMT mice display a higher frequency of infected and reactivating splenocytes, a phenotype which may indicate a greater propensity toward reactivation in non-B cell reservoirs and/or a requirement for antibody-mediated control of virus reactivation (Weck et al. 1999a). Despite the contraction in the frequency of infected cells that occurs following the initial establishment of latent infection, levels of antiMHV68 serum IgG are first detectable at around 2 weeks and continue to rise following infection (Stevenson and Doherty 1999). Previous studies have demonstrated that MHV68-immune serum from infected animals confers protection from infection in naïve animals (Gangappa et al. 2002a; Gargano et al. 2008); this would suggest that serum anti-MHV68 IgG may have some role in limiting lytic replication during long-term latent infection. However, the role of antibody in controlling MHV68 replication is perhaps a phenomenon only relevant in vivo, as mixing B cells from infected wild-type mice with those from mMT mice in ex vivo reactivation assays does not ameliorate the mMT hyperreactivation phenotype (Weck et al. 1999a). Interestingly, recent data have demonstrated that although MHV68-specific IgG titers remain constant in aged mice (>18 months) relative to younger controls, sera from aged mice provides less-efficient virus neutralization and reduced protection during passive immunization of naïve mice against de novo MHV68 infection (Yager et al. 2010). This reduction in neutralizing ability does not translate into reduced control of latent infection, however, as aged mice exhibit no obvious signs of viral recrudescence or other pathology associated with aberrant virus reactivation. This is in apparent contrast to the increased frequency of gammaherpesvirusassociated malignancies reported in elderly humans, yet the authors argue that (1) the direct evidence supporting a link between aging and most gammaherpesvirusrelated tumors is not strong; and (2) the increased incidence in the elderly is negligible considering >90% of the world population harbors gammaherpesvirus infection. Therefore, the role of anti-MHV68 humoral immunity in long-term latency and tumorigenesis is still only partially understood. CD8+ T cells. Cell-mediated immune functions are key in the host response to viral pathogens. As discussed above in the context of the mK3 protein, the classic antiviral adaptive immune response centers around the recognition of viral antigens derived from intracellular processes and presented to cytotoxic CD8+ T lymphocytes (CTLs) via MHC class I molecules. The requirement for a strong CTL response in controlling MHV68 infection has been demonstrated by several groups using both transgenic mice lacking mature CD8+ T cells, as well as CD8-depleted animals. For the latter, BALB/c mice whose CD4+ T cells were depleted prior to and during infection exhibit delayed but eventual clearance of virus from the lung, while CD8-depleted mice fail to clear virus from the lung or spleen and eventually succumb

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to infection (Ehtisham et al. 1993). Similarly, C57B1/6b2m−/− mice deficient in CD8+ T cells exhibit exaggerated, unresolved splenomegaly and ongoing lytic replication in the spleen, which persists even at 6 weeks postinfection (Coppola et al. 1999). These studies clearly indicate the importance of CD8+ CTLs in limiting lytic viral replication, a critical barrier to the establishment of a normal latent infection. Ex vivo depletion analyses have further demonstrated that the majority of cell cytotoxicity results from the action of CD8+ T cells rather than CD4+ T cells (Topham et al. 2001) (although CD4+ CTLs most likely play a role in controlling latent infection as discussed in the following section). Alternatively, another study showed that C57BL/6 CD8+ T-cell-deficient mice remain healthy after infection, while concomitant CD4+ T-cell-deficiency consistently led to mortality (Stevenson et al. 1999b). Therefore, the role of CD8+ T cells in limiting MHV68 infection seems to be strain dependent and relies in part on the presence of CD4+ T cell help. Characterizations of MHV68-specific CD8+ T cell populations were initially performed during the establishment of latency and have been subsequently completed for mice infected long-term as well. The kinetics and nature of both CD8+ and CD4+ T cell expansions during the acute phase of infection bear strong similarities to those observed during the initial stages of infectious mononucleosis in humans newly infected with EBV. Infected mice exhibit a massive expansion of CD8+ T cells during the first few weeks of infection. The number of MHV68-specific CD8s in the lung begins to decline by day 15, but the spleen and MLN still experience a massive influx of lymphocytes (Stevenson and Doherty 1998). Many of these cells bear markers of activation, but a significant portion are naïve (CD62Lhi), a cellular profile similar to that seen in the blood of IM patients and attributed to activation of both MHV68-specific effector cells as well as bystander activation of non-MHV68specific lymphocytes. Concomitant with the resolution of lytic replication, total numbers of CD8+ T cells decline after 4 weeks. The exception to this pattern is the Vb4+ CD8+ T cell population discussed previously. Cells in this population begin to expand around the third week of infection and persist at a disproportionately high frequency (as high as 50% of all CD8s) throughout the duration of infection. This expanded population is entirely distinct from the virus-specific CD8s dominating the early stages of latency as they are clonal, not specific for MHV68-derived antigens, and exhibit an effector-memory phenotype even long after the presence of detectable lytic replication (Evans et al. 2008; Flano et al. 2004; Stevenson et al. 1999a; Tripp et al. 1997). When both Vb4+ and Vb8+ populations were transferred into MHV68-infected mice, both populations proliferated in 2-week-infected, while only Vb4s proliferated in 10-week infected mice (Flano et al. 2004). This suggests that the Vb4+ CD8+ T cell expansion is induced and maintained by a factor specifically produced during latent infection. B cells and reactivation competence also appear to play a role, as mMT mice and mice infected with reactivation-incompetent viruses fail to develop robust Vb4+ CD8+ T cell populations (Brooks et al. 1999). In further support of an association with latency, the presence or absence of Vb4s does not affect lytic replication, as depleting Vb4s before adoptive transfer does not alter frequency of infected cells in spleen, nor does transfer of Vb4s into infected animals (Flano et al. 2004). However, the latter results must be interpreted with some caution since a subsequent study showed that the sole available

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anti-Vb4+ antibody is incapable of depleting the expanded Vb4+ CD8+ T cell population (but does mask their subsequent detection) (Evans et al. 2008). CD4+ T cells. One of the most dramatic phenotypes associated with MHV68 infection is the development of marked splenomegaly that is coincident with the resolution of lytic infection in the lung and establishment of latent infection in the spleen. As discussed above, this aspect of infection is shared with acute EBV infection and is most likely a reflection of polyclonal B and T cell activation in response to viral infection. In the above T cell-depletion experiments, mice depleted of CD8+ T cells still develop splenomegaly comparable to undepleted controls upon infection, while spleens from CD4+ T cell-depleted mice do not increase in size or cell number (Brooks et al. 1999; Ehtisham et al. 1993; Usherwood et al. 1996a). Mice depleted of CD4+ T cells exhibit a delayed kinetics in resolving acute virus replication in the lung – clearing virus by day 13 postinfeciton – while CD8-depleted mice fail to clear lytic virus by this time (Ehtisham et al. 1993). This suggests CD8+ T cells play the primary role in controlling early infection and become activated effector cells even in the absence of CD4+ T cell help. However, separate CD4+ T-cell depletion analyses reveal the absence of an expanded Vb4+ CD8+ T cell population following the establishment of latency in mice depleted of CD4+ T cells (Brooks et al. 1999). It is possible that although they are marginally involved in the direct resolution of early lytic infection in the lung, CD4+ T cells, and perhaps mechanisms underlying CD4+ T cell-induced splenomegaly, are critical in setting the stage for particular cellmediated and humoral immune responses observed during latent MHV68 infection. This notion is corroborated by analyses of MHV68 infection in mice lacking molecules required for receiving CD4+ T cell costimulation. Ligation of CD40 on B cells by CD40-ligand (CD40L)-expressing T cells is important for development, survival, and differentiation of normal B cells. Experiments using mixed bone marrow chimeras demonstrated that MHV68 long-term latency is preferentially established in CD40-sufficient B cells, while it is progressively lost from CD40−/− B cells – suggesting that this interaction between T and B cells is important for maintaining the latently infected B cell population (Kim et al. 2003). CD40–CD40L interactions are also required for the development of splenomegaly (Brooks et al. 1999), further highlighting the role of CD40L-expressing CD4s in normal MHV68 pathogenesis. By contrast, infection of CD40−/− mice is largely unaltered from infection of wildtype mice, with viral genomes detectable in IgD-negative memory B cells even at 3 months postinfection (Willer and Speck 2005). EBV LMP1 mimics many aspects of CD40 signaling in B cells; although MHV68 does not encode a direct homolog of LMP1, it is interesting to note that CD40−/− mice still harbor MHV68 genomes in IgD- negative B cells, suggesting that the virus may in fact be able to overcome the requirement for CD40 to establish latency in class-switched memory B cells. In these studies, however, CD40−/− mice displayed evidence of persistent infection in the lung long after initial infection. Nascent infection resulting from ongoing lytic replication, therefore, likely contributes to the high frequency of infection in CD40−/− animals and may explain the apparent discrepancy of these results with those obtained using mixed bone marrow chimeric mice. Conflicting evidence exists regarding the role of CD40-mediated CD8+ cytolytic activity, but given the delayed

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clearance of virus from the lung in CD4-depleted mice, there is a possibility that CD40L supplied by CD4+ T cells is required for CD8+ effector functions during the initial adaptive immune response to MHV68 in the lung. Ligation of CD28 on B cells and CD8+ T cells by CD4+ T cells is also important for humoral and cellular immune responses. However, in contrast to CD40-deficient mice, CD28−/− mice develop wild-type splenomegaly and are able to control MHV68 infection (Lee et al. 2002; Lyon and Sarawar 2006). Splenocytes from CD28−/− mice exhibited attenuated IFNg responses when stimulated ex vivo at day 7 postinfection, but produced levels comparable to wild type by day 15 (Lee et al. 2002). Perturbations in the humoral immune response were also observed in CD28−/− with respect to antibody isotypes present in infected animals. As mentioned above, these data collectively indicate that costimulatory signals provided by CD4+ T cells are important in shaping normal cellular and humoral immune responses and creating the ideal environment for establishment and maintenance of long-term latency in an infected host.

Adaptive Immune Response in Controlling Malignant Disease Numerous studies have documented the characteristics of MHV68 infection in immunocompromised mice. Not surprisingly, many immune defects result in increased pathology, such as the multifibrotic organ disease observed in IFNgR−/− mice or the uncontrolled, systemic viremia and subsequent mortality associated with loss of a type I interferon response in IFNabR−/− mice (Barton et al. 2005). While these results are not unexpected in virally challenged mice lacking either arm of an interferon response, what is more remarkable is the ability of mice with humoral immune defects to effectively control viral infection and achieve latent infection without significant pathology. For example, despite the involvement of B cells in the maintenance of long-term latency, mMT mice (B cell-deficient) are still able to support latent infection (Weck et al. 1996, 1999a). These mice display a defect in acute splenic infection, yet the frequency of infected splenocytes during long-term latency is higher than that in wild-type controls. Reactivation is also enhanced at later time postinfection, suggesting a role for the humoral immune response in controlling virus reactivation, a notion supported by the perpetual rise in serum MHV68-specific antibody in infected mice. Although these mice lack the gene required to generate a pre-B cell receptor to allow B cell maturation, it is possible that some mature B lymphocytes are circulating in the animal. However, the clearest indication of the host immune components involved in controlling MHV68associated tumorigenesis comes from studies involving mice with T cell defects, not only RAG−/− and nude mice, which are deficient in mature T cell populations, but also BALB/c b2m−/− mice which lack functional CD8+ T cells. MHV68 has been implicated in the causation of lymphoproliferative disease in these models, as well as in IFNgR−/− mice, providing strong evidence that T cells and their effector functions play an integral role in controlling MHV68-associated tumorigenesis. b2m−/− mice (LPD and ALH). As discussed above, MHV68 infection of BALB/c b2m−/− mice results in atypical lymphoid hyperplasia (ALH) and lymphoproliferative

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disease (LPD) (Tarakanova et al. 2005). While these mice are CD8+ T-cell-deficient and prone to developing tumors, MHV68 infection both exacerbates this propensity and reduces the time to develop disease (relative to mock-infected controls). Development of proliferative disease appears to require an intact v-cyclin and v-bcl2, supporting the hypothesis that ALH and LPD are either directly or indirectly induced by wild-type MHV68 infection (Tarakanova et al. 2008). Importantly, BALB/c mice did not develop disease following infection, strongly suggesting that cells absent in BALB/c b2m−/− mice are important for limiting lymphomagenesis in a wild-type infection. BALB/c mice do not develop a robust Vb4+ CD8+ population, suggesting that background and, therefore T cell repertoire may be involved in preventing malignant disease (Tripp et al. 1997). Interestingly, while the incidence of LPD in BALB/c b2m−/− mice infected with an M1-null virus was similar to wildtype virus, the severity of the disease was increased in mice infected with mutant virus (Tarakanova et al. 2008). As discussed above, M1 is required for the expansion of an effector-memory-like Vb4+ CD8+ T cell population hypothesized to produce IFNg that suppresses virus reactivation. The Vb4+ expansion requires both CD4+ T cells, B cells, and MHC class II (Flano et al. 2000), yet MHC is not required for M1 to activate Vb4+ CD8+ hybridomas (Coppola et al. 1999; Evans et al. 2008). This suggests that the requirement for MHC class II seen in an earlier study is due more to its importance for the development of a full CD4+ T cell repertoire which can facilitate the maturation of B cells that support latent infection and elicit the Vb4+ CD8+ expansion via production of secreted M1 protein. Despite defective positive T cell selection in the thymus, a majority of C57BL/6 b2m−/− mice still develop an expanded Vb4+ CD8+ T cell population following MHV68 infection (Coppola et al. 1999). Along these lines, B2−/− 129/Pas mice in this study did not develop disease. Other strain-specific differences most likely play a role in regulation aspects of infection in these animals, but the exaggerated pathology in M1-null infected BALB/c b2m−/− mice indirectly implies the involvement of an M1-induced cell population capable of limiting lymphoproliferative disease. IFNgR−/− mice (lymphoid granulomatosis). Most pathology in immune-compromised mice has been shown to require v-cyclin, presumably reflecting a requirement for viral replication in the onset of disease. Yet a recent study carefully examining lung specimens from IFNgR−/− mice demonstrated that although ongoing viral replication is only detected in the lungs of mice infected with wild-type virus, mice infected with either wild-type or v-cyclin-null virus develop angiocentric infiltrates, exhibit hallmarks of prolonged inflammation, and develop pulmonary B cell lymphomas (Lee et al. 2009). Cells within these tumors were shown to be MHV68-positive, and latent viral transcripts were detected in lung tissue from mice infected with either virus, suggesting that this pathology, unlike MHV68-mediated fibrosis or arteritis, is associated with latent, not lytic, infection in IFNgR−/− mice. This provides evidence to support an association of latent gammaherpesvirus infection with tumorigenesis in immunocompromised hosts as a specific consequence of a deregulated immune response in the context of MHV68 infection. Although CD8+ T cells are typically the population to which most cytolytic activity is ascribed, the existence of cytolytic (CTL) CD4+ T cells has been reported for

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many years (Brown 2010). The contribution of CD4+ CTLs in the control of viral infection and tumorigenesis is becoming increasingly well appreciated. In particular, EBV-specific CD4+ CTLs isolated from infected individuals have the ability to lyse EBV transformed B cells, and CTL CD4+ T cells generated in vitro have been used therapeutically to treat EBV+ malignancies or transplant recipients infected with EBV (Adhikary et al. 2006; Heller et al. 2006). A recent report demonstrated that MHV68-infected mice harbor CD4+ T cells bearing a cytolytic phenotype (reduced CD27 expression) (Stuller and Flano 2009). When isolated from infected C57BL/6 mice, this population degranulated upon stimulation with MHV68 gp150 on isotypematched MHC class I. Furthermore, these cells could induce cytolysis of virus-pulsed IAb expressing fibroblasts, while cells from BALB/c mice could not. CTL CD4+ T cells were also demonstrated to have cytolytic activity in vivo. The implications of this study are significant given the well-established association of gammaherpesvirus-associated lymphoproliferative disease and immunosuppression. In particular, EBV and KSHV-associated lymphomas occur at much higher incidence in AIDS patients whose CD4+ T cells are greatly compromised. It is, therefore, entirely possible that CTL CD4+ T cells specific for gammaherpesvirus-infected cells are essential for controlling lymphomagenesis in infected hosts. When this population is diminished, cytolytic control of gammaherpesvirus-antigen-expressing cells is affected, and growth and expansion of these cells may proceed unchecked. The antigen specificity of both CD8+ and CD4+ CTL T cells is also a subject of interest. Tetramer analyses of MHV68-specific CD8+ T cells have been performed to identify the kinetics of virus-specific CD8+ T cell responses during various phases of MHV68 infection (Freeman et al. 2010; Husain et al. 1999; Usherwood et al. 2000). During acute EBV infection, a high percentage of CD8+ T cells are specific for epitopes derived from EBV lytic antigens, while CD8s specific for latency-associated antigens are less prolific (Khanna et al. 1992; Murray et al. 1992). This balance shifts during latent infection, however, as lytic cycle-specific cells diminish while the frequency of latency-specific cells remains constant. These cells most likely play a role in controlling virus reactivation in EBV+ individuals, as studies with MHV68 have demonstrated that CD8+ T cells specific for the latency-associated M2 antigen can reduce the latent viral load during the initial establishment of latent infection in BALB/c mice (Usherwood et al. 2000). Subsequent studies have demonstrated that vaccination with M2, as well as other latent and lytic antigens, can reduce latent viral load during early infection (Hoegh-Petersen et al. 2009; Stewart et al. 1999; Usherwood et al. 2001) but do not affect long-term latent viral load. One of these studies identified CD4+ CTLs that can respond to stimulation with a lytic epitope (gp150) (Stewart et al. 1999) and other studies of MHV68-specific CD8+ T cells have also demonstrated the existence of CD8+ T cells specific for lytic antigens (Freeman et al. 2010; Stewart et al. 1999). Studies characterizing the CD8+ T cell response to two previously identified epitopes in C57BL/6 were elaborated upon in this recent study identifying several new MHV68 epitopes (Freeman et al. 2010). The CD8+ T cell responses to MHV68 antigens largely follow two distinct kinetic patterns defined most likely by the major stages of MHV68 infection: acute lytic replication, latency amplification, and reactivation from latency.

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The results of these studies strongly suggest that the host immune response is exquisitely sensitive to alterations in antigen expression in latently infected cells throughout the lifetime of the infected host. Gammaherpesviruses have most certainly evolved to exploit the B cell arm of the immune system to perpetuate longterm latent infection. The broad repertoire of CD8+ T cells, and most likely CD4+ T cells, specific for MHV68 antigens is most likely a strategic mechanism on the part of the virus to use a healthy host immune system to control the waxing and waning of latent infection that is an essential part of latency maintenance. When this finely tuned system is perturbed by coinfection or immune dysfunction, the result may be unchecked “latency-promotion,” a transcriptional program demonstrated to have transforming potential in EBV-infected lymphocytes. Further studies addressing the role of the immune system in controlling MHV68 infection and tumorigenesis will be important to address this issue.

Implications and Application of MHV68 System for Study of Human Gammaherpesvirus-Related Tumorigenesis In line with key pathogenic features shared with its human gammaherpesvirus family members, evidence discussed in the above sections supports a role for MHV68 in tumorigenesis in mice. However, as mentioned in the introduction, the area of gammaherpesvirus-mediated tumorigenesis has not yet experienced the full benefit of the MHV68 model system because of (1) the inability to transform primary murine B cells, and (2) the absence of a robust MHV68 tumor model. However, there are promising developments on the horizon that appear likely to remove these barriers. Once these systems are in place, key aspects of gammaherpesvirus-related disease can be addressed. Some of the outstanding questions include the following: • What is the precise role of individual viral genes in lymphomagenesis? Are there complementary or combinatorial effects? • What is the role of host proteins in controlling gammaherpesvirus-related oncogenesis? • What is the role of the host immune system in controlling gammaherpesvirusrelated oncogenesis? • What are the consequences of age-related immune senescence on control of lymphomagenesis? • How do repeated pathogenic assaults shape/alter the lymphocyte repertoire? Do these changes affect gammaherpesvirus-related tumorigenesis? • How does immunosuppression, particularly loss of CD4+ T cell function, facilitate malignant disease? • What therapies will be most effective to treat/prevent gammaherpesvirus-associated disease?

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The MHV68 system is a readily manipulable animal model; the entire viral genome has been cloned into a bacterial artificial chromosome, thereby allowing the easy generation of recombinant viruses to study the functions of viral genes. Similarly, the availability of numerous transgenic mice, coupled with the faithful recapitulation of key features of gammaherpesvirus infection in these animals, provide opportunity to study host aspects in the development of gammaherpesvirusassociated malignancies – including, but not limited to, known tumor suppressors and promoters and components of the host immune system. In addition, several mouse models have been developed to study the effects of aging and senescence in mice, and one can envision how these systems may be used to answer questions regarding the increased incidence of lymphoma in elderly individuals. Studies of infectious disease in mice are moving into the realm of coinfection, trying to establish relationships between the carriage of chronic pathogens and acute infection. Since MHV68 infection is maintained for the lifetime of the infected host, it provides an attractive model to examine the effects of subsequent or preexisting infection on gammaherpesvirus-related disease. Murine systems have long been used in the study of transplant medicine with respect to gaining insights into compatibility and rejection, and MHV68 has been used in these models to understand the pathogenesis of PTLD. As this field advances, the MHV68 system provides a rich resource for the evaluation of new therapies and their consequences with regard to disease in immunosuppressed patients. Finally, with an MHV68 tumor model in place, chemoor immune-therapeutic treatments for gammaherpesvirus-associated lymphomas can be developed and tested in tumor-bearing mice. Humanized mice bearing human CD20 recapitulate the cytolytic effects of rituximab treatment, used to manage PTLD in transplant recipients, and M3, a chemokine-binding protein not discussed in this chapter, has been used to enhance the efficacy of oncolytic therapy in a rat model of hepatocellular carcinoma (Wu et al. 2008). In conclusion, MHV68 is a well-established system to study aspects of gammaherpesvirus pathogenesis. Its contribution to the study of gammaherpesvirus-associated tumorigenesis is in its infancy but has the potential to grow substantially upon the development of appropriate model systems. Once in place, the ready genetic manipulation of both the virus and the host, as well as the ability to study salient features of host cell biology, should allow important new discoveries regarding the intimate relationship between viral pathogenesis, host immune responses, and the delicate balance between successful long-term latency and the genesis of malignant disease.

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Sinclair AJ, Palmero I, Holder A, Peters G, Farrell PJ (1995) Expression of cyclin D2 in EpsteinBarr virus-positive Burkitt’s lymphoma cell lines is related to methylation status of the gene. J Virol 69:1292–1295 Sinha S, Colbert CL, Becker N, Wei Y, Levine B (2008) Molecular basis of the regulation of Beclin 1-dependent autophagy by the gamma-herpesvirus 68 Bcl-2 homolog M11. Autophagy 4:989–997 Sodhi A, Montaner S, Patel V, Zohar M, Bais C, Mesri EA, Gutkind JS (2000) The Kaposi’s sarcoma-associated herpes virus G protein-coupled receptor up-regulates vascular endothelial growth factor expression and secretion through mitogen-activated protein kinase and p38 pathways acting on hypoxia-inducible factor 1alpha. Cancer Res 60:4873–4880 Song MJ, Hwang S, Wong WH, Wu TT, Lee S, Liao HI, Sun R (2005) Identification of viral genes essential for replication of murine gamma-herpesvirus 68 using signature-tagged mutagenesis. Proc Natl Acad Sci USA 102:3805–3810 Steed A, Buch T, Waisman A, Virgin HW (2007) Gamma interferon blocks gammaherpesvirus reactivation from latency in a cell type-specific manner. J Virol 81:6134–6140 Steed AL, Barton ES, Tibbetts SA, Popkin DL, Lutzke ML, Rochford R, Virgin HW (2006) Gamma interferon blocks gammaherpesvirus reactivation from latency. J Virol 80:192–200 Stevenson PG, Belz GT, Altman JD, Doherty PC (1999a) Changing patterns of dominance in the CD8+ T cell response during acute and persistent murine gamma-herpesvirus infection. Eur J Immunol 29:1059–1067 Stevenson PG, Cardin RD, Christensen JP, Doherty PC (1999b) Immunological control of a murine gammaherpesvirus independent of CD8+ T cells. J Gen Virol 80(Pt 2):477–483 Stevenson PG, Doherty PC (1998) Kinetic analysis of the specific host response to a murine gammaherpesvirus. J Virol 72:943–949 Stevenson PG, Doherty PC (1999) Non-antigen-specific B-cell activation following murine gammaherpesvirus infection is CD4 independent in vitro but CD4 dependent in vivo. J Virol 73:1075–1079 Stevenson PG, May JS, Smith XG, Marques S, Adler H, Koszinowski UH, Simas JP, Efstathiou S (2002) K3-mediated evasion of CD8(+) T cells aids amplification of a latent gamma-herpesvirus. Nat Immunol 3:733–740 Stewart JP, Micali N, Usherwood EJ, Bonina L, Nash AA (1999) Murine gamma-herpesvirus 68 glycoprotein 150 protects against virus-induced mononucleosis: a model system for gammaherpesvirus vaccination. Vaccine 17:152–157 Stuller KA, Flano E (2009) CD4 T cells mediate killing during persistent gammaherpesvirus 68 infection. J Virol 83:4700–4703 Suarez AL, van Dyk LF (2008) Endothelial cells support persistent gammaherpesvirus 68 infection. PLoS Pathog 4:e1000152 Sunil-Chandra NP, Arno J, Fazakerley J, Nash AA (1994) Lymphoproliferative disease in mice infected with murine gammaherpesvirus 68. Am J Pathol 145:818–826 Tarakanova VL, Kreisel F, White DW, Virgin HW (2008) Murine gammaherpesvirus 68 genes both induce and suppress lymphoproliferative disease. J Virol 82:1034–1039 Tarakanova VL, Leung-Pineda V, Hwang S, Yang CW, Matatall K, Basson M, Sun R, PiwnicaWorms H, Sleckman BP, Virgin HW (2007) Gamma-herpesvirus kinase actively initiates a DNA damage response by inducing phosphorylation of H2AX to foster viral replication. Cell Host Microbe 1:275–286 Tarakanova VL, Suarez F, Tibbetts SA, Jacoby MA, Weck KE, Hess JL, Speck SH, Virgin HW (2005) Murine gammaherpesvirus 68 infection is associated with lymphoproliferative disease and lymphoma in BALB beta2 microglobulin-deficient mice. J Virol 79:14668–14679 Thomson RC, Petrik J, Nash AA, Dutia BM (2008) Expansion and activation of NK cell populations in a gammaherpesvirus infection. Scand J Immunol 67:489–495 Thornburg NJ, Kulwichit W, Edwards RH, Shair KH, Bendt KM, Raab-Traub N (2006) LMP1 signaling and activation of NF-kappaB in LMP1 transgenic mice. Oncogene 25:288–297 Tibbetts SA, van Dyk LF, Speck SH, Virgin HW (2002) Immune control of the number and reactivation phenotype of cells latently infected with a gammaherpesvirus. J Virol 76:7125–7132

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Topham DJ, Cardin RC, Christensen JP, Brooks JW, Belz GT, Doherty PC (2001) Perforin and Fas in murine gammaherpesvirus-specific CD8(+) T cell control and morbidity. J Gen Virol 82:1971–1981 Tripp RA, Hamilton-Easton AM, Cardin RD, Nguyen P, Behm FG, Woodland DL, Doherty PC, Blackman MA (1997) Pathogenesis of an infectious mononucleosis-like disease induced by a murine gamma-herpesvirus: role for a viral superantigen? J Exp Med 185:1641–1650 Tschopp J, Thome M, Hofmann K, Meinl E (1998) The fight of viruses against apoptosis. Curr Opin Genet Dev 8:82–87 Uchida J, Yasui T, Takaoka-Shichijo Y, Muraoka M, Kulwichit W, Raab-Traub N, Kikutani H (1999) Mimicry of CD40 signals by Epstein-Barr virus LMP1 in B lymphocyte responses. Science 286:300–303 Upton JW, Speck SH (2006) Evidence for CDK-dependent and CDK-independent functions of the murine gammaherpesvirus 68 v-cyclin. J Virol 80:11946–11959 Upton JW, van Dyk LF, Speck SH (2005) Characterization of murine gammaherpesvirus 68 v-cyclin interactions with cellular cdks. Virology 341:271–283 Usherwood EJ, Meadows SK, Crist SG, Bellfy SC, Sentman CL (2005) Control of murine gammaherpesvirus infection is independent of NK cells. Eur J Immunol 35:2956–2961 Usherwood EJ, Ross AJ, Allen DJ, Nash AA (1996a) Murine gammaherpesvirus-induced splenomegaly: a critical role for CD4 T cells. J Gen Virol 77(Pt 4):627–630 Usherwood EJ, Roy DJ, Ward K, Surman SL, Dutia BM, Blackman MA, Stewart JP, Woodland DL (2000) Control of gammaherpesvirus latency by latent antigen-specific CD8(+) T cells. J Exp Med 192:943–952 Usherwood EJ, Stewart JP, Nash AA (1996b) Characterization of tumor cell lines derived from murine gammaherpesvirus-68-infected mice. J Virol 70:6516–6518 Usherwood EJ, Ward KA, Blackman MA, Stewart JP, Woodland DL (2001) Latent antigen vaccination in a model gammaherpesvirus infection. J Virol 75:8283–8288 van Dyk LF, Hess JL, Katz JD, Jacoby M, Speck SH, Virgin HI (1999) The murine gammaherpesvirus 68 v-cyclin gene is an oncogene that promotes cell cycle progression in primary lymphocytes. J Virol 73:5110–5122 van Dyk LF, Virgin HW, Speck SH (2003) Maintenance of gammaherpesvirus latency requires viral cyclin in the absence of B lymphocytes. J Virol 77:5118–5126 van Dyk LF, Virgin HW, Speck SH (2000) The murine gammaherpesvirus 68 v-cyclin is a critical regulator of reactivation from latency. J Virol 74:7451–7461 Verma SC, Lan K, Robertson E (2007) Structure and function of latency-associated nuclear antigen. Curr Top Microbiol Immunol 312:101–136 Verzijl D, Fitzsimons CP, Van Dijk M, Stewart JP, Timmerman H, Smit MJ, Leurs R (2004) Differential activation of murine herpesvirus 68- and Kaposi’s sarcoma-associated herpesvirusencoded ORF74 G protein-coupled receptors by human and murine chemokines. J Virol 78:3343–3351 Virgin HW, Latreille P, Wamsley P, Hallsworth K, Weck KE, Dal Canto AJ, Speck SH (1997) Complete sequence and genomic analysis of murine gammaherpesvirus 68. J Virol 71:5894–5904 Virgin HW, Presti RM, Li XY, Liu C, Speck SH (1999) Three distinct regions of the murine gammaherpesvirus 68 genome are transcriptionally active in latently infected mice. J Virol 73:2321–2332 Vockerodt M, Morgan SL, Kuo M, Wei W, Chukwuma MB, Arrand JR, Kube D, Gordon J, Young LS, Woodman CB, Murray PG (2008) The Epstein-Barr virus oncoprotein, latent membrane protein-1, reprograms germinal centre B cells towards a Hodgkin’s Reed-Sternberg-like phenotype. J Pathol 216:83–92 Wakeling MN, Roy DJ, Nash AA, Stewart JP (2001) Characterization of the murine gammaherpesvirus 68 ORF74 product: a novel oncogenic G protein-coupled receptor. J Gen Virol 82:1187–1197 Wang GH, Garvey TL, Cohen JI (1999) The murine gammaherpesvirus-68 M11 protein inhibits Fas- and TNF-induced apoptosis. J Gen Virol 80(Pt 10):2737–2740

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Weck KE, Barkon ML, Yoo LI, Speck SH, Virgin HI (1996) Mature B cells are required for acute splenic infection, but not for establishment of latency, by murine gammaherpesvirus 68. J Virol 70:6775–6780 Weck KE, Dal Canto AJ, Gould JD, O’Guin AK, Roth KA, Saffitz JE, Speck SH, Virgin HW (1997) Murine gamma-herpesvirus 68 causes severe large-vessel arteritis in mice lacking interferon-gamma responsiveness: a new model for virus-induced vascular disease. Nat Med 3:1346–1353 Weck KE, Kim SS, Virgin HI, Speck SH (1999a) B cells regulate murine gammaherpesvirus 68 latency. J Virol 73:4651–4661 Weck KE, Kim SS, Virgin HI, Speck SH (1999b) Macrophages are the major reservoir of latent murine gammaherpesvirus 68 in peritoneal cells. J Virol 73:3273–3283 White DW, Keppel CR, Schneider SE, Reese TA, Coder J, Payton JE, Ley TJ, Virgin HW, Fehniger TA (2010) Latent herpesvirus infection arms NK cells. Blood 115:4377–4383 Willer DO, Speck SH (2005) Establishment and maintenance of long-term murine gammaherpesvirus 68 latency in B cells in the absence of CD40. J Virol 79:2891–2899 Willer DO, Speck SH (2003) Long-term latent murine Gammaherpesvirus 68 infection is preferentially found within the surface immunoglobulin D-negative subset of splenic B cells in vivo. J Virol 77:8310–8321 Wu L, Huang TG, Meseck M, Altomonte J, Ebert O, Shinozaki K, Garcia-Sastre A, Fallon J, Mandeli J, Woo SL (2008) rVSV(M Delta 51)-M3 is an effective and safe oncolytic virus for cancer therapy. Hum Gene Ther 19:635–647 Wu TT, Usherwood EJ, Stewart JP, Nash AA, Sun R (2000) Rta of murine gammaherpesvirus 68 reactivates the complete lytic cycle from latency. J Virol 74:3659–3667 Yager EJ, Kim IJ, Freeman ML, Lanzer KG, Burkum CE, Cookenham T, Woodland DL, Blackman MA (2010) Differential impact of ageing on cellular and humoral immunity to a persistent murine gamma-herpesvirus. Immun Ageing 7:3 Yang TY, Chen SC, Leach MW, Manfra D, Homey B, Wiekowski M, Sullivan L, Jenh CH, Narula SK, Chensue SW, Lira SA (2000) Transgenic expression of the chemokine receptor encoded by human herpesvirus 8 induces an angioproliferative disease resembling Kaposi’s sarcoma. J Exp Med 191:445–454 Yao Z, Fanslow WC, Seldin MF, Rousseau AM, Painter SL, Comeau MR, Cohen JI, Spriggs MK (1995) Herpesvirus Saimiri encodes a new cytokine, IL-17, which binds to a novel cytokine receptor. Immunity 3:811–821 Yu F, Feng J, Harada JN, Chanda SK, Kenney SC, Sun R (2007) B cell terminal differentiation factor XBP-1 induces reactivation of Kaposi’s sarcoma-associated herpesvirus. FEBS Lett 581:3485–3488

Chapter 13

Marek’s Disease Virus-Induced T-Cell Lymphomas Mark S. Parcells, Joan Burnside, and Robin W. Morgan

Marek’s Disease Marek’s disease (MD) describes pathologies induced by an avian alphaherpesvirus, subfamily Mardivirus, called Marek’s disease virus (MDV) (Osterrieder et al. 2006). MD presents as a combination of immunosuppressive, inflammatory, and lymphoproliferative lesions in chickens. Common signs include paralysis, skin leukosis, dermatitis, cachexia, neurological signs (ataxia, torticollis, etc.), stunting, and death. Common lesions of MD are inflammatory nerve and brain lesions, bursal and thymic atrophy, gross visceral lymphomas, and skin leukosis. MD is caused by infection with MDV1 strains, and signs are observed in chickens typically between 10 and 49 days postinfection depending on the challenge strain, the challenge dose, breed susceptibility, and vaccination status. Because of its ubiquitous presence in commercial chicken operations, its stability, the large numbers of chickens produced annually for consumption, and the fact that vaccination is not sterilizing, MD is considered to be the most prevalent clinically diagnosed cancer in the animal kingdom.

M.S. Parcells (*) Department of Animal and Food Sciences, University of Delaware, Newark, DE 19716, USA Department of Biological Sciences, University of Delaware, 052 Townsend Hall, 531 South College Avenue, Newark, DE 19716, USA e-mail: [email protected] J. Burnside • R.W. Morgan Department of Animal and Food Sciences, University of Delaware, Newark, DE 19716, USA

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History MD was initially described as paralysis that was associated with inflammation of the major nerves in egg-laying chickens (Marek 1907). This condition, also referred to as range paralysis, was found to be associated with the formation of lymphomas in 1929, and was termed Neurolymphomatosis gallinarum (Pappenheimer et al. 1929a, b). MD was described sporadically for the next thirty years, but a fundamental change in virulence of field strains noted in the early 1960s coupled with the expansion of commercial poultry husbandry catalyzed more aggressive studies on the pathogenesis of MDV (Biggs et al. 1966) as well as isolation of the causative agent in cell culture (Churchill 1968; Churchill and Biggs 1968; Solomon et al. 1968; Witter et al. 1969). Since the identification of acute MDV strains, at least two notable increases in virulence of MDV1 field strains have occurred (Witter 1997). The evolution of MDV1 field strains of increased virulence has been apparently mediated through vaccine use. In the early 1980s, following the near ubiquitous use of herpesvirus of turkeys (HVT) as the vaccine of choice in the USA (Okazaki et al. 1970), very virulent MDVs (vvMDVs) evolved (Schat et al. 1982a; Witter 1983). Strains of this pathotype (MD5, RB-1B) cause rapid lymphoma formation and overcome the vaccinal protection elicited by HVT. Isolation of a nonpathogenic herpesvirus from chickens, termed MDV2 (type strain SB-1), allowed the formulation of a bivalent vaccine (HVT in combination with MDV2), which showed increased efficacy against vvMDV challenge (Calnek et al. 1983; Chang et al. 1983, 1984; Witter et al. 1984). This bivalent vaccine remains in widespread use throughout much of the world. In the early 1990s, field strains began to emerge that could overcome the protection elicited by bivalent vaccination (Rosenberger et al. 1997; Witter 1997). These strains, termed very virulent plus MDVs (vv + MDVs) cause more severe lesions within a shorter time frame. These lesions include rapid and severe neurological disorders (Gimeno et al. 1999, 2002), acute dermatitis or “red-leg,” stunting, and tumors with necrotic centers (Montiel 1995; Olmeda-Miro 1996; Olmeda-Miro et al. 1996). Continued ubiquitous vaccination, the anticipation of further evolution of MDV1 virulence, and the rapidity and reproducibility of T-cell lymphoma induction make MDV a very attractive model for the study of herpesvirus lymphomagenesis, virulence evolution, and anticancer vaccines.

Pathogenesis Historically, the pathogenesis of MDV1 strains has been characterized according to four phases; namely, early cytolytic infection, latency, secondary cytolytic infection, and transformation (Calnek 1986). In this model, entry is via inhalation of infectious dander, and early virus replication takes place in B-cells and macrophages recruited to the lung epithelium (Baigent and Davison 2004). Replication takes

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place in the primary lymphoid organs (bursa of Fabricius, thymus), which undergo lymphoid depletion and atrophy due to apoptosis of infected cells (Morimura et al. 1995, 1996, 1997). The spleen then becomes a major site of virus replication and undergoes hyperplasia followed by necrosis. As the host encounters this early cytolytic infection, an induced innate response begins to impair virus replication, and the virus enters latency primarily in CD4+ T-cells (Shek et al. 1983). Interferons and other soluble factors yet to be defined mediate the induction of latency (Buscaglia and Calnek 1988; Buscaglia et al. 1988). Several lines of evidence implicate macrophages in the early response to MDV lytic infection. First, cytokine expression associated with early lytic replication includes proinflammatory cytokines IL-1a, IL-1b, IL-6, IL-8, as well as type I and II IFNs and iNOS (Davison and Kaiser 2004; Jarosinski et al. 2005a; Kaiser et al. 2003). Second, induction of iNOS expression in macrophages positively correlates with both genetic resistance and vaccine-induced protection (Djeraba et al. 2002b, d). Third, myelomonocytic growth factor (MGF) provides some level of protection against MD in the absence of vaccination (Djeraba et al. 2002a, c). Fourth, chickens infected with highly virulent strains of MDV undergo transient paralysis and brain edema, which are directly linked to monocytic perivascular cuffing and high levels of proinflammatory cytokine expression (Abdul-Careem et al. 2006; Jarosinski et al. 2005a; Swayne et al. 1988, 1989). During the latent phase of infection, MDV lytic expression is suppressed and latently infected cells proliferate and disseminate virus to peripheral sites. As the innate response maintaining latency wanes, MDV begins to reactivate at secondary sites (Schwann cells, feather follicle epithelium, etc.). During this time, transformed T-cells begin to accumulate within the host. This distinction of separable phases varies according to host and virus strain factors. Chickens challenged after 1–2 weeks of age typically undergo rapid early cytolytic infection within a few days followed by latency by 7–10 days and secondary cytolytic infection by 14–21 days postinfection. Lymphomagenesis usually occurs 4–6 weeks postinfection using vvMDV challenge, but may vary according to the inherent susceptibility or resistance of chickens to MD.

Oncogenes Encoded by MDV The efficiency of MDV-mediated tumor formation (80–95% in naturally exposed, unvaccinated chickens), suggested that MDV encodes a potent oncogene or oncogenes. In MDV-transformed cells, a cluster of transcripts mapping to the repeats flanking the unique long region of the virus (TRL and IRL) were implicated due to their continuous expression in latency and transformed cells (Fig. 13.1). Of genes expressed in MDV-induced lymphomas and cell lines established from tumors, only one gene, meq (for Marek’s EcoRI-Q-encoded protein) fulfills several of the criteria consistent with being an oncogene (Kung et al. 2001). First, Meq is consistently expressed in tumors and cell lines derived from tumors (Jones et al. 1992), and

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Fig. 13.1 Transformation-associated genes of Marek’s disease virus. Panel (a) depicts the MDV genome and location of the unique and repeated sequences. The approximate location and orientation of the UL36 (major tegument protein, MTP) gene is shown. Panel (b) shows an amplified map of the internal repeat long (IRL) with pathogenicity-associated genes LORF12, pp38, HEP, pp14, RLORF9, Meq, RLORF5a, RLORF4, vIL8 and vTR. The lytic origin of replication (ORIL), internal ribosomal entry sites (IRES) for the expression of pp14 and RLORF9 gene products and miRNA coding regions (blue box) are shown. Color-coding of ORFs depicts an association with pathogenicity. Meq is shown in red, as it is essential for MDV-mediated transformation. ORFs in pink are associated with pathogenicity or oncogenicity as their deletion partially attenuates MDV but does not prevent transformation. Panel (c) shows the pattern of Meq protein expression during in vivo infection of spleen cells over four weeks (VR1, -2, -3, and -4), and MD-induced tumor and cell line established from an MD-induced tumor (UD35, transformed by RB1B). As controls for full-length and spliced products, macrophage cell line HTC (Rath et al. 2003) was transfected with Meq, Meq/vIL8 (M8), and Meq/vIL8Δexon3 (M8Δ3) expression vectors. Protein samples were separated by SDS-PAGE, electroblotted, and probed with a rabbit polyclonal antibody to the amino terminus of Meq (Lee et al. 2003). As a control for protein loading, the blot was probed with a murine monoclonal antibody to GAPDH (Sigma). As controls for lytic infection, the blot was subsequently probed with a rabbit anti-US1 protein polyclonal antibody (Parcells et al. 1994), and viremia data for each sample is given in PFU/million spleen cells. Panel (d) depicts the full length and variant forms of Meq reported in the literature. The sizes of each protein in terms of number of amino acids and calculated molecular sizes are given at the right

inhibition of its expression decreases proliferation of these cell lines (Levy et al. 2005; Xie et al. 1996). Second, expression of Meq in cell lines (rat and chicken fibroblast lines) induces many aspects of transformation, including proliferation, apoptosis resistance, colony formation in soft agar (Levy et al. 2005; Liu et al. 1998)

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as well as microtumors in a chick embryo-based model (Levy et al. 2005). Third, deletion of Meq from the MDV genome, and even mutation of one particular binding motif, is sufficient to block tumorigenesis (Brown et al. 2006; Lupiani et al. 2004). Although Meq has not been shown to induce IL-2 independence in a chicken T-cell model (as no IL-2 dependent chicken T-cell lines currently exist), it has been shown to bind the IL-2 promoter in vivo (Levy et al. 2003). Although necessary, Meq is not sufficient for MDV transformation, since Meq is also encoded and expressed by attenuated MDV1 strains that are nononcogenic (Ajithdoss et al. 2009). Consequently, discussion of MDV1 transformation includes a major focus on Meq, but also includes other gene products that contribute to lymphoma development and progression.

Meq Meq is a basic leucine zipper (b-ZIP) protein having characteristics of several viral oncoproteins including v-Jun (Levy et al. 2005), HBZ of HTLV-I (Reinke et al. 2010), Tat of HIV (Kung et al. 2001; Liu and Kung 2000), and EBNA-3C of EpsteinBarr virus (EBV) (Brown et al. 2006; Hickabottom et al. 2002). Meq is encoded as a 339-amino-acid unspliced open reading frame in very virulent and very virulent plus pathotypes of MDV (vv, vv+MDV). Mild and virulent MDVs (m/vMDVs) from the 1960s and 1970s encode a larger form of Meq (398-amino acids) having reiterations of a C-terminal, proline-rich repeat (PRR) domain (Shamblin et al. 2004). Most of the biochemical, localization, and functional analyses of Meq have concentrated on the 339-amino acid form, and this will be the focus of the present discussion.

Meq Localization and Dynamics The full-length, unspliced Meq protein functions as a transcription factor (Qian et al. 1995) having rapid nuclear mobility indicative of this function (Anobile et al. 2006). Meq partitions to nucleolar and coiled body (CB) subnuclear domains as well as the nucleoplasm in transfected and infected cells (Liu et al. 1997). Its nuclear and nucleolar localizations are mediated by basic regions 1 and 2 (BR1 and BR2) in the amino terminus of the protein (Fig. 13.2), with BR2 being most efficient in mediating nucleolar localization (Liu et al. 1997). Meq also encodes a nuclear export signal and shuttles out of the nucleus during cell cycle progression (Anobile et al. 2006; Kung et al. 2001). The functional relevance of the nuclear exclusion is unknown but is mediated by phosphorylation of serine 42 (S42), which lies between basic regions 1 and 2 in the amino terminus of Meq (Liu et al. 1999b). S42 is a substrate of cyclindependent kinase 2 (CDK-2), and while the functional significance of these interactions is not clear, it is possible that Meq mediates interactions of CDK-2 with a

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Fig. 13.2 Comparative domains of Meq proteins. The picture above shows comparative domain maps of Meq, Meq/vIL8, and Meq/vIL8 exon3 proteins. The activities associated with each of the proteins are provided in the list at left, with the relative prominence of the activity with the protein shown in bold. Activities for each domain are also depicted by bars, below. Domain abbreviations are PLDLS canonical CtBP-1-binding site, BR1 and BR2 basic regions 1 and 2, ZIP leucine zipper domain, LACHE Rb-binding domain (consensus sequence LxCxE), TA transactivation domain. Light green bars in exon 2 depict putative secondary CtBP-1-binding domains. Dark green bars show putative SUMOylation sites in exon 3

number of its cellular substrates (i.e., retinoblastoma, Rb) or in targeted degradation of these factors through recruitment of SKP-2 and the Skp, Cullin, F-box containing complex (SCF complex) (Kung et al. 2001; Liu et al. 1999a).

Meq Dimerization Partners Meq can form homo- and heterodimers, and apparently both dimerization forms are essential for MDV-mediated transformation (Brown et al. 2009; Suchodolski et al. 2009, 2010). Meq has been shown to heterodimerize with c-Jun, CREB, ATF-1, -2, -3, and Fra-2 (Levy et al. 2005). Using a coiled-coil array, a number of other b-ZIP proteins were found to dimerize with Meq, including ATF-2, Jun-B, Jun-D, and NFIL3 (Reinke et al. 2010). Among these interactions, the association of Meq and c-Jun has been thoroughly characterized. When associated with cJun, Meq appears to stabilize the c-Jun protein and potentiate its signaling (Levy et al. 2005), which was shown through the upregulation of known v-Jun targets, including Hb-EGF, JTAP-1 and JAC. The importance of Meq-c-Jun interactions was further demonstrated

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through the interference of Meq transformation via downregulation of c-Jun expression (Levy et al. 2005). c-Jun not only affects cellular proliferation and antiapoptotic pathways (see Meq target genes, below) but also mediates the migration of CD4+ T-cells via the upregulation of stem cell factor (SCF) (Katiyar et al. 2007). Consequently, the interaction of Meq and c-Jun could be important not only for the suppression of apoptosis and induction of proliferation, but for increased mobility of latently infected CD4+ T-cells. This increased mobility/metastasis expression has also been suggested by proteomic data obtained using an MDV-transformed cell line, MDCC-UA01 (Buza and Burgess 2007).

Meq-Binding Proteins In addition to its ability to dimerize through its coiled-coil, leucine zipper domain, Meq has been found to bind numerous cellular proteins, including cell cycle regulators CDK-2, p53 and Rb (Kung et al. 2001) as well as repression complex protein CtBP-1 (Brown et al. 2006) and HSP-70 (Zhao et al. 2009a). Reports at recent meetings have implicated Meq binding to p300/CBP (H.-J. Kung, personal communication), prostate apoptosis response 4 (Par-4) (V. Nair, personal communication), and SKP-2 (Kim et al. 2010). The most defined of these interactions is the binding to CtBP-1, which is essential for MDV oncogenicity (Brown et al. 2006). CtBP-1 is the founding member of a family of proteins involved in transcriptional repression, lipid metabolism, and Golgi vesicle fission (Chen et al. 2009). In animal cells, CtBP-1 functions in both the nucleus and nucleolus as a dimer and recruits a large complex of proteins to repress genetic loci (Chinnadurai 2007) based on the DNA-binding proteins with which it interacts. CtBP-1 does not bind to DNA directly, but is recruited to loci through DNA-binding proteins that contain the motif PLDLS (proline–leucine– aspartic acid–leucine–serine). Proteins recruited to repress transcription include histone deacetylases (HDACs), small ubiquitin-like modifier (SUMO), E2-conjugating (UBC9), and E3-ligase (HPC2) enzymes, H3-K9 histone methyltransferases (G9a/HMTase1), and H3-K4 histone demethylases (LSD1) (Chinnadurai 2007). Consequently, CtBP-1 represses transcription of select genes through alteration of local chromatin structure. CtBP-1 shares homology with NAD/NADH-dependent, D-isomer-specific 2-hydroxy acid dehydrogenases (D2-HDHs), and has been shown to possess this activity in vitro. The substrate for CtBP-1 in vivo appears to be 2-keto-4-methylthiobutyrate, an intermediate in the methionine salvage pathway (Chinnadurai 2007). Binding of NADH mediates dimerization of CtBP-1 and binding to PLDLS proteins. Therefore, CtBP-1 has the ability to serve as a sensor for the redox potential within the cell and binds PLDLS-containing proteins when in the NAD(H)-bound form (Chen et al. 2009). Several of the genes targeted for CtBP-1 repression are adhesion molecules including E-cadherin (Chinnadurai 2009). The expression of CtBP-1 is induced by hypoxia, a condition associated with the high metabolic activity

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and NAD(H) levels within tumor cells. Therefore, repression of cellular adhesion molecules contributes to tumor cell motility and metastatic potential during tumorigenesis (Chinnadurai 2009). In the case of the adenovirus E1A protein, a loss in CtBP-1-binding through mutation of the PLDLS domain resulted in greater E1Amediated malignant transformation, including the ability to cause metastatic tumors in vivo. The binding of a CtBP-1 by E1A, therefore, actually decreases the transforming potential of E1A. As a repressor, CtBP-1 downregulates E-cadherin and contributes to the epithelial–mesenchymal transition (EMT) associated with metastasis (Chinnadurai 2009). In addition, proapoptotic genes p21, Bax, Noxa, and PTEN are targets of CtBP-1 repression. PTEN negatively regulates AKT (PKB) and, therefore, repression of PTEN results in sustained phosphorylation of AKT, a kinase directly involved in cell survival. Additionally, the inhibitor of cyclin kinase p16 (p16/Ink4a) and alternative reading frame (ARF) pathway, which modulate Rb function, are repressed by CtBP-1. CtBP-mediated repression of these factors permits cell cycle progression; however, in response to UV damage, CtBP is phosphorylated and targeted for proteosomal degradation, thus relieving its repression of tumor suppressor and apoptotic pathways. In addition to repressing adhesion and proapoptotic gene loci, CtBP-1 is a key regulator of cellular differentiation. In CD4+ T-cells, IL-4 expression is downregulated by CtBP-1, directly affecting the differentiation of naïve CD4+ T cells into TH1 cells (Kitamura et al. 2009). Thus, CtBP-1 can regulate proliferative, apoptotic, and developmental signaling within cells through the selective repression of targeted genetic loci. Finally, in Caenorhabditis elegans, genetic deletion or targeted downregulation of CtBP-1 increased worm lifespan (Chen et al. 2009). Genes selectively repressed by CtBP-1 are included or are part of the insulin-like growth factor (IGF)/lipid metabolism axis and repression resulted in lower levels of lipid metabolism. Interestingly, alteration of lipid metabolism is a hallmark of MD and has been described since the early 1970s (Fabricant et al. 1981; Fujii et al. 1988; Haider and Ringen 1970; Hajjar 1986). The molecular basis of the observed changes in lipid metabolism, therefore, may be due to the effects of Meq on selective CtBP-1 binding in MDV-transformed cells. Meq binds CtBP-1 through a PLDLS domain in its amino terminus (Brown et al. 2006; Hickabottom et al. 2002). Since Meq binds to different DNA sequences based on dimerization partner usage (Qian et al. 1996), it likely recruits CtBP-1 to specific loci during latency and transformation. Indeed, proapoptotic genes downregulated by Meq include Fas and DAP5 (Levy et al. 2005). Moreover, cells transformed by MDV show a distinct regulatory T-cell (Treg) immunophenotype (Buza and Burgess 2007; Shack et al. 2008), suggesting that this expression profile contributes to lymphoma progression. Since the Meq-CtBP-1 interaction is essential for MDV-mediated tumorigenesis (Brown et al. 2006), it is likely that this interaction is directly involved in repressing the sensitivity of the latently infected T-cell to apoptosis, while also causing the differentiation of the T-cell to a Treg expression pattern.

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Meq Target Genes Genes upregulated by Meq in a fibroblast cell line model include several proliferation-associated genes (JAC, JTAP-1, HB-EGF), but also antiprotein Bcl-2 and TGFb-signaling regulatory protein c-Ski (Levy et al. 2005). The effect of Meq on DF-1 cell growth was directly related to its interaction with c-Jun, as downregulation of either Meq or c-Jun using gene-specific siRNAs decreased cellular proliferation. Another major class of Meq-responsive genes in this fibroblast model, as well as a class of proteins upregulated in an MDV-transformed T-cell line, are cell adhesion/cytoskeleton regulators (Buza and Burgess 2007; Levy et al. 2005). This alteration in cell shape and movement-associated gene expression was interpreted to indicate increased mobility and metastatic potential and differed from superantigenactivated T-cells (Buza and Burgess 2008). In MDV-induced tumors and cells lines, Marek’s tumor-associated surface antigens (MATSAs) have been described since the 1970s as being indicative of malignant transformation by MDV (Horie et al. 1991; Ikuta et al. 1980, 1981, 1984; Lee et al. 1983; Matsuda et al. 1977; McColl et al. 1987; Murthy and Calnek 1979; Witter et al. 1975). Several of these were found to be activation-associated lymphocyte antigens (McColl et al. 1987), but to date, only one has been cloned and identified, CD30, the chicken homolog of the Reed–Sternberg antigen of EBV-associated Hodgkin’s lymphoma (Burgess et al. 2004). The CD30 promoter was directly upregulated by Meq, which directly linked Meq to expression of at least one MATSA (Burgess et al. 2004). The importance of CD30 expression to the immunophenotype of MDV-transformed cells and its contribution to disease progression are described in recent articles elsewhere (Buza and Burgess 2007, 2008; Shack et al. 2008).

Splice Variants of Meq In addition to the full-length version of the Meq protein, there are at least two spliced gene products that result in the fusing the first 100 amino acids of Meq to exons 2 and 3 of viral interleukin 8 (vIL8) (Figs. 13.1 and 13.2) (Kumar et al. 2010; Peng and Shirazi 1996; Peng et al. 1995). These spliced forms, termed Meq/vIL8 and Meq/ vIL8Δexon3 generate smaller proteins (212 and 149 amino acids, respectively) and have starkly different nuclear mobility than full-length Meq (Anobile et al. 2006). The smaller variants also localize to the nucleus, nucleolus, and CBs (Anobile et al. 2006) and were originally thought to serve as negative regulators of Meq (Peng and Shirazi 1996), since they lack transcriptional activation functions. However, the spliced forms do not form heterodimers with Meq as determined by fluorescence resonance energy transfer (FRET) and colocalization studies (Anobile et al. 2006). The spliced forms are expressed in vivo, primarily during latency, in primary lymphomas and derived cell lines (Fig. 13.1) (Kumar et al. 2010). Based on chromatin IP (ChIP) studies, the smaller variants were found to bind to the MDV genome and exert

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potent transcriptional repression activity. Despite their lack of transactivation, both variants induced proliferation of fibroblast and macrophage cell lines, while only one form could block staurosporine-induced apoptosis (Kumar et al. 2010). Examination of exons 2 and 3 of MDV vIL8 shows that two putative secondary binding sites exist for CtBP-1 (NRPTGLPII and RRTEIIFA/SL) bearing homology to the E1A conserved region 3 of adenovirus (consensus RXNTGDXEXL) (Bruton et al. 2008). Consequently, variants of Meq may contribute to the transformation of T-cells through interactions with CtBP-1 and repression of select cellular loci as well as silencing of lytic promoters within the latent MDV genome. The multiple forms of Meq observed in the MDV-induced lymphoma and cell line (Fig. 13.1c) may also indicate differential modification of the spliced forms, compared to full-length Meq. Examination of Meq, Meq/vIL8, and Meq/vIL8Δexon3 using modification prediction software shows two putative type II sumoylation motifs (IKKLER and RTRKENL, where K denotes the target lysine for sumoylation) in exon 3 of Meq/ vIL8 (http://sumosp.biocuckoo.org/), which could account for one or more of the protein species seen between Meq/vIL8 and Meq (Fig. 13.1c). Since both Meq/vIL8 and Meq/vIL8Δexon3 maintain the CtBP-1-binding domain of Meq (PLDLS), and CtBP-1 serves as a recruitment platform for sumoylation factors (Kuppuswamy et al. 2008), these Meq splice variants may very well serve as substrates for sumoylation. Alternatively, these proteins may bind sumoylated proteins via a sumoylation interaction motif (SIM) in a manner similar to EBNA3C of EBV. The full transforming properties of EBNA3C require the both CtBP- and SIM domains (Lee et al. 2009).

Phenotype of a Meq Deletion Mutant Deletion of Meq from the genome of Md5, a vvMDV strain, resulted in a marked decrease in virus replication in vivo and the complete attenuation of tumorigenicity (Lupiani et al. 2004). This deletion virus, MD5DMeq, replicated within lymphoid organs (spleen, bursa, and thymus) to near wild-type levels for approximately one week and then rapidly declined in titer to being barely detectable by four weeks postinoculation (Lupiani et al. 2004) and (Parcells et al., unpublished). Several reports on the use of this virus, Md5ΔMeq, as a vaccine have demonstrated it to be highly protective, despite this limited replication (Lee et al. 2010; Su et al. 2010). We have found that in SPF leghorn chickens, Md5ΔMeq induces an acute inflammatory response and profound thymic atrophy, suggesting that protection is mediated by a loss of target cells (T-cells) during infection (Parcells et al., unpublished). Target cell loss was also found to be the means by which another candidate vaccine, RM-1, which has an LTR-insertion in the MDV genome, reduced tumor incidence upon subsequent challenge (Liu et al. 2001; Witter et al. 1997). RM-1 has an insertion of ~600 bp of the LTR from reticuloendotheliosis virus (REV) located between the ICP4 and SORF2 genes (Jones et al. 1996). Insertion at this locus upregulated the expression of SORF2, but may also affect the regulation of ICP4 during latency. Since Meq binds to and represses the expression of MDV lytic

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promoters (Levy et al. 2003), including the ICP4 promoter, the similarity in replication and thymic atrophy mediated by both MD5ΔMeq and RM-1 suggests that Meq may serve as an important switch between lytic and latent infections. In summary, Meq appears to have an important function during the lytic phase of infection in which it appears to block the massive lytic replication within the thymus and mitigate the associated proinflammatory response to MDV lytic replication.

Viral Telomerase RNA A noncoding RNA having structure and function homologous to a telomerase RNA (TR) is encoded in the RL segments of the MDV genome adjacent to the L and S component repeats (IRL–IRS and TRL–TRS) (Fragnet et al. 2003). This virus-encoded TR (vTR) is expressed during MDV lytic and latent infections as well as in MDVtransformed cell lines (Trapp et al. 2006). Biochemically, Viral Telomerase RNA (vTR) functions as a TR in telomerase in vitro assays and out-competes the chicken cellular TR for telomerase reverse transcriptase (cTERT) (Fragnet et al. 2003; Fragnet et al. 2005). Deletion of vTR from the MDV genome does not abolish oncogenicity, but does affect lymphoma frequency, size, and progression (Trapp et al. 2006). Over-expression of vTR induced cellular proliferation, decreased anchorage dependence, increased colony formation in soft agar, and increased expression of integrin alpha V, suggesting that it functions above and beyond its telomerase activity. The promoter regulating the expression of vTR in the MDV genome has recently been found to be three times more active than the chicken cellular TR promoter (Chbab et al. 2010) as measured by pRT-PCR. This increased expression of vTR was found to be important to efficient MDV transformation (Chbab et al. 2010). In a recent study, mutations that abolished the ability of vTR to complement telomerase activity did not affect the transformation efficiency of MDV, suggesting function(s) independent of telomerase activity (Osterrieder, personal communication). Since increases in cellular telomerase activity accompany T-cell activation (Bodnar et al. 1996), the sustained high expression of MDV vTR apparently not only serves to maintain telomere length in proliferating, latently infected T-cells but also contributes other functions (i.e., migration, metastasis) to developing lymphomas.

Viral Interleukin 8 Within the block of genes unique to MDV1 strains, an interleukin 8 homolog (vIL8) was identified that affects MDV pathogenicity (Cortes and Cardona 2004; Cui et al. 2004, 2005; Parcells et al. 2001). An interesting aspect of vIL-8 is that its intron–exon junctions are very similar to those of the chicken cellular IL-8 proteins (9E3/CEF4, aka cCAF, and K60), suggesting that it was captured by MDV1 from the chicken genome (Parcells et al. 2001). Nevertheless, vIL-8 has several features

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that distinguish it from chicken cellular IL-8 proteins. vIL-8 lacks the glutamic acid–leucine–arginine (ELR) motif important for heterophil (neutrophil in mammals) chemoattraction, which is replaced with an aspartic acid–lysine–arginine (DKR) motif. In addition, vIL-8 also contains a larger third exon that is markedly divergent from the cellular homologs (Parcells et al. 2001). In addition, vIL-8 is a chemo-attractant for chicken PBMCs and macrophages (Parcells et al. 2001; Parcells, unpublished), but lacks angiogenic activity (Cui and Lee, unpublished). Deletion of vIL-8 in its entirety (Cortes and Cardona 2004; Cui et al. 2004; Parcells et al. 2001) or exon I only (which contains the signal peptide for secretion) (Jarosinski and Schat 2007) results in an MDV that has decreased lytic infection and an accompanying decrease in oncogenicity. A vIL-8 knockout virus was found to protect against vv+MDV challenge, but retained a low level of inherent oncogencity, making this virus unsuitable as an MD vaccine (Cui et al. 2005). A cell line established from an RB1BvIL8ΔsmGFP-induced tumor, MDCC-UA20, showed constitutive expression of the marker gene smGFP, was notably lymphocytoid (as opposed to lymphoblastoid), and replicated poorly in culture (Parcells et al. 2001), suggesting a putative role for vIL-8 or portions of vIL-8 in maintaining T-cell transformation (see Spliced forms of Meq, above). The vIL-8 gene of a BAC version of the vaccine strain CVI988 is truncated compared to the vIL-8 genes of virulent MDVs (CVI988 vIL-8 is 121 vs. 134 amino acids for Md5, RB1B), further suggesting a role in MDV pathogenicity (Spatz et al. 2007); however, this truncated version was not identified in CVI988 vaccine strains.

Viral Ubiquitin-Specific Protease Recently, MDV, like herpes simplex virus (HSV), has been found to encode a ubiquitin-specific protease domain within the major tegument protein (MTP) encoded by the UL36 gene (Jarosinski et al. 2007). MTP is a very large (3,348 amino acids for MD5), multidomain protein expressed late during infection that acts in an immediate-early fashion during subsequent rounds of infection. Ubiquitin ligases and proteases are key components of important regulatory networks within cells in which the abundance of signaling intermediates, enzymes, chaperones, and sensing proteins, etc., is regulated through their targeted degradation. During the course of viral infection, cells may become less permissive to viral replication through the targeted degradation of proteins, and hence, some viruses, notably herpesviruses, have evolved enzymes for the targeted removal of ubiquitin. Through alignment of MTPs encoded by other herpesviruses, a Ubiquitin-Specific Protease (USP) enzymatic signature was identified within a 322-amino-acid segment of the MTP of MDV1 (Jarosinski et al. 2007). This signature contained cysteine, aspartic acid, histidine, and glutamic acid residues essential for function as a USP. Herpesvirus USPs fall into the USP7 family of deubiquitinases (DUBs) and have a cysteine essential to catalysis (Komander et al. 2009). Mutation of this cysteine to alanine at position 98 resulted in complete loss of USP activity (Jarosinski et al. 2007). Mutant viruses

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having this mutation (C98A-1 and -2) showed a slight replication deficiency in cell culture and replicated at near wild-type levels for one week postinfection in vivo but showed a marked decrease in replication after this time, and both mutant viruses induced tumors in only 10–20% of the infected chickens (Jarosinski et al. 2007). Of the tumors induced by in the mutant-infected groups (C98A-1 and -2), several were found to have reversion mutations, reestablishing the cysteine at position 98. Consistent with a role in transformation, UL36 was found to be transcribed in MDVtransformed cell lines as well as during lytic replication.

Additional Gene Products Contributing to Oncogenesis RLORF4 Between Meq and the vIL-8 coding sequences of the MDV genome, there are two open reading frames RLORF4 and RLORF5a (aka L1) encoded in the same orientation as both Meq and vIL-8. RLORF4 encodes a methionine and cysteine-rich ORF of 142 amino acids predicted to localize to the ER and the mitochondrial and/or plasma membrane based on PSORT-II prediction (http://psort.ims.u-tokyo.ac.jp/form2.html). Several attenuated strains of MDV were found to have deletions of this ORF (Jarosinski et al. 2003), and targeted deletion of RLORF4 resulted in a marked attenuation of the BAC-based RB1B strain of MDV (Jarosinski et al. 2005b). The RLORF4 deletion mutant also showed decreased plaque size in cell culture, and although no protein product has been demonstrated for this ORF, the gene is transcribed during infection (Jarosinski et al. 2005b). Based on the high degree of splicing within this region of the genome (Jarosinski and Schat 2007), RLORF4 or portions of RLORF4 may represent an as yet undescribed splice variant of Meq (see Western blot, Fig. 13.1c). Upstream and adjacent to RLORF4, RLORF5a also appears to be expressed, at least transcriptionally (Jarosinski et al. 2005b). Originally identified in cDNAs cloned from the MDV-transformed cell line MDCC-CU41, RLORF5a (aka L1) encodes a 107-amino acid open reading frame, with no homology to known proteins, that is predicted to localize to mitochondrial membranes (http://psort.ims.u-tokyo.ac.jp/ form2.html). A small portion of RLORF5a was found to be among several splice variants cloned by 3¢-RACE that included exons II and III of vIL-8 (Jarosinski and Schat 2007). Deletion of RLORF5a did not affect virus replication in cell culture or in vivo and did not result in attenuation of the virus (Jarosinski et al. 2005b).

Phosphoprotein 14 and RLORF9 In the initial screening of the MDV genome for oncogenes, several research groups searched for differences in gene expression between oncogenic strains and their attenuated derivatives (Bradley et al. 1989a, b; Ross et al. 1993). A cluster of RNAs

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encoded at the MDV lytic origin of replication were found to change with serial passage in cell culture, and several cDNAs were cloned from this region (Hong and Coussens 1994; Hong et al. 1995; Peng et al. 1992). Transcripts encoding phosphoprotein 14 (pp14) are expressed with immediate early kinetics and differentially spliced yielding 86- and 94-amino acid open reading frames, respectively, that share a common 71-amino acid second exon (Hong and Coussens 1994; Hong et al. 1995). The two ORFs are predicted to have both nuclear and cytoplasmic localization but the proteins appear to be primarily cytoplasmic (Hong et al. 1995). Both proteins have numerous putative serine/threonine phosphorylation motifs and a prominent PEST (proline–glutamic acid–serine/threonine) domain in the common C-terminus, suggesting that they undergo rapid turnover. The discrepancy between the predicted size of the proteins (9 and 10 kDa, predicted) and 14 kDa (observed) could not be linked to phosphorylation or glycosylation (Hong and Coussens 1994; Hong et al. 1995). Consequently, these proteins are likely to have additional, yet uncharacterized, posttranslational modifications (Fig. 13.3). Immediately downstream of pp14 and encoded in a bicistronic transcript is RLORF9, which predicts an unspliced open reading frame of 108 amino acids (Tahiri-Alaoui et al. 2009b). Expression of RLORF9 is regulated posttranscriptionally via an internal ribosomal entry site (IRES) between the second exon of pp14 and RLORF9. Deletion of this IRES abolishes translation of RLORF9 but was found to not affect MDV pathogenesis (Tahiri-Alaoui et al. 2009b). In more recent work, a second IRES has been identified in the 5¢ leader of the longer pp14 transcript, and that this form also could regulate RLORF9 expression (Tahiri-Alaoui et al. 2009a). Several lines of evidence suggest that pp14 is important to MDV replication and/ or pathogenicity. First, pp14 species are expressed with immediately-early kinetics to very high levels during lytic infection (Liu et al. 2006), suggesting important regulatory and/or host cell interaction functions (Hong and Coussens 1994; Hong et al. 1995). Second, serial passage with attenuation affects expression of pp14, suggesting a role in pathogenicity. Finally, the encoding of two IRES sequences within pp14 transcripts suggests that translation of these proteins is essential in the face of cellular interferon responses. The combination of IRES sequences, cytoplasmic localization, and the prominent PEST domains in their C-termini, in fact, suggest that these proteins may be involved in the regulation of the intracellular host immune response through the targeted degradation of specific proteins. The functions of these proteins await further characterization.

MDV-Encoded MicroRNAs MDV microRNAs (miRNAs) were first described following 454 Life Sciences pyrosequencing of MDV1 strain RB1B-infected CEF (Burnside et al. 2006). These miRNAs were found to cluster to two distinct regions of the MDV genome. One cluster lies near the meq gene, and another maps in the LAT/ICP4 region. These

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Fig. 13.3 Colocalization of Meq proteins with CtBP-1. Cell line HTC (Rath et al. 2003) was cotransfected with Meq protein eYFP (Meq-eYFP, top; Meq/vIL8-eYFP, middle; Meq/vIL8Δexon3-eYFP, bottom) and chicken CtBP-1-eCFP expression vectors (Anobile et al. 2006). Transfected cells were fixed with 2% paraformaldehyde at 24 h posttransfection and stained with DAPI prior to imaging. Images were acquired using a Nikon TE-2000 microscope with epifluorescence, a Roper CCD camera and NIS-Elements image acquisition and analysis software (http:// www.nis-elements.com/). Representative fields are shown for colocalization of proteins. Arrows of the same color (white, yellow, blue, green, and pink) denote common cells imaged with respective filters. Overlays at right show colocalization of proteins within nuclei of transfected cells. Note that Meq spliced proteins show very dense nuclear localization with CtBP-1

results were confirmed and extended following sequencing of a small RNA library prepared from MSB-1 cells (Waidner et al. 2009; Yao et al. 2007, 2008). Since the MSB-1 cells used also harbored MDV2, this study included identification of MDV2 miRNAs. MDV2 miRNAs were found to cluster in a 4.2-kb repeat region encoding R-LORF2 to R-LORF3, with one additional miRNA positioned in the 3¢ end of the ICP4 gene. MicroRNAs of herpesvirus of turkeys (HVT) have been reported based on analysis of deep-sequencing libraries of HVT-infected CEF (Waidner et al. 2009). While there is no sequence conservation among the miRNAs of MDV1,

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14 miRs

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Fig. 13.4 Comparative genomic locations of MDV miRNAs. Genome maps of MDV1, MDV2, and HVT are shown with the approximate genomic locations of their respective miRNAs. The number of miRNAs reflects data found in miRBase Release 15 (April 2010) (http://mirbase.org) and does not include passenger or star strands as separate miRNAs

MDV2, and HVT, for all of these herpesviruses, the general genomic location of the miRNAs is conserved (Waidner et al. 2009) (see Fig. 13.4). At present, mirBase lists 14 MDV1, 18 MDV2, and 17 HVT miRNAs. Expression of miRNAs has been confirmed, in general, by Northern blot analysis or qPCR (Burnside et al. 2006; Xu et al. 2008; Yao et al. 2007). By and large, patterns of expression reflect meq and LAT expression; however, expression during lytic infection of CEF is also detected (Burnside et al. 2006; Yao et al. 2007). Expression of mdv1-miR-M4 was particularly high in MDV-induced tumors and represented approximately 72% of all sequences matching the MDV genome from deep sequencing analysis (Morgan et al. 2008). In some cases, the miRNA precursors were detected. The relative expression of the 3p and 5p arms of mdv1-miR-M2 and mdv1-miR-M8 differed between MSB-1 lymphoblastoid cells and lytically infected CEF, but the significance of this differential processing remains to be understood (Burnside et al. 2006). Since all of the Gallid (Infectious Laryngotracheitis [ILTV], MDV1, MDV2) and Meleagrid (HVT) herpesviruses encode miRNAs, their presence in and of itself does not signal oncogenic potential. However, because the targets and functions of miRNAs are unknown, a role in the manifestation of oncogenicity remains possible, and even likely, given that they are present in many undifferentiated cells and that their expression has, in some cases, been linked to oncogenicity (Esquela-Kerscher and Slack 2006; Garzon et al. 2009). One approach to determining if MDV1 miRNAs contribute to oncogenic potential has been to determine if expression of various MDV1 miRNAs is correlated with relative oncogenic potential of a variety of MDV1 strains that have been characterized over the years (Morgan et al. 2008). Sequencing of PCR products derived from the meq and LAT miRNA cluster regions and comparing the sequence data to the Md5 reference sequence (AF243438) revealed that no SNPs within miRNA coding sequences are associated with relative oncogenicity (Morgan et al. 2008).

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Sequencing of the putative promoter for the meq miRNAs did reveal at least one potentially interesting SNP, but its significance awaits more detailed promoter characterization (Lagasse, preliminary results). Overall, miRNAs appear to be under stringent selective pressure for sequence conservation. A second approach to examining oncogenic potential of MDV1 miRNAs has been to compare miRNA expression among tumors induced by vv versus vv+ viruses or among various lymphoblastoid cells. When small RNA samples from MDV-induced tumors were examined, meq miRNAs were found to clearly accumulate to a greater degree in tumors induced by vv+ MDV (615K, aka T. King) compared to tumors induced by the vv strain RB1B (Morgan et al. 2008). This differential expression was specific for the meq miRNA cluster and was not observed for LAT miRNAs. In addition, this difference could not be explained by general differential transcription of the meq gene between tumors induced by 615K or RB1B, since qPCR revealed that meq mRNA levels were similar in these samples. Seven MDV1-induced lymphoblastoid cell lines have been compared to an MDV1-negative REV-T-transformed CD4+ cell line (AVOL-1) and an MDV1negative avian leukosis virus (ALV) HPRS F42 strain transformed B-cell line (HP45) with regard to micRNA expression signatures (Yao et al. 2009). All MDV1 lymphoblastoid cell lines showed MDV1 miRNA expression as expected, although some variation in relative expression of specific miRNAs was noted. With regard to host miRNAs, downregulation of miR-155, miR-150, and miR223 was noted, with downregulation of miR-150 and miR-223 also being observed in AVOL-1 (MDV1negative) cells. A third approach to determining if MDV1 miRNAs contribute to oncogenic potential has been to look for orthologs among other oncogenic viruses. MDV1miR-M4 shares a seed sequence (TAATGCT) with gga-miR-155, which is considered an oncomir (Morgan et al. 2008; Zhao et al. 2009b). In addition, Kaposi’s sarcoma herpesvirus (KSHV) encodes a miRNA, kshv-miR-K12-11, which shares an identical seed sequence (Gottwein et al. 2007; Skalsky et al. 2007). In addition, mdv1-miR-M32 shares a seed sequence with gga-miR-221 (Morgan et al. 2008). miR-155 is interesting in that it is expressed in activated B and T cells, and it is important for B-cell development and germinal center development (Rodriguez et al. 2007). A key role for miR-155 is the downregulation of proinflammatory protein expression during the immune response (Ceppi et al. 2009). miR-155 is expressed in many human malignancies, including B-cell lymphomas (Eis et al. 2005), breast, lung, and colon cancers (Volinia et al. 2006), and it is expressed in avian leukosis virus (ALV)-induced B-cell lymphomas (Tam et al. 2002). In humans, miR-155 is processed from exon 3 of BIC (B-cell integration cluster) RNA, an RNA that enhances c-Myc-associated lymphomagenesis. Overexpression of miR-155 in transgenic mice induces B-cell lymphoma formation (Costinean et al. 2006). However, in MDV-induced T-cell lymphomas, gga-miR-155 is not overexpressed, but mdv1-miR-M4 accumulates to a greater degree than any other MDV1 miRNA. Thus, it appears that in MDV-induced T-cell lymphomas, mdv1-miR-M4 may alleviate the need for gga-miR-155 overexpression in inducing and/or maintaining the transformed phenotype (Morgan et al. 2008).

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The search for mdv-miR-M4/miR-155 targets has revealed a long list of potential targets based on results obtained using a variety of target prediction software (Zhao et al. 2009b). A few targets have been validated using luciferase-based reporter assays, including PU.1, CEBPß, HIVEP2, BCL2L13, and PDCD6 (Lambeth et al. 2009). In human dendritic cells (DC), an important set of targets for miR-155 are those induced through Toll-like receptor signaling (Ceppi et al. 2009). Treatment of DCs with IL-1ß, TNF, IL-6, etc. resulted in a rapid increase miR-155 expression and concomitant downregulation of signaling molecules Pellino-1 and TAB2. Consequently, miR-155 is part of a negative feedback loop in the downregulation of proinflammatory signaling. For the PU.1 target, evidence has been presented that chicken macrophage cell line HD11 shows reduced expression of endogenous PU.1 when transfected with miR-M4 or miR-155 (Lambeth et al. 2009). Although not shown directly for mdv1miR-M4, BACH1/Brip1 is likely to be a target since it is recognized by both miR-155 and KSHV miR-K12-11 (Gottwein et al. 2007; Skalsky et al. 2007). BACH1 is a basic leucine zipper transcription factor that participates in a variety of key cellular functions such as hypoxia and DNA repair (Cantor et al. 2001; Reichard et al. 2008). miR-221, which shares a seed sequence with mdv1-miR-M32 (Morgan et al. 2008), has been shown to target p27Kip1, a negative regulator of the cell cycle that, when downregulated, promotes cancer cell proliferation (Fornari et al. 2008). GgamiR-221 and gga-miR-222 have been shown by Northern blotting to be upregulated in MSB-1 cells compared to CEF or splenocytes (Lambeth et al. 2009). In vitro luciferase reporter assays have indicated that p27Kip1 has the potential to be specifically targeted by gga-miR-221 and gga-miR-222 in CEF and MSB-1 cells (Lambeth et al. 2009). In addition, Western blotting and retroviral mediated expression of antagomirs to gga-miR-221 and gga-miR-222 were used to demonstrate an inverse relationship between p27Kip1 protein levels and gga-miR-221 and gga-miR-222 levels, with the gga-miR-221 effect being the stronger of the two (Lambeth et al. 2009). A fourth approach to associating MDV1 miRNA expression with oncogenicity has been to construct mutants that fail to express miRNAs and/or to construct recombinant HVT strains that express MDV1 miRNAs. These mutants are works in progress, and their phenotypes in cell culture and in vivo will be interesting to dissect. In our laboratory, a recombinant HVT expressing mdv-miR-M4 shows improved growth characteristics in vitro and in vivo suggesting that miR-M4 can provide replicative assistance. Since this recombinant displays no tumorigenicity or pathogenicity, mdv1-miR-M4 should not be considered an oncomir in and of itself (Anderson et al., unpublished results).

A Developing Model of MDV-Mediated Lymphomagenesis Based on our current understanding of the expression patterns, molecular and biochemical functions of MDV gene products, we have developed a model describing important events in MD-lymphomagenesis (Fig. 13.5). Latency and transformation

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UL36-USP+ mdv1-M4-miR+ Hi

Meq Meq/vIL8Hi Meq/vIL8Δexon3+

CD4Hi TCR-2 (αVβ1) or TCR-3 (αVβ2) MHC-IIHi CD25+

vTR+

Genome State (Integrated?)

Genome Integrated

10 – 14 dpi

~ 15 + dpi

Latently-infected TH cell

Transformed TREG cell

Fig. 13.5 A developing model of MDV-mediated Lymphomagenesis. The diagram above comprises our relative understanding of MDV-mediated transformation. T-cells become activated following early cytolytic infection and latently infected at 10–14 dpi. Factors affecting the transition of latently infected to transformed are currently unknown but are correlated with increased expression of Meq splice variants, viral miRNA expression, as well as sustained expression of vTR, UL36 and other products. Multiple MDV genome integration events may serve as a catalyst for sustained, high-level expression of these gene products, as integration is common to MD lymphomas and derived cell lines. Sustained expression of spliced Meq products likely affects the T-cell immunophenotype through CtBP-1-mediated repression of specific loci giving rise to the transformed Treg cell. Expression of factors from these cells shapes the tumor microenvironment as well as suppresses antitumor responses. Transformed cells are also latently infected from which virus can reactivate. Although highly efficient at transformation, MDV is believed to induce multiple-monoclonal tumors as opposed to polyclonal transformation events (Delecluse and Hammerschmidt 1993; Delecluse et al. 1993)

are closely related, as depicted in Fig. 13.5. Events distinguishing latency from transformation are not known, but likely reflect differences in expression level and the accumulation of secondary mutations in proliferating lymphocytes.

Initiating Events One of the key initiating events in MD lymphomagenesis is the effect of lytic replication on immune surveillance. MDVs that have limited lytic replication (e.g., pp38 and vIL-8 deletion mutants) are capable of retaining oncogenicity, but at a much reduced level (Parcells et al. 2001; Reddy et al. 2002). It is, therefore, likely that the primary immunological damage elicited by early MDV replication is important to

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tumor development. In the Cornell model for MD, early replication in B-cells activates T-cells and allows them to become infected (Calnek 1986, 1992). Expression of vIL-8 elicits macrophages to sites of lytic replication and drives the virus latent through expression of interferons, NO, etc. Establishment of latency is required, but not sufficient, for transformation. MDV establishes latency and transforms CD4+, TCR-2+, and TCR-3+ T-cells exclusively. No MDV-associated B-cell or TCR-1+ (gd) T-cell tumors have been identified (Parcells and Burgess 2008; Schat et al. 1982b). In our findings regarding Meq and Meq splice variant expression (Fig. 13.1c), we see that Meq variants are detected at three weeks (VR3), but not at 2 weeks postinfection (VR2), a time frame consistent with latency. Stages of latency have been described previously for MDV (Volpini et al. 1995), with cells latently infected at one week being more sensitive to induced viral antigen expression than after two weeks. Coupled with our observation of Meq expression (Fig. 13.1c), we hypothesize that the later, more repressed stage of latency reflects increased expression of spliced forms of Meq. In our model, therefore, Meq and Meq variants repress viral lytic gene expression (ICP4, pp38, etc.) through recruitment of repression complexes by CtBP-1. These complexes block apoptosis and maintain cellular proliferation through interaction with cell cycle regulators Rb and p53 and repression of proapoptotic signals via interaction with CtBP-1 (and other factors, i.e., Par-4). Additional latent gene products vTR, USP (UL36), and pp14 maintain telomerase activity and alter cellular signal transduction. During latency, Meq induces the upregulation of MATSAs, including CD30, which likely have important consequences to lymphoma progression. In addition, host differences in cytokine responses likely contribute to initial tumor promotion events through the TH2 polarization of the immune response (Heidari et al. 2008; Kaiser et al. 2003). MDV-encoded miRNAs, notably mdv1-miR- M4, likely modulates proinflammatory signaling in latently infected T-cells and its sustained expression likely contributes to further lymphoma progression. Accessory roles of MDV1 miRNAs would be in the balance between lytic replication and latency, as MDV lytic gene mRNAs (ICP4, ICP27, pp38, etc.) are likely targets for these micros, but these targets have yet to be demonstrated.

Lymphoma Progression A key step in tumor progression is the sustained Meq-mediated blocking of T-cell anergy/apoptosis by downregulation of proapoptotic gene expression (through interactions with CtBP-1) and alteration of TGFß signaling (via upregulation of c-Ski). Since Meq unspliced and spliced forms are highly expressed in tumors and lymphomas, it is likely that the spliced forms code for proteins that are involved primarily in repression complexes, while Meq homo- and heterodimers function in the targeted transactivation of cellular genes as well as in a positive feedback loop on expression of itself and its spliced forms. This model is supported by the lack of colocalization of Meq and the spliced Meq proteins (Anobile et al. 2006) and the inherent activities of the proteins (Kumar et al. 2010).

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During lymphoma progression, Meq-cJun heterodimers increase proliferation, mobility and apoptosis resistance of cells that form initial tumor masses, perhaps through upregulation of adhesion molecules via vTR etc. Meq-induced CD30hiexpressing cells begin to shape the tumor microenvironment and begin to shift to a Treg immunophenotype. This is likely accomplished through the signaling mediated by CD30 engagement and the selective repression of T-cell expression to differentiate latently infected and transformed cells to a Treg immunophenotype. Alternatively, MDV may selectively target Treg cells for transformation as these are localized to distinct thymic regions and undergo different thymic programming. In either case, cells expressing high levels of CD30 shape the tumor microenviroment to block cell-mediated responses to tumor cells. MD tumor cells are somewhat resistant to CTL killing, as noted in the selection of REV-based cell lines for studying CTL responses to MDV (Omar and Schat 1997; Omar et al. 1998; Weinstock and Schat 1987). As lymphomas become larger and begin to become hypoxic, increased CtBP-1 expression and dimerization would drive further Meq–CtBP complex formation, repressing intracellular adhesion molecule expression and thus driving increased mobility/metastasis. vTR contributes to progression through upregulation and perhaps increased turnover of integrins, contributing to increased migration and also increased resistance to anoikis. Sustained MDV1 miRNA expression likely contributes to progression through the targeting of cell cycle regulatory and immune signaling pathways within transformed cells, based on their homology to human and murine counterparts. Although MDV is highly efficient at transformation, MDV-induced tumors are monoclonal, as determined through integration site analysis (Delecluse and Hammerschmidt 1993; Delecluse et al. 1993). The efficiency of MDV-mediated transformation, however, suggests that multiple monoclonal events may be present per chicken, and those that progress most rapidly give rise to the large frank lymphomas associated with MDV. Acknowledgments The authors would like to thank Drs. Venugopal Nair, Klaus Osterrieder, and Hsing-Jien Kung for the sharing of unpublished data and critical reading of select sections.

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Pappenheimer AW, Dunn LC, Cone V (1929b) Studies on fowl paralysis (Neurolymphomatosis gallinarum). I. Clinical features and pathology. J Exp Med 49:63–86 Parcells MS, Burgess SC (2008) Immunological aspects of Marek’s disease virus (MDV)-induced Lymphoma Progression. In: Kaiser HE, Nasir A (eds) Selected aspects of cancer progression: metastasis, apoptosis and immune response, vol 11. Springer, The Netherlands, pp 169–191 Parcells MS, Anderson AS, Cantello JL, Morgan RW (1994) Characterization of Marek’s disease virus insertion and deletion mutants that lack US1 (ICP22 homolog), US10, and/or US2 and neighboring short-component open reading frames. J Virol 68(12):8239–8253 Parcells MS, Lin SF, Dienglewicz RL, Majerciak V, Robinson DR, Chen HC, Wu Z, Dubyak GR, Brunovskis P, Hunt HD, Lee LF, Kung HJ (2001) Marek’s disease virus (MDV) encodes an interleukin-8 homolog (vIL-8): characterization of the vIL-8 protein and a vIL-8 deletion mutant MDV. J Virol 75(11):5159–5173 Peng Q, Shirazi Y (1996) Characterization of the protein product encoded by a splicing variant of the Marek’s disease virus Eco-Q gene (Meq). Virology 226(1):77–82 Peng F, Bradley G, Tanaka A, Lancz G, Nonoyama M (1992) Isolation and characterization of cDNAs from BamHI-H gene family RNAs associated with the tumorigenicity of Marek’s disease virus. J Virol 66(12):7389–7396 Peng Q, Zeng M, Bhuiyan ZA, Ubukata E, Tanaka A, Nonoyama M, Shirazi Y (1995) Isolation and characterization of Marek’s disease virus (MDV) cDNAs mapping to the BamHI-I2, BamHI-Q2, and BamHI-L fragments of the MDV genome from lymphoblastoid cells transformed and persistently infected with MDV. Virology 213(2):590–599 Qian Z, Brunovskis P, Rauscher F 3rd, Lee L, Kung HJ (1995) Transactivation activity of Meq, a Marek’s disease herpesvirus bZIP protein persistently expressed in latently infected transformed T cells. J Virol 69(7):4037–4044 Qian Z, Brunovskis P, Lee L, Vogt PK, Kung HJ (1996) Novel DNA binding specificities of a putative herpesvirus bZIP oncoprotein. J Virol 70(10):7161–7170 Rath NC, Parcells MS, Xie H, Santin E (2003) Characterization of a spontaneously transformed chicken mononuclear cell line. Vet Immunol Immunopathol 96(1–2):93–104 Reddy SM, Lupiani B, Gimeno IM, Silva RF, Lee LF, Witter RL (2002) Rescue of a pathogenic Marek’s disease virus with overlapping cosmid DNAs: use of a pp 38 mutant to validate the technology for the study of gene function. Proc Natl Acad Sci USA 99(10):7054–7059 Reichard JF, Sartor MA, Puga A (2008) BACH is a specific repressor of HMOX1 that is inactivated by arsenite. J Biol Chem 283:22363–22370 Reinke AW, Grigoryan G, Keating AE (2010) Identification of bZIP interaction partners of viral proteins HBZ, MEQ, BZLF1, and K-bZIP using coiled-coil arrays. Biochemistry 49(9):1985–1997 Rodriguez A, Vigorito E, Clare S, Warren MV, Couttet P, Soond DR, van Dongen S, Grocock RJ, Das PP, Miska EA, Vetrie D, Okkenhaug K, Enright AJ, Dougan G, Turner M, Bradley A (2007) Requirement of bic/microRNA-155 for normal immune funcion. Science 316:608–611 Rosenberger JK, Cloud SS, Olmeda-Miro N (1997) Avian tumor virus symposium, Reno, NV Ross N, Binns MM, Sanderson M, Schat KA (1993) Alterations in DNA sequence and RNA transcription of the Bam HI-H fragment accompany attenuation of oncogenic Marek’s disease herpesvirus. Virus Genes 7(1):33–51 Schat KA, Calnek BW, Fabricant J (1982a) Characterisation of two highly oncogenic strains of Marek’s disease virus. Avian Pathol 11(4):593–605 Schat KA, Chen CL, Shek WR, Calnek BW (1982b) Surface antigens on Marek’s disease lymphoblastoid tumor cell lines. J Natl Cancer Inst 69(3):715–720 Shack LA, Buza JJ, Burgess SC (2008) The neoplastically transformed (CD30hi) Marek’s disease lymphoma cell phenotype most closely resembles T-regulatory cells. Cancer Immunol Immunother 57(8):1253–1262 Shamblin CE, Greene N, Arumugaswami V, Dienglewicz RL, Parcells MS (2004) Comparative analysis of Marek’s disease virus (MDV) glycoprotein-, lytic antigen pp 38- and transformation antigen Meq-encoding genes: association of meq mutations with MDVs of high virulence. Vet Microbiol 102(3–4):147–167

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Shek WR, Calnek BW, Schat KA, Chen CH (1983) Characterization of Marek’s disease virus-infected lymphocytes: discrimination between cytolytically and latently infected cells. J Natl Cancer Inst 70(3):485–491 Skalsky RL, Samols MA, Plaisance KB, Boss IW, Riva A, Lopez MC, Baker HV, Renne R (2007) Kaposi’s sarcoma-associated herpesvirus encodes an otholog of miR-155. J Virol 81:12836–12845 Solomon JJ, Witter RL, Nazerian K, Burmester BR (1968) Studies on the etiology of Marek’s disease. I. Propagation of the agent in cell culture. Proc Soc Exp Biol Med 127(1):177–182 Spatz SJ, Petherbridge L, Zhao Y, Nair V (2007) Comparative full-length sequence analysis of oncogenic and vaccine (Rispens) strains of Marek’s disease virus. J Gen Virol 88(Pt 4):1080–1096 Su S, Li Y, Sun A, Zhao P, Ding J, Zhu H, Cui Z (2010) Protective immunity of a meq-deleted Marek’s disease virus against very virulent virus challenge in chickens. Wei Sheng Wu Xue Bao 50(3):380–386 Suchodolski PF, Izumiya Y, Lupiani B, Ajithdoss DK, Gilad O, Lee LF, Kung HJ, Reddy SM (2009) Homodimerization of Marek’s disease virus-encoded Meq protein is not sufficient for transformation of lymphocytes in chickens. J Virol 83(2):859–869 Suchodolski PF, Izumiya Y, Lupiani B, Ajithdoss DK, Lee LF, Kung HJ, Reddy SM (2010) Both homo and heterodimers of Marek’s disease virus encoded Meq protein contribute to transformation of lymphocytes in chickens. Virology 399(2):312–321 Swayne DE, Fletcher OJ, Schierman LW (1988) Marek’s disease virus-induced transient paralysis in chickens: alterations in brain density. Acta Neuropathol (Berl) 76(3):287–291 Swayne DE, Fletcher OJ, Schierman LW (1989) Marek’s disease virus-induced transient paralysis in chickens: demonstration of vasogenic brain oedema by an immunohistochemical method. J Comp Pathol 101(4):451–462 Tahiri-Alaoui A, Matsuda D, Xu H, Panagiotis P, Burman L, Lambeth LS, Petherbridge L, James W, Mauro V, Nair V (2009a) The 5¢ leader of the mRNA encoding the Marek’s disease virus serotype 1 pp 14 protein contains an intronic internal ribosome entry site with allosteric properties. J Virol 83(24):12769–12778 Tahiri-Alaoui A, Smith LP, Baigent S, Kgosana L, Petherbridge LJ, Lambeth LS, James W, Nair V (2009b) Identification of an intercistronic IRES in a Marek’s disease virus immediate early gene. J Virol 83(11):5846–5853 Tam W, Hughes SH, Hayward WS, Besmer P (2002) Avian bic, a gene isolated from a common retroviral site in avian leukosis virus-induced lymphomas that encodes a noncoding RNA, cooperates with c-myc in lymphomagenesis and erythroleukemogenesis. J Virol 76:4275–4286 Trapp S, Parcells MS, Kamil JP, Schumacher D, Tischer BK, Kumar PM, Nair VK, Osterrieder N (2006) A virus-encoded telomerase RNA promotes malignant T cell lymphomagenesis. J Exp Med 203(5):1307–1317 Volinia S, Calin GA, Liu CG, Ambs S, Cimmino A, Petrocca F, Visone R, Iorio M, Roldo C, Ferracin M, Prueitt RL, Yanaihara N, Lanza G, Scarpa A, Vecchione A, Negrini M, Harris CC, Croce CM (2006) A microRNA expression signature of human solid tumors defines cancer gene targets. Proc Natl Acad Sci USA 103(7):2257–2261 Volpini LM, Calnek BW, Sekellick MJ, Marcus PI (1995) Stages of Marek’s disease virus latency defined by variable sensitivity to interferon modulation of viral antigen expression. Vet Microbiol 47(1–2):99–109 Waidner LA, Morgan RW, Anderson AS, Bernberg EL, Kamboj S, Garcia M, Riblet SM, Ouyang M, Isaacs GK, Markis M, Meyers BC, Green PJ, Burnside J (2009) MicroRNAs of Gallid and Meleagrid herpesviruses show generally conserved genomic locations and are virus-specific. Virology 388(1):128–136 Weinstock D, Schat KA (1987) Virus specific syngeneic killing of reticuloendotheliosis virus transformed cell line target cells by spleen cells. Prog Clin Biol Res 238:253–263

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Witter RL (1983) Characteristics of Marek’s disease viruses isolated from vaccinated commercial chicken flocks: association of viral pathotype with lymphoma frequency. Avian Dis 27(1):113–132 Witter RL (1997) Increased virulence of Marek’s disease virus field isolates. Avian Dis 41(1):149–163 Witter RL, Burgoyne GH, Solomon JJ (1969) Evidence for a herpesvirus as an etiologic agent of Marek’s disease. Avian Dis 13(1):211–214 Witter RL, Stephens EA, Sharma JM, Nazerian K (1975) Demonstration of a tumor-associated surface antigen in Marek’s disease. J Immunol 115(1):177–183 Witter RL, Sharma JM, Lee LF, Opitz HM, Henry CW (1984) Field trials to test the efficacy of polyvalent Marek’s disease vaccines in broilers. Avian Dis 28(1):44–60 Witter RL, Li D, Jones D, Lee LF, Kung HJ (1997) Retroviral insertional mutagenesis of a herpesvirus: a Marek’s disease virus mutant attenuated for oncogenicity but not for immunosuppression or in vivo replication. Avian Dis 41(2):407–421 Xie Q, Anderson AS, Morgan RW (1996) Marek’s disease virus (MDV) ICP4, pp 38, and meq genes are involved in the maintenance of transformation of MDCC-MSB1 MDV-transformed lymphoblastoid cells. J Virol 70(2):1125–1131 Xu H, Yao Y, Zhao Y, Smith LP, Baigent SJ, Nair V (2008) Analysis of the expression profiles of Marek’s disease virus-encoded microRNAs by real-time quantitative PCR. J Virol Methods 149(2):201–208 Yao YX, Zhao YG, Xu HT, Smith LP, Lawrie CH, Sewer A, Zavolan M, Nair V (2007) Marek’s disease virus type 2 (MDV-2)-encoded microRNAs show no sequence conservation with those encoded by MDV-1. J Virol 81(13):7164–7170 Yao Y, Zhao Y, Xu H, Smith LP, Lawrie CH, Watson M, Nair V (2008) MicroRNA profile of Marek’s disease virus-transformed T-cell line MSB-1: predominance of virus-encoded microRNAs. J Virol 82(8):4007–4015 Yao Y, Zhao Y, Smith LP, Lawrie CH, Saunders NJ, Watson M, Nair VK (2009) Differential expression of miRNAs in Marek’s disease virus-transformed T-lymphoma cell lines. J Gen Virol 90(Pt 7):1551–1559 Zhao Y, Kurian D, Xu H, Petherbridge L, Smith LP, Hunt L, Nair V (2009a) Interaction of Marek’s disease virus oncoprotein Meq with heat shock protein 70 in lymphoid tumour cells. J Gen Virol 90(Pt 9):2201–2208 Zhao Y, Yao Y, Xu H, Lambeth L, Smith LP, Kgosana L, Wang X, Nair V (2009b) A functional MicroRNA-155 ortholog encoded by the oncogenic Marek’s disease virus. J Virol 83(1):489–492

Chapter 14

Polyomaviruses and Cancer Ole Gjoerup

Introduction Polyomaviruses are deceptively simple because of their small genomes and limited coding potential, yet they provide us with highly sophisticated model systems for understanding basic cellular processes. The power of studying them lies in their targeting of the most central players and mechanisms for proliferation control. Studies of polyomaviruses have allowed us to make significant advances in deciphering the molecular basis of cancer. Thus, critical signaling elements and pathways that have been elucidated with the help of these viruses include the p53 tumor suppressor, pRB (retinoblastoma) function in cell cycle control, protein phosphatase 2A (PP2A) function in regulation of cell proliferation, as well as discovery of tyrosine kinases and phosphoinositide 3-kinase (PI3K) (Linzer and Levine 1979; Lane and Crawford 1979; DeCaprio et al. 1988, 1989; Pallas et al. 1990; Eckhart et al. 1979; Whitman et al. 1985). These seminal discoveries were made by direct biochemical analysis of immunoprecipitates of the polyomaviral T antigens. In summary, the polyomaviruses have provided us insight to cellular signaling of general applicability, extending far beyond the realm of viruses. Moreover, studies of polyomavirus gene products have been instrumental not just for the understanding of molecular mechanisms related to cancer development, but also for understanding of enhancers and transcriptional regulation, splicing and polyadenylation, and DNA replication and nuclear translocation. A fundamental principle of polyomaviruses is that they operate by encoding proteins that bind and alter the key host proteins. Their interaction with a cellular protein might lead to its functional inactivation, activation, or redirection toward a different target (for example, an associated kinase or ubiquitin ligase). As is discussed, there appears to be a convergence on a small number of cellular target

O. Gjoerup (*) Cancer Virology Program, University of Pittsburgh Cancer Institute, Research Pavilion Suite 1.8, 5117 Centre Avenue, Pittsburgh, PA 15213, USA e-mail: [email protected] E.S. Robertson (ed.), Cancer Associated Viruses, Current Cancer Research, DOI 10.1007/978-1-4614-0016-5_14, © Springer Science+Business Media, LLC 2012

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proteins, notably pRB, p53, Hsc70, p300/CBP, and PP2A, that nearly all of the polyomaviruses encode one or more gene products to interact with. In addition, there are multiple other binding proteins, where functionality of the interaction is not fully established, and their role may not be universal for all the polyomaviruses but perhaps allowing more specialized functions. The intention of this chapter is to introduce the reader to this important family of viruses and to emphasize the defining characteristics of the polyomaviruses, including their conserved as well as unique features. Since most of the viruses discussed here are reviewed individually in the following chapters, the focus is on the larger picture and how they compare with each other in mechanisms and biology.

History and Discovery The founding member of the polyomaviruses, murine polyomavirus (MPyV), was identified by Ludwik Gross in 1953 when, searching for cell-free transmission of leukemia, he found a filterable agent capable of inducing salivary gland tumors in newborn mice (Gross 1953). This new virus became the archetypal member of the Polyomaviridae family. Polyomavirus is so named because of the ability of MPyV to induce solid tumors at many different sites. Shortly thereafter, Sweet and Hilleman discovered simian vacuolating virus 40 (also known as “simian virus 40” or “SV40”) as a contaminant in monkey kidney cells used to grow the poliovirus vaccine (Sweet and Hilleman 1960). Subsequent studies estimate that as many as 100 million people may have been inadvertently exposed to vaccine possibly containing live SV40 virus, mainly between 1955 and 1963, as part of the poliovirus vaccination program (Garcea and Imperiale 2003). Due to the lack of convincing epidemiological and serological evidence, SV40 has not been conclusively linked to human malignancies, although it has been proposed to be causally associated with mesotheliomas, osteosarcomas, choroid plexus brain tumors, and non-Hodgkin’s lymphoma (Poulin and DeCaprio 2006a). Nevertheless, a lot of questions remain. Most recent surveys indicate that SV40 is not a human polyomavirus, but it might be present at low prevalence in the population, and the behavior of SV40 in humans has not been clarified (Garcea and Imperiale 2003). In 1971, the first human polyomaviruses, BKV and JCV, were discovered in urine from a renal transplant patient and brain tissue of a patient with progressive multifocal leukoencephalopathy (PML), respectively (Padgett et al. 1971; Gardner et al. 1971). BK and JC refer to patient initials. Amazingly, technological advances have allowed the identification of six additional human polyomaviruses within the last 3 years. In 2007, Karolinska Institute virus (KIV) and Washington University virus (WUV) were discovered in symptomatic pediatric respiratory specimens (Gaynor et al. 2007; Allander et al. 2007). These viruses were detected by treatment of the specimens with endonuclease, which digests host DNA, but not viral DNA protected within virions, followed by high-throughput shotgun sequencing of all DNAs present. There is currently no evidence linking KIV and WUV to cancer or non-neoplastic disease (Dalianis et al. 2009).

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In 2008, Merkel cell polyomavirus (MCV) was detected by digital transcriptome subtraction (DTS) analysis of Merkel cell carcinoma (MCC) samples (Feng et al. 2008). MCC is a rare but highly aggressive type of skin cancer of neuroendocrine origin. The incidence of MCC is highly elevated in immunosuppressed patients suggesting an infectious etiology. DTS involves preparing a library of sequences of the entire transcriptome from a sample, followed by comparing and subtracting in silico known human sequences from all available databases (Feng et al. 2007). The remaining sequences after rigorous subtraction potentially belong to novel viruses and may be identified as such by shared homology with known families of viruses. Accumulating evidence strongly supports a causal role for MCV in about 80% of MCC patients, and this is now becoming widely accepted, thus providing the most conclusive direct link so far between a polyomavirus and human malignancy. In 2010, rolling circle amplification of DNA from normal human skin samples identified an additional human polyomavirus-designated HPyV6 (Schowalter et al. 2010). Several isolates of variant MCV genomes were identified in the same search. A PCR-based search with degenerate primers for additional polyomaviruses in skin samples further led to discovery of HPyV7. Finally, in 2010, another polyomavirus, trichodysplasia spinulosa-associated polyomavirus (TSV), was discovered by rolling circle amplification from facial spicules of a heart transplant patient with the extremely rare disease trichodysplasia spinulosa (van der Meijden et al. 2010). Phylogenetic analysis revealed that it is most closely related with orangutan polyomavirus and MCV. Taken together, BKV, JCV, KIV, WUV, MCV, HPyV6, HPyV7, and TSV constitute the eight currently known human polyomaviruses. Moreover, lymphotropic polyomavirus (LPV), also known as African green monkey polyomavirus, which was discovered in 1979, could also be of relevance to human cancer (zur Hausen and Gissmann 1979; Pawlita et al. 1985). There is long-standing evidence that a human counterpart to LPV exists, but it has not been identified yet. Thus, different groups have found serological evidence that an LPVlike human virus circulates in 15–20% of the human population (Brade et al. 1981; Kean et al. 2009).

Phylogenetic Comparison of Polyomaviruses At least 23 different polyomaviruses have been found so far with hosts ranging from birds, bats, rabbits, and rodents to primates (see Dalianis and Garcea (2009) for a comprehensive list). Phylogenetic analysis, based on an early gene product, LT, or a late gene product, VP1, divides polyomaviruses into separate clades (Fig. 14.1). Within the SV40 group, there is close homology among SV40, BKV, JCV, and SA12 when comparing either LT or VP1. However, KIV and WUV, while very similar to each other, are more distantly related to the rest of the SV40 group. Especially the VP1 proteins of KIV and WUV exhibit significant sequence divergence from the remainder of the SV40 group and cluster more closely with HPyV6 and HPyV7.

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Fig. 14.1 Phylogenetic analysis of polyomavirus LT and VP1 protein sequences. Phylogenetic trees were generated for the LT and VP1 protein sequences from 19 polyomaviruses: crow, goose, finch, budgerigar fledgling disease polyomavirus (BFPyV), bovine polyomavirus (BPV), bat, LPV, MCV, hamster polyomavirus (HaPyV), murine pneumotropic virus (MptV), MPyV, BKV, simian agent 12 (SA12), JCV, SV40, KIV, WUV, HPyV6, and HPyV7. Sequences were aligned with the ClustalX program, and the alignment was imported into TreeView for generation of phylograms

Interestingly, MCV is belonging to a separate clade of MPyV and is most similar with LPV (Feng et al. 2008). Generally, the avian polyomaviruses cluster together and form their own clade. Bovine polyomavirus (BPV), murine pneumotropic virus (MptV, also known as Kilham strain), and little brown bat polyomavirus are outliers and do not readily fit into the clades of MPyV, SV40, or avian polyomaviruses.

Genome Organization Genomes of the polyomaviruses range in size from 5 to 5.4 kb of circular, doublestranded, covalently closed DNA. The DNA is present in complex with cellular histones, with the exception of histone H1, thus resembling cellular genomes (minichromosomes). Viral particles consist of nonenveloped icosahedral capsids with a 40–45-nm diameter. Viral genomes are divided into three parts: the early region encodes the early transcript, which is expressed before the onset of viral DNA replication (see Fig. 14.2 for a schematic drawing of SV40 and MPyV genomes). Conversely, the late region produces the late transcript, which is predominantly expressed after the onset of viral replication. The noncoding regulatory region (NCRR),

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Fig. 14.2 Overview of SV40 and MPyV genome organization. The genomes of SV40 (5,243 bp) and MPyV (5,297 bp) are outlined, including the early region that encodes the T antigens and the late region that encodes the capsid proteins VP1-3 (VP4 is unique to SV40). Shown are the open reading frames that are generated through alternative splicing. An miRNA known to target the early message is also included. The core origin of replication is indicated (“ori”). Some of the differences to be noted are the existence of an agnoprotein generated from the leader of the SV40 late message, as well as the unique MT of MPyV. The tiny T antigen of MPyV was not included in the drawing

consisting of the viral replication origin and transcriptional control elements, is intertwined between these. Early and late promoters are in close proximity with the origin of replication. The transcriptional control elements in the NCRR of various polyomaviruses were recently reviewed (White et al. 2009). Early and late transcripts are of approximately equal length but encoded on opposite strands of the viral genomes. The early transcript is differentially spliced to generate 2–5 early proteins (Fig. 14.3). Large T antigen (LT) and small t antigen (ST) are expressed by all the known polyomaviruses, although for some like KIV and WUV, these have only been predicted by open reading frame analysis. These proteins are referred to as T antigens because they were first identified using antibodies from tumor-bearing animals. LT and ST have yielded profound insights to biological processes when used in model systems. Besides LT and ST, a variable number of accessory T antigens are expressed from the early transcript. These include the 17k T antigen of SV40 (Zerrahn et al. 1993), the T¢165, T¢136, and T¢135 of JCV (Trowbridge and Frisque 1995), the truncT of BKV (Abend et al. 2009b), the 57kT of MCV (Shuda et al. 2008), and the tiny T of MPyV (Riley et al. 1997). Because of the splicing arrangement, all of the early viral transcripts share exon 1 at the N-terminus, whereas the C-terminus is unique. The role of the auxiliary T antigens is not well-understood, although in the case of JCV they have been demonstrated to contribute to efficient viral replication (Prins and Frisque 2001). SV40 17k exhibits minimal transforming activity (Zerrahn et al. 1993), but its role during the viral life cycle remains to be demonstrated. MPyV and hamster polyomavirus are unique in that they also encode a middle

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Fig. 14.3 The splicing patterns of various polyomavirus T antigens. The splicing patterns are shown for all the T antigens from MCV, JCV, BKV, SV40, and MPyV. The different reading frames are outlined by different color rectangles, whereas the broken line illustrates introns. The N-terminus of each T antigen is shared (exon 1), but the C-terminus is most often unique (except 57kT and T¢165). TT refers to tiny T antigen

T antigen (MT), which is their principal transforming protein (reviewed in Fluck and Schaffhausen 2009; Schaffhausen and Roberts 2009). How MT evolved during evolution is an intriguing question. The late message encodes at a minimum three capsid proteins, known as VP1, VP2, and VP3. Only for SV40 has a VP4 been reported as well (Daniels et al. 2007). Both alternative splicing and internal translation, the late message being bicistronic, generate the various capsid proteins. While VP1 is the main capsid protein, the minor components VP2/3 are also required for encapsidation. VP1 can spontaneously assemble into virus-like particles (VLPs) when expressed individually. Since VP1 is the major capsid protein, it is frequently used to develop serologic assays to detect the polyomaviruses and estimate their prevalence in the human population. While we think of VP1 as being inert with regard to transformation-related signaling, it is worth noting that interaction of VP1 with the cell is known to trigger activation of c-myc and c-fos genes (Zullo et al. 1987), which have been associated with transformation, and also recently poly(ADP-Ribose) polymerase 1 (PARP-1), caspases, phospholipase Cg1 (PLCg1), and Akt-1 (Butin-Israeli et al. 2010). VP4 is believed to be important for egress of the viral genomes at the end of the lytic cycle (Daniels et al. 2007). Leader sequences of the late transcripts from SV40, BKV, and JCV are known to also encode an agnoprotein, which has not been detected for most of the other polyomaviruses (Ng et al. 1985; Khalili et al. 2005). The agnoprotein contributes

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to viral assembly and maturation but is not contained in the virions. Several polyomaviruses (SV40, BK, JC, MCV, MPyV) encode a miRNA from the late transcript probably by a read-through mechanism (Sullivan et al. 2005, 2009; Seo et al. 2008). The general function of this miRNA is to cleave the early message late in infection, thus downregulating its expression and escaping immune surveillance by cytotoxic T cells (Sullivan et al. 2005).

Viral Life Cycle Polyomaviruses are internalized by interaction of the VP1 capsid protein with sialic acids on specific gangliosides acting as receptors. Known receptors are gangliosides GD1b and GT1b for BKV, GT1b and the serotonin receptor 5HT2AR for JCV, GM1 for SV40, and GT1b for MCV (Tsai et al. 2003; Low et al. 2006; Elphick et al. 2004; Erickson et al. 2009). The polyomaviruses mainly enter cells through caveolae that are lipid rafts invaginating from the plasma membrane; however, JCV passes through clathrin-dependent pathways (Eash et al. 2006). The viruses then traffic through the perinuclear ER to the nucleus, where uncoating occurs, followed by early transcription of the viral genome via host RNA polymerase II. After translation of LT, it subsequently binds GAGGC repeats within the origin and initiates melting and unwinding of the core origin, followed by recruitment of host replication factors, like polymerase a/primase. LT is a prototype initiator of replication with intrinsic ability to bind DNA specifically, assemble into single or double hexamers, and unwind DNA using its ATP-dependent helicase activity (reviewed in Fanning and Knippers 1992; Simmons 2000). LT also has an ability to modulate transcription, which is important during infection, where it represses early transcription while enhancing late viral gene expression. After viral replication commences, the late genes are expressed and their protein products assemble into capsids that encapsulate the viral genome. A full lytic cycle, in the case of SV40, spans approximately 72 h. Polyomaviruses normally have a narrow host range and restricted cell-type tropism. SV40 only productively infects monkey cells and MPyV only murine cells. These restrictions are believed to be in part mediated by the species-specific interactions between LT and polymerase a that are critical for a productive initiation of viral replication (Schneider et al. 1994). For example, JCV prefers to grow in fetal glial cells (Feigenbaum et al. 1987); however, it is not strictly neurotropic, as it can replicate in many other cell types, including human embryonic kidney (Major et al. 1992). LPV preferentially grows in B-lymphoblastoid cells. Other polyomaviruses, like SV40 and MPyV, in particular, replicate in a broad range of cell types. Polyomaviruses productively infect their natural (permissive) host, resulting in cell lysis and release of virions. Tumors are very rarely caused in the natural host because the lytic cycle kills the cell. These viruses can also establish persistent infections that rarely cause tumors or other diseases, except in immunocompromised or newborn animals. SV40 does not induce disease in rhesus monkeys, except when these are immunosuppressed through simian immunodeficiency virus infection (Horvath et al. 1992).

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Conversely, nonpermissive cells elicit an abortive infection, meaning that viral entry and early gene expression occur normally, but somehow there is a block in viral replication and late gene expression. At high multiplicities of infection, the whole cell population can transiently become transformed, but as the cells divide and the viral genome is diluted out, only rare transformants grow out in which the viral genome is integrated. It is important to emphasize that oncogenic transformation upon integration of these viruses can be viewed as a “biological accident”, since it is a dead-end event preventing multiplication of the virus (Shuda et al. 2008). Human cells are considered to be semipermissive for SV40, meaning that low levels of replication and virus production occur. Not just species dictates the outcome of a polyoma infection, but also the cell type. Thus, SV40 productively infects human fibroblasts, whereas in mesothelial cells the outcome is a persistent infection without cell lysis, thereby facilitating oncogenic transformation (Bocchetta et al. 2000).

General Properties Despite 50 years of intense studies, some fundamental questions of polyomavirus biology continue to elude us. What cells are initially infected? How is the virus disseminated between tissues? Is there a major virus reservoir in certain tissues that are persistently infected? How is the virus transmitted between individuals? These are all questions for which we have still incomplete answers. Most of the human polyomaviruses are widespread in nature and cause lifelong infections that are asymptomatic or subclinical, except in immunosuppressed individuals. In other words, these viruses appear to be most often kept in check by the immune system. Individuals contact BKV, JCV, and MCV in early childhood or adolescence leading to seroconversion (Gjoerup and Chang 2010; Kean et al. 2009). Approximately 50–80% of the human population is seropositive for BKV and JCV (Egli et al. 2009; Knowles et al. 2003; Carter et al. 2009). Preliminary tests indicate that MCV, KIV, and WUV are equally prevalent (Kean et al. 2009; Carter et al. 2009). Transmission routes have not been definitively established (Gjoerup and Chang 2010). Fecal/oral and oral transmissions have been proposed for BKV and JCV, whereas KIV, WUV, and MCV may occur through the respiratory system. Interestingly, tonsillar DNA can be shown to contain genomic DNA from most of the polyomaviruses, suggesting a potential entry point. JCV infects tonsillar cells but is circulated for dissemination via B lymphocytes, which is how JCV is believed to cross the blood–brain barrier and reach the central nervous system. The kidney is considered a key site of persistence for BKV and JCV, and these viruses are consistently associated with viruria, especially upon reactivation. Another site of persistent infection for BKV is the urinary tract. BKV and JCV can also be detected in blood.

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LT Induces Cell Cycle Progression from G0/G1 → S Phase The polyomaviruses have very limited coding capacity that they have exploited maximally by utilization of alternative splicing. Since these viruses depend extensively on the cellular replication machinery and nucleotide pool, they must reprogram the host environment to support viral DNA replication by inducing cell cycle progression from quiescence into S phase (Hatanaka and Dulbecco 1966; Dickmanns et al. 1994). This is especially important, since viruses like SV40 are believed to normally infect terminally differentiated kidney epithelial cells that are withdrawn from the cell cycle. This bypass of normal proliferation control mechanisms associated with unscheduled DNA synthesis in a viral replication setting, when uncoupled from a complete lytic cycle, sends a cell on the path to malignancy (Dickmanns et al. 1994). Accordingly, activities of LT required for promotion of cell cycle progression are overlapping or identical with those required for oncogenic transformation. The LT mitogenic effect is believed, at a minimum, to be dependent on binding of negative growth control elements, such as pRB family members and p53. The DnaJ domain and an uncharacterized function in the origin-binding domain (OBD) also appear to contribute to efficient induction of cell cycle progression (Dickmanns et al. 1994). Finally, based on lessons from the large DNA tumor viruses (Epstein–Barr virus and Kaposi’s sarcoma-associated herpesvirus), it should be emphasized that targeting of pRB and p53 to drive cell cycle progression may not be the only rationale from the virus point of view. Evidence indicates that tumor suppressors like p53 play key roles in innate immunity; for example, interferons a and b are known to boost the p53 response (Moore and Chang 2003; Takaoka et al. 2003). Thus, it remains possible that polyomaviruses also target these tumor-suppressor proteins to evade antiviral signaling through innate immunity and as an unfortunate by-product, breaks down our antitumor defenses. Currently, there is little known about the polyomaviruses and interferon signaling.

Non-neoplastic Disease Association BKV and JCV are linked to nonmalignant human disease with high morbidity and mortality. BKV is, mainly in bone marrow transplants, associated with hemorrhagic cystitis, which is an inflammation of the bladder, and also polyomavirus-associated nephropathy, which is a primary cause of kidney transplant rejection (Imperiale 2000; Jiang et al. 2009). It is believed to be immune suppression that triggers reactivation. PML is a fatal neurodegenerative disease caused when JCV, upon reactivation, destroys the oligodendrocytes, which are the cells that produce myelin (Imperiale 2000; Maginnis and Atwood 2009; Khalili et al. 2006; Tan and Koralnik 2010; Major et al. 1992). PML is predominant in patients with a compromised immune system, for example AIDS patients, of which around 5% develop PML. Recent attention to PML and JCV has come from the observation that treatment

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with the multiple sclerosis drug Natalizumab apparently on rare occasion can cause JCV reactivation and PML in patients (Kleinschmidt-DeMasters and Tyler 2005; Langer-Gould et al. 2005). Natalizumab is a monoclonal antibody against a4 integrin. It may facilitate JCV reactivation and PML by blocking migration of leukocytes across the blood–brain barrier to the site of reactivation.

Assays for Transformation There are multiple assays by which the transformed phenotype can be scored. Each of these assays measures different, although often related, parameters. For each assay, the general principle is that one measures the ability to overcome normal restraints to cell proliferation, an ability, which is shared by most tumor cells. As is discussed below, these restraints can be in the form of a limited life span, absence of or limiting growth factors, and anchorage dependence or contact inhibition. These are all restrictions that are believed to be imposed on nascent tumor cells in vivo, and that they most overcome to further develop. There are additional properties relevant for a malignancy, but these are not easily measured outside of an animal, for example invasiveness, metastasis, and impact of the immune system.

Immortalization Primary cells grown in culture have a finite life span. After a certain number of passages, cells become permanently growth arrested, also referred to as senescent. However, both SV40 and MPyV LT have been demonstrated to efficiently immortalize rodent cells in culture, meaning that the cells can be serially passaged indefinitely. SV40 LT appears to require both its pRB and p53 binding sites for immortalization, whereas MPyV LT immortalizes via binding of pRB family members (Tevethia et al. 1998; Larose et al. 1991). Immortalization is generally considered to be the first step toward tumorigenesis, although there may be instances where it is not a prerequisite (Freund et al. 1992). Human cells are fundamentally different with regard to cell immortalization perhaps in part because their life span in culture is much longer than that of rodent cells. Whereas rodent cells can spontaneously immortalize at some low frequency, this does not occur with human cells. Furthermore, human cells do not have constitutive telomerase activity, which means that every time cells divide, their telomeres shorten, unless the telomerase gene (hTert) is turned on. At a certain point, the telomeres are shortened beyond a threshold, and cell death occurs by a cellular process known as “crisis.” In most cell types, SV40 LT does not upregulate telomerase expression. Accordingly, LT does not immortalize human cells, but it does extend their life span (Neufeld et al. 1987). After the extended life span, most cells undergo crisis, but extremely rarely clones grow out that are immortal. Conversely, LT cooperates with hTert to efficiently immortalize human cells (Counter et al. 1998; Zhu et al. 1999).

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Transformation Besides immortalization, several assays that measure the transformed phenotype are relevant for in vivo tumorigenesis. While normal cells require growth factors that are present in serum for their proliferation, transformed cells can often grow under low or no serum conditions, making this one important assay. Also, normal cells are contact inhibited, meaning that cell proliferation is arrested when a confluent monolayer is formed. However, transformed cells are able to overcome these restrictions, thus allowing dense foci of cells to grow out on top of the monolayer. The focus formation assay, often performed in rodent cells, has been instrumental for the genetic analysis of functions needed for T antigen-mediated transformation. However, this assay is predominantly carried out with fibroblast, not epithelial, cells, reflecting some intrinsic limitations. A related assay to focus formation is to determine saturation density by establishing growth curves. Because transformed cells are not contact inhibited, they continue proliferating on top of a packed monolayer and thus reach higher saturation densities than normal cells. Another important transformation assay relates to loss of anchorage dependence and is referred to as the soft agar assay. Most normal cells need to adhere to a solid surface of a culture vessel, and if they are suspended in semisolid medium like agar, they will growth arrest but remain viable for weeks. However, many transformed cells have acquired anchorage-independent growth. This assay is considered a more stringent assay for transformation than focus formation and frequently correlates well with tumor induction in animals. While focus formation assays are often performed by direct transfection of T antigen expression vectors, the soft agar assay most often employs cell lines that stably express T antigen(s). The best correlate for tumorigenesis is not surprisingly considered to be a direct analysis of tumor formation in susceptible animals.

SV40 Large T Antigen Since SV40 LT is the most highly studied of the polyomavirus LTs, it is introduced first as the prototype, followed by other family members. SV40 LT is a truly remarkable, highly multifunctional nuclear protein living a double life as both an initiator of viral replication and an oncogene. Several reviews on LT have been previously published (Ahuja et al. 2005; Cheng et al. 2009; Fanning and Knippers 1992; Pipas 1992, 2009; Manfredi and Prives 1994; Gjoerup and Chang 2010). The oncogenic activities are a by-product of LT stimulation of S-phase entry in order to prepare cells for viral replication and of blocking antiviral signaling through innate immunity. Replication and transformation activities of LT were shown early on to be genetically separable from each other. Most of the functions of LT are mediated by interaction with critical host proteins. Strikingly, most polyomaviral LT proteins have converged on a small number of common targets (Hsc70, pRB family, p53, and possibly p300/ CBP) that are discussed.

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Fig. 14.4 Schematic diagram of SV40 LT. The main domains and binding sites contained in LT are illustrated. The DnaJ domain at the N-terminus mediates binding of the Hsc70 chaperone dependent on the canonical sequence “HPDK.” Downstream of the DnaJ domain, LT contains separate binding sites for Cul7, Bub1, and IRS1. Pocket proteins (pRB, p107, p130) bind to LT dependent on an “LxCxE” consensus sequence. The nuclear localization signal (NLS) is a requirement for nuclear translocation of LT. The OBD mediates not just binding to the viral origin of replication, but also binding of a number of factors, including Nbs1, RPA, and components of the transcriptional machinery. LT has a C2H2 Zn2+ binding element important for hexamer assembly. The C-terminus consists of an AAA+ type ATP-dependent helicase (“ATP” outlines the minimal ATPase domain), which is interspersed with a bipartite binding site for p53 that in turn bridges binding of p300/CBP coactivators. At the extreme C-terminus, LT contains a host range (HR) function and binding site for the F-box protein FBW7

LT is highly modular in structure, with discrete regions corresponding to the various functions (see Fig. 14.4 for a diagram of LT with domains and major binding proteins). At its N-terminus, within the first 70 amino acids, LT contains a DnaJ domain, which serves to recruit chaperones like Hsc70 (Kelley and Landry 1994; Campbell et al. 1997; Srinivasan et al. 1997). The consensus motif found in all DnaJ domains is “HPDK,” which is conserved in nearly all polyomavirus LT and ST proteins. LT binding to Hsc70 leads to stimulation of its ATPase activity, which is in turn used to act on a chaperone substrate (Sullivan and Pipas 2002). For SV40 LT, it is known that the DnaJ domain is required for disruption of repressive complexes between p107/p130 pocket proteins and E2F family members, as well as p130 degradation (Srinivasan et al. 1997; Stubdal et al. 1996, 1997; Zalvide et al. 1998). The DnaJ domain must be present in cis with the pRB family binding site. DnaJ mutants, depending on the nature of the mutation, exhibit variable defects in different assays for transformation. A role of the DnaJ domain in viral replication and virion production has also been reported (Campbell et al. 1997; Spence and Pipas 1994). At the N-terminus, following the DnaJ domain, LT contains binding sites in close proximity for the Cul7 member of the SCF (skp1, cullin, F-Box)-type ubiquitin ligase complex as well as the Bub1 mitotic checkpoint kinase (Kohrman and Imperiale 1992; Ali et al. 2004; Cotsiki et al. 2004). Cul7 binds LT dependent on residues 69–83, and its binding has been linked to SV40 transformation of rodent cells (Kasper et al. 2005). Bub1 requires amino acids 89–97 to bind LT, which has been shown to be important for rat-1 focus formation (Cotsiki et al. 2004). This region of LT contains the sequence motif “WExWW,” which is highly conserved in a subset of the polyomaviruses. LT binding to Bub1 furthermore elicits tetraploidy and a DNA damage response (Hein et al. 2009). Bub1 is a prime candidate for LT’s

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ability to cause chromosomal instability. For both Cul7 and Bub1, it is unclear whether binding occurs with other polyomaviral LTs. Downstream of the Cul7/Bub1 binding sites, the region from residues 101–115, and in particular the “LxCxE” motif contained within, is required for interaction with pRB family members. The founding member of this family is the retinoblastoma protein, pRB, which is a key cellular tumor suppressor mutated in the pediatric tumor of the same name. Adenovirus E1A was first shown to bind pRB (Whyte et al. 1988), quickly followed by SV40 LT (DeCaprio et al. 1988). Now we know that every polyomavirus LT is likely to bind pRB family members, given the absolute conservation of the LxCxE motif. LT binding to the pRB family is linked to induction of S-phase entry, which is likely why it is being targeted. For nearly all transformation in vitro, as well as tumor induction in transgenic models, the pRB binding site is essential (DeCaprio et al. 1988; Kalderon and Smith 1984; Chen and Paucha 1990; Manfredi and Prives 1994). Structurally, pRB is composed of two domains, A and B, that together form a pocket conformation critical for tumor suppression (Burkhart and Sage 2008), hence the name “pocket proteins”. There are two other family members, p107 and p130. These share structural and functional properties but have not been shown to be bona fide tumor suppressors like pRB. They are nevertheless very important for cell cycle regulation. The ability of LT to transform depends not just on overcoming pRB, but also on p107/p130 inactivation (Sullivan et al. 2000; Stubdal et al. 1997; Zalvide et al. 1998). The primary function of the pRB family is to associate with and repress E2F family members, which are important transcription factors (Morris and Dyson 2001). LT, by binding pRB family members, allows derepression of E2F target genes (reviewed in DeCaprio 2009). The pRB family/E2F circuit regulates a broad range of cellular target genes involved in cell cycle (DNA replication, mitosis), DNA repair, development, and apoptosis. Normally, pRB family members are regulated by cyclin-dependent kinases that phosphorylate them in a cell cycle-dependent manner, thus causing release from E2F at the appropriate phase of the cell cycle. LT also binds to insulin receptor substrate 1 (IRS1), with the binding site overlapping the LxCxE (Fei et al. 1995; Yu and Alwine 2008). IRS1 binding studies were prompted by initial observations that LT cannot transform insulin-like growth factor I receptor (IGF-IR)-deficient cells (D’Ambrosio et al. 1995; DeAngelis et al. 2006). IRS1 is a major downstream target of IGF-IR. LT causes relocalization of IRS1 from the cytoplasm into the nucleus. Binding of IRS1 appears to be associated with Akt activation and protection from apoptosis (Yu and Alwine 2008). Immediately downstream of the LxCxE is located a classic nuclear localization signal, the first one to be studied closely (Kalderon et al. 1984; Lanford and Butel 1984), and following it, the OBD, which resides in the next 130 amino acids. The OBD is by itself highly multifunctional with roles in transcriptional activation, an uncharacterized activity contributing to transformation (Dickmanns et al. 1994; Kalderon and Smith 1984), binding sites for replication protein A (RPA) and Nbs1 (Weisshart et al. 1998; Wu et al. 2004), besides the major role in site-specific DNA binding. The central part of LT contains a Zn2+ coordination element, which is required for hexamer assembly (Loeber et al. 1991). This element is highly conserved between polyomaviruses.

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The C-terminus, spanning approximately residues 350–650, contains the ATP-dependent helicase region interspersed with a bipartite p53 binding site (Kierstead and Tevethia 1993). LT possesses key activities required for initiation of viral replication (reviewed in Fanning and Knippers 1992; Simmons 2000). It assembles as a single or double hexameric complex at the viral origin by specific binding to GAGGC repeats contained within a palindrome. The ATP-dependent helicase activity is then used to unwind the origin DNA, and subsequently LT recruits replication enzymes like polymerase a/primase, RPA, and topoisomerase I to initiate viral DNA replication. The p53 protein was first discovered as a LT binding protein in 1979 (Lane and Crawford 1979; Linzer and Levine 1979). Soon, it was realized that the majority of viral proteins target it for inactivation. p53 is a transcription factor that can turn on the expression of genes involved in either cell cycle arrest or apoptosis (Levine and Oren 2009; Vousden and Prives 2009). It is a master regulator that responds to genotoxic stress of various kinds like DNA damage or oncogenic stress. A role in the interferon response indicates contributions to innate immunity as well (Takaoka et al. 2003). While initially perceived as an oncogene because mutant forms of the gene had been studied, it is now clear that p53 is instead a tumor suppressor (Baker et al. 1990; Finlay et al. 1989; Eliyahu et al. 1989; Malkin et al. 1990; Donehower et al. 1992). It has been dubbed “guardian of the genome” (Lane 1992). Strikingly, it is the most mutated gene in human cancer, with greater than 50% of all tumors harboring p53 mutation or loss (Hollstein et al. 1994). LT binds the site-specific DNA-binding domain of p53 and prevents it from contacting DNA, thereby blocking target gene activation (Bargonetti et al. 1992; Jiang et al. 1993; Segawa et al. 1993; Mietz et al. 1992). It is generally believed that LT binding to pRB family members triggers unscheduled DNA synthesis, which is sensed by the cell. To prevent an apoptotic response to this, p53 must be inactivated. The binding of p53 is required for immortalization of rodent cells but not always strictly required for oncogenic transformation (Kierstead and Tevethia 1993; Tevethia et al. 1998; Zhu et al. 1991; Srinivasan et al. 1997). Interestingly, and somewhat paradoxically, LT not only binds and inactivates p53 but also potently stabilizes it (Oren et al. 1981; Reich et al. 1983). Accumulating evidence indicates that the stabilized form of p53 has acquired a gain of function in transformation possibly because LT in complex with p53 turns on a unique set of genes (Deppert et al. 1989; Hermannstadter et al. 2009; Tiemann and Deppert 1994a, b; Bocchetta et al. 2008). Several naturally occurring mutants of p53 also exhibit a gain of function in transformation (Dittmer et al. 1993; Cadwell and Zambetti 2001), making studies of the LT-induced gain of function relevant for these nonviral oncogenic mechanisms. As it was first shown for adenovirus E1A, LT also binds the transcriptional coactivators/histone acetyltransferases p300 and CBP (Eckner et al. 1996). These large scaffolds are considered potential tumor suppressors, function in a variety of different biological processes, and are targeted by many viral oncoproteins (Gayther et al. 2000; Goodman and Smolik 2000; Iyer et al. 2004). Binding is mainly indirect, bridged by p53, but a direct contact between LT and p300/CBP has also been demonstrated (Lill et al. 1997; Borger and DeCaprio 2006; Ahuja et al. 2009).

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This mutant, defective in p300/CBP binding, is also defective in transformation (Ahuja et al. 2009). Binding of p300/CBP has been suggested to underlie the p53 gain of function because LT effectively gains access to these via p53 (Borger and DeCaprio 2006; Hermannstadter et al. 2009). Like E1A, LT also binds the p400 protein (Lill et al. 1997), which is a member of the SWI2/SNF2 family of chromatinremodeling factors. While for E1A this is required for transformation (Fuchs et al. 2001), in the case of LT this has not been elucidated. The extreme C-terminus of LT contains two separate functions. The host range function is conserved only among SV40, BKV, and JCV and allows SV40 to grow in certain cell lines like CV-1 but is not required for growth in others like BSC40 (Pipas 1985). Host range function is likely mediated by binding to a cellular protein, which has yet to be identified (Poulin and DeCaprio 2006b). The extreme C-terminus also binds the F-box protein FBW7 via a phosphodegron that mimics the consensus normally found in FBW7 substrates (Welcker and Clurman 2005). FBW7 is the substrate-recognizing component of SCF complexes that target cellular proteins for proteasomal degradation. Although LT was shown to mislocalize FBW7 from nucleoli to the nucleoplasm and slightly increased cyclin E levels, this region of LT is not required for transformation in the context of full-length LT. Therefore, its function remains unknown.

Non-SV40 LT Proteins While SV40 LT is a versatile transforming protein, which can potently induce focus formation in cultured cells and a wide variety of tumors when expressed transgenically, other polyomavirus LT proteins appear less potent. MPyV LT, because it strikingly lacks p53 binding, has no independent transforming activity, but it does immortalize primary rodent cells dependent on pRB family binding (Larose et al. 1991). MPyV LT is a replication initiator with DnaJ domain, site-specific DNA binding, and ATP-dependent helicase activities similar to SV40. MPyV LT has an insert sequence of ~120 amino acids between the end of first exon and the beginning of the OBD, which corresponds to the coding sequence for MT in a different reading frame (Pipas 1992). Even though it does not bind p53, it appears to have evolved an independent binding mode for p300/CBP (Cho et al. 2001). BKV, and JCV in particular, have lower transformation potential in vitro than SV40 (Abend et al. 2009a; Harris et al. 1996; Frisque et al. 2006). This is in part due to the restricted tropism causing lower LT expression in many cultured cells, but also because of reduced binding affinities for pRB and p53 (Bollag et al. 1989; Harris et al. 1996, 1998b). For BKV, it has been demonstrated that there is not enough LT to sequester pRB and the DnaJ domain plays a major role in pRB family inactivation (Harris et al. 1996, 1998b). BKV early region can immortalize and transform embryonic fibroblasts from mouse, rat, and hamster. Transformation of human embryonic kidney cells is not efficient and often abortive, but coexpression with c-myc or oncogenic Ras leads to full transformation.

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JCV can transform human fetal glial cells and also primary hamster brain cells, but the efficiency is not high (Frisque et al. 2006). Mainly JCV transforms cells of neural origin. This is because the NCRR directs glial-specific transcription of the early region. The host range is mainly restricted by the NCRR because the promoter/ enhancer region within the NCRR contains binding sites for transcription factors that are expressed in a tissue-specific manner. The promoter/enhancer region is known to undergo numerous rearrangements in the form of mutations, deletions, and duplications. These rearrangements are more frequent in disease states, but their exact relationship with pathogenesis and how they are generated remain unresolved questions (Newman and Frisque 1999). JCV LT, apparently uniquely, binds to b-catenin (Gan and Khalili 2004), a key protein in the Wnt pathway, and the putative tumor-suppressor neurofibromatosis type 2 (NF2) (Shollar et al. 2004), which might contribute to its tumor induction potential. At present, there are no reported in vitro transformation systems for KIV, WUV, or MCV.

MPyV Middle T Antigen MT is uniquely encoded only by MPyV and hamster polyomavirus. MT is required for an efficient MPyV life cycle, which reflects contributions at several levels (reviewed in Fluck and Schaffhausen 2009). It is required for VP1 phosphorylation and capsid assembly, but also plays a key role in regulation of viral transcription and replication. The latter effects are mediated by MT activation of AP1 and PEA3 (ETS family) binding sites within the MPyV enhancer (Chen et al. 1995). MPyV differs from SV40 in its absolute dependence on the enhancer for viral replication (de Villiers et al. 1984), whereas for SV40 the core origin suffices. Presumably, the MPyV enhancer allows for the broad tissue tropism of this virus. MT is always required for MPyV transformation and often sufficient (reviewed in Fluck and Schaffhausen 2009; Schaffhausen and Roberts 2009; Ichaso and Dilworth 2001). Its transformation mechanism is fundamentally different from that of other T antigens. Notably, MT is a potent transforming protein in established cell lines. However, transformation of primary cells requires cooperation with LT or ST (Rassoulzadegan et al. 1982, 1983; Asselin et al. 1983, 1984; Lomax and Fried 2001). Efficient transformation of human cells in cooperation with a dominant negative p53 has also been demonstrated (Utermark et al. 2007). Due to a hydrophobic sequence at its C-terminus, MT is membrane-localized (Ito 1979). This includes localization both at the plasma membrane as well as internal membranes and cytoskeletal elements. Membrane localization is essential for its transforming activity (Carmichael et al. 1982). MT has often been envisioned to mimic a constitutively active growth factor receptor (like Her2/Neu) because it acts by assembling signaling molecules that activate downstream cytoplasmic signaling cascades. A schematic drawing of MT is outlined in Fig. 14.5.

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Fig. 14.5 Schematic drawing of MPyV MT. The N-terminus contains a DnaJ domain wherein lies determinants also for binding of TAZ. MT may be viewed as a mimic of a constitutively active growth factor receptor, membrane-localized through its C-terminal hydrophobic anchor sequence. MT binds to PP2A and uses it to build a scaffold leading to recruitment and activation of PTKs like Src, Fyn, and Yes that in turn phosphorylate MT on the major sites Y250, Y315, and Y322. Each of these phosphorylation site acts as a docking platform for a major signal generator. Y250 signals through binding of the Shc adaptor protein to mediate activation of the Ras/MAPK pathway. Y315 recruits the p85 subunit of PI3K to activate downstream signaling through Akt and Rac. Y322 is important for recruitment of PLCg1, which generates diacylglycerol (DAG) and inositol trisphosphate (IP3) that in turn can release Ca2+ and activate protein kinase C (PKC). Shown are also the S257 phosphorylation site involved in 14-3-3 recruitment and the proline-rich region (PPP) implicated in transformation via an unknown mechanism

MT contains a DnaJ domain at its N-terminus whose function per se has not been linked with transformation (Campbell et al. 1995); however, it is required for overall folding of the protein and binding of more C-terminal interactors. The extreme N-terminus (amino acids 2–4) has been found to bind a protein called TAZ, which is playing a role in transcriptional regulation and regulated protein degradation through the b-TrCP E3 ubiquitin ligase complex (Tian et al. 2004). Binding of TAZ appears to be important for transformation, but since the mutation affects all three T antigens, it remains uncertain wherein the defect lies. Similarly to ST, MT also binds to PP2A (Pallas et al. 1990), but the signaling outcome is different because of MT localization at the membrane (Mullane et al. 1998). Binding of MT to PP2A is absolutely required to build a scaffold for recruitment of protein tyrosine kinases (PTKs) like c-Src, c-Fyn, and c-Yes (Courtneidge and Smith 1983), reviewed in (Fluck and Schaffhausen 2009). C-Src is the cellular counterpart of the retroviral oncogene v-Src. Importantly, tyrosine phosphorylation was first discovered as a major signaling mechanism by studying MT immunoprecipitates (Eckhart et al. 1979). How PP2A mechanistically contributes to PTK recruitment is not clear and further confounded by the observation that catalytic activity of PP2A is not required (Ogris et al. 1999). Moreover, it remains unclear if PP2A and PTKs act on targets outside the MT complex.

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The recruitment of PTKs is critical to MT transformation. MT binding leads to the activation of PTK activity, and in turn PTKs phosphorylate the major sites Y250, Y315, and Y322 on MT. Each of these tyrosine phosphorylation site constitutes a docking site for a major signaling molecule. Y250 is part of the sequence NPTY, which is required for binding to the PTB domain of the adaptor molecule Shc (Dilworth et al. 1994; Campbell et al. 1994). Shc can signal to another adaptor molecule, Grb2, which in turn can associate with and activate the Ras guanine nucleotide exchange factor Sos. Sos activation leads to Ras activation and activation of the downstream MAPK signaling cascade. Grb2 can also, alternatively, signal to the Gab1 docking molecule, which is linked to PI3K signaling. The Y250 site can have a dramatic effect on MT transformation in murine cells, but in other contexts like human cell transformation assays, there is no effect (Utermark et al. 2007). This reflects the importance of different signaling circuits required for transformation in different cell systems. The Y315 site connects MT with the important signaling molecule, PI3K (Whitman et al. 1985; Kaplan et al. 1986). In fact, PI3K was first discovered in studies of MT immunoprecipitates (Whitman et al. 1985). PI3K consists of a regulatory p85 subunit and a catalytic p110 subunit. Y315 is part of a YMPM motif, which binds to the SH2 domains of p85. Thus, MT recruits PI3K to the membrane, which stimulates its activity. PI3K causes production of PIP3 and the membrane recruitment of proteins with specific pleckstrin homology domains. Downstream targets of PI3K include PDK1, Akt, and Rac. Akt is a kinase that protects against cell death and contributes to transformation. Rac is a small GTP-binding protein involved in cytoskeletal organization, which also contributes to MT transformation (Urich et al. 1997). The Y315F mutant exhibits a striking defect in many, but not all, cell transformation and tumor induction systems. In human cells, MT transformation is abrogated (Utermark et al. 2007). Also, it was demonstrated that knockout of p110a abrogates MT transformation of mouse embryo fibroblasts, proving that it is essential for MT oncogenesis (Utermark et al. 2007). The discovery of PI3K through its association with MT is especially significant given the recent findings of frequent activating p110a mutations in human cancers of breast, colon, prostate, and liver (Samuels et al. 2004). PI3K inhibitors are in clinical trials and may provide an efficient therapeutic for certain cancers (Wong et al. 2010; Liu et al. 2009). The Y322 site, part of the sequence YLDI, is required for binding of PLCg1 (Su et al. 1995). The effects of signaling through PLCg1 have been more difficult to discern than through Shc and PI3K. A Y322F mutant is not defective under normal growth conditions, but does exhibit a defect in transformation, when the assay is conducted under reduced serum conditions (Su et al. 1995). Consistent with binding of PLCg1, an increase in inositol trisphosphate can be observed. This can potentially lead to protein kinase C (PKC) activation and release of Ca2+. Two other regions of MT are of interest. The S257 phosphorylation site is implicated in recruitment of 14-3-3 proteins (Cullere et al. 1998). While the S257 mutant does not show a phenotype in cell culture assays for transformation, there is a striking deficiency of this mutant virus to specifically induce salivary gland tumors (Cullere et al. 1998). The mechanism involved is not known. Furthermore, a proline-rich

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region between amino acids 332–347 plays an unknown role in transformation (reviewed in Fluck and Schaffhausen 2009; Schaffhausen and Roberts 2009). It appears that all the known binding proteins are recruited normally, yet mutants in this region fail to transform. Finally, it should be emphasized that MT transgenic models, especially the MMTV-MT mammary carcinoma model (Guy et al. 1992), continues to inform us about metastasis, the role of stromal–epithelial interactions for tumor progression, and the contribution of different genetic backgrounds for carcinogenesis.

Small T Antigen ST is not strictly required for the viral life cycle but can stimulate viral replication to various extents depending on the polyomavirus (Sleigh et al. 1978; Cicala et al. 1994; Berger and Wintersberger 1986; Kwun et al. 2009). While ST is not sufficient to transform cells, it can cooperate with LT for transformation of certain cell types, where LT is not sufficient, notably human cells (de Ronde et al. 1989; Chang et al. 1985; Hahn et al. 1999; Porras et al. 1996; Yu et al. 2001). ST expression by itself does allow cells to grow to a higher saturation density (Cherington et al. 1986; Noda et al. 1986). ST can also cooperate with LT to allow rodent cell lines to grow independent of anchorage (Bouck et al. 1978; Sleigh et al. 1978; Jog et al. 1990; Mungre et al. 1994; Bikel et al. 1986). Much emphasis has centered on the remarkable demonstration that human cells can be fully transformed by defined genetic elements, such as combinations of LT, ST, hTert, and oncogenic H-Ras (Hahn et al. 1999). These observations have definitively highlighted a critical function of ST in oncogenesis. The function of the ST DnaJ domain has not been elucidated (Boyapati et al. 2003). ST is localized in both the cytoplasm and the nucleus. The unique region contains two CxCxxC clusters that coordinate Zn2+ and confer conformational stability (Turk et al. 1993). These are highly conserved in most of the polyomaviruses, with the exception of the avian (Pipas 1992). Virtually, all of the known functions of ST, including its contributions to transformation, can be attributed to its binding of the important cellular serine–threonine phosphatase PP2A (Rundell and Parakati 2001; Sablina and Hahn 2008; Skoczylas et al. 2004). PP2A is a heterotrimeric enzyme composed of a scaffold subunit (A), a regulatory subunit (B), and a catalytic subunit (C). It is a diverse class of enzymes, since there are 2 different A subunits, 2 C subunits, and 17 B subunits (reviewed in Sablina and Hahn 2008). While it is generally believed that ST binds the AC holoenzyme and displaces or prevents the binding of B subunits, thus causing overall inactivation of PP2A activity (Pallas et al. 1990; Yang et al. 1991), there are also some examples of substrates, where ST promotes their dephosphorylation (Yang et al. 1991, 2007; Yu et al. 2005). Interestingly, in human transformation assays, the effect of ST can be partially mimicked by specific knockdown of the B56g subunit, highlighting its importance (Chen et al. 2004). Significantly, mutations in PP2A subunits have been discovered in certain human cancers, consistent with a tumor-suppressor function (Sablina and Hahn 2008; Arroyo and Hahn 2005).

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The binding of ST to PP2A leads to a panoply of outcomes associated with increased proliferation and disruption of the actin cytoskeleton (reviewed in Rundell and Parakati 2001; Sablina and Hahn 2008; Skoczylas et al. 2004). Some of the important downstream targets are kinases like Akt, MAPK, and PKCz (Yuan et al. 2002; Rodriguez-Viciana et al. 2006; Sontag et al. 1993, 1997), as well as stabilization of the proto-oncogene c-myc (Yeh et al. 2004). There is also clear promotion of the G1-S cell cycle transition likely mediated through activation of the cyclin A and cyclin D1 promoters combined with the degradation of the cdk inhibitor p27kip1 (Sontag et al. 1993; Howe et al. 1998; Porras et al. 1999; Skoczylas et al. 2005; Watanabe et al. 1996). ST is a somewhat promiscuous transcriptional activator (Loeken et al. 1988). Microarray experiments with SV40 ST indicate that it activates some genes in a non-PP2A-dependent manner, but the exact nature of other functions is not known (Moreno et al. 2004). Although polyomaviral ST proteins other than SV40 are not as well-characterized, the general theme of PP2A binding appears to hold true. MPyV ST also induces proliferation alone (Mullane et al. 1998) or together with LT (Ogris et al. 1992), and it can cooperate with MT for transformation or tumor induction (Asselin et al. 1983, 1986). The complementation of MT for transformation of REF52 or primary cells by ST has been reported to reflect a disconnection of p19ARF, which MT induces, from p53 induction (Lomax and Fried 2001). The exact mechanism involved is unclear, but ST must bind PP2A to overcome p19ARF signaling to p53 (Moule et al. 2004). Whether this mechanism operates with other ST proteins remains to be shown. MPyV ST can also induce cell death in growing cells but protect against apoptosis when cells are starved, in both cases dependent on PP2A binding (Andrabi et al. 2007). Toggling between these two opposing outcomes likely has to do with Akt phosphorylation at its two activating sites T308 and S473, which is in turn regulating downstream substrate selection. Both JCV and MCV ST also bind PP2A and stimulate viral DNA replication (Bollag et al. 2010; Kwun et al. 2009). Surprisingly, it was recently shown that JCV ST has 2 LxCxE motifs and is capable of binding members of the pRB family (Bollag et al. 2010). Little is known about BKV ST, except that it appears to bind PP2A (Rundell et al. 1981).

Polyomavirus-Induced Tumors in Animals Polyomavirus tumor induction in animals can be determined in either of the three different ways: inoculation of virus into sites of a susceptible animal, transgenic expression of LT, or the whole early region, with a tissue-specific promoter or injection of cells expressing the T antigens into nude mice to produce xenografts. Early studies clearly demonstrated the ability of SV40 to induce tumors in newborn hamsters. Different tumors are produced dependent on the route of inoculation. Intracranial injection of SV40 resulted in ependymomas (Kirschstein and Gerber 1962), whereas intravenous injection of hamsters yielded leukemias, lymphomas,

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and sarcomas (Diamandopoulos 1972). Intrapleural injection produced mesothelial tumors in weanling hamsters (Cicala et al. 1993). Transgenic expression of SV40 in mice using its natural promoter potently induces choroid plexus tumors of the brain epithelium (Brinster et al. 1984). Interestingly, transgenic expression of LT can induce a spectrum of different responses ranging from no stimulation of proliferation, hyperplasia, dysplasia, and carcinoma to fullblown invasive or metastatic phenotypes. A wide variety of other tumors have been induced by transgenic expression of SV40 LT alone, or in combination with ST, when driven by various tissue-specific promoters (reviewed in Ahuja et al. 2005; Saenz Robles and Pipas 2009). Transgenic model systems have been developed for intestine, eye, pancreas, prostate, salivary gland, stomach, lung, and liver in addition to brain to study SV40-induced tumorigenesis. Tumorigenesis invariably depends on binding of pRB family members, whereas the requirement for p53 binding is highly variable. Frequently, expression of a truncated N-terminal fragment of LT (1–136 or 1–121), which carries the DnaJ and pRB binding sequences, is sufficient to trigger tumorigenesis (Chen et al. 1992; Symonds et al. 1994; Bennoun et al. 1998; Rathi et al. 2007). Collectively, these model systems have provided extraordinary insight to the development of cell type-specific tumors, and together these studies have underscored the importance of cell context for tumor progression. BKV and, especially, JCV have also been shown to be highly oncogenic in animals (reviewed in White et al. 2005). JCV induces brain tumors in Golden Syrian hamsters, but astrocytomas, glioblastomas, and neuroblastomas in owl and squirrel monkeys. Monkeys are nonpermissive for JCV, and the virus is integrated in the tumors. JCV has also been reported to induce neuroectodermal tumors in hamsters and rats. Conversely, transgenic expression of JCV early region results in adrenal neuroblastomas, neuroectodermal tumors, as well as tumors originating from the cerebellum and pituitary neoplasia. Interestingly, dysmyelination of the central nervous system was also observed, reminiscent of PML. JCV is suspected of playing a role in human medulloblastomas, glioblastomas, and astrocytomas. Linking BKV and JCV to human cancer is difficult since they are ubiquitous in the human population and most of the proposed neoplasias, where a causal link might exist, are not characterized by integration of the viral genome (Abend et al. 2009a; Maginnis and Atwood 2009). BKV is oncogenic in young or newborn rodents, but the efficiency of tumorigenesis depends on inoculation route (White et al. 2005). Transgenic expression of the BKV early region also results in tumors, but the spectrum is different from JCV. Mainly hepatocellular carcinomas, renal adenocarcinomas, thymomas, or lymphomas are observed with BKV.

Genome Destabilization Much accumulated evidence indicates that LT can induce genomic instability manifested in both structural and numerical chromosome aberrations (Ray et al. 1990, 1992, 1998; Ray and Kraemer 1993; Woods et al. 1994; Stewart and Bacchetti 1991;

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Friedrich et al. 1992; Chang et al. 1997; Schaefer et al. 1993; Levine et al. 1991; Sargent et al. 1997; Ewald et al. 1996). This has been demonstrated to occur rapidly after LT expression, thus preceding the transformed phenotype and excluding it as a secondary effect of transformation (Chang et al. 1997; Ray et al. 1990). Most of these experiments have been performed with SV40 LT, but limited studies of BKV and JCV LT suggest that they exhibit similar characteristics with regard to induction of chromosome instability (Neel et al. 1996; Darbinyan et al. 2007; Trabanelli et al. 1998; Theile and Grabowski 1990; Ricciardiello et al. 2003). Cytogenetic analysis indicates that LT-expressing rodent or human cells often are tetraploid or aneuploid and harbor structural alterations, such as chromosome breaks, fragments, rings, double minutes, translocations, as well as frequent dicentric chromosomes, perhaps arising from telomeric fusion (Chang et al. 1997; Woods et al. 1994; Stewart and Bacchetti 1991; Ray et al. 1992). Furthermore, LT is mutagenic (Theile and Grabowski 1990), recombinogenic (St-Onge et al. 1990; Xia et al. 1997), and causes sister chromatid exchanges (Nichols et al. 1978), along with frequent micronuclei formation (Hein et al. 2009). Interestingly, expression of the first 147 amino acids of LT appears to induce genome destabilization with wild-type efficiency (Woods et al. 1994). Moreover, binding to pocket proteins was found not to be essential for the phenotype. It was reported that interference with mitotic checkpoints might contribute toward the induced genomic instability (Chang et al. 1997). While the exact molecular mechanism underlying genome destabilization remains to be elucidated, the binding of LT to the mitotic kinase Bub1 is a good candidate (Cotsiki et al. 2004). Bub1 is primarily involved with the spindle checkpoint, which is a cellular quality control mechanism that monitors bivalent attachment of microtubules to the kinetochores at the metaphase-to-anaphase transition of mitosis (Meraldi and Sorger 2005; Perera et al. 2007; Williams et al. 2007). Failure of Bub1 to act can result in chromosomal instability, aneuploidy, chromosome missegregation, and enhanced tumorigenesis (Cahill et al. 1998; Jeganathan et al. 2007; Kawashima et al. 2010; Meraldi and Sorger 2005; Perera et al. 2007; Schliekelman et al. 2009; Tang et al. 2004; Baker et al. 2009). LT binding to Bub1 results in attenuation, but not complete inactivation, of the spindle checkpoint (Cotsiki et al. 2004). Strikingly, while wild-type LT induces tetraploidy in ~20% of normal human fibroblasts, the Bub1 binding mutant of LT is completely defective in this regard (Hein et al. 2009). Of further interest, LT point mutant analysis has correlated Bub1 binding with transformation of Rat-1 fibroblasts (Cotsiki et al. 2004). Notably, tetraploidy has recently been demonstrated in a p53-deficient background to promote tumor development (Fujiwara et al. 2005). A key unanswered question is whether LT induction of genomic instability contributes to, or even drives, long-term tumorigenesis. In the case of human papillomavirus (HPV), expression of the viral oncoproteins E6 and E7 is not sufficient to drive tumorigenesis; genomic instability is required as well (Hurlin et al. 1991). This likely explains a long latency period between the initial infection and the occurrence of cervical cancer. For polyomaviruses, the evidence is more tenuous, although a number of observations suggest a role.

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SV40 LT is a highly versatile oncoprotein but not particularly potent when compared with MT or oncogenic H-Ras. Even when LT is delivered into every single cell using a retroviral vector, only few transformed foci grow out, suggesting that additional events may be required (Jat et al. 1986). When LT is expressed transgenically in the mouse salivary gland using a tetracycline-regulatable promoter, hyperplasia and polyploidy can be reversed after 4 months of age, but not after 7 months (Ewald et al. 1996). Moreover, while most cell clones expressing temperaturesensitive mutants of LT can be reversed to a normal phenotype at the nonpermissive temperature, the A-type transformants remain neoplastic following LT inactivation after temperature shift (Rassoulzadegan et al. 1978; Seif and Martin 1979). Finally, although stable expression of an origin-defective SV40 genome in keratinocytes failed to elicit a tumorigenic response in nude mice, 46 passages of these cells in vitro allowed the induction of invasive squamous cell carcinomas (Brown and Gallimore 1987). Taken together, these observations suggest that LT expression might induce chromosomal aberrations that contribute toward tumorigenesis, for example through loss of tumor-suppressor genes or oncogene copy number gains.

Induction of DNA Damage Responses Recent evidence illustrates that viruses like polyoma not only induce S phase in quiescent cells, but that they have also targeted the DNA damage response (DDR) to regulate their own replication (Dahl et al. 2005; Hein et al. 2009; Shi et al. 2005; Zhao et al. 2008; Boichuk et al. 2010). It is a relatively recent discovery that most viruses interact with components of the DDR to stimulate or inhibit it (reviewed in Chaurushiya and Weitzman 2009; Lilley et al. 2007). Adenoviruses appear to mainly inactivate the DDR by targeting the Mre11-Rad50-Nbs1 (MRN) DDR sensor complex (Stracker et al. 2002). In contrast, the polyomaviruses activate the DDR and exploit it (Dahl et al. 2005; Shi et al. 2005; Zhao et al. 2008; Boichuk et al. 2010). The DDR can be divided into two major branches. The ATM kinase responds to DNA doublestrand breaks, whereas ATR kinase responds to replication-associated single-stranded DNA lesions. MPyV was demonstrated to activate the ATM pathway upon infection, and loss of ATM led to reduced replication and virus yield (Dahl et al. 2005). It was proposed that maintaining an S/G2-phase environment via ATM activation might cause the replication enhancement (Dahl et al. 2005). JCV was shown to induce both the ATM and ATR pathways, thus eliciting a G2 checkpoint-mediated arrest and increased genome replication (Orba et al. 2010). SV40 infection of monkey kidney cells also results in ATM/ATR and Chk1/2 activation (Zhao et al. 2008; Shi et al. 2005; Okubo et al. 2003; Boichuk et al. 2010). ATM signaling was shown to promote viral replication in part via phosphorylation of LT on Ser120, which plays a regulatory role in replication (Shi et al. 2005). After SV40 infection of human or monkey cells, LT colocalizes with activated ATM, the phosphorylated form of histone H2AX (g-H2AX) and the MRN complex in nuclear

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foci corresponding to replication centers (Zhao et al. 2008; Shi et al. 2005; Boichuk et al. 2010). ATM signaling was shown to be important for establishment of replication centers and for proteasomal degradation of the MRN complex via the Cul7 ubiquitin ligase late in infection (Zhao et al. 2008). Interestingly, not just SV40 virus, but even LT expression in the absence of a functional origin, induces multiple DDR signaling responses in normal human fibroblasts via different LT domains (Boichuk et al. 2010; Hein et al. 2009). LT induces large nuclear foci of g-H2AX/53BP1, a hallmark of the DDR, via Bub1 binding (Hein et al. 2009). In addition, other downstream ATM/ATR targets are activated, including Chk1, Chk2, and p53, in the latter case leading to its stabilization. LT induces overt DNA damage, in part double-strand breaks, as demonstrated in comet assays (Boichuk et al. 2010). Besides g-H2AX/ 53BP1 DDR foci, LT also, via distinct domains, induces foci of FancD2 and Rad51, consistent with their activation on chromatin. FancD2 is a key member of the Fanconi anemia family, which is characterized by enhanced susceptibility to interstrand cross-links probably due to a role in repair of stalled or collapsed replication forks (Moldovan and D’Andrea 2009; Howlett et al. 2005). Fanconi anemia patients suffer from genomic instability and are predisposed to cancer. Rad51 is essential for homologous recombinationmediated DNA repair and acts to enhance SV40 replication (Boichuk et al. 2010). Exactly what LT domains or functions trigger the distinct responses involving FancD2 and Rad51 remains to be shown. It seems reasonable to hypothesize that LT induces a form of replication stress by deregulating the firing and dynamics of cellular replication origins. These extensive perturbations of DDR signaling are likely to influence oncogenesis perhaps in part through effects on genomic stability and generation of aneuploidy. Traditionally, the DDR is viewed as an early barrier to tumorigenesis (Bartkova et al. 2005). Loss of several of the genes involved in the DDR is associated with genetic predisposition to human malignancy, for example ATM, Nbs1, BRCA1, and FancD2. This is consistent with fibroblasts from Fanconi anemia patients being hypersensitive to SV40-mediated transformation (Todaro et al. 1966; Liu et al. 1996). However, various reports indicate that the stabilized form of p53, presumably a by-product of DDR activation (Hein et al. 2009), can acquire a gain of function in transformation (Deppert et al. 1989; Hermannstadter et al. 2009; Bocchetta et al. 2008). The 17k gene product, when rendered pRB binding deficient, can induce both premature cellular senescence and DDR activation (Gjoerup et al. 2007; Hein et al. 2009), two cellular responses that have previously been linked together in the context of oncogene-induced senescence (Bartkova et al. 2006; Di Micco et al. 2006). Interestingly, senescence induction can cause the acquisition of the senescent-associated secretory phenotype, which entails secretion of proinflammatory cytokines that might promote tumor progression (Rodier et al. 2009). Hence, senescence is a double-edged sword with regard to malignancy (Davalos et al. 2010). The exact contribution of DDR signaling to polyomavirusinduced transformation and tumorigenesis has not been established but is likely to depend on the viral oncoprotein and cellular context.

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Concluding Remarks In the last 3 years, we have witnessed a revitalization of the polyomavirus field with the discovery of six new human polyomaviruses, including MCV, which appears to be directly linked to human cancer causality (Feng et al. 2008). Technological advances have allowed us to make these new virus discoveries. They provide us with a framework of expectations for what the next several years might bring: Are there yet more undiscovered human polyomaviruses out there? And more importantly, how do we decisively determine if they are associated with human disease, if their prevalence is widespread? For viruses that might act as cofactors in carcinogenesis, causality is ever more challenging to prove (Harris et al. 1998a). Indeed, here, the example of MCV may guide future explorations. First, a cancer was chosen with a likely infectious etiology because of an increased incidence in immunodeficient patients. Second, an infectious agent was identified to be MCV based on DTS technology (Feng et al. 2008). Third, it was demonstrated that the MCV genome is monoclonally integrated in most of the MCC samples, indicating that integration preceded tumor formation. Fourth, the integration event invariably was accompanied with point mutations in the LT-coding sequence leading to loss of the helicase domain but retention of DnaJ, pRB binding domains, and ST coding (Shuda et al. 2008). This is consistent with observations from other polyomaviruses, where it was also found that viral replication functions like the helicase activity are deleterious in the context of tumor formation, perhaps due to localized collision of replication forks and DNA damage responses at the integration site (Israel et al. 1980; Lania et al. 1981). This observation establishes that MCV is not just a “passenger virus” replicating well in tumors. Fifth, in the infected tumors, T antigen is expressed only in the tumor cells (Shuda et al. 2009). Sixth, it was demonstrated that knockdown of the truncated MCV LT and ST with short hairpin RNA (shRNA) leads to growth arrest, or cell death, but only in MCV-positive MCC samples (Houben et al. 2010). Collectively, this is providing strong evidence for a continuous requirement for MCV T antigens in maintaining the tumor. Despite the recent progress on MCV, there is still much that we do not understand. MCV, as well as cutaneous polyomaviruses HPyV6 and HPyV7, was detected in skin samples along with many cutaneous HPVs, perhaps illustrating a closer kinship with HPVs than previously anticipated (Schowalter et al. 2010). Virions are shed from the skin. Is the life cycle, like HPV, also tied to epidermal differentiation? Indeed, it has not been possible to establish a cell culture system yet where KIV, WUV, and MCV can be grown likely because we are missing the right cell type or culturing conditions. Can recombination events occur, either between different polyomaviruses coinhabiting the same cell or even with HPVs? Hybrid genomes between the polyomaviruses and papillomaviruses can apparently be created occasionally by recombination. Woolford et al. discovered a new virus in papillomas and carcinomas of bandicoots, whose early region encodes T antigens resembling those from polyomaviruses, whereas the late region encodes capsid proteins similar to those of HPV (Woolford et al. 2007).

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Furthermore, it becomes important to know if there are different serotypes of MCV that can be categorized as high risk versus low risk for MCC, similarly to HPV and cervical cancer. Moreover, have the tumor mutants of MCV acquired any gains in transformation properties relative to the wild-type T antigens? MCV is present in many tissues; however, it is only associated with MCC. Why are some tissues apparently more susceptible to tumorigenesis than others? The observations for SV40, BKV, and JCV also suggest extensive perturbations of DNA damage responses and chromosome stability. Is this a common phenomenon of the polyomaviruses, and more importantly, does it contribute to long-term tumorigenesis? The recent dramatic increase in discovery of new polyomaviruses, and the first solid link to human cancer etiology, reinforces our beliefs that these viruses continue to serve as important model systems for the development and progression of cancer.

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Stubdal H, Zalvide J, Campbell KS, Schweitzer C, Roberts TM, DeCaprio JA (1997) Inactivation of pRB-related proteins p130 and p107 mediated by the J domain of simian virus 40 large T antigen. Mol Cell Biol 17(9):4979–4990 Su W, Liu W, Schaffhausen BS, Roberts TM (1995) Association of Polyomavirus middle tumor antigen with phospholipase C-gamma 1. J Biol Chem 270(21):12331–12334 Sullivan CS, Pipas JM (2002) T antigens of simian virus 40: molecular chaperones for viral replication and tumorigenesis. Microbiol Mol Biol Rev 66(2):179–202 Sullivan CS, Cantalupo P, Pipas JM (2000) The molecular chaperone activity of simian virus 40 large T antigen is required to disrupt Rb-E2F family complexes by an ATP-dependent mechanism. Mol Cell Biol 20(17):6233–6243 Sullivan CS, Grundhoff AT, Tevethia S, Pipas JM, Ganem D (2005) SV40-encoded microRNAs regulate viral gene expression and reduce susceptibility to cytotoxic T cells. Nature 435(7042):682–686 Sullivan CS, Sung CK, Pack CD, Grundhoff A, Lukacher AE, Benjamin TL, Ganem D (2009) Murine Polyomavirus encodes a microRNA that cleaves early RNA transcripts but is not essential for experimental infection. Virology 387(1):157–167 Sweet BH, Hilleman MR (1960) The vacuolating virus, S.V. 40. Proc Soc Exp Biol Med 105:420–427 Symonds H, Krall L, Remington L, Saenz-Robles M, Lowe S, Jacks T, Van Dyke T (1994) p53-dependent apoptosis suppresses tumor growth and progression in vivo. Cell 78(4):703–711 Takaoka A, Hayakawa S, Yanai H, Stoiber D, Negishi H, Kikuchi H, Sasaki S et al (2003) Integration of interferon-alpha/beta signalling to p53 responses in tumour suppression and antiviral defence. Nature 424(6948):516–523 Tan CS, Koralnik IJ (2010) Progressive multifocal leukoencephalopathy and other disorders caused by JC virus: clinical features and pathogenesis. Lancet Neurol 9(4):425–437 Tang Z, Sun Y, Harley SE, Zou H, Yu H (2004) Human Bub1 protects centromeric sister-chromatid cohesion through Shugoshin during mitosis. Proc Natl Acad Sci USA 101(52):18012–18017 Tevethia MJ, Lacko HA, Conn A (1998) Two regions of simian virus 40 large T-antigen independently extend the life span of primary C57BL/6 mouse embryo fibroblasts and cooperate in immortalization. Virology 243(2):303–312 Theile M, Grabowski G (1990) Mutagenic activity of BKV and JCV in human and other mammalian cells. Arch Virol 113(3–4):221–233 Tian Y, Li D, Dahl J, You J, Benjamin T (2004) Identification of TAZ as a binding partner of the polyomavirus T antigens. J Virol 78(22):12657–12664 Tiemann F, Deppert W (1994a) Immortalization of BALB/c mouse embryo fibroblasts alters SV40 large T-antigen interactions with the tumor suppressor p53 and results in a reduced SV40 transformation-efficiency. Oncogene 9(7):1907–1915 Tiemann F, Deppert W (1994b) Stabilization of the tumor suppressor p53 during cellular transformation by simian virus 40: influence of viral and cellular factors and biological consequences. J Virol 68(5):2869–2878 Todaro GJ, Green H, Swift MR (1966) Susceptibility of human diploid fibroblast strains to transformation by SV40 virus. Science 153(741):1252–1254 Trabanelli C, Corallini A, Gruppioni R, Sensi A, Bonfatti A, Campioni D, Merlin M, Calza N, Possati L, Barbanti-Brodano G (1998) Chromosomal aberrations induced by BK virus T antigen in human fibroblasts. Virology 243(2):492–496 Trowbridge PW, Frisque RJ (1995) Identification of three new JC virus proteins generated by alternative splicing of the early viral mRNA. J Neurovirol 1(2):195–206 Tsai B, Gilbert JM, Stehle T, Lencer W, Benjamin TL, Rapoport TA (2003) Gangliosides are receptors for murine polyoma virus and SV40. EMBO J 22(17):4346–4355 Turk B, Porras A, Mumby MC, Rundell K (1993) Simian virus 40 small-t antigen binds two zinc ions. J Virol 67(6):3671–3673 Urich M, Senften M, Shaw PE, Ballmer-Hofer K (1997) A role for the small GTPase Rac in polyomavirus middle-T antigen-mediated activation of the serum response element and in cell transformation. Oncogene 14(10):1235–1241

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Chapter 15

Polyomavirus SV40: Model Infectious Agent of Cancer Janet S. Butel

Introduction Polyomavirus SV40 is a small DNA-containing tumor virus that was identified 50 years ago. It gained immediate scientific attention because of its unexpected presence in poliovaccines of the time. SV40 has been a valuable tool for discovery of fundamental processes in cell biology as its limited genetic content makes it dependent on cellular machinery for virus replication. These discoveries have provided insights into mechanisms that are at the root of cell transformation and neoplasia. SV40 earned its status as a model infectious agent of cancer because of its ease of culture and quantitation in vitro, its capacity to transform cells in culture, and the susceptibility of small animal models to infection and tumor induction. It is also the most tractable polyomavirus and often serves as the representative in studies of fundamental properties of the family. This review will focus on selected features of the SV40 system that have contributed to our understanding of cancer, including elements of viral biology that make it a tumor virus, factors that affect its oncogenic potential, mechanistic insights gained from animal models, and its status as a potential human pathogen.

The SV40 Viral Genome and Virus-Encoded Proteins SV40 is the type species member of the family Polyomaviridae. Polyomaviruses are small, nonenveloped, icosahedral DNA viruses (Imperiale and Major 2007). Their genomes consist of a single copy of double-stranded, circular, supercoiled DNA

J.S. Butel (*) Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030, USA Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, TX 77030, USA e-mail: [email protected] E.S. Robertson (ed.), Cancer Associated Viruses, Current Cancer Research, DOI 10.1007/978-1-4614-0016-5_15, © Springer Science+Business Media, LLC 2012

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Fig. 15.1 Genome organization of polyomavirus simian virus 40, strain 776. The circle represents the circular SV40 DNA genome. Nucleotide numbers begin and end at the origin (Ori) of viral DNA replication (0/5,243). Unique Bgl-1 and Sfi-1 sites flank the Ori. A unique EcoRI site is shown at map unit 0/1. Boxed arrows indicate the open reading frames that encode the viral proteins. Arrowheads point in the direction of transcription; the beginning and end of each open reading frame is indicated by nucleotide numbers. The regulatory region is shown at the top; Ori, origin of DNA replication; 21-bp repeats, GC-rich SP1 binding sites and location of the SV40 packaging signal (SES); 72-bp, tandemly repeated 72-bp sequences within the enhancer. Numbers above the diagram identify nucleotide positions that border specific regulatory region segments. From Lednicky and Butel (2010)

about 5 kb in length (Fig. 15.1). Although there appears to be only one serotype of SV40, genetic strains exist that can be distinguished by nucleotide differences in the early region. Isolates also display differences in the regulatory region of the viral genome (Stewart et al. 1998; Lednicky and Butel 2001; Forsman et al. 2004).

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Fig. 15.2 Regulatory region DNA sequence profiles of SV40 viral ioslates. Shown are examples of simple and complex regulatory regions. Landmarks are as identified in the legend to Fig. 15.1. The rearranged enhancer region of SVPML-1 is represented with boxes labeled 38, 23, 20 and a shaded box referring to nucleotides 252–263. Nucleotide numbers are based on that of SV40-776. Origins of strains: SVCPC, human tumor; SV40-776, adenovirus vaccine; Baylor, oral poliovaccine; VA45-54, monkey kidney cells; SVPML-1, human brain (Forsman et al. 2004). Modified from Stewart et al. (1998)

Viral Regulatory Region The nontranslated regulatory region contains a single origin (Ori) of DNA replication and elements controlling transcription and replication. The regulatory region can be divided into the Ori, the GC-rich 21-bp repeat region containing Sp1 binding sites (part of the early promoter), the enhancer containing the 72-bp element, and a region containing the late promoter/initiator (Fig. 15.2) (Lednicky and Butel 2001; Yaniv 2009). Different strains of SV40 possess variations in the structure of the regulatory region. Among most laboratory strains there are duplications and rearrangements involving the 72-bp element (complex, 2E), whereas viruses detected in natural infections in monkeys or humans often contain no duplications in the enhancer (simple, archetypal, 1E) (Ilyinskii et al. 1992; Lednicky et al. 1998; Newman et al. 1998; Stewart et al. 1998). A 1E SV40 sequence also was found within a natural SV40-adenovirus 7 hybrid virus (Lanford et al. 1986).

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Regulatory region rearrangements can arise within a given host; in rhesus monkeys immunosuppressed due to simian immunodeficiency virus infection, SV40 versions with simple or complex regulatory regions but a common T-antigen (T-ag) variable domain were detected (Lednicky et al. 1998). Although viral regulatory region rearrangements are commonly observed in SV40-infected immunocompromised monkeys, those changes do not appear to be required for induction of progressive multifocal leukoencephalopathy disease in those animals (Dang et al. 2005). Numerous examples of complex rearrangements in the viral regulatory region from various sources have been described (Lednicky et al. 1998; Butel et al. 1999; Lednicky and Butel 2001; Sroller et al. 2008). As the regulatory region is not invariant, it cannot be used for strain identification purposes. In addition to these large structural changes, several single-nucleotide polymorphisms have been detected in the regulatory region that are useful in distinguishing different isolates (Butel et al. 1999; Lednicky and Butel 2001). Viruses with complex regulatory regions tend to replicate better in tissue culture than those with simple regulatory regions (Lednicky et al. 1995b). In low-passage, uncloned virus stocks of two laboratory strains (Baylor, VA45-54), both simple and complex versions of the regulatory region could be detected whereas higher passage stocks of both strains contained only a complex version (Lednicky and Butel 1997). This indicates that 2E viruses were selected during passage in tissue culture cells, presumably due to superior growth, and explains why other laboratory strains also have complex regulatory region structures. It has been suggested from studies with mouse polyomavirus that 2E viruses may induce more acute disease whereas 1E viruses produce low-grade, benign infections (Shadan and Villarreal 1993). It can be envisioned that the host immune response might more readily eliminate the better-replicating 2E virus, while allowing the survival of the 1E strains found in most natural infections.

Viral Late Proteins SV40 encodes three late structural viral proteins (VP1–3) (Fig. 15.1). The major capsid protein, VP1, contains 362 amino acids and forms the pentameric capsomeres that make up the surface of the virus particle. Viruses initiate infection by VP1 binding to specific receptors (GM1 gangliosides and major histocompatibility class I molecules) on the surface of host cells (Breau et al. 1992; Tsai et al. 2003); many cell types express these receptors, allowing for a broad cellular tropism for the virus. The VP1 gene of SV40 is very highly conserved and cannot be used for genotyping. Most SV40 strains share an identical VP1 amino acid sequence, although SV40-776 contains a nucleotide polymorphism that changes VP1 amino acid 83 from aspartic acid (Asp) to glutamic acid (Glu), a conservative change. VP1 residue 83 does not interact with the ganglioside receptor of SV40 (Neu et al. 2008) and has no effect on induction or detection of neutralizing antibody. Perhaps antigenic variation is limited

SV40 and Cancer

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Fig. 15.3 Functional domains of SV40 large T-ag. Specific regions assigned to functional activities or as binding sites for target proteins are indicated below the horizontal bar representing the T-ag molecule. Numbers given are the amino acid residues using the numbering system for SV40-776. Regions are indicated as follows. Small t-ag and 17KT common: region of large T-ag encoded in the first exon; the amino acid sequence in this region is common to large T-ag, small t-ag, and 17KT. DnaJ domain: region required for binding the chaperone heat shock protein hsc70; the HPDK motif is required for binding hsc70. pRb/p107/p130 binding: region required for binding of the Rb tumor suppressor protein, and the Rb-related proteins p107 and p130; the LXCXE motif is required for binding these proteins. NLS: contains the nuclear localization signal (PKKKRKV). Ori DNA binding: minimal region required for binding to SV40 Ori DNA to initiate viral DNA replication. Helicase: region required for full helicase activity. Pola binding: region required for binding to DNA polymerase a-primase; necessary for viral DNA replication. Zn finger: region which binds zinc ions. p53 binding: regions required for binding the p53 tumor suppressor protein. ATP binding/ATPase: region containing the ATP binding site and ATPase catalytic activity. Variable domain: region containing amino acid differences among viral strains and used for genotyping. Host range: region defined as containing the host range and Ad helper functions. Cul7 binding, Bub1 binding, Nbs1 binding, and Fbw7 binding: regions required for binding of Cul7, Bub1, Nbs1, and Fbw7 proteins, respectively. The circles containing a P above the bar indicate sites of phosphorylation found on large T-ag expressed in mammalian cells. S indicates a serine and T indicates a threonine residue. Modified from McNees and Butel (2008)

due to restrictions imposed by capsid symmetry, allowing negligible deviation in the sequence of VP1 and no change in serotype. There are also two late nonstructural proteins that are not present in mature virions. The agnoprotein has a maturation function that facilitates virus assembly; VP4 induces cell lysis late in infection.

The Large T-Antigen Early Protein SV40 encodes two major early nonstructural proteins, which share 82 N-terminal amino acids as a result of alternative splicing of viral transcripts. The large T-ag of SV40 contains 708 amino acids and is a highly multifunctional protein that comprises a series of functional domains (Fig. 15.3). It is an essential replication protein for the virus. T-ag activities and functions expressed during the viral life cycle include initiation of viral DNA replication, ATPase and helicase activities,

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autoregulation of early transcription, and induction of late transcription. T-ag undergoes post-translational modifications, including phosphorylation, N-terminal acetylation, O-glycosylation, poly-ADP-ribosylation, palmitylation, and adenylation. The sites of phosphorylation are clustered near the ends of the molecule, with the majority of residues being serines (Fig. 15.3). Unlike many oncoproteins, T-ag is not phosphorylated at tyrosine residues. Dysregulation of Cell Cycle T-ag functions to prepare a cellular environment conducive to viral replication. This is necessary as the polyomavirus genome is too small to encode homologs for components of the cellular replication machinery. Rather, the viral early proteins target cellular proteins to exert control over essential cellular processes, with the end result of forcing quiescent cells to enter into S phase and thus providing factors needed for cellular DNA replication. Viral DNA replication can then proceed. Binding of T-ag to cellular tumor suppressor proteins is key to expression of viral effects on host cells, both for producing a permissive environment for viral replication and for inducing cell transformation (Butel and Lednicky 1999; Butel 2000; Arrington and Butel 2001; SáenzRobles et al. 2001; Sullivan and Pipas 2002; Ahuja et al. 2005; Levine 2009; Pipas 2009; Gjoerup and Chang 2010). Particular T-ag–cell protein interactions are described in a following section “Cellular Proteins Targeted by SV40 T-Antigen”. Maintenance of Transformation The essential role of T-ag in maintenance of cellular transformation in vitro was established using temperature-sensitive (ts) mutants of the A gene protein (T-ag) (Brugge and Butel 1975; Martin and Chou 1975; Tegtmeyer 1975; Noonan et al. 1976). Mouse, hamster, and human cells were transformed by early region mutants (tsA) of SV40. When cultured at the permissive temperature, the cells exhibited the phenotype of transformed cells, including elevated saturation density, colony formation on plastic and in soft agar, and increased uptake of hexose. However, when grown at the high, restrictive temperature, the cells reverted to the phenotype of normal, untransformed cells. When the mutant-transformed cells were shifted down from the high temperature to the permissive temperature, they formed transformed foci, indicating that the cells were alive and that the phenotype was reversible. Wild-type (WT) virus-transformed cells exhibited transformed characteristics under both conditions. This showed that the continual expression of the viral T-ag protein was required to maintain the transformed phenotype (Brugge and Butel 1975). Cytoplasmic T-Antigen An odd adenovirus-SV40 hybrid carrying a defective SV40 genome was discovered in 1964 in a strain of adenovirus 7 that had been adapted to grow in monkey kidney cells;

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Fig. 15.4 Localization of SV40 T-ag in monkey kidney cells as revealed by indirect immunofluorescence. (a) Typical nuclear localization of T-ag in an SV40 hamster tumor cell line. (b) Green monkey kidney cells infected with hybrid virus PARA(2cT)-adenovirus 7, showing the retention of T-ag in the cytoplasm. This mutant resulted in the identification of the SV40 NLS and shows that nuclear translocation of T-ag is dependent on the nuclear localization signal. (c) Nuclear T-ag expression in an SV40-immortalized green monkey kidney cell line. (d) Same cells as in panel c showing cytoplasmic localization of T-ag 24 h after infection with the cytoplasmic mutant hybrid virus, showing that the cT-ag was dominant-acting and prevented the movement of WT T-ag into the nucleus

the adenovirus stock had been contaminated with SV40 that was removed by passage in the presence of SV40 antiserum. The hybrid virus was recognized by the induction of synthesis of SV40 T-ag that could be prevented by neutralization with adenovirus antiserum but not by SV40 antiserum (Huebner et al. 1964; Rapp et al. 1964; Rowe and Baum 1964). Three of 112 doubly plaque-purified clones from the parental hybrid virus were found to induce the synthesis of SV40 T-ag that was retained in the cytoplasm (Butel et al. 1969), in striking contrast to the typical nuclear localization of T-ag (Fig. 15.4a, b). It was later shown that an SV40 DNA insert containing the entire SV40 early region had replaced the adenovirus 7 fiber gene in the hybrid virus (Lanford et al. 1986). An SV40 mutant virus [SV40(cT)] was constructed from the cytoplasmic hybrid virus. This construct was defective for replication, but could be propagated in COS-1 cells. Sequence analysis revealed a single point mutation at nucleotide 4434 which would convert a positively charged lysine at residue 128 of T-ag to a neutral asparagine,

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Fig. 15.5 Effect of T-ag on localization of p53 in transformed mouse cells. (Panels a and b) BALB-3T3 mouse cells transformed by WT SV40. (Panels c and d) BALB-3T3 cells transformed by the SV40(cT) cytoplasmic construct. Panels a and c were stained for SV40 T-ag and panels b and d were stained for p53 by indirect immunofluorescence. (a) WT T-ag localized in the nucleus. (b) Nuclear p53 in WT transformed cells. (c) T-ag induced by the cytoplasmic mutant present in the cytoplasm. (d) p53 retained in the cytoplasm in SV40(cT) mutant-transformed cells. Note that the subcellular localization of p53 matched that of T-ag. From Lanford et al. (1985a)

a finding that resulted in the identification of the SV40 nuclear localization signal (NLS; Fig. 15.3) (Lanford and Butel 1984). A mutagenesis approach independently identified the SV40 NLS (Kalderon et al. 1984). The discovery of the SV40 NLS was of broad interest to the fields of virology and cell biology. The cytoplasmic mutant of SV40 was competent to transform established mouse 3T3 cells at an efficiency comparable to that of WT SV40, whereas its transforming ability was substantially reduced in primary mouse embryo fibroblasts under stringent growth conditions (Lanford et al. 1985b). The cT-ag transformants displayed similar growth properties to those of WT transformants, showing that it is possible for T-ag to mediate and maintain transformation without translocating to the nucleus (Fig. 15.5a, c). This property may be related to the observation that p53 was also retained in the cytoplasm of SV40(cT)-transformed cells, suggesting that the cT-ag was sequestering p53 and preventing its function (Fig. 15.5b, d). The transport-defective form of T-ag was dominant acting and blocked the translocation of WT T-ag to the nucleus in infected and transformed cells (Lanford and Butel 1980, 1984) (Fig. 15.4c, d).

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The SV40(cT) mutant was able to induce tumors in newborn hamsters, but at a much reduced frequency compared to WT virus and after extended latent periods. Cells in the tumors that arose expressed T-ag in the cytoplasm (Lanford et al. 1985b). This same cT-ag mutant was able to produce rapidly appearing brain tumors in transgenic mice (Section “Animal Models and Insights into SV40 Biology”). The functions necessary for tumor formation in inoculated newborn hamsters that were dispensable in transgenic mice are unclear.

T-Antigen Variable Domain There is a variable domain at the C-terminus of T-ag (T-ag–C) encompassing residues 622–708 (Fig. 15.3). SV40 strains can be distinguished on the basis of nucleotide differences in this region, including polynucleotide insertions and deletions as well as single nucleotide changes, which often would change predicted amino acids (Stewart et al. 1996). In contrast, the first 622 residues of T-ag are completely conserved (Fig. 15.6a). Phylogenetic analysis established that SV40 strains can be grouped into clades (genogroups) based on the sequence of the T-ag variable domain (Forsman et al. 2004) (Fig. 15.6b). Analysis based on the T-ag–C was highly congruent with whole-genome analysis. (A segment of the regulatory region (nucleotides 29–246) was excluded from the whole-genome analysis because genetic changes can occur within that region during viral growth in vivo.) Several strains were outliers and did not map to one of the clades, suggesting that other viral genogroups may occur. The existence of different SV40 genogroups raised the question of whether viral strains may differ in biological properties. This intriguing possibility was answered affirmatively in the hamster animal model (Section “Animal Models and Insights into SV40 Biology”). The viral encoded microRNAs (miRNAs) target early viral transcripts within this region (Sullivan et al. 2005) (Fig. 15.1). The SV40 miRNAs accumulate at late times during infection in cultured cells and mediate cleavage of early viral mRNA, thereby reducing expression of viral tumor antigens. At the extreme C-terminus of large T-ag, embedded within the variable domain, is the host range/adenovirus (Ad) helper function (hr/hf) domain (amino acids 682–709) (Fig. 15.3). A C-terminal fragment of T-ag can relieve the Ad replication block in monkey cells (Cole et al. 1979) by an unknown mechanism. The hr function was identified because T-ag C-terminal deletion mutants grew very poorly in the CV-1 monkey kidney cell line, but grew well in the BSC-1 and Vero monkey kidney cell lines (Pipas 1985; Tornow et al. 1985). Although viral DNA was replicated to near WT levels in all three cell types (Pipas 1985; Stacy et al. 1989), virions produced by the hr/hf mutants did not assemble properly, seemingly due to an inability to add VP1 to the 75S assembly intermediates (Spence and Pipas 1994). Interestingly, not all primate polyomavirus large T-ags contain an hr domain. In addition to SV40, those with an hr domain include JCV, BKV, and SA12, whereas LPV, KIV, WUV, and MCV apparently lack such a domain. The precise function of the hr domain and the explanation for this difference among viral species are not known.

a

SV40-776

SVCPC No amino acid changes

SV40-Baylor No amino acid changes

VA45-54 No amino acid changes

SVPML-1 No amino acid changes

b Clade C

H388(98)

OST9 (97)

I508(98)

EP14 (95)

Clade B Baylor(56)

K661(98) Rh911(62)

GM00637H (99)

CPP15 (95)

SVCPC (95)(SVMEN (84), NHL-8 (02), Men-99 (03))

60 74

777(62) PML-1

(72)

62

777*(79) N128(65)

62

69

T302(98)

MC028846B(55) (NHL-28 (02), NHL-170 (02), NHL-416(02))

62

84

PA-57(61)

OST3 (97)

100

OST2

776(60)

(97)

6593(98)

GN13 (95) VA45-54(60) 776*(60) H328(98)

Clade A

Fig. 15.6 SV40 large T-antigen variable domain. (a) Examples of diversity among variable domains at the C-terminus of T-ag (T-ag–C). The rectangles represent the amino acid sequence of the protein. Viral strains are identified on the left. Sequence differences in the T-ag–C compared to SV40-776 are indicated above the rectangle on the right using the SV40-776 numbering system. Numbers below the rectangle are actual amino acid numbers for that particular protein. Hatch marks indicate regions of complete identity with SV40-776. The open portion indicates the sequence variability in the variable domain among strains. From Stewart et al. (1996). (b) Phylogenetic tree for SV40 based on T-ag–C sequences. Unrooted consensus tree of 1,000 bootstrap replicates of available T-ag–C sequences, generated by maximum-parsimony analysis. Conventions for labels are as follows: monkey isolates = roman type; vaccine isolates = bold-face type; human isolates = underscored italic type. Sequences in parentheses are from independent sources, but are the same as the sequence preceding the parentheses. Year of sample origin/isolation/detection is indicated in superscript in parentheses. From Forsman et al. (2004)

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The Small t-Antigen Early Protein The second major early nonstructural protein, small t-antigen (t-ag), contains 174 residues, the first 82 of which are in common with large T-ag (Fig. 15.1). Through its unique region, small t-ag complexes with cellular protein phosphatase-2A (PP2A), a serine/threonine phosphatase that regulates many biological processes in mammalian cells. PP2A contains three subunits: a scaffold A subunit, a catalytic C subunit, and a regulatory B subunit, the latter of which is displaced by t-ag. This has the effect of modifying the substrate specificity of PP2A activity or inhibiting its enzyme activity. Small t-ag is able to induce proliferation of cells. It enhances transformation by large T-ag, although its precise contribution is unclear (Khalili et al. 2008; Gjoerup and Chang 2010). Tumors induced in hamsters by a t-ag deletion mutant of SV40 often arose in lymphoid tissue and not in all tissues affected by WT virus, suggesting that small t-ag may be required to stimulate proliferation of quiescent cells in vivo (Carbone et al. 1998). Small t-ag is essential for SV40 transformation of normal human cells (Hahn et al. 2002). Complete transformation of several types of human cells (fibroblasts, mammary epithelial cells and kidney epithelial cells) required the combination of SV40 large T-ag (disruption of pRb and p53 pathways), small t-ag (alteration of PP2A functions), telomerase (hTERT), and oncogenic H-ras (stimulation of growth signals). Thus, five regulatory pathways must be altered for transformation of those normal human cells, and SV40 is able to affect three of them, indicating the genetic underpinnings that make SV40 a strongly oncogenic virus.

Cellular Proteins Targeted by SV40 T-Antigen T-ag forms complexes with several cellular proteins in order to carry out its multiple functions (Fig. 15.3). Some of the molecules targeted as part of the viral replication program can also contribute to cell transformation, making T-ag an oncoprotein and endowing SV40 with cancer-causing potential. Recent reviews have described these protein interactions in detail (Sullivan and Pipas 2002; Ahuja et al. 2005; DeCaprio 2009; Fanning and Zhao 2009; Levine 2009; Pipas 2009; Gjoerup and Chang 2010; Rathi and Pipas 2010).

DnaJ Domain and hsc70 The T-ag amino terminus contains a DnaJ (or J) domain (Srinivasan et al. 1997; Sullivan et al. 2000; Sullivan and Pipas 2002; Ahuja et al. 2005; Gjoerup and Chang 2010). DnaJ proteins are molecular chaperones that, together with members of the DnaK class of chaperone proteins, bind substrate proteins and modify them in some way, such as refolding or disrupting protein complexes. The J domain of T-ag (amino acids 1–70) binds cellular heat-shock protein hsc70 and stimulates its

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ATPase activity. It is thought that the T-ag J domain functions in the disruption of Rb-E2F complexes, using energy provided by the hsc70 ATPase activity. A functional J domain is necessary for SV40 viral replication and for many instances of cell transformation.

Rb Family of Tumor Suppressor Proteins A separate domain of T-ag (amino acids 102–115) binds to retinoblastoma (Rb)related tumor suppressor proteins (pRb, p107, p130/pRb2) through its LXCXE motif. The first recognition of an interaction between an oncogene and an antioncogene involved adenovirus E1A and pRb (Whyte et al. 1988), followed quickly by the finding that SV40 T-ag also bound pRb (DeCaprio et al. 1988). The Rb protein functions as a transcriptional repressor and is regulated by phosphorylation throughout the cell cycle. T-ag selectively complexes with the underphosphorylated form of pRb found in the G0/G1 phase of the cell cycle and not the more highly phosphorylated forms present in the later stages of the cell cycle; in so doing, T-ag abolishes G1/S checkpoint control. This causes unscheduled dissociation of pRb:E2F complexes and release of E2F to activate expression of growth-stimulatory genes (Sáenz-Robles et al. 2001; Sullivan and Pipas 2002; Ahuja et al. 2005; Javier and Butel 2008; DeCaprio 2009; Pipas 2009; Gjoerup and Chang 2010). T-ag–pRb binding is required for SV40 transformation to occur. There are two other Rb-related proteins, p107 and p130, that also bind T-ag at the same LXCXE site. Presumably, inactivation of p107 and p130 are also important in SV40 transformation. There are multiple E2F proteins and it appears that different ones are preferentially bound by the individual Rb family members. pRb binds activating molecules E2F1–3, whereas p107 and p130 bind to the repressors E2F4–5. T-ag disrupts this pathway by binding the Rb proteins and blocking their ability to regulate E2Fs (Ahuja et al. 2005; DeCaprio 2009; Gjoerup and Chang 2010). Many human tumors contain mutations in the Rb pathway.

p53 Tumor Suppressor Protein A region toward the C-terminus of T-ag contains the bipartite binding site (amino acids 350–450 and 533–626) for the p53 tumor suppressor protein. The p53 protein was discovered as a cellular protein in complex with SV40 T-ag (Lane and Crawford 1979; Linzer and Levine 1979; Levine 2009). However, a decade of research was required to establish p53 as a tumor suppressor and not an oncogene, culminating in the generation of p53 knock-out mice that were highly prone to spontaneous tumors (Donehower et al. 1992). The early studies were confounded because some p53 clones contained gain-of-function mutations and behaved differently, and it was not clear which was the WT version. The p53 protein acts to detect stress signals that

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would affect the fidelity of DNA replication, such as DNA damage, hypoxia, heat shock, spindle damage, virus infection, and oncogene activation (Levine 2009). It then either pauses the cell in late G1 (cell cycle arrest) to permit DNA repair, directs the cell to terminate cell division (senescence), or initiates programmed cell death via the apoptotic pathway. SV40 T-ag binding sequesters p53, abolishing its function and allowing infected cells to survive to complete virus replication. That may also allow cells with genetic damage to survive and proliferate, resulting in T-ag-expressing cells that accumulate genomic mutations (Sáenz-Robles et al. 2001; Ahuja et al. 2005; Javier and Butel 2008; Levine 2009; Pipas 2009; Gjoerup and Chang 2010). T-ag binding stabilizes p53 and extends its half-life so that there are large amounts of p53 in T-ag-expressing cells. In contrast, WT p53 is present in normal cells at very low levels with a short half-life of less than 30 min. It has been envisioned that T-ag blocks p53 sequence-specific DNA binding and transcriptional activation. However, it is still not clear if T-ag binding inactivates all p53 activities, equivalent to a p53-null environment, or if some p53 activity might be retained or if a gain-of-function might be generated. The T-ag–p53 interaction is clearly important for virus replication and cell transformation, with T-ag mutants unable to bind p53 being defective for cell transformation (Kierstead and Tevethia 1993). However, studies from transgenic mouse systems have revealed that in certain specific cell types p53 inactivation is not a requirement for SV40 transformation (Section “Animal Models and Insights into SV40 Biology”). The power of the p53 tumor suppressor system is illustrated by the fact that at least half of human cancers contain alterations in the p53 protein or the p53 pathway.

Other Targets Several other cellular proteins have been identified as potential targets for SV40 T-ag. However, additional characterizations are necessary to understand the role of these interactions in viral replication and/or transformation. There are adjacent binding sites in the amino terminus of T-ag for Cul7 (residues 69–83) and Bub1 (residues 89–97). Cul7 is part of an E3 ubiquitin ligase that is involved in proteasomal degradation of proteins (Ali et al. 2004). Deletion mutants suggest that Cul7 binding may be involved in SV40 transformation. Bub1 is a mitotic spindle checkpoint protein. The T-ag–Bub1 interaction compromises the spindle checkpoint and may contribute to chromosomal instability in SV40-transformed cells (Cotsiki et al. 2004). If the T-ag–Bub1 interaction is abolished, SV40-induced focus formation in Rat-1 cells appears inhibited. However, the role Bub1 may play in SV40 transformation is unclear. T-ag reportedly binds Nbs1, the Nijmegen breakage syndrome protein 1, a component of the MRN (Mrell/Rad50/Nbs1) complex, through the origin-binding domain (residues 147–259) (Digweed et al. 2002; Wu et al. 2004). The MRN complex functions in DNA repair. It was reported that the number of nuclear repair foci following irradiation of cells was reduced in the presence of T-ag. Fbw7 is

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another cellular protein reported to interact with T-ag (Welcker and Clurman 2005). Fbw7 is part of the cellular ubiquitination machinery that binds to a motif (phosphodegron) that is present at the C-terminus of T-ag (residues 682–708). Whether these interactions with Nbs1 or Fbw7 contribute to genomic instability or transformation by SV40 is unknown. T-ag associates with p300/CBP, also called E1A binding protein p300/CREB binding protein, two closely related transcriptional coactivators that interact with multiple transcription factors and increase target gene expression. Thus, they are involved in many cellular processes. The association of p300/CBP with T-ag is indirect, with p53 serving as a bridge (Poulin et al. 2004). p300/CBP possess intrinsic histone acetyltransferase activity and acetylate T-ag on K697 in a p53-dependent process. The biological effects of T-ag interactions with p300/CBP on virus replication or cell transformation have not been established.

Animal Models and Insights into SV40 Biology Small animal models are invaluable for studies of the biology of infectious agents. Steps in the viral life cycle and consequences of infection, including development of neoplasia, can be evaluated in the context of differentiated cell types, complex tissues, and host responses. Animal models for SV40 include Syrian golden hamsters and mice.

Hamster Model of SV40 Infection and Oncogenesis Syrian golden hamsters (Mesocricetus auratus) have long been recognized as the tumor model for SV40 (Butel et al. 1972; Butel and Lednicky 1999; Butel 2000). The oncogenic potential of SV40 was demonstrated soon after its discovery by inoculation of newborn hamsters (Eddy et al. 1962; Girardi et al. 1962). Eddy had previously shown that rhesus monkey kidney cells contained an oncogenic substance able to induce tumors in newborn hamsters (Eddy et al. 1961) and subsequently identified the factor as SV40. Several host and viral factors influence the development of SV40-mediated neoplasia, including age at the time of infection, route of inoculation, dose of inoculum, and viral genetic variation. The frequency of tumor development is high in animals infected as newborns, but is reduced in older animals. This suggests that the host immune response is capable of controlling tumor development, but can be overwhelmed. Environmental carcinogens can be evaluated in this system; asbestos and SV40 infection cooperate as co-carcinogens in causation of malignant mesotheliomas in hamsters (Kroczynska et al. 2006). The types of tumors that develop in SV40-infected hamsters vary, depending on the route of inoculation (which determines tissue exposures). Subcutaneous inoculation usually results in sarcomas at the site of injection; intrapleural and intraperitoneal

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inoculation leads to a high proportion of mesotheliomas (Cicala et al. 1993; Vilchez et al. 2004; Sroller et al. 2008); intracerebral inoculation produces ependymomas (Kirschstein and Gerber 1962); and intravenous inoculation induces a spectrum of tumors, especially lymphomas, mesotheliomas, and osteosarcomas (Diamandopoulos 1973; Carbone et al. 1989; McNees et al. 2009). These studies showed that SV40 has a broad tissue tropism with the capacity to transform a variety of target cell types. In addition, tumors induced in hamsters turned out to predict the types of human tumors later found to be associated with SV40 (Section “Human Infections and Cancer Associations”).

Influence of Viral Regulatory Region on Tumor Induction and Vertical Transmission Recent studies have established that the viral regulatory region is a genetic determinant of SV40 oncogenic potential whereas the T-ag variable domain exerts negligible influence (Sroller et al. 2008). These two genomic regions were described above as being sites of variation among SV40 isolates. A panel of recombinants was constructed that mixed different regulatory regions and T-ag–C variable domains. Interestingly, the parental and recombinant viruses displayed similar transforming frequencies in vitro in mouse cells (Sroller et al. 2008). However, when 21-day-old weanling hamsters were inoculated intraperitoneally, the oncogenic potential in vivo of the viruses ranged from 83% to 0% (Fig. 15.7). Significantly more animals developed tumors after exposure to viruses with simple (1E) regulatory regions than to those with complex (2E) regulatory regions. In contrast, the T-ag–C variable domain exhibited no particular patterns related to oncogenicity. As the SV40 variants showed no differences in transforming activities in vitro, the in vivo results must have reflected the consequences of variable virus–host interactions. One possible explanation is that the viruses with complex regulatory regions replicate more abundantly and are recognized and cleared more efficiently by the host’s immune response than are the slower-replicating viruses (those with simple regulatory regions). This concept is supported by observations with mouse polyoma virus and lymphocytic choriomeningitis virus, which showed that slower-growing viruses elicited milder host responses and were more apt to persist long-term (Rochford and Villarreal 1991; Bocharov et al. 2004). More residual persistently infected cells by the SV40 1E variants could account for their higher level of subsequent tumor development. An experiment designed to examine vertical transmission of SV40 in hamsters, rather than tumor formation, supported the interpretation that relative levels of viral replication affect biological outcomes (Patel et al. 2009). Pregnant hamsters inoculated intraperitoneally with SV40 strains containing complex regulatory regions transmitted virus to their offspring more frequently than those infected with simple enhancer viruses (p < 0.001). Virus was detected in maternal kidney and spleen tissues in some animals for at least 24 days after infection. These data suggested that SV40 strains with complex regulatory regions replicated to higher levels in the pregnant hamsters than 1E viruses, increasing the likelihood of transmission to their litters.

392

J.S. Butel 100

Tumor development (%)

90 Regulatory region 1E

80 70

2E

60 50 40 30 20 10

C

Ba

y( 1 PC E) -P SV ML C CPC PC C -Ba PC y 77 776 6( C 1E) PC 77 -VA 6VA Bay 45 77 -54 677 VA 6 77 (2E 6- ) C Ba PC y 77 (2E 6- ) P SV ML P co ML nt ro ls

0

SV40 parental and recombinant viruses

Fig. 15.7 Influence of the SV40 regulatory region on tumor induction in hamsters. Weanling hamsters were inoculated intraperitoneally with parental and recombinant viruses and observed for 1 year for tumor development. Each bar shows the tumor incidence in the group inoculated with the virus identified below the bar. Black bars indicate viruses with simple regulatory regions (1E); hatched bars indicate viruses with complex regulatory regions (2E). Viruses with simple regulatory regions were more oncogenic than those with complex regulatory regions. The difference between the two groups (1E vs. 2E) was statistically significant (66/152, 43% vs. 18/155, 12%; p = 0.0001). From Sroller et al. (2008)

Other lines of evidence also indicate that SV40 can infect and replicate in hamsters. First, many virus-inoculated but tumor-free animals develop antibodies to SV40 T-ag, with more responders among animals inoculated with 2E viruses as compared to 1E viruses (Sroller et al. 2008; McNees et al. 2009). SV40 T-ag is not a component of the virus particle, but rather is synthesized in infected cells. Second, infectious SV40 was recovered from many (39%) tumor cell lines established from primary tumors that arose months after virus infection of the hamsters (Sroller et al. 2008). This is in keeping with earlier studies that had reported recovery of infectious virus from SV40-induced hamster tumor cells (Sabin and Koch 1963; Black and Rowe 1964; Gerber 1964; Boyd and Butel 1972; Lednicky and Butel 1999). Third, SV40 regulatory region rearrangements were detected in several SV40 hamster tumors induced by 1E viruses (Sroller et al. 2008). The mechanism of rearrangement is unproven, but it has been suggested that it reflects a recombination event during viral DNA replication (Yogo and Sugimoto 2001).

Model of SV40 Pathogenesis of Infection and Disease These hamster data showed that SV40 strains differ in biological properties and suggested that the risk of disease may depend on the viral isolate causing an infection.

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No infection

Infection — Mucosal surface (respiratory, gastrointestinal) (?)

Initial replication (lymphoid tissue, GI tract) (?) SV40 genetic variation?

Dissemination (blood)

Eradication

Viral load?

SV40 microRNA? Secondary organs (kidney, liver, lung, spleen, tonsil, brain)

Persistence Viral load?

Excretion (urine, feces)

Disease (cancer)

Fig. 15.8 Model of SV40 pathogenesis of infection and disease. This model proposes that attributes of persistent infections established by SV40 during early stages of infection are key factors in determining the relative risk of subsequent development of disease. As viral infection occurs and spreads to secondary organs, the host response attempts to eradicate the infection. Factors influencing this process are proposed to be SV40 genetic variation, SV40 microRNA, and viral load. “Viral load” refers to the number of persistently infected cells in the host; the more that survive elimination are predicted to produce an increased chance of subsequent tumor development. Proposed steps in the pathogenesis of infection are based on observations from hamsters and humans. Question marks indicate details of the model that have not been experimentally proven

Those insights inspired the development of a model of SV40 pathogenesis of infection and disease (Fig. 15.8). The model envisions that the risk of virus-induced disease is dependent on the particular characteristics of a persistent infection, with those characteristics determined by the virus–host interaction during the acute phase of infection in each given host. A significant feature is predicted to be the viral load, which would likely reflect the absolute number of persistently infected cells in the host. This hypothesis can be tested experimentally in Syrian golden hamsters as they are both an infection and a tumor model for SV40. Studies could be designed to identify cell types that support virus replication, as well as sites of long-term persistence. Genetic factors that do not affect virus replication in vitro, such as the viral miRNA, could be examined for effects in vivo. General principles that emerge would in all likelihood be applicable to other polyomavirus systems. Cancer viruses establish persistent infections in susceptible hosts and a better understanding of the origin and evolution of persistently infected cells prior to the initiation of transformation would help clarify the risk of development of virus-associated cancer.

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Mouse Model of Cellular Immune Responses to SV40 T-Antigen Tumor-bearing hamsters usually develop antibodies to SV40 T-ag, as do many virus-infected animals without tumors (Sroller et al. 2008). Virus-inoculated rabbits and monkeys also produced T-ag antibody responses (Rapp et al. 1967; Vonka et al. 1967; Tevethia 1970). Such observations showed that SV40 T-ag is a strongly immunogenic protein and that development of antibodies to T-ag is a sign of SV40 infection and not necessarily a sign of neoplasia (Tevethia 1990; Tevethia and Schell 2001). Immunological studies of cell-mediated immunity in hamsters are difficult because of the limited knowledge of hamster immunology and the lack of genetically inbred lines. Although mice fail to develop tumors after SV40 inoculation and do not support SV40 replication, they have been valuable models for defining basic mechanisms of cytotoxic T lymphocyte (CTL) responses to SV40 proteins.

CTL Epitopes on T-Antigen Tevethia and colleagues have identified four distinct CTL epitopes for T-ag in mice. These epitopes were mapped using deletion mutants and synthetic peptides to the following T-ag residues: epitope I, residues 206–215; epitope II/III, 223–231; epitope IV, 404–411; and epitope V, 489–497. Three of the epitopes are H-2Dd restricted, whereas one (IV) is H-2Kb restricted. Epitope V is immunorecessive (Tevethia 1990; Deckhut et al. 1992; Lippolis et al. 1995; Mylin et al. 1995; Schell and Tevethia 2001; Tevethia and Schell 2001) (Fig. 15.9). Four additional epitopes have been identified: residues 362–384 (Förster et al. 1995), 499–507 (Newmaster et al. 1998), 529–543 (Schell TD 2010, personal communication), and 560–568 (Zheng et al. 2002). These are H-2Ak, H-2Kd, H-2Ab, and H-2Kk restricted, respectively. These multiple CTL determinants provide effective immunosurveillance against the outgrowth of SV40 tumor cells in mice. In fact, CD8+ CTLs against a single T-ag epitope (404–411) were able to control progression or cause regression of T-aginduced pancreatic tumors and brain tumors in vivo in two different transgenic systems (Otahal et al. 2006; Tatum et al. 2008; Yorty et al. 2008). However, tumor cells that escape CTL immunosurveillance are known to emerge. In vitro selections of SV40transformed mouse cells using individual epitope-specific CTL clones resulted in CTL-resistant cell populations. Genetic analyses revealed that the resistant cells expressed T-ag with point mutations or deletions that affected the respective CTLrecognition epitope. A multiplicity of escape mutations were identified, with residue substitutions more common than sequence deletions. Thus, single residue changes within CTL recognition sites are sufficient to abrogate transformed cell destruction by particular CTL clones (Lill et al. 1992; Mylin et al. 2007). These studies revealed a mechanism for tumor cell escape from immunosurveillance while allowing for maintenance of cell transformation. They also suggested potential complications of certain immunotherapy strategies.

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H-2b-restricted epitopes 1

82 83

I

Residues: Sequence:

206-215

II/ III

223-231

708

IV

V

404-411

489-497

529-543

SAINNYAQKL CKGVNKEYL VVYDFLKC QGINNLDNL NEYSVPKTLQARFVK

MHC restriction:

H-2Db

H-2Db

H-2Kb

H-2Db

H-2Ab

HLA-restricted epitopes 1

82 83

708

Residues:

138-146

281-289

Sequence:

FPSELLSFL

KCDDVLLLL

MHC restriction:

HLA-B7

HLA-A2.1

285-293 VLLLLGMYL HLA-A2.1

577-585 LMLIWYRPV HLA-A2.1

Fig. 15.9 Relative location of murine H-2b-(top) and human HLA-(bottom) restricted epitopes in SV40 T-ag recognized by CD8+ T cells. Roman numerals in top diagram indicate the designations for the corresponding MHC class I-restricted epitopes. Arabic numbers indicate the amino acid positions within the T-ag sequence. Only the epitopes found in C57BL/6 mice are shown in the top panel. See text for details. Courtesy of T.D. Schell and S.S. Tevethia

As the T-antigens of polyomaviruses SV40, JCV and BKV are related, the possibility of CTL epitope cross-reactivity was examined. CTL clones specific for epitope II/III reacted with both JCV- and BKV-transformed cells, identifying a particular epitope that is found on the T-antigens of all three polyomaviruses. In contrast, epitope V was specific for SV40. Particular CTL clones against epitope I and epitope IV of SV40 recognized JCV T-ag as well, but not BKV T-ag (Tevethia et al. 1998). Thus, CTL reagents exist that can discriminate among the T-antigens of SV40, JCV and BKV, whereas others show that cross-reactivity exists for certain epitopes in mice. Four epitopes have been identified on T-ag that are recognized in humans: residues 138–146 (Coleman et al. 2008), residues 281–289 (Schell et al. 2001), residues 285– 293 (Bright et al. 2002), and residues 577–585 (Velders et al. 2001) (Fig. 15.9). The first is HLA-B7 restricted and the others are HLA-A2.1 restricted. The T cell responses against epitopes 138–146 and 285–293 were found in patients with malignant pleural mesothelioma and were capable of recognizing and lysing T-ag-expressing target cells in vitro (Bright et al. 2002; Coleman et al. 2008). It is apparent that the human T cell responses discovered to date are against epitopes different from those identified in mice. More work is needed to identify epitopes on T-ag that can be recognized by other HLA types in humans. Both epitopes 138–146 and 281–289 were shown to be specific for SV40 and not shared with JCV or BKV T-ag (Schell et al. 2001; Coleman et al. 2008). Whether other T-ag epitopes recognized in humans might cross-react with

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those of JCV or BKV and what effect such immunity might have on SV40 infections in humans are unknown. It is possible, for example, that pre-existing cross-reactive immunity might dampen specific antibody responses to SV40.

SV40 MicroRNA and CTL Evasion To experimentally test if specific CD8+ T cell clones could recognize and attack SV40-infected cells, TC-7 monkey kidney cells were transfected with murine H2-Kb and H2-Db class I antigens, allowing murine CTL clones against SV40 T-ag epitopes to be utilized. Following infection of the modified TC-7 cells with SV40, exposure to T-ag-specific CTL clones significantly reduced viral yields (Bates et al. 1988). This showed that CTLs that target SV40 T-ag epitopes can mediate the elimination of virus-infected cells. Subsequently, this system was applied to address the possibility that SV40 miRNAs function to facilitate CTL evasion by infected cells. It was found that cells infected with a virus unable to express viral miRNAs were more susceptible to CTL-mediated lysis and produced more interferon gamma than companion cultures infected with WT SV40 (Sullivan et al. 2005). Perhaps this mechanism to delay or reduce elimination of infected cells by the host facilitates virus production or establishment of persistently infected cells; it would seem it might also help the survival of transformed cells if they were to arise in an infected host and express any viral miRNA.

Transgenic Mouse Models of SV40 T-Antigen Effects The development of transgenic mouse technology added a new dimension to studies of viral oncogenes (Hanahan 1989; Adams and Cory 1991; Van Dyke 1994). It became possible to analyze tumor virus proteins in host animals to complement studies with cultured cells in vitro. Tissue-specific promoters could be used to examine viral gene effects on different cell types, mutated genes could be tested to dissect viral transforming functions, and different host genetic backgrounds could be utilized to expand insights into host responses in vivo. SV40 T-ag was the first viral oncoprotein expressed in transgenic mice; under the control of its natural regulatory region the virus induced brain tumors in the choroid plexus of the animals (Brinster et al. 1984). Since that beginning, SV40 T-ag expression has been directed to many different tissues and cell types by the use of tissue-specific promoters. Transgenic models have contributed to fundamental principles related to SV40 T-ag transformation and tumor development (Lednicky and Butel 1999; Butel 2000; Ahuja et al. 2005; Pipas 2009; Sáenz Robles and Pipas 2009; Gjoerup and Chang 2010). These general principles include the following: T-ag is a potent oncoprotein able to induce neoplasms in a variety of tissues, T-ag can stimulate resting cells to enter the cell cycle and proliferate, T-ag inhibition of the Rb and p53 pathways is central to the SV40 transformation process, and additional genetic changes beyond viral

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oncogene expression are necessary for tumor formation. Transgenic studies also have shown that there are exceptions to those general rules and that T-ag expression may exhibit tissue- and cell-type-specific effects (Ahuja et al. 2005; Pipas 2009; Sáenz Robles and Pipas 2009; Gjoerup and Chang 2010). Thus, another basic principle is that cell context is important for expression of T-ag-mediated transforming events. Several examples are described to illustrate the significance of transgenic systems in broadening an understanding of viral carcinogenesis.

Rb and p53 Pathways in SV40 Transformation The effects of the Rb and p53 pathways on T-ag tumor induction was first analyzed in the choroid plexus model. The choroid plexus epithelium lines the ventricles of the brain and maintains a blood–cerebrospinal fluid barrier. A truncated protein containing only the first 121 amino acids of T-ag and unable to bind p53 (T121) induced choroid plexus tumors, but at a lower rate than WT T-ag, and the tumors that appeared grew more slowly and displayed many apoptotic cells compared to tumors expressing full-length T-ag. If the Rb-binding sequence were mutated, the T121 T-ag fragment failed to induce tumors, showing the absolute requirement for T-ag inhibition of the Rb pathway in this system (Chen and Van Dyke 1991; Chen et al. 1992; Symonds et al. 1993; Sáenz-Robles et al. 1994). The observed apoptosis was p53-dependent because when the amino terminal truncated mutant was expressed on a p53-deficient background, the tumors that arose showed negligible apoptosis and grew rapidly (Symonds et al. 1994). The interpretation was that T-ag binds and inactivates pRb proteins, inducing cell proliferation and unscheduled DNA synthesis. This triggers a p53 response poised to eliminate the damaged cells via apoptosis, which T-ag blocks by binding and inactivating p53. Thus, WT T-aginduced tumors were fast-growing and aggressive whereas those induced by T-ag unable to inactivate p53 were slow-growing and apoptotic. Transgenic mice were produced with the cytoplasmic T-ag mutant of SV40 to explore the effects of transport-defective T-ag in vivo. A transgenic line was established using the natural viral promoter with the result that animals died regularly of choroid plexus tumors at an early age (81 days) (Pinkert et al. 1987). T-ag expressed in the brain tumor cells was localized in the cytoplasm. We can speculate that cT-ag was able to readily induce brain tumors because it could interact with p53 (and presumably with Rb proteins) in the cytoplasm, thereby avoiding an apoptotic response. The rapid tumorigenicity by the cT-ag mutant was somewhat unexpected as the virus was crippled for transformation of primary cells in culture (Section “The SV40 Viral Genome and Virus-Encoded Proteins”). This study illustrated that transformation assays in vitro may fail to accurately reflect the oncogenic process in vivo, as different cell types are involved and the surrounding environments are different. Transgenic systems have revealed that there are cell types in which inactivation of p53 is not a requirement for SV40 transformation. These include pancreatic acinar cells and intestinal enterocytes. In the pancreatic model, full-length T-ag and an

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N-terminal fragment of T-ag (T127) unable to bind p53 behaved comparably when expressed under the control of the rat elastase-1 promoter (Tevethia et al. 1997). Both WT T-ag and the truncated mutant induced pancreatic acinar carcinomas. Intestinal enterocytes represent the predominant specialized cell type in the epithelium of the small intestine. SV40 T-ag expression under the control of the rat intestinal fatty acid binding promoter results in hyperplasia that over time progresses to dysplasia (Markovics et al. 2005; Sáenz-Robles et al. 2007; Rathi et al. 2009). N-terminal truncated mutants of T-ag (T121, N136) induced hyperplasia similar to WT T-ag, but there was less frequent progression to dysplasia. In addition, there did not appear to be stabilization of p53 by full-length T-ag in the enterocytes and T-ag–p53 complexes could not be detected in the cells. In contrast, pRb inactivation was essential for SV40 transformation in this system. T-ag mutants unable to bind Rb proteins or lacking a functional J domain had no phenotypic effects on the enterocytes. Finally, recent gene expression studies confirmed that the Rb-E2F pathway is the primary T-ag target in enterocytes and that the p53 pathway is not important (Rathi et al. 2009). Using whole-mouse genome arrays to compare mRNA levels, the N136 truncation mutant was found to upregulate essentially the same genes as WT T-ag.

Loss of Dependence on T-Antigen in Transformed Cells Although T-ag is expressed in most tumors, there are indications that the continual expression of T-ag is not always required to maintain the transformed cell phenotype. Time-sensitive reversal of T-ag-induced hyperplasia in the salivary gland was demonstrated in transgenic mice using a tetracycline-responsive gene expression system (Ewald et al. 1996). When T-ag was expressed, extensive ductal hyperplasia was evident by 4 months of age. If T-ag expression were silenced at 4 months, the hyperplasia was reversed. However, if T-ag was not silenced until 7 months of age, the hyperplasia persisted in the absence of T-ag expression. This demonstrated that SV40-transformed cells may over time accumulate sufficient genetic changes to lose their dependence on T-ag expression. SV40 T-ag targeted to the liver by the regulatory elements of the human a1antitrypsin gene was expressed in nearly all hepatocytes (Sepulveda et al. 1989). The transgenic animals showed reproducible liver changes with predictable kinetics (hyperplastic, dysplastic, neoplastic) and developed liver tumors by 10 weeks of age. The tumors arose as nodules on a background of dysplasia, showing that additional events beyond T-ag expression were needed for cellular progression to neoplasia. There was considerable variation in the proportion of T-ag-positive cells in and among tumor nodules, with some nodules being negative for T-ag. This observation suggested that cellular genetic changes may accumulate and make the continued presence of T-ag dispensable for tumor progression.

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An SV40 T/t-Antigen Gene Signature A recent report provided a powerful demonstration of the applicability of SV40 transgenic mouse studies to human cancer (Deeb et al. 2007). An integrated SV40 T/t-antigen gene expression signature was identified from transgenic models of breast, lung, and prostate cancer. This gene signature reflected primarily the expression of genes regulating cell replication, proliferation, DNA repair, and apoptosis and was distinctive from those of several other oncogenes (Ras, Her2/neu, Myc, and PyMT). This gene signature identified a subset of aggressive human breast, lung, and prostate carcinomas with poor prognosis and was highly predictive of human breast cancer prognosis. A comparison of the genes upregulated by T-ag in mouse intestinal enterocytes found that a majority (78%) overlapped with the SV40 T/t signature (Rathi et al. 2009). This observation indicates that SV40 T-ag affects the same molecular pathways in different tissues to mediate transformation. Other A transgenic mouse model directed SV40 T-ag expression to mesothelial cells using the mesothelin promoter. Members of a transgenic line exhibited a low level of spontaneous tumor development, but when exposed to asbestos developed more rapidly growing, invasive mesotheliomas than the control nontransgenic mice (Robinson et al. 2006). This system showed co-carcinogenicity between SV40 and asbestos in vivo. Those results complemented similar findings of synergism between SV40 and asbestos in Syrian hamsters in vivo and in tissue culture studies involving primary human and hamster mesothelial cells (Kroczynska et al. 2006). The results were supportive also of observations made in humans (Procopio et al. 2000; Cristaudo et al. 2005). These reports showed that both transgenic and nontransgenic animal models can be used to examine potential co-carcinogen effects on a virus-induced cancer, an underexplored area in viral carcinogenesis. The potential role of carcinogens on induction of other human neoplasias linked to SV40 warrants investigation.

Human Infections and Cancer Associations SV40 was discovered as a contaminant of early poliovaccines in 1960 (Sweet and Hilleman 1960). The vaccines had been prepared using primary cultures of rhesus monkey kidney cells, some of which harbored natural persistent infections by SV40. The virus produced no discernible cytopathic effects in the rhesus cells and its presence went unrecognized. It was only when African green monkey kidney cells were used that cellular changes (cytoplasmic vacuolization) developed and the virus was detected. By that time both contaminated inactivated poliovaccines (IPV)

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(“Salk” vaccines) and live, attenuated oral poliovaccines (OPV) (“Sabin” vaccines), as well as some contaminated adenovirus vaccines, had been administered to millions of people. Residual infectious SV40 survived the inactivation procedures used to prepare the killed vaccines, whereas there was no inactivation step to decrease viral viability in the oral vaccines (Fraumeni et al. 1963; Shah and Nathanson 1976; Stratton et al. 2003). The presence of SV40 in the human population today, as well as its potential role as a human pathogen, will be considered here.

Human Exposures to SV40-Contaminated Vaccines The IPVs were widely used starting in 1955, both in the US and other countries. The chance of IPV contamination by SV40 depended on several factors, including the frequency of viral infection in the monkeys, the levels of viral infection in their kidneys, and whether culture methods for vaccine production used kidneys from a single monkey or pooled organs from multiple animals. The higher the titer of contaminating SV40, the higher the likelihood of residual infectious virus surviving formalin inactivation. Most vaccine lots were not tested for SV40, but in two vaccine batches that were tested retrospectively live virus was in the range of 103 infectious units per ml. Estimates are that up to 30% of IPV lots contained live SV40 and that by 1961 approximately 10–30 million of the 98 million vaccinated in the US were exposed to live virus (Shah and Nathanson 1976). Rhesus macaques, the natural hosts for SV40, were generally used for vaccine production. Other species, such as cynomolgus macaques, African green, and patas monkeys, could be readily infected by contact with an infected rhesus. As many juvenile animals were held in gang cages, this provided opportunities for SV40 to be transmitted from an infected animal to uninfected ones and productive infections established before their kidneys were harvested. The time span during which potentially contaminated poliovaccines were used was from 1954 to early 1963. Vaccines were supposed to be free from SV40 after June 1961, but previous lots of vaccine were not withdrawn from the market and could have been used until 1963 (Shah and Nathanson 1976). However, recent evidence suggests that the Russian OPV likely was contaminated until the late 1970s (Cutrone et al. 2005) and there is uncertainty about the status of the OPVs used in some other countries. Work was proceeding during the 1950s on derivation of attenuated poliovirus strains for a live vaccine as debate raged about the relative merits of killed and live poliovaccines. Large-scale field trials were carried out from 1958 to 1960 for two candidate live vaccines developed by Dr. Albert Sabin and by Lederle Laboratories, respectively. These trials were not carried out in the US because widespread use of the Salk vaccine had produced antibodies in many persons. The candidate OPVs were grown in monkey kidney cells and were presumably contaminated with SV40. Virus titers in the oral vaccine were as high as 104–106 infectious units per ml (Shah and Nathanson 1976; Hilleman 1998; Rollison and Shah 2001). In fact, the Baylor

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strain of SV40 was originally recovered from a Type II Sabin OPV from 1956 and other SV40 strains were isolated from monkey kidney cells in use at the time (Forsman et al. 2004). Sequences of recognized SV40 strains were detected recently in a Russian OPV seed stock used until the late 1970s (Cutrone et al. 2005). Sabin’s vaccines were field tested in Mexico (Ramos-Alvarez and Gomez-Santos 1961) and very widely in the USSR (Chumakov 1961; Sabin 1985). The Russians prepared OPV based on Sabin’s attenuated viral strains and over 100 million persons were vaccinated in Russia, East Germany, Czechoslovakia, Hungary, Romania, Bulgaria, Latvia, and other countries in 1959–1960 (Sabin 1985, 1991). Sabin vaccines were also tested in Houston, Texas and in Cincinnati, Ohio (Melnick 1960; Sabin et al. 1961; Sabin 1985). The Lederle strains were tested in large trials in Central and South America, including in Costa Rica, Nicaragua, Colombia, and Uruguay (Pan American Sanitary Bureau 1959, 1960; Cabasso et al. 1960). Those trials involved about one million people, including many children. In the US in 1958 and 1959, the Lederle vaccine was tested in Minnesota and New England in about 1,400 people and in a large trial in Dade County, Florida (~400,000 people) (Pan American Sanitary Bureau 1959, 1960). The Koprowski candidate vaccine, based on the same viral strains as the Lederle vaccine, was tested in over 12 million people in Poland, Croatia, and the Belgian Congo (Plotkin 2001; Koprowski 2006). In 1961, the Type I Sabin vaccine was licensed in the US following an evaluation that concluded it to be safer and less virulent than the Lederle vaccine. Type II vaccine was approved later in 1961 and Type III in March 1962. A trivalent vaccine became available in 1963. Another source of human exposure to SV40 was contaminated adenovirus vaccines used to vaccinate military recruits against epidemic acute respiratory disease (Rapp et al. 1964; Lewis 1998; Rollison et al. 2004). Several different human adenoviruses (types 1–5 and 7) had been “adapted” to grow in rhesus monkey kidney cells in the 1950s for vaccine development purposes before it was known that adenoviruses grow extremely poorly in those cells unless coinfected with SV40. It was subsequently discovered that the vaccines were contaminated with live SV40 or with hybrid adenovirus-SV40 recombinants (Section “The SV40 Viral Genome and Virus-Encoded Proteins”). Several hundred thousand military recruits received the contaminated vaccines between 1957 and 1961 (Shah and Nathanson 1976; Lewis 1998). The widely studied SV40 strain 776 was isolated from an adenovirus type 1 vaccine seed stock (Forsman et al. 2004).

Predictions of Contemporary Human Infections by SV40 Among all the potential exposures to SV40-contaminated vaccines, it is unknown which individuals actually received contaminated vaccines, how much infectious SV40 was present in each contaminated lot, or who among the exposed was successfully infected by SV40. While there is strong evidence that SV40 human infections

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are occurring today, the distribution and prevalence of those infections are unclear. There are also numerous findings of the presence of SV40 markers in selected human cancers, but those positive results are counterbalanced by reports of negative findings. This inconsistency among studies has inhibited progress in the assessment of SV40 as a potential human pathogen. A model is proposed here to provide both a rationale for the maintenance of SV40 infections in restricted geographic locations and a possible explanation for the conflicting reports in the literature of SV40 detections in humans. The model is based on virus biology and contaminated vaccine usage. The key observation relevant to viral transmission is that polyomaviruses, including SV40, can be found in stool samples, in addition to urine. This suggests that the viruses can be transmitted by the fecal-oral route, as well as presumably by a urine-oral route (Vanchiere et al. 2005a, b, 2009; Bialasiewicz et al. 2009; Wong et al. 2009). A model of SV40 spread among humans can be predicated on the pattern of poliomyelitis epidemiology before vaccination. The polio system accurately predicted the epidemiology of hepatitis A virus (HAV) and provided a model for understanding Helicobacter pylori. In developing countries, polio infections transmitted by the fecal-oral route were common in infants in whom the infection caused mild or no disease due to the presence of maternal antibodies; older children and adults were then immune for life. In regions and populations where standards of sanitation and household hygiene improved, fewer polio infections occurred in infants and young children, reducing the overall prevalence of infection and resulting in larger numbers of susceptible older individuals in whom poliovirus infections were less common but sometimes caused paralytic poliomyelitis. This led to a change in disease pattern from endemic to epidemic poliomyelitis (Walton and Melnick 1955; Paul 1971; Melnick 1985; Sabin 1991). With HAV, also transmitted by the fecal-oral route, the prevalence of viral antibodies reflects the level of sanitation in the individual’s living environment. Under conditions of overcrowding, lack of clean water, and inadequate systems for disposal of human waste (situations that are more common in developing countries), HAV causes infections that occur early in life in most persons and are usually subclinical (high prevalence rates). In areas with high levels of sanitation, many individuals reach adulthood without exposure to the virus (low prevalence rates) and when infection occurs in a susceptible adult, clinically apparent hepatitis is more common. Even in developed countries, such as the US, there can be pockets of high infection rates, reflecting local differences in living standards or customs (Feinstone and Gust 2002). Similarly, a high prevalence of antibodies to H. pylori in a given population group is a marker for poor sanitation in their living conditions (Graham 1991; Nurgalieva et al. 2002). The application of the polio/HAV/H. pylori model to SV40 predicts that human infections are likely to be maintained by fecal-oral transmission in settings of overcrowding, poor sanitation, and unclean water sources. In regions and populations with access to clean water and high levels of sanitation, such as in the US and other developed countries, the prevalence of SV40 infections today would be expected to be very low, reflecting reduced opportunities for fecal-oral transmission. The model further predicts that SV40 infections will be distributed unevenly geographically and among population groups with different standards of living or levels of hygiene.

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It is envisioned that more human infections were seeded by the use of contaminated OPV as compared to IPV because of a more natural route of exposure (oral rather than intramuscular) and the higher titers of contaminating infectious SV40. A higher dose of inoculum would probably have resulted in increased numbers of successful infections among the vaccinees. As field trials with contaminated OPV in the Western hemisphere were carried out in locations in Mexico and in Central and South America rather than in the US, poor sanitation conditions in some areas in those regions could have provided opportunities for maintenance of recurring cycles of SV40 infections. These predictions concerning the importance of sample and subject selection probably explain, in large part, the inconsistency among reports of SV40 in humans, with discrepant findings reflecting the demographic backgrounds of the populations from which specimens were obtained. To analyze SV40 infections or SV40-associated cancers in humans, studies should involve regions or populations in which the virus is present. This model provides guidance as to the potential geographic areas and populations to consider in future study designs.

Evidence of Human Infections by SV40 A review of the evidence involving noncancer samples indicates that SV40 human infections occur today, at relatively low prevalence, establishing that this monkey virus has crossed over to humans (Butel and Lednicky 1999; Vilchez and Butel 2004; Baranova and Carbone 2010; Butel 2010). The earliest indication that SV40 could establish infections in humans was reported in 1962; 19% of newborns and 15% of infants 3- to 6-months of age at the time of receiving contaminated OPV were found to excrete infectious SV40 in their stools for up to 5 weeks (Melnick and Stinebaugh 1962). Modern studies have employed molecular assays to detect viral sequences. SV40 excretion was detected in about 8% of pediatric stool samples from Houston, Texas, and less frequently in adult stools (Vanchiere et al. 2005a, 2009). SV40 DNA has been found in urine samples from children and, on rare occasions, in urines from adults (Li et al. 2002a, b; Milstone et al. 2004; Vanchiere et al. 2005b; Thomas et al. 2009). Viral sequences have also been detected in tissue samples: in renal biopsies from patients with kidney disease (Butel et al. 1999; Li et al. 2002b; Milstone et al. 2004), in normal liver tissues from mesothelioma patients (Comar et al. 2007), and in lymphoid specimens from healthy children (Patel et al. 2008; Comar et al. 2010). SV40 DNA has been found at low frequencies in peripheral blood cells from healthy individuals. Reports have involved subjects from the US (David et al. 2001; Li et al. 2002a), Europe (Martini et al. 1996, 2002; Paracchini et al. 2005; Heinsohn et al. 2009; Pancaldi et al. 2009), Japan (Yamamoto et al. 2000; Nakatsuka et al. 2003), Egypt (Zekri et al. 2007), and Russia (Lapin and Chikobava 2009). In an Italian study, viral differences in regulatory region sequences were noted across birth cohorts (Paracchini et al. 2005). Studies (n = 20) using polymerase chain reaction-based

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assays for SV40 sequences in healthy subjects were reviewed recently and the prevalence rates ranged from 25% to 0% (Paracchini et al. 2006). The studies were highly heterogeneous, perhaps because those samples often represented convenience controls for cancer studies. These results establish the presence of SV40 in humans, both in people of an age who might have received contaminated vaccines and in those born after vaccines were supposed to be SV40-free. They also demonstrate the nephrotropic, lymphotropic, and gastrointestinal properties of the virus. It is likely that the kidney serves as a reservoir of persistent infections, while it is possible that lymphoid and/or gastrointestinal tissues may be additional sites. However, the DNA-based studies have been too limited in scope to establish the distribution of viral infections in different areas of the world. Serology studies are a common approach to establishing the prevalence of a viral infection, with neutralization assays being the most specific serological measure of viral antibodies. Studies using plaque reduction or microtiter infectivity assays have detected SV40 neutralizing antibodies in target groups in the US and the United Kingdom, with positive results ranging from 2% to greater than 10%. Seroprevalences in most cases were 5% or less (Butel and Lednicky 1999; Minor et al. 2003; Shah et al. 2004; Vilchez and Butel 2004; Butel 2010). Samples from two central European countries, Hungary and the Czech Republic, where early vaccine use was well documented, also had low overall frequencies of SV40 antibody, although a striking observation was a higher prevalence of antibodies in females compared to males in both countries, reaching 15.6% in Hungary (Butel et al. 2003). A study in the central Asian country of Kazakhstan, known to have used contaminated Russian vaccine, found an overall seroprevalence of 4.9%, but with higher frequencies among older ethnic Kazakhs (8.5%) and younger ethnic Russians (9.7%) (Nurgalieva et al. 2005). Unfortunately, SV40 neutralization assays are time-consuming and labor-intensive and cannot be applied to large population surveys. Enzyme immunoassays can be used to test large numbers of samples for serum antibodies. All antibodies that are able to bind to virus particles or soluble capsid proteins are measured in these assays, including those that are non-neutralizing and those that recognize cross-reactive epitopes on BKV, JCV, and SV40. Using this methodology, competition assays with BKV and JCV particles reduced SV40 reactivity in screened sera to an overall seroprevalence of about 2% (Shah et al. 2004; Kean et al. 2009). These results provide evidence of SV40 human infections at low rates in the human groups tested. However, several limitations of the reported studies should be considered. First is the general lack of knowledge of the human immune response to SV40 (Section “Mouse Model of Cellular Immune Responses to SV40 T-Antigen”). Cross-reactive antibodies or cytotoxic lymphocytes to BKV or JCV may reduce specific responses to SV40 infection. SV40 antibody titers in humans are usually low, perhaps reflecting a weak specific response. Another factor is that SV40 antibodies reportedly wane over time, lowering apparent seroprevalences (Lundstig et al. 2005). Finally, the majority of such studies have been carried out in countries with high standards of living, some of which had limited exposure to contaminated OPV. It would be informative to perform serological surveys in populations predicted to be at higher risk of SV40 infections.

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Association of SV40 with Human Cancer SV40 is a potent cancer-causing virus with the ability to transform many cell types from different species, including cells of human origin. There is evidence that SV40 infections occur in humans, but whether the virus may be playing an etiological role in human cancer remains controversial. Numerous studies have investigated possible links of SV40 with human tumors and obtained positive findings. However, other studies have failed to detect evidence of SV40 in cancer samples. These studies have been reviewed (Butel and Lednicky 1999; Shah 2000; Arrington and Butel 2001; Rollison and Shah 2001; Stratton et al. 2003; Vilchez and Butel 2004; White and Khalili 2004; Poulin and DeCaprio 2006; Rollison 2006; Martini et al. 2007; Baranova and Carbone 2010; Butel 2010). Tumor types most commonly associated with SV40 markers are mesotheliomas, lymphomas, brain tumors, and osteosarcomas. Interestingly, these are the most common types of tumors induced in hamsters by SV40. Controlled studies based on polymerase chain reaction assays involving the first three tumor types, in which control samples were analyzed in parallel, are summarized (Table 15.1). Although SV40 was detected significantly more frequently in cancer than control specimens for all three systems, the percent positivity among studies ranged from 100% to 0%. This lack of reproducibility has raised questions about the significance of viral detection. A variety of technical factors have been suggested that might explain the inconsistency among reports, including sample selection, sample processing, laboratory contamination, DNA quality, DNA quantity, and assay sensitivity. However, a more important factor may be the geographic source of the specimens analyzed. A recent study addressed the hypothesis that the presence of SV40 in lymphomas can vary depending on the populations sampled, even when uniform technical procedures are applied. Archival specimens from two hospitals in Houston, Texas having patient populations with significantly different demographics were compared. SV40 DNA was detected more often in lymphomas from the public hospital (23%) as compared to those from the veterans’ hospital (3%; p < 0.0001) (Toracchio et al. 2009). A possible explanation for those differences is that many immigrants in Houston from Mexico and Central America access the public hospital for medical care and some individuals may have emigrated from high SV40 prevalence locales. Studies need to be designed that focus on regions predicted to be high prevalence areas for SV40 infections. One study has been described in which samples from Costa Rica, such a predicted area, were analyzed. SV40 sequences were detected in 24% of non-Hodgkin lymphomas and not in the control samples (Meneses et al. 2005). SV40 T-ag was expressed in many of the DNA-positive cancers as detected by immunohistochemistry (Fig. 15.10). The T-ag reactions in the human lymphomas were less intense as compared to that of SV40 hamster tumors and a lower proportion of cells in the lymphomas stained positively. This suggests that T-ag stability or accumulation differs in the human tumor cells compared to rodent tumors. T-ag expression has been reported also in studies involving brain tumors (Bergsagel et al. 1992; Zhen et al. 1999), AIDS-related lymphomas (Vilchez et al. 2005), and mesotheliomas (Arrington and Butel 2001; Baranova and Carbone 2010).

Table 15.1 Detection of SV40 DNA by PCR in controlled studies of human cancers a SV40 DNA by PCR Range in % No. positive/No. tested (%) positivity of Type of cancer No. of studies Cancer P value cancer specimens Controlb Mesothelioma 18 269/671 (40.1) 19/474 (4.0) <0.001 100–0 Lymphoma 16 389/2,033 (19.1) 39/1,207 (3.2) <0.001 56–0 Brain 9 152/912 (16.7) 49/455 (10.8) 0.007 100–2 a Adapted from Butel (2010). See review for original citations b Many of the SV40-positive control samples were peripheral blood samples from noncancer patients or healthy individuals

Fig. 15.10 SV40 T-ag expression in non-Hodgkin lymphomas detected by immunohistochemistry. (a) T-ag expression in SV40-induced hamster tumor cells. (b and c) T-ag expression in SV40 DNA-positive diffuse large B cell lymphomas from Costa Rica. (d) No T-ag expression detected in an SV40 DNA-negative lymphoma from Costa Rica. (e and g) T-ag expression in SV40 DNApositive AIDS-related lymphomas from Houston, Texas. (f) No T-ag expression detected in an SV40 DNA-negative reactive lymph node from an HIV-infected patient. Original magnification for all panels (except f), ×100. Panel f, ×40. From Vilchez et al. (2005) and Meneses et al. (2005)

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A recent study compared osteosarcomas from Hungary and from Germany and found SV40 DNA at high levels in 74% of Hungarian samples and at low levels in 22% of German tumors (Heinsohn et al. 2009). The authors concluded that SV40 prevalence varies in different geographical regions. Complementary lines of evidence have been described, in addition to the polymerase chain reaction-based surveys of tumors and detections of antigen expression, that indicated the presence of SV40 in human tumors. Examples include recovery of an infectious isolate of a novel strain of SV40 from a pediatric brain tumor (Lednicky et al. 1995a), methylation of specific tumor suppressor genes in SV40 DNA-positive mesotheliomas and lymphomas (Shivapurkar et al. 2004; Suzuki et al. 2005; Amara et al. 2007), detection of SV40 sequences in the meningioma of a scientist with a risk of laboratory exposure identical to those of the exposure source (Arrington et al. 2004), and derivation of T-ag-positive oligodendroglial cell lines from an SV40 DNA-positive brain tumor (Kim et al. 2009). Epidemiological support for a link between SV40 infections and human cancer is currently lacking (Strickler et al. 1998; Shah 2000; Rollison and Shah 2001; Rollison et al. 2004; Rollison 2006). However, because of the unique history of human exposures to SV40, the reported studies have had limitations. These include not knowing how many in an “exposed” group were actually infected, the possibility that members of control “unexposed” groups were infected, the lack of assays able to reliably identify those who were infected, and the possible involvement of unknown cofactors (Vilchez et al. 2003; Dang-Tan et al. 2004; Rollison 2006). Importantly, no epidemiological study has focused on populations exposed to SV40-contaminated OPV in regions likely to have maintained a high prevalence of SV40 infections. It is challenging to establish an etiological role for a virus in human cancer, especially when the geographic distribution of human infections is unknown, specific biomarkers of infection have not been established, only a subset of a given type of tumor may be involved, unknown cofactors may be contributing factors, and criteria for causality are not standardized (Butel 2000, 2010; Pagano et al. 2004). It is possible that the viral presence in tumors reflects persistent infections in those tissues, rather than a tumor-inducing relationship (a so-called “passenger” virus). However, given the wealth of information about the oncogenic capabilities of SV40 and the fact that human cells can be infected and transformed by SV40, it is difficult to imagine that humans with SV40 infections would be completely resistant to virusmediated carcinogenesis. Among the major tumor types linked to SV40, the molecular and biochemical data from studies of tissue culture cells, animal models, and human specimens, in aggregate, are strongest for malignant mesotheliomas that SV40 is playing a causal role in a subset of tumors. In this example, the virus is probably functioning as a cofactor with asbestos (Baranova and Carbone 2010; Butel 2010). It is important to determine the biological significance of SV40 association with human tumors. A causative role in certain cancers would have a significant public health impact. Research efforts are warranted on a variety of topics, including the following. Seroprevalence surveys should be conducted to establish the extent of infections in predicted geographically limited high-prevalence areas. As only subsets of tumors will presumably be related to SV40, biomarkers need to be developed to

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recognize virus-positive tumors. Investigations should consider novel molecular mechanisms underlying SV40 effects in human tumors, recalling that exceptions have been described to conventional principles of polyomavirus transformation (Sections “The SV40 Viral Genome and Virus-Encoded Proteins” and “Animal Models and Insights into SV40 Biology”). The effect of the immune response on viral persistence and tumor progression in humans awaits characterization. The documented synergy between SV40 and asbestos in mesotheliomas suggests that the role of co-carcinogens in other SV40-associated cancers should be investigated. Virusassociated tumors might respond uniquely to specific therapies, prompting the design of novel approaches, while a vaccine could be considered to prevent the development of cancers having a viral component. Finally, the geographic distribution of SV40 infections should be monitored to recognize if some event were to cause the infection to spread to new populations or regions. Scientific progress inevitably raises new questions in any biological system. There is every expectation that SV40 will continue to be an outstanding model infectious agent of cancer and contribute novel insights into the process of carcinogenesis in the future, just as it has in the past. Acknowledgments I thank Drs. T.D. Schell and S.S. Tevethia for providing Fig. 15.9. Research in my laboratory was supported in part by grants CA104818 and CA134524 from the National Institutes of Health.

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Chapter 16

BK Polyomavirus and Transformation Tina Dalianis and Hans H. Hirsch

BK Virus Discovery Polyomavirus (PyV) infections are linked to malignant transformation ever since the first isolation of the mouse PyV as a transmissible agent causing tumors in newborn or immunosuppressed mice (for review, see [Atkin et al. 2009]). The concepts have been confirmed by numerous elegant studies for the mouse PyV (MPyV) and for the simian virus 40 (SV40) that was identified as a contaminant of poliovirus and adenovirus vaccines in the early 1960s (Stewart et al. 1958; Sweet and Hilleman 1960). Thereby, MPyV and SV40 have become prototypes of DNA tumor viruses (Atkin et al. 2009; Ramqvist and Dalianis 2009). Given these circumstances, the detection of cells with atypical nuclei in the urine of a kidney transplant patient with ureteric stenosis was even more intriguing in 1971 when PyV-bearing intranuclear inclusions were revealed by electron microscopy (Gardner et al. 1971). This suggested the hypothesis that a human polyomavirus (HPyV) might play a role in human diseases and possibly cancer. The viral isolate was called BK virus (BKV) after the patient’s initials B.K. and further experimental studies were initiated to characterize its properties in vitro, in animal models, and in human disease.

T. Dalianis Department of Oncology-Pathology, Karolinska Institutet, Cancer Center Karolinska R8:01, Karolinska University Hospital, 171 76 Stockholm, Sweden H.H. Hirsch (*) Department of Biomedicine, Clinical and Transplantation Virology, Institute for Medical Microbiology, University of Basel, Petersplatz 10, CH-4003 Basel, Switzerland Infectious Disease and Hospital Epidemiology, University Hospital Basel, Basel, Switzerland e-mail: [email protected] E.S. Robertson (ed.), Cancer Associated Viruses, Current Cancer Research, DOI 10.1007/978-1-4614-0016-5_16, © Springer Science+Business Media, LLC 2012

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BK Virus Genome and Life Cycle BKV belongs to genus polyomavirus in the family of polyomaviridae which is today separated from the previous common category of papilloma, polyoma, and vacuolating viruses summarized as papovaviridae (Johne et al. 2011). The viral particles are of icosahedral symmetry with a diameter of 40–45 nm and contain the circular double-stranded genome of approximately 5.1 kb. The genome organization of the polyomaviridae is highly conserved, but there are certain species of this family that group closer together due to higher homology. Thus, BKV is closest related to simian SV40 and human JC virus, the etiologic agent of progressive multifocal leukoencephalopathy, a rare, mostly fatal brain disease in immunocompromised patients (Padgett et al. 1971). The BKV genome can be divided into three regions. • The noncoding control region (NCCR) of approximately 0.4 kb that bears the origin of replication and the promoter/enhancer sequences controlling viral gene expression. • The viral early gene region of approximately 2.3 kb encoding the regulatory proteins called large tumor antigen (LTag) and small tumor antigen (sTag). Both proteins are generated by alternative splicing of a primary transcript (started from the NCCR in one direction) and then localize to nucleus and cytoplasm, respectively. • The viral late gene region of approximately 2.4 kb encoding the capsid proteins VP1, VP2, and VP3 as well as the BKV agnoprotein. These proteins are also generated by alternative splicing from a primary transcript (started from the NCCR in the opposite direction), and then localize to the nucleus for capsid assembly and the cytoplasm, respectively (Rinaldo et al. 1998). Initial studies have investigated the BKV life cycle in diverse, mostly nonhuman cell lines, where the focus was on the characterization of the transforming potential. The emergence of BKV nephropathy in kidney transplants has generated considerable interest in nontransformed human cells (Hanssen Rinaldo et al. 2005) as well as in the host cell targeted by this disease (Bernhoff et al. 2008; Gosert et al. 2008; Low et al. 2004). In the latter cells, BKV are able to replicate with high-efficiency releasing by host cell lysis, 10,000–100,000 viral particles per cell within 48–72 h post infection (hpi). The primary host cell specificity is mediated by the attachment of the major capsid protein VP1 to glycosylated surface structures, whereas VP2 and VP3 play a role in packaging of the viral genome and likely stabilize the three-dimensional virion structure. The binding sugar moieties have been identified on membrane lipids, such as gangliosides. BKV capsids interact with an alpha (2,3)-linked sialic acid linkage present of the gangliosides GD1b and GT1b (Dugan et al. 2006; Low et al. 2006). Additionaly proteinaceous molecules may serve as secondary receptors and facilitate PyV entry. Thus, BKV appears to interact with a 55-kDa molecule yet to be identified and is then taken up by endocytosis of caveolae (Dugan et al. 2006; Neu et al. 2009). From the caveosome, the endoplasmic reticulum (ER) is reached,

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where uncoating and delivery of the episomal viral genome to the nucleus occur (Neu et al. 2009). The VP1 protein is also the determinant of the six major BKV serotypes that have been described and is the major target of neutralizing antibody activities (Jin et al. 1993). The secondary host cell specificity of BKV is mediated at the level of viral gene expression from the NCCR (Watanabe and Yoshiike 1986). In line with its predilection for the renourinary tract, BKV infects, and efficiently replicates in, primary human renal proximal tubular epithelial cells (RPTECs). RPTECs have been wellcharacterized by Imperiale and colleagues (Low et al. 2004) and proved to be an important model for the study of host cell responses (Abend et al. 2010; Grinde et al. 2007) and antiviral activities (Bernhoff et al. 2008; Bernhoff et al. 2010; Rinaldo et al. 2010; Rinaldo and Hirsch 2007). However, BKV detected in healthy individuals around the world is characterized by a linear architecture of the NCCR (Flaegstad et al. 1991; Markowitz et al. 1991; Negrini et al. 1991) and replicates slowly in RPTECs (Gosert et al. 2008; Olsen et al. 2009). By contrast, BKV variants bearing rearranged NCCR are rapidly selected in vitro (Johnsen et al. 1995) which includes the original BKV isolate described by Gardner (Gardner et al. 1971). Thus, the wellcharacterized BKV (Dunlop) is characterized by a rearranged (rr)-NCCR. Recent data revealed that BKV rr-NCCR variants also emerge in vivo in the blood of kidney transplant patients with prolonged BKV-associated nephropathy. These (rr)- NCCRs confer increased LTag expression and accelerated replication in vitro (Gosert et al. 2008). Thus, the NCCR is an important determinant of the secondary host cell specificity and pathology by controlling the expression of the early viral proteins, LTag and sTag, and by determining the effective switch to late gene expression and lytic replication. The LTag and sTag are key regulatory proteins of roughly 700 and 170 aa that permit PyV replication through different direct and indirect mechanisms. The indirect mechanisms involve the interaction with host cell proteins to create an optimal metabolic environment for the efficient biosynthesis of the viral progeny. The direct mechanisms consist of binding to the viral genome to recruit the host cell replication complex and to mediate the switch to viral late gene expression. The key elements of the indirect LTag functions have been discussed in great detail elsewhere in this book. They include the inactivation of the tumor-suppressor protein functions of the retinoblastoma family pRB, p103 and p107. Thereby, the cell cycle control is abrogated and the host cell is shifted into a proliferative G2/S state, where enzymes and building blocks become abundant. Binding of LTag to p53 is thought to counteract apoptosis which may be triggered by accumulating viral DNA fragments and disintegrating host cell DNA as well as by the metabolic exhaustion. Binding to p53 may also activate cellular genes, like insulin-like growth factor (IGF)-1 expression and signaling, and potentially affect gene expression by stabilizing regulators, like p300/CBP and Mdm2 (Reiss et al. 2006). The sTag presumably provides additional cytoplasmic proliferation signals by inactivating the protein phosphatase 2A (PP2A) that negatively regulates activating signal transduction along the MAP kinase pathway. The DNA-binding domain and the ATPase/helicase activity of LTag are crucial for melting the viral DNA and recruiting proteins to replication fork, including the host cell DNA polymerase. With the switch to late gene expression and capsid

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Episomal DNA

Signals

Latency

Early gene expression small T antigen Large T antigen Host cell activation Cell cycle shift G2/S Anti-apoptosis uncoupling

Late gene expression VP-1,2,3 capsids Productive BKV infection Cytopathic cell lysis

No late gene expression no virion assembly Abortive BKV infection Oncogenic transformation

Fig. 16.1 Scheme of BKV infection, replication or transformation

assembly, the late phase of the viral life cycle is initiated. The role of the nonstructural agnoprotein is presently unresolved. Homologous agno proteins are currently only found for SV40 and JCV. Its dominant cytoplasmic expression late in the viral life cycle of BKV suggests a regulatory role in virion assembly and release, but a multitude of other functions including oncogenic transformation have been discussed. The efficient release of progeny virions from the nucleus requires host cell lysis which naturally counteracts tumor development. Hence, the uncoupling of early gene expression from late gene expression must be postulated as a key component of oncogenic transformation by PyVs including BKV. This can be envisioned on a functional level, where the NCCR is intact, but late gene expression cannot occur in a given host cell because of its state or differentiation on a genetic level via mutations, rearrangements, or disruption by genomic integrations that affect VP1 expression or truncate the LTag downstream of the LxCxE domain involved in pB binding (Fig. 16.1).

Cytopathic BKV Disease Initial clinical studies in kidney transplant recipients suggested that BKV might play a role in the stenosis of the alloureter and proposed a role of corticosteroids in the reactivation of BKV in this setting (Coleman et al. 1978; Gardner et al. 1984). Although the role of steroids has been confirmed in some later studies (Hirsch et al.

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2002; Hirsch et al. 2010), other patient and immunosuppression-related factors must be involved as well. Early histopathology studies reported biopsy findings consistent with virally induced interstitial nephritis that was difficult to be distinguished from acute cellular rejection (Gardner et al. 1984; Mackenzie et al. 1978). Subsequently, this complication was virtually nondiagnosed (Prince et al. 2008) and reemerged only when the calcineurin inhibitor cyclosporine-A was widely substituted by tacrolimus in the late 1990s (Binet et al. 1999; Randhawa et al. 1999). Currently, BK polyomavirus-associated nephropathy (PyVAN) is encountered in 1–10% of kidney transplant patients, and more than half of the affected patients are at risk for premature graft failure (Hirsch 2010; Ramos et al. 2009). The patients are clinically asymptomatic despite a high urine BKV loads of >7 log10 genome equivalents (geq)/mL and the shedding of “decoy cells” (Funk et al. 2008). High-level viruria precedes the onset of detectable plasma BKV loads and histological evidence of nephropathy (Hirsch et al. 2002; Nickeleit et al. 2000). With increasing viral cytopathic damage and denudation of the infected tubuli, inflammation increases and laboratory signs of progressive graft failure appear in more than 80% (Drachenberg et al. 2007, 2004; Hirsch et al. 2002). In the absence of effective antivirals, the judicious reduction of immunosuppression at the onset of BKV viremia has become the treatment of choice in many centers (Hirsch and Randhawa 2009). Thereby, viremia was cleared in adult and pediatric patients, and the risk of graft loss was reduced to less than 10% (Ginevri et al. 2007; Hardinger et al. 2010; Schaub et al. 2010). On the other hand, in patients with persistent, high-level BKV replication, viral variants with rr-NCCR emerged in vivo that were associated with 20-fold higher plasma BKV loads and more advanced PyVAN in the renal allograft (Gosert et al. 2008). The rr-NCCRs were shown to consist of different deletions, duplications, and complex combinations in the promoter/enhancer elements (Gosert et al. 2008; Olsen et al. 2006). Reporter gene constructs and recombinant viruses demonstrated that the rr-NCCR variants increased the viral early gene expression the viral replication capacity in vitro as compared to the naturally found archetype virus (Gosert et al. 2008; Olsen et al. 2009). Studies in allogenic hematopoietic stem cell transplant recipients provided evidence for a role of high-level BKV replication in the late-onset, postengraftment hemorrhagic cystitis (Arthur et al. 1986; Bedi et al. 1995). The urine BKV loads are also above 7 log10 geq/mL, but unlike in kidney transplantation the affected patients are clinically symptomatic with severe signs of cystitis, immobilizing pain, macrohematuria with clots, and even postrenal failure that requires bladder irrigation and urologic intervention (Hirsch 2010). Although previously seen more frequently, current estimates suggest that this complication occurs in 5–15% of patients (Erard and Hirsch 2009; Hirsch 2010). The risk factors include myeloablative conditioning often involving cyclophosphamide and total body irradiation, unrelated donors, graft-versus-host disease, and increasing BKV loads in the blood (Erard et al. 2005; Giraud et al. 2008a). The pathogenesis seems to involve a combination of factors, where BKV replication leads to a denudation of the urothelial mucosa predamaged by the toxic conditioning and an immune reconstitution inflammatory syndrome (IRIS) response following engraftment of the allogenic stem cells (Hirsch 2010).

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Transforming BKV Activity in Experimental Systems A role for BKV in oncogenic transformation has been suggested early after the reports of its initial isolation in 1971 (Gardner et al. 1971; Padgett et al. 1971; Walker et al. 1973). Initial in vitro studies indicated that BKV could not be cultured in rodent cells, but mostly induced an altered phenotype or outright transformation, depending on the cell type and its underlying lesions (for review, see [Barbanti-Brodano et al. 2006]). Further in vitro studies could link the transforming contribution of BKV to the viral early gene region encoding the BKV LTag and the sTag (Harris et al. 1996) and the ability to cooperate with other oncogenic alterations (Harris et al. 1998a, b). Defective BKV genomes were linked to the transforming potential, and particular cell types like the pancreas proved to be effectively targeted in rodent- and humanderived cells (van der Noordaa et al. 1986; Watanabe et al. 1979; Yogo et al. 1980). Animal studies in rodents showed that, diverse tumors could be induced as had been previously shown for MPyV or SV40 (Shah et al. 1977). Furthermore, animal studies in rodents also showed that diverse tumors could be induced by BKV, including brain tumors and sarcomas, as had been observed for MuPyV and SV40 (Ramqvist and Dalianis 2009; zur Hausen 2008). Depending on the route of inoculation, the tumor type and penetrance varied considerably. In addition, the immune system needed to be either immature or experimentally depleted prior to the virus challenge. In hamsters, for example, little tumorigenicity by BKV was observed in subcutaneous sites, whereas direct intracerebral inoculation was followed by tumor formation in more than 80% of the animals (Uchida et al. 1976). Different types of tumors were observed in these experiments, including neuroblastoma, ependymoma, pancreatic cancer, and sarcomas of the connective or osteogenic tissues. When an intravenous application was chosen, prior immunosuppression was needed for tumor formation in hamsters and this was done by the application of lymphocytedepleting antisera or the use of high-dose steroids (Corallini et al. 1982). However, primary human cells appeared to be more refractory to the transforming effects of the BKV early genes (Portolani and Borgatti 1978; Shah et al. 1976). In contrast, cells with chromosomal abnormalities were more susceptible in line with the presence of cooperative alterations in oncogene or tumor-suppressor gene products.

BK Virus in Human Cancers Despite the suggestive mechanistic evidence from experimental models, the role of BKV in human malignancies is controversial (Grossi et al. 1981). The high seroprevalence and frequent detection of BKV in healthy individuals by sensitive molecular tools is noted (Egli et al. 2008), since this complicates study design and requires a rigorous reevaluation of the published data. However, the possibility of an oncogenic role of BKV in human cancer is captured in single case reports of urothelial malignancies and renal tubular malignancies, typically in the setting of kidney transplantation as illustrated by the following three cases.

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• A 53-year-old simultaneous kidney–pancreas transplant patient was diagnosed with metastatic urothelial carcinoma 1 year after losing his kidney transplant to BK-PyVAN while the pancreas transplant was functioning well (Geetha et al. 2002). In situ hybridization identified BKV. Immunohistochemistry revealed positive staining for LTag and co-staining for p53, but adjacent nondysplastic urothelium or adjacent stroma was negative. LTag and p53 staining was also positive in the metastases. Sequencing of the p53 gene demonstrated the absence of stabilizing mutations, implicating the BKV LTag binding in p53 overexpression. The workup of this case is exemplary and the association of BKV LTag and malignancy is highly suggestive. However, molecular-genetic analyses are missing, and it is formally possible that BKV had been infecting the urothelial carcinoma only after its genesis (“passenger”) rather than contributing to its actual condition. On the other hand, prolonged, high-level BKV replication and PyVAN in severely immunodeficient patients are known to allow the emergence of genetic BKV variants with rr-NCCR which may or may not support lytic replication (Gosert et al. 2008; Myhre et al. 2010). This may in turn also create opportunities for otherwise rare events and thereby favor malignant transformation, as proposed earlier (Hirsch and Steiger 2003). • A 40-year-old simultaneous kidney pancreas transplant recipient was diagnosed with a BKV LTag-positive metastasizing renal cell carcinoma after developing BK-PyVAN treated with intravenous cidofovir (Narayanan et al. 2007). • A pediatric kidney transplant patient developing a BKV LTag–p53 positive collecting duct carcinoma after BK-PyVAN (Emerson et al. 2008). While the similarity between these cases is intriguing, there are, however, also two other reports. • The first is a 10-year-old kidney transplant recipient developing a renal cell carcinoma of donor origin after BK-PyVAN that was BKV negative by in situ hybridization (Kausman et al. 2004). This case raises questions whether or not the hybridization was analytically false negative and should have been complemented by immunohistochemistry for LTag. Alternatively, the results were conceptually false negative due to a “hit-and-run” mechanism. Where by BKV contributed to an earlier stage, but was then lost. • The second case is an 80-year-old man with past colon cancer and significant steroid exposure for bullous pemphigus (Loghavi and Bose 2010). The patient was diagnosed with a papillary urothelial carcinoma supposedly superinfected with BKV (“passenger”) since only a fraction of cells were LTag positive (Loghavi and Bose 2010). However, the “hit-and-run” remains as alternative hypothesis for the BKV-negative cancer tissue and requires further investigations. Together, these cases illustrate the caveats which need to be addressed in larger studies by stringent, quite extensive protocols which best include several complementing diagnostic procedures. Clearly, PCR analysis of tissues may be one element which can not be sufficient to draw conclusions without ancillary techniques. The study by Weinreb and colleagues (Weinreb et al. 2006) identified kidney transplant patients with urine cytology in their pathology database. They found an

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increased odds ratio of 3.42 (95% confidence interval 1.79–6.55; p < 0.001) for urothelial cancers among 123 patients with “decoy cell” shedding compared to 3,617 patients without “decoy cell” shedding (Weinreb et al. 2006). Although this data does not directly implicate a direct contribution of PyV to the malignancy, “decoy cells” may serve as noninvasive biomarker of increased risk in patients with high-level replications. Given the high rate of spontaneous BKV shedding in healthy controls, it is not surprising that smaller studies searching for BKV by more sensitive techniques like PCR were frequently not conclusive (Fioriti et al. 2003; Knoll et al. 2003). The study by Herawi and colleagues (Herawi et al. 2006) is an interesting extension of earlier studies. They identified 732 kidney transplant patients with “decoy cell” shedding in a retrospective cohort from 1988 to 2003. For 33 of them, biopsies were available which included the diagnosis of urothelial carcinoma in 21 cases. LTag staining was positive in seven cases (30%) including two cases (10%) with strong signals. Using tissue microarrays for a random sample of 79 low-grade papillary urothelial neoplasias, only patchy LTag and p53 co-staining was detected (0.63% or 2 of 316 tumor spots). Similarly, Roberts and colleagues (Roberts et al. 2008) interrogated the local pathology registry for urothelial cancers in patients without and with a history of renal transplantation. All of the 20 cases without transplantation were negative for LTag, whereas 1 of 8 transplanted cases had a positive immunohistochemistry for LTag in tumor cells while adjacent cells were negative (Roberts et al. 2008). Taken together, urine cytology may identify patients at increased risk for urothelial abnormalities including cancer. A role of BKV in the genesis of urothelial cancers is possible, and high-level urine viral loads or “decoy cells” may serve as a risk factor, but sofar, this association has not been documented with sufficient stringency. The role of BKV in prostate cancer has also been addressed by several recent studies. Using broad-range molecular approaches, Sfanos et al. detected BKV in 1% of prostate carcinoma tissue, Epstein–Barr virus in 8%, and large number of different bacterial species (Sfanos et al. 2008). The data illustrate that urogenital tissues may be a habitat of diverse agents and emphasize the need for specific studies to unravel pathologic contributions (Sfanos and Isaacs 2011). The development of prostate cancer appears to proceed along several histologically defined presentations called proliferative inflammatory atrophy (PIA), prostatic benign intraepithelial neoplasia (PIN), and prostatic carcinoma. Molecular studies suggested that there are low expression levels of relevant tumor-suppressor genes, including the p27 cyclin-dependent kinase inhibitor and the phosphatase and tensin homologue PTEN, and mutations in the p53 and pRB1 are generally rare (Gonzalgo and Isaacs 2003). The Imperiale group investigated the possibility that BKV infection could contribute to prostate cancer progression by interfering with p53 and pRB1 (Das et al. 2004, 2008). Using PCR and in situ hybridization on biopsy tissues, BKV DNA was detected in the epithelium of benign ducts and in PIA lesions in more than 70% of cases. In tissue sections and by selective laser capture microscopy, LTag was shown to colocalize with wild-type p53 in the cytoplasm of PIN and PIA cells, whereas a mixture of wild-type and mutant p53 was found in carcinoma cells. The LTag colocalization in the cytoplasm with the wild-type p53 suggested that sequestration was more important than the nuclear functions of this interaction, including those required for viral replication

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and late gene expression. Expression of VP1 was absent arguing that BKV early gene expression was indeed uncoupled from late gene expression. However, the absence of LTag in the carcinoma lesions has been counterintuitive and interpreted as an indication for a “hit-and-run” mechanism: LTag exerts its proliferative and antiapoptotic effects, and spawns further genetic alterations through chromosomal instability during early stages in transition to cancer. The loss of LTag in the carcinoma indicates that LTag is no longer conferring proliferative advantage, and may even become a target of cellular immunity. Thus, further independent studies still need to corroborate the role of BKV in the progression to prostate cancer and to elucidate appropriate interventions, including vaccination and immunotherapy (Abend et al. 2009; Provenzano et al. 2006). There is extensive literature on the detection of BKV DNA in other cancers reviewed recently (Abend et al. 2009). These include colorectal tumors, lymphomas, pancreatic cancer, brain tumors, and a range of sarcomas, and the interpretation of the results is controversial. On the other hand, no evidence for BKV sequences was found in a case series of 38 non-UV light-associated melanomas, thereby arguing against a role of these PyVs as an alternative genetic insult in this kind of malignancy (Giraud et al. 2008b).

Conclusions and Outlook The last decade has witnessed a significant change in our understanding of PyV prevalence, biology, and pathology, particularly in humans. Thus, the role of BKV in PyV-associated nephropathy after kidney transplantation is now accepted as a frequent complication which is being met by national and international recommendations for screening and early intervention. The role of BKV in human malignancies is suggested by its oncogenic potential in vitro, in experimental animal models, but only limited evidence is available from human studies. Lytic BKV replication seems to be a major biological safeguard against oncogenic disease, but it is conceivable that immunocompromised patients with high-level BKV replication could be at an increased risk for abortive infection that leads to functional or genetic uncoupling of early and late gene expression. However, distinguishing a role of BKV as “driver” versus “passenger” and “innocent bystander” needs to be stringently addressed in future studies.

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Chapter 17

Polyomavirus JC and Human Cancer: Possible Role of Stem Cells in Pathogenesis Kamel Khalili, Martyn K. White, Jennifer Gordon, and Barbara Krynska

Introduction JC virus (JCV) is a ubiquitous human virus and a member of Polyomaviridae family of DNA tumor viruses, which also includes BK virus and the well-known Simian virus 40 (SV40) (Imperiale and Major, 2007). JCV is the etiologic agent of progressive multifocal leukoencephalopathy (PML) (Åström et al. 1958; Padgett et al, 1971), most frequently seen in patients with a disrupted immune system. In addition to its causative role in PML, there is also experimental and clinical evidence for the role of JCV in cancer. JCV DNA sequences, and expression of the viral oncoprotein T-antigen, were found in a variety of human neural and nonneural tumors (see Table 17.1). JCV T-antigen, the main regulatory protein encoded by the virus, has well-established oncogenic potential in experimental animals and strong cell transforming ability in vitro (Khalili et al. 2003a, b), suggesting that JCV-mediated cellular transformation may be involved in neoplasia. However, despite a plethora of data supporting the oncogenic nature of JCV, whether JCV has a causal role in human cancer remains controversial. Attempts to understand the latency and dissemination of this virus indicate that the bone marrow plays an essential role in harboring persistent virus (Marshall and Major 2010). Given the growing role of stem cells in the pathogenesis of human diseases, in particular cancer, an interesting question is what role stem cells from the bone marrow may have in the pathogenesis of JCV-associated diseases. In this review, we highlight the migratory nature of

K. Khalili (*) • M.K. White • J. Gordon Department of Neuroscience and Center for Neurovirology, Temple University School of Medicine, 3500 N. Broad Street, Philadelphia, PA 19140, USA e-mail: [email protected] B. Krynska Center of Neural Repair and Rehabilitation and Shriners Hospitals Pediatric Research Center, Temple University School of Medicine, 3500 N. Broad Street, Philadelphia, PA 19140, USA Department of Neurology, Temple University School of Medicine, 3500 N. Broad Street, Philadelphia, PA 19140, USA E.S. Robertson (ed.), Cancer Associated Viruses, Current Cancer Research, DOI 10.1007/978-1-4614-0016-5_17, © Springer Science+Business Media, LLC 2012

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Table 17.1 Association of JC virus and human cancer Tumor type Reference Oligodendroglioma Rencic et al. (1996), Caldarelli-Stefano et al. (2000), Del Valle et al. (2002a) Astrocytoma Boldorini et al. (1998), Caldarelli-Stefano et al. (2000), Del Valle et al. (2001b) Medulloblastoma Krynska et al. (1999b) Ependymoma Caldarelli-Stefano et al. (2000), Del Valle et al. (2001b) Glioblastoma Del Valle et al. (2001b, 2002b) Colorectal carcinoma Laghi et al. (1999), Enam et al. (2002), Ricciardiello et al. (2000), Nosho et al. (2009), Morikawa et al. (2011) B-cell lymphoma Del Valle et al. (2004) Esophageal carcinoma Del Valle et al. (2005) Gastric cancers Shin et al. (2006), Yamaoka et al. (2009), Ksiaa et al. (2010) Anal carcinoma Ramamoorthy et al. (2010)

bone marrow-derived stem cells and evidence on the recently discovered broad differentiation potential of nonhematopoietic stem cells from the bone marrow and discuss the possible relationship between JCV and bone marrow-derived stem/progenitor cells in relation to viral latency, distribution, and oncogenic capacity.

JCV: Latency, Reactivation, and Cellular Tropism In PML, JCV replication in the brain causes the cytolytic destruction of oligodendrocytes, the myelin-producing cells, leading to lesions of demyelination and subsequent death of the patient. Antibodies to JCV are highly prevalent in human populations worldwide indicating that JCV infection is common and widespread (reviewed in White and Khalili 2011). The virus appears to infect most people during childhood but may then enter a latent state, where JCV replication is low and asymptomatic. When the immune system is disrupted, most commonly in people with HIV/AIDS or receiving immunosuppressive drug treatment, JCV emerges from latency to become reactivated in the CNS leading to PML (Berger 2003). Before AIDS, PML was very rare (Brooks and Walker 1984), but then increased dramatically (Berger et al. 1987). PML is characterized by foci of myelin loss, enlarged oligodendrocytes with viral inclusion bodies and bizarre astrocytes, resembling oncogenically transformed cells (Åström et al. 1958). The transmission of JCV from person to person may be fecal/oral and involve an archetype form of the virus (JCVCY), which is excreted in urine and found in sewage (Bofill-Mas et al. 2003). Isolates of JCV from PML brain tissue have multiple rearrangements (deletions, duplications, and point mutations) in the control region (NCCR) and are known as “PML-type” JCV, e.g., Mad-1. In contrast, archetype JCV is present in the kidney of normal individuals and may replicate at low levels in renal tubular epithelial cells. The relationship of archetype to PML type is complex and was reviewed recently (White and Khalili 2011). Notably, PML-type JCV has recently

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been detected in bone marrow of individuals without PML (Tan et al. 2009). Many other tissues are reported to harbor latent JCV, including bone marrow and brain (reviewed by White and Khalili 2011). Interestingly, while the kidney harbors archetype JCV, latent JCV in the normal brain and bone marrow is of the “PML type.”

JCV and Oncogenic Potential: Studies on the Molecular Mechanisms JCV encodes three capsid proteins, VP1, VP2, and VP3, in the late region, which are only expressed in permissive cells during viral DNA replication. JCV has two early proteins, large T-antigen (T-Ag) and small t-antigen (t-Ag), expressed in both permissive and nonpermissive cells. In nonpermissive cells, these cause cell transformation via their dysregulation of cellular processes. Additionally, another viral protein, agnoprotein, also dysregulates cellular processes (Khalili et al. 2004). JCV T-Ag interacts with cellular proteins facilitating transition into S phase, and thus transformation. T-Ag is a 688 aa nuclear phosphoprotein, which interferes with two key tumor suppressors, pRb and p53 (Krynska et al. 1997). Aberrant stimulation of the cell cycle drives oncogenesis (White and Khalili 2004; White et al. 2005; Del Valle et al. 2008). Interaction of T-Ag with pRb activates E2F, advancing the cell cycle (White and Khalili 2006), while its interaction with p53 may block p53 protection against DNA damage and transformation. JCV T-Ag also affects other signaling proteins, including IRS-1 (Lassak et al. 2002; Khalili et al. 2003a, b) and b-catenin, which it directly binds causing nuclear translocation and enhanced c-myc and cyclin D1 expression (Enam et al. 2002; Gan and Khalili 2004; Bhattacharyya et al. 2007). JCV T-Ag also associates with NF2 (Sholler et al. 2004). The JCV early region also expresses the 172 aa small t-Ag, which shares its N terminus with T-Ag but has a unique C terminus resulting from alternative splicing. JCV t-Ag may function through its interaction with phosphatase PP2A (Khalili et al. 2008; Sariyer et al. 2008). Another regulatory protein, agnoprotein, is encoded in the late region (Khalili et al. 2004) and is a 71 aa protein with a characteristic perinuclear subcellular localization as well as a small amount detected in the nucleus (Del Valle et al. 2000). Agnoprotein dysregulates the cell cycle (Darbinyan et al. 2002) and impairs cellular responses to DNA damage (Darbinyan et al. 2004). Importantly, JCV induces mutations in cellular DNA, which may contribute to transformation (White et al. 2006). Thus, JCV infection can induce DNA damage, impair DNA repair, increase chromosome ploidy, and elevate the levels of gH2AX and Rad51 (Darbinyan et al. 2007).

Cell Culture and Animal Models of JCV Tumorigenesis JCV transforms cells in culture, especially glial cells, such as primary cultures of human fetal glial cells and primary hamster brain cells. JCV-transformed cells exhibit the so-called transformed phenotype characterized by loss of contact inhibition,

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an ability to grow in soft agar, serum independence, morphological changes, multinucleation, plasminogen activator production, and enhanced glucose uptake and are tumorigenic when transplanted in vivo (Del Valle et al. 2001a, 2008). JCV is highly oncogenic in laboratory animals and was found to induce multiple types of tumors when injected into the brains of newborn Golden Syrian hamsters (Walker et al. 1973). Notably, JCV is the only human virus that is able to induce solid tumors in nonhuman primates and has been found to cause astrocytomas, glioblastomas, and neuroblastomas in owl and squirrel monkeys, which develop 16–24 months after intracerebral inoculation of JCV (Del Valle et al. 2001a, 2008). The analysis of tumor tissue revealed the expression of the JCV early protein T-antigen, but no evidence of viral replication or persistent infection could be detected. Similarly, inoculation of JCV into the brains of newborn rats resulted in induction of undifferentiated neuroectodermal tumors in the cerebrum of 75% of experimental animals (Ohsumi et al. 1985, 1986). These studies indicate that expression of T-antigen in the absence of viral lytic infection may lead to cellular transformation. Among the most interesting observations on the oncogenecity of JCV are studies on several lines of transgenic mice which contained the entire gene for JCV T-antigen under the control of its own promoter. As shown in this animal model system, in the absence of virus and therefore viral replication, T-antigen is capable of transforming cells and induces a variety of tumors. Earlier studies by Small et al. (1986) using the Mad-4 viral promoter driving T-antigen expression reported adrenal neuroblastomas while Franks et al. (1996) generated transgenic mice that developed solid mesenteric tumors with tumor cells exhibiting characteristics of primitive epithelial/ neuroectodermal origin. Transgenic mice with the JCV early region under the control of Mad-4 promoter can also develop tumors arising from the pituitary gland (Gordon et al. 2000) and some of these animals developed solid masses arising from the soft tissues surrounding the salivary gland, the sciatic nerve, and along the extremities that are histologically compatible with malignant peripheral nerve sheath tumors, rare neoplasms that occur in individuals with neurofibromatosis (Shollar et al. 2004). In another series of transgenic animal studies using the archetype or kidneyderived isolate of JCV as a source of transgene, a wide range of tumors, including primitive neuroectodermal tumors (PNETs), medulloblastomas, pituitary tumors, malignant peripheral nerve sheath tumors, adrenal neuroblastomas, and glioblastoma multiforme, were identified (Krynska et al. 1999a; Pina-Oviedo et al. 2007). Some features of these experimental tumors resemble JCV-associated human cancers discussed below, i.e., the nonpermissive nature of the JCV infection, high T-Ag expression in tumor cells, and lack of late capsid protein expression or replication.

Association of JCV with Human Malignancies There are many reports from a number of different laboratories of the association of JCV with human cancer either by virtue of the occurrence of the cancer concomitantly with PML or by the molecular detection of viral footprints in neoplastic cells

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(see Table 17.1). The first indication for the association of JCV with tumors of the CNS came from reports of brain tumors being found in patients with coexistent PML as exemplified by a case reported by Richardson (1961) who first described PML. A patient with chronic lymphocytic leukemia and PML was found to have an oligodendroglioma on postmortem examination. Other reported cases have been reviewed by White et al. (2005). The finding of brain tumors associated with PML is consistent with a role for JCV in tumor development, but it is also possible that the cancer develops secondarily to the immunosuppression that leads to PML. JCV has also been reported to be associated with neural tumors in patients without PML. Rencic et al. (1996) reported JCV DNA detected by PCR in tumor tissue from a patient with an oligoastrocytoma. The PCR product was confirmed as JCV by DNA sequencing and JCV RNA and T-Ag protein were detectable in the tumor by primer extension analysis, Western blot, and immunohistochemistry, respectively, i.e., JCV gene expression occurred in the tumor. Del Valle et al. 2001b examined 85 samples of various tumors of glial origin for JCV DNA and T-Ag expression and found that 57–83% of tumors were positive. In other studies, JCV has been reported to be associated with CNS lymphoma, glioblastoma multiforme, oligoastrocytoma, oligodendroglioma, medulloblastoma, and xanthoastrocytoma (reviewed by Del Valle et al. 2001a; White et al. 2005). As well as the CNS, JCV has also been reported in tumors outside of the CNS. JCV DNA sequences have been detected in normal tissue samples taken from the upper and lower human gastrointestinal tract and in colon cancer (Laghi et al. 1999; Ricciardiello et al. 2000). In a study of malignant epithelial tumors of the large intestine, expression of JCV T-Ag and agnoprotein was found in about half of the samples (Enam et al. 2002) but not in any of the samples of normal gastrointestinal epithelial tissue. Similar results were reported for the upper GI tract. JCV DNA was found in 11 of 13 normal esophageal biopsies (85%) and 5 of 5 esophageal carcinomas (100%). By immunohistochemistry, JCV T-Ag expression was detected in 10 of 19 carcinomas (53%) and agnoprotein in 8 (42%) while none of 51 normal, benign, or premalignant esophageal samples expressed viral proteins (Del Valle et al. 2005). In other studies JCV T-Ag was detected in colon and rectal cancer cases evaluated by immunohistochemistry (Morikawa et al. 2011), gastric cancers (Shin et al. 2006; Yamaoka et al. 2009; Ksiaa et al. 2010), and anal carcinoma (Ramamoorthy et al. 2010). Thus, JCV is associated with tumors throughout the GI tract. Other studies have begun to define molecular differences between JCV T-Ag-positive and -negative tumors. For example, Nosho et al. (2009) detected JCV T-Ag expression by immunohistochemistry in 271 (35%) of 766 colorectal cancers and T-Ag expression was significantly associated with p53 expression (P < 0.0001), p21 loss (P < 0.0001), chromosomal instability (P < 0.0001), nuclear b-catenin (P = 0.006), and microsatellite instability (P < 0.0001), but not with PIK3CA mutation. Recently, the association of JCV with tumors was reexamined using immunocytochemistry of commercially available tissue arrays, including colon adenocarcinomas. Expression of both T-Ag and agnoprotein, but not capsid proteins, was found in some, but not all, tumors of neural and nonneural origin. Notably, more than 40%

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of human glioblastomas and greater than 30% of colon adenocarcinomas were found to express viral proteins (Del Valle and Khalili 2010). Despite the accumulating reports of an association of JCV with human cancer, there have also been reports that failed to detect JCV DNA in panels of human tumors. For example, Herbarth et al. (1998) failed to amplify JCV DNA from 30 brain tumors classified as oligodendroglioma or astrocytomas and 22 human glioma cell lines and did not detect T-Ag expression by Northern blot. This and other negative studies and possible technical issues that may be responsible for these disparities have recently been reviewed (Del Valle et al. 2008). It remains unknown why studies for JCV DNA in normal and tumor tissues have yielded such variable results and the extent of association of JCV with human tumors is still an area of controversy.

JCV and Stem Cells: Brain and Bone Marrow The hypothesis has been proposed that JCV persistently infects hematopoietic stem/ progenitor cells in the bone marrow and circulating B cells, which arise from the hematopoietic lineage, most likely transport the virus to the sites of latency or lytic replication in the brain (Marshall and Major 2010). It is believed that in the brain the hematopoietic cells can infect neighboring oligodendrocytes leading to lytic infection of oligodendrocytes and the development of PML and/or abortive infection of astrocytes and neurons possibly resulting in the development of tumors (Khalili et al. 2003a, b). However, recent studies have suggested that non-PML brain tissue may also harbor the latent virus (reviewed by White and Khalili 2011). The biology and molecular characteristics of JCV have been the subject of intense research; however, the initiating event, i.e., the clinical status of patients and at what stage of cell differentiation the virus can infect cells in the brain, is largely unknown. Interestingly, human fetal brain-derived progenitor cells with the ability to differentiate into neuronal or astrocytic lineage have been shown to support viral infection in culture (Hou et al. 2006). Additionally, it has been demonstrated that oligodendrocyte progenitors derived from a human embryonic stem cell line could be infected by JCV (Schaumburg et al. 2008). However, whether JCV infects progenitor or stem cells in the brain of PML patients has not been shown. Given the strong presence of JCV in both the brain and the bone marrow, an interesting question is whether stem cells play a role in vivo and how the stem cells from the bone marrow and brain may be involved in the pathogenesis of JCV-associated diseases. Adult bone marrow contains hematopoietic stem and progenitor cells that generate all of the major blood cell lineages (Till and McCulloch 1961, 1964; Morrison et al. 1995). The involvement of the hematopoietic lineage in trafficking of the JC virus to the brain is supported by data showing that infected, circulating B lymphocytes can carry the virus to the brain and studies showing the presence of JCV DNA in B lymphocytes isolated from a PML patient (Houff et al. 1988; Tornatore et al. 1992). Furthermore, the infection of CD34-positive hematopoietic cell lines, and primary CD34-positive hematopoietic progenitor cells isolated from human fetal liver,

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demonstrated the susceptibility of hematopoietic cells to JCV infection (Monaco et al. 1996). The bone marrow has been reported to be a site of JCV latency, where neurotropic transformation of the virus could possibly take place, as the regulatory region of JCV identified in bone marrow aspirates correlates with the “PML-type” regulatory region of JCV found in the brain (Tan et al. 2009). However, recent studies in the context of cases of PML occurring in multiple sclerosis patients show that CD34-positive cells mobilized to the circulation by natalizumab do not contain JCV DNA (Warnke et al. 2011). Thus, while JCV DNA can be extracted from bone marrow aspirates, and bone marrow has been reported to be an important site of JCV latency, the phenotype of the cell in the bone marrow and the phenotype of the cell in the blood seeding JCV throughout different organs of the human body remain unknown. New studies show that in addition to stem/progenitor cells of the hematopoietic lineage, the bone marrow contains a heterogenous population of nonhematopoietic stem and progenitor cells that have the ability to differentiate into a variety of nonhematopoietic tissues (Prockop 1997; Jiang et al. 2002; Kucia et al. 2004, 2006a; Keene et al. 2003). These cells exist during development and persist in small numbers in adults (Pittenger et al. 1999; Colter et al. 2000; Kucia et al. 2006a). Some of the nonhematopoietic cells may enter the circulation and traffic throughout different organs, where they may be involved in the maintenance of homeostasis and/or repair of solid organs (Prockop et al. 2003; Sasaki et al. 2008; Mezey et al. 2003; Cogle et al. 2004). These cells can be mobilized from the bone marrow into peripheral blood and are actively recruited to sites of injury, further suggesting a role for these cells in tissue maintenance and repair (Rochefort et al. 2006; Sasaki et al. 2008; Kucia et al. 2006b; Paczkowska et al. 2009). However, in pathological situations, bone marrow-derived cells can also contribute to the pathogenesis of diseases, such as atherosclerosis (Sata et al. 2002), gastric cancer (Houghton et al. 2004), osteosarcoma (Stark et al. 1986; Berger et al. 2008), or childhood leukemia (Greaves 1999). The presence of JCV in the bone marrow suggests potential scenarios in which nonhematopoietic bone marrow cells may be involved in infection with JCV. In pathological situations, bone marrow-derived multipotent nonhematopoietic cells harboring JCV could be recruited to different tissues, where they potentially could undergo neoplastic conversion into tumorigenic cells and contribute to the development of tumors. Thus, we propose that in addition to the hematopoietic fraction of the bone marrow, the nonhematopoietic subpopulation of bone marrow cells can be another potential source of JCV latency, spreading, and ultimately oncogenic transformation.

Pathogenesis of JCV: Why Nonhematopoietic Bone Marrow-Derived Stem Cells? The role of stem cells in the pathogenesis of human diseases, in particular the development of cancer, has recently gained a lot of attention. Cancer is viewed as a stem cell disease because of accumulating data suggesting that cancers are maintained by a

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small population of cancer cells functioning as stem-like cells, as well as evidence that stem cells are a possible origin of some cancer stem cells (Reya et al. 2001; Dontu et al. 2003; Singh et al. 2004; Xie 2009). There are several indications that some JCV T-Ag-associated malignancies may in fact originate in primitive nonhematopoietic bone marrow-derived stem cells that can migrate and are often found in multitude of tissues. As mentioned above, the nonhematopoietic stem-like cells coexist in the bone marrow and may be a possible source of neural and nonneural cell types (Lepski et al. 2010 Sasaki et al. 2008). It is well-known that JCV has a strong association with the bone marrow and the nervous system (reviewed by White and Khalili 2011), and JCV T-Ag is associated with neural and nonneural tumors (see Table 17.1). Thus, these unique bone marrow-derived stem cells, which have the ability to migrate and a potential to transition into nonneural and neural-like cells in homed tissues, may be involved in JCV dissemination and pathogenesis. From the cancer development point of view, recent studies have shown that tumors may arise from the transformation of normal stem/progenitor cells into tumorigenic cancer stem cells, which are responsible for the initiation, maintenance, and recurrence of tumors after therapy (Reya et al. 2001; Singh et al. 2004; Xie 2009). Given that JCV induces neuronal and glial tumors in animal models and has been associated with these types of tumors in humans, one may anticipate that neural stem or progenitor cells may be the target of T-Ag-induced transformation in the brain. However, other evidence demonstrates the association of JCV with peripheral neuroblastic tumors in animal models and nonneural types of cancers, including colorectal cancers and cancers of the gastrointestinal tract in humans, which further suggests alternative models of JCV dissemination throughout different organs and other cells of origin of T-Ag-induced malignancies. Because of the widespread nature of JCV, one source of ubiquitous stem cells may be the bone marrow-derived nonhematopoietic stem cells that can spill into the circulatory system from the bone marrow and ultimately reside in various tissues (Rochefort et al. 2006; Sasaki et al. 2008; Paczkowska et al. 2009). It is possible that JCV-infected nonhematopoietic stem cells trafficking through the body could take up residence in the brain and give rise to cells that can undergo lytic infection, i.e., oligodendrocytes leading to development of PML, or alternatively can give rise to neurons or astrocytes that undergo transformation and generate astrocytic or neuronal types of brain tumors (Fig. 17.1). The concept that mobile bone marrow-derived multipotent stem cells, with the ability to give rise into brain and other cell types (Rochefort et al. 2006; Sasaki et al. 2008; Paczkowska et al. 2009; Lepski et al. 2010), may be the common cell of origin of T-Ag-related malignancies perhaps could explain the association of JCV with different types of tumors found in the brain and non-CNS tissues. In addition to the above considerations, support for a stem cell origin of T-Aginduced malignancies comes from the fact that signaling pathways (WNT, p53, pRB) and mechanisms by which T-Ag may lead to cellular transformation and development of tumors in animal models appear to be essential for the development and regulation of stem cells (Molofsky et al. 2004; Yang and Hinds 2007; Xie 2009). Taken together, these data suggest that T-Ag expression may potentially dysregulate pathways of stem cell self-renewal and contribute to neoplastic transformation of these cells.

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Fig. 17.1 JCV infection and dissemination from the bone marrow. Hypothesis for JCV infection of nonhematopoietic stem cells from the bone marrow in dissemination of JC virus in the host and pathogenesis of JC virus-induced diseases. Bone marrow harbors latent JC virus, where viral DNA replication may occur sporadically and at a low level establishing a persistent long-lived infection. Nonhematopoietic cells harboring JC virus can enter the circulation and transport the virus to the brain and other organs. Once in the brain, the bone marrow-derived stem cells may differentiate into cells permissive for viral replication, i.e., oligodendrocytes, or can lead to the infection of native oligodendrocytes, both of which result in lytic infection and the development of PML. Alternatively, nonhematopoietic stem cells may differentiate into neuronal or astrocytic cells which are non- or semipermissive for JCV lytic infection, in which initiation of T-antigen expression can induce oncogenic transformation leading to the development of glial or neuronal tumors in the brain. JC virus can also be transported to other organs via nonhematopoietic stem cells from the bone marrow leading to the development of non-CNS tumors (not shown)

Transformation of Bone Marrow-Derived Nonhematopoietic Cells by JCV T-Ag An important question related to the T-Ag-induced transformation of nonhematopetic bone marrow-derived cells is whether these cells can be transformed by T-Ag and can generate tumorigenic cells. Recently, new data from our group has shown that nonhematopoietic cells from the bone marrow, commonly described as mesenchymal stem cells or multipotent stromal cells, can be transformed by JCV T-Ag in culture. When T-Ag-transformed cells were propagated as xenografts in the flanks of nude mice, they developed tumors. Histologically, the tumors were heterogenous with mesenchymal, neural, and neural crest characteristics (Del Valle et al. 2010). It is assumed that oncogenesis is a reversal of ontogeny in cell development (Rapp et al. 2008). Thus, our data is particularly interesting as recent reports suggest that nonhematopoietic mesenchymal cells from the bone marrow may be of neural crest

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origin (Miller 2007). Notably, our data show T-Ag-mediated oncogenic transformation of nonhematopoietic bone marrow cells with multipotent capacities that are tumorigenic in vivo. Phenotypical heterogeneity of tumors that developed in the flank suggests that such multipotent cells transformed with JCV T-Ag may be the origin of some T-Ag-associated neural and nonneural tumors, in particular when influenced by the microenvironment of surrounding host tissues. Thus, these data provide this data provides a novel stem cell model of T-Ag-induced oncogenic transformation that may lend some support for the aforementioned hypothesis that some T-Agassociated tumors may originate from primitive bone marrow-derived multipotent cells that persist in adults and have the ability to differentiate into variety of nonhematopoietic cell types. Our model is supported by observations made by others, in which various tumor types developed after subcutaneous injection of transformed bone marrow-derived cells (Liu et al. 2006), and evidence from animal models of gastric cancers showing that epithelial tumors may arise from bone marrow-derived cells during chronic infection (Houghton et al. 2004).

Conclusions JCV transformation and tumorigenesis are well-established in cell culture and experimental animal models while their role in human cancer remains contentious. JCV exerts a variety of molecular effects, including the action of the oncogenic protein, T-Ag, on the tumor suppressors p53 and pRb as well as the induction of DNA damage, as described above. The nature of JC viral DNA in tumors is unknown. JCV may be present in stem cells in the bone marrow and perhaps be carried via the blood stream to the brain and other organs. Notably, JCV in the bone marrow is rearranged in the noncoding region, i.e., “PML-type” configuration, which is in contrast to the reservoir of JCV in the kidney, which has an archetypal configuration. Interestingly, some JCV-associated tumors contain viral DNA with “PML-type” sequences suggesting that the rearrangements necessary for pathogenesis of PML may also occur for virus that becomes associated with tumors. It is important to stress that studies have found that JCV VP1 is not expressed in JCV-associated tumors, indicating that viral replication is not occurring. Expression of JCV T-Ag and the more recently identified agnoprotein is usually robust in tumor cells. In the case of T-Ag, expression was found to occur in some, but not all, of the tumor cells containing JCV DNA in JCV-associated human medulloblastoma (Khalili et al. 1999; Krynska et al. 1999b). This is also found in JCV-transgenic mice, where immunohistochemical analysis of JCV-induced mouse medulloblastomalike tumors showed a heterogenous population of T-positive and T-negative cells (Krynska et al. 2000). The molecular and cellular processes that drive the heterogeneity of T-Ag expression among the tumor cells are not yet known. However, this phenomenon is very interesting in the light of the cancer stem cell hypothesis of tumor

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development and raises questions as to whether JCV T-Ag is expressed in the fraction of cancer stem cells that replicate to drive the growth of tumor. In view of the reports on the association of JCV with a broad variety of tumors and the accumulating evidence maintaining that cancer may originate from transformation of stem cells, we raise several important considerations related to the pathogenesis of JCV: (1) stem/progenitor cells may be a target of JCV T-Ag-induced oncogenic transformation; (2) we envision that ubiquitous nonhematopoietic bone marrow-derived stem/progenitor cells, which have been postulated to give rise to various cell types, including brain, may not only be the origin of some of T-Ag-associated cancers, but also play a role in dissemination of JCV to the brain and other organs. Therefore, we hypothesize that potential persistent infection of mobile nonhematopoietic bone marrow cells with JCV can contribute to viral dissemination into the CNS. Notably, JCV in the bone marrow is rearranged in the noncoding region, i.e., “PML-type” configuration (Tan et al. 2009). In the brain, nonhematopoietic bone marrow-derived cells harboring JCV may undergo differentiation toward permissive for viral replication in oligodendrocytes that may result in the development of PML or may undergo differentiation toward cells of neuronal or astrocytic type, which are non/semipermissive for viral replication, where T-Ag expression may potentially induce oncogenic transformation and generation of cancer stem cells leading to the development of tumors in the brain (Fig. 17.1). Another possibility is that in the brain JCV may infect resident neural stem/progenitor cells, which differentiate into major cell types in the brain. Infected oligodendrocytes undergo lytic infection resulting in destruction of the host cell and demyelination seen in PML while astrocytic cells allow expression of T-antigen but cannot complete the lytic cycle, leading to cellular transformation and tumor formation (Fig. 17.2). These two models are not mutually exclusive. However, we are aware that this hypothesis requires experimental evidence to support our concept. We propose that the research exploring the oncogenic potential of JCV T-Ag that is focused on novel in vitro and in vivo models of T-Ag-induced oncogenic transformation with the contribution of adult stem/progenitor cells from the bone marrow and brain and the identification of cancer stem cells in T-Ag-associated cancers, in particular human cancers, is necessary in order to better understand the role of JCV T-Ag in cancer. In summary, available data suggest that JCV is a ubiquitous virus that usually exists in a latent state as an asymptomatic infection of the bone marrow and/or brain and perhaps spreads by intermittent and subclinical infection events. JCV involvement in tumors may be caused by very rare abortive infection event giving rise to tumorigenic cells driven to grow progressively by T-Ag expression. A better understanding of these processes may lead to advances in understanding the role of JCV in human cancer. Acknowledgments We wish to thank members of the Department of Neuroscience and Center for Neurovirology for their continued support, insightful discussion, and sharing of ideas.

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Fig. 17.2 JCV infection and transformation of neural stem cells. Bone marrow-derived stem cells can traffic to the brain, where they infect resident neural stem cells. Neural stem cells infected with JCV differentiate along the glial lineage into either oligodendrocytes, which are fully permissive for viral replication, or into astrocytic cells which are semipermissive allowing for T-antigen expression but not viral replication. Infected oligodendrocytes undergo lytic infection resulting in destruction of the host cell and demyelination seen in PML while astrocytic cells allow expression of T-antigen but cannot complete the lytic cycle, leading to cellular transformation and tumor formation

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Bofill-Mas S, Clemente-Casares P, Major EO et al (2003) Analysis of the excreted JC virus strains and their potential oral transmission. J Neurovirol 9:498–507 Boldorini R, Caldarelli-Stefano R, Monga G et al (1998) PCR detection of JC virus DNA in the brain tissue of a 9-year-old child with pleomorphic xanthoastrocytoma. J Neurovirol 4:242–245 Brooks BR, Walker DL (1984) Progressive multifocal leukoencephalopathy. Neurol Clin 2:299–313 Caldarelli-Stefano R, Boldorini R, Monga G et al (2000) JC virus in human glial-derived tumors. Hum Pathol 31:394–395 Cogle CR, Yachnis AT, Laywell ED et al (2004) Bone marrow transdifferentiation in brain after transplantation: a retrospective study. Lancet 363:1432–1437 Colter DC, Class R, DiGirolamo CM et al (2000) Rapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrow. Proc Natl Acad Sci USA 97:3213–3218 Darbinyan A, Darbinian N, Safak M et al (2002) Evidence for dysregulation of cell cycle by human polyomavirus, JCV, late auxiliary protein. Oncogene 21:5574–5581 Darbinyan A, Siddiqui KM, Slonina D et al (2004) Role of JCV agnoprotein in DNA repair. J Virol 78:8593–8600 Darbinyan A, White MK, Akan S et al (2007) Alterations of DNA damage repair pathways resulting from JCV infection. Virology 364:73–86 Del Valle L, Khalili K (2010) Detection of human polyomavirus proteins, T-antigen and agnoprotein, in human tumor tissue arrays. J Med Virol 82:806–811 Del Valle L, Croul S, Morgello S et al (2000) Detection of HIV-1 Tat and JCV capsid protein, VP1, in AIDS brain with progressive multifocal leukoencephalopathy. J Neurovirol 6:221–228 Del Valle L, Gordon J, Assimakopolou M et al (2001a) Detection of JC virus DNA sequences and expression of the viral regulatory protein, T-antigen, in tumors of the central nervous system. Cancer Res 61:4287–4293 Del Valle L, Gordon J, Ferrante P et al (2001a) JC virus in experimental and clinical brain tumorigenesis. In: Khalili K and Stoner GL (eds) Human polyomaviruses: molecular and clinical perspective. New York Wiley-Liss Inc., New York pp. 409–430 Del Valle L, Enam S, Lara C et al (2002a) Detection of JC polyomavirus DNA sequences and cellular localization of T-antigen and agnoprotein in oligodendrogliomas. Clin Cancer Res 8:3332–3340 Del Valle L, Delbue S, Gordon J et al (2002b) Expression of JC virus T-antigen in a patient with MS and glioblastoma multiforme. Neurology 58:895–900 Del Valle L, Enam S, Lara C et al (2004) Primary central nervous system lymphoma expressing the human neurotropic polyomavirus, JC virus, genome. J Virol 78:3462–3469 Del Valle L, White MK, Enam S et al (2005) Detection of JC virus DNA sequences and expression of viral T antigen and agnoprotein in esophageal carcinoma. Cancer 103:516–527 Del Valle L, White MK, Khalili K (2008) Potential mechanisms of the human polyomavirus JC in neural oncogenesis. J Neuropathol Exp Neurol 67:729–740 Del Valle L, Pina-Oviedo S, Perez-Liz G et al (2010) Bone marrow-derived mesenchymal stem cells undergo JCV T-antigen mediated transformation and generate tumors with neuroectodermal characteristics. Cancer Biol Ther 9; 286–294 Dontu G, Al-Hajj M, Abdallah WM, Clarke MF, Wicha MS (2003) Stem cells in normal breast development and breast cancer. Cell Prolif 36(Suppl 1):59–72 Enam S, Del Valle L, Lara C et al (2002) Association of human polyomavirus JCV with colon cancer: evidence for interaction of viral T-antigen and beta-catenin. Cancer Res 2:7093–7101 Franks RR, Rencic A, Gordon J et al (1996) Formation of undifferentiated mesenteric tumors in transgenic mice expressing human neurotropic polyomavirus early protein T-Antigen. Oncogene 12:2573–2578 Gan DD, Khalili K (2004) Interaction between JCV large T-antigen and beta-catenin. Oncogene 23:483–490 Gordon J, Del Valle L, Otte J et al (2000) Pituitary neoplasia induced by expression of human neurotropic polyomavirus, JCV, early genome in transgenic mice. Oncogene 19:4840–4846

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Sariyer IK, Khalili K, Safak M (2008) Dephosphorylation of JC virus agnoprotein by protein phosphatase 2A: inhibition by small t antigen. Virology 375:464–479 Sasaki M, Abe R, Fujita Y et al (2008) Mesenchymal stem cells are recruited into wounded skin and contribute to wound repair by transdifferentiation into multiple skin cell type. J Immunol 180:2581–2587 Sata M, Saiura A, Kunisato A et al (2002) Hematopoietic stem cells differentiate into vascular cells that participate in the pathogenesis of atherosclerosis. Nat Med 8:403–409 Schaumburg C, O’Hara BA, Lane TE et al (2008) Human embryonic stem cell-derived oligodendrocyte progenitor cells express the serotonin receptor and are susceptible to JC virus infection. J Virol 82:8896–8899 Shin SK, Li MS, Fuerst F et al (2006) Oncogenic T-antigen of JC virus is present frequently in human gastric cancers. Cancer 107:481–488 Shollar D, Del Valle L, Khalili K et al (2004) JCV T-antigen interacts with the neurofibromatosis type 2 gene product in a transgenic mouse model of malignant peripheral nerve sheath tumors. Oncogene 23:5459–5467 Singh SK, Hawkins C, Clarke ID et al (2004) Identification of human brain tumour initiating cells. Nature 432:396–401 Small JA, Khoury G, Jay G et al (1986) Early regions of JC virus and BK virus induce distinct and tissue-specific tumors in transgenic mice. Proc Natl Acad Sci USA 83:8288–8292 Stark A, Aparisi T, Ericsson JL (1986) Human osteogenic sarcoma: fine structural localization of adenosine triphosphatase. Ultrastruct Pathol 10:145–155 Tan CS, Dezube BJ, Bhargava P et al (2009) Detection of JC virus DNA and proteins in the bone marrow of HIV-positive and HIV-negative patients: implications for viral latency and neurotropic transformation. J Infect Dis 199:881–888 Till JE, McCulloch EA (1961) A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radiat Res 14:213–222 Till JE, McCulloch EA, Siminovitch L (1964) A stochastic model of stem cell proliferation, based on the growth of spleen colony-forming cells. Proc Natl Acad Sci USA 51:29–36 Tornatore C, Berger JR, Houff SA et al (1992) Detection of JC virus DNA in peripheral lymphocytes from patients with and without progressive multifocal leukoencephalopathy. Ann Neurol 31:454–462 Walker DK, Padgett BL, ZuRhein GM et al (1973) Human papovavirus (JC): induction of brain tumors in hamsters. Science 81:674–676 Warnke C, Smolianov V, Dehmel T et al (2011) CD34+ progenitor cells mobilized by natalizumab are not a relevant reservoir for JC virus. Mult Scler 17:151–156 White MK, Khalili K (2004) Polyomaviruses and human cancer: molecular mechanisms underlying patterns of tumorigenesis. Virology 324:1–16 White MK, Khalili K (2006) Interaction of retinoblastoma protein family members with large T-antigen of primate polyomaviruses. Oncogene 25:5286–5293 White MK, Khalili K (2011) Pathogenesis of progressive multifocal leukoencephalopathy – revisited. J Infect Dis 203:578–586 White MK, Gordon J, Reiss K et al (2005) Human polyomaviruses and brain tumors. Brain Res Rev 50:69–85 White MK, Skowronska A, Gordon J et al (2006) Analysis of a mutant p53 protein arising in a medulloblastoma from a mouse transgenic for the JC virus early region. Anticancer Res 26:4079–4092 Xie Z (2009) Brain tumor stem cells. Neurochem Res 34:2055–2066 Yamaoka S, Yamamoto H, Nosho K, Taniguchi H, Adachi Y, Sasaki S, Arimura Y, Imai K, Shinomura Y (2009) Genetic and epigenetic characteristics of gastric cancers with JC virus T-antigen. World J Gastroenterol 15:5579–5585 Yang HS, Hinds PW (2007) pRb-mediated control of epithelial cell proliferation and Indian hedgehog expression in mouse intestinal development. BMC Dev Biol 7:6

Chapter 18

Merkel Cell Polyomavirus David Schrama and Jürgen C. Becker

Polyomaviruses The first polyomaviruses have been identified in mice in the early 1950s, i.e., the K virus [now known as the murine pneumotropic virus] and the murine polyomavirus (Kilham 1952; Gross 1953). The name polyomavirus, however, was first given in 1958 due to its ability to produce a variety of solid tumors (Stewart et al. 1958). In the species list 2009 version 7 of the International Committee on Taxonomy of Viruses (ICTV), 13 different species are listed. Among them, simian vacuolating virus 40 (SV40) might be the most prominent member. SV40 was found infecting rhesus monkey kidney cells used for the production of polio vaccines (Sweet and Hillemann 1960) raising health concerns due to the widespread administration of the polio vaccine and the tumorigenicity of SV40 in experimental models. Notably, despite intense scientific effort, a causal relationship between SV40 and human cancers could not yet been established. However, these studies on SV40 contributed largely to our understanding of viral oncogenesis and cellular biology (Cheng et al. 2009). Of the 13 species of polyomavirus listed by ICTV in 2009, 4 are human species. Two of these are the JC polyomavirus (JCV) and BK polyomavirus (BKV) associated with progressive multifocal leukoencephalopathy in immunocompromised hosts and nephropathy in renal transplant recipients, respectively. Two others were isolated from respiratory samples in symptomatic pediatric patients and named after the institutions where they were identified, i.e., the Karolinska Institute virus (KIV) and Washington University virus (WUV). Within the list of the ICTV, however, the newest human members are not yet included. Indeed, in the last 2 years, four new

D. Schrama • J.C. Becker (*) Division of General Dermatology, Department of Dermatology, Medical University of Graz, Auenbruggerplatz 8, A-8036 Graz, Austria e-mail: [email protected] E.S. Robertson (ed.), Cancer Associated Viruses, Current Cancer Research, DOI 10.1007/978-1-4614-0016-5_18, © Springer Science+Business Media, LLC 2012

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Fig. 18.1 Appearance and structure of a polyomavirus icosahedral capsid and MCV genome. (a) The viral capsid is composed of 72 pentamers of the major capsid protein VP1 that contacts 72 copies of the minor capsid proteins VP2/3 arranged on an icosahedral lattice. The appearance of SV40 serves as an example. (b) Map of MCV genome organization. VP3 is not depicted, since currently there is no indication that VP3 is generated by internal translation from the MCV genome. However, the MCV genome encodes a pre-miRNA (MCV-mir-M1) which is expressed from the late strand, MCV-mir-M1 lies antisense to the early transcripts, and can negatively regulate early transcripts’ expression. The mature sequences are MCV-mir-M1 5p and MCV-mir-M1 3p. In MCC, the MCV miRNA probably plays no role as the tumors are likely only competent to express early gene products, and the miRNA is presumably only expressed during lytic infection when the late genes are expressed (Seo et al. 2009)

species were reported (Feng et al. 2008; Schowalter et al. 2010; van der Meijden et al. 2010): Merkel cell polyomavirus (MCV or MCPyV), human polyomavirus-6 (HPyV6) and HPyV7, and trichodysplasia spinulosa-associated polyomavirus (TSV). Thus, currently, eight human polyomaviruses have been described. Polyomaviruses are nonenveloped, 40–45-nm icosahedral capsids containing approximately 5 kb of double-stranded, circular DNA. The DNA closely associated with histones is packaged into chromatin-resembling cellular genomes (minichromosomes). The polyomavirus genome is almost evenly divided into an early and a late region encoded on opposite strands (Fig. 18.1). Gene expression is regulated by a noncoding, transcriptional control region, which also harbors the origin of DNA replication. Transcription of the early region from the early promoter starts immediately upon entry and uncoating of the genome. Early message is differentially spliced to encode at least two and up to five proteins in the respective polyomaviruses; the large tumor (T) antigen (LT) and small T antigen (sT) are invariantly expressed in all polyomaviruses. The T antigens are the oncogenically transforming proteins as they initiate viral DNA replication. After the onset of viral DNA replication, the late region is expressed from the late promoter. From the late message, three to four capsid proteins VP1, VP2, VP3, and VP4 are generated by differential splicing and internal translation. In this regard, VP3, and VP4 when present, is generated by internal translation of VP2 from an in-frame methionine (Met) codon. Since MCV lacks the conserved Met-Ala-Leu motif that forms the amino-terminus of all previously described polyomavirus VP3 proteins, MCV may encode only VP1 and VP2, but not a functional VP3 protein (Pastrana et al. 2009) (Fig. 18.1).

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Polyomaviruses replicate their genomes in the nucleus of the host cell. Since they encode only a few proteins, polyomaviruses heavily rely on cellular proteins for both DNA replication and gene transcription.

Merkel Cell Carcinoma and MCV Detection In 1972, Toker described five patients with unusual skin tumors characterized by histologically anastomosing trabeculae and cell nests (Toker 1972). In 1982, Rywlin suggested to name this tumor Merkel cell carcinoma (MCC) after the possible cell of origin, the Merkel cell, which functions as slowly adapting mechanoreceptor in the basal layer of the epidermis (Rywlin 1982; Maricich et al. 2009). For a long time, Merkel cells were believed to be derived from the neural crest. Thus, the hypothesis from several groups that MCC may arise from primitive totipotent epidermal stem cells, able to differentiate along different cell lines, was contradictory to the initial hypothesis (Albores-Saavedra et al. 2009). Recently, however, the report that mammalian Merkel cells do not develop from neural crest progenitors but rather from epidermal stem cells conciliated these two hypotheses (Van Keymeulen et al. 2009; Morrison et al. 2009). Notably, MCCs express both neuroendocrine and epithelial markers. MCC is an uncommon cutaneous malignancy, with age-adjusted incidence rates of 0.18–0.41 per 100,000 persons. MCCs are most often localized on the sun-exposed skin of Caucasians older than 50 years. The mean age of patients at the time of initial diagnosis is about 75 years (Albores-Saavedra et al. 2009). In a study of 6,700 MCC patients, 66% were diagnoses with local, 27% with nodal, and 7% with distant metastatic diseases (Lemos et al. 2010). The initial stage largely affects 5-year survival rate with 64% for patients with local, 39% for patients with regional, and 18% for patients with distant metastatic disease (Lemos et al. 2010). MCC occurs much more frequently in severely immunosuppressed populations caused, for example, by immune-suppressive drugs in organ transplant patients, lymphoma, or HIV infection (Rollison et al. 2010). Noteworthy, many cancers with infectious etiologies are more often observed in the context of immunosuppression, such as Kaposi’s sarcoma, lymphomas, cervical, oropharyngeal, and penile cancers in HIV-infected individuals (Engels et al. 2008) and nonmelanoma skin cancers, lymphomas, and cancers of the oral cavity, vulva, and vagina in organ transplant recipients (Adami et al. 2003). Thus, the observation that MCC is also more common in immunocompromised individuals first raised the hypothesis that MCC may also have an infectious origin. Indeed, Feng and coworkers were able to provide evidence for a possible viral oncogenesis of MCC (Feng et al. 2008). They applied the digital transcriptome subtraction (DTS) technique to specifically seek for an infectious agent in MCC. DTS involves transcriptome sequencing followed by in silico subtraction to exclude human from candidate viral transcripts (Feng et al. 2007). Pooling cDNA libraries made from four MCC lesions, a DTS candidate was detected demonstrating a significant degree of similarity to the LT of the African

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green monkey lymphotropic polyomavirus (LPV). The complete genome of 5,387 bp was subsequently sequenced by PCR walking using primers designed from the DTS viral transcript [Figure 1; (Feng et al. 2008)] After their initial identification of the MCV genome, they verified MCV presence in eight of ten MCCs. In six of them, they even could detect the viral genome integrated into the human genome. Importantly, the patterns of integration suggest that MCV infection/integration occurs before the clonal expansion of the tumor cells. Meanwhile, MCV presence in MCCs has been generalized to geographically diverse populations with about 80% of MCCs positive for MCV (reviewed in Rollison et al. 2010). The fraction of MCV-negative MCCs suggests that MCC is a heterogenous disease having at least two pathoetiologies. Interestingly, until recently, MCV’s closest relatives known to date were LPV from some African green monkeys and ChPyV from one chimpanzee. This was fortified by a report of Leendertz et al. identifying two new groups of polyomaviruses in chimpanzees and gorillas, demonstrating that the great apes – the closest phylogenetic relatives of humans – are infected with such polyomaviruses, which are by far the closest known relatives to MCV (Leendertz et al. 2010). Several serological and molecular studies have meanwhile established that MCV is prevalent in the general population (Schowalter et al. 2010). Indeed, in a recent study of Weiland et al., MCV was found in anogenital, oral samples (31%), and eyebrow hairs (50%) of HIV-positive men as well as in 8 of 13 forehead swabs (62%) of healthy controls suggesting a widespread distribution of MCV (Wieland et al. 2009). Moreover, infection with MCV is common: 42–77% of subjects from the general population have antibodies to capsid proteins of MCV. For example, among 166 healthy blood donors from the USA, IgG antibodies against MCV VP1 and VP2 proteins were observed in 107 (64%) (Kean et al. 2009). Interestingly, the anti-VP1 antibody titers were significantly higher in patients with MCV-positive MCC as compared to patients with MCV-negative tumors or healthy controls. In a separate study of 1,501 healthy adult blood donors, the prevalence of IgG antibodies against MCV VP1 was 46% (Tolstov et al. 2009). Although MCV prevalence increases in an age-dependent manner, initial exposure to MCV seems to occur already at very young ages, with seroprevalences of 20, 30, and 40% for children aged 1–5, 5–10, and 10–15 years, respectively (Kean et al. 2009). In contrast to VP antibodies, antibodies recognizing MCV T antigens are relatively specifically associated with MCC and almost not detectable in healthy individuals. Moreover, although they do not effectively protect against disease progression, they may serve as a clinically useful indicator of disease status (Paulson et al. 2010). Given the widespread presence of MCV, its mere detection in most of the MCC cases is certainly only a first indication for the involvement of this virus in the etiology of these cases. It could also be just a bystander virus. For example, MCV presence has been infrequently observed in other, non-MCC cancers – albeit often with a lower copy number of virus per genome (Becker et al. 2009a; Murakami et al. 2011; Andres et al. 2009). In experimental models, polyomaviruses induce tumors upon integration of the viral genome into the host genome (Poulin and DeCaprio 2006). Integration might render the viral infection oncogenic by disrupting viral genes, by creating virus–cell fusion proteins, or by dysregulating expression of

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cellular oncogenes or tumor-suppressor genes. Therefore, it is important that the initial report of MCV by Feng and colleagues not only noted an integration in six of the eight MCV-positive MCC samples, but that the banding pattern on Southern blots also indicated a monoclonal integration (Feng et al. 2008). Subsequent studies confirmed clonal integration in ten out of ten MCV-positive tumors examined (Sastre-Garau et al. 2009). Notably, integration sites varied between different MCC tumors suggesting that this event is likely to occur at random sites in the host genome. Since primary and metastatic tumor samples of one MCV-positive MCC patient exhibited an identical integration pattern, MCV integration most likely occurs before tumor metastasis. Thus, these observations suggest that infection and subsequent integration precede clonal expansion of tumor cells (Fig. 18.2). This assumption should translate into at least one MCV copy per cell. Indeed, several studies reported average copy numbers of more than one MCV genome per cell (Shuda et al. 2009; Laude et al. 2010; Houben et al. 2010a). However, Bhatia et al. reported that many of the MCV-positive tumors had less than 1 virus copy per 300 cells (Bhatia et al. 2010b), and certainly in most studies there are at least some samples with a low copy number. Future studies have to clarify whether those samples lost the virus, are only contaminated by MCV, or that an infected cell can contribute to transformation of neighboring uninfected cells by paracrine mechanisms as suggested by Bhatia et al. (2010b).

Small and Large T Antigens, the Potential Oncogenic Proteins Since MCV integrate – just like SV40 in experimental models (Pipas 2009) – at different sites into the host genome, it seems unlikely that destruction of certain host genes by integration contributes to the etiology of MCC. Indeed, the prime candidates for a transforming activity of MCV are the early proteins, i.e., small and large T antigens, which are referred to as tumor antigens because they were originally detected using antibodies from tumor-bearing animals. The functions of MCV’s sT and LT have not yet been unraveled, as only a short period has passed since the discovery of MCV. In contrast, much time and effort have been spent on the characterization of SV40 T antigens. Thus, the following functional data refers mostly to findings on SV40 biology. The involved mechanisms, however, are probably similar for MCV, especially as MCV T antigens contain most of the respective domains (Fig. 18.3). As mentioned before, polyomaviruses rely heavily on the cellular replication machinery to replicate their genome because of their small genome size. Indeed, by reprogramming the host cell cycle, they abrogate the quiescent state and induce progression into S phase. Thereby, they create a suitable environment for viral replication. Importantly, the molecules targeted by the virus to promote unscheduled DNA replication or to inhibit innate immune signaling in the setting of viral replication are often the same as those involved in oncogenesis. In this regard, viral infection leads to transformation of a range of cultured rodent and human cells and induces tumors in newborn hamsters (Pipas 2009).

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Fig. 18.2 Proposed viral carcinogenesis of Merkel cell carcinoma. GT1b has been identified as a putative host cell receptor for MCV (Erickson et al. 2009). Polyomaviruses must enter host cells by endocytosis and navigate through various intracellular compartments, where they undergo sequential conformational changes which enable them to uncoat and deliver the DNA genome into the nucleus (Tsai and Qian 2010). In MCCs, the MCV genome might get integrated into the host genome, cause transformation, and is then found clonally integrated in all MCCs of one patient. The interference of TA function might be a future target of MCC therapy, since knockdown in MCV-positive MCC cell lines caused growth arrest and cell death

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Fig. 18.3 MCV small and large T antigen domains. The T antigen region of MCV is depicted (upper row). Through alternative splicing, the LT (middle row) and sT proteins are generated. Integrated MCV genomes generally harbor a mutation or deletion truncating the LT (truncated area is indicated by arrow). Only those domains likely to be involved in carcinogenesis are depicted with the respective cellular protein partners

Only SV40 constructs containing the early region, i.e., sT and LT, are sufficient for transformation while those encoding only the late region do not transform (Pipas 2009). By binding to the viral origin of DNA replication and recruiting cellular replication factors, LT promotes DNA synthesis and hence plays a key role in regulating the viral life cycle. As a multifunctional nuclear phosphoprotein, LT also prepares the cell for replication by stimulating cell cycle progression from G0/G1 into S phase. The latter function is likely to be the main contributor to oncogenic transformation. Notably, transfection of origin-deficient SV40 genomic DNA significantly enhances in vitro transformation of human cells, suggesting that viral replication presents an obstacle to stable transformation (Small et al. 1982). Importantly, in nearly all tumor-derived MCV genomes sequenced, missense mutations or deletions in the early region result in the expression of truncated LT antigens while MCV strains derived from non-MCC tissues encode full-length LT (Shuda et al. 2008; Sastre-Garau et al. 2009). These mutations truncating either the origin-binding domain (OBD) or the helicase domain eliminate the ability of MCV to replicate. Until recently, these MCC LT mutations have not been detected in MCV present outside of MCC tumors, and thus are signature mutations (zur Hausen 2008). In a recent publication analyzing 70 chronic lymphocytic leukemia (CLL) cases, however, an LT deletion in the helicase gene has been described in 6 of 19 MCV-positive CLLs. This observation suggests that MCV might have a role of MCV in a subset of CLL cases, although the integration of this virus into the host genome has yet to be proven (Pantulu et al. 2010). Thus, the loss of replication activity of MCV LT integrated in Merkel cell tumor cells provides further evidence that MCV is not simply a “passenger virus” which happens to grow well in MCC cells. Besides the OBD and helicase/ATPase domains required for viral replication, MCV LT contains additional conserved features of other polyomavirus LT proteins,

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the conserved region 1 (CR1), the DnaJ domain, and the retinoblastoma (RB) tumor-suppressor protein family-binding motif LXCXE (Fig. 18.3, Shuda et al. 2008). In addition, for the p53/DNA-binding region, there is also a homology of about 50% with the other polyomaviruses (Johnson 2010). In the adenovirus 1A, CR1 is required for the cell-transforming E1A region to bind RB (Berk 2005). In SV40, however, crystallographic data suggest that the CR-1-like region is buried in the hydrophobic core indicating that the function of CR1 might be different in polyomaviruses LT (Sullivan and Pipas 2002). The DnaJ domain, present in both sT and LT, is a molecular chaperone which recruits DnaK family chaperones (i.e., the heat-shock protein family) to perform functions, such as protein folding, protein transport, or remodeling of protein complexes. In this regard, polyomavirus DnaJ domain binds the constitutively expressed HSC70 (Whalen et al. 2005). It seems that the DnaJ domain is critical for stimulation of viral replication and to enhance oncogenic transformation by functionalinactivating RB members (Campbell et al. 1997; Pipas 1998). Notably, the contribution of the DnaJ domain to LT transformation seems variable. For example, DnaJ activity is unnecessary for anchorage-independent growth and for immortalization of mouse embryo fibroblasts, but required for promotion of growth in low serum and high saturation density (Stubdal et al. 1997). One major domain for the transformation activity of LT is the RB family-binding motif LXCXE. Indeed, interaction of T antigen with the RB family of proteins is essential for transformation. It is currently believed that LT must block the growthsuppressive functions of RB proteins in order to induce transformation. RB is a negative regulator of cell proliferation by repressing the E2F transcription factor which in turn regulates the expression of genes required for entry and progression of the cell cycle. By binding to the hypo- or underphosphorylated form of RB, LT disrupts pRB/E2F complexes (Fig. 18.4a). As a consequence, many E2F target genes are activated or derepressed leading to cell cycle progression (Pipas 2009). For MCV, cotransfection of LT mutants with RB in 293 cells followed by immunoprecipitation confirmed MCV LT binding to RB through the LXCXE motif (Shuda et al. 2008). The RB/E2F signaling pathway is of such central significance for the control of cell proliferation that it is assumed that its regulation is disturbed in practically all tumors. In MCC, however, loss of RB seems to be a rare event (Becker et al. 2009b). Thus, MCV present in the majority of MCC cases might contribute to a disturbed regulation of this important pathway. To elicit transformation, LT may also act on another critical cellular target, i.e., the tumor suppressor p53, for which a multitude of functions have been described. For example, the antiproliferative activity of p53 plays a significant role in the avoidance of tumor development. Probably, the most important mechanism to control p53 in normal proliferating cells involves the control of protein stability via ubiquitination by the p53-specific ubiquitin ligase mdm2 and subsequent proteasomal degradation; oncogenic stress results in the induction of the tumor-suppressor protein p14ARF, which binds to mdm2, inactivates it, and thus prevents degradation of p53. The p21 protein is a critical downstream target of p53 that mediates cell cycle arrest by inhibiting cyclin-dependent kinases, RB phosphorylation, and

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Fig. 18.4 Possible mechanisms by which MCV’s T antigens might contribute to transformation. (a) Large T antigen (LTA) might exert transforming capacity by binding RB in combination with the chaperone protein hsc70 leading to the release of E2F. Unbound E2F transcription factor mediates cell cycle progression. Another mechanism might be by binding and inactivating the tumor suppressor p53. However, since this domain is often lost in MCV integrated into the host genome, this pathway might be negligible. (b) Small T antigen (sT) binds to PP2A displacing the regulatory B subunit. This modified sT/PP2A complex might act on many different pathways. For example, it prevents the dephosphorylation and inactivation or degradation, respectively, of AKT and Myc

cell cycle progression. Experiments with SV40 have demonstrated that LT/p53 complexes can be observed. LT binds p53 within its core DNA-binding domain leading to loss of target gene activation. Notably, mutated LTs not capable of binding p53 were defective in transformation (Peden et al. 1998). As mentioned before,

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MCV LT demonstrates only limited homology to the other polyomavirus in the p53 DNA-binding region. Moreover, LT-truncating mutations often affect the p53-binding region (Shuda et al. 2008). Thus, p53 binding capacity might be not very relevant for MCV in the etiology of MCC. In accordance, alterations of the p53 pathway independent of the MCV virus have been observed (Becker et al. 2009b). In contrast to LT, for sT, only a few domains have been described. Moreover, sT cannot transform cells by itself; however, it can cooperate with LT to fully transform cells. sT codes for the same DnaJ domain as LT, but its function or target has not been elucidated nor has it been implicated in sT-mediated transformation (Boyapati et al. 2003). Indeed, sT activities can be largely attributed to the binding of the serine– threonine protein phosphatase PP2A, which appears to be the major kinase phosphatase in eukaryotic cells that downregulates activated protein kinases (Fig. 18.4b) (Millward et al. 1999). PP2A is a heterotrimeric enzyme family composed of a scaffold A subunit, a regulatory B subunit, and a catalytic C subunit. There are 2 different A subunits, 2 C subunits, and at least 17 B subunits that can assemble together into >100 different holoenzyme complexes (Sablina and Hahn 2008). sT interacts with PP2A by either preventing the binding of B subunits or replacing them leading in most cases to an inhibition of enzyme activity (Cho et al. 2007). Since PP2A regulates many different kinases, probably only a fraction of the targets of PP2A potentially involved in sT-induced tumorigenesis have been identified to date. For example, SV40 sT can activate AKT signaling through activation of PI3K, contributes to stabilization of Myc by inhibiting PP2A-dependent dephosphorylation, and inhibits cap-dependent translation (Cheng et al. 2009). For MCV’s role in the etiology of MCC, only indirect evidence is available. Indeed, results of classical transformation assays for MCV T antigens have not yet been published. Certainly, given the time since the discovery of MCV and the impact it had on the scientific community, it seem conceivable that the transforming capacity of the T antigens might be not very high in standard assays. One source of indirect evidence for involvement of MCV in MCC is studies comparing MCVnegative and -positive MCC cases. In this regard, two groups have demonstrated that p53 and KIT expression in tumors was associated with the absence of MCV DNA or lower copy number, respectively. Moreover, higher copy numbers of MCV DNA was directly associated with the presence of RB suggesting that the molecular pathogenesis of MCC is multifactorial (Bhatia et al. 2010a; Waltari et al. 2010). In contrast, we and others did not observe significant differences between viruspositive and -negative MCC cases (Handschel et al. 2010; Houben et al. 2010a). However, we recently demonstrated that expression of the oncogenic T antigens is mandatory for the maintenance of MCV-positive MCC cell lines; upon knock down of T antigens by shRNA, MCV-positive MCC cells displayed growth arrest and partially died (Houben et al. 2010b). Since the knockdown did not differentiate between sT and LT, their exact roles have still to be untangled. Nevertheless, this observation is the first direct experimental evidence that TA expression is necessary for the maintenance of MCV-positive MCC and provides a further line of evidence that MCV is the infectious cause of MCV-positive MCC.

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Conclusion The recent findings that (a) MCV is integrated into the host genome in the majority of MCC cases, (b) integrated MCV genome encode truncated LT, and (c) MCVpositive cell lines are dependent on T antigen expression strongly imply that MCV is the etiological agent in a major subset of MCC cases. The exact mechanism, however, has still to be unraveled. But since the discovery of MCV has boosted the research activity on MCC, this will probably sooner or later translate into the knowledge of the signaling pathways involved in the pathogenesis of this tumor.

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Feng H, Taylor JL, Benos PV, Newton R, Waddell K, Lucas SB, Chang Y, Moore PS (2007) Human transcriptome subtraction by using short sequence tags to search for tumor viruses in conjunctival carcinoma. J Virol 81:11332–11340 Feng H, Shuda M, Chang Y, Moore PS (2008) Clonal integration of a polyomavirus in human Merkel cell carcinoma. Science 319:1096–1100 Gross L (1953) A filterable agent, recovered from Ak leukemic extracts, causing salivary gland carcinomas in C3H mice. Proc Soc Exp Biol Med 83:414–421 Handschel J, Muller D, Depprich RA, Ommerborn MA, Kubler NR, Naujoks C, Reifenberger J, Schafer KL, Braunstein S (2010) The new polyomavirus (MCPyV) does not affect the clinical course in MCCs. Int J Oral Maxillofac Surg 39(11):1086–1090 Houben R, Schrama D, Alb M, Pfohler C, Trefzer U, Ugurel S, Becker JC (2010a) Comparable expression and phosphorylation of the retinoblastoma protein in Merkel cell polyoma viruspositive and negative Merkel cell carcinoma. Int J Cancer 126:796–798 Houben R, Shuda M, Weinkam R, Schrama D, Feng H, Chang Y, Moore PS, Becker JC (2010b) Merkel cell polyomavirus-infected Merkel cell carcinoma cells require expression of viral T antigens. J Virol 84:7064–7072 Johnson EM (2010) Structural evaluation of new human polyomaviruses provides clues to pathobiology. Trends Microbiol 18:215–223 Kean JM, Rao S, Wang M, Garcea RL (2009) Seroepidemiology of human polyomaviruses. PLoS Pathog 5:e1000363 Kilham L (1952) Isolation in suckling mice of a virus from C3H mice harboring Bittner milk agent. Science 116:391–392 Laude HC, Jonchere B, Maubec E, Carlotti A, Marinho E, Couturaud B, Peter M, Sastre-Garau X, Avril MF, Dupin N, Rozenberg F (2010) Distinct merkel cell polyomavirus molecular features in tumour and non tumour specimens from patients with merkel cell carcinoma. PLoS Pathog 6(8):e1001076 Leendertz FH, Scuda N, Cameron KN, Kidega T, Zuberbuhler K, Leendertz SA, Couacy-Hymann E, Boesch C, Calvignac S, Ehlers B (2010) African great apes are naturally infected with polyomaviruses closely related to Merkel cell polyomavirus. J Virol 85(2):916–924 Lemos BD, Storer BE, Iyer JG, Phillips JL, Bichakjian CK, Fang LC, Johnson TM, LiegeoisKwon NJ, Otley CC, Paulson KG, Ross MI, Yu SS, Zeitouni NC, Byrd DR, Sondak VK, Gershenwald JE, Sober AJ, Nghiem P (2010) Pathologic nodal evaluation improves prognostic accuracy in Merkel cell carcinoma: analysis of 5823 cases as the basis of the first consensus staging system. J Am Acad Dermatol 63:751–761 Maricich SM, Wellnitz SA, Nelson AM, Lesniak DR, Gerling GJ, Lumpkin EA, Zoghbi HY (2009) Merkel cells are essential for light-touch responses. Science 324:1580–1582 Millward TA, Zolnierowicz S, Hemmings BA (1999) Regulation of protein kinase cascades by protein phosphatase 2A. Trends Biochem Sci 24:186–191 Morrison KM, Miesegaes GR, Lumpkin EA, Maricich SM (2009) Mammalian Merkel cells are descended from the epidermal lineage. Dev Biol 336:76–83 Murakami M, Imajoh M, Ikawa T, Nakajima H, Kamioka M, Nemoto Y, Ujihara T, Uchiyama J, Matsuzaki S, Sano S, Daibata M (2011) Presence of Merkel cell polyomavirus in Japanese cutaneous squamous cell carcinoma. J Clin Virol 50(1):37–41 Pantulu ND, Pallasch CP, Kurz AK, Kassem A, Frenzel L, Sodenkamp S, Kvasnicka HM, Wendtner CM, Zur HA (2010) Detection of a novel truncating Merkel cell polyomavirus large T antigen deletion in chronic lymphocytic leukemia cells. Blood 116(24):5280–5284 Pastrana DV, Tolstov YL, Becker JC, Moore PS, Chang Y, Buck CB (2009) Quantitation of human seroresponsiveness to Merkel cell polyomavirus. PLoS Pathog 5:e1000578 Paulson KG, Carter JJ, Johnson LG, Cahill KW, Iyer JG, Schrama D, Becker JC, Madeleine MM, Nghiem P, Galloway DA (2010) Antibodies to Merkel Cell Polyomavirus T Antigen Oncoproteins Reflect Tumor Burden in Merkel Cell Carcinoma Patients. Cancer Res 70(21):8388–8397 Peden KW, Srinivasan A, Vartikar JV, Pipas JM (1998) Effects of mutations within the SV40 large T antigen ATPase/p53 binding domain on viral replication and transformation. Virus Genes 16:153–165

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Pipas JM (1998) Molecular chaperone function of the SV40 large T antigen. Dev Biol Stand 94:313–319 Pipas JM (2009) SV40: Cell transformation and tumorigenesis. Virology 384:294–303 Poulin DL, DeCaprio JA (2006) Is there a role for SV40 in human cancer? J Clin Oncol 24:4356–4365 Rollison DE, Giuliano AR, Becker JC (2010) New virus associated with merkel cell carcinoma development. J Natl Compr Canc Netw 8:874–880 Rywlin AM (1982) Malignant Merkel-cell tumor is a more accurate description than trabecular carcinoma. Am J Dermatopathol 4:513–515 Sablina AA, Hahn WC (2008) SV40 small T antigen and PP2A phosphatase in cell transformation. Cancer Metastasis Rev 27:137–146 Sastre-Garau X, Peter M, Avril MF, Laude H, Couturier J, Rozenberg F, Almeida A, Boitier F, Carlotti A, Couturaud B, Dupin N (2009) Merkel cell carcinoma of the skin: pathological and molecular evidence for a causative role of MCV in oncogenesis. J Pathol 218:48–56 Schowalter RM, Pastrana DV, Pumphrey KA, Moyer AL, Buck CB (2010) Merkel cell polyomavirus and two previously unknown polyomaviruses are chronically shed from human skin. Cell Host Microbe 7:509–515 Seo GJ, Chen CJ, Sullivan CS (2009) Merkel cell polyomavirus encodes a microRNA with the ability to autoregulate viral gene expression. Virology 383:183–187 Shuda M, Feng H, Kwun HJ, Rosen ST, Gjoerup O, Moore PS, Chang Y (2008) T antigen mutations are a human tumor-specific signature for Merkel cell polyomavirus. Proc Natl Acad Sci USA 105:16272–16277 Shuda M, Arora R, Kwun HJ, Feng H, Sarid R, Fernandez-Figueras MT, Tolstov Y, Gjoerup O, Mansukhani MM, Swerdlow SH, Chaudhary PM, Kirkwood JM, Nalesnik MA, Kant JA, Weiss LM, Moore PS, Chang Y (2009) Human Merkel cell polyomavirus infection I. MCV T antigen expression in Merkel cell carcinoma, lymphoid tissues and lymphoid tumors. Int J Cancer 125:1243–1249 Small MB, Gluzman Y, Ozer HL (1982) Enhanced transformation of human fibroblasts by origindefective simian virus 40. Nature 296:671–672 Stewart SE, Eddy BE, Borgese N (1958) Neoplasms in mice inoculated with a tumor agent carried in tissue culture. J Natl Cancer Inst 20:1223–1243 Stubdal H, Zalvide J, Campbell KS, Schweitzer C, Roberts TM, DeCaprio JA (1997) Inactivation of pRB-related proteins p130 and p107 mediated by the J domain of simian virus 40 large T antigen. Mol Cell Biol 17:4979–4990 Sullivan CS, Pipas JM (2002) T antigens of simian virus 40: molecular chaperones for viral replication and tumorigenesis. Microbiol Mol Biol Rev 66:179–202 Sweet BH, Hillemann MR (1960) The vacuolating virus, S.V. 40. Proc Soc Exp Biol Med 105:420–427 Toker C (1972) Trabecular carcinoma of the skin. Arch Dermatol 105:107–110 Tolstov YL, Pastrana DV, Feng H, Becker JC, Jenkins FJ, Moschos S, Chang Y, Buck CB, Moore PS (2009) Human Merkel cell polyomavirus infection II. MCV is a common human infection that can be detected by conformational capsid epitope immunoassays. Int J Cancer 125: 1250–1256 Tsai B, Qian M (2010) Cellular entry of polyomaviruses. Curr Top Microbiol Immunol 343:177–194 van der Meijden E, Janssens RW, Lauber C, Bouwes Bavinck JN, Gorbalenya AE, Feltkamp MC (2010) Discovery of a new human polyomavirus associated with trichodysplasia spinulosa in an immunocompromized patient. PLoS Pathog 6:e1001024 Van Keymeulen A, Mascre G, Youseff KK, Harel I, Michaux C, De GN, Szpalski C, Achouri Y, Bloch W, Hassan BA, Blanpain C (2009) Epidermal progenitors give rise to Merkel cells during embryonic development and adult homeostasis. J Cell Biol 187:91–100 Waltari M, Sihto H, Kukko H, Koljonen V, Sankila R, Bohling T, Joensuu H (2010) Association of Merkel cell polyomavirus infection with tumor p53, KIT, stem cell factor, PDGFR-alpha and survival in Merkel cell carcinoma. Int J Cancer 129(3):619–628

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Chapter 19

Human Papillomaviruses and Cancer Jianxin You and Susanne Wells

Introduction Papillomaviruses (PVs) are small DNA tumor viruses that induce a diverse range of benign and malignant epithelial lesions in the infected host (Howley and Lowy 2001). Over 140 human PV (HPV) types have been identified to date. Depending on the potential for inducing malignant transformation, these viruses are further classified into high-risk and low-risk HPVs (Howley and Lowy 2001). Persistent infection with high-risk HPVs are recognized as the major cause of cervical cancer (zur Hausen 2002), which is the second most common cancer among women worldwide and the leading cause of death from cancer among women in developing countries; approximately 500,000 cases and 275,000 deaths are reported annually. Over 97% of cervical cancers contain high-risk HPV genomic DNA and express the viral oncogenes E6 and E7, thus providing a direct link between HPV infection and carcinogenesis. In this chapter, we summarize the HPV life cycle and oncogenic events that contribute to the role of HPV in carcinoma development. Most of our current knowledge derives from studies on cervical cancers, but HPV has also been implicated in other anogenital cancers, as well as head and neck cancers (HNCs), and skin cancers (Giuliani et al. 2007; McKaig et al. 1998; Pfister 2003; Vernon et al. 1998). In the last section of the chapter, we discuss emerging developments on the understanding of the role of HPV in HNCs.

J. You (*) Department of Microbiology, University of Pennsylvania School of Medicine, 3610 Hamilton Walk, Philadelphia, PA 19104, USA e-mail: [email protected] S. Wells Division of Hematology/Oncology, Cincinnati Children’s Hospital, 3333 Burnet Avenue, MLC 7013, TCHRF, Room S7-206, Cincinnati, OH 45229, USA E.S. Robertson (ed.), Cancer Associated Viruses, Current Cancer Research, DOI 10.1007/978-1-4614-0016-5_19, © Springer Science+Business Media, LLC 2012

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The Differentiation-Dependent Papillomavirus Life Cycle Productive Viral Life Cycle PVs contain circular double-stranded DNA genomes of approximately 8,000 bp within an icosahedral capsid. The viral genome encodes eight open reading frames (ORFs), including six early genes (E1, E2, E4, E5, E6, and E7) and two late genes (L1 and L2) (Doorbar 2006) (Fig. 19.1). HPVs have a specific tropism for squamous epithelial cells and must infect cells within the dividing basal layer. The productive life cycle of HPV is intimately tied to the differentiation status of the host squamous epithelium (Howley and Lowy 2001) (Fig. 19.2). Various phases of the HPV life cycle

Fig. 19.1 HPV16 genome organization and functions of HPV gene products. The HPV16 genome contains 7,904 bp and encodes eight open reading frames (ORFs). The six early ORFs, E1, E2, E4, E5, E6, and E7, are expressed from either p97 (early promoter) or p670 (late promoter) at different stages of host epithelial cell differentiation. The late ORFs, L1 and L2, are expressed from p670. All the viral genes are transcribed from only one strand of the double-stranded circular DNA genome. Functions of the viral gene products are also shown

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Fig. 19.2 The productive HPV life cycle. HPVs have specific tropism for squamous epithelial cells. They infect cells within the dividing basal epithelial layer through microabrasions. The HPV life cycle is strictly tied to the differentiation program of the host keratinocyte. After infection, viruses establish themselves as episomes at 50–100 copies per infected basal cell and replicate with the host DNA once per cell cycle. The viral oncoproteins E6 and E7 expressed in the basal cells modify the cell cycle to retain the differentiating host keratinocytes in a state that allows viral genome replication. Expression of low levels of E1, E2, E4, and E5 allows maintenance of the viral genome in the suprabasal cells. Viral genome amplifies in the upper epithelial layers, where the expressed late genes L1 and L2 serve as structural proteins to encapsidate the amplified viral genomes. Virions can then be sloughed off the surface of the host epithelium

are controlled through tightly regulated activation of the early and late viral promoters as the infected basal cell migrates toward the epithelial surface (Doorbar 2006; Hebner and Laimins 2006). Productive infection is achieved through different viral proteins playing specific roles at distinct phases of the viral life cycle (Fig. 19.2). Within infected basal cells, viral genomes are established and maintained as extrachromosomal elements (episomes) that replicate along with host DNA (Fig. 19.2). In the basal epithelium where initial stages of the viral life cycle takes place, the early viral promoter is transcribed to express the viral E1, E2, E6, and E7 genes (Hummel et al. 1992; Ozbun and Meyers 1998). The establishment of the viral genome as a stable episome in the basal epithelia cells requires expression of the viral E1 and E2 proteins. The papillomavirus E2 is a sequence-specific DNAbinding protein involved in viral DNA replication, episome maintenance, and viral transcription (Hebner and Laimins 2006). It consists of an N-terminal transcriptional activation domain linked to a C-terminal DNA-binding/dimerization domain by a flexible hinge (McBride et al. 1991). The C-terminus of E2 is involved in interactions with viral replication factor E1 and also recognizes a palindromic motif [AACCg(N4)cGGTT] in the noncoding region of the viral genome (Dell et al. 2003).

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The multiple functions of E2 rely on its sequence-specific recognition of a number of E2-binding sites (E2BSs) within papillomavirus genomes. E2 initiates viral genome replication by loading the viral helicase E1 onto the origin of replication, which in turn recruits cellular proteins necessary for DNA replication (Conger et al. 1999; Han et al. 1999; Howley and Lowy 2001; Loo and Melendy 2004; Masterson et al. 1998; Mohr et al. 1990). This allows the viral episome to be maintained at low copy numbers in the basal epithelium. During mitosis, E2 ensures accurate partitioning of the replicated viral genomes to daughter cells by tethering them to host mitotic chromosomes (Bastien and McBride 2000; Ilves et al. 1999; Lehman and Botchan 1998; Piirsoo et al. 1996; Skiadopoulos and McBride 1998). In the differentiating host epithelium, E2 also contributes to the tight regulation of the viral oncogene transcription to create a cellular environment conducive to completion of the productive viral infectious cycle. E2 binds to four E2BSs in the long-control region (LCR) of the HPV genome in a cooperative manner that results in either activation (at low levels of E2) or repression (at high concentrations of E2) of E6 and E7 expression from the early promoter (Bouvard et al. 1994; Doorbar 2006; Steger and Corbach 1997). E6 and E7 are the primary viral oncogenes, which promote cell growth through a variety of mechanisms, including inactivation of the p53 and pRb tumor suppressors, respectively (Doorbar 2006; Dyson et al. 1989; Huibregtse et al. 1991; Munger et al. 1989; Scheffner et al. 1993). The dose-dependent ability of E2 to either repress or activate early viral gene expression is thought to result from differential affinities of E2 for its various binding sites (Hines et al. 1998). At low concentration, E2 binds the primary binding site distal to the HPV promoter and leads to promoter activation (Steger and Corbach 1997). As E2 level increases, occupancy of the remaining sites proximal to the promoter leads to transcription repression of the early promoter through displacement of other cellular transcription factors (Demeret et al. 1994; Dong et al. 1994; Steger and Corbach 1997; Tan et al. 1994). Suprabasal cells normally exit the cell cycle and differentiate to produce the protective barrier of the skin (Madison 2003). The strict control of the viral early promoter allows the minimum expression of E6 and E7 that is needed to drive cells into S phase for viral genome replication while preventing the inopportune overexpression of these viral oncoproteins that leads to dysplasias and carcinomas. As discussed below, the cellular factors that contribute to the tightly regulated viral transcription are not very well-understood. As HPV-positive cells differentiate, the late promoter is activated leading to the expression of the two late-gene products, capsid proteins L1 and L2, and the high-level amplification of the viral genome (Doorbar 2006; Hebner and Laimins 2006). In the highly differentiated suprabasal cells, the replicated viral DNA genomes are packaged into newly formed capsids and infectious progeny virions are released from the cell (Howley and Lowy 2001) (Fig. 19.2). Besides E1, E2, E6, and E7 proteins, the viral E5 protein also plays important role in the productive stage of the viral life cycle (Fehrmann et al. 2003; Genther et al. 2003). HPV E5 is a transmembrane protein that resides predominantly in the

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endoplasmic reticulum. Association of E5 protein with the vacuolar proton ATPase delays the process of endosomal acidification (Disbrow et al. 2005; Hwang et al. 1995; Straight et al. 1995), contributing to an increase in epidermal growth factormediated receptor signaling and the maintenance of a cellular environment for viral replication in the upper epithelial layers (Crusius et al. 2000). In addition, E4 accumulates in the cells at the time of viral genome amplification and contributes to multiple facets of the papillomavirus life cycle (Nakahara et al. 2005; Peh et al. 2004; Wilson et al. 2005). The E4 proteins encoded by several HPV types have been shown to cause cell cycle arrest in G2 and to antagonize E7-mediated cell proliferation (Davy et al. 2002; Knight et al. 2004; Nakahara et al. 2002). However, the role of E4 in genome amplification is poorly understood (Doorbar 2006). E4 can also disrupt the keratin network to facilitate the efficient release of the assembled virions from the cornified envelope at the cell surface (Bryan and Brown 2000; Doorbar et al. 1991; Wang et al. 2004).

Abortive Viral Infection The incidence of cervical cancer development from an HPV-infected lesion is low, with most infections limited by the host immune system (Parkin et al. 2005). Highgrade cervical neoplasia usually arises in individuals who maintain persistent active infection for years or decades following initial exposure and fail to resolve their infection. In these situations, the continuous stimulation of S-phase entry and cell proliferation by E7, coupled with the loss of p53-mediated DNA repair pathways resulting from E6 expression, leads to the accumulation of additional genetic mutations in the cellular genome that eventually lead to cancer (Doorbar 2006). The identification of cervical lesions as flat condyloma, low-grade squamous intraepithelial neoplasia lesions (LSIL), high-grade squamous intraepithelial neoplasia lesions (HSIL), or invasive cervical cancer has allowed the detection of molecular changes in the normal epithelial cell differentiation that occur after viral infection (Doorbar 2006). In productive lesions, cells are driven into S phase only in the lower epithelial layers and virus production at the epithelial surface relies on the ordered and timely expression of viral gene products (Middleton et al. 2003; Peh et al. 2002). The viral protein expression patterns in low-grade cervical lesions resemble the patterns found in productive lesions with viral coat proteins usually detected in cells at the epithelial surface (Middleton et al. 2003). However, the timing of viral gene expression becomes progressively disturbed during neoplastic progression. In high-grade neoplasia, the proliferative phase becomes more extensive with viral genome amplification occurring closer to the epithelial surface, and with viral capsid protein synthesis abolished (Middleton et al. 2003). These changes reflect an abortive infection in which viral gene expression becomes deregulated and the productive stages of the virus life cycle can no longer be properly supported (Doorbar 2006).

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HPV-Persistent Latent Infection High-risk HPV-infected cells progress to invasive cancer only after they have accumulated substantial cytogenetic changes needed for malignant progression. The cellular events that cause this transition do not occur until many years after the initial infection, supporting the concept that persistent infection with highrisk HPVs is required for the malignant progression of HPV pathogenic lesions. HPVs establish persistent infection by maintaining their genomes as episomes that replicate along with host DNA in infected basal epithelial cells (Howley and Lowy 2001). The functions of the high-risk HPV E6 and E7 oncoproteins are essential for the viral genome to be stably maintained in the infected cells (Thomas et al. 1999). It was postulated that these oncoproteins may create a suitable cellular environment for HPV maintenance through abrogating the cellular checkpoints that block the long-term retention of viral episomes (Kadaja et al. 2009b). In addition, to establish persistence in host cells, some PVs have evolved a strategy for hitchhiking on cellular mitotic chromosomes. This mechanism ensures that the replicated viral episomes are retained inside the nuclei of dividing host cells and faithfully partitioned to the daughter cells during mitosis. For many papillomaviruses, this noncovalent association of viral DNA with chromosomes is mediated by the viral E2 protein. Bovine papillomavirus type 1 (BPV1) genomes persist as stable episomes in transformed rodent cells, and have been used as a model system to establish the role of E2 in viral genome maintenance (Bastien and McBride 2000; Ilves et al. 1999; Law et al. 1981; Lehman and Botchan 1998; Skiadopoulos and McBride 1998). E2 and BPV1 episomes are closely associated with mitotic chromatin in dividing cells (Ilves et al. 1999; Lehman and Botchan 1998; Skiadopoulos and McBride 1998). Long-term maintenance of viral episomes requires BPV1 E2 binding to the multiple E2BSs within a cis-minichromosome maintenance element (MME) of the viral genome (Piirsoo et al. 1996). These studies demonstrate that BPV1 E2 facilitates viral genome segregation by anchoring viral episomes to mitotic chromosomes. The cellular bromodomain protein Brd4 has been postulated to serve as a host chromatin adaptor for BPV1 E2 (You et al. 2004). Brd4 is a member of the BET family proteins that harbor double bromodomains (Dey et al. 2000). It binds to acetylated histone H3 and H4 on host chromatin through its bromodomains and becomes associated with mitotic chromosomes (Dey et al. 2003). Brd4 binds BPV1 E2 through its C-terminal domain and tethers the E2/viral episome complex to host mitotic chromosomes to ensure the faithful partitioning of replicated viral episomes during cell division (You et al. 2004, 2005). Further studies demonstrated that the Brd4–E2 interaction bridges a number of animal and human PV episomes onto mitotic chromosomes for viral genome maintenance (Abbate et al. 2006; Baxter et al. 2005; Brannon et al. 2005; Cardenas-Mora et al. 2008; Ilves et al. 2006; McBride et al. 2004; McPhillips et al. 2005), establishing this complex as a potential

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therapeutic target for PV infections. Through these interactions, Brd4 also contributes to E2-mediated transcriptional activation and repression of the viral oncogenes (Ilves et al. 2006; Lee and Chiang 2009; McPhillips et al. 2006; Schweiger et al. 2006; Senechal et al. 2007; Wu et al. 2006). The structure of the N-terminal domain of HPV16 E2 in complex with the C-terminus of Brd4, thus, offers a molecular platform for developing antiviral compounds to block E2–Brd4 interaction during HPV latent infection (Abbate et al. 2006). The molecular mechanism involved in maintaining E2 and papillomavirus episome persistence in dividing host cells appears complex. A number of additional mitotic cellular factors have been identified as interacting proteins for E2. Among them, ChlR1, a DNA helicase that functions in sister chromatid cohesion, appears essential for loading the papillomavirus E2 protein onto mitotic chromosomes during early mitosis (Parish et al. 2006). Other proteins, such as MKlp2 and TopBP1, have been shown to interact and colocalize with E2 during different phases of mitosis (Donaldson et al. 2007; Yu et al. 2007). It is conceivable that Brd4 and the identified additional cellular receptors may interact either sequentially or simultaneously with E2 and the viral episome to constitute a tethering cascade/complex for viral genome maintenance and segregation in latently infected cells. Different modes of papillomavirus anchoring on mitotic chromosomes have also been reported. A wide range of papillomaviruses have adopted the strategy of tethering their genomes to host chromosomes, but individual viruses appear to use different chromosomal targets. A recent localization analysis of 13 different animal and human E2 proteins from 7 papillomavirus genera have shown that E2-mediated tethering of viral genomes to mitotic chromosomes is a common strategy shared by these papillomaviruses. However, different viruses bind to different regions of the host chromosomes during mitosis. Unlike BPV1 E2 whose nonspecific binding to all mitotic chromosomes is reflected by small speckles associated with Brd4, several other E2 proteins appear to localize to more specific regions of mitotic chromosomes (Oliveira et al. 2006). For instance, the alpha-papillomavirus E2 proteins only closely associate with prophase and telophase chromosomes while binding to the pericentromeric region of metaphase chromosomes (Oliveira et al. 2006). In contrast, the HPV8 E2 binds as large speckles at the pericentromeric regions of chromosomes (Oliveira et al. 2006). Further analysis indicates that the HPV8 E2 protein targets the short arms of acrocentric mitotic chromosomes and interacts with the repeated ribosomal DNA loci of host mitotic chromosomes. In addition to mitotic chromosomes, some high-risk HPV type E2 proteins have been shown to associate with mitotic spindles to enable the partitioning of HPV origincontaining plasmids as minichromosomes in transfected cells (Van Tine et al. 2004). The molecular mechanisms by which high-risk HPVs adopt the cellular machinery to maintain persistent infection in host cells remain an interesting topic for future research. A complete understanding of the HPV tethering mechanism is bound to uncover new targets for the development of novel therapeutic strategies to cure latent HPV infection.

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Molecular Events Contributing to Malignant Progression of HPV-Positive Lesions HPV Integration and Cervical Cancers HPV DNA is maintained episomally in benign, precancerous lesions, but integration into the cellular genome is frequently observed in cervical cancers and effectively eliminates the viral life cycle (Pett and Coleman 2007). It has been suggested that integration of the HPV genome into the host cell chromosome may be a critical event in the development of most cervical cancers (Peitsaro et al. 2002). Although HPV integration can occur randomly throughout the genome, several studies have indicated a preference for integration occurring within common fragile sites (Thorland et al. 2000, 2003). Variable portions of the viral genome are present in the integrants, but common features include sustained expression of E6/E7 under the control of their viral promoter as well as loss of the viral E2 gene (Baker et al. 1987; Corden et al. 1999; Durst et al. 1985; Howley and Lowy 2001; Jeon et al. 1995; Jeon and Lambert 1995b; Park et al. 1997; zur Hausen 2000) (Fig. 19.3). As described above, HPV E2 is also a transcription repressor that can directly bind to and repress the viral E6/E7 promoter of high-risk HPVs (Demeret et al. 1997; Romanczuk et al. 1990; Tan et al. 1992; Thierry and Howley 1991). The loss of E2 expression derepresses the viral oncogene E6 and E7 expression to reduce p53 and pRb expression, which in turn stimulates cellular proliferation (Dyson et al. 1989; Gonzalez et al. 2001; Munger et al. 1992, 2004; Scheffner et al. 1990). As such, disruption of the E2 gene has been mechanistically linked to malignant progression of HPV-associated cancers (Howley and Lowy 2001). In cervical cancer cell lines containing integrated HPV DNA and a disrupted E2 gene, reintroduction of E2 represses the HPV E6/E7 promoter and reverses cellular immortality (Dowhanick et al. 1995; Francis et al. 2000; Goodwin and DiMaio 2000; Hwang et al. 1993; Thierry and Yaniv 1987) (Fig. 19.3). It has been suggested that E2 represses the E6/E7 promoter by binding its cognate sites proximal to the promoter and displacing other cellular transcription factors (Demeret et al. 1994; Dong et al. 1994; Tan et al. 1994). In addition, E2 could function as an active repressor by preventing the assembly of a functional preinitiation complex on the viral chromatin template (Dostatni et al. 1991; Wu and Chiang 2007; Yan et al. 2010). A small proportion of human cervical cancers harbor exclusively episomal viral DNA (Guo et al. 2007; Hudelist et al. 2004; Matsukura et al. 1989; Park et al. 1997), suggesting that viral integration is not necessarily required for cancer development. A postulated intermediate state in which transcriptionally silent integrants coexist with episomal genomes has been described in clinical lesions. Because E2 expressed from episomal genomes can repress oncogene expression from the integrant (Bechtold et al. 2003; Demeret et al. 1997; Romanczuk et al. 1990; Tan et al. 1992), episome loss and clonal selection for integrants are likely important steps during cervical carcinogenesis (Pett and Coleman 2007; Pett et al. 2004). HPV integration has been shown to confer a selective growth advantage compared to cells with

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Fig. 19.3 Repression of HPV oncogenes by the E2 protein. Shown is a schematic representation of a fragment of high-risk HPV genome integrated into the host genome. Integration of HPV DNA into the host genome frequently results in disruption of the E2 gene, which normally inhibits the production of E6 and E7 proteins of high-risk HPVs. Loss of E2 expression derepresses the expression of E6 and E7, which function as oncogenes to promote tumor growth and malignant transformation by inhibiting p53 and pRb, respectively. E6 protein binds p53 via E6-associated protein (E6AP), which ubiquitinates p53, targeting it for degradation by proteasome. The binding of E7 to pRb releases E2F for the transcription of its responsive genes to promote cell cycle progression. In cervical cancer cell lines containing integrated HPV DNA and a disrupted E2 gene, reintroduction of E2 represses E6 and E7 expression, thereby inhibiting their effects on p53 and pRb and reversing cellular immortality [Modified from Thierry (2009)]

episomal HPV copies (Jeon and Lambert 1995b) perhaps at least in part due to the stabilization of E6 and E7 mRNAs expressed from the viral integrants (Jeon and Lambert 1995a).

The Contribution of HPV Oncogenes to Human Cervical Cancers High-risk HPV E6 and E7 proteins are universally expressed in human HPV-positive cancers in vivo and function cooperatively in vitro, findings that have defined them as the primary viral oncogenes. The viral E5 protein can further contribute to HPV malignancy (DiMaio and Mattoon 2001), and carries independent carcinogenic potential in transgenic mouse models (Maufort et al. 2007, 2010). Viral oncogenic potential is also reflected in the ability of high-risk, but not low-risk, HPV genomic DNA to immortalize primary human keratinocytes in vitro (zur Hausen 2000). In order to progress to transformed phenotypes, either the coexpression of defined oncogenes or extended passaging in tissue culture is required (DiPaolo et al. 1989; Durst et al. 1989; Hurlin et al. 1991; Pei et al. 1993).

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Since the specific activity of high-risk HPV E6 and E7 has consistently been associated with cervical cancers, deregulated expression of the viral oncogenes is considered a predisposing factor in the development of HPV-associated cervical cancers. The E7 oncogene appears to play a dominant role in cell culture studies; highrisk HPV E7, but not E6, is capable of immortalizing primary human keratinocytes on its own. A critical role for E7 has also emerged in E6 and E7 transgenic mouse models of cervical cancer (Jabbar et al. 2009; Riley et al. 2003) and HNC (Strati and Lambert 2007). Many protein–protein interactions have been reported between the high-risk HPV oncoproteins and host cellular factors, at least some of which contribute to oncogenic E6 and E7 activities (Jones and Wells 2006). E7 binds and degrades Rb-related pocket proteins and neutralizes the cyclindependent kinase inhibitors p21 and p27 through direct interactions (Funk et al. 1997; Jones et al. 1997; Zerfass-Thome et al. 1996). Deregulation of these molecules is critical for HPV-mediated S-phase induction in terminally differentiated cells. One important consequence of these E7 functions is the activation of E2F transcriptional activity, the uncontrolled expression of genes that regulate G1/S cell cycle progression and subsequent inappropriate entry of differentiating keratinocytes into S phase (Banerjee et al. 2006; Cheng et al. 1995). The maintenance of an S-phase milieu conducive to viral replication is further supported by the binding of E7 to a transcriptionally repressive E2F family member E2F6 (McLaughlin-Drubin et al. 2008) and to chromatin-modifying histone deacetylases (HDACs) independent of Rb association (Longworth and Laimins 2004b; Longworth et al. 2005). The function of the viral E6 protein complements that of E7 with regard to cancer progression. To counteract the growth arrest or apoptosis that would normally occur in response to E7-mediated inappropriate cell proliferation, the high-risk HPV type E6 forms a tripartite complex with p53 and the cellular ubiquitin ligase E6AP (E6-associated protein), which causes proteasome-mediated p53 degradation (Huibregtse et al. 1991; Kao et al. 2000; Scheffner et al. 1990). This compromises the p53-mediated DNA damage response and allows the accumulation of secondary mutations in the host genome that eventually lead to the development of cervical cancers. In addition, in vitro studies establish that E6 inhibits p300mediated acetylation on both p53 and nucleosomal core histones, thereby converting p53–p300 from an activating complex to a chromatin repressor for p53-dependent transcription (Thomas and Chiang 2005). E6 also transcriptionally and posttranscriptionally upregulates the human telomerase catalytic subunit, an enzyme that is frequently activated in human tumors (Galloway et al. 2005; Katzenellenbogen et al. 2007, 2009; Liu et al. 2005, 2009). The high-risk E6 proteins further contribute to the development of metastatic tumors through binding and stimulating the degradation of several cellular targets that contain PDZ motifs, which are thought to be involved in the regulation of cell growth and attachment (Nguyen et al. 2003a, b; Zeitler et al. 2004).

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HPV Oncogenic Activities, Host DNA Damage Response, and Genomic Instability Deregulated expression of the high-risk HPV oncogenes is a critical event for the progression of HPV-positive lesions. High-risk HPV E6/E7 activities fulfill some, but not all, classical criteria for cellular transformation as described by Hanahan and Weinberg (2000). These include inactivation of the p53 and Rb tumor suppressors and elevated telomerase activity. Various approaches to specifically inhibit E6 and E7 have resulted in cellular growth arrest of HPV-positive cancer cells (Alvarez-Salas et al. 1998; Hall and Alexander 2003; Hu et al. 1995; Venturini et al. 1999; von Knebel Doeberitz et al. 1992; Watanabe et al. 1993). However, in order to achieve bona fide transformed potential, E6/E7 immortalized cells require additional “hits.” Similarly, in high-risk HPV-infected cells, substantial cytogenetic changes are needed for progression to invasive tumors. The acquisition of additional oncogenic events is greatly stimulated by genome instability, which occurs upon the individual and particularly the joint expression of high-risk E6 and E7 by a multitude of molecular mechanisms. Over 20 years ago, expression of high-risk HPV E6 and E7 protein was shown to stimulate integration of exogenous DNA (Kessis et al. 1996). Such cells harbor DNA breaks, which can ultimately result in the acquisition of anaphase bridges and catastrophic breakage-fusion-bridge cycles. Doublestranded HPV DNA intermediates might arise from integrated HPV DNA and during the normal life cycle as a result of “onion-skin” type and/or rolling circle DNA replication (Flores and Lambert 1997; Kadaja et al. 2009a; Mannik et al. 2002). Additionally, DNA damage responses arise as a result of expression of the HPV replication machinery and the high-risk HPV oncoproteins (Kadaja et al. 2007). For example, HPV16 gene expression has been shown to induce both numerical and structural chromosome instability during HPV-associated carcinogenesis (Duensing and Munger 2002). Genomic instability and the resulting multipolar mitoses, aneuploidy, and chromosomal rearrangements are early hallmarks of HPVassociated cancers, which are specific to the high-risk HPV types. In fact, they occur even in the presence of viral episomal HPV genomic DNA and prior to integration (Duensing et al. 2001a), suggesting that they play a causal role in the development of malignancies. Molecular cross talk between DNA strand breaks, DNA damage signaling, HPV gene products, and transformation is likely complex and occurs at multiple levels. The detection and repair of cellular DNA strand breaks depend upon three major kinases and members of the phosphoinositide 3 kinase-like kinase (PIKK) family: ataxia telangiectasia mutated (ATM), ATM and Rad3 related (ATR), and DNAdependent protein kinase (DNA-PK). Each can phosphorylate a histone H2A variant H2AX on serine 139 rapidly following DNA damage. The resulting gH2AX is a marker of damaged sites, which recruits the DNA repair machinery and further amplifies the DNA damage signal. ATM is best known for its ability to respond to double-strand breaks and to phosphorylate itself on serine 1981 as well as downstream targets, such as the Chk2 kinase. ATR is largely activated by single-stranded

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DNA breaks, such as from UV exposure and replication stress, and phosphorylates Chk1 and other targets. High-risk E6 protein interferes with the recovery of stalled replication forks in a p53-independent manner and through the deregulation of Chk1 (Chen et al. 2009). E6 expression also leads to abnormal centrosome numbers after prolonged cell passaging likely through the inactivation of p53. High-risk HPV E7 independently causes centrosome abnormalities (Duensing et al. 2001b; Duensing and Munger 2004; Schaeffer et al. 2004). These occur rapidly as a result of centrosome overduplication within a single S phase. In contrast to normal centrosome duplication, E7-deregulated centrosome duplication is dependent upon high CDK2 activity and is at least partially independent of the retinoblastoma protein family (Duensing et al. 2000, 2001a, 2008; Duensing and Duensing 2008; Duensing and Munger 2002, 2003). Expression of high-risk, but not low-risk, HPV E7 additionally stimulates the activation of the Fanconi anemia pathway which is crucial for the maintenance of genome stability (Hoskins et al. 2008; Spardy et al. 2007), and forces the degradation of claspin, an important regulator of the upstream ATR DNA damage sensor of replication stress (Spardy et al. 2009). High-risk HPV E6 and E7 also cause polyploidy through bypassing mitotic and postmitotic checkpoints (Heilman et al. 2009; Liu et al. 2007; Thomas and Laimins 1998; Thompson et al. 1997). These events may cooperate to further stimulate cellular genome instability, viral integration and rearrangement, and ultimately progression to malignancy. Virally induced activation of cellular DNA damage responses has emerged as a common theme in the literature, and can serve to either support viral replication as in the case of SV40, HSV, or HIV or to inhibit replication as in the case of adenovirus (Lilley et al. 2007; Sinclair et al. 2006). ATM is a particularly interesting molecular modifier of viral DNA replication: its activity is stimulated by both HSV and adenovirus. However, while HSV replication exploits ATM activation, ATM activity obstructs adenoviral replication and the virus thus evolved to degrade specific DNA repair components to circumvent the cellular DNA damage response. With regards to the HPV life cycle, however, our understanding of the effects of specific DNA damage-signaling responses has lagged behind. This is likely due to technical reasons that relate to the difficulties of studying the HPV life cycle, which is tightly linked to the keratinocyte differentiation program (Doorbar 2005; Longworth and Laimins 2004a). At least some aspects of the viral replicative cycle must be analyzed in 3D models of differentiated epidermis, such as organotypic epithelial rafts. Manipulation of DNA damage responses and viral replication, then, need to be interpreted in the context of geographically distinct keratinocytes, which maintain varying degrees of endogenous proliferation and HPV replication. An elegant recent report highlights the specific importance of ATM activation by HPV31 episomes in the productive HPV replicative cycle (Moody and Laimins 2009). ATM, Chk2, and gH2AX phosphorylation were found elevated in HPV31-positive keratinocyte populations and organotypic epithelial rafts. The specific inhibition of either ATM or Chk2 did not affect viral maintenance over time, but greatly reduced the assembly of viral replication centers as well as productive viral amplification in calciumdifferentiated cells. Interestingly, the authors further showed that Chk2 was required for caspase 7 cleavage, which together with caspase 3 was previously implicated in

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cleavage of the viral E1 protein and productive replication. These observations link the HPV-activated DNA damage response directly to the viral replication machinery, and represent the first step in elucidating the role of DNA damage signaling in the HPV life cycle. Whether specific ATM inhibitors might then be useful clinically for the treatment of HPV infections remains to be determined.

The Role of HPV in Head and Neck Cancer High-risk HPV infection is the primary risk factor for cervical cancer. Studies also suggest that HPVs may play a role in many other types of anogenital cancers and skin cancers (Giuliani et al. 2007; Parkin 2006; Pfister 2003; Vernon et al. 1998). The implication of HPV in these other types of cancers has been covered in great details in these reviews (Giuliani et al. 2007; Pfister 2003; Vernon et al. 1998). Studies have also found that oral HPV infection is a strong risk factor for HNCs (McKaig et al. 1998). In this section of the chapter, we focus on the emerging development on the molecular understanding of HPV in HNCs. HNC comprises the sixth most common malignancy worldwide, with an incidence of approximately 500,000 and an associated mortality of over 300,000 per year (Parkin et al. 2005). Treatment regimen often approximate the limits of patient tolerance, and more than half of patients who receive radiation therapy suffer from debilitating acute toxicity in the form of mucositis, pharyngitis, and/or severe dermatitis. HNC survivors often experience poor quality of life from disfiguring surgeries and permanent tissue damage following radiation and chemotherapy. Despite advances for locoregional control, overall cure rates have shown little improvement in decades. Early detection, targeted delivery of radiation and chemotherapy, and therapy de-escalation based on biomarkers need to be pursued for improved outcomes (Corry et al. 2010). These renewed efforts must be accompanied by molecular research into the mechanisms of HNC development, metastasis, and treatment response. HNCs arise from the oral cavity, oro-, naso-, and hypopharynx as well as the larynx. The majority of HNCs are squamous cell carcinomas (SCCs), which originate from keratinocytes in the pluristratified squamous epithelium lining of the upper aerodigestive tract. Alcohol and tobacco consumption have long been recognized as major risk factors. Additionally, an etiological association with HPV was first experimentally suggested by Syrjanen et al. (1983), and has been further substantiated and reported for approximately one quarter of HNSCCs, predominantly those originating in the oropharynx (Braakhuis et al. 2004; D’Souza et al. 2007a, b; Dai et al. 2004; Gillison et al. 2000; Gillison and Shah 2001, 2003; Herrero et al. 2003; Kreimer et al. 2004a, b; Schwartz et al. 1998; Smith et al. 2004; van Houten et al. 2001; Wiest et al. 2002; zur Hausen 2009). Transmission of carcinogenic HPV types occurs predominantly via sexual contact, including oral sex and open-mouthed kissing (D’Souza et al. 2009). Approximately 90% of HPV-positive HNCs harbor viral sequences encoded by the high-risk HPV16 type (Capone et al. 2000; Gillison et al. 2000; Kreimer et al. 2005; Munoz et al. 2003; Nasman et al. 2009), a finding that is

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in contrast to a broader spectrum of HPV types in cervical cancer, such as 18, 31, and 45. HPV-positive tumors can be considered a distinct disease when compared to HPV-negative HNCs. In agreement with sexual transmission as the predominant route of HPV infection, HPV-positive tumors are strongly associated with sexual behavior. In contrast, HPV-negative HNCs are linked to alcohol and tobacco use. Recent clinical evidence additionally suggests that HPV-positive compared to HPV-negative tumor status is associated with good prognosis in patients treated with radiotherapy and perhaps even in patients that received surgery alone (Ang et al. 2010; Fischer et al. 2010). It is likely that incomplete inactivation of p53 and Rb pocket proteins in HPV-positive cells as opposed to their deletion or mutation in HPV-negative cells confers an inherently less-malignant and more therapy-responsive phenotype. Viral protein immunogenicity may further contribute to a positive outcome (Lowy and Munger 2010; Spanos et al. 2009). However, precise molecular mechanisms mediating differential biologies and outcomes are likely diverse and remain to be defined. Molecular comparisons of HPV-positive and -negative HNCs should also consider differences at the level of the originating cell status, expression, and activity of specific viral proteins, including but not limited to E6 and E7 and unique microRNA, transcriptome, and proteome profiles. As discussed earlier, in general, viral integration stimulates but is not necessarily required for cervical cancer development. The physical state of HPV DNA in HNSCCs has not been extensively examined yet, but may depend upon the site of infection and transformation (Koskinen et al. 2003; Mellin et al. 2000; Venuti et al. 2000). Integrated genomes have been detected in SCCs from oral, tonsillar, and laryngeal carcinomas, but usually at a frequency of less than 50%. This indicates that, similar to cervical cancers, HPV integration into host cell DNA may not be a necessary requirement for malignant transformation but that the proportion of SCCs harboring exclusively viral episomes may be greater in HNC compared to cervical cancer. It is tempting to speculate that HPV infection and transformation in the head and neck region occur by mechanisms similar to that in the cervix. However, the preponderance of HPV16 and a lesser contribution of HPV to the overall disease burden might suggest tissue-specific differences which could in turn affect HPV infection, maintenance, replication, and/or virus production in a virus type-specific manner. This could be due to intrinsic differences between cervical versus oropharyngeal keratinocytes or the result of differential immunological control of virus infection in the cervical versus oropharyngeal tract. Models of HPV transformation in the human head and neck region are now needed to compare and contrast molecular mechanisms that drive HNC malignancy. The results promise to yield new and exciting insights into the biology of HPV.

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Concluding Remarks HPVs establish persistent infection by maintaining their genomes as episomes in infected cells. Papillomavirus hitchhikes on host mitotic chromosomes for transmitting its genetic materials across the cell cycle to ensure accurate segregation of the replicated viral episomes to the daughter cells during host cell division. In the meantime, the virus hijacks the cellular machinery to facilitate viral transcription and efficient propagation of viral genomes. Through its association with a diverse range of host cellular factors, papillomavirus subverts the normal cellular control of proliferation to benefit its own survival and to induce malignant progression of host cells. Studies from recent years have greatly improved our understanding of HPV productive infection and associated cancer progression. However, how different cellular environmental and signaling events influence the viral gene expression during epithelial differentiation remains an important question for future study in order to fully understand the consequences of HPV infection in humans. The application of HPV vaccine in cervical cancer prevention has been discussed in depth elsewhere (Marra et al. 2009). It is worth noting that currently available HPV vaccines protect against up to four major types of cancer-causing strains. However, the vaccines do not protect against many other HPV types, are costly, and are not useful for those who are already infected with the virus. As described above, besides the deregulated expression of high-risk HPV oncogenes, substantial cytogenetic changes in host genome are needed for high-risk HPV-infected cells to progress to invasive tumors. Accumulation of secondary genetic changes usually occurs during the years or even decades of a precancerous state after the initial infection. Alternative therapeutic approaches are, therefore, needed to cure ongoing infections. A greater understanding of the cellular factors and events that regulate persistent latent papillomavirus infection provides a point of departure for developing new therapeutic compounds to abrogate critical virus–host interaction and viral presence. Mechanistic insights into the functions of the viral oncogenes that account for the tumorigenic nature of HPV-associated diseases also offer new strategies to prevent papillomavirusinduced human cancers. Comparing and contrasting these oncogene activities in cervical cancer to other HPV-associated tumors determine whether similar or distinct treatments are ultimately needed.

References Abbate EA, Voitenleitner C, Botchan MR (2006) Structure of the papillomavirus DNA-tethering complex E2:Brd4 and a peptide that ablates HPV chromosomal association. Mol Cell 24:877–889 Alvarez-Salas LM, Cullinan AE, Siwkowski A, Hampel A, DiPaolo JA (1998) Inhibition of HPV16 E6/E7 immortalization of normal keratinocytes by hairpin ribozymes. Proc Natl Acad Sci USA 95:1189–1194

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Peh WL, Middleton K, Christensen N, Nicholls P, Egawa K, Sotlar K, Brandsma J, Percival A, Lewis J, Liu WJ, Doorbar J (2002) Life cycle heterogeneity in animal models of human papillomavirus-associated disease. J Virol 76:10401–10416 Peh WL, Brandsma JL, Christensen ND, Cladel NM, Wu X, Doorbar J (2004) The viral E4 protein is required for the completion of the cottontail rabbit papillomavirus productive cycle in vivo. J Virol 78:2142–2151 Pei XF, Meck JM, Greenhalgh D, Schlegel R (1993) Cotransfection of HPV-18 and v-fos DNA induces tumorigenicity of primary human keratinocytes. Virology 196:855–860 Peitsaro P, Johansson B, Syrjanen S (2002) Integrated human papillomavirus type 16 is frequently found in cervical cancer precursors as demonstrated by a novel quantitative real-time PCR technique. J Clin Microbiol 40:886–891 Pett M, Coleman N (2007) Integration of high-risk human papillomavirus: a key event in cervical carcinogenesis? J Pathol 212:356–367 Pett MR, Alazawi WO, Roberts I, Dowen S, Smith DI, Stanley MA, Coleman N (2004) Acquisition of high-level chromosomal instability is associated with integration of human papillomavirus type 16 in cervical keratinocytes. Cancer Res 64:1359–1368 Pfister H (2003) Chapter 8: human papillomavirus and skin cancer. J Natl Cancer Inst Monogr 31:52–56 Piirsoo M, Ustav E, Mandel T, Stenlund A, Ustav M (1996) Cis and trans requirements for stable episomal maintenance of the BPV-1 replicator. EMBO J 15:1–11 Riley RR, Duensing S, Brake T, Munger K, Lambert PF, Arbeit JM (2003) Dissection of human papillomavirus E6 and E7 function in transgenic mouse models of cervical carcinogenesis. Cancer Res 63:4862–4871 Romanczuk H, Thierry F, Howley PM (1990) Mutational analysis of cis elements involved in E2 modulation of human papillomavirus type 16 P97 and type 18 P105 promoters. J Virol 64:2849–2859 Schaeffer AJ, Nguyen M, Liem A, Lee D, Montagna C, Lambert PF, Ried T, Difilippantonio MJ (2004) E6 and E7 oncoproteins induce distinct patterns of chromosomal aneuploidy in skin tumors from transgenic mice. Cancer Res 64:538–546 Scheffner M, Werness BA, Huibregtse JM, Levine AJ, Howley PM (1990) The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53. Cell 63:1129–1136 Scheffner M, Huibregtse JM, Vierstra RD, Howley PM (1993) The HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53. Cell 75:495–505 Schwartz SM, Daling JR, Doody DR, Wipf GC, Carter JJ, Madeleine MM, Mao EJ, Fitzgibbons ED, Huang S, Beckmann AM, McDougall JK, Galloway DA (1998) Oral cancer risk in relation to sexual history and evidence of human papillomavirus infection. J Natl Cancer Inst 90:1626–1636 Schweiger MR, You J, Howley PM (2006) Bromodomain protein 4 mediates the papillomavirus E2 transcriptional activation function. J Virol 80:4276–4285 Senechal H, Poirier GG, Coulombe B, Laimins LA, Archambault J (2007) Amino acid substitutions that specifically impair the transcriptional activity of papillomavirus E2 affect binding to the long isoform of Brd4. Virology 358:10–17 Sinclair A, Yarranton S, Schelcher C (2006) DNA-damage response pathways triggered by viral replication. Expert Rev Mol Med 8:1–11 Skiadopoulos MH, McBride AA (1998) Bovine papillomavirus type 1 genomes and the E2 transactivator protein are closely associated with mitotic chromatin. J Virol 72:2079–2088 Smith EM, Ritchie JM, Summersgill KF, Klussmann JP, Lee JH, Wang D, Haugen TH, Turek LP (2004) Age, sexual behavior and human papillomavirus infection in oral cavity and oropharyngeal cancers. Int J Cancer 108:766–772 Spanos WC, Nowicki P, Lee DW, Hoover A, Hostager B, Gupta A, Anderson ME, Lee JH (2009) Immune response during therapy with cisplatin or radiation for human papillomavirus-related head and neck cancer. Arch Otolaryngol Head Neck Surg 135:1137–1146

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Spardy N, Duensing A, Charles D, Haines N, Nakahara T, Lambert PF, Duensing S (2007) The human papillomavirus type 16 E7 oncoprotein activates the Fanconi Anemia (FA) pathway and causes accelerated chromosomal instability in FA cells. J Virol 81(23):13265–13270 Spardy N, Covella K, Cha E, Hoskins EE, Wells SI, Duensing A, Duensing S (2009) Human papillomavirus 16 E7 oncoprotein attenuates DNA damage checkpoint control by increasing the proteolytic turnover of claspin. Cancer Res 69:7022–7029 Steger G, Corbach S (1997) Dose-dependent regulation of the early promoter of human papillomavirus type 18 by the viral E2 protein. J Virol 71:50–58 Straight SW, Herman B, McCance DJ (1995) The E5 oncoprotein of human papillomavirus type 16 inhibits the acidification of endosomes in human keratinocytes. J Virol 69:3185–3192 Strati K, Lambert PF (2007) Role of Rb-dependent and Rb-independent functions of papillomavirus E7 oncogene in head and neck cancer. Cancer Res 67:11585–11593 Syrjanen K, Vayrynen M, Castren O, Mantyjarvi R, Pyrhonen S, Yliskoski M (1983) Morphological and immunohistochemical evidence of human papilloma virus (HPV) involvement in the dysplastic lesions of the uterine cervix. Int J Gynaecol Obstet 21:261–269 Tan S-H, Gloss B, Bernard H-U (1992) During negative regulation of the human papillomavirus-16 E6 promoter, the viral E2 protein can displace Sp1 from a proximal promoter element. Nucleic Acids Res 20:251–256 Tan SH, Leong LE, Walker PA, Bernard HU (1994) The human papillomavirus type 16 E2 transcription factor binds with low cooperativity to two flanking sites and represses the E6 promoter through displacement of Sp1 and TFIID. J Virol 68:6411–6420 Thierry F (2009) Transcriptional regulation of the papillomavirus oncogenes by cellular and viral transcription factors in cervical carcinoma. Virology 384:375–379 Thierry F, Howley PM (1991) Functional analysis of E2-mediated repression of the HPV18 P105 promoter. New Biol 3:90–100 Thierry F, Yaniv M (1987) The BPV1-E2 trans-acting protein can be either an activator or a repressor of the HPV18 regulatory region. EMBO J 6:3391–3397 Thomas MC, Chiang CM (2005) E6 oncoprotein represses p53-dependent gene activation via inhibition of protein acetylation independently of inducing p53 degradation. Mol Cell 17:251–264 Thomas JT, Laimins LA (1998) Human papillomavirus oncoproteins E6 and E7 independently abrogate the mitotic spindle checkpoint. J Virol 72:1131–1137 Thomas JT, Hubert WG, Ruesch MN, Laimins LA (1999) Human papillomavirus type 31 oncoproteins E6 and E7 are required for the maintenance of episomes during the viral life cycle in normal human keratinocytes. Proc Natl Acad Sci USA 96:8449–8454 Thompson DA, Belinsky G, Chang TH, Jones DL, Schlegel R, Munger K (1997) The human papillomavirus-16 E6 oncoprotein decreases the vigilance of mitotic checkpoints. Oncogene 15:3025–3035 Thorland EC, Myers SL, Persing DH, Sarkar G, McGovern RM, Gostout BS, Smith DI (2000) Human papillomavirus type 16 integrations in cervical tumors frequently occur in common fragile sites. Cancer Res 60:5916–5921 Thorland EC, Myers SL, Gostout BS, Smith DI (2003) Common fragile sites are preferential targets for HPV16 integrations in cervical tumors. Oncogene 22:1225–1237 van Houten VM, Snijders PJ, van den Brekel MW, Kummer JA, Meijer CJ, van Leeuwen B, Denkers F, Smeele LE, Snow GB, Brakenhoff RH (2001) Biological evidence that human papillomaviruses are etiologically involved in a subgroup of head and neck squamous cell carcinomas. Int J Cancer 93:232–235 Van Tine BA, Dao LD, Wu SY, Sonbuchner TM, Lin BY, Zou N, Chiang CM, Broker TR, Chow LT (2004) Human papillomavirus (HPV) origin-binding protein associates with mitotic spindles to enable viral DNA partitioning. Proc Natl Acad Sci USA 101:4030–4035 Venturini F, Braspenning J, Homann M, Gissmann L, Sczakiel G (1999) Kinetic selection of HPV 16 E6/E7-directed antisense nucleic acids: anti-proliferative effects on HPV 16-transformed cells. Nucleic Acids Res 27:1585–1592

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Venuti A, Manni V, Morello R, De Marco F, Marzetti F, Marcante ML (2000) Physical state and expression of human papillomavirus in laryngeal carcinoma and surrounding normal mucosa. J Med Virol 60:396–402 Vernon SD, Unger ER, Reeves WC (1998) Human papillomaviruses and anogenital cancer. N Engl J Med 338:921–922 von Knebel Doeberitz M, Rittmuller C, zur Hausen H, Durst M (1992) Inhibition of tumorigenicity of cervical cancer cells in nude mice by HPV E6-E7 anti-sense RNA. Int J Cancer 51:831–834 Wang Q, Griffin H, Southern S, Jackson D, Martin A, McIntosh P, Davy C, Masterson PJ, Walker PA, Laskey P, Omary MB, Doorbar J (2004) Functional analysis of the human papillomavirus type 16 E1=E4 protein provides a mechanism for in vivo and in vitro keratin filament reorganization. J Virol 78:821–833 Watanabe S, Kanda T, Yoshiike K (1993) Growth dependence of human papillomavirus 16 DNApositive cervical cancer cell lines and human papillomavirus 16-transformed human and rat cells on the viral oncoproteins. Jpn J Cancer Res 84:1043–1049 Wiest T, Schwarz E, Enders C, Flechtenmacher C, Bosch FX (2002) Involvement of intact HPV16 E6/E7 gene expression in head and neck cancers with unaltered p53 status and perturbed pRb cell cycle control. Oncogene 21:1510–1517 Wilson R, Fehrmann F, Laimins LA (2005) Role of the E1–E4 protein in the differentiation-dependent life cycle of human papillomavirus type 31. J Virol 79:6732–6740 Wu SY, Chiang CM (2007) The double bromodomain-containing chromatin adaptor Brd4 and transcriptional regulation. J Biol Chem 282:13141–13145 Wu SY, Lee AY, Hou SY, Kemper JK, Erdjument-Bromage H, Tempst P, Chiang CM (2006) Brd4 links chromatin targeting to HPV transcriptional silencing. Genes Dev 20:2383–2396 Yan J, Li Q, Lievens S, Tavernier J, You J (2010) Abrogation of the Brd4-positive transcription elongation factor B complex by papillomavirus E2 protein contributes to viral oncogene repression. J Virol 84:76–87 You J, Croyle JL, Nishimura A, Ozato K, Howley PM (2004) Interaction of the bovine papillomavirus E2 protein with Brd4 tethers the viral DNA to host mitotic chromosomes. Cell 117:349–360 You J, Schweiger MR, Howley PM (2005) Inhibition of E2 binding to Brd4 enhances viral genome loss and phenotypic reversion of bovine papillomavirus-transformed cells. J Virol 79:14956–14961 Yu T, Peng YC, Androphy EJ (2007) Mitotic kinesin-like protein 2 binds and colocalizes with papillomavirus E2 during mitosis. J Virol 81:1736–1745 Zeitler J, Hsu CP, Dionne H, Bilder D (2004) Domains controlling cell polarity and proliferation in the Drosophila tumor suppressor Scribble. J Cell Biol 167:1137–1146 Zerfass-Thome K, Zwerschke W, Mannhardt B, Tindle R, Botz JW, Jansen-Durr P (1996) Inactivation of the cdk inhibitor p27KIP1 by the human papillomavirus type 16 E7 oncoprotein. Oncogene 13:2323–2330 zur Hausen H (2000) Papillomavirus causing cancer: evasion from host-cell control in early events in carcinogenesis. J Natl Cancer Inst 92:690–698 zur Hausen H (2002) Papillomaviruses and cancer: from basic studies to clinical application. Nat Rev Cancer 2:342–350 zur Hausen H (2009) Papillomaviruses in the causation of human cancers – a brief historical account. Virology 384:260–265

Chapter 20

Tumorigenesis by Adenovirus Type 12 E1A Hancheng Guan and Robert P. Ricciardi

Introduction All human adenoviruses, of which there are 51 known serotypes, are capable of transforming mammalian cells including those of human and rodent origin. Two viral immediate early gene products E1A and E1B, which are encoded on the far left end of the linear viral genome, are responsible for cell transformation. E1A stimulates the cell cycle via its binding and interference of key cellular regulators including retinoblastoma protein (Rb) and p300/CBP, thus leading to constitutive cellular DNA synthesis and the loss of cell cycle regulation. In turn, through its interaction with the tumor suppressor p53, E1B serves to block cell growth arrest and apoptosis incurred by E1A-stimulated unconstrained cellular DNA synthesis and damage. The net result is immortalization and continued cell proliferation, i.e., transformation. In adenovirustransformed cells, E1A and E1B genes are normally integrated into the chromosomes. Transformation by adenovirus can be fulfilled directly by transfection of E1A and E1B into mammalian cells in vitro or by infection of nonpermissive cells (e.g., those of rodents) with the virus. It is noted that adenovirus transformation requires the two viral genes to be expressed above a certain threshold. Transformation by human adenoviruses has been the subject of several recent reviews (Ricciardi 1999; Gallimore and Turnell 2001; Williams et al. 2004; Hohlweg et al. 2004; Endter and Dobner 2004; Chinnadurai 2004) and is not discussed in detail here.

H. Guan Department of Microbiology, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA R.P. Ricciardi (*) Department of Microbiology, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA Abramson Cancer Center, University of Pennsylvania School of Medicine, Levy Research Building, 4010 Locust Street, Philadelphia, PA 19104, USA e-mail: [email protected] E.S. Robertson (ed.), Cancer Associated Viruses, Current Cancer Research, DOI 10.1007/978-1-4614-0016-5_20, © Springer Science+Business Media, LLC 2012

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While all human adenoviruses have the ability to transform cells in culture as described above, only a small group of serotypes have the additional capability to generate tumors in immunocompetent adult rodents. Among the tumorigenic strains of human adenovirus, adenovirus type 12 (Ad12) is the most intensively studied. An important feature related with Ad12 tumorigenesis is the ability of the viral transformed cells to escape immune surveillance and destruction (reviewed in Williams et al. 2004; Hohlweg et al. 2004; Ricciardi et al. 2006). Ad12 E1A oncoprotein (E1A-12) has been shown to be the key determinant of tumorigenicity (Bernards et al. 1982; Sawada et al. 1988). As reviewed below, Ad12 tumorigenesis relies on the ability of E1A-12 to mediate evasion of cytolysis by CTLs and NK cells. Some of the mechanisms for these immune escape strategies mediated by E1A-12 have recently become disclosed. Moreover, a new insight into the involvement of E1A-12 induced expression of neuronal and tumor-related genes in Ad12 tumorigenesis has been revealed.

Ad12 Tumorigenesis Requires MHC Class I to Be Shutoff: A Means for CTL Avoidance About five decades ago, Ad12 was first found to generate tumors in newborn Syrian Hamsters (Huebner et al. 1962; Trentin et al. 1962). However, many other human adenoviruses including the well-studied Ad5 were shown to be nontumorigenic. It was later discovered that adenoviruses that were classified as nontumorigenic could be made to induce tumors in immunosuppressed rats or mice (Gallimore 1972; Bernards et al. 1983). These studies suggested that Ad12 generates tumors in immunocompetent adult rodents by providing the tumor cells with a means of avoiding cytolysis by CTLs. This notion was supported by the finding that cell surface expression of major histocompatibility complex (MHC) class I molecules is dramatically reduced on Ad12 transformed cells (Schrier et al. 1983; Eager et al. 1985). MHC class I molecules are glycoproteins belonging to the immunoglobulin superfamily. MHC class I molecules are expressed on the surfaces of most mammalian cells and play an essential role in “tagging” cells that express foreign (e.g., viral) proteins for immune destruction by CTLs. The class I molecules accomplish this by presenting nonself proteins, in the form of processed peptides, to the cell surface where they can be recognized by CTLs to trigger lysis of the target cell. However, cells that express very low surface levels of MHC class I molecules are able to escape from recognition and lysis by CTLs (Zinkernagel and Doherty 1979; reviewed in Garcia-Lora et al. 2003). As stated above, reduced expression of MHC class I molecules on the cell surface provides Ad12-transformed cells with a powerful strategy for immune evasion. Significantly, this reduction in MHC class I surface expression occurs with all of the haplotypes such as H2-K, -D, and -L in mouse (Eager et al. 1985). By contrast, substantial levels of each of the class I glycoproteins are expressed on the surfaces of nontumorigenic Ad5-transformed cells (Eager et al. 1985). It is noted that as with Ad5-transformed cells, Ad12 tumorigenic cells have intact MHC class I genes, since stimulation of the interferon inducible element in their promoters by interferon-gamma

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(IFN-g) can activate MHC class I expression in these tumorigenic cells (Eager et al. 1985; Kimura et al. 1986). It is well documented that reduced cell surface levels of MHC class I antigens contribute to CTL escape and tumorigenesis by Ad12-transformed cells. A study in which influenza virus-infected Ad12-transformed cells were challenged with influenza-specific CTLs revealed resilience of the cells to lysis, whereas Ad5transformed control cells were susceptible to the lysis (Yewdell et al. 1988). Significantly, treatment of the influenza virus-infected Ad12-transformed cells with IFN-g induced an increase of surface MHC class I molecules and renewed CTL lysis of the cells (Yewdell et al. 1988). This demonstrates that downregulation of MHC class I surface antigen expression on Ad12-transformed cells enables evasion of cytolysis by self-restricted CTLs. As a consequence, Ad12transformed cells can continuously proliferate to generate tumors in rodents with intact immune systems. Consistent with these results, another study showed that expression of an exogenous MHC class I gene in Ad12-transformed cells could abrogate tumorigenicity (Tanaka et al. 1985). It was also shown that IFN-g induced transient expression of MHC class I antigens in Ad12-transformed cells is sufficient to reduce their tumorigenicity in immunocompetent rodents (Hayashi et al. 1985). In addition, subcutaneous administration of IFN-g following the introduction of a tumorigenic dose of Ad12-transformed cells completely hindered the development of tumors in rodents (Hayashi et al. 1985). Importantly, cells from freshly excised tumors generated by Ad12-transformed cells were found to exhibit diminished MHC class I antigens (Eager et al. 1985). These data clearly demonstrate a direct association between Ad12 tumorigenesis and diminution of MHC class I surface expression.

E1A-12 Is Solely Responsible for MHC Class I Shutoff Although cell transformation by Ad12 requires both E1A-12 and E1B-12 genes, only the E1A-12 gene is responsible for cell surface MHC class I reduction (Schrier et al. 1983; Vasavada et al. 1986). This was first demonstrated by expressing E1A-12 protein as the only product of Ad12 in a human cell line (Vasavada et al. 1986). This E1A-12 expressing cell line exhibited dramatic reduction in class I HLA levels of each haplotype (HLA, A, B, and C) compared with a matched control cell line. Another study using hybrid cell lines of Ad12and Ad5-transformed cells also revealed that MHC class I surface antigens of the hybrid cells were reduced to the same extent as that of their parental Ad12transformed cells (Ge et al. 1994). This indicates that E1A-12 is able to reduce the high surface class I levels of Ad5-transformed cells. Indeed, a direct stable introduction of E1A-12 into Ad5-transformed cells gave rise to reduced expression of MHC class I antigens on the cell surface (Guan et al. 2008). Together, these findings demonstrate that E1A-12 actively mediates the dramatic reduction of MHC class I surface antigens.

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E1A-12 Mediates MHC Class I Shutoff at the Transcription Level The low amount of MHC class I surface antigens on Ad12 transformed cells were found to be directly related to a drastic reduction in the class I mRNA levels (Schrier et al. 1983; Eager et al. 1985; Vasavada et al. 1986; Liu et al. 1996). Significantly, as revealed by nuclear run-on experiments, diminished transcription is responsible for these reduced levels of MHC class I mRNAs (Ackrill and Blair 1988; Friedman and Ricciardi 1988). This suggested that the class I promoter is negatively regulated in Ad12 tumorigenic cells. Extensive mutational analyses identified that the class I promoter enhancer region (located between nucleotides −205 and −159 upstream of the transcription start site) is the targeted element responsible for diminished MHC class I transcription (Ge et al. 1992). This enhancer, when appended to the class I basal TATA box promoter (located between nucleotides −37 and +1), strongly stimulated transcription in Ad5-transformed cells, but not in Ad12-transformed cells (Ge et al. 1992). As discussed below, modulation of transcription factor binding to the class I enhancer region by E1A-12 accounts for transcription diminution of all MHC class I genes. The MHC class I enhancer comprises two DNA regulatory elements, referred to as R1 and R2 (Kimura et al. 1986). The R1 site contains a recognition sequence for the transcription activator NF-kB, whereas the R2 sequence has a recognition sequence for nuclear hormone receptors including the transcription repressor COUPTFII. As detailed below and depicted in Fig. 20.1, E1A-12 prevents NF-kB binding and transactivation activity at the class I enhancer R1 site, while concurrently mediating the binding of the nuclear hormone repressor COUP-TFII to the R2 site.

E1A-12 Prevents NF-kB (p50/p65) Binding to the Class I Enhancer R1 Site as well as Its Transactivation Activity by Blocking Phosphorylation of p50 and p65 NF-kB is a transactivator composed predominantly of two subunits p50 and p65. The p50 subunit is largely responsible for DNA binding, whereas the p65 subunit is accountable for transactivation through its C-terminal activation domain. NF-kB is the master regulator of immune responsive genes including the class I alleles (reviewed in Xiao and Ghosh 2005). Under normal circumstances, NF-kB is retained in the cytoplasm via its association with inhibitor IkB. Upon stimulation by cytokines such as TNF-a, IkB is phosphorylated by IkB kinase (IKK) and subsequently ubiquitinated and degraded by the 26S proteasome (reviewed in Baldwin 1996; Pahl 1999; Scheidereit 2006). This leads to the release and translocation of NF-kB to the nucleus where it binds to target genes and activates transcription. In both Ad5 nontumorigenic and Ad12 tumorigenic cells, NF-kB constitutively translocates to the nucleus and is similarly abundant (Liu et al. 1996). Yet, only

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Ad5 non-tumorigenic cells

493 E1A-5 PKAc 50 65

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NCoR HDAC1/8 COUP -TFII COUP -TFII R2 SITE

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Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac

Fig. 20.1 Model of E1A-12-mediated repression of MHC class I transcription. Upper panel: Active MHC class I transcription in Ad5 nontumorigenic cells. The ability of NF-kB (p50/p65) to bind DNA and stimulate transcription requires phosphorylation of each subunit. Specifically, NF-kB is phosphorylated by the catalytic subunit of PKA (PKAc) at serine-337 of p50 and serine 276 of p65. It is noted that E1A-5 can bind PKAc but not NF-kB. The R2 site of the class I enhancer is not occupied by the COUP-TFII repression complex and the chromatin is acetylated (Ac), indicative of active transcription. Lower panel: Downregulated MHC class I transcription in Ad12 tumorigenic cells. Binding of E1A-12 to both NF-kB and PKAc prevents access of PKAc to p50 and p65, thereby prohibiting phosphorylation and binding of NF-kB (p50/p65) to the R1 site. By contrast, E1A-12 mediates the binding of the COUP-TFII repression complex to the R2 site. This repression complex includes homodimers of COUP-TFII, which directly bind the R2 site, the nuclear corepressor NCoR, the histone deacetylases HDAC1 and HDAC8, and E1A-12. The HDACs act to deacetylate chromatin (→Ac), indicative of inactive transcription. The recruitment or stabilization of HDAC1 and HDAC8 to the repression complex is dependent on the presence of E1A-12

nuclear NF-kB in Ad12 tumorigenic cells fails to bind the R1 site of the class I enhancer (Liu et al. 1996). Exchange of NF-kB p50 and p65 subunits between Ad5and Ad12-transformed cells clearly revealed that the p50 subunit is largely responsible for the NF-kB DNA binding deficiency in Ad12 tumorigenic cells (Kushner and Ricciardi 1999). Essentially, p50 proteins from Ad12 tumorigenic cells fail to support DNA binding when reconstituted with the p65 subunit from Ad5 nontumorigenic cells. By contrast, NF-kB composed of p50 from Ad5-transformed cells and p65 from

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either cell strongly binds DNA. It was discovered that p50 from Ad12 tumorigenic cells is hypophosphorylated, whereas this subunit is highly phosphorylated in Ad5transformed cells (Kushner and Ricciardi 1999). This finding supports the notion that phosphorylation of p50 is critical for DNA binding (Li et al. 1994). Indeed, phosphatase treatment of p50 from Ad5-transformed cells abolished p50 DNA binding activity (Kushner and Ricciardi 1999). As indicated above, due to the hypophosphorylation of p50, NF-kB fails to bind to the class I enhancer even though it is abundant in nuclei of Ad12 tumorigenic cells (Kushner and Ricciardi 1999). To address how phosphorylation of p50 affects its DNA binding activity, extensive mutagenesis of p50 was conducted to define which phospho-residues of p50 are essential for DNA binding. This led to the finding that serine residue 337 is critical for DNA binding of p50 (Hou et al. 2003). This serine residue was determined to be phosphorylated by the protein kinase A catalytic subunit (PKAc) and its phosphorylation by this kinase is required for p50 DNA binding (Guan et al. 2005). It is interesting to note that this discovery provides another example by which viral related studies often lead to understanding natural host mechanisms, in this case, the manner by which DNA binding of NF-kB is regulated by phosphorylation. The major question regarding the mechanism of how E1A-12 prevents DNA binding of NF-kB is becoming clearer. It has been recently discovered that while both E1A-12 and E1A-5 can physically interact with PKAc, only E1A-12 is able to bind p50 (Guan et al. 2008; Guan and Ricciardi, unpublished). Significantly, the binding of E1A-12 to p50 prevents PKAc from phosphorylating serine-337, whereas the kinase activity of PKAc is not affected by E1A-12 (Guan et al. 2008). These data suggest that E1A-12, PKAc and p50 could form a tri-complex, in which E1A-12 may block p50 from gaining access to the catalytic site of PKAc. Since PKAc is essential to cell survival, then E1A-12 in this context would target only a single substrate of the kinase, i.e., serine-337 of p50. Interestingly, by binding to the p65 subunit, E1A12 also prevents PKAc from phosphorylating serine-276 in a similar manner (Guan et al. 2008; Jiao et al. 2010). Phosphorylation of p65 serine-276 by PKAc is important for NF-kB transactivation (Zhong et al. 1997, 1998, 2002). It has been proposed that phosphorylation of p65 serine-276 by PKAc induces a conformational change that releases an intramolecular masking of the C-terminal transactivation domain by the N-terminal region, thus leading to enhanced binding by the coactivator CBP/ p300 (Zhong et al. 1998). E1A-12 deletion analyses demonstrate that the N-terminal 40 residues are sufficient to bind p65 and block serine-276 phosphorylation (Jiao et al. 2010). This very N-terminal region is also capable of binding the p50 subunit (Jiao et al., unpublished data), which is likely to block p50 phosphorylation as well. In summary, one mechanism employed by E1A-12 to downregulate transcription from the class I promoter is to prevent NF-kB from binding to the enhancer R1 site and stimulating transcription. Specifically, E1A-12, especially the N-terminal 40-residue region, impedes the ability of PKAc to phosphorylate NF-kB at serine-337 of p50 and serine-276 of p65 (Fig. 20.1). As a consequence, NF-kB DNA binding and transactivation activities are inhibited, MHC class I transcription is downregulated, and MHC class I antigen expression on the cell surface is diminished.

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E1A-12 Mediates Repression of MHC Class I Transcription by Recruiting COUP-TFII/HDAC to the R2 Site of the Class I Enhancer The R2 site of the MHC class I enhancer contains a recognition sequence for nuclear hormone receptors (Kimura et al. 1986). Ad12 tumorigenic cells were shown to display a strong binding activity to the R2 site, whereas such binding activity is absent in Ad5-transformed cells (Ge et al. 1992; Kurshner et al. 1996). In Ad12 tumorigenic cells, this strong binding activity to the R2 site is correlated with repression of the class I promoter (Ge et al. 1992; Kralli et al. 1992). It was determined that the nuclear hormone orphan receptor COUP-TFII is responsible for the R2 site binding activity (Liu et al. 1994; Smirnov et al. 2001). COUP-TFII, which is known to function mainly as a transcriptional repressor (Zhou et al. 2000), occupies the R2 site as a homodimer in Ad12 tumorigenic cells (Liu et al. 1994). Interestingly, this is the only known instance in which COUP-TFII binds to the well-studied MHC class I enhancer to repress transcription. It was discovered that COUP-TFII, via its C-terminal domain, forms a repression complex at the R2 site with HDAC (histone deacetylase) and N-CoR (Smirnov et al. 2000). Histone deacetylation by HDAC in the class I promoter causes chromatin condensation, which results in transcription repression of the target gene. Since there is COUP-TFII binding to the R2 site only in Ad12 tumorigenic cells, this explains why the R2 site exerts its repression on MHC class I transcription in Ad12 tumorigenic cells, but not in Ad5 nontumorigenic cells. In Ad12 tumorigenic cells, what accounts for COUP-TFII binding to the R2 site of the class I enhancer? It was shown that Ad12 tumorigenic cells contain elevated levels of COUP-TFII mRNA and protein when compared with Ad5 nontumorigenic cells (Smirnov et al. 2001). E1A-12 is involved in upregulating COUP-TFII expression. Obviously, this explains in one way how E1A-12 mediates repression at the R2 site of the class I enhancer. Significantly, in addition to upregulating COUPTFII, E1A-12 was found to be an integral component of the COUP-TFII/HDAC complex (Zhao et al. 2003). Essentially, the physical presence of E1A-12 in this complex is necessary for transcriptional repression of MHC class I (Zhao and Ricciardi 2006). It is noted that E1A-12 does not bind to DNA directly, but functions through protein–protein interactions (reviewed in Flint and Shenk 1997). A recent ChIP (chromatin immunoprecipitation) assay, a method used to determine specific proteins that bind to targeted DNA region, revealed that HDAC1 and/or HDAC8 reside in the COUP-TFII repression complex at the class I enhancer R2 site (Zhao and Ricciardi 2006). Elimination of E1A-12 from this COUP-TFII repression complex by antisense oligonucleotides also removes HDAC1 and HDAC8 from the complex, which results in an increase in both chromatin acetylation (a hallmark of active transcription) of the class I enhancer and expression of MHC class I antigens on the cell surface (Zhao and Ricciardi 2006). These data strongly indicate that E1A-12 mediates MHC class I repression via recruiting (or stabilizing) HDAC1 and HDAC8 to the COUP-TFII repression complex.

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In summary, E1A-12 mediates MHC class I repression via the enhancer R2 site. E1A-12 not only upregulates COUP-TFII expression and binding activity to the R2 site but also acts to recruit (or stabilize) HDAC1 and HDAC8 to the COUP-TFII repression complex, thus resulting in chromatin condensation of the class I gene (Fig. 20.1). As a consequence of these E1A-12-mediated activities at the enhancer R2 site, MHC class I transcription is repressed and the cell surface levels of MHC class I antigens are dramatically diminished.

E1A-12-Mediated Regulation of Both NF-kB and COUP-TFII/ HDAC Binding Provides a FAIL-SAFE Mechanism to Ensure MHC Class I Repression What is the advantage for E1A-12 to employ two distinct mechanisms, i.e., inhibition of NF-kB DNA binding and transactivation plus enhancement of COUP-TFII/ HDAC repression, for MHC class I shutoff? It is likely that E1A-12 uses these two mechanisms to ensure that MHC class I antigens on the cell surface do not become induced by physiological fluctuations. For example, under normal cell growth conditions, E1A-12 is able to prevent nuclear NF-kB DNA binding and transactivation by blocking phosphorylation of both p50 and p65 as described above (Hou et al. 2003; Guan et al. 2005, 2008; Jiao et al. 2010). However, cytokines such as TNF-a and IL-1b are able to induce a new pool of nuclear NF-kB that is capable of DNA binding in Ad12 tumorigenic cells, while the preexisting pool of hypophosphorylated NF-kB remains unable to bind DNA (Hou et al. 2002). This indicates that while E1A-12 constitutively blocks phosphorylation of the preexisting pool of NF-kB, this mechanism is not always sufficient to completely prevent phosphorylation of a massive new pool of cytokine-induced NF-kB. Nevertheless, the binding of COUP-TFII/ HDAC repressor complex to the R2 site should be able to override the cytokineinduced NF-kB DNA binding and transactivation activity, thus keeping MHC class I shutoff. Indeed, TNF-a and IL-1b fail to enhance MHC class I transcription as long as the COUP-TFII/HDAC complex binds to the class I enhancer (Hou et al. 2002). Only when the repressive effect of COUP-TFII/HDAC is relieved, a pronounced activation of MHC class I transcription by NF-kB following cytokine treatment is observed (Hou et al. 2002). As expected, treatment of Ad12 tumorigenic cells with both cytokines and HDAC inhibitor TSA leads to a significant increase in the level of cell surface MHC class I antigens (Hou et al. 2002). Further evidence that COUPTFII repression dominates over NF-kB activation in MHC class I transcription regulation is obtained from NF-kB knockout cells, in which activation by exogenous NF-kB is completely blocked by exogenous COUP-TFII (Smirnov et al. 2001). In summary, Ad12 tumorigenic cells apparently benefit from the collateral effects of COUP-TFII/HDAC repressor complex and hypophosphorylated NF-kB in downregulating MHC class I transcription (Fig. 20.1). These dual functions provide a stringent “FAIL-SAFE” mechanism to ensure that transcription of MHC class I genes remains shutoff under oscillating physiological conditions. In the event that NF-kB

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is activated by cytokines and other stimulators, COUP-TFII/HDAC will still maintain repression of MHC class I transcription, thus ensuring immune escape and survival of tumorigenic Ad12 cells. Alternatively, if COUP-TFII/HDAC repressor complex loses its binding to the class I enhancer or the repression activity under certain physiological conditions, then hypophosphorylation of NF-kB will ensure that this activator fails to bind DNA and stimulate MHC class I transcription. We propose that to stringently protect cells from lysis by CTLs, E1A-12 coevolved dual functions that serve to block NF-kB DNA binding and transactivation at the class I enhancer R1 site, while simultaneously recruit the COUP-TFII/HDAC repressor complex to the R2 site.

E1A-12 Mediates Escape from Natural Killer Cells by Downregulating NK Activating Ligands E1A-12 Confers Resistance to NK Cell Lysis NK cells of the innate immune system serve as the first line of defense. These effectors of innate immunity respond to the integration of inhibitory and activating signals from target cells (Garcia-Lora et al. 2003; Makrigiannis and Anderson 2003; Lodoen and Lanier 2005; reviewed in Lanier 2005). It is well known that loss of MHC class I antigen expression on target cells relieves the NK inhibitory receptor and leads to lysis of target cells. Yet, surprisingly, Ad12 tumorigenic cells that have diminished MHC class I surface antigens are also resistant to lysis by NK cells (Sawada et al. 1985; Kenyon and Raska 1986). The E1A-12 gene of tumorigenic Ad12 specifically confers this resistance to NK lysis (Sawada et al. 1985; Kenyon and Raska 1986). Could it be that E1A-12 has found a way to downregulate cell surface expression of both activating ligands and MHC class I surface antigens as a means of avoiding NK and CTL lysis? Recent evidence (below) suggests that E1A-12 does employ this strategy to escape both innate (NK) and cell-mediated (CTL) immunity.

E1A-12 Reduces Expression of Activating Ligands that Trigger NK Lysis NKG2D is a major activating receptor expressed on the surface of murine NK cells. The NKG2D receptor on NK cells recognizes activating ligands (RAE1-a, -b, -g, -e, MULT1, and H60) that are upregulated on infected and transformed target cells. By contrast, MHC class I molecules expressed on target cells engage inhibitory receptors on NK cells. Because MHC class I molecules function as recognition elements for CTLs but as ligands for inhibitory receptors on NK cells, they play diametric roles in CTL versus NK cell-mediated lysis (Kenyon and Raska 1986;

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Fig. 20.2 Mechanism by which Ad12 tumorigenic cells escape lysis by NK cells. Top panel: Modulation of NK lysis of target cells depends on the balance of inhibitory (e.g., MHC class I) and activating ligands. Middle panel: Loss of inhibitory MHC class I favors lysis. Bottom panel: Ad12 tumorigenic cells have diminished MHC class I expression yet escape NK cells due to E1A-12mediated reduction of all NKG2D activating ligands. See text for details

reviewed in Lanier 2005). Ultimately, the deciding factor as to whether the NK cell should kill the target cell is somewhat complex in that lysis is modulated by the balance of inhibitory and activating signals (Fig. 20.2, top panel). Indeed, when inhibitory MHC class I molecules are downregulated, the engagement of NKG2D receptor and activating ligands enables lysis (Fig. 20.2, middle panel). Sometimes, however, the activating signals are sufficiently elevated to cause lysis, even though surface MHC class I is expressed. This apparently is the case in nontumorigenic Ad5-transformed cells that express MHC class I antigens. Here, E1A-5 has been shown to sensitize the nontumorigenic Ad5 cells to NK lysis, by binding to p300 and upregulating the RAE-1 activating ligand that is recognized by the NKG2D activating receptor (Cook et al. 1996; Routes et al. 2005). Presumably, in Ad5 nontumorigenic cells, the activating ligand expression (e.g., RAE-1) dominates the inhibitory MHC class I signal. Contrary to expectations, the almost complete diminution in MHC class I expression on Ad12 tumorigenic cells does not make them sensitive to NK lysis. Rather, Ad12 tumorigenic cells are highly resistant to NK lysis and E1A-12 has been shown to be responsible for conferring this resistance (Sawada et al. 1985; Kenyon and Raska 1986; Heyward et al., unpublished).

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Recent findings now reveal that E1A-12 mediates resistance to NK lysis by inhibiting expression of all of the NKG2D activating ligands on the cell surface (Heyward et al., unpublished). This paninhibition is depicted in Fig. 20.2 (bottom panel), which indicates that on Ad12 cells, there is lack of both inhibitory MHC class I ligand as well as all of the NKG2D activating ligands. Further studies demonstrated that the level of mRNA for each NKG2D activating ligand is also greatly reduced (Heyward et al., unpublished). Thus, the diminished levels of NKG2D activating ligands on the surface of Ad12 tumorigenic cells directly correlate with the dramatically downregulated mRNA expression of NKG2D activating ligands. Based on general knowledge that E1A proteins regulate gene expression of multiple promoters, it is likely that E1A-12 may target regulatory transcription factor complexes that are common to all of the NKG2D activating ligands. Currently, the regulatory domains of the NKG2D activating ligand promoters are poorly characterized (Nomura et al. 1996; Nausch et al. 2006). However, a comparison of the NKG2D activating ligand promoter sequences revealed binding sites for NF-kB and COUP-TFII (Heyward and Ricciardi, unpublished), which, of course, are the targets of E1A-12-mediated repression of the class I promoter as described above. Intriguingly, should the binding activities of NF-kB and COUP-TFII in the NKG2D activating ligand promoters also be affected, this would suggests that E1A-12 uses a single mechanism to coordinately enable escape of Ad12 tumorigenic cells from both innate (NK) and cell-mediated (CTL) immunity.

E1A-12 Induces Expression of Neuronal and Tumor-Related Genes While E1A-12-mediated downregulation of MHC class I and NK ligands essentially contributes to viral tumorigenesis via enabling immune escape from CTLs and NK cells, other cellular genes mediated by the E1A-12 protein likely also play a critical role in Ad12 tumorigenesis. Therefore, a broader view of Ad12 tumorigenesis may be better addressed by understanding of E1A-12-mediated global changes in gene expression. Recent studies using microarray have revealed that E1A-12 is involved in modulating numerous genes that are implicated in various cell functions including the cell cycle, transcriptional regulation, signal transduction, immune response, and tumor invasiveness (Guan et al. 2003, 2009). Of special interest among those genes regulated by E1A-12 are a number of neuronal and tumor-related genes whose expression is induced by E1A-12 in Ad12 tumorigenic cells (Guan et al. 2009). Compared with E1A-5 and the counterparts of other nontumorigenic adenoviruses, E1A-12 contains a unique “Spacer” region that is composed of 20 amino acids between the conserved regions CR2 and CR3 (Fig. 20.3). This Spacer region is essential for Ad12 tumorigenesis (reviewed in Ricciardi 1999, 2006; Williams et al. 2004). Deletion of the Spacer or alteration of even a single amino acid in the Spacer can abolish Ad12 tumorigenesis (reviewed in Williams et al. 2004; Ricciardi et al. 2006). However, Ad12 Spacer mutants retain the ability to repress MHC

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Fig. 20.3 E1A-12 oncoprotein. Depicted is the largest spliced form of the E1A-12 protein (266 residues) showing four conserved regions (CR1, CR2, CR3, CR4), and the unique 20-residue Spacer (S) region. Indicated are the functions encoded by each domain

class I expression as well as to transform cells in culture (reviewed in Ricciardi 1999, 2006; Williams et al. 2004). A recent microarray study has revealed that the Spacer, along with other regions in E1A-12, plays a critical role in upregulating a number of neuronal and tumor-related genes including N-MYC, ROBO1, and neuronal protein 3.1 (Guan et al. 2009). Specifically, this microarray compared gene expression profiles between Ad12 cell lines with only a single amino acid difference in E1A-12: one cell line expressed wt E1A-12 and was tumorigenic, whereas the other cell line expressed a point-mutant of E1A-12 and was nontumorigenic. Thus, the differential expression in cellular genes could be accounted for by a singleamino acid substitution in E1A-12. The fact that the E1A-12 spacer is essential for Ad12 tumorigenesis suggests that some neuronal genes it upregulates are likely involved in the viral tumorigenic process. For example, N-MYC is known to possess cell-transforming and oncogenic functions (Ingvarsson 1990; Kawagoe et al. 2007); moreover, the transmembrane receptor ROBO1, whose normal function involves axon path finding and neuronal migration (Kidd et al. 1998; Zallen et al. 1998; Brose et al. 1999), has been reported to participate in mediating glioma cell migration (Mertsch et al. 2008). Importantly, ROBO1 has been found to be highly expressed in several nonneuronal tumors including hepatocellular carcinoma (Ito et al. 2006) and colorectal cancer (Grone et al. 2006). In accord with a potential nexus between neuronal gene expression and E1A-12-mediated tumorigenesis is the reduction of MHC class I surface antigens on neuronal cells as well as a requirement of COUP-TFII for neuronal development (Qiu et al. 1994). Particularly relevant to these molecular studies is the observation that Ad12-induced tumors exhibit mesenchymal and neuronal characteristics (reviewed in Hohlweg et al. 2004). It is, thus, intriguing to speculate that Ad12 may usurp the functions of certain neuronal genes to promote tumorigenesis. It is noted that the induction of neuronal gene expression is not limited to Ad12 tumorigenic cells, but is relatively common in tumor cells. For example, it has been found that several nonneuronal tumors including breast, ovarian,

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colorectal, and pancreatic cancers all display aberrant expression of neuronal genes (Chen et al. 1997; Garber et al. 2001; Albert and Darnell 2004; Zhang et al. 2005; Grone et al. 2006). While the real functions and the involvement of tumorassociated neuronal genes in cancer formation has yet to be further elucidated, it is possible that the neuronal gene expression may provide the tumor cells with some survival advantage, e.g., immune privilege and relatively high motility as found with neuronal cells.

Neuronal Gene Induction by E1A-12 Involves the Inhibition of the Neuronal Repressor NRSF and the Stimulation of Certain Neuronal Transactivators Like many other cellular genes, neuronal genes are regulated by both transcription repressors and activators. In nonneuronal cells, however, neuronal genes are normally repressed by the master neuronal regulator NRSF (neuron-restrictive silencer factor), also known as REST (repressor element 1-silencing transcription factor) (reviewed in Coulson 2005; Majumder 2006). NRSF consists of two N- and C-terminal repressor domains (RD1 and RD2) and nine C2H2 (Cys2His2) zinc-finger motifs, as well as lysine- and proline-rich domains (Fig. 20.4). The two repressor domains serve as scaffolds for formation of distinct, large repressor complexes via recruitment of multiple corepressors such as Sin3, HDAC, and CoREST (reviewed in Coulson 2005; Majumder 2006). NRSF recognizes and binds to cis-acting DNA sequences called RE1 (repressor element 1) found in over a thousand neuronal genes. NRSF is rarely or not expressed in neuronal cells, but is widely expressed in nonneuronal cells. NRSF was highly expressed in both Ad5- and Ad12-transformed rat and mouse kidney cells. Surprisingly, NRSF was barely present in the nuclei of these cells, but located mainly in the cytoplasm (Guan et al. 2009). This indicates that the neuronal repression function of NRSF is compromised in the viral transformed cells. Interestingly, NRSF was able to efficiently translocate into the nucleus in these cells, but was unable to accumulate in the nucleus, even when the cells were treated with the nuclear export inhibitor leptomycin B (Guan and Ricciardi, unpublished data). By contrast, treatment of the adenovirus-transformed cells with the proteasome inhibitor MG-132 significantly increased nuclear accumulation of NRSF (Fig. 20.5). These data indicate that in the viral transformed cells, the loss of NRSF in the nucleus is not due to its nuclear entry blockage or enhanced nuclear export, but rather rapid degradation by proteasomes. Proteasomal degradation first requires the target protein to be covalently modified by ubquitination (reviewed in Finley 2009; Schrader et al. 2009). Our most recent data indicated that NRSF is ubiquitnated in the nuclei of both Ad5- and Ad12transformed cells, and this ubiquitination can be blocked by PYR-41, an inhibitor of ubiquitin-activating enzyme E1 (Guan and Ricciardi, unpublished data). Importantly, E1A is likely responsible for promoting NRSF ubiquitination, since knockdown of E1A in the viral transformed cells lessened the nuclear presence of ubiquitinated

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Fig. 20.4 Schematic representation of NRSF. NRSF consists of two N- and C-terminal repressor domains (RD1 and RD2), nine zinc-finger motifs, and lysine- and proline-rich domains. RD1 and RD2 can recruit distinct corepressors to form large repressor complexes for transcription silencing. The nine zinc fingers are numbered. Zinc fingers 1–8 form the DNA binding domain, whereas zinc finger 9 is required for corepressor recruitment by RD2

Fig. 20.5 Degradation of NRSF by proteasomes in the nucleus. Ad12-transformed cells were either left untreated or treated with proteasome inhibitor MG-132, followed by confocal immunofluorescent microscopy using an antibody against NRSF (green). Nuclei were stained with DAPI (blue)

NRSF, which is much larger in size than the cytoplasmic un-ubiquitinated form (Guan and Ricciardi, unpublished data). Recently, two research groups have identified two adjacent, but distinct degrons near the C-terminal RD2 (Guardavaccaro et al. 2008; Westbrook et al. 2008). Both degrons are recognized by the ubiquitin E3 ligase SCFb-TRCP and can elicit NRSF degradation by proteasomes. We found that deletion or mutation of either of the two degrons eliminated NRSF degradation in the nuclei of Ad12-transformed cells, showing that both degrons are required for E1A-mediated proteasomal degradation of NRSF (Guan and Ricciardi, unpublished). These data strongly indicate that E1A is involved in stimulating ubiquitination and proteolysis of NRSF in the nucleus, thus relieving NRSF silencing effect on neuronal genes. The rapid proteasomal degradation of NRSF in both Ad5- and Ad12transformed cells should serve as a means by which the viral oncoprotein E1A

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overcomes repression of neuronal genes. It is noted that by interacting with the NRSF corepressor CoREST, the ICP0 protein of herpes simplex virus 1 is able to mediate nuclear export of NRSF to the cytoplasm (Gu et al. 2005; Gu and Roizman 2007). While the relief of NRSF repression should enable both Ad5- and Ad12transformed cells to express neuronal genes, neuronal gene expression was found to occur only in Ad12 tumorigenic cells. What accounts for this difference? In addition to the relief of NRSF repression, the induction of neuronal genes by E1A-12 would further require the activation of neuronal promoters. Based on sitedirected mutagenesis analysis in the promoter region of neuronal gene a-internexin, our data clearly demonstrate that neuronal gene induction by E1A-12 (but not E1A-5) is dependent on the binding of certain nuclear factors especially basic helix-loop-helix (bHLH) transactivators to the promoter (Guan et al. 2009). In strong contrast, neuronal promoter binding by these factors was not found in nontumorigenic Ad5-transformed cells (Guan et al. 2009). Yet, further study is needed to identify these nuclear factors and how their DNA binding activities are differentially regulated by E1A12 and E1A-5.

Involvement of E1A-12-Mediated NRSF Degradation and Neuronal Gene Induction in Ad12 Tumorigenesis Apart from being a key neuronal repressor, NRSF has recently been identified as a tumor suppressor in nonneuronal cells (Westbrook et al. 2005, 2008). Significantly, knockdown of NRSF in epithelial cells was able to cause cell transformation (Westbrook et al. 2005). In accordance with this, NRSF dysfunction and/or deregulation with concurrent aberrant neuronal gene expression have been implicated in human cancers (reviewed in Coulson 2005; Majumder 2006). The dual role of NRSF in repressing both neuronal genes and tumor formation in nonneuronal cells implies that there is a connection between aberrant neuronal gene expression and tumor formation. However, it has yet to be determined that repression of neuronal genes by NRSF in nonneuronal cells really contributes to tumor suppression. In terms of Ad12 tumorigenesis, a related question that remains to be addressed is whether E1A12-mediated NRSF proteasomal degradation or neuronal gene induction or both are essential for cell transformation and tumorigenesis by the virus. In light of the fact that the nontumorigenic E1A-5 can also induce NRSF proteasomal degradation but not neuronal gene expression, it is intriguing to speculate that the loss of NRSF repression is implicated in viral transformation, whereas the neuronal gene induction is likely a forward step involving tumorigenesis. In summary, E1A-12, as with its nontumorigenic adenovirus counterparts including E1A-5, is capable of reprogramming cellular gene expression and transforming cells. Uniquely, E1A-12 is able to mediate MHC class I shutoff, which enables Ad12 tumorigenic cells to escape from CTL-mediated cytolysis. Also, E1A-12 is likely involved in mediating evasion of cytolytic killing by NK cells through the downregulation of NKG2D activating ligands in Ad12 tumorigenic cells. Importantly,

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Ad12 tumorigenesis requires the function encoded by the unique 20-amino acid Spacer between the conserved regions CR2 and CR3. This Spacer, together with other regions in E1A-12, plays a critical role in inducing neuronal and tumor-related genes. Neuronal gene induction by E1A-12 requires not only the relief of NRSF repression via ubiquitin-mediated proteolysis in the nucleus but also the activation of neuronal promoters by certain transactivators. The E1A-12-mediated proteasomal degradation of NRSF, as well as the induction of neuronal and tumor-related genes by the oncoprotein, likely plays an important role in Ad12 tumorigenesis. Acknowledgment We wish to acknowledge Grant CA29797 from the National Institutes of Health (to R. P. R.).

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Chapter 21

Overview of Hepatitis Viruses and Cancer Timothy M. Block, Jinhong Chang, and Ju-Tao Guo

Introduction The liver is one of the body’s largest organs, is part of the digestive system, and is heavily vascularized, as illustrated in Fig. 21.2. Primary cancer of the liver and bile duct is increasing in incidence in the world and in the USA. Hepatocellular carcinoma (HCC), the most common primary liver cancer, is increasing in incidence both worldwide and in the USA. In the world, HCC is now responsible for an estimated 600,000 deaths annually and, as indicated in Table 21.1, is now the third or fourth most common cause of cancer death in the world, accounting for as much as 13% of all cancer mortality. In the USA, HCC is the fastest rising cancer in annual incidence and accounts for approximately 19,000 deaths per year and is now the tenth most common cause of cancer death (Howlader et al. 2011). Thus, while many cancers are declining in incidence and mortality, HCC is on the rise in the developed countries (Fig. 21.1). Despite the overwhelming evidence supporting that chronic infections with HBV and HCV are the primary cause of HCC (Block et al. 2003; Tsai and Chung 2010), the molecular pathways by which the viral infections lead to the development of HCC remain largely elusive. Curiously, although both HBV and HCV infect hepatocytes, the parenchyma cells of the liver, and establish life-long persistent infections under certain circumstances, they are virologically distinct, in many aspects. Most obviously, HBV is a

T.M. Block (*) Department of Microbiology and Immunology, Drexel University College of Medicine, Pennsylvania Biotechnology Center, Doylestown, PA, USA Hepatitis B Foundation, Pennsylvania Biotechnology Center, Doylestown, PA, USA e-mail: [email protected] J. Chang • J.-T. Guo Department of Microbiology and Immunology, Drexel University College of Medicine, Pennsylvania Biotechnology Center, Doylestown, PA, USA E.S. Robertson (ed.), Cancer Associated Viruses, Current Cancer Research, DOI 10.1007/978-1-4614-0016-5_21, © Springer Science+Business Media, LLC 2012

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Table 21.1 Leading causes of cancer death in the world

Organ Lung Stomach Colorectal Liver Breast

Deaths per year 1,300,000 803,000 639,000 610,000 519,000

The five leading causes of death due to cancer, worldwide. Note that there is significant geographic variation for liver, stomach, and lung. Based on World Health Organization statistics (2010), Parkin (2006), and El-Serag et al. (2001)

a

HCC Etiology Worldwide Other 22% HBV 53%

HCV 25%

b

HCC Etiology in the US (2004) Alcohol, No HBV, HCV 9%

HBV+HCV 4%

HBV 16%

HCV 55%

Fig. 21.1 Etiologies of hepatocellular carcinoma (HCC). The percentage of HCC estimated to be attributed to each of the indicated, major, etiologies: alcohol, hepatitis B virus (HBV), hepatitis C virus (HBV), and “other” in the world (a) and the USA (b) is shown. References: Howlader et al. (2011), Miller et al. (2008), El-Serag et al. (2001), and Parkin (2006)

pararetrovirus that harbors a DNA genome, but replicates via reverse transcription of an RNA intermediate. Although it is not essential for viral replication, HBV DNA often integrates into host chromosomes, which may directly transform hepatocytes or induce instability of chromosomal DNA (Seeger and Mason 2000). On the contrary, HCV is an RNA virus that belongs to the hepacivirus genus of the family Flaviviridae (Tellinghuisen et al. 2007).

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Multiple HBV and HCV proteins have been implicated in the disruption of cellular signaling pathway that lead to unchecked cell growth (Table 21.3). However, the most prominent driving force for the development of HCC in HBV and HCV chronically infected livers is thought to be the sustained cycles of hepatocyte necrosis–inflammation–regeneration, driven by interaction between virus infection and host antiviral immune response (Nakamoto et al. 1998). This chapter highlights the biological property of HBV, HCV, and HCC from the virological and molecular cell biological perspectives. In-depth discussion on HBV and HCV biology is provided in subsequent chapters.

Epidemiology As summarized in Table 21.1, HCC is the fifth most common cancer and the third leading cause of cancer death worldwide (El-Serag and Rudolph 2007). Its incidence and geographic distribution are largely governed by its prominent etiology, the chronic infections with HBV or HCV. Thus, HCC is most common in the areas where HBV (and to a lesser extent, HCV) are epidemic, especially East and SouthEast Asia, and sub-Saharan Africa (Davis et al. 2008). In addition to chronic HBV and HCV infections (Marrero 2006), there are other risk factors for HCC development, most importantly, a family history of HCC, chronic alcohol consumption, and ingestion of aflatoxin B1-contaminated food. There is an increasing appreciation for the fact that combinations of these risk factors, when present in the same individual, greatly increase the probability of HCC occurrence. For example, those who are chronically infected with HBV have a lifetime risk of HCC of between 15 and 20%, with an odds ratio of ~13.7, and the HCC usually occurs after the fourth or fifth decade (Donato et al. 1998). However, those with chronic hepatitis B who are either alcoholic or coinfected with HCV have a lifetime risk of HCC of greater than 20%, and the HCC appears to declare itself much earlier in life (Aravalli et al. 2008; Davis et al. 2008).

Table 21.2 HCC incidence distribution by age in the USA Age at diagnosis Percentage of all diagnosed at that age Under 20 0.3 20–34 0.7 35–44 2.4 45–54 15 55–64 21.7 65–74 23.5 75–84 25.8 85 and older 10.5 Age at which HCC is diagnosed, as a percentage of all HCC diagnosed, in the USA (Howlader et al. 2011 Miller et al. 2008)

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As shown in Table 21.2 and illustrated in Fig. 21.3, the majority of HCC cases occur in older people. This observation reflects the fact that HCC is often associated with several decades of HBV/HCV chronic infection. However, HCC occurs occasionally in young people and children, usually in a setting of chronic HBV infection (Table 21.2). In contrast to adult HCC, which is usually developed in the cirrhotic liver, the pediatric HCC is commonly developed in liver that is in the absence of cirrhosis. This observation implies that pediatric and adult HCC may develop through distinct mechanisms. Alternatively, perhaps childhood HCC may occur in individuals with specific genetic (or environmental) factors that accelerate HCC development.

Hepatitis B Virus HBV is the prototype member of Hepadnaviridae family and contains relaxed circular (rc) partially double-stranded DNA (3.2 kb in length) genome with its DNA polymerase protein covalently attached to the 5¢ terminus of minus strand DNA. The HBV genome contains four open reading frames. Because of overlapping coding regions and proteolytic processing reactions, the virus specifies a total of seven viral proteins from these ORFs, which include DNA polymerase, nucleocapsid (core) protein and a secreted viral protein HBeAg, three envelope proteins (surface antigens), and the “X” protein (Seeger and Mason 2000). One of the most intriguing biological features of HBV is that the viral genomic DNA is replicated via protein-primed reverse transcription of an RNA intermediate called pregenomic (pg) RNA in the cytoplasmic nucleocapsids (Summers and Mason 1982). However, unlike classical retroviruses, the integration of hepadnavirus genomic DNA into host cellular chromosomes is not an obligatory step in its life cycle. Instead, a nuclear episomal covalently closed circular (ccc) DNA is formed from the rcDNA genome in nucleocapsids, either from incoming virions during initial infection or from the pool of progeny nucleocapsids formed in the cytoplasm during replication (Tuttleman et al. 1986; Wu et al. 1990). Those two pathways culminate in the formation of a regulated steady-state population of 10–50 cccDNA molecules per infected cell (Beck and Nassal 2007; Newbold et al. 1995; Seeger and Mason 2000). The cccDNA exists as a minichromosome in the nucleus and serves as the template for the transcription of viral RNAs (Zoulim 2005). The stability of this key replication intermediate is still in debate, but a continued productive hepadnavirus infection clearly requires a persistent population of cccDNA as the source of viral RNAs for viral replication and production of virions (Moraleda et al. 1997; Tuttleman et al. 1986; Wu et al. 1990; Zhang et al. 2003). Concerning the molecular mechanisms by which chronic HBV infection causes HCC, the following gene products or virological property of HBV have been implicated in the hepatocarcinogenesis process.

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Viral Load Virion DNA is what is conventionally detected by serum assay and reported in laboratory assessments of people chronically infected, and referred to as “viral load”. Approximately 15–40% of all people with chronic HBV infection eventually develop liver cirrhosis, hepatic decompensation, and HCC. An elevated serum HBV DNA level (³10,000 copies/mL) is a strong risk predictor of HCC independent of HBeAg, serum alanine aminotransferase level, and liver cirrhosis (Chen et al. 2006a, b). Consistently sustained suppression of HBV replication with antivirals can significantly reduce the likelihood of cirrhosis and HCC development (Yuen et al. 2007). These observations imply that high level of HBV replication is one of the dominant driving forces for HCC development.

Integration of HBV DNA into Host Chromosomes Despite the fact that HBV DNA integration into host chromosome is not required for viral replication, it does occur randomly in infected hepatocytes. The frequency of duck hepatitis B virus (DHBV) DNA integration has been estimated to be at least one viral genome per 103–104 cells by 6 days postinfection of ducklings (Yang and Summers 1999). Interestingly, the frequency of integrated woodchuck hepatitis virus (WHV) DNA in chronically infected woodchucks was found to be one or two orders of magnitude higher than that in transiently infected woodchucks, implying that integration and other genomic damage accumulate over the duration of infection (Summers and Mason 2004). Analysis of integrated viral DNA sequences and genetic studies indicated that the most likely precursor of integrated viral DNA is the double-stranded linear (dsl) DNA, a replication product of in situ priming of plus strand DNA (Bill and Summers 2004). Owing to the disruption of the circular genome, integrated HBV DNA is able to be transcribed into functional mRNAs for envelope proteins, but not 3.5 kb precore mRNA and pgRNA, and thus would be unable to support production of infectious virus. HBV DNA integration has been reported to occur in a large fraction of HCC tumors derived from people chronically infected with HBV, most notably in clonal patterns. It was hypothesized that HBV DNA integration could activate cellular protooncogene by insertion activation mechanism and thus contribute to the development of HCC. However, this hypothesis has been confirmed only in WHV-infected woodchucks, in which WHV DNA were found to integrate close to members of the myc oncogene family (Fourel et al. 1990). In human HCC, HBV DNA integration is largely random and only in a few cases, close to important cell growth regulatory genes (Dejean et al. 1986; Wang et al. 1990). Despite this observation, viral DNA integration may play a role in HCC tumor genesis by promoting instability of chromosomal DNA in virally infected cells.

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X Protein HBV X is a 17 kDa, 155-amino-acid protein. It is called “X protein” or HBx because of uncertainty about its function in a natural viral infection. WHV X protein is not essential for WHV DNA replication in cultured cells, but is required for its infectivity in vivo (Chen et al. 1993; Zoulim and Seeger 1994). Recent studies reported by Schneider and colleagues indicated that HBx protein promotes viral DNA synthesis in HBV genome transfected HepG2 cells via immobilizing intracellular calcium and thereby activating Pyk and FAK-mediated signaling pathways (Bouchard et al. 2001, 2003). The role of the HBx protein in HCC development has been suggested by several studies, but the molecular mechanism remains controversial (Bouchard and Schneider 2004).

Truncated Forms of Pre-S2/S Proteins and Large Envelope Protein HBV specifies three envelope glycoproteins called large (L, LHBs), middle (M, MHBs) and small (S, SHBs or more commonly abbreviated as HBsAg) surface proteins. The molecular weights of the three envelope proteins as found in the infectious virion and subviral particles, are 23 and 26 kDa (for SHBs), 30, 33, and 36 kDa (for MHBs) and 36 and 39 kDa (for LHBs), the size varying with the degree of glycosylation (40). While LHBs is translated from 2.4 kb mRNA, MHBs and SHBs are translated from 2.1 kb mRNA by using different starting codons (Seeger and Mason 2000). The truncated form of the pre-S2/S proteins are occasionally found to be expressed in HCC from rearranged integrated viral genomes. It had been shown that these proteins were able to transactivate cellular oncogenes, such as c-myc and c-fos (Schluter et al. 1994) and activate PKC and c-Raf-1/MEK/ERK pathway (Hildt et al. 2002). In addition, it is well documented that overexpression of LHBs activates endoplasmic reticulum (ER) stress (Xu et al. 1997), which can further induce oxidative DNA damage and genomic instability and thus promotes hepatocarcinogenesis (Hsieh et al. 2004). Indeed, it had been elegantly demonstrated in a transgenic mice model that overexpression of LHBs in hepatocytes induced HCC (Chisari et al. 1989).

Hepatitis C Virus HCV is a member of the family Flaviviridae, which also includes some well-known human and animal pathogens, such as yellow fever virus, West Nile virus, Dengue virus, and bovine viral diarrhea virus (Choo et al. 1989). These viruses have in common a single-stranded, positive-sense RNA genome carrying one long open reading frame that is flanked by nontranslated regions (NTR). The HCV genome has a length of

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about 9,600 nucleotides and encodes an approximately 3,000-amino-acid-long polyprotein that is proteolytically processed into ten polypeptides (Reed and Rice 2000). Three of them are structural proteins required for capsid formation (core) and assembly into enveloped viral particles (E1 and E2). Four virus-encoded proteins are enzymes including cysteine and serine proteases (NS2 and NS3), an ATP-dependent helicase (NS3), and a RNA-directed RNA polymerase (NS5B). The functions of the remaining three polypeptides, p7, NS4B, and NS5A, for viral replication are not yet known. The 5¢ NTR spans about 340 nucleotides and harbors the cis-elements for viral RNA replication and an internal ribosomal entry site (IRES) directing the translation of viral polyprotein. The 3¢ NTR has a highly conserved sequence element (3¢ X) that is essential for viral RNA replication (Tellinghuisen et al. 2007). HCV replication begins with sequential interactions of the virion particle with its cellular receptor and coreceptor molecules on cell surface, including CD81, class B scavenger receptor (SRBI), claudin-1 and occludin, followed by entry into hepatocyte via endocytosis (Evans et al. 2007; Ploss et al. 2009). Viral genomic RNA is released into the cytoplasm upon fusion of viral envelope and endosomal membrane and serves as a template for translation of viral polyproteins. Accumulation of viral proteins induces rearrangements of ER membranes that form the locales for replication of viral RNA. Minus strand RNA synthesis leads to the formation of a double-stranded RNA molecule that bears a promoter at the 3¢ end of minus strand RNA required for amplification of plus strands by a semiconservative mechanism (Tellinghuisen et al. 2007). During the early phase of the infection, the progeny plus strand RNA is transported to ribosomes for additional rounds of translation and replication of viral RNA. But late in infection, the RNA is packaged into virion particles by viral core and envelope proteins on the surface of lipid droplets and secreted out from infected cells (Miyanari et al. 2007). Apparently, unlike HBV, HCV is unable to reverse transcribe its RNA genome and thus to integrate it into the host chromosome. However, there is ample evidence suggesting that multiple HCV proteins, particularly core and NS5A protein, evoke host cellular responses that may contribute to HCC development.

Core Protein The mature form of HCV core contains approximately 174aa that is separated into two structural domains. The domain I, encompassing N-terminal 122aa, is highly basic and responsible for RNA binding and capsid assembly. The domain II encompasses the C-terminal part of HCV core protein that is hydrophobic and mediates interactions with lipids and membrane proteins. Besides to serve as an essential structural component of HCV, the core protein appears to have diverse functions and interact with many cellular proteins that may lead to hepatocarcinogenesis. For examples, HCV core has been demonstrated to bind to tumor suppressor proteins, such as p53 and pRb (Ray et al. 1997). In addition, HCV core is able to upregulate the expression of TGF-b and VEGF and activates multiple signal transduction

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pathways, including PKC, RB/E2F1, ASK1-JNK/p38, and ERK (Hassan et al. 2009). Consistent with its pleiotropic function on cellular signal transduction pathways, overexpression of HCV core protein, alone, in Huh7 cells induces the expression of more than 300 cellular genes with most of the induced genes involved in cell growth and oncogenic signaling (Fukutomi et al. 2005).

NS5A Protein Two forms of NS5A protein, termed p56 and p58, can be distinguished by their electrophoretic mobility. While the p56 is a basally phosphorylated protein, the p58 is hyperphosphorylated within a serine-rich region in the center of the protein. The hyperphosphorylation of NS5A has been demonstrated to regulate HCV RNA replication (Evans et al. 2004). Besides being an essential component of HCV RNA replication complex and playing a critical role in directing viral RNA onto the surface of lipid droplets for virion assembly, NS5A protein has been attributed to an array of cellular functions, including inhibition of interferon response and apoptosis, modulation of signal transduction and gene transcription, induction of cell transformation, and ROS production (Macdonald and Harris 2004). Like the core protein, NS5A can also directly bind to p53 and inhibit its transcriptional transactivation activity (Majumder et al. 2001). Moreover, our own study reveals that NS5A can activate PI3K/Akt/mTOR signal transduction pathway and promote HBV replication in HBV and HCV coinfected hepatocytes (Guo et al. 2007).

Steatosis and Oxidative Stress Chronic HCV infection is characterized by prevalence of steatosis and increased oxidative stress. Steatosis is independently associated with the development of HCC in patients with HCV-related cirrhosis (Pekow et al. 2007). A recent transgenic mice study demonstrates that overexpression of HCV core protein in hepatocytes induces severe steatosis in a PPAR-alpha-dependent manner and results in HCC development in 35% of HCV core transgenic mice bearing homozygote wild-type PPAR-alpha genes (Tanaka et al. 2008). Increased oxidative stress in chronically HCV-infected liver may result from HCV induced steatosis, ER stress and immune-cell-mediated oxidative bursts. Both HCV core and NS5A proteins have been implicated in induction of oxidative stress and ROS production. ROS has been shown to be able to activate multiple signal transduction pathways, such as MAPK, PI3K, NFkB, and Wnt/b-catenin, and thus modulate many cellular functions, including gene expression, cell adhesion, cell metabolism, cell proliferation as well as apoptosis. Most importantly, ROS can induce oxidative DNA damage, which in turn increases chromosomal aberrations associated with cell transformation.

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Liver

Hepatic Bile Duct

Stomach Gall Bladder Pancreas Common Bile Duct

Small Intestine

Fig. 21.2 The digestive system with a focus on the liver. Illustration of the liver, its two lobes, and how it resides within the human digestive track

Molecular Pathogenesis of HCC Molecular Pathways of HCC Hepatocarcinogenesis is a complex multistep process involving a number of genetic and epigenetic alterations, which result in the activation of cellular oncogenes and/or the inactivation of tumor suppressor genes, and dysregulation of multiple signal transduction pathways (Farazi and DePinho 2006; Tsai and Chung 2010). As indicated by the age distribution of the disease (Fig. 21.2 and Table 21.2), when it occurs, it is usually after decades of chronic infection and often preceded by necroinflammatory liver disease (Fig. 21.3). However, it has been difficult to identify common genetic changes in more than 20–30% of tumors, suggesting that HCCs are genetically heterogeneous (Branda and Wands 2006). As discussed in the above sections, although cell culture studies have suggested that multiple HBV and HCV proteins are able to interact with key components of cellular signal transduction pathways and lead to unchecked cell growth, their roles as viral oncogenes to promote HCC development in vivo have not been firmly established. Instead, as illustrated in Fig. 21.4, regardless the etiology, malignant transformation of hepatocytes most likely results from the sustained

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HCC, in US, Incidence Distribution by Age 30.00% 25.00% 20.00% 15.00% Series1

10.00% 5.00% 0.00% Under 20-34 35-44 45-54 55-64 65-74 75-84 85 and older 20

Relative amount of Morbidity/Mortality

Fig. 21.3 Distribution of the incidence of HCC by age in the USA. The percentage of ~18,000 cases of HCC diagnosed at each of the ages indicated by histograms is shown (Miller et al. 2008)

Time of infection

HCC Active hepatitis Inactive hepatitis Acute hepatitis

0

10

20 30 40 50 Years of chronic infection

60+

Fig. 21.4 Relative progression of different clinical conditions associated with chronic hepatitis virus, as a function of decades of infection. Dramatization of the likelihood of moving from one clinical state to another increases with period of time of infection (based on Block et al. 2003). Progression from one step to the other is not necessary, but is the most common, natural history of the disease

cycles of increased liver cell death induced by chronic liver injury and regeneration in the condition of inflammation and oxidative DNA damage over a long period of time, usually several decades (Nakamoto et al. 1998).

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In case of chronic hepatitis B and hepatitis C, as illustrated in Fig. 21.4, the virally infected hepatocytes can be targeted and killed by viral specific cytotoxic T lymphocytes (CTLs) that recognize epitopes derived from various viral proteins and presented by the MHC I molecules on the surface of infected hepatocytes. However, such immune response is insufficient to eliminate viral infection and killed hepatocytes are replaced by proliferation of residual hepatocytes (Chisari and Ferrari 1995). This cycle of immunorecognition-killing (liver destruction) and regeneration repeats itself over years. Indeed, there is well-documented evidence suggesting that the rate of hepatocyte turnover is accelerated in HBV and/or HCV-infected individuals. Proliferation of hepatocytes in an environment of inflammation is prone to accumulate mutations, which ultimately leads to transformation and hepatocarcinogenesis.

Inherited and Acquired Mutations After chronic infection with HBV or HCV, having a close family history of HCC is probably the greatest risk factor for developing the disease (Hoofnagle 2004; Zhang et al. 2010). HCC susceptibility genes have been elusive, but there is a growing interest in pursuing genes whose heritable lesions may be associated with HCC risk (Zhang et al. 2010). Indeed, multiple genetic and epigenetic alterations have been documented to associate with HCC. So far, comparative genomic hybridization studies have revealed frequent chromosomal gains in 1q, 6p, 8q, 11q, and 17q, and losses in 1p, 4q, 8p, 13q, and 17p (Farazi and DePinho 2006). Interestingly, a recent genome-wide association study identified one intronic SNP (rs17401966) in KIF1B on chromosome 1p36.22 that is highly associated with HBV-related HCC. In addition to KIF1B, the association region also encodes two other potential tumor-related genes, UBE4B and PGD, and thus confers susceptibility to HBV-related HCC (Zhang et al. 2010). Furthermore, p53 inactivation and mutation seems to be a common pathogenic pathway in HBV- and HCV-induced HCC. HBx, HCV core, and NS5A proteins have all been shown to bind p53 and inhibit its function. Analyses of HBV and HCV-related HCCs have also shown that p53 mutations can be found in 43% of patients with advanced HCC (Minouchi et al. 2002). It has been shown by biochemical analysis and genome-wide gene expression studies that multiple signal transduction pathways are altered in HCC by viral proteins, virus-induced cellular responses, or unidentified mechanisms, and thus may contribute to hepatocarcinogenesis. These pathways include Insulin/IGF-1/ IRS-1/MAPK, Wnt/Frizzeled/b-catenin, Ras/Raf/MAPK, Janus kinase (JAK)/ signal transducer and activator of transcription (STAT), phosphatidylinositol 3-kinase (PI3K)/Akt, Hedgehog and growth factors such as epidermal growth factor, and transforming growth factor-b (TGF-b) pathways. Interestingly, a recent integrative transcriptome analysis stratifies HCC into three subclasses, each correlated with clinical parameters as well as biological mechanism known to be operative in the

Table 21.3 Selected pathways and molecular targets associated with HBV and HCV-induced HCC Target HBV HCV References Wnt/b-catenin Yes Yes Colnot et al. (2004), Hickman and Helin (2002), Taniguchi et al. (2002) P53 Yes Unknown Azechi et al. (2001) pRB Yes Unknown Satoh and Kaziro (1992), Yoshida et al. (2006) MAP kinases Yes Yes Bai et al. (2003) Cytokines Yes Yes Budhu et al. (2006) P16 Yes Yes Satoh and Kaziro (1992) Frizzled Unknown Yes Colnot et al. (2004) Pathways and molecular targets that have been reported to be altered (in some cases, mutated, in some cases upregulated, in other cases downregulated, depending on the pathway or target) in people with HBV and HCC-associated HCC are shown. For most of the targets, alterations have been reported for both HBV and HCV-associated HCC. Citations supporting the associations are shown

T a

A

B

Cancer

C

b Biological Event Infection, antigen presentation

Clinical Event

In-apparent infection

Chronic liver damage, hepatocyte regeneration

Hepatitis, fibrosis, cirrhosis

Genetic alterations

Transformation

Pre-malignant masses, then HCC

Fig. 21.5 Idealization of the molecular and cellular events occurring which are thought to lead to the pathogenesis of chronic viral infection and, ultimately, HCC. (a). Events thought to happen at the cellular level, where (a): T lymphocytes attack infected hepatocytes expressing viral antigens, followed by (b): destruction of the infected cells which are replaced by “scar” tissue, leading to fibrosis and cirrhosis accompanied by continuous regeneration of new hepatocytes (c): some of which make acquire mutations, leading to transformation and cancer. (b): The progression of pathogenesis, explaining the natural history of HCC, from the biological perspective and the clinical manifestation of these events, is shown (based on Block et al. 2003)

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pathogenesis of HCC (Hoshida et al. 2009). For examples, the subclass 1(S1) was characterized by TGF-b induced activation of the WNT signaling pathway, S2 was characterized by high level alpha-fetoprotein expression as well as MYC and AKT activation, and S3 was associated with hepatocyte differentiation, featured by hepatocyte-like gene expression profile, smaller tumor and better prognosis (Fig. 21.3). Such a molecular classification of HCC is helpful in understanding the HCC pathogenesis and development of targeted therapeutic intervention of HCC. A summary of some of the prooncogenes disrupted by HBV and HCV is shown in Table 21.3 and Fig. 21.5.

Role of MicroRNA in Hepaocarcinogenesis MicroRNAs (miRNAs) are small noncoding RNAs of approximately 22 nts in length. They are derived from cellular or viral transcripts and bind to their target mRNAs in a sequence-specific manner, resulting in either mRNA cleavage or translational repression (Ambros 2004; Bartel 2004; Jackson and Standart 2007). MicroRNAs have been shown to play a fundamental role in regulating gene expression and thus modulating multiple cellular functions. Aberrant expression of several miRNAs was found to be involved in human hepatocarcinogenesis. For examples, upregulation of mir-221 and mir-21 could promote cell cycle progression, reduce cell death and favor angiogenesis and invasion (Meng et al. 2007; Pineau et al. 2010). Moreover, the most abundant miRNA in the liver, miR-122, is involved in cellular stress response, regulation of cholesterol metabolism and required for HCV replication in hepatocytes in vitro and in vivo (Bhattacharyya et al. 2006; Chang et al. 2004; Esau et al. 2006; Jopling et al. 2005; Lanford et al. 2010). miR-122 is also considered to have the potential as tumor suppressor, since downregulation of miR-122 has been found to correlate with hepatocarcinogenesis (Chang et al. 2004; Chang and Taylor 2008; Coulouarn et al. 2009; Kutay et al. 2006).

Detection and Management of HCC Currently, surgical resection and liver transplantation are the best choices to treat HCC. However, the five-year survival rate in people with resection and/or liver transplantation is less than 30% (Roncalli et al. 2007). Chemoembolization (TACE), ethanol (Percutaneous), cryotherapy, and radiofrequency thermoablation are being used as alternatives to resection (Block et al. 2008; Marrero 2006). Recurrences are common and recovery from such interventions can be compromised. Medically, there have been even fewer satisfactory options. The recent approval of sorafenib for the advanced HCC treatment has been received with great anticipation. However, its efficacy is extremely limited, increasing overall survival by only a few months (Llovet 2007). The poor prognosis and therapeutic outcomes of HCC is thought mainly as the result of failure to diagnose the cancer at its early and treatable stage. Hence, early

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detection of the cancer is generally considered to be critical to improve outcome (Hoofnagle 2004; Marrero 2006; Seki et al. 2000; Sherman 2005). Since the prominent etiology of HCC is chronic hepatitis B followed by hepatitis C (Di Bisceglie et al. 1998, 2003), there is a clear and identifiable risk population. This allows for a realistic and focused cancer screening (Block et al. 2007). Moreover, since patients with cirrhosis (with or without HBV or HCV infection) are at significantly increased (between 10- and 100-fold) risk of developing HCC, this subpopulation receives the closest attention (Marrero 2006). Current methods for HCC detection are either unreliable or impractical. Liver biopsy is still considered to be the definitive standard to assess the degree of liver damage in people with chronic hepatitis, although it is usually to be avoided where HCC is suspected (Block et al. 2007; Lok and McMahon 2001). However, monitoring by noninvasive methods is currently performed by physical assessment, ultrasound imaging of the liver, and analysis of serum with a panel of markers including liver function tests and platelet counts (Lok and McMahon 2001; Lok et al. 2001). Advanced imaging is costly and even ultrasound detection is expensive and operator dependent. It also usually requires at least a 1–2 cm tumor mass to be present. Unfortunately, most scans are performed when the HCC is at a late stage and prognosis is very poor (Brechot 1987; Hoofnagle and Di Bisceglie 1997). Since early surgical intervention is the best hope for patient survival (Block et al. 2003; Lok and McMahon 2001; Lok et al. 2001), accurate detection of early stage HCC is necessary to identify the need for intervention. For smaller lesion, 1–2 cm in diameter, i.e., those that are the target of HCC screening, radiology is less sensitive, but similarly biopsy is less accurate as well. This is because of difficult inaccurate needle placement, and because pathological interpretation in these small lesion is controversial and difficult. In this situation, a diagnostic biomarker would be very helpful. Liver function lab values (serum tests) are used to assess liver damage but are of dubious value in diagnosis and detection of HCC (Bruix et al. 2001). Many of the constituents of the liver function test panels used to evaluate disease status (i.e., degree of liver damage), such as alanine aminotransferase (ALT) levels (Imbert-Bismut et al. 2001; Myers et al. 2003; Poynard et al. 2002), vary throughout the course of chronic hepatitis and are of limited use in early detection of HCC (Sherman 2001). Since there is a correlation between elevated levels of alpha fetoprotein (AFP) and the occurrence of HCC, determination of AFP levels is often included as a serum marker of disease (Aoyagi et al. 1998; Buamah et al. 1984; Lok and McMahon 2001; Lok et al. 2001; McMahon et al. 2000). AFP, a 72,000 Da liver derived glycoprotein with a function that may be analogous to albumin, has been used for detection of HCC since 1968 (Alpert et al. 1968). AFP as a sole indicator of HCC is of limited value, often being elevated in the absence of serious disease or not elevated when cancer is present or at an early stage (Sherman 2001). Nevertheless, even the limited correlation between AFP and HCC underscores the potential of serum as a source of biomarkers of liver disease. The assay for fucosylated AFP (AFP-L3), as used in the clinical setting, is a bit complicated and requires specialized equipment. However, its commercialization by Wako Diagnostics proves that these matters can be overcome. Since the sensitivity of AFP-L3 in detecting

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Chronic HBV or HCV ?Viral proteins

Cytokines (necro-inflammation)/ Ligands

(extracellular) Cytokine receptors/wnt receptor

PI3-K-Activation

Akt activation mTOR

E-cadherin/b-catenin

PTEN ?Viral proteins

b-catenin

(intra-cellular)

p53 ROS

Anti-apoptosis

Invasion

Metastasis

pRB, p16, myc

Fig. 21.6 Effect of chronic hepatitis virus and other insults to the hepatocytes on pathways and protooncogenes, whose dysregulation is associated with HCC. Illustration of some of the enzymes, pathways, polypeptides, and protooncogene products, whose functions have been reported to be disrupted (activated or inactivated) in either HBV or HCV associated HCC. ROS (reactive oxygen species). Shaded oval, nucleus. The listing of pathways and polypeptides is not complete, but is intended to represent some of the more prominent targets. Based on Choudhari et al. (2007)

Tumor Mass Size

Larger Tumor Masses

Less Differentiated

TGFb, Wnt Activated

MYC, AKT Activated

Smaller Tumor Masses

Retains Hepatocyte Phenotype

CTNNB1 Variable

AFP Positive

Poorer Survivor

Better Survivor

Fig. 21.7 Model that uses molecular and biomarker information to subclassify HCC. Based on model proposed by Hoshida et al. (2009)

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early stage HCC is only approximately 50%, although promising, more work or combinations with additional biomarkers are needed. There have been significant efforts to discover and use new serum markers for the early detection of HCC (Wright et al. 2007). For example, des-gamma-carboxy prothrombin (DCP) has received a great deal of attention as a biomarker of HCC, so has glypican, serum glycan, and several other circulating biomarkers as reviewed in (Block et al. 2008). However, none of these prospects has held up as superior or offering advantages to current methods in multicentered, diverse, and large blinded studies (Block et al. 2008; Marrero et al. 2009). Serum markers of HCC, as well as the molecular analysis of the tumors, have been used to predict outcome, and this is becoming more and more fashionable. For an example of how molecular classification has been proposed for use in clinical outcome prediction and care, see Figs. 21.6 and 21.7.

Future Challenges HCC is a growing problem in the world. Although with clear etiology and in-depth understanding of HBV and HCV virology, the molecular pathogenesis of HCC remains largely elusive. Clinically, there are also unmet needs for the early detection and therapeutic intervention of HCC. Therefore, many challenges and opportunities exist in this field. For examples, development of novel antiviral drugs or other therapeutic agents that cure chronic HBV and HCV infections will ultimately prevent HCC development. In addition, detailed understanding of the genetic (or epigenetic) lesions of HCC, signal transduction pathways involved in HCC development, host–virus interaction, antiviral immune response and liver inflammation, cell of origin, and evolution of hepatocarcinogenesis will provide a roadmap for the development of HCC therapeutics. Needless to say, successful treatment of HCC relies on the discovery of biomarkers to identify early-stage HCC as well as those at greatest risk of developing HCC. Acknowledgments The authors would like to thank Ms. Erica Litschi for help in manuscript and figure preparation. The authors acknowledge support from National Cancer Institute and National Institute of Allergy and Infectious Diseases, and the Hepatitis B Foundation and The Commonwealth of Pennsylvania.

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Seeger C, Mason WS (2000) Hepatitis B virus biology. Microbiol Mol Biol Rev 64(1):51–68 Seki S, Sakaguchi H, Kitada T, Tamori A, Takeda T, Kawada N, Habu D, Nakatani K, Nishiguchi S, Shiomi S (2000) Outcomes of dysplastic nodules in human cirrhotic liver: a clinicopathological study. Clin Cancer Res 6(9):3469–3473 Sherman M (2001) Surveillance for hepatocellular carcinoma. Semin Oncol 28(5):450–459 Sherman M (2005) Hepatocellular carcinoma: epidemiology, risk factors, and screening. Semin Liver Dis 25(2):143–154 Summers J, Mason WS (1982) Replication of the genome of a hepatitis B-like virus by reverse transcription of an RNA intermediate. Cell 29:403–415 Summers J, Mason WS (2004) Residual integrated viral DNA after hepadnavirus clearance by nucleoside analog therapy. Proc Natl Acad Sci USA 101(2):638–640 Tanaka N, Moriya K, Kiyosawa K, Koike K, Gonzalez FJ, Aoyama T (2008) PPARalpha activation is essential for HCV core protein-induced hepatic steatosis and hepatocellular carcinoma in mice. J Clin Invest 118(2):683–694 Taniguchi K, Roberts LR, Aderca IN, Dong X, Qian C, Murphy LM, Nagorney DM, Burgart LJ, Roche PC, Smith DI, Ross JA, Liu W (2002) Mutational spectrum of beta-catenin, AXIN1, and AXIN2 in hepatocellular carcinomas and hepatoblastomas. Oncogene 21(31):4863–4871 Tellinghuisen TL, Evans MJ, von Hahn T, You S, Rice CM (2007) Studying hepatitis C virus: making the best of a bad virus. J Virol 81(17):8853–8867 Tsai WL, Chung RT (2010) Viral hepatocarcinogenesis. Oncogene 29(16):2309–2324 Tuttleman JS, Pourcel C, Summers J (1986) Formation of the pool of covalently closed circular viral DNA in hepadnavirus-infected cells. Cell 47:451–460 Wang J, Chenivesse X, Henglein B, Brechot C (1990) Hepatitis B virus integration in a cyclin A gene in a hepatocellular carcinoma. Nature 343(6258):555–557 Wright LM, Kreikemeier JT, Fimmel CJ (2007) A concise review of serum markers for hepatocellular cancer. Cancer Detect Prev 31(1):35–44 Wu TT, Coates L, Aldrich CE, Summers J, Mason WS (1990) In hepatocytes infected with duck hepatitis B virus, the template for viral RNA synthesis is amplified by an intracellular pathway. Virology 175(1):255–261 Xu Z, Jensen G, Yen TS (1997) Activation of hepatitis B virus S promoter by the viral large surface protein via induction of stress in the endoplasmic reticulum. J Virol 71(10):7387–7392 Yang W, Summers J (1999) Integration of hepadnavirus DNA in infected liver: evidence for a linear precursor. J Virol 73(12):9710–9717 Yoshida T, Hisamoto T, Akiba J, Koga H, Nakamura K, Tokunaga Y, Hanada S, Kumemura H, Maeyama M, Harada M, Ogata H, Yano H, Kojiro M, Ueno T, Yoshimura A, Sata M (2006) Spreds, inhibitors of the Ras/ERK signal transduction, are dysregulated in human hepatocellular carcinoma and linked to the malignant phenotype of tumors. Oncogene 25(45):6056–6066 Yuen MF, Seto WK, Chow DH, Tsui K, Wong DK, Ngai VW, Wong BC, Fung J, Yuen JC, Lai CL (2007) Long-term lamivudine therapy reduces the risk of long-term complications of chronic hepatitis B infection even in patients without advanced disease. Antivir Ther 12(8):1295–1303 Zhang YY, Zhang BH, Theele D, Litwin S, Toll E, Summers J (2003) Single-cell analysis of covalently closed circular DNA copy numbers in a hepadnavirus-infected liver. Proc Natl Acad Sci USA 100(21):12372–12377 Zhang H, Zhai Y, Hu Z, Wu C, Qian J, Jia W, Ma F, Huang W, Yu L, Yue W, Wang Z, Li P, Zhang Y, Liang R, Wei Z, Cui Y, Xie W, Cai M, Yu X, Yuan Y, Xia X, Zhang X, Yang H, Qiu W, Yang J, Gong F, Chen M, Shen H, Lin D, Zeng YX, He F, Zhou G (2010) Genome-wide association study identifies 1p36.22 as a new susceptibility locus for hepatocellular carcinoma in chronic hepatitis B virus carriers. Nat Genet 42(9):755–758 Zoulim F (2005) New insight on hepatitis B virus persistence from the study of intrahepatic viral cccDNA. J Hepatol 42(3):302–308 Zoulim F, Seeger C (1994) Woodchuck hepatitis virus X protein is required for viral infection in vivo. J Virol 68:2026–2030

Chapter 22

Hepadnaviruses and Hepatocellular Carcinoma William S. Mason

Introduction Hepadnaviruses are a family of small, enveloped, DNA viruses that productively infect hepatocytes, the major cell type of the liver. The prototype virus of this family is hepatitis B virus (HBV) (Blumberg et al. 1967), which infects humans and higher primates. Closely related viruses are found in the Woolly Monkey (Lanford et al. 1998), woodchuck (Summers et al. 1978b) and Beechey ground squirrel (Marion et al. 1980). More distantly related viruses are found in ducks, geese, herons, storks, and cranes (Guo et al. 2005; Mason et al. 1980; Prassolov et al. 2003; Pult et al. 2001; Sprengel et al. 1988; Wang et al. 1980). These two groups of viruses are assigned to the genus orthohepadnavirus and avihepadnavirus, respectively. All hepadnaviruses are able to cause transient infections, with recovery and immunity to re-infection, as well as chronic, life-long infections. Chronic infections by the orthohepadnaviruses can cause hepatocellular carcinoma (HCC), while avihepadnavirus infections do not. The lifetime risk of HCC is ~25% in humans chronically infected with HBV, higher in Beechey ground squirrels chronically infected with ground squirrel hepatitis virus (GSHV), and virtually 100% in woodchucks chronically infected with woodchuck hepatitis virus (WHV). A vaccine to prevent primary HBV infection and associated HCC has been available for almost 30 years (Blumberg and London 1982; McAuliffe et al. 1980); the vaccine is not useful for therapy of chronic infections. It was hoped for some time that studies with the woodchuck and ground squirrel models would reveal how chronic infection with HBV leads to human liver cancer. This has not, so far, been the case. HCC in woodchucks is often associated with integration of viral DNA into host DNA, typically near to N-myc (Bruni et al. 1999, 2006; Fourel et al. 1990, 1994) and, much less often, C-myc (Wei et al. 1992). Overexpression of these genes then occurs, probably through the action of the WHV

W.S. Mason (*) Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA e-mail: [email protected] E.S. Robertson (ed.), Cancer Associated Viruses, Current Cancer Research, DOI 10.1007/978-1-4614-0016-5_22, © Springer Science+Business Media, LLC 2012

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transcriptional enhancer (Flajolet et al. 1998; Ueda et al. 1996) or, in some cases, to a more profound effect of integration on chromosome structure in the vicinity of the gene, due to disruption of nuclear matrix attachment regions (Bruni et al. 2004; Shera et al. 2001). The fact that all transformed cells in a tumor contain the same integrant, and that the nearby oncogene is typically over-expressed, implies that the integration of viral DNA was required for emergence of the woodchuck tumor, was probably causative, and perhaps sufficient (Renard et al. 2000). In contrast, amplification of the C-myc gene, unassociated with integration of GSHV DNA, is found in 25–50% of ground squirrel HCCs (Hansen et al. 1993; Transy et al. 1992, 1994). Integrations near C-myc and N-myc were not found with GSHV. Considering that both hosts are rodents/ground squirrels and are infected by closely related viruses, this dichotomy is unexpected. It suggests that there might not be a simple relationship between HBV DNA integration and HCC in humans. In fact, although exceptions have been found in which integration has activated host genes which are probably important in development of the HCC, including erbA, retinoic acid receptor beta, and cyclin A (Dejean et al. 1986; Murakami et al. 2005; Paterlini-Brechot et al. 2003; Wang et al. 1990), studies of human HCCs have failed, in most cases, to reveal a clear role for the sites of integration of HBV DNA (Murakami et al. 2005; Paterlini-Brechot et al. 2003). This was particularly disappointing since virtually all human HCCs contain integrated DNA, an observation which indicated that these tumors resulted from clonal expansion of cells that took place at some time after the integration event (Brechot et al. 1980; Shafritz and Kew 1981). While activation of a host oncogene by nearby insertion of an HBV enhancer or promoter sequence does not so far explain the production and clonal expansion of malignant cells to produce most human HCCs, the idea that integration of HBV DNA plays a major role has persisted. For instance, as mentioned above, the possibility that integration activates gene expression by altering chromosome structure (Bruni et al. 2004; Shera et al. 2001) has been proposed. Another hypothesis, which has received more attention, but again without conclusive results, is that one or more normal or aberrant viral gene products, expressed from the integrated viral DNA, are oncogenic. As discussed below, there is substantial indirect support for this notion, but there remain many difficulties in connecting experimental observations in model systems with human HCC. Another pathway to human HCC, which has received a great deal of attention, but without definitive conclusions, is persistent injury to the liver caused by the host immune response. It is clear that the immune response to infected hepatocytes, mediated by antiviral cytotoxic T lymphocytes, is a feature of HBV chronic infections. This response, along with cell death, activates macrophages, leading to free radical generation by these cells, as well as in cells targeted by antiviral cytokines such as TNFa, which is secreted by activated macrophages. This appears to lead to increased hepatocyte levels of hydroxyguanosine, an oxidized version of guanosine capable of base pairing with adenosine (Hagen et al. 1994) to create mutations in host DNA. In addition, persistent killing causes tissue scarring, which leads to fibrosis and, ultimately, cirrhosis. Cirrhosis severely disrupts the arrangement of hepatocytes and blood flow through the liver, and may be, along with HBV infection and free radical generation, an important risk factor for HCC.

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Finally, persistent liver regeneration, following genotoxic events effecting hepatocyte DNA (e.g., free radical formation and DNA mutation), will increase the risk of HCC (Marongiu et al. 2008). That is, mutation of hepatocyte DNA provides an essential event that initiates the progression to HCC, but would be ineffective without the promoting event of liver regeneration (Maeda et al. 2005). Unfortunately, the importance of these and other factors in the progression to HCC in HBV carriers remains unresolved. HBV is a serious public health concern because so many people worldwide are chronically infected with the virus. The WHO estimates there are ~350 million HBV carriers worldwide and that 60 million of these may die prematurely from HCC and/or cirrhosis (Lavanchy 2004). Resolving the association between chronic infections and HCC is particularly difficult because infections are very prolonged. Chronic HBV infections typically begin at birth (neonatal transmission) or in the first year of life. As a result, the duration of an infection is generally the same as a patient’s age. HCC may appear at any age (Chang et al. 2009), but the incidence peaks after the age of 30 (Beasley 1982; Beasley et al. 1981; Ganem and Prince 2004; Liaw 2009; Yim and Lok 2006). Intervention and study of younger patients that have not developed HCC is problematic because HBV infection in these individuals is generally asymptomatic (Lok and McMahon 2007) until significant fibrosis and cirrhosis have developed, by which time the progression to HCC may also be well advanced. In the following sections, possible events in the progression to HCC are discussed. The focus of this review is the role of HBV, and the host response to this virus, in the progression to HCC. For additional information on HCC, the reader is referred to recent articles on the role of environment carcinogens (Groopman et al. 2008; Wild and Gong 2010), the molecular changes that distinguish HCCs from surrounding liver (Connolly et al. 2008; Lee et al. 2004; Neuveut et al. 2010; Thorgeirsson and Grisham 2002; Zhang et al. 2009), the occurrence and role of cancer stem cells (Rountree et al. 2009; Sun et al. 2008; Yamashita et al. 2010), and a relatively new area of investigation, possible HBV genotype differences in the risk of HCC (Yuen et al. 2009). The reader should also be aware of the existence of hepatitis delta virus (HDV), a subviral satellite of HBV that requires HBV envelope proteins in order to spread. HDV is a viroid-like agent with an RNA genome that can super-infect or co-infect HBV carriers. HDV infection of HBV carriers increases the severity of chronic liver disease and the risk of HCC (Tamura et al. 1993; Verme et al. 1991).

The Biology of Chronic HBV Infection Although the course of chronic infection can vary widely, a generalized picture has emerged from clinical studies. These studies indicate that infections may pass through several stages, including an early, immuno-tolerant stage lasting 20–30 years or more, an immune clearance stage, an inactive carrier stage, and a reactivation stage (Liaw 2009; Yim and Lok 2006) (Fig. 22.1). It should be kept in mind that these changes may occur without the patient’s awareness, as advanced cirrhosis and

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W.S. Mason HCC incidence Cirrhosis and HCC (may occur at any age, incidence increases in middle age) Reactivation phase (Virus titers and inflammation increase) Inactive phase (little or no HBV replication or inflammation) Immune-clearance phase (Virus titer declines with bouts of acute inflammation) Immuno-tolerant phase

Age (years): 0

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Fig. 22.1 Stages of chronic hepatitis B following perinatal infection. Chronic hepatitis B is now considered to have four phases, an immunotolerant phase in which there is little or no inflammation or fibrosis, an immune-clearance phase in which there may be one or more bouts of hepatitis associated with a decline in viremia as the immune response attempts to clear the infection, an inactive carrier phase in which virus titers are low and there is little or no inflammation, and in some cases, a reactivation phase, in which virus titers and the immune response elevate, with increased liver inflammation and damage. As noted, these phases may not always be present or, if they are, may not be apparent to a carrier that is not actively followed in a clinical setting. HCC and cirrhosis may occur at any age, but generally show dramatic increases after the age of 30 (Figure adapted from Beasley 1982; Liaw 2009; Wang et al. 2010; Yim and Lok 2006). Chronic HBV infection initiated in adult life probably lacks an immunotolerant phase (Liaw 2009)

HCC often produce the first outward symptoms of chronic liver disease which are apparent to a carrier (Evans et al. 1998). Thus, the different stages of infection are largely defined from clinical monitoring and experience, rather than from populationbased studies (Evans et al. 1998). In addition, the concept of an immuno-tolerant phase is based on the absence of overt signs of liver damage, including significant fibrosis and cirrhosis of the liver and elevated levels of liver enzymes in the blood. Despite its name, ongoing liver damage, which is not apparent by these assays, is probably a feature of the immuno-tolerant phase of infection. The liver is made up of a variety of cell types, of which the major is the hepatocyte, which constitutes ~70% of the cell population of the healthy liver. Human hepatocytes are arranged primarily in 1-cell thick plates surrounded by fenestrated sinusoidal endothelial cells. Blood flowing through the sinusoids, across the surface of the endothelial cell layer, exchanges non-cellular components with hepatocytes. This occurs through the fenestrations in the endothelial cells, as well as by directional transport across the endothelial cell layer. Fixed tissue macrophages (Kupffer cells), also part of the sinusoidal lining, are found in association with the endothelial cell layer, where they play a major role in response to a variety of microbial infections. Ito cells (hepatic stellate cells), which can produce collagen in response to liver injury, are found between the endothelial cell layer and hepatocytes, and are responsible for development of fibrosis and cirrhosis in response to chronic liver injury (e.g., caused by chronic HBV infections). HBV infection results from exposure to blood or tissues of HBV-infected patients that leads to entry of virus into the blood stream. From there it is carried to hepatocytes, the only well-established site of HBV infection and reproduction

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in humans. It remains unclear if HBV accesses hepatocytes directly by passing through the fenestrations in the endothelial layer, or is taken up first by the endothelial lining cells and from there passed to hepatocytes (Breiner et al. 2001). Once a hepatocyte(s) is infected, HBV spreads through the liver with a doubling time of about 4 days (Wieland et al. 2004b), based on studies with chimpanzees, which are HBV susceptible. Since the human liver contains ~5 × 1011 hepatocytes, infection of the entire liver starting from a single infected hepatocyte could take as long as 4 months (Asabe et al. 2009). Remarkably, in many cases, and in all those infections which become chronic, infection of the entire hepatocyte population actually occurs. It is inferred that in these cases the HBV infection does not elicit a significant innate immune response and that the adaptive response is slow to develop (Wieland et al. 2004a). Even with transient infections that involve, at their peak, the entire hepatocyte population, adaptive responses may not be apparent until days or weeks after the entire population is infected (Asabe et al. 2009; Kajino et al. 1994; Summers et al. 2003; Wieland et al. 2004a, b). If this adaptive response is strong, the infection will clear, typically over the course of a few weeks (Wieland et al. 2004b). If not, the infection will become chronic. Chronic liver disease then results from a persistent adaptive immune response that results in an accelerated rate of hepatocyte killing and compensatory regeneration, as compared to the uninfected liver. The normal rate of hepatocyte death and replacement is probably around 0.01% per day, but can be 10 to >100-fold higher in chronic infections (e.g., Mason et al. 1998; Nowak et al. 1996). The extent of hepatocyte turnover varies in proportion to the degree of hepatic inflammation and, as a result, can be very different at different times during the course of a chronic infection. The belief that the immune response plays an important role in the outcome of chronic HBV infections came from early studies in animal models, which suggested that productive hepadnavirus infections are not cytopathic (e.g., Halpern et al. 1983; Jilbert et al. 1988). These early observations have been supported by a variety of later observations, including the persistence of infected hepatocytes during antiviral therapy with inhibitors of viral DNA synthesis (e.g., Werle-Lapostolle et al. 2004; Zhu et al. 2001). These infected hepatocytes should have quickly vanished if the infection was cytocidal, but they generally persist for months or years after therapy begins. A separate line of evidence that the immune response plays an essential role in chronic HBV infections, which may lead to HCC, comes from a study of HBV transgenic mice. Immune system reconstitution with syngeneic, non-transgenic lymphocytes reactive to HBV envelope proteins led to chronic liver disease and HCC (Nakamoto et al. 1998). However, while this shows that a host immune response to products of an HBV transgene can lead to HCC, the actual role of the antiviral immune response to a natural HBV infection in the progression to HCC has remained elusive. Thus, the following generalized view of the immune response to HBV infections has emerged. Typically, especially in cases that become chronic or that cause clinically apparent transient infections, virus spreads to the entire hepatocyte population. Hepatocyte infection, per se, is both productive and non-cytopathic. The adaptive immune response, particularly mediated by CT8(+) T lymphocytes (Asabe et al. 2009;

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Thimme et al. 2003; Wieland et al. 2004b), then activates, causing an acute hepatitis. This response generally abates after several weeks, whether or not the infection has cleared. HBV clearance presumably depends upon the strength of the immune response. The residual immune response that persists in chronic carriers is the cause of liver disease, cirrhosis, and HCC (Liaw 2009; Lok and McMahon 2007; Rehermann et al. 1996b; Yim and Lok 2006). Interestingly, even if the infection is resolved, a residual immune response persists, and presumably serves, along with antiviral antibodies, to prevent future infections, as well as reactivation of the original infection (Rehermann et al. 1996a). Reactivation may occur from very small amounts of residual virus that persist in a patient who has recovered from a transient infection (Hoofnagle 2009; Mulrooney-Cousins and Michalak 2007; Reaiche et al. 2010; Rehermann et al. 1996a; Yotsuyanagi et al. 1997); the mechanism of persistence of these residual infections is unclear. Reactivation is usually associated with, and presumably the result of, immuno-suppression.

Origin of Hepatocellular Carcinomas The progression to HCC and the nature of the cells that ultimately undergo neoplastic transformation are still a matter of debate. Some investigators have suggested that HCC in HBV carriers arise from hepatocyte progenitor/oval cells (Rogler 1991; Roskams 2006), while others have felt that HCCs in HBV carriers may arise from differentiated hepatocytes (Thorgeirsson and Grisham 2002). This remains a difficult problem to resolve. Hepatocytes constitute a self-renewing population and are clearly able to divide to compensate for the death of other hepatocytes. However, it is also clear that hepatocyte progenitor cells, although rare, do exist, and are able to expand and differentiate to replace dying hepatocytes during acute and chronic liver injury, particularly when the injury is caused by agents which prevent mature hepatocytes from fulfilling this role (Evarts et al. 1987; Hsia et al. 1992; Libbrecht et al. 2000; Oertel et al. 2008), but also to some extent during chronic hepatitis B. The strongest argument that HCCs in adults arise from mature hepatocytes comes from the fact that HCCs almost always contain integrated HBV DNA and that hepatocytes are the only liver cell, in human liver, that have been unambiguously demonstrated to be infected by HBV. One early study detected high level viral envelope protein accumulation in the putative hepatocyte progenitor cell population (Hsia et al. 1994) and this study is often cited to support the conclusion that HBV associated HCC is of progenitor cell origin. We are unaware of any subsequent study that has re-visited these early results. Interestingly, while neonatal infection does not typically lead to childhood HCC, exceptions do occur (Chang et al. 2009; Chen 2009). It has been suggested that many HCCs in HBV-infected children have a progenitor cell origin, while those in adults, an hepatocyte origin (Ward et al. 2010). More work is needed to test this idea. In brief, it is not clear how the issue of HCC origin can be resolved from deductive experiments, since HCC cells share many properties with hepatocytes as well as

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with hepatocyte progenitor cells. As noted above, with some caveats, the best evidence for a hepatocyte origin is the presence of integrated HBV DNA in the tumor cells.

Hepadnavirus Replication HBV is a small DNA virus with a genome size of ~3.2 kbp (Fig. 22.2). The virus encodes only seven proteins. Among these are five structural proteins, including a nucleocapsid protein subunit (core protein), three envelope proteins, and a reverse transcriptase, and two nonstructural proteins, known as hepatitis B virus e-antigen (HBeAg) and X (HBx). HBeAg is a glycosylated, C-terminally truncated, secreted version of the nucleocapsid subunit which is thought to help the virus evade the host immune response, at least early in infection (Chen et al. 2005). HBeAg-negative

HBV Genome ~3200 bp

(+) (-)

Fig. 22.2 The rcDNA genome of HBV. The HBV genome is a relaxed circular DNA with a complete minus strand, incomplete plus strand, and a short cohesive overlap between the 5¢ ends of the two DNA strands. A protein, the viral reverse transcriptase (Pol), which is also the primer for minus strand synthesis, remains covalently bound to the DNA during virus maturation (filled circle). The RNA primer of 2nd strand (plus strand) synthesis remains attached to the 5¢ end of the plus strand (thin line). All viral open reading frames (ORFs) are in the same direction, though out of frame where they overlap. Each viral protein has its own mRNA, transcribed from promoters upstream of the respective coding region, except for Pol, which is translated from the mRNA (the pregenome) for the core protein (see text; Seeger et al. 2006, for details). The pregenome, which is also the template for reverse transcription of minus strand DNA, is illustrated for comparison to the structure of the rcDNA viral genome

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HBV strains often arise late in chronic infection, suggesting that this immune evasion role of HBeAg is eventually lost. HBx is required for virus replication (Keasler et al. 2009; Xu et al. 2002; Zhang et al. 2001; Zoulim et al. 1994) and is able to activate transcription of both HBV (Spandau and Lee 1988; Twu and Schloemer 1987) and host mRNAs (Aufiero and Schneider 1990; Avantaggiati et al. 1993; Balsano et al. 1991; Mahe et al. 1991; Twu et al. 1993). The exact role of this gene remains obscure. Finally, although the reverse transcriptase is a virus structural protein that is packaged in nascent viral nucleocapsids, cytoplasmic (Yao et al. 2000) and secreted (Cao et al. 2009) forms of the polymerase have also been described. Their functional significance has not yet been established. The genome of HBV is a relaxed circular DNA (rcDNA), which is held in a circular conformation by a short cohesive overlap between the 5¢ ends of the two DNA strands (Fig. 22.2). One strand is always complete within the virus, while the other is always incomplete, so that the genome is actually only partially double stranded. The complete strand is the template for viral RNA synthesis and thus is of negative polarity, while the incomplete strand is of plus polarity. When the virus infects a hepatocyte (Fig. 22.3), the plus strand is completed and the ends of each strand are ultimately ligated to form a covalently closed circular DNA (cccDNA), which is found in the nucleus of infected cells. cccDNA is associated with histones (Levrero et al. 2009; Newbold et al. 1995) to form the transcriptional template of the virus, from which all viral mRNAs needed for virus replication are ultimately transcribed. cccDNA does not replicate but, instead, is produced, from rcDNA synthesized in the cytoplasm of infected hepatocytes (Tuttleman et al. 1986a, b). Among the various viral RNAs, the second largest is the pregenome, with a terminal redundancy of ~120 nts (Fig. 22.2); a larger mRNA, the mRNA for HBeAg, is only a few nts longer than the pregenome. The pregenome is the template for translation of both the viral nucleocapsid protein (core protein) and the viral DNA polymerase (reverse transcriptase), as well as the template for reverse transcription to synthesize new viral DNA. Because the open reading frame (ORF) for the polymerase is downstream (and overlapping with) that of the core protein on the pregenome mRNA, core protein is the major translation product. Polymerase, which is in a different translation reading frame than core, is translated following ribosomal shunting (Sen et al. 2004) to the polymerase ORF, which is thought to be a rare event as compared to translation initiation at the upstream core AUG. When the polymerase is translated, it often binds to a stem-loop structure, epsilon, located in the 5¢ copy of the terminal redundancy of its own mRNA (Junker-Niepmann et al. 1990). This polymerase/pregenome complex is then packaged, along with host chaperones (Hu and Seeger 1996; Hu et al. 1997), into icosahedral nucleocapsid shells composed of 240 or 180 copies of the viral core protein (Bringas 1997; Crowther et al. 1994; Wynne et al. 1999; Zlotnick et al. 1997). Reverse transcription of the pregenome takes place within the nucleocapsids (Summers and Mason 1982) to form new viral rcDNA (Fig. 22.3). Once the plus strand is partially complete, the nucleocapsids interact with the viral envelope proteins and bud into the endoplasmic reticulum (ER) to form virions (Wei et al. 1996), which are ultimately transported and secreted from the infected hepatocytes into the blood stream.

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Endothelial Cells Virus rcDNA genome 90%

Virus DSL DNA genome 10%

Integration into host DNA cccDNA host DNA Transcription of cccDNA

Packaging and reverse transcription of the pregenome

Hepatocyte

Fig. 22.3 HBV life cycle in hepatocytes. Blood enters the liver and passes through sinusoidal spaces lined with hepatic endothelial cells. HBV present in the blood infects hepatocytes, probably after passing through fenestrations in the endothelial cells. The virus envelope is removed and nucleocapsids transported to the nucleus, where rcDNA is released and converted to cccDNA. cccDNA serves at the template for six viral mRNAs, including the pregenome, the template for reverse transcription. The pregenome enters the cytoplasm and is translated into core (nucleocapsid subunit) and pol proteins. When pol is made, it binds to the 5¢ end of its own mRNA and the complex is packaged into nucleocapsids, where viral DNA synthesis takes place to form new rcDNA and DSL DNA. Early in infection, newly made viral DNA is transported to the nucleus to amplify the cccDNA copy number to about ~5–50 per cell. Further cccDNA synthesis is then blocked and newly made DNA is assembled into viruses by interaction of nucleocapsids with viral envelope proteins, budding into the ER, and export into the blood stream. Virus with linear DNA can initiate the infection pathway, but may not be able to complete it, since genetic information is lost during cccDNA formation via illegitimate recombination between the ends of the DSL DNA. DSL DNA also has a propensity to integrate into host DNA, again by illegitimate recombination

Nucleocapsids can also migrate to the nucleus (Rabe et al. 2009; Tuttleman et al. 1986a) to form more cccDNA (Fig. 22.3). In addition, nucleocapsids lacking viral DNA (Burrell et al. 1982) typically accumulate in the nucleus in HBV-infected cells, a phenomenon not found with other hepadnaviruses. HBV core protein was reported a number of years ago to associate with, and hypothesized to regulate,

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Pregenome DSL-DNA

X-ORF

Envelope ORF PreS1 PreS2

S S M L

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Fig. 22.4 The structure of double-strand, linear virus DNA (DSL DNA). Reverse transcription of the minus strand always begins near the 3¢ end of the pregenome in the terminal redundancy, and continues to its 5¢ end, to create a full length minus strand with a short, ~9 base, terminal repeat. Normally, plus strand synthesis initiates near the 5¢ end of the minus strand, creating the cohesive overlap that holds the two strands in a circular conformation. About 10% of the time, plus strand synthesis begins, instead, at the 3¢ end of the minus strand, from an RNA primer, to create a double-stranded linear DNA (DSL DNA) (c.f., Fig. 22.2). DSL DNA is the most common substrate for integration of HBV into host DNA (see text for discussion)

the transcription of HBV cccDNA (Bock et al. 2001). Support for this hypothesis, or for core protein regulation of host gene transcription, has not yet been established. rcDNA is the primary product of HBV DNA replication. However, as the result of an occasional replication error, about 10% of new viral DNA is linear and double stranded (Staprans et al. 1991), beginning at the 5’ end of pregenome RNA (Fig. 22.4). Double stranded Linear genomes (DSL DNA) that enter the nucleus can form cccDNA via a process of illegitimate recombination between the free ends (Yang et al. 1996b; Yang and Summers 1998). This cccDNA is typically defective as a result of sequence loss during recombination. More interestingly, it can also undergo illegitimate recombination to integrate into host DNA (Bill and Summers 2004; Gong et al. 1999; Yaginuma et al. 1987; Yang and Summers 1999). A second form of linear DNA also seems to serve, less frequently, as a substrate for cccDNA formation via recombination and for integration into host DNA. This DNA is probably formed by strand displacement synthesis through the cohesive overlap of rcDNA (Mason et al. 2010; Yang et al. 1996b). Because the promoters for the pregenome and for the PreCore mRNA are located downstream of their respective genes in the smaller double-stranded linear DNA (Fig. 22.4), only the envelope and X proteins could be expressed from this smaller DNA after integration into host DNA. All viral proteins might be expressed after integration of the larger linear HBV DNA. Although most HCCs contain integrated HBV DNA, expression of core protein has been found in only about 15% of HCCs, and then, only in a minority of cells in the tumor (Hsu et al. 1989). Envelope protein is detected in about 30% of HCCs and, in a larger fraction of tumor cells than typical of core (Hsu et al. 1989). HBx expression has been observed in ~20–50% of HCCs (Seo et al. 1997; Su et al. 1998) but, like core, only in a small fraction of cells in a tumor (Su et al. 1998). As discussed later, envelope and/or X proteins expressed from integrated DNA have been hypothesized, based on studies in model systems, to have a role in HCC through their proposed ability to either initiate, promote, or fully transform hepatocytes. However, their expression may also make these hepatocytes targets of the antiviral immune

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response, so that expression might decline in tumors that were, at least hypothetically, initiated by these viral gene products (Hsu et al. 1989). This might explain why, not all tumors, or tumor cells, express these viral proteins.

Changes in the Virus and in the Liver During Chronic HBV Infection At the peak of transient infections and early in chronic infections, virtually every hepatocyte is infected, and virus titers in the blood stream are typically between 109 and 1010 per ml. Moreover, productive infection by HBV appears to be non-cytopathic and the hepatocyte population to be self-renewing. These two points might lead to the inference that chronic infections are associated with high titer production of HBV throughout their course. Surprisingly, rather than remaining high, virus titers actually decline over time, often by several orders of magnitude (Evans et al. 1998). The decline in virus titers appears to be associated with a decline in the fraction of infected hepatocytes (Burrell et al. 1984; Gowans et al. 1981; Mason et al. 2008, 2009; Volz et al. 2007) and in the amount of virus replication per infected hepatocyte (Volz et al. 2007). These observations suggest that there is a change over time in the ability of the hepatocyte population to support HBV replication. Dramatic changes in the virus population may also occur over time, with the emergence of mutant virus, often with mutations that would make them replication defective, as predominant forms of circulating HBV (e.g., Gunther et al. 1999; Marinos et al. 1996; Preikschat et al. 2002; Yuan et al. 1998, 1999). These and other mutant strains of HBV presumably survive in the hepatocyte population because of complementation with wild type virus or because a lost virus function is no longer required for virus survival. For instance, if horizontal, cell-to-cell spread of virus were no longer required once a chronic infection were established, virus envelope determinants essential for infectivity might no longer be essential. The fact that the hepatocyte population is largely self-renewing means that death of an infected hepatocyte is often compensated by division of another, also infected, hepatocyte, and presumably no extra-cellular spread of virus is required in this situation to maintain the infected cell number. The host immune response is undoubtedly a factor in the changing virus population, since deletions of immunodominant epitopes have been documented (Chen et al. 2006; Gunther et al. 1998; Ji et al. 2009; Lee et al. 1996). Perhaps the most well described change in the virus population is the loss of HBeAg, which occurs in many patients due to replacement of the predominant wild type strain of HBV by viruses that are no longer able to make HBeAg. This may be due either to a decline in transcription of the HBeAg mRNA, as a result of mutations in the promoter for the PreCore mRNA, or to stop codon or frame shift mutations in the signal sequence for this secreted protein (Brunetto et al. 1999; Okamoto et al. 1994; Parekh et al. 2003; Sato et al. 1995; Yu and Mertz 1996). HBeAg is thought to induce immune tolerance to the core protein early in infection, particularly perinatal infection from

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infected mothers to their newborn, but to lose this function later, so that its elimination is now actually beneficial for the survival of HBV-infected hepatocytes (Frelin et al. 2009; Milich et al. 1993). In brief, immune selection for less immunogenic variants of HBV is probably a major factor in the emergence of mutant viruses. The possibility of vertical transmission during mitosis may facilitate virus survival even if there is a loss of a function essential for production of infectious virus. Thus, it is possible that virus evolution during the course of a chronic infection occurs to a significant extent at the cellular level, with selective survival of hepatocyte lineages that have evolved, within themselves, less immunogenic strains of HBV. Surprisingly, it seems that the hepatocyte population also changes over time, declining in its ability to support HBV infections. This is strongly suggested by the reduction in the fraction of infected hepatocytes as a patient ages. It is assumed, as noted, that 100% of hepatocytes are infected early in an infection, at least for the first few years. However, this fraction can decline to 30% or less. Since the patients often remain viremic, the decline seems to reflect resistance of these hepatocytes to HBV infection. The origin of these hepatocytes and a molecular basis for their resistance to HBV is unknown. However, precedents for their emergence come from studies of chronic liver injury with a genetic basis (e.g., as reviewed by Alison et al. 2009; Marongiu et al. 2008). It has been found in a number of different studies that any source of chronic injury and death that affects all hepatocytes, or at least the vast majority, leads to repopulation of the liver by rare hepatocytes that are resistant to the toxicity. For instance, Chisari and colleagues studied transgenic mice that over-expressed the HBV large envelope protein, L, in hepatocytes (Chisari et al. 1989). Over-expression of L leads to its accumulation in the hepatocyte ER, in the form of 22 nm diameter rod-like structures. This accumulation leads to ER proliferation, producing so-called ground-glass hepatocytes (based on their appearance in stained tissue sections) and, ultimately, leads to hepatocyte death. The livers of mice that had the highest levels of L expression, and of hepatocyte injury, were eventually re-populated by hepatocytes that did not transcribe L mRNA and/or had acquired deletions in the transgene (Chisari et al. 1989; Crawford et al. 2006). Repopulation in this and other models of hepatocyte injury merely requires a differential survival advantage for a minor subpopulation of cells that can proliferate to become the predominant hepatocytes in the liver (Alison et al. 2009; Marongiu et al. 2008; Mason et al. 2008). It has been argued that the resistant cells, at least in the L over-expression model, may actually be derived from hepatocyte progenitor cells (Crawford et al. 2006); this may not be a fundamentally distinct issue if these cells then mature to hepatocytes, which have the capacity for self-renewal. Likewise, hepatocytes with a primary resistance to HBV would have a survival advantage in an HBV carrier since they would not be targets of the antiviral immune response, and could clonally expand to repopulate the liver of HBV carriers. The basis for HBV resistance could be at a variety of different levels. For instance, virus-free hepatocytes may be a subset that is especially sensitive to inhibition of HBV infection by antiviral cytokines. Also, it is not directly known if HBV-free hepatocytes constitute a fixed or fluctuating population that is responding to local conditions in hepatic lobules. The population would appear to be stable,

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since HBV titers tend to return to baseline levels after withdrawal of antiviral therapy (Dienstag et al. 1995), rather than to the high titers (>109 virions per ml) characteristic of new infections. A second possible source of resistance to infection is expression of envelope proteins from viral DNA that has randomly integrated into host DNA. cccDNA synthesis has been shown, with duck hepatitis B virus (DHBV), to be negatively regulated by viral envelope proteins (Lenhoff et al. 1998; Summers et al. 1990). In brief, according to this model, envelope proteins, if sufficiently abundant, lead to incorporation of all newly made rcDNA into virus, so that little is left to form cccDNA. With less cccDNA in the cell, there is presumably less viral mRNA synthesis and envelope protein production, and more opportunity for newly made viral DNA to be transported to the nucleus, rather than exported as virus particles. In theory, production of envelope proteins from integrated DNA could shut down this negative feedback loop and permanently suppress cccDNA synthesis. These envelope proteins might also induce super-infection resistance, so that cells freed of cccDNA are not re-infected. It should be noted, however, that attempts to show that envelope proteins negatively regulate HBV cccDNA formation, as shown with DHBV, have been difficult primarily because cell culture systems for HBV do not support significant cccDNA copy number amplification (Ling and Harrison 1997). In addition, it is unclear if virus-free hepatocytes, most appropriately defined by a lack of DNA replication intermediates and cccDNA (Fig. 22.3), typically express HBV envelope proteins from integrated DNA. The major difference between these two possible modes of resistance to HBV infection, or at least productive infection, is their origin. The first involves selection of rare, mutant, or epigenetically altered hepatocytes that are intrinsically resistant to HBV. HBV infection plays only an indirect role in their emergence, by establishing an environment that is toxic to normal HBV-susceptible hepatocytes. The second source of resistance is a direct result of HBV infection and DNA integration, and presumably, could be initiated in any hepatocyte which was infected by HBV. Studies with woodchuck hepatitis virus have revealed that integration occurs in at least ~0.1% of hepatocytes during a transient infection (Summers et al. 2003). Higher levels of integrated viral DNA have been found during chronic infections in human, chimpanzees, and woodchucks (Brechot et al. 1981a, b; Mason et al. 2005, 2009, 2010; Shafritz et al. 1981). Experiments to distinguish these two explanations for HBV-free hepatocytes in chronically infected patients, or to define alternative explanations, have not yet been reported. An additional major point, mentioned above, is that the virus population and hepatocyte population may both evolve during a chronic infection as a result of immune selection against viral antigens, leading to clonal expansion not only of virus resistant hepatocytes but also of hepatocytes that are infected with less immunogenic strains of HBV (Frelin et al. 2009; Mason et al. 2008). Thus, whatever its cause, hepatocyte repopulation, if it occurs to a significant level as suggested, should also lead to a significant genetic narrowing of the hepatocytes population (Mason et al. 2009), a known HCC risk factor (Marongiu et al. 2008). In addition, as discussed in the next section, expression of viral proteins has been proposed to have a more direct role in HCC.

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Hepatocyte Evolution and Liver Cancer in Patients with Chronic HBV Infection Introduction The notion that HBV encodes oncogenes is widely cited and may actually be correct, but this notion is almost entirely dependent upon interpretation of data collected using model systems. The obvious problem is that HBV infection does not transform hepatocytes; rather, decades of chronic infection are often needed to produce a cell capable of unregulated growth to form a tumor (Ganem and Prince 2004). Therefore, no HBV protein is able to transform normal hepatocytes, and any oncogenic ability, if it exists, is dependent upon other slowly evolving changes in the hepatocyte and/or virus population, presumably extremely rare, since only one or a few independent tumors will arise in a patient’s lifetime. Indeed, while HBx and expression of aberrant envelope proteins have been reported to activate expression of a wide variety of cellular genes (Koike 2009; Lupberger and Hildt 2007), changes in host gene expression, prior to the adaptive immune response, were not detected in a serial biopsy study of transiently infected chimpanzees (Wieland et al. 2004a). We are unaware of any studies showing that in vivo HBV infection, by itself, modifies expression of host genes in infected hepatocytes. Short-term studies in liver cell lines, typically derived from non-HBV related HCCs, may reveal what a viral protein is capable of doing, without proving that it actually does this in infected hepatocytes, or revealing what the consequences might be. Studies in transgenic mice, where it is possible to target viral gene expression to hepatocytes, would seem to be more biologically relevant. Even here, however, there are significant problems of interpretation. For instance, HBx expression reportedly leads to HCC in some but not all strains of HBx transgenic mice (Kim et al. 1991; Madden and Slagle 2001; Slagle et al. 1996). Interestingly, in those mice that did not develop HCC due to HBx expression, HBx was still found to facilitate liver carcinogenesis. For instance, diethylnitrosamine (DEN)-induced HCC was facilitated by the presence of an HBx transgene in mice in which HBx, alone, did not cause HCC (Slagle et al. 1996). HCC was also facilitated by the introduction of an HBx transgene in C-myc transgenic mice. These latter findings support the idea that HBx is pro-carcinogenic, even if not fully oncogenic. In contrast, later studies using mice transgenic for the entire viral genome revealed an increased sensitivity to DEN-mediated HCC that was not dependent upon HBx expression (Zheng et al. 2007). There is no explanation for these different outcomes, except that other viral proteins (e.g., envelope) are also pro-carcinogenic. Nor, for that matter, is it clear that mediators of rodent HCC will behave similarly in human HBV carriers. As noted earlier, the two rodent models of chronic HBV infection, ground squirrels and woodchucks, display different profiles of HCC induction from each other and from humans. In summary, it remains possible that HBV causes HCC simply by stimulating a chronic immune response to the hepatocyte population, enhancing cell injury, mutagenesis, death, and regeneration over a very long time period. Alternatively,

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HCC may be a result of this prolonged period of injury, death, and regeneration working in combination with viral proteins that directly alter cellular metabolism and, as a result, play a more direct role in cellular transformation.

Hepatocyte Regeneration and HCC As mentioned above, studies in mouse models and in humans with metabolic diseases of the liver (e.g., hereditary tyrosinemia) have suggested that progression to HCC is paralleled in almost every case by death of most of the hepatocytes that are initially present, and their replacement by clonal expansion of the remainder (see Alison et al. 2009; Marongiu et al. 2008, for review). This occurs because the agent which causes HCC, whether endogenous (i.e., hereditary) or exogenous (e.g., a chemical), also has a major toxic effect on the hepatocyte population. As a result, most hepatocytes die and clonal expansion of rare hepatocytes with resistant phenotypes, some of which are pre-neoplastic and progress to HCC (Marongiu et al. 2008), is necessary for host survival. Thus, as in chemical carcinogenesis, liver regeneration is necessary to promote carcinogenesis that is initiated by mutation of a hepatocyte or hepatocyte progenitor cell (Maeda et al. 2005). Of course, this does not explain HCC at the molecular level, but is at least the starting point of a clearly defined biological process ending in HCC. Thus, it seems reasonable to assume that the same hepatocyte repopulation happens in HBV carriers and leads to, or at least promotes the process leading to HCC. In particular, the clonal expansion of rare hepatocytes that are unable or have lost the capacity to support productive HBV infections. As a result, these hepatocytes would not be efficient targets for killing by the host’s antiviral immune response and, therefore, may have the ability to repopulate the liver at the expense of HBV susceptible hepatocytes. Two types of observation support the idea of clonal hepatocyte repopulation, in HBV carriers, due to killing of productively infected hepatocytes and expansion of rare hepatocytes that are not productively infected: (1) A decline in the fraction of hepatocytes that support virus replication, to 30% or less, is a characteristic feature of HBV infection. This has typically been noted in late stages of infection, reflecting the clinical focus on patients who are in the immune clearance, inactive carrier, or reactivation stages of the disease (Burrell et al. 1984; Gowans et al. 1981; Mason et al. 2008, 2009; Volz et al. 2007), when biopsy or surgical specimens are more likely to be taken. Support for the histologic observations comes from qPCR assays of liver biopsies. These assays show that in late stages of infection, there is often insufficient cccDNA, the viral transcriptional template, for it to be present in all hepatocytes (Volz et al. 2007). (2) Clonal expansion has been observed in hepatocytes in non-cirrhotic liver of chronically infected woodchucks, chimpanzees, and humans, to produce large clones of 1,000 to 50,000 or more cells (Mason et al. 2005, 2009, 2010). In these experiments, viral DNA, which can integrate at random sites in host DNA, was used as a marker of cell lineages (Bill and Summers 2004;

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Gong et al. 1996, 1999; Summers et al. 2003; Yang and Summers 1999). Determining the number of copies of an integrant in a liver fragment by inverse PCR indicates how much clonal expansion of these hepatocytes occurred following the integration event (Mason et al. 2005). The fact that these hepatocytes contain integrated DNA means that they were, prior to expansion, susceptible to HBV uptake. There are several other reasons to think this repopulation could actually occur, and progress to HCC. (1) Hepatocytes are, to a large extent, members of a closed and self-renewing population. The population may therefore evolve under selective pressure, with emergence of initially rare hepatocytes to become the predominant cell type (e.g., Alison et al. 2009; Chisari et al. 1989; Marongiu et al. 2008; Ponder 1996); (2) Foci of altered hepatocytes (FAH), which develop during chronic infection and are considered to be pre-neoplastic, generally do not contain replicating virus, even when surrounded by productively infected hepatocytes (Abe et al. 1988; Fausto 2004; Govindarajan et al. 1990; Radaeva et al. 2000; Rogler 1991; Sell et al. 1987; Toshkov et al. 1990; Yang et al. 1993; Yang et al. 1996a; Yeh et al. 2001); (3) HCCs, like FAH, are generally not productively infected. As noted earlier, expression of core and HBx are seen in only a minority fraction of tumors, and only in a minor fraction of cells within those tumors; (4) It has been reported that up to 50% of HCCs may occur in non-cirrhotic HBV carriers (Bosch et al. 2005), though the number may be several fold lower (Beasley 1988). This is consistent with the notion of extensive hepatocyte repopulation even in non-cirrhotic patients. Thus, hepatocyte repopulation in HBV carriers to evade the host immune response, or some other unknown factor, may occur, as a risk factor for HCC which precedes cirrhosis. An apparently distinct source of clonal hepatocyte repopulation related, only indirectly, to immune killing of hepatocytes, is well documented in late-stage HBV patients with cirrhosis. In particular, 50% or more of cirrhotic nodules are clonal (Aoki and Robinson 1989; Furuya et al. 1988; Mashal et al. 1993; Paradis et al. 1998; Piao et al. 1997; Robinson et al. 1990; Yeh et al. 2001) and often have a pre-neoplastic appearance. This clonality presumably reflects a selection for rare mutant and/or epigenetically altered hepatocytes that are able to survive the disruption of lobular architecture and blood flow that occurs within the nodules. Cirrhosis has long been known to be an epidemiological risk factor for HCC (Beasley 1988). The histologic findings suggest that cirrhosis and HCC are not merely coincident outcomes of chronic HBV infection. In brief, clonal hepatocyte repopulation, which appears to promote emergence of HCC in liver diseases of non-viral origin, also takes place in HBV carriers, in cirrhotic nodules and also in non-cirrhotic livers. Clonal repopulation in cirrhotic livers is likely a direct consequence of changes in hepatic structure and restrictions in blood and lymph flow in cirrhotic nodules. Clonal repopulation in non-cirrhotic livers likely results from immune evasion of the antiviral host response. In both cases, rare hepatocytes expand clonally, presumably because of genetic or epigenetic changes which allow them to survive in an otherwise toxic environment. The fact that hepatocytes are self-renewing argues against the necessity of a progenitor cell origin for these rare cells, although some have argued that hepatocyte regeneration during

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chronic liver disease has a major progenitor cell component (Libbrecht et al. 2000). Whether the changes which allow survival of these hepatocytes are also, in some cases, responsible for HCC, is unknown, though clearly, in most cases, they are not. A reasonable hypothesis is that these early, pre-HCC, changes are necessary but not sufficient for transformation of normal hepatocytes to carcinoma cells. Other factors required for full transformation may include viral protein expression from integrated DNA and/or additional mutations in regulatory cell proteins or destabilization of host chromosomes (Barash et al. 2010; Lee et al. 2009; Pineau et al. 2008).

HBx and HCC Interest in HBx as an HBV oncogene has been persistent since the detection of this small ORF in the viral DNA sequence and the discovery of its ability to trans-activate expression of viral and cellular genes in culture. Indeed, early reports indicated that HBx could transform immortalized cells in culture (Shirakata et al. 1989) and later studies with transgenic mouse models implied that HBx could act alone (Kim et al. 1991; Koike et al. 1994), or as a co-factor with C-myc (Shirakata et al. 1989) or with DEN (Lee et al. 1990; Slagle et al. 1996) to transform mouse liver hepatocytes. However, extension of these findings to HBV patients has been problematic. HBx clearly does not cause rapid transformation of hepatocytes, even in the mouse models, nor is there any evidence that infection per se alters hepatocyte behavior. Moreover, its putative oncogenic potential is limited. While up to 25% of HBV carriers may develop HCC, the majority do not. Understanding the role of HBx in infection and HCC is compounded by the wide range of activities and mechanisms of action that have been reported. Some selected examples are illustrated in Table 22.1. What remains unclear is whether the different reported activities of HBx have a common basis. A recent report suggests that HBx binds to the DDB1 subunit of the CUL4-DDB1 ubiquitin ligase and blocks binding of receptors molecules to the DDB1, the adaptor subunit of the ligase (Li et al. 2010). The effect would, in theory, be to alter expression of pathways that are regulated through this E3 ubiquitin ligase. These newer binding results are consistent with early reports that HBx inhibits DNA repair by binding to DDB1, which is also a component of the DNA repair machinery. It is difficult, however, to reconcile binding to DDB1 with all the diverse effects of HBx expression, since binding to DDB1 is apparently not required for its function as a transcription transactivator (Wentz et al. 2000). Moreover, although in some cases controversial (Madden and Slagle 2001), other binding partners, including the tumor suppressor P53 (Elmore et al. 1997; Feitelson and Duan 1997; Greenblatt et al. 1997), and mitochondrial HSP60 (Tanaka et al. 2004; Zhang et al. 2005a), have been reported in the literature. In brief, it has not been apparent how to prove that HBx has a direct role in human HCC. The major problem is that no studies to date show that HBx is needed for maintenance of the transformed state. Thus, detection of HBx expression in some HCCs does not prove any expression is essential; in fact, as noted earlier, the majority

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Table 22.1 Some reported activities of HBxa Activates viral gene expression Colgrove et al. (1989) and Spandau and Lee (1988) Activates host gene expression Aufiero and Schneider (1990), Bock et al. (2008), Cohen et al. (2010), Cross et al. (1993), Twu and Schloemer (1987), Wang et al. (1998), Yoo et al. (1996), Yu et al. (2005), and Zhang et al. (2005b, c) Modifies/activates host transcription Barnabas et al. (1997), Kong et al. (2000, 2003), Lucito factors and Schneider (1992), Maguire et al. (1991), Rui et al. (2006), Seto et al. (1990) and Shamay et al. (2002) Binds to RNA polymerase Dorjsuren et al. (1998), Haviv et al. (1998), Lin et al. complex (1997) and Qadri et al. (1995) Modifies/inhibits E3 ubiquiton Li et al. (2010) ligase by binding to DDB1 subunit Causes HCC in mice Kim et al. (1991) Collaborates with DEN and Becker et al. (1998), Madden et al. (2001), Sitterlin et al. C-myc to cause HCC in mice (2000), Slagle et al. (1996) and Zhu et al. (2004) Inhibits nucleotide Becker et al. (1998), Capovilla and Arbuthnot (2003), excision repair Groisman et al. (1999), Jaitovich-Groisman et al. (2001), Ji et al. (2009), Mathonnet et al. (2004) and Qadri et al. (1996) Anti-apoptotic Elmore et al. (1997), Pan et al. (2001) and Wang et al. (1995) Binds to and inactivates/inhibits Elmore et al. (1997), Feitelson and Duan (1997), tumor suppressor P53; represses Greenblatt et al. (1997) and Lee and Rho (2000) P53 transcription Induces cellular proliferation Madden et al. (2001) Transforms immortalized mouse Shirakata et al. (1989) cells Disrupts intercellular and matrix Lara-Pezzi et al. (2001a, b) adhesion Promotes fibrosis/cirrhosis via Feitelson et al. (2009) and Martin-Vilchez et al. (2008) paracrine activation of hepatic stellate cells Induces polyploidy Rakotomalala et al. (2008) and Studach et al. (2009) Induces chromosome instability Kim et al. (2008) and Martin-Lluesma et al. (2008) Shifts TGF-beta signaling to Murata et al. (2009) pro-oncogenic pathway Pro-apoptotic Bergametti et al. (1999), Liang et al. (2007), Schuster et al. (2000), Shirakata and Koike (2003), Su and Schneider (1997), Tanaka et al. (2004), and Terradillos et al. (1998, 2002) Induces cell cycle arrest Bouchard et al. (2001a), Cheng et al. (2009), Friedrich et al. (2005), Gearhart and Bouchard (2010), Lee et al. (2002), Park et al. (2000) and Sirma et al. (1999) Binds to mitochondria and induces Bouchard et al. (2001b), Chen and Siddiqui (2007), calcium release, increases Clippinger and Bouchard (2008), Henkler et al. (2001), Li et al. (2008), Lim et al. (2010), Rahmani reactive oxygen species, et al. (2000), Shirakata and Koike (2003), Tanaka et al. activates NF-kB and Stat-3, (2004), Waris et al. (2001) and Zhang et al. (2005a) induces cell death a A sampling of publications illustrating some of the major activities and functions ascribed to HBx

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of tumors do not express detectable levels of HBx, and even those that do, express it in only a minority of the tumor cells. Future analyses of putative HCC stem cells may help to resolve this issue. Perhaps the most consistent finding, in model systems, is that HBx inhibits nucleotide excision repair (Table 22.1), although possible disagreements exist as to the molecular mechanism and the target. Binding to DDB1 would suggest inhibition of global genome nucleotide excision (NER) repair, in which DDB1 participates in the recognition of distortions in the DNA helix. Based on these data, HBx would not be expected to inhibit transcription-coupled NER, which generally occurs when RNA polymerase II is stalled due to DNA damage. However, HBx has also been reported to bind to downstream components that are common to both transcriptioncoupled and global NER, suggesting that both pathways are inhibited. While many reported functions of HBx (inhibition of apoptosis; late G1 inhibition of cell cycle progression) have some obvious function [e.g., survival of infected hepatocytes; prevention of passage through the cell cycle, which reduces virus replication (Jansen et al. 1993)], the advantage to the virus of inhibiting NER remains to be determined and perhaps is only coincident with some other consequence of binding to the DDB1 component of the ubiquitin ligase. Based on these findings, a simple model is that HBx initiates carcinogenesis by facilitating accumulation of DNA damage via reactive nitrogen and oxygen species (Gehrke et al. 2004) and that the immune response promotes carcinogenesis by accelerating hepatocyte regeneration (Maeda et al. 2005). Other reported functions of HBx may also increase the risk of HCC (see Table 22.1), though inhibition of NER would seem the most obvious direct effect. It should be noted that, despite the still limited amount of data, the claim has often been made that HBx is expressed in HCCs. This claim would be consistent with the hypothesis that HBx has a role in maintaining the transformed state of tumor cells. Moreover, the promoter and most of the HBx gene, except for the last few amino acids, is present in the linear HBV DNA which is the putative precursor to most integrations (Fig. 22.3). Therefore, the presence and expression of a carboxy terminal truncated HBx from many integrated DNAs would not be surprising. A problem in assuming that HBx has a role in maintaining cellular transformation is that wild type HBx has been found by several groups to induce cell cycle arrest in model systems (Bouchard et al. 2001b; Cheng et al. 2009; Gearhart and Bouchard 2010; Lee et al. 2002; Park et al. 2000; Sirma et al. 1999). A resolution to this difficulty may exist in the reports that a carboxy terminal truncated X ORF, found in at least some HCCs, yields an HBx that has lost the ability to induce cell cycle arrest, and has an enhanced ability to transform cells in culture (Ma et al. 2008; Tu et al. 2001). This raises the possibility that HBx has at least two roles in HCC, inhibition of NER leading to accumulation of host mutations early in infection (i.e., initiation) and direct promotion of pre-neoplastic/neoplastic cells late in infection. Again, it needs to be noted that truncated HBx, whatever its role in HCC, is not a typical oncogene because generation of a partially truncated HBx gene is an expected feature of viral DNA integration, but very few HCCs ever arise. More importantly, expression of HBx in most HCCs is not, as noted, well documented, and the idea

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that HBx, or a truncated HBx, maintains the transformed state, contradicts at least some studies looking at HBx expression in tumors (Su et al. 1998). Therefore, like inhibition of NER, the data on other activities of HBX support the conclusion that it is not required once a cell is cancerous, but may be involved in initiation and/or promotion of carcinogenesis. Finally, a significant effect of HBx expression could, in theory, be entirely indirect. The wild type HBV X protein expressed by replicating HBV has been claimed to be pro-apoptotic and to induce cell cycle arrest in late G1 (Bouchard et al. 2001a; Cheng et al. 2009; Gearhart and Bouchard 2010; Lee et al. 2002; Park et al. 2000; Sirma et al. 1999). This inhibition is readily overcome in vivo, at least partially, to the extent that infected hepatocytes do divide to replace dying hepatocytes during the course of a chronic infection (Wolf and Michalopoulos, 1992). It remains possible, however, that hepatocytes that do not express HBx (e.g., because they are unable to express HBV) have a greater probability than infected hepatocytes of dividing to replace dying hepatocytes. This, like selective immune killing of infected hepatocytes, should contribute to clonal hepatocyte repopulation, an HCC risk factor (Marongiu et al. 2008), although one inversely related to the functional activity of HBx.

Viral Envelope Proteins and HCC The three envelope proteins of HBV, encoded by a single ORF, define a nested set of proteins (Figs. 22.2 and 22.4). The smallest, S, is at the C terminus. Addition of 55 more amino acids to S creates M. The additional sequences in M are referred to as PreS2. M is not required for assembly of infections virus (Bruss and Ganem 1991; Fernholz et al. 1993; Santantonio et al. 1992). Addition of another ~120 amino acids to M defines L, with the extra sequences referred to as PreS1. These three proteins combine to form the viral envelope, with S the most, and L, the least abundant component. During synthesis of L at the ER, the PreS1 domain of only about 50% of the L protein molecules is translated into the ER. Those PreS1 sequences in the ER (and ultimately, on the outer surface of virions), are involved in virus recognition of target hepatocytes, while those on the cytoplasmic face appear to recruit virus nucleocapsids for budding into the ER to form virions (Bruss and Vieluf 1995; Ostapchuk et al. 1994). Interestingly, HBV synthesizes much more S, M, and L envelope protein than are needed to produce progeny virus. The excess is assembled and secreted from hepatocytes, mostly as 22 nm diameter spheres and, to a smaller extent, rods. The titer of these surface antigen (HBsAg) particles in sera is typically at least 100– 1,000 fold greater than virus titers. The reason for the over-production of envelope proteins is unclear. Analysis of spheres and rods purified from sera revealed that the spheres are made up primarily of S and, to a lesser extent, M and perhaps a small amount of L, while rods contain S, M, and L (Heermann et al. 1984). As noted earlier, over-expression of the large envelope protein, L, leads to accumulation of 22 nm rods in the endoplasmic reticulum (ER) (Chisari et al. 1987; McLachlan et al. 1987; Ou and Rutter 1987).

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This is associated with the induction of ER proliferation to give hepatocytes a ground glass appearance (Gerber et al. 1975). ER proliferation may be a unique response to L that is only indirectly related to the ER stress response (Foo and Yen 2000), which is also attributed to L overproduction (see below). Ground glass hepatocytes (GGH) generally accumulate toward the end of chronic liver disease, rather than early, or during transient infections, and have mostly been studied in non-tumorous liver samples acquired from patients with HCC. Two types of GGH have been distinguished, in diseased liver, based on differences in the pattern of intracellular accumulation of viral envelope proteins detected by immunohistochemistry and light microscopy. One type generally occurs singly while the other type, in clusters. Those GGH occurring singly are referred to as type I GGH while those in clusters type II GGH. Su and colleagues have suggested that type II GGH result from expression of a mutant form of L protein with a deletion in the PreS2 region (Fan et al. 2000), while type I GGH result from expression of a mutant form of L with a deletion in PreS1 (Wang et al. 2003, 2006). They have also suggested that type II GGH are pre-neoplastic. This suggestion is supported by the fact that type II GGH occur as clusters of cells, which is consistent with (but does not prove) the notion that the clusters arose by hepatocyte proliferation. Thus, a major issue is whether envelope proteins, particularly L, are also pro-oncogenic. In fact, interest in envelope proteins as potential oncogenes started with the early report that a C-terminal truncated form of the M protein, originally detected in integrated viral DNA from an HCC, could trans-activate host promoters, including C-myc, C-fos, and C-raf (Caselmann et al. 1997; Hildt et al. 2002; Kekule et al. 1990; Lauer et al. 1992). This activity was not found in M itself and required deletion of the carboxy terminal hydrophobic domain of S, including about 90 amino acids. The authors suggested the mutant M protein was produced from viral DNAs that had lost sequences during integration and that the necessary truncation might be present in at least 25% of HCCs (Lauer et al. 1992). This truncated protein would not normally be produced during virus replication. However, later studies from this group suggested that L protein, like truncated M, also functions as a transcriptional transactivator (Hildt et al. 1996). That is, the transactivation function first ascribed to truncated M protein may reflect a normal function of L that is sometimes essential during virus replication (Foo and Yen 2000; Hildt et al. 1996). At high enough levels, expression of L leads to hepatocyte death (Huang and Chisari 1995). Presumably related to this, L protein expression also leads to an ER stress response (unfolded protein response), which may lead to apoptosis if not relieved by some means; e.g., increased production of chaperones involved in protein folding. Interestingly, cell culture studies have suggested that ER stress also increases transcription from the HBV M/S promoter, with increased production of M and S envelope proteins contributing to relief of ER stress. Presumably, more M and S protein reduced formation of 22 nm rods in favor of virus and 22 nm spherical particles, which are more readily secreted (Huang et al. 2005; Xu et al. 1997) from the cell. Thus, at least in cell culture studies, L protein expression appears to induce an ER stress response that has a feedback role in balancing expression of the three viral envelop proteins, so that accumulation of potentially toxic levels of L protein in the ER does not occur.

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This raises an obvious question, are GGH always associated with accumulation of variants forms of L protein that no longer cause ER stress, despite their accumulation to high levels in the ER and the induction of ER proliferation. At present, there is no answer. With the exception of several recent studies from Su and colleagues, reviewed in (Hsieh et al. 2007), this issue has not received a great deal of attention, and it remains unclear if HBV variants in type II GGH are the cause or only passengers in these focal proliferations. This is a problem because HBV mutants with deletions in PreS1 and PreS2 are known to accumulate in the liver during chronic infections (Chen et al. 2006, 2007; Fernholz et al. 1993; Gunther et al. 1999). Their appearance late in infection is thought to be the result of immune pressure leading to loss of T and B cell epitopes, which is also true for the PreS2 deletion mutation found in type II GGH (Hsieh et al. 2007). Thus, the connection between the PreS2 mutation and putative clonal hepatocyte proliferation to form clusters of type II GGH might be coincidental rather than causative. One attempt to ascribe a role for the PreS2 deletion mutants of L, in HCC, has focused on a role in oxidative DNA damage, in the form of 8-hydroxyguanine, which can lead to insertion of an deoxyadenosine and mutation of host DNA (Hsieh et al. 2004). In support of this idea, expression of mutant L protein was shown to modestly upregulate 8-oxoguanine glycosylase, a protein involved in single nucleotide excision/repair (Hsieh et al. 2004). Enhanced expression of, for instance, vascular endothelial growth factor-A (VEGF-A), in type II GGH, and in Huh7 cells transfected with the mutant L, has also been reported (Yang et al. 2009). The mutant L protein has also been reported to enhance proliferation of Huh7 cells (Yang et al. 2009). (Huh7 is a cell line derived from a hepatocellular carcinoma (Nakabayashi et al. 1982), probably from an hepatitis C virus carrier). Up-regulation of cyclin A and induction of anchorage independent growth of HH4 (Tang et al. 2007), a line of human hepatocytes immortalized by transduction of HPV E6/E7, has also been claimed (Wang et al. 2005). Both VEGF-A and cyclin A were reported by this group to be upregulated in type II GGH (Wang et al. 2005; Yang et al. 2009). A problem with all these studies is uncertainty about the fraction of HCCs that express either the carboxy terminal-truncated M protein or an L protein with a deletion in PreS2, or even the wild type envelope proteins. One study, for instance, suggested that only 30% of HCCs express envelope protein (Hsu et al. 1989). And, as noted above, it is unclear if expression of the PreS2 deletion mutant of L is always associated with hepatocyte proliferation to form, for instance, type II GGH, or if the GGH are merely a carrier of a common HBV variant. This might occur if the mutation eliminated a major T cell epitope, giving the mutant-infected hepatocytes a selective survival advantage, compared to hepatocytes infected with wt HBV. In summary, as with HBx, available data do not provide a clear picture of the role of HBV envelope proteins in carcinogenesis. These proteins are clearly needed to make virus, and it is not surprising that their expression, particularly of L, may alter normal host functions, particularly involving the ER, since virus and surface antigen particles are formed by budding into the ER. On the other hand, there is so far no data/evidence on changes in host gene expression, in hepatocytes, that occur simply in response to in vivo infection. Finally, any role of envelope proteins in promoting

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hepatocyte proliferation must be subtle, at least for most of an infection, because hepatocyte proliferation appears tightly regulated in order to maintain liver size and function. Mutant viral proteins, even if more oncogenic in model systems than the wild type, are likely to be under this same restriction, since HBV has a high mutation rate, but HCC normally does not arise for decades. Thus, as with HBx, any effect of wild or mutant envelope proteins in cellular transformation might also depend upon the accumulation of mutations in host DNA that synergize with these changes in the virus population. These host mutations must be extremely rare, considering the large size of the hepatocyte population (~5 × 1012 cells) from which they arise and the long delay between infection and HCC.

Overview Chronic HBV infection has been known, for almost 40 years, to cause HCC (Beasley et al. 1981; Myerson et al. 1971; Nayak et al. 1977; Simons et al. 1971; Smith and Francis 1972; Summers et al. 1978a; Teres et al. 1971; Tong et al. 1971; Velasco et al. 1971; Vogel et al. 1972), and HBV’s mode of replication has been known for nearly 30 (Summers and Mason 1982). The fact that HBV replicates via reverse transcription led to the idea that HBV might also cause cancer like a retrovirus, particularly avian leucosis viruses, which were shown in the early 1980s to transform B cells by insertion of viral DNA in the vicinity of the C-myc gene (Hayward et al. 1981). This hypothesis was spectacularly successful with the woodchuck model of HBV. However, results with HBV itself have been disappointing, and a clear role for HBV DNA integration remains, for most HCCs, elusive. Part of the problem may be that woodchuck and human hepato-carcinogenesis may have fundamental differences and that human HCC may require many more changes in the liver, including not just persistent hepatocyte death and regeneration, but oxidative DNA damage and mutation, insertion of viral DNA into host DNA, ER stress, and in some cases, expression of proteins from integrated viral DNA. DNA damage (initiation) and regeneration (promotion) would presumably lead to some HCCs without any other risk factors being involved. However, a number of additional risk factors appear to exist, at least hypothetically, including hepatocyte repopulation by HBV resistant cells, due to persistent immune killing of infected hepatocytes, cirrhosis, and expression of viral proteins that may not be oncogenes in normal hepatocytes, but may assume this role in the highly selected hepatocyte population that emerges during the course of a chronic infection. Unfortunately, current results do not yet indicate how to chemotherapeutically treat and destroy HCCs. An additional problem is that new tumors may arise de novo even after effective surgical removal of an existing tumor. As with chronic hepatitis, a driving factor in recurrence appears to be the antiviral immune response, with associated hepatocyte death and regeneration (Budhu et al. 2006; Hoshida et al. 2008). Thus, a major emphasis has been given to cancer prevention. This has been approached in two ways, vaccination to prevent infection and antiviral therapy to

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slow disease progression. Therapy has been successful in slowing or reversing fibrosis and the progression to HCC (Chien and Liaw 2008; Lai et al. 1998; Liaw et al. 2004), but for most patients, is not curative, and therefore cannot be withdrawn without a risk of virus rebound and a bout of immune-mediated acute hepatitis (Chien and Liaw 2003; Honkoop et al. 2000). In addition, permanent suppression of HBV will not totally reduce the risk of HCC, which can occur even during the inactive phase of HBV infection (Chen et al. 2010), when virus productive is virtually undetectable. An additional and major problem with the most common therapy, nucleoside analog inhibitors of viral DNA synthesis, is the appearance of drugresistant variants that replace wild type virus as prevalent quasi-species (Locarnini and Mason 2005). Even with these patients, drug withdrawal can be problematic as the reemergence of the wild type virus, which generally replicates better than the drug-resistant strains, can also lead to acute hepatitis (Fung et al. 2005). It is hoped that these drug therapy issues will be less serious as the standard of treatment changes from single to multi drug regimens. It is also hoped that a multi-drug regimen will allow treatment of younger patients, in the immune-tolerant phase of infection, who are not currently candidates for antiviral therapy by recent guidelines (Lok and McMahon 2007), in part because they do not have overt liver disease, but equally, because they do not show a sustained response to monotherapy. Monotherapy generally shows a sustained response only in patients with active inflammatory disease to assist in HBV elimination before drug-resistant virus emerges. In summary, studies over the last 25 years have given a large number of insights into how HBV gene products might cause HCC, but have not provided clear evidence that the any viral protein is needed once an HCC has emerged. The implication is that HBV proteins, like all the other factors that contribute to HCC, provide only part of a very complex picture of carcinogenesis. Once the tumor has emerged, disease progression is largely refractory to chemotherapy that targets the metabolism of tumor cells. The most effective treatments are surgical resection or chemo-ablation of the HCC (e.g., injection of alcohol into the tumor) (Colombo and Sangiovanni 2002; Teratani et al. 2002), but these procedures are only efficacious with small HCCs, which are unlikely to be detected unless an HBV carrier is actively monitored. The best approaches to preventing deaths from HBV-associated cirrhosis and HCCs are (1) vaccination, (2) effective antiviral therapies, and (3) ablation of small tumors. Vaccination is clearly the most effective. The expense of post-infection therapies, and the difficulties in identifying and monitoring carriers make post-infection treatments impractical on a worldwide basis. This may change as better, and less expensive, treatments for HBV carriers, and more effective treatments for patients with advanced liver disease and HCC become available. Acknowledgements I am grateful to Drs. Christoph Seeger and John M. Taylor for a critical reading of this manuscript.

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Chapter 23

Hepatitis C Virus and Hepatocellular Carcinoma Brett Lindenbach

Introduction Liver cancer is the sixth most common form of cancer and the third most common cause of cancer death worldwide (Parkin et al. 2005). Hepatocellular carcinoma (HCC) accounts for a vast majority – 84% – of all liver cancers, and a large proportion of HCC cases – for instance, up to 70% of cases in Japan – are attributable to hepatitis C virus (HCV) infection (Tanaka et al. 2002; Yoshizawa 2002; Umemura et al. 2009). HCV is a major cause of acute and chronic liver disease, infecting between 130 and 170 million people (Lavanchy 2009). Once HCV was identified in 1989 as the major causative agent of non-A, non-B viral hepatitis (Choo et al. 1989), it soon became apparent that chronic HCV infection is linked with HCC (Bruix et al. 1989; Colombo et al. 1989). In addition, HCV infection is frequently associated with lymphoproliferative disorders, including non-Hodgkin’s B-cell lymphomas (Dustin and Rice 2007). In the ensuing years, much has been learned about the links between HCV and cancer, yet numerous questions remain. This chapter summarizes our current understanding of HCV and HCC, with a focus on clinical and molecular virological perspectives.

Clinical Perspective Epidemiological Link Between HCV and HCC While the link between HCC and chronic HBV infection was well-known (Chap. 21), the link between HCC and chronic HCV infection emerged only once diagnostic

B. Lindenbach (*) Section of Microbial Pathogenesis, Yale University School of Medicine, 354C Boyer Center for Molecular Medicine, 295 Congress Avenue, New Haven, CT 06536-0812, USA e-mail: [email protected] E.S. Robertson (ed.), Cancer Associated Viruses, Current Cancer Research, DOI 10.1007/978-1-4614-0016-5_23, © Springer Science+Business Media, LLC 2012

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tools for HCV infection became available. Soon thereafter, research teams in Italy and Spain showed that 65–75% of HCC patients had serological evidence of HCV infection, frequently accompanied by cirrhosis (Bruix et al. 1989; Colombo et al. 1989). These correlations were upheld in other populations, including North America, Europe, and Japan (Liang et al. 1993; Deuffic et al. 1999; Nagao et al. 2004). We now know that chronic HCV infection is a major risk factor for developing HCC (El-Serag 2002). One early study estimated that HCV infection increases the relative risk for developing HCC by 25-fold when compared to HCV-negative controls (Resnick and Koff 1993). Similarly, a more recent meta-analysis estimated that HCV-positive patients have a 17-fold higher relative risk for developing HCC than HCV-negative controls (Donato et al. 1998). The development of HCV-related HCC is a slow process, typically occurring 25–30 years after infection, and usually after the development of advanced liver fibrosis and cirrhosis (Castells et al. 1995; Tong et al. 1995). This slow progression, as well as the fact that many people are unaware of their HCV-infected status, has made it difficult to quantify the rate of progression to HCC in HCV-infected individuals. Nevertheless, a published review of the literature identified a few welldesigned and controlled studies that ameliorated such variables and patient selection biases (El-Serag 2002). Based on this analysis, it is estimated that 3–35% of HCVpositive individuals develop cirrhosis within 25 years after infection and up to 3% of HCV-positive individuals develop HCC within 30 years after infection (El-Serag 2002). It should be noted that numerous studies have shown that once cirrhosis develops, the annual rate of developing HCC is between 1 and 4%, and has been reported as high as 7% in Japan (Chiba et al. 1996; Bruno et al. 1997; Gordon et al. 1998; Serfaty et al. 1998; Degos et al. 2000). Additional risk factors that contribute to the development of HCC in HCV-positive individuals include coinfection with HBV, heavy alcohol consumption, older age, race, and male sex (Donato et al. 1997, 1998, 2002; Degos et al. 2000; Freeman et al. 2001).

Antiviral Therapy and HCC HCV infection was initially treated with interferon alpha, but the rate of sustained virologic response (SVR; i.e., undetectable serum viral load for 6 months post treatment) with interferon monotherapy is low, around 10–15% (Hoofnagle and di Bisceglie 1997). As of this writing, the current standard of treatment for chronic HCV infection is a rigorous 24- or 48-week combined regimen of pegylated interferon alpha and ribavirin (Manns et al. 2001). This combination therapy is expensive and difficult for patients, but does show a high rate of SVR for some but not all viral genotypes (described in the section “Experimental Systems to Study HCV”). In addition, a series of specifically targeted antiviral therapies for HCV (STAT-C), which seek to target mechanisms essential for viral replication and should improve HCV SVR rates, are currently in preclinical and clinical stages of development (Pereira and Jacobson 2009).

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Since chronic HCV infection contributes to the development of HCC, does successful treatment of HCV reduce the development of HCC? Answering this question is complicated by the fact that interferon has both antiviral and antitumor activities. While some studies have suggested that interferon or interferon/ribavirin combination therapy has preventative effects (reviewed in Liang and Heller 2004), most of these were retrospective or uncontrolled studies. In one prospective, controlled, and randomized study conducted in Japan, interferon-treated HCV patients had a significantly lower incidence of HCC than untreated HCV-positive controls, 4% vs. 38% £7 years of follow-up (Nishiguchi et al. 1995). However, it is notable that this control group exhibited an unusually high incidence of HCC. Another prospective, controlled, and randomized study conducted in France found that interferon-treated HCV patients had a slightly reduced incidence of HCC, 12% vs. 23% over 3 years of follow-up, but this was not statistically significant (Valla et al. 1999). A prospective, controlled, but nonrandomized study conducted in Italy showed that interferon treatment had a significant effect on the incidence of HCC, 2.6% vs. 9.8% over £3 years of follow-up (Mazzella et al. 1996). Interestingly, there was no difference in HCC incidence between patients who had an SVR vs. nonresponders, which suggested that these latter results may reflect the antitumor properties of interferon rather than its antiviral effect. More recently, the large, prospective, controlled, and randomized HALT-C trial failed to show an effect of long-term, low-dose interferon “maintenance” therapy on clinical outcomes, including the incidence of HCC, in patients that did not previously respond to interferon plus ribavirin (Di Bisceglie et al. 2008; Lok et al. 2009). These results suggested that successful reduction of viral loads may be necessary to prevent HCC.

Contribution of HCV to the Etiology of HCC Cancer development is a multistage process that requires multiple cellular checkpoints to be bypassed. These include continued proliferation, sustained immortality, evasion of growth inhibition, avoidance of cell death mechanisms, induction of angiogenesis, altered energy metabolism, evasion of immune surveillance, and acquisition of an invasive phenotype (Hanahan and Weinberg 2011). While there is a clear link between chronic HCV infection and HCC, it is not fully understood how HCV contributes to the etiology of HCC. HCV does not acutely transform cells, for example, like small DNA tumor viruses (Chaps. 13–19). A prevailing hypothesis is that chronic HCV infection indirectly contributes to the development of HCC by setting up a long-term inflammatory cascade that leads to hepatocyte turnover, advanced liver fibrosis (cirrhosis), and generation of DNAdamaging reactive oxygen intermediates (Liang and Heller 2004). While HCV can induce cirrhosis, and cirrhosis is a risk factor for developing HCC regardless of HCV infection status, some HCV-infected people develop HCC in the absence of cirrhosis (De Mitri et al. 1995; Lok et al. 2009). Moreover, there is evidence that some HCV gene products may directly contribute to cellular transformation (see the section “HCV Tropism”). Thus, HCV infection may promote cancer development through both direct and indirect mechanisms.

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Virological Perspective Experimental Systems to Study HCV HCV is an enveloped, positive-strand RNA virus, classified as a separate genus in the family Flaviviridae (Lindenbach et al. 2007). As such, HCV is distantly related to the classic flaviviruses, such as yellow fever, dengue, and West Nile viruses, as well as to the ruminant-infecting pestiviruses. Within the hepacivirus genus, HCV exhibits a large genetic diversity that can be classified into seven major genotypes and numerous subtypes. It is notable that genotypes 1a and 1b are the most prevalent worldwide and tend to respond poorly to interferon monotherapy or combination therapy. HCV has been difficult to study because the virus does not readily grow in cell culture and because animal models for HCV infection are limited to chimpanzees or to immunodeficient mice containing human liver grafts. Therefore, many early virological studies examined the structure and function of viral gene products by overexpressing them in various cell systems. The first HCV genetic systems became available when functional cDNA clones were constructed and shown to give rise to RNAs that are infectious in chimpanzees (Kolykhalov et al. 1997; Yanagi et al. 1997). The next major breakthrough came from the construction of selectable, genotype 1a and 1b HCV replicons and genomes that could replicate after transfection into the human hepatoma cell line Huh-7 (Lohmann et al. 1999; Blight et al. 2000, 2002). These systems provided genetically tractable tools to dissect the intracellular aspects of the viral replication cycle in cell culture. However, for reasons that are not entirely clear, infectious virus was not produced in these early replication systems (Blight et al. 2002; Pietschmann et al. 2002). More recently, a functional genotype 2a cDNA clone was shown to replicate and produce high-titer infectious virus in cell culture (Lindenbach et al. 2005; Wakita et al. 2005; Zhong et al. 2005; Pietschmann et al. 2006). In addition, infectious genotype 1a and 1b culture systems do now exist but yield very low titers (Yi et al. 2006; Pietschmann et al. 2009).

HCV Tropism HCV is hepatotropic, and it is likely that many of the difficulties in culturing this virus stem from the fact that human hepatocytes are extremely difficult to maintain in culture (Bhatia et al. 1999; Khetani and Bhatia 2008). It should be emphasized that the inability to study untransformed, primary hepatocytes in vitro also makes it difficult to experimentally assess the contribution of HCV infection to cellular transformation and oncogenesis. Nevertheless, recent advances in the long-term culture of primary hepatocytes may now allow these questions to be addressed (Khetani and Bhatia 2008; Ploss et al. 2010; Podevin et al. 2010). So, if HCV directly contributes to cellular transformation, are transformed HCC cells infected with HCV in vivo? The answer to this question remains elusive due to

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difficulties in detecting HCV in situ within human liver samples. However, recent advances in sensitive and specific antigen detection by using two-photon fluorescent microscopy and semiconducting quantum dots have revealed that HCV infects 7–20% of hepatocytes, typically within clusters, within frozen liver samples from chronically infected patients (Liang et al. 2009). In one sample from the study of Liang et al., HCV was abundantly found in a serial section adjacent to a cancerous lesion, although it could not be resolved whether the infected cells were malignant (Liang et al. 2009). These intriguing results beg for further analysis. In addition to hepatotropism, HCV RNA has been found in extrahepatic compartments, most notably associated with B lymphocytes (Dustin and Rice 2007). However, definitive evidence for replication in these cells is elusive, as replicating forms of the viral genome were not detected by using highly sensitive and validated quantitative methods (Lanford et al. 1995). One likely explanation is that HCV particles may associate with B cells via interaction with CD81, a known HCV coreceptor, or through interaction of immune complexes with Fc receptors. Despite these caveats, one group has provided evidence that Epstein–Barr virus-transformed B cells derived from chronically infected HCV patients contain signs of HCV infection (Sung et al. 2003). Interestingly, these cells appeared to exhibit a fivefold increased rate of mutation in the cellular BCL-6, p53, and ß-catenin proto-oncogenes (Machida et al. 2004). Intriguing as these results may be, replication of HCV in peripheral blood mononuclear cells has been difficult to reproduce in other laboratories (Marukian et al. 2008).

Molecular Biology of HCV The HCV genome is a monopartite, 9.6-kb coding-sense RNA that encodes a single, large, open reading frame (Fig. 23.1a). Translation of this genome produces a large polyprotein that is processed by viral and cellular proteases to produce ten distinct viral proteins (Fig. 23.1b). The N-terminal region of the genome encodes the structural (i.e., virion associated) proteins: core, which presumably forms the viral nucleocapsid, and envelope glycoproteins E1 and E2, which mediate virion binding and fusion during the initial steps of virus infection. The remainder of the polyprotein encodes nonstructural (NS) proteins: p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B. By definition, NS proteins are not incorporated into infectious virions, but are expressed within infected cells and coordinate the intracellular aspects of the viral life cycle.

HCV Proteins and Carcinogenesis Several HCV proteins have been implicated in cellular transformation by the fact that they interact with cell cycle regulators or tumor-suppressor proteins, as summarized below. However, most of these studies were performed outside the context

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Fig. 23.1 HCV genome (a) and polyprotein products (b)

of authentic viral infection, so it is difficult to assess how relevant these observations are to carcinogenesis in patients. To better understand the role of HCV proteins in physiologically relevant conditions, numerous transgenic mice have been constructed. One mouse line that expressed the entire HCV polyprotein under a liver-specific promoter on the C57BL/6 genetic background developed liver steatosis (fatty liver disease) and a low but discernible frequency of liver cancer (Lerat et al. 2002). Interestingly, a parallel transgenic line expressing the core-p7 region did not develop cancer, suggesting that the viral NS proteins could contribute to carcinogenesis (Lerat et al. 2002). One caveat to these mouse studies is that transgene insertion sites can contribute to the phenotype, and no current study has systematically shown that knockdown or inhibition of HCV protein expression in transgenic mice reverses the observed phenotype.

The HCV Core Protein The HCV core protein is synthesized at the endoplasmic reticulum, trafficked to lipid storage droplets, and incorporated into virus particles (Moradpour et al. 1996; Miyanari et al. 2007). This pattern of intracellular trafficking may contribute to the dysregulation of lipid metabolism and the intracellular accumulation of lipids known as hepatic steatosis (Abid et al. 2005; Jhaveri et al. 2008). Heterologous overexpression of core protein reportedly affects numerous cellular pathways that are relevant to cancer. However, conflicting reports abound, which could reflect differences in experimental systems and/or differential effects of overexpression.

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In some reports, core promotes apoptosis (Zhu et al. 1998; Honda et al. 2000a, b; Chou et al. 2005) while in other studies core inhibits apoptosis (Ray et al. 1998; Lu et al. 1999; Sacco et al. 2003). Furthermore, core reportedly inhibits the p53 tumor-suppressor pathway (Ray et al. 1998) while other studies suggest that core activates this pathway (Lu et al. 1999; Otsuka et al. 2000; Kao et al. 2004; Siavoshian et al. 2004). Core is also reported to reduce transcription of retinoblastoma (Rb) tumor-suppressor protein and enhance expression of E2F-1 and Mad2, leading to inhibited mitotic spindle checkpoint function and polyploidy (Machida et al. 2009). Core expression can also lead to the accumulation of reactive oxygen species, induce oxidative stress, and activate antioxidant gene expression (Li et al. 2002; Okuda et al. 2002; Korenaga et al. 2005). Finally, core can interact with Smad3, a modulator of transforming growth factor-ß signaling, and thereby inhibit its antitumor activities (Cheng et al. 2004; Pavio et al. 2005; Battaglia et al. 2009). To better understand the role of core in vivo, several core-expressing transgenic mouse lines have been characterized. One mouse line that expressed high levels of HCV core protein under a liver-specific promoter in the C57BL/6 genetic background developed liver steatosis and HCC at a high rate (Moriya et al. 1997, 1998). However, the expression levels of core in these mice were likely much higher than those found in HCV infections (McGivern and Lemon 2011). Similarly, another mouse line constitutively expressing core-E1-E2 in the B6C3F1 genetic background developed liver steatosis and a variety of hepatic and nonhepatic tumors (Naas et al. 2005). In contrast, other core or core-E1–E2 transgenic mice did not show increased cancer development (Kawamura et al. 1997; Pasquinelli et al. 1997; Lerat et al. 2002; Kamegaya et al. 2005), but core and core-E1-E2 transgenes could sensitize mice to a chemical carcinogen (Kamegaya et al. 2005).

The HCV NS3-4A Enzyme Complex NS3-4A is a membrane-bound enzyme complex that has serine protease and RNA helicase activities encoded by NS3. NS4A serves as a cofactor for these activities and serves to anchor the NS3-4A complex to cellular membranes. The NS3-4A serine protease is responsible for cleaving the NS3/4A, NS4A/4B, NS4B/5A, and NS5A/B junctions during viral polyprotein processing (Steinkühler 2004). In addition, the NS3-4A serine protease downregulates innate antiviral immune activation by cleaving the MAVS/IPS-1/ Cardif and TRIF signal transduction proteins (Li et al. 2005a, b; Meylan et al. 2005). NS3 has been shown to prevent apoptosis and can interact with and inhibit p53 (Ishido et al. 1998; Kwun et al. 2001; Deng et al. 2006; Tanaka et al. 2006). Mutations that prevent these activities map to the serine protease domain of NS3 (Deng et al. 2006; Tanaka et al. 2006), although serine protease activity is not required to inhibit p53 activity (Kwun et al. 2001). However, two contrasting reports suggested that expression of NS3 or NS4A induces apoptosis independent of serine protease activity (Prikhod’ko et al. 2004; Nomura-Takigawa et al. 2006). In addition, NS3-4A can inhibit cellular response to DNA damage through interaction with Chk2 and ATM (Ariumi et al. 2008; Lai et al. 2008).

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The HCV NS5A Phosphoprotein NS5A is a homodimeric, RNA-binding phosphoprotein of unknown function. However, NS5A is essential for HCV RNA replication and virus particle assembly. In addition, NS5A has been found to interact with numerous cellular proteins, including the key regulators of apoptosis, innate immunity, and tumor suppression. Expression of NS5A in cultured cells has variably been found to inhibit apoptosis (Gale et al. 1999; Lan et al. 2002), induce oxidative stress (Gong et al. 2001), and promote anchorage-independent growth (Ghosh et al. 1999). The antiapoptotic activity of NS5A may be due to interaction with p53 and inhibition of p53-mediated apoptosis (Majumder et al. 2001; Lan et al. 2002; Qadri et al. 2002). In addition, NS5A can bind to and activate PI3K, ultimately leading to stabilization of ß-catenin, a key regulator of cell growth (Street et al. 2004, 2005). NS5A may also directly bind and stabilize ß-catenin (Park et al. 2009; Milward et al. 2010). Transgenic mice expressing NS5A in the C57BL/6 × CBA/J genetic background showed a high level of liver steatosis and HCC (Wang et al. 2009); however, NS5A expression was not oncogenic in two other independently derived transgenic mouse lines with the FVB genetic background (Majumder et al. 2003).

The HCV NS5B RNA Polymerase NS5B is the RNA-dependent RNA polymerase responsible for replication of the viral genome. In addition, NS5B can interact with Rb and target it for E6AP-mediated ubiquitylation and degradation by the proteasome (Munakata et al. 2005, 2007; McGivern et al. 2009). Amazingly, the region of NS5B that interacts with Rb overlaps with the active site of the polymerase (Munakata et al. 2005). NS5B mutations that block Rb interaction were found to cause profound defects in replication but did not inhibit polymerase activity, suggesting that Rb degradation is important for HCV RNA replication (McGivern et al. 2009). However, these mutants were not rescued by Rb knockdown, suggesting that the NS5B–Rb interaction itself may be important.

Summary and Conclusions The epidemiological evidence strongly indicates that HCV contributes to the development of HCC. However, HCC usually arises only after decades of HCV infection, and likely involves both direct and indirect mechanisms. The purely reductionistic approach has identified candidate HCV genes that could promote direct oncogenic effects. However, we currently do not yet know how these observations relate to HCC development in authentic infections. Clearly, the recent advances in HCV and hepatocyte culture systems will help to clarify some of these outstanding issues.

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Qadri I, Iwahashi M, Simon F (2002) Hepatitis C virus NS5A protein binds TBP and p53, inhibiting their DNA binding and p53 interactions with TBP and ERCC3. Biochim Biophys Acta 1592(2):193–204 Ray RB, Steele R, Meyer K et al (1998) Hepatitis C virus core protein represses p21WAF1/Cip1/ Sid1 promoter activity. Gene 208(2):331–336 Resnick RH, Koff R (1993) Hepatitis C-related hepatocellular carcinoma. Prevalence and significance. Arch Intern Med 153(14):1672–1677 Sacco R, Tsutsumi T, Suzuki R et al (2003) Antiapoptotic regulation by hepatitis C virus core protein through up-regulation of inhibitor of caspase-activated DNase. Virology 317(1):24–35 Serfaty L, Aumaitre H, Chazouilleres O et al (1998) Determinants of outcome of compensated hepatitis C virus-related cirrhosis. Hepatology (Baltimore, MD) 27(5):1435–1440 Siavoshian S, Abraham JD, Kieny MP et al (2004) HCV core, NS3, NS5A and NS5B proteins modulate cell proliferation independently from p53 expression in hepatocarcinoma cell lines. Arch Virol 149(2):323–336 Steinkühler C (2004) Hepacivirin. In: Barrett AJ, Rawlings ND, Woessner JF (eds) Handbook of proteolytic enzymes, 2nd edn. Elsevier, London, pp 1773–1779 Street A, Macdonald A, Crowder K et al (2004) The hepatitis C virus NS5A protein activates a phosphoinositide 3-kinase-dependent survival signaling cascade. J Biol Chem 279(13):12232–12241 Street A, Macdonald A, McCormick C et al (2005) Hepatitis C virus NS5A-mediated activation of phosphoinositide 3-kinase results in stabilization of cellular beta-catenin and stimulation of beta-catenin-responsive transcription. J Virol 79(8):5006–5016 Sung VM, Shimodaira S, Doughty AL et al (2003) Establishment of B-cell lymphoma cell lines persistently infected with hepatitis C virus in vivo and in vitro: the apoptotic effects of virus infection. J Virol 77(3):2134–2146 Tanaka Y, Hanada K, Mizokami M et al (2002) A comparison of the molecular clock of hepatitis C virus in the United States and Japan predicts that hepatocellular carcinoma incidence in the United States will increase over the next two decades. Proc Natl Acad Sci USA 99(24):15584–15589 Tanaka M, Nagano-Fujii M, Deng L et al (2006) Single-point mutations of hepatitis C virus NS3 that impair p53 interaction and anti-apoptotic activity of NS3. Biochem Biophys Res Commun 340(3):792–799 Tong MJ, el-Farra NS, Reikes AR et al (1995) Clinical outcomes after transfusion-associated hepatitis C. N Engl J Med 332(22):1463–1466 Umemura T, Ichijo T, Yoshizawa K et al (2009) Epidemiology of hepatocellular carcinoma in Japan. J Gastroenterol 44(Suppl 19):102–107 Valla DC, Chevallier M, Marcellin P et al (1999) Treatment of hepatitis C virus-related cirrhosis: a randomized, controlled trial of interferon alfa-2b versus no treatment. Hepatology (Baltimore, MD) 29(6):1870–1875 Wakita T, Pietschmann T, Kato T et al (2005) Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat Med 11(7):791–796 Wang AG, Lee DS, Moon HB et al (2009) Non-structural 5A protein of hepatitis C virus induces a range of liver pathology in transgenic mice. J Pathol 219(2):253–262 Yanagi M, Purcell RH, Emerson SU et al (1997) Transcripts from a single full-length cDNA clone of hepatitis C virus are infectious when directly transfected into the liver of a chimpanzee. Proc Natl Acad Sci USA 94(16):8738–8743 Yi M, Villanueva RA, Thomas DL et al (2006) Production of infectious genotype 1a hepatitis C virus (hutchinson strain) in cultured human hepatoma cells. Proc Natl Acad Sci USA 103(7):2310–2315 Yoshizawa H (2002) Hepatocellular carcinoma associated with hepatitis C virus infection in Japan: projection to other countries in the foreseeable future. Oncology 62(Suppl 1):8–17 Zhong J, Gastaminza P, Cheng G et al (2005) Robust hepatitis C virus infection in vitro. Proc Natl Acad Sci USA 102(26):9294–9299 Zhu N, Khoshnan A, Schneider R et al (1998) Hepatitis C virus core protein binds to the cytoplasmic domain of tumor necrosis factor (TNF) receptor 1 and enhances TNF-induced apoptosis. J Virol 72(5):3691–3697

Chapter 24

Human and Animal Retroviruses: HIV-1 Infection Is a Risk Factor for Malignancy Amy M. Hayes and Kathleen Boris-Lawrie

Comparison of the Distinct Cancer Mechanisms of Simple RNA Tumor Viruses, Complex Human Transforming Retrovirus, and Human Immunodeficiency Virus Retrovirus Can Activate Cellular Proto-oncogenes At the advent of modern molecular biology, the genetic basis of cancer was discovered by the study of retroviruses. Study of infectious cancers in birds and mice discovered cellular proto-oncogenes and their transduction and mutation by transforming retroviruses (Weiss et al. 1982). The hallmark features of cell transformation were defined, a host of oncogenes were characterized, and the riveting story of retrovirus insertional mutagenesis, transduction, recombination, mutation, and quasispecies evolution was revealed by cell and molecular biologists (Chaps. 27 and 28). The paradigm was revealed that retroviruses may be acutely transforming or slowly transforming, lack a detectable disease end point, or lead to immunodeficiency of the infected host, as first observed for the sheep lentivirus, Visna maedi (Fan 1997).

A.M. Hayes • K. Boris-Lawrie (*) Center for Retrovirus Research, Ohio State University, Columbus, OH 43210, USA Center for RNA Biology, Ohio State University, Columbus, OH 43210, USA Comprehensive Cancer Center, Ohio State University, Columbus, OH 43210, USA Department of Veterinary Biosciences, Ohio State University, Columbus, OH 43210, USA e-mail: [email protected] E.S. Robertson (ed.), Cancer Associated Viruses, Current Cancer Research, DOI 10.1007/978-1-4614-0016-5_24, © Springer Science+Business Media, LLC 2012

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Viral Oncoproteins Can Cause Transformation Directly The first human retrovirus, human T-lymphotropic virus type 1 (HTLV-1), was discovered in 1980 (Poiesz et al. 1981) and ultimately revealed a distinct molecular mechanism for retrovirus carcinogenesis. Several decades after initial infection with HTLV-1, a minority of infected individuals present with an aggressive monoclonal tumor that is refractory to chemotherapy. HTLV-1 induces cell transformation, but not by activation of cellular proto-oncogene. Instead, the virus-encoded transcriptional transactivator Tax is an oncoprotein necessary and sufficient to transform cells, which disrupts cell biology via multiple mechanisms (Chap. 26). The transforming ability of HTLV-1 Tax is similar in principle to polyomavirus T antigen, adenovirus E1A, and the human papillomavirus (HPV) E6/E7 oncoproteins, which also dysregulate cell biology by multiple mechanisms (chapters in this volume). In comparison to the genetically simpler retroviruses that infect avian and murine species, HTLV-1 possesses a genetically complex genome structure. HTLV-1 encodes the structural and enzymatic genes common to all retroviruses and additional open reading frames for regulatory and accessory proteins. Two small open reading frames encode the Tax and Rex nucleic acid-binding proteins that transactivate viral gene expression. Alternatively spliced mRNAs encode accessory proteins that are dispensable in cell culture, but required for viral persistence in patient infections. Human immunodeficiency virus type 1 (HIV-1) shares this genetically complex structure (Chap. 32). However, HIV-1 does not cause cell transformation nor activate cellular proto-oncogenes, which may be an adaptation to survival in humans. Instead, HIV-1 infection is a risk factor for cancers caused by other transforming viruses, which are less well-adapted to human infection. HIV-1 augments the pathophysiological drivers of cell transformation and this interface presents a natural model to decipher the fundamental connection between cellular immunity and progression to malignancy.

HIV-1-Associated Malignancies Are Prognostic Indicators of Acquired Immunodeficiency Syndrome Large cohort studies have documented an increased risk of several cancers in HIV-1infected individuals (Clifford et al. 2005; Engels et al. 2008; Guiguet et al. 2009; Mbulaiteye et al. 2003). HIV-1 infection is most prominently associated with three malignancies: Kaposi’s sarcoma (KS); non-Hodgkin’s lymphoma (NHL); and invasive cervical carcinoma (ICC). Each is attributable to coinfection with a slowly transforming human oncogenic virus. The immune depletion and cytokine signaling in HIV-1 infection promotes the progression to neoplasia. KS is associated with human herpesvirus 8 (HHV-8) (Moore and Chang 1995); some NHLs are associated with Epstein–Barr virus (EBV) (Thorley-Lawson et al. 2004); and ICC is associated with HPV (Lorincz et al. 1987). For an HIV-1-infected individual, presentation

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Standard incidence ratio 0

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Fig. 24.1 HIV-1 infection is associated with an increase in the incidence of multiple types of cancer. The incidence of selected malignancy is increased in HIV-1-positive patients relative to the general population. AIDS-defining malignancies (black bar) are prognostic of AIDS and related to coinfection with transforming oncogenic virus. Non-AIDS-defining malignancies (gray bar) are associated with environmental factors that include viral coinfection and behavioral risks. Standardized incidence ratio (SIR) is the cancer incidence in an HIV-1-positive cohort living in the USA in the period from 1991 to 2002 relative to general population. These SIRs were obtained in the 5 years after HIV-1 registration by Engels et al. (2008). HAART was introduced in 1996. HAART status was not tracked herein although two-thirds of the cohort was recruited in the HAART era

of KS, NHL, or ICC is a prognostic indicator of progression to acquired immunodeficiency syndrome (AIDS). Accordingly, KS, NHL, and ICC are designated AIDS-defining malignancies. A cohort study of patients in the first 5 years after HIV-1 registration revealed that their risk of KS is 1,000-fold greater compared to the general population, which is largely free of KS [Fig. 24.1, standardized incidence ratio (SIR) 1,300] (Engels et al. 2008). The risk of NHL is also increased (SIR 7) (Engels et al. 2008). The two most common forms of NHL in HIV-1-infected individuals are Burkitt’s lymphoma (BL) (SIR 15) and diffuse large B-cell lymphoma (DLBL) (SIR 10) (Engels et al. 2008). Primary central nervous system lymphoma (PCNSL) is unusual in the general population and exhibits an SIR of 250 among individuals with HIV-1 (MacMahon et al. 1991). The increased incidence of ICC represents progression of cervical dysplasia attributable to persistence of HPV infection (SIR 3, Fig. 24.1). Additionally, some non-AIDS-defining cancers exhibit increased incidence ratios in persons with HIV-1; the SIRs of five neoplasms are provided in Fig. 24.1.

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Time Fig. 24.2 The risk of AIDS-defining malignancy increases with continuous decline in CD4+ T-cell count below 500 cells/mL. In the course of infection with HIV-1, CD4+ T-cell counts decrease over a period of years to the level diagnostic of acquired immunodeficiency (AIDS, <200 cells/mL). The risk of the AIDS-defining malignancies begins to increase as CD4+ T-cell count declines below 500 cells/mL (Mbulaiteye et al. 2003; Guiguet et al. 2009). Dashed lines indicate the typical CD4+ T-cell count at diagnosis of indicated malignancy. Diagnosis of Burkitt’s lymphoma and invasive cervical carcinoma is often before the onset of AIDS (Mbulaiteye 2003), whereas diffuse large B-cell lymphoma (Lim et al. 2005) and Kaposi’s sarcoma (Gallafent et al. 2005) are commonly diagnosed at CD4+ counts £100 cells/mL and primary central nervous system lymphoma is observed in profoundly immunocompromised individuals (Hoffmann et al. 2001)

These malignancies are associated with viral and behavioral risk factors. Oral/ pharyngeal and anal cancers are associated with HPV (Melbye and Frisch 1998; Gillison et al. 2000); hepatocellular carcinoma (HCC) is associated with hepatitis C virus (HCV) (Saito et al. 1990); and Hodgkin’s lymphoma (HL) is associated with EBV infection (Pallesen et al. 1991) that is predominant worldwide. Oral, liver, and lung cancers are associated with tobacco and alcohol use. The increase in SIR for these cancers may be due to a direct contribution of HIV-1 infection to cancer progression or due to an increase in risk behaviors in the HIV-1-infected population.

HIV-1-Associated Immunodepletion Heightens the Risk for Malignancy A CD4+ T-cell count below 500 cells/mL and sequential declines significantly increase the risk for the development of AIDS-defining malignancies (Fig. 24.2) (Mbulaiteye et al. 2003; Jellinger and Paulus 1995; Cingolani et al. 2000).

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Malignancies with the greatest increase in SIR are KS and three NHL subtypes (Fig. 24.1) (Mbulaiteye et al. 2003). In an American cohort, CD4+ T-cell count did not significantly increase the risk of ICC or the non-AIDS-defining malignancies (Mbulaiteye et al. 2003). In a French cohort, presentation of CD4+ T-cell count below 500 cells/mL was correlated with increased risk of KS and NHL and also HL, lung cancer, liver cancer, and anal cancer (Guiguet et al. 2009).

Treatment of HIV-1 Infection As a Means to Mitigate Concurrent Malignancy HAART Has Changed the Spectrum of HIV-1-Associated Malignancy The premise of current antiretroviral therapy is to inhibit two or more viral enzymes necessary for the hallmark steps in the retrovirus replicative cycle (Broder 2009). The first reverse transcriptase inhibitor was developed in the 1960s, though its potential was not recognized at that time. The first inhibitor of HIV-1 protease became available in 1996; quickly, innovative combination regimens were tested. Designated “highly active antiretroviral therapy” (HAART), a typical combination is a protease inhibitor and two nucleoside reverse transcriptase inhibitors. The principle behind HAART is that the simultaneous application of three drugs decreases the ease of evolving resistance. A quasispecies resistant to one of the three drugs is likely to be vulnerable to the other two, and does not experience positive selection. However, comparative analysis of the dose–response curve slope for class-specific anti-HIV drugs has produced a new explanation for the success of HAART. Siliciano and colleagues determined that the log reduction in single-round infectivity at clinical drug concentrations is strongly influenced by slope and varies by >8 logs for anti-HIV drugs (Shen et al. 2008). Observation of a slope >1 was characterized for nonnucleoside reverse transcriptase inhibitors, protease inhibitors, and fusion inhibitors. The results suggest that the efficacy of HAART regiments is attributable to the superior therapeutic threshold of class-specific anti-HIV-1 drugs. Since 1996, HAART has been the standard of treatment for HIV-1 in developed nations, and has dramatically stabilized the immune status of HIV-infected persons. Large cohort clinical studies to measure the association of AIDS onset, CD4 count, HAART treatment, and cancer presentation have documented corresponding reductions in the incidence rates of KS and NHL (Engels et al. 2008; Clifford et al. 2005). However, cohort comparison from 1991 to 1995 or 1996 to 2002 revealed that the incidence of some non-AIDS-defining cancers has increased. Incidence of nonAIDS-defining cancers, including HCC, oral/pharyngeal cancer, lung cancer, anal cancer, Hodgkin’s lymphoma, and nonmelanoma skin tumors, increased from 31 to 58% (Engels et al. 2008). Possible explanations are prolonged survival with exposure to multiple risk factors (including viral coinfections and behavioral factors), sustained marginal immunosuppression, or both (Engels et al. 2008).

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Combination of HAART with Chemotherapy Stabilizes Patient Prognosis Temporary depletion of T lymphocytes is a generic outcome of chemotherapy (Mackall 2000). Typically, CD4+ lymphocyte levels are more affected by chemotherapy than are CD8+ lymphocyte levels (Mackall et al. 1997) and their recovery to pretreatment levels can be prolonged to beyond a year after treatment cessation (Hakim et al. 1997). Especially implicated is cyclophosphamide therapy, which is part of the standard treatment regimen for NHL (Gershwin et al. 1974). Chemotherapy-induced depletion of T lymphocytes is of great concern for HIV-1infected patients. However, immune stabilization by HAART has profoundly improved the prognosis of standard chemotherapy regimens for HIV-1-infected patients with cancer. A study of 18 patients with NHL (16 DLBL, 1 BL, 1 peripheral T-cell lymphoma) and 2 with HL examined viral load during HAART plus bleomycin, etoposide, methotrexate, vincristine, prednisolone/cyclophosphamide and doxorubicin (BEMOP/CA) or infusional cyclophosphamide, doxorubicin, and etoposide (CDE) (Powles et al. 2002). HAART during chemotherapy suppressed an increase in the viral load and was associated with rapid recovery of CD4+ lymphocytes to baseline in the month after cessation of chemotherapy (Powles et al. 2002). A subsequent retrospective study compared DLBL and BL survival with HAART chemotherapy (Lim et al. 2005). Before HAART, patients with NHL had a median survival time of approximately 6–8 months (Lim et al. 2005). After HAART, median survival time for DLBL patients increased to 43 months, although there was no change in survival for BL patients (Lim et al. 2005). In the case of PCNSL, HAART addition to chemotherapy dramatically increased survival. Patients without HAART exhibited a median survival of 52 days while patients on HAART had not reached median survival at 667 days (Skiest and Crosby 2003). While the risk of NHL has decreased by approximately a factor of 5 with modern HAART (Carrieri et al. 2003), the potential for developing NHL poses a greater risk of mortality than the other AIDS-defining malignancies. In a study conducted in 2000 of HIV-1-infected patients, NHL was responsible for 29% of deaths due to malignancy while KS was responsible for 15% (Bonnet et al. 2004). Primary effusion lymphoma and PCNSL remain consistently fatal diagnoses despite HAART (Bonnet et al. 2004).

Drug Interactions Demand Management of HAART and Chemotherapy Regiments While HAART has facilitated more effective chemotherapy, drug interactions pose a new challenge to clinical treatment of cancer in AIDS patients. In HAART formulations, protease inhibitors and nonnucleoside reverse transcriptase inhibitors modulate the efficiency of the cytochrome P450 (CYP) pathway. The pathway component

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CYP34A has broad substrate specificity and is active in the metabolism of multiple drugs. However, HIV-1 protease inhibitors typically inhibit CYP34A, which can lead to reduced metabolism for the chemotherapeutic. The protease inhibitor ritonavir has little anti-HIV-1 activity but strongly inhibits CYP34A. Thus, ritonavir is a useful “boosting agent” in antiretroviral therapy to enable administration of lower dosages of drugs metabolized by this enzyme (Merry et al. 1997). However, inhibition of CYP34A by ritonavir can alter metabolism of chemotherapy drugs, which can result in toxicity when prescribed with anticancer vinca alkaloids (Cingolani et al. 2010) and docetaxel (Mir et al. 2010). Conversely, the nonnucleoside reverse transcription inhibitors nevirapine and efavirenz increase CYP34A activity. Therefore, chemotherapy drugs metabolized by this enzyme may not reach therapeutic levels if these drugs are prescribed simultaneously (Mounier et al. 2009). Generally, adverse drug interactions can be avoided by adjustment of the HAART regimen and dosage (Mounier et al. 2009).

Long-Term Control of HIV-1 Is Achievable by Selective Bone Marrow Reconstitution HIV-1 enters a susceptible cell by interaction with the CD4 receptor and one of the two coreceptor molecules, CCR5 or CXCR4 (Choe et al. 1996; Feng et al. 1996). Homozygous mutation of the CCR5 coreceptor is sufficient to confer resistance to infection by HIV-1 (Marmor et al. 2001). Recently, bone marrow transplantation was performed from a donor homozygous for a 32 base pair deletion in the CCR5 gene, which encodes an inactive receptor (Hutter et al. 2009). The transplant recipient was an acute myeloid leukemia patient with HIV-1 infection. The treatment has produced remission of the leukemia and the HIV-1 viral load has been below detectable limits for over 20 months without further antiviral therapy (Hutter et al. 2009). Although a minority of the viral quasispecies of this patient had been observed to be CXCR4 tropic, virus load has not rebounded and the patient may experience a durable cure (Allers et al. 2011). In principle, this clinical outcome validates the significant therapeutic utility of downregulation of coreceptors. It is conceivable that reduced viral replication and viral load are necessary to permit a robust antiviral response capable of preventing resurgence of infection. Designated as drug-induced vaccine effect, studies led by Ruprecht and by Mathes have documented this in primate and feline models (Ruprecht et al. 1990; Mathes et al. 1992). Alternatively, the rigorous ablation therapy may have completely eliminated any virus reservoir. Current studies are addressing the source of long-lived reservoirs of HIV-1 persistence that are sufficient for eventual recovery of detectable HIV-1 viral load. While transplantation therapy is unlikely to become useful for treatment of concurrent HIV-1 infection and leukemia due to the rarity of appropriate donors, there is interest in developing a similar therapy involving transduced lymphocytes expressing anti-CCR5 shRNA (Qin et al. 2003). Autologous stem cell transplantation with these transduced lymphocytes could potentially simultaneously treat HIV-1 and NHL (Gabarre et al. 2000).

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Viral Coinfection Contributes to Malignancy in HIV-1 Infection Presentation of Kaposi’s Sarcoma Led to Discovery of AIDS HHV-8 is the Kaposi’s sarcoma-associated herpesvirus (KSHV). In most cases, HHV-8 causes no overt disease. However, HHV-8 infection can be associated with KS, NHL subtypes, and Castleman’s disease; the particular outcome is related to cell type infected and expression pattern of virus-encoded interleukin 6 (vIL-6) (Staskus et al. 1999). Coinfection with HIV-1 and HHV-8 produces a 50% increase in the 10-year risk of KS (Martin et al. 1998) and an SIR of 1,300 compared to HIV-1uninfected people in the USA (Engels et al. 2008). KS was originally described in elderly Mediterranean men as a disease with an indolent course (Kaposi 1872). KS is observed in immunosuppressed organ-transplant recipients (Penn 1979) and in Africa (Lothe 1960), where HHV-8 infection is widespread by mother-to-child transmission (Bourboulia et al. 1998). However, in the USA, HHV-8 is primarily sexually transmitted (Kedes et al. 1996). HHV-8 is not common in other areas of the world (Ablashi et al. 1999). Clinical presentation of KS was instrumental in the discovery of AIDS, and the eventual discovery of HIV-1 as its causative agent. In 1981, eight cases of KS in homosexual men were ascribed to a sexually transmitted causative agent (Hymes et al. 1981). In 1982, another unusual appearance of aggressive KS was attributed to “an acquired immunoregulatory defect, and one or more infectious agents” (Friedman-Kien et al. 1982). While the discovery of HIV-1 followed in 1983 (Gallo et al. 1983; Barre-Sinoussi et al. 1983), HHV-8 eluded researchers until discovery by Moore, Chang, and coworkers in 1994 (Chang et al. 1994). Treatment of HIV-1 infection with HAART has decreased the risk of developing KS by about a factor of 5 (Carrieri et al. 2003; Jones et al. 2000).

Synergistic Viral–Host Interactions of HIV-1 and HHV-8 Exacerbate the Severity of Kaposi’s Sarcoma KS is a complex proliferative mucocutaneous lesion, characterized by hypervascularization, which often first appears on the skin but may affect other mucous membranes, including the lungs and gastrointestinal tract. KS exhibits the histology of a benign hyperproliferative lesion, although it is frequently designated a sarcoma (Brooks 1986). Sarcomas are clonal expansions of transformed cells while KS typically is polyclonal and multifocal (Gill et al. 1998; Delabesse et al. 1997). However, later in the disease, lesions may become monoclonal (Rabkin et al. 1995, 1997). KS lesions consist of spindle cells, which are sparse in the initial lesions, endothelial cells, and infiltrating leukocytes (Flore et al. 1998). As summarized in Fig. 24.3, HHV-8 infection of cells provides paracrine stimulation that prolongs the survival of uninfected bystander cells (Flore et al. 1998). While HHV-8 can infect dendritic cells, monocytes, and endothelial cells, the spindle morphology develops only in HHV-8-infected endothelial cells (Grossmann et al. 2006).

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HIV-1 infected lymphocytes secrete Tat

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IFN IL-1 TNF

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Fig. 24.3 Multiple points of virus–host interface exacerbate Kaposi’s sarcoma in patients coinfected with HHV-8 and HIV-1. Kaposi’s sarcoma lesions are a mixture of HHV-8-infected spindle cells, endothelial cells, and infiltrating leukocytes. In coinfection, HHV-8 and HIV-1 exhibit synergistic virus–host interactions that result in cellular proliferation and angiogenesis. In HHV-8-infected spindle cells, expression of interleukin-6 (IL-6) and viral IL-6 (vIL-6) induces expression of vascular endothelial growth factor (VEGF), which induces proliferation of spindle cells and angiogenesis of endothelial cells. This upregulation of VEGF is amplified by IL-6 secreted from circulating HIV-1-infected leukocytes (indicated by linear provirus). Secreted HIV-1 Tat induces leukocytes to infiltrate endothelium and to release interferon-g (IFN-g), interleukin-1 (IL-1), and tumor necrosis factor-a (TNF-a). These cytokines induce basic fibroblast growth factor (bFGF) expression in spindle cells, and Tat enhances release of bFGF from matrix-associated heparin sulfate by competing for binding sites. Redundant paracrine signaling by VEGF and bFGF contributes to proliferation of both spindle cells and endothelial cells. HHV-8 viral G-protein-coupled receptor (vGPCR) cooperates with HIV-1 Tat in posttranscriptional activation of nuclear factor of activated T cells 1 (NFAT-1) and NFAT-2. These transcription factors control the expression of cytokines and growth factors contributing to spindle cell proliferation. In sum, viral coinfection reinforces local hyperproliferation and hypervascularization, exacerbating Kaposi’s sarcoma

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Cytokines, especially IL-6, have a key role in tumorigenesis in KS. HHV-8 alters the cytokine environment through expression of a virus-encoded cytokine homolog and manipulates expression of endogenous cytokines. Cells infected by HHV-8 express viral homolog vIL-6 (Nicholas et al. 1997) and vFLIP, which induces IL-6 expression (Grossmann et al. 2006). Additionally, HHV-8 encodes microRNA that upregulates IL-6 and IL-10 (Nicholas et al. 1997). The virally encoded miR-K12-3 and miR-K12-7 target the mRNA of a negative regulator of IL-6 and IL-10, the liver-enriched inhibitory protein isoform of CCAAT/enhancer-binding protein (Qin et al. 2010). Expression of IL-6 and vIL-6 results in proliferation of HHV-8-infected cells (Miles et al. 1990) and angiogenesis (Cohen et al. 1996), attributable to the ability of IL-6 to upregulate vascular endothelial growth factor (VEGF). HHV-8 also encodes homologs of macrophage inhibitory protein (MIP): vMIP-I (Nicholas et al. 1997), vMIP-II (Weber et al. 2001), and vMIP-III (Stine et al. 2000). These b-chemokine family members aid HHV-8 evasion of cytotoxic T lymphocytes (CTLs) by shifting the T-cell response from a Th1 type toward a Th2 type. Increased severity of KS in HIV-1 coinfection is attributable to paracrine stimulation of proliferation of KS lesion spindle cells and uninfected endothelial cells by secreted Tat and cytokines (Fig. 24.3). HIV-1 infection of peripheral blood mononuclear cells (PBMCs) leads to expression of the Tat regulatory protein. Within HIV1-infected cells, Tat provides essential transcriptional transactivation of viral gene expression (Charnay et al. 2009; Coffin et al. 1997) and suppresses microRNAmediated translational repression of HIV-1 (Bennasser et al. 2005; Qian et al. 2009). Tat secreted from HIV-1-infected cells manipulates cytokine expression by leukocytes (Ott et al. 1998; Izmailova et al. 2003). The activity of Tat on the miRNA signatures in leukocytes and in circulating plasma microvesicles (Hunter et al. 2008) and the definition of the biological processes modulated by these miRNA are areas ripe for investigation (Houzet et al. 2008). The activity of Tat reinforces at multiple points the pathophysiological interface of KSHV with the host cell (Fig. 24.3). Secreted Tat induces chemotactic migration and invasion of the endothelium by leukocytes (Lafrenie et al. 1996) and fosters growth within the tumor microenvironment. HHV-8-infected spindle cells produce basic fibroblast growth factor (bFGF), and cytokines released by HIV-1-infected cells upregulate bFGF further, which bolsters cell proliferation and angiogenesis (Ensoli et al. 1994). HIV-1 Tat (Rautonen et al. 1994; Zauli et al. 1993) and Nef (Olivetta et al. 2003) cause increase in serum IL-6 levels (Honda et al. 1990), which promotes HIV-1 replication (Poli et al. 1990). HHV-8 also upregulates IL-6 in infected cells and expresses vIL-6, and the further augmentation in IL-6 caused by HIV-1 coinfection may contribute to greater severity of KS. Tat itself induces invasion and proliferation of spindle cells and endothelial cells through multiple paracrine mechanisms (Fig. 24.3). Tat activates cellular integrins, stimulates VEGF-A receptor fetal liver kinase-1 (Flk-1), activates NFAT-1 and -2, boosts levels of soluble bFGF, stimulates expression of matrix metalloproteases (MMPs), and induces lytic replication of HHV-8. These interactions contribute to angiogenesis and spindle cell proliferation. Tat interacts with cellular integrins through an arginine, glycine, aspartic acid (RGD) peptide, promoting adherence of

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HHV-8-infected cells and uninfected endothelial cells (Barillari et al. 1993). Tat signaling through the VEGF-A receptor Flk-1 enhances spindle cell proliferation (Ganju et al. 1998) and induces angiogenesis in endothelial cells (Albini et al. 1996). Furthermore, Tat provides paracrine stimulation of the phosphatidylinositol 3-kinase–RAC-a serine/threonine-protein kinase (PI3K/Akt) pathway that activates the phosphorylation and nuclear translocation of NFAT-1 and NFAT-2 in HHV-8infected cells, contributing to upregulation of cytokines and growth factors (Pati et al. 2003). The activation of NFAT-1 and NFAT-2 by Tat is augmented by the activity of the viral G-protein-coupled receptor (vGPCR) expressed by HHV-8-infected spindle cells (Fig. 24.3) (Guo et al. 2004). This receptor is orthologous to the interleukin 8 receptor CXCR2 and signals in the absence of bound ligand. The outcome of this signaling event is the posttranscriptional activation of transcription factors NF-kB, NFAT-1, and NFAT-2 in the infected cell (Pati et al. 2003) and activation of hypoxiainducible factor-1a (HIF-1a) resulting in transcriptional activation of VEGF (Sodhi et al. 2000). The consequences of the redundant upregulation of cytokines and growth factors, such as VEGF, by HIV-1 and HHV-8 are proliferation and neovascularization of the KS lesion. Tat provides another redundant activity with HHV-8 by increasing soluble bFGF in the cellular matrix (Barillari et al. 1999) (Fig. 24.3). Tat competitively binds the heparin sulfate proteoglycans of the cellular matrix, which increases the concentration of soluble bFGF to induce cell proliferation (Fig. 24.3). Furthermore, bFGF in spindle and endothelial cells induces the expression of MMPs, and secreted Tat further stimulates production of MMPs (Toschi et al. 2001; Meade-Tollin et al. 1999). Tat activates membrane-type-1 MMP, which binds and activates soluble MMP-2, and inhibits secretion of the membrane-bound tissue inhibitor of metalloproteinase-1 and -2 (Toschi et al. 2001). These activities provide redundant signals for proliferation of HHV-8-infected cells and angiogenesis in endothelial cells (Barillari et al. 1999). The paracrine activity of Tat on endothelial cells (Ensoli et al. 1990) induces lytic replication of HHV-8 (Zeng et al. 2007), attributed to induction of oncostatin M, hepatocyte growth factor/scatter factor, and interferon-g (Mercader et al. 2000). In sum, HIV-1 and HHV-8 coinfection results in multiple synergistic virus–host interactions that foster the tumor microenvironment. KS is the malignancy most responsive to HAART (Carrieri et al. 2003). However, in AIDS patients of the developed nations, 15% of cancer fatalities remain attributable to KS (Bonnet et al. 2004).

HHV-8 and HIV-1 Coinfection Is Associated with Presentation of Rare Lymphoma NHL in HIV-1 infection is most often associated with coinfection with EBV. However, a rare DLBL is observed in patients coinfected with HIV-1 and HHV-8: primary effusion lymphoma (PEL) (Cesarman et al. 1995; Carbone et al. 1996). In some

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patients, EBV infection may contribute to PEL (Horenstein et al. 1997). Among patients not infected with HIV-1, PEL makes up only 0.3% of NHL, but among patients with HIV-1 it makes up 4% of NHL (Gaidano and Carbone 2001). Prognosis of PEL is poor even in the absence of HIV-1 infection, with a median survival of 6 months (Chen et al. 2007). PEL is refractory to both HAART and chemotherapy. Similar to the mechanism of KS, IL-6 and other cytokines coordinately stimulate proliferation of tumor cells and the replication of HIV-1 in PBMC, which promotes immunodepletion (Honda et al. 1990; Rautonen et al. 1994; Zauli et al. 1993).

HHV-8 and HIV-1 Coinfection Is Associated with Multicentric Castleman’s Disease Multicentric Castleman’s disease (MCD) is not a cancer, but a lymphoproliferative disorder resulting in the accumulation of cytokine-secreting B cells. Castleman’s disease in the absence of HIV-1 infection is attributed to dysregulation of IL-6 and presents in unicentric or multicentric forms. In the context of HIV-1 infection, MCD is associated with HHV-8 infection (Soulier et al. 1995) and is augmented by IL-6 upregulation similarly to KS. This coinfection enhances MCD morbidity and mortality. MCD is frequently a precursor to HHV-8-positive NHL (Oksenhendler et al. 2002; Dupin et al. 2000). Rituximab has become a standard therapy for MCD and depletes proliferating B cells (Clifford and Demierre 2005). However, rituximab may induce proliferation of KS lesions by reactivation of HHV-8 (Pantanowitz et al. 2008), an important consideration since 75% of patients with HIV-1-associated MCD develop concurrent KS (Oksenhendler et al. 1996). Unlike PEL, MCD may respond to treatment with HAART alone (Lee et al. 2010). In the pre-HAART era, mortality was 70–85% (Oksenhendler et al. 1996). In the post-HAART era, MCD mortality is reduced to about 30% (Mylona et al. 2008). However, for unknown reasons, the incidence of MCD has risen during the HAART era, from 2.3 (95% CI 0.02–4.2) per 10,000 patients years to 8.3 (95% CI 4.6–12.6) per 10,000 patient years (Powles et al. 2009).

HIV-1 and EBV Coinfection Is Prominent in B-Cell Lymphoma NHL was classified as an AIDS-defining malignancy in 1987 by the Centers for Disease Control and Prevention (Centers for Disease Control and Prevention 1987). NHL is not a discrete disease, but a heterogenous group of over 40 different cancers, as classified by the World Health Organization International Agency for Research on Cancer in 2008 (The International Agency for Research on Cancer 2008). The NHLs occurring in HIV-1 patients overlap with the classifications of the general population, but are more likely to be high-grade B-cell lymphomas. In persons not

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infected by HIV-1, most NHLs are EBV negative while most NHLs occurring with HIV-1 infection are EBV positive. The two most common forms of NHL among people with HIV-1 are BL and DLBL, both associated with EBV (Engels et al. 2008). Individuals with HIV-1 also are more likely to develop unusual lymphomas, such as PCNSL, which is almost universally associated with EBV (MacMahon et al. 1991). During the clinical progression of HIV-1, lower CD4+ T-cell counts are associated with EBV-associated NHL (Fig. 24.2) (Jellinger and Paulus 1995; Cingolani et al. 2000). The majority of adults worldwide are infected with EBV, which is a ubiquitous B-lymphotropic herpesvirus (human herpesvirus-4) that establishes lifelong infection (Henle and Henle 1979). Normally, EBV infects naïve B cells, induces differentiation to memory B cells, then becomes latent, and escapes immune detection. Upon differentiation of a memory cell into a plasma cell, EBV replication resumes. EBV is postulated to contribute to lymphoma when non-naïve cells are infected, and instead of escaping the cell cycle by differentiation into memory B cells continue to replicate (Thorley-Lawson and Gross 2004). In vitro infection of B cells by EBV leads to the induction of somatic hypermutation and accumulation of mutations in proto-oncogenes p53 and B-cell lymphoma 6 (Epeldegui et al. 2007). Genomic instability can result from the expression of two nuclear antigens (EBNA-1 and EBNA-3C) and latent membrane protein-1 (LMP-1) (Gruhne et al. 2009). EBNA-1 leads to the production of DNA-damaging reactive oxygen species by upregulating NADPH oxidase 2 (Nox2) while LMP-1 abrogates the G2 checkpoint (Gruhne et al. 2009). EBNA-3C inactivates the mitotic spindle checkpoint, potentially leading to aneuploidy (Gruhne et al. 2009). Thus, infection of non-naïve B cells followed by unrestricted replication and accrual of activated oncogenes and aneuploidy contribute to the development of NHLs. The major role of HIV-1 in the development of all forms of NHL appears to be immune dysfunction.

IL-10 Has a Prominent Role in HIV-1-Related NHL A second role for HIV-1 in the development of NHL is the dysregulation of IL-10, in particular in subtypes of B-cell NHL, including BL (Ogden et al. 2005), largecell lymphoma (Marsh et al. 1995), mantle cell lymphoma (Visser et al. 2000), and chronic lymphocytic leukemia (Fayad et al. 2001). In AIDS-related B-cell lymphoma, serum IL-10 has been found to be elevated prior to the development of lymphoma, and is associated with a particular polymorphism in the IL-10 promoter (Breen et al. 2003). The risk for B-cell NHL was shown to be dependent upon polymorphisms in the IL-10 gene while other cytokines were not implicated (Wong et al. 2010). HIV-1 infection results in increased IL-10 serum levels (Ameglio et al. 1994) while treatment with HAART decreases serum IL-10 levels (Stylianou et al. 1999), suggesting a potential role for HIV-1 dysregulation of IL-10 in NHL.

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HIV-1 Contributes to NHL by Impairing Anti-EBV Cytotoxic T Lymphocytes A longitudinal study of a small sample of 13 individuals with HIV-1 and EBV coinfection showed a decrease in the population of HIV-1-specific CTL over the study period while levels of EBV-specific CTL typically remained steady (Kersten et al. 1997). However, in four out of five patients that developed diffuse large-cell lymphoma, EBV-specific CTL dropped in conjunction with a rise in EBV viral load (Kersten et al. 1997). A more recent study found similar trends, but additionally that in patients who developed NHL CTL gradually lost the ability to produce IFN-g in response to EBV even as CTL levels remained unchanged, and this loss paralleled a trend of decreasing CD4+ T-cell count (van Baarle et al. 2001). Others have found that EBV-specific CTL in people with HIV-1 infection and CD4+ counts below 350/mL have 1.5-fold decreased polyclonality and recognize fewer EBV epitopes (Legoff et al. 2004). As part of the ongoing immune dysregulation caused by HIV-1 infection, EBV CTL response may be impaired, leading to loss of immune control of EBV and eventual NHL.

HIV-1 and HPV Coinfection Contributes to Cervical, Anogenital, and Oropharyngeal Malignancies The primary cause of ICC is infection with HPV strains HPV-16 or HPV-18 (Lorincz et al. 1987). HPV infects undifferentiated keratinocytes, and viral replication begins upon differentiation and ceases when cells exit the cell cycle. The early 6 (E6) and E7 viral oncogenes of HPV induce deregulation of cell growth. E6 promotes ubiquitin-mediated degradation of p53 (Scheffner et al. 1990) while E7 binds retinoblastoma proteins and promotes their ubiquitin-mediated degradation (Dyson et al. 1989), releasing E2F/DP-1 transcription factors. While the HPV genome is initially present in nuclei as an episome, integration into the host genome can occur. Integration typically disrupts the reading frame of E2, which represses transcription of E6 and E7. Disruption of E2 is associated with a worse prognosis (Kalantari et al. 1998), usually attributed to loss of control of E6 and E7 expression; however, cervical intraepithelial neoplasia can develop in the presence of an intact E2 (Sathish et al. 2004). Another contributing factor is posttranscriptional upregulation of E6 and E7 by the deletion of an AU-rich element in the 3¢ untranslated region (Jeon and Lambert 1995). An increase in E6 and E7 mRNA stability engenders a cell growth advantage (Jeon et al. 1995) and permits increased progression to neoplasia (Kulmala et al. 2006; Hudelist et al. 2004). HIV-1 infection increases a woman’s risk of development of squamous intraepithelial lesions by fivefold compared to sociodemographically matched HIV-1negative women (Ellerbrock et al. 2000). HIV-1-infected women exhibit more aggressive and treatment-refractory cervical cancer than women free of HIV-1

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(Maiman et al. 1993). The increased severity of cervical carcinoma in HIV-1 infection is attributed to impaired immune function. High HIV-1 viral load and low CD4+ T-cell counts are associated with higher frequency and greater severity of HPV lesions (Davis et al. 2001; Cardillo et al. 2001; Harris et al. 2005). Unlike KS and NHL, the frequency of occurrence of ICC has decreased only modestly from treatment with HAART (International Collaboration on HIV and Cancer 2000). Without HAART treatment, premalignant lesions fail to regress and are associated with greater HIV-1 viral load and lower CD4+ T-cell count (Ahdieh-Grant et al. 2004) while HAART treatment contributes to regression of premalignant lesions (AhdiehGrant et al. 2004; Heard et al. 2002). However, HPV infection may persist with HAART even as lesions regress (Heard et al. 1998; Minkoff et al. 2001). Persistence of HPV infection is associated with the development of cervical cancer (Rodriguez et al. 2008). The impact of HIV-1 on the duration of productive infection by HPV is controversial. Some studies have found delayed clearance of HPV infection (Sun et al. 1997; Moscicki et al. 2004; Ahdieh et al. 2001) while others found little difference in times to clearance of HPV infection (Koshiol et al. 2006). Besides its role in impairing immune function, HIV-1 coinfection may contribute directly to HPV replication through stimulation by Tat. HIV-1-infected cells can be detected within cervical carcinoma lesions (Vernon et al. 1994) and Tat can induce expression of E6 and E7 in tissue cultures of HPV-16-infected oral keratinocytes, resulting in cellular proliferation (Kim et al. 2008). In HPV-18-infected HeLa T4 cells, exposure of cells to extracellular Tat induced transcription of E1 and late 1 (L1) and a rise in L1 capsid protein levels (Dolei et al. 1999). Therefore, HIV-1 coinfection may promote reactivation of HPV. The possible modulation of miRNA signatures by HIV-1 gene products and their role in the reactivation process remains to be determined. Nuovo and colleagues have identified a strong inverse correlation between microRNA-125b and productive HPV infection (Nuovo et al. 2010). MicroRNA-125b is abundantly expressed at the site of infection, the transformation zone epithelia. Marked reduction in miR-125b was observed in productively infected cells and correlated with expression of the HPV late gene product, L2. HIV-1 infection induces marked downregulation of microRNA-125b in lymphocytes (Hayes et al. 2011) and may promote productive HPV infection by a paracrine signaling mechanism. The role of HPV infection in other cancers is being increasingly appreciated. Of greatest note for HIV-1 infection is HPV-associated anal squamous cell carcinoma (ASCC). HIV-1 infection is associated with a greater risk of ASCC (Fig. 24.2) (Engels et al. 2008). Similarly to ICC, the impact of HAART on HPV-associated ASCC is controversial (Bower et al. 2004; Hawes et al. 2002; Wilkin et al. 2004). Oropharyngeal cancer is associated with HPV infection (Gillison et al. 2000) and the incidence of oropharyngeal cancer is increased in HIV-1 infection (Fig. 24.2) (Engels et al. 2008). Environmental factors, such as a higher incidence of smoking in the HIV-1-positive sample, may contribute to this increase, but severity of disease has also been found to increase with increasing immunosuppression (Gillison 2009). Similarly to CIN and ASCC, treatment with HAART did not affect the incidence of oropharyngeal cancer (Gillison 2009).

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HIV-1 and HCV Coinfection Leads to More Rapid Progression of Liver Fibrosis Persistent HCV infection can result in liver fibrosis and the eventual development of HCC (Saito et al. 1990). Coinfection by HCV and HIV-1 is a growing concern, since they share similar transmission routes. HCV is easily spread through sharing needles for injection drug use, and also may be spread through sexual practices, especially among those who are already infected with HIV-1 (Tohme and Holmberg 2010). HIV-1 has a strong influence upon the course of HCV infection, leading to greater HCV viremia (Beld et al. 1998; Eyster et al. 1994) and more rapid fibrosis (Benhamou et al. 1999). Another study found fibrosis rate increased with increasing HIV-1 viral load or with CD4+ T-cell counts of <500 cells/mL (Brau et al. 2006). HAART has been shown to counteract this more rapid progression of fibrosis, producing similar fibrosis progression to patients not infected by HIV-1 (Brau et al. 2006). Consistent with this finding of more rapid fibrosis, patients with HIV-1 and HCV coinfection develop cirrhosis more rapidly than those without HIV-1, with 15% of patients with coinfection developing cirrhosis by 10 years after HCV infection compared to 2.6% in those with only HCV infection (Soto et al. 1997). Furthermore, patients coinfected with HCV and HIV-1 develop HCC earlier than those uninfected by HIV-1, with patients with coinfection developing HCC at 18 ± 3 years compared to 28 ± 11 years for patients with only HCV (p < 0.05) (Garcia-Samaniego et al. 2001). HCVassociated end-stage liver disease is an increasing cause of mortality in people with HIV-1 and is a leading non-AIDS cause of death (Salmon-Ceron et al. 2005; Rosenthal et al. 2003; Macias et al. 2002; Weber 2006). While HIV-1 coinfection appears to increase the severity of HCV infection, HCV infection may also impact the clinical progression of HIV-1 infection. Patients infected with HCV and HIV-1 have a greater risk of progression to AIDS or death, with a hazard ratio of 1.7 (Greub et al. 2000). The authors also found that in patients coinfected with HCV and HIV-1 there was a 21% lower likelihood of an increase of at least 50 cells/ mL in CD4+ T-cell counts after 1 year of HAART (Greub et al. 2000). A complicating factor is the high frequency of intravenous drug use among people with HIV-1 and HCV coinfection, which also may contribute to adverse effects (Greub et al. 2000). The advent of HAART has caused a decline in the rate of death in those infected with HIV-1 by a factor of 10 (Palella et al. 1998; Mocroft et al. 2002). The increase in life span of those infected with HIV-1 permits chronic conditions to exert their effect. The outcome in the case of HCV infection is an increase in the rates of morbidity and mortality due to liver disease. This unintended consequence presents a new clinical challenge in the management of HIV-1 infection.

Cancer Risk Is Not Elevated in HIV-1 and HTLV-1 Coinfection Coinfection with HIV-1 and HTLV-1 or HTLV-2 commonly occurs with intravenous drug use (Casoli et al. 2007). Since adult T-cell leukemia due to HTLV-1 infection

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develops after decades, prolonged survival of HIV-1-infected patients due to HAART may reveal increased cancer risk in coinfected individuals, whereas prior to HAART, coinfected individuals would have succumbed to AIDS. Interestingly, while patients with HIV-1 and HTLV-1 coinfection have higher CD4+ T-cell counts, this does not appear to be associated with decreased immunosuppression (Schechter et al. 1997). However, HTLV-1 coinfection does not result in increased HIV-1 viral load (Harrison et al. 1997). Moreover, HIV-1 and HTLV-1 coinfection does appear to be associated with greater HTLV-1-related morbidity, with increased risk of myelopathy (Harrison and Schechter 1998). The alternative tropism of HTLV-2 may have a protective effect against HIV-1 progression. HTLV-1 infects CD4+ cells while HTLV-2 infects CD8+ cells and causes their clonal expansion. HTLV-2 also induces the expression of CCR5-binding chemokines (Lewis et al. 2000). Competition by these chemokines with HIV-1 virions for CCR5 binding plus clonal expansion of HIV-suppressing CTL could contribute to slower HIV-1 infection progression (Casoli et al. 2007).

Perspectives on the Future In the clinic, increased risk of malignancy is a feature of HIV-1 infection. Screening of cancer patients for HIV-1 infection is not the current standard of care even though cancer is an AIDS-defining condition (Chiao et al. 2010). In 2006, the Centers for Disease Control and Prevention recommended HIV-1 screening as a standard test offered in all health care settings, given with patient choice to “opt out.” Presently, this practice is typically limited to emergency departments and pregnant women. However, detection of HIV-1 infection has the potential to significantly improve clinical outcomes in an HIV-1-infected cancer patient. Treatment by HAART in synch with appropriate management of chemotherapeutic regiments is important to manage clinical outcome. In the biomedical research setting, HIV-1 provides a natural model system to investigate the fundamental connection between the human immune response and neoplastic progression. The admixture of innate, adaptive, and cell-mediated responses to HIV-1 during coinfection with EBV, HHV-8/KSHV, or HPVs provides a vast, interrelated network of intricate interactions that can culminate in perilous cell growth. Connections are wired between and among cell lineages by cytokine signaling, paracrine interactions, and secondary effects of immune disbalance. Two frontiers of ongoing exploration are apparent and their paths are likely to converge. Progress is underway to characterize the innate response to HIV-1 and other chronic virus infections, including HCV. Notably, coinfection provides an added dimension to view the innate response. A less-developed frontier is to define host microRNA signatures of HIV-1 and other chronic virus infections, to determine their potential interfaces, and to understand their contribution to neoplastic growth. The convergence of these paths is likely to build new bridges from the bench to the bedside.

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Acknowledgments We appreciate the assistance of Mr. Tim Vojt on the illustrations, Dr. Kate Hayes-Ozello for comments on the document, and the support of the National Institutes of Health for National Cancer Institute RO1CA108882 and P30CA100730 to KBL; P01CA16058 to the Ohio State University Comprehensive Cancer Center.

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Chapter 25

HTLV-1 and Oncogenesis Chou-Zen Giam

Introduction Human T-lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia/lymphoma (ATL), a malignancy of CD4+ T cells that develops in 3–5% of infected individuals over a course of several decades (Taylor and Matsuoka 2005; Matsuoka and Jeang 2007). It was first isolated in 1980 from Hut102, a T cell line established from a T-cell lymphoma patient (Poiesz et al. 1980; Gallo 2005). Shortly after its discovery, HTLV-1 became causally linked to ATL, a leukemia first described in 1977 in Japan and etiologically associated with a virus (Uchiyama et al. 1977; Takatsuki 2005). Epidemiological studies indicate that HTLV-1 infection is endemic in parts of the Caribbean basin, the southern islands of Japan − Kyushu and Okinawa, parts of Africa, South America, and the Pacific islands of Melanesia and Papua New Guinea (Gessian et al. 1991; Wong Staal and Gallo 1985). To date, HTLV-1 remains the only retrovirus associated with human malignancy. HTLV-1 infection is highly cell-associated and largely T-cell tropic. The virus preferentially infects and transforms CD4+ T cells. HTLV-1 is transmitted predominantly via breast milk, sex, and transfusion of blood products containing HTLV-1-infected cells. The diseases caused by HTLV-1, namely, adult T-cell leukemia (ATL), HTLV1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), HTLV-1 uveitis, and other inflammatory skin diseases have their etiologies in the dysregulated proliferation of T-cells and/or ensuing immune dysfunctions. Presently, there is no effective treatment for HTLV-1-associated diseases (Uozumi 2010; Lezin et al. 2007).

C.-Z. Giam (*) Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814, USA e-mail: [email protected] E.S. Robertson (ed.), Cancer Associated Viruses, Current Cancer Research, DOI 10.1007/978-1-4614-0016-5_25, © Springer Science+Business Media, LLC 2012

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HTLV-1 Genome and Replication HTLV-1 is a complex delta retrovirus that encodes in addition to viral structural proteins, MA (matrix), CA (capsid), NC (nucleocapsid), RT (reverse transcriptase), PR (protease), IN (integrase), SU (surface) and TM (transmembrane) glycoproteins, six accessory proteins: Tax (transactivator encoded by the X region), Rex (regulator of gene expression encoded by the X region), HBZ (HTLV-1 bZip protein), p12I, p13II, and p30II. HTLV-1 expresses eight major mRNA species in the sense orientation and 1 mRNA species in the antisense orientation (Fig. 25.1). The unspliced HTLV-1 mRNA, like that of other retroviruses, serves as the genomic RNA and the mRNA that encodes Gag (MA-CA-NC) and Gag-Pol (MA-CA-NC–RT-IN) polyproteins. The major singly spliced mRNA encodes the envelope (Env) glycoprotein precursor that is cleaved by a cellular protease to produce the SU and TM proteins. Tax and Rex are encoded by a doubly spliced mRNA. Tax is a potent activator of HTLV-1 mRNA transcription, while Rex regulates the transport of the unspliced and the singly spliced or incompletely spliced viral mRNA to the cytoplasm. In the early stage of viral replication, cellular transcription factors activate the synthesis of viral mRNA, which is first doubly spliced to produce Tax/Rex transcript.

Fig. 25.1 Genomic organization of HTLV-1 proviral DNA and viral mRNA trasnscripts. The open reading frames for Gag, Pro, Pol, Env, Tax, Rex, p12I, p30II, p13II, and HBZ genes are as indicated. The splice donor and acceptor sites in HTLV-1 mRNAs are marked by black and gray triangles. The nucleotide positions of the splice sites are indicated

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Tax, in turn, augments viral mRNA transcription, while the accumulation of Rex causes viral mRNA to be diverted to the unspliced and incompletely spliced forms for the production of viral structural proteins necessary for virus assembly. P30II preferentially sequesters Tax/Rex mRNA in the nucleus, thus downregulating Tax/Rex expression (Nicot et al. 2004), while HBZ antagonizes the transactivation functions of Tax to dampen viral mRNA transcription (Gaudray et al. 2002; Lemasson et al. 2007; Basbous et al. 2003). By downregulating Tax expression and Tax activities respectively, p30II and HBZ are thought to modulate viral replication and possibly facilitate the establishment of viral latency. The roles p12I and p13II play in viral life cycle are not fully understood.

HTLV-1 Accessory Proteins and Their Functions HTLV-1 Tax Functions Tax is a 40-kDa protein that resides in both the nucleus and cytoplasm and has both nuclear and cytoplasmic functions.

Tax Activates Viral mRNA Transcription Tax is a potent activator of HTLV-1 viral mRNA transcription. The mechanism by which Tax activates viral transcription is depicted in Fig. 25.2. The viral transcriptional enhancer in the U3 region of the long terminal repeats (LTR) consists of three imperfect 21-bp repeats, each containing a cAMP response element (CRE) core flanked by 5¢ G-rich and 3¢ C-rich sequences. The CREs in the 21-bp repeats are bound by the cellular basic domain-leucine zipper (bZip) transcription factors – CREB and ATF-1, and possibly other CREB/ATF-like proteins. CREB/ATF-1 and the 21-bp repeats, in turn, recruit Tax into stable ternary complexes (Armstrong et al. 1993; Wagner and Green 1993; Yin and Gaynor 1996a; Zhao and Giam 1991; Suzuki et al. 1993) in which Tax binds the basic domains of CREB/ATF-1 (Adya et al. 1994; Yin et al. 1995; Baranger et al. 1995; Perini et al. 1995) and makes contacts with the DNA minor groove of the G/C-rich sequences that flank the CRE, thus achieving the exquisite DNA sequence specificity of Tax-mediated LTR transactivation (Paca Uccaralertkun et al. 1994; Yin and Gaynor 1996b; Lenzmeier et al. 1998; Tang et al. 1998; Kimzey and Dynan 1998, 1999; Lundblad et al. 1998; Datta et al. 2000). In the context of the ternary Tax-CREB/ATF-1-21 bp repeat complex, Tax further recruits transcriptional coactivators, CREB binding protein (CBP)/p300 and, possibly, p300-CBP associated factor (P/CAF) for potent gene activation (Kwok et al. 1996; Lenzmeier et al. 1998; Harrod et al. 1998, 2000; Bex et al. 1998; Jiang et al. 1999). Recent studies have shown that transcriptional coactivator, transducers of regulated CREB (TORCs), also participate in the high-order nucleoprotein

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616 HTLV-I 21-bp TaxREs: AAGGCCC TGACGTGT CCCCCT TAGGCTC TGACGTCT CCCCCC CAGGCGT TGACGACA ACCCCT

CREB/ATF-1 Nucleosome 21bp-repeat

Tax TORC Interactions with other cellular factors?

P/CAF

CBP/p300

Fig. 25.2 Activation of HTLV-1 transcription by Tax. The viral transcriptional enhancer in the U3 region of the long terminal repeats (LTR) consists of three imperfect 21-bp repeats, each containing a cAMP response element (CRE) core flanked by 5¢ G-rich and 3¢ C-rich sequences. The CREs in the 21-bp repeats are bound by the cellular basic domain-leucine zipper (bZip) transcription factors – CREB and ATF-1. CREB/ATF-1 and the 21-bp repeats, in turn, recruit Tax into stable ternary complexes in which Tax binds the basic domains of CREB/ATF-1 and makes contacts with the DNA minor groove of the G/C-rich sequences that flank the CRE, thus achieving the exquisite DNA sequence specificity of Tax-mediated LTR transactivation. In the context of the ternary TaxCREB/ATF-1-21bp repeat complex, Tax further recruits transcriptional coactivators, CREB binding protein (CBP)/p300, TORCs, and p300-CBP associated factor (P/CAF) for potent gene activation. Recent data indicate that Tax establishes a nucleosome-free region in the LTR

complex assembled on the 21-bp repeats (Siu et al. 2006; Nyborg et al. 2009). This complex creates a nucleosome-free region in the viral LTR that facilitates mRNA transcription (Sharma and Nyborg 2008). Whether ATP-dependent chromatin remodeling factors such as BRG1 is recruited by Tax to the HTLV-1 promoter is a matter of debate (Zhang et al. 2006; Easley et al. 2010). In the presence of Tax, gene expression driven by multiple copies of the 21-bp repeat element can increase up to 100-fold or higher.

Tax Activates IKK/NF-kB HTLV-1-transformed cell lines are known to express in abundance a wide variety of cytokines and cytokine receptors including IL2 receptor alpha chain. This is largely because Tax potently and constitutively activates NF-kB. NF-kB/Rel family of transcription factors are controlled by inhibitory I-kB proteins – I-kBa, I-kBb, and the

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I-kB-like domains in NF-kB1 and NF-kB2 – that sequester NF-kB/Rel in the cytoplasm as multiprotein complexes (for reviews, see Hayden and Ghosh 2008). Upon activation by extracellular stimuli such as interleukin-1 (IL-1), tumor necrosis factor-a (TNF-a), bacterial lipopolysaccharide (LPS), or Tax, I-kBa and I-kBb become serine phosphorylated by I-kB kinase (IKK). This marks them for polyubiquitination and rapid degradation by proteasome-mediated proteolysis. The degradation of I-kB results in heightened nuclear levels of NF-kB and increased expression of a plethora of cellular genes under NF-kB regulation, including the genes of many cytokines and their receptors, adhesion molecules, and immune modulators (Beg and Baldwin 1993; Liou and Baltimore 1993; Siebenlist et al. 1994). Dysregulation and/or hyperactivation of the NF-kB/I-kB regulatory pathway as caused by chromosomal translocation (Neri et al. 1991), oncogene transduction (Gilmore 1992), viral infection (as in the case of HTLV-I), or targeted gene disruption (Beg et al. 1995; Klement et al. 1996) leads to cancers of the hematopoietic cells or chronic inflammatory diseases. IKK, the Ser/Thr kinase that phosphorylates I-kBa and targets it for lysine-48 (K48) polyubiquitination and proteasome degradation, is the focal point of multiple signaling pathways that lead to NF-kB activation. The IKK holoenzyme consists of two catalytic subunits, IKKa and IKKb, together with a regulatory subunit, IKKg/ NF-kB essential modulator (NEMO, referred to as NEMO herein). Tax activates IKK constitutively (Sun et al. 1994; Good and Sun 1996; Sun and Ballard 1999; Xiao and Sun 2000; Chu et al. 1998, 1999). This is due primarily to a direct interaction between Tax and NEMO (Chu et al. 1999; Jin et al. 1999; Xiao and Sun 2000), which results in constitutive activation of IKKa and IKKb, degradation of all I-kBs, and activation of both classical and alternative NF-kB pathways (Chu et al. 1999; Jin et al. 1999; Xiao and Sun 2000). While NEMO is essential for IKK activation, the mechanism by which NEMO controls IKK activity remains to be fully elucidated. Both IKKb and NEMO undergo extensive Lys-63 (K63) polyubiquitination mediated by E3 ubiquitin ligases such as TRAF2 and TRAF6, and a E2 ubiquitinconjugating enzyme, UBC13/UBE2V1 heterodimer (Deng et al. 2000). K63 polyubiquitination is essential for IKK activation. It is thought that K63 polyubiquitin recruits the TGF-b activated kinase 1 (TAK1) by interacting with TAB2, the ubiquitin receptor subunit of TAK1, to phosphorylate and activate IKK (Leibovitz et al. 1973; Xia et al. 2009). TAK1 has also been reported to interact with Tax and to be necessary for Tax-mediated NF-kB activation (Wu and Sun 2007). As might be expected, Tax is extensively modified posttranslationally by phosphorylation, ubiquitination, and sumoylation (Peloponese et al. 2004; Lamsoul et al. 2005). The activating posttranslational modifications of NEMO and IKK, including K63 polyubiquitination of NEMO and monoubiquitination of IKK, are highly induced by Tax (Carter et al. 2005). Recent data suggest that UBC13/UBE2N associates with both Tax and NEMO and is critical for their K63 polyubiquitination and Taxmediated IKK activation (Shembade et al. 2007). The importance of K63 polyubiquitination of NEMO in IKK/NF-kB activation has been challenged recently, however (Tokunaga et al. 2009; Iwai and Tokunaga 2009). Deletion of UBC13 gene in mice

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did not affect NF-kB activation significantly (Yamamoto et al. 2006a, b). Furthermore, head-to-tail linear polyubiquitination of NEMO by the linear ubiquitin-chain assembly complex (LUBAC), which consists of two RING finger proteins HOIL-1L and HOIP, has been shown to be critical for NF-kB activation via the TNF-a and IL-1b signaling pathways (Tokunaga et al. 2009). Whether and how Tax interacts with the E2 and E3 enzymes that carry out either K63 or linear polyubiquitination of NEMO would be topics of significant interest. Finally, Tax also activates transcription of serum-response factor-regulated genes by directly interacting with SRF (Fujii et al. 1992). Tax is thought to be the key viral factor responsible for ATL development.

Rex Functions HTLV-1 Rex is a 27 kDa nuclear phosphoprotein (Younis and Green 2005). It binds viral RNAs via an RNA element known as the Rex-response element (RxRE) that resides in the R region of the viral mRNA (Younis and Green 2005). The argininerich region in the NH2-terminus of Rex functions as an RNA-binding motif. Rex also contains both nuclear localization and nuclear export signals and shuttle between the nucleus and the cytoplasm. Like HIV Rev, Rex forms homooligomer/ polymer as a part of its interaction with RNA (Younis and Green 2005).

p12I, p13II, and p30II Functions As indicated in Fig. 25.1, the region between Env and Tax/Rex open reading frames contains multiple genes in both directions: p12I, p13II, and p30II in the sense orientation and HBZ in the antisense orientation. p12I, a membrane-associated protein, appears to play a role in enhancing T cell activation and signaling (Ding et al. 2003), although contradictory findings have also been described (Fukumoto et al. 2007). A recent article has reported p12I-mediated downregulation of ICAM-1 and ICAM-2, which is thought to mitigate autologous natural killer cell cytotoxicity for the infected CD4+ T cells (Banerjee et al. 2007). The p13II is an inner mitochondrial membrane protein with antiproliferation activity (Silic-Benussi et al. 2004). Its role in HTLV-1 infection and replication is not entirely clear at present. The antiproliferation activity of p13II appears to be related to an interaction with farnesyl pyrophosphate synthetase and alteration of Ras-mediated apoptosis in T lymphocytes (Hiraragi et al. 2005). P30II is a nuclear and nucleolar protein that functions as a posttranscriptional modulator of viral replication (Nicot et al. 2004). Data suggest that p30II retains the doubly spliced Tax/Rex mRNA in the nucleus and thereby downmodulates viral gene expression by reducing the levels of Tax and Rex (Nicot et al. 2004). It has also been reported to interact with CBP/p300 and interfere with LTR transactivation by Tax (Zhang et al. 2001).

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HBZ Functions As shown in Fig. 25.1, the 3¢ region of HTLV-1 encodes an mRNA of opposite polarity to the major HTLV-1 mRNA transcript. This antisense transcript spans the open region that resides between the env and the tax/rex ORFs, and encodes a basic domain-leucine zipper protein known as the HTLV-1 b-Zip protein (HBZ) (Gaudray et al. 2002). The HBZ transcript exists in both unspliced and singly spliced forms, with the latter representing the dominant species. The spliced HBZ mRNA transcript does not contain sequences complementary to the Tax/Rex mRNA and, therefore, is not expected to affect Tax/Rex mRNA by RNA interference (Cavanagh et al. 2006; Satou et al. 2008). The singly spliced HBZ mRNA encodes a 30 kDa basic domainleucine zipper protein (Gaudray et al. 2002; Clerc et al. 2008). Earlier studies have indicated that HBZ could downregulate Tax-mediated transactivation of the HTLV-1 LTR by binding CREB/ATF (Gaudray et al. 2002; Lemasson et al. 2007; Basbous et al. 2003). Interaction between HBZ and CBP/p300 also contributes to downregulation of Tax-mediated LTR transactivation (Clerc et al. 2008). HBZ also interacts with other bZip proteins including Jun and Jun D (Basbous et al. 2003). Most recently, HBZ has been shown to selectively inhibit the classical NF-kB pathway by binding p65 RelA to prevent NF-kB binding to DNA and by facilitating ubiquitinmediated degradation of RelA (Zhao et al. 2009). Most intriguingly, the HBZ mRNA is widely expressed in ATL cells in contrast to the Tax/Rex mRNA (Saito et al. 2009); and the HBZ mRNA, but not the HBZ protein, has been reported to stimulate cell proliferation. This raises interesting questions about HBZ’s role in ATL maintenance (Satou et al. 2008). The p12I, p13II, and p30II proteins all have been reported to be critical for HTLV-1 infection in vivo in a rabbit model (Collins et al. 1998; Hiraragi et al. 2006; Silverman et al. 2004). These earlier results, however, should be interpreted with caution because mutations introduced into all three open reading frames could have also altered the HBZ coding sequence.

Cell-Associated Transmission of HTLV-1 HTLV-1 infection is highly cell associated. Cell-to-cell contact is required for the infection of naïve cells. It has been shown that the spread of HTLV-1 occurs through “virological synapses” (Igakura et al. 2003) formed between integrin LFA1 of virusproducing cells and intracellular adhesion molecule ICAM1 of target cells (Barnard et al. 2005; Liu et al. 2006). The ubiquitously expressed glucose transporter 1 (GLUT1) serves as the receptor for HTLV-1 (Manel et al. 2003). Other cell surface molecules such as heparin sulfate proteoglycan and neuropilin-1 appear to facilitate viral attachment and infection as well (Lambert et al. 2009). Curiously, while cells of many types can be infected by HTLV-1, most are not capable of spreading the infection further, suggesting that they lack critical cellular factors important for becoming competent HTLV-1 donors. Recent data have indicated that free HTLV-1 particles

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are assimilated by dendritic cells and transmitted to target cells in biofilm-like extracellular viral assemblies (Jones et al. 2008; Pais-Correia et al. 2010). While the T cell tropism of HTLV-1 seems to go against the fact that its receptor is ubiquitously expressed, the dependence of HTLV-1 transmission on dendritic cells, which interact intimately with CD4+ T cells, may account in part for this tissue tropism.

Natural History of HTLV-1 Infection Because the levels of cell-free HTLV-1 virus in infected individuals are low, serum antibody titer (to viral antigens) and proviral load are used to monitor the natural history of HTLV-1 infection. De novo viral infection of naïve cells and clonal expansion of infected cells contribute to the HTLV-1 proviral load in an infected person. The former requires reverse transcription and synthesis of viral antigens and, thus, is likely to be accompanied by higher antibody responses; the latter is driven by increased division/proliferation of “latently” infected cells and is usually not accompanied by increased viral antigens or an increased antibody response. The proviral loads in asymptomatic adults with HTLV-1 infection are, in general, stable over the years and are maintained at around 5,000–9,000 copies/105 PBMCs, while those for HAM/TSP patients are generally higher at around 22,500 copies/105 PBMCs (Yamano et al. 2002). Studies of ATL patients in Japan by Okayama et al. have shown that an increase in proviral DNA load frequently preceded the onset of ATL (Okayama et al. 2004).

Development of ATL Oligoclonal Expansion of HTLV-1-Infected Cells Analyses of the clonality of HTLV-1 proviral DNA by inverse PCR have revealed that during seroconversion the clonality of the infected T cells in a person infected through spouse is heterogeneous and remains unstable for several years (Tanaka et al. 2005). By contrast, major clones were common in long-term HTLV-1 carriers. Longitudinal study of ATL is difficult logistically for the obvious reasons that ATL develops decades after viral infection and its frequency of occurrence is low. Progression to ATL is associated with higher proviral load, advanced age, family history of ATL, and opportunity for HTLV-1 testing (Iwanaga et al. 2010). ATL cells show monoclonal patterns of HTLV-1 proviral DNA integration and T cell receptor beta chain gene rearrangement (Maeda et al. 1985). Longitudinal studies of HTLV-1 carriers have indicated that virus-infected cells initially undergo polyclonal or oligoclonal expansion (Okayama et al. 2004). Major HTLV-1-infected cell clones are maintained in carriers for many years. The infected cell clone that contains the same site of proviral integration as the leukemic cells can be detected 2–8 years

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prior to the development of ATL. These results implicate two major events critical to the onset of ATL: (a) the initial oligoclonal expansion of virus-infected T cells and (b) their subsequent evolution and acquisition of additional malignant potentials (Okayama et al. 2004). Tax is thought to promote the initial clonal expansion of specific infected cells. Its expression and viral transcription, however, are frequently silenced or greatly attenuated in most ATL cells (60%) due to mutations and/or methylation of the viral LTR, or other epigenetic changes (Matsuoka et al. 1997; Takeda et al. 2004; Taniguchi et al. 2005). Thus, the many oncogenic activities of Tax are not absolutely needed for maintaining malignancy. This contrasts with the oncoproteins of small DNA viruses such as human papilloma virus E6 and E7 proteins whose constant expression is needed to drive cancer cell proliferation. It is thought that the oncogenic activities of Tax are eventually supplanted through acquisition of genetic or epigenetic changes that activate IKK/NF-kB, the Jak/Stat pathway, and/or the PI3K-Akt pathway during the evolution of ATL (Tomita et al. 2006; Takemoto et al. 1997; Fukuda et al. 2005). Importantly, the HTLV-1 HBZ mRNA transcript appears to be widely expressed in ATL cells (Saito et al. 2009). How HBZ impacts ATL development remains to be fully elucidated. Intriguingly, HBZ mRNA has been reported to stimulate T cell proliferation recently (Satou et al. 2006). Thus, HBZ may serve as the viral determinant for ATL maintenance (Fig. 25.3).

Tumor Suppressors and ATL The role of tumor suppressors in ATL development has been reviewed previously (Hatta and Koeffler 2002). One of the most prominent features of ATL cells is the frequent loss of p16INK4a and p15INK4b, especially from acute/lymphomatous ATL (Yamada et al. 1997; Hatta and Koeffler 2002; Oshiro et al. 2006). Loss of pRb occurs occasionally (Hatta and Koeffler 2002), and mutations in p53 gene occur in some (30–40%) but not all acute/lymphomatous ATL cases, while genetic alterations in p18INK4c, p19INK4d, p21Cip1/Waf1, and p27Kip1 are rare (Morosetti et al. 1995; Oshiro et al. 2006). Stabilization and functional inactivation of p53 by Tax has been described (Takemoto et al. 2000; Pise-Masison et al. 2000, 2001) and may contribute to the maintenance of wild-type p53 gene in HTLV-1-infected cells. The loss or inactivation of tumor suppressors correlates with genetic and chromosomal instability in ATL cells.

Genomic Instability in ATL Cells Unlike cells of other leukemia, ATL cells, especially those of the acute type, are often aneuploid with complex numerical and structural chromosomal abnormalities. No single specific chromosomal gain, loss, translocation, deletion, or rearrangement has been associated with ATL, however. Fujimoto et al. had previously reported

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a

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c

Fig. 25.3 IKK/NF-kB activation by Tax. IKK is the focal point of multiple pathways that lead to the activation of NF-kB. The core IKK enzyme consists of two highly homologous catalytic subunits a and b of 85 kDa and 87 kDa in sizes, respectively, and a 48-kDa regulatory subunit, NEMO. Both IKK-a and IKK-b contain NH2-terminal kinase domains followed by leucine zippers (LZ) and helix-loop-helix (HLH) domains that mediate protein-protein interactions important for IKK oligomerization and kinase activity. NEMO also contains extensive helical regions and leucine zipper domains that are involved in posttranslational modifications and protein-protein interactions. It has been proposed that stimulation of cells with proinflammatory cytokines such as TNF-a leads to the assembly of TNF receptor (TNFR), TRAF2, and RIP1 complex, and the K63 polyubiquitination of NEMO and RIP1 by TRAF2, a E3 ligase, in association with a E2 Ub conjugating enzyme UBC13/UBE2N. IKK is recruited to the K63 polyubiquitin chain of RIP1 via the ubiquitin-binding domain of NEMO. NEMO, in turn, becomes K63 polyubiquitinated and recruits TAK1 (TGFb-activated kinase 1) by interacting with its K63 polyubiquitin-binding subunits (TAB1/TAB2). TAK1 then phosphorylates and activates IKK (a). Recent data, however, indicate that IKK activation results from linear polyubiquitination of NEMO by a E3 ligase known as the linear ubiquitin chain assembly complex (LUBAC) consisting of HOIL-1L and HOIP RING finger proteins. Linear polyubiquitin recruits TAK1, IKK (by interacting with NEMO), or other IKK kinases to phosphorylate and activate IKK (b). Activated IKK phosphorylates I-kBa and causing it to be K48 polyubiquitinated by the SCFbTrCP E3 ubiquitin ligase and targeted for proteasomemediated degradation. In the absence of its inhibitor, NF-kB translocates to the nucleus and activates genes involved in innate immunity, inflammation, and cell survival (c). Tax directly binds NEMO and activates IKK. The mechanism by which Tax activates IKK via NEMO binding is not clear at present, but may involve the K63 or linear polyubiquitin assembly systems

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trisomy 3, trisomy 7, a partial deletion of 6q, and abnormalities of 14q11 in ATL cells (Fujimoto et al. 1999). Oshiro et al. has recently shown distinct chromosomal alterations in acute ATL lymphoma type versus leukemia type, with frequent gains at 1q, 2p, 4q, 7p, and 7q, and losses of 10p, 13q, 16q, and 18p in the lymphoma type, and a gain of 3/3p in the acute type (Oshiro et al. 2006). CARMA1, a critical subunit of the CARMA1-Bcl10-Malt1 complex that mediates I-kB kinase activation, was suggested to be a potential target for recurrent 7p22 amplification in the lymphoma type (Oshiro et al. 2006). Frequent homozygous deletion of 9p21.3 implicates the loss of tumor suppressors p16INK4a (CDKN2A) and p15INK4b (CDKN2B) as a critical determinant in ATL evolution (Yamada et al. 1997; Hatta and Koeffler 2002; Oshiro et al. 2006). Gain of the 3p26-q26 region characteristic of acute ATLL may involve the phosphatidylinositol 3-kinase catalytic subunit b gene (PIK3CB2) located at 3q22.3 (Oshiro et al. 2006), which has been implicated in T cell proliferation and the development of the flower cell morphology of ATL ells (Fukuda et al. 2005). As Tax compromises DNA damage repair, causes DNA damage, and induces mitotic abnormalities to cause both genetic and chromosomal instabilities, it is conceivable that Tax is directly responsible for the genetic changes seen in ATL cells. The roles of these common gains and losses of genetic information during ATL evolution remain to be elucidated. Whether a single multistep pathway initiated by HTLV-1 infection can adequately explain ATL development is also not clear at present.

In Vitro T-Cell Transformation by HTLV-1 Similarities and Differences Between HTLV-1-Transformed Cells and ATL Cells Transformation of primary T cells by HTLV-1 in cell culture shows many of the characteristics of ATL development albeit with important differences especially with regards to Tax expression, alterations of cyclin-dependent kinase inhibitors, and tumorigenic potentials in the severe combined immunodeficiency (SCID) mouse model (Liu et al. 2002; Richard et al. 2001). A comparison between HTLV1-transformed T cells and ATL cells is shown in Table 25.1. Their differences are likely to be due to the absence of an antiviral immune response and the continual inclusion of IL-2 in cell culture for in vitro cell transformation. After infection by HTLV-1 in culture, specific clones of primary T cells can continue to proliferate indefinitely in an IL-2-dependent manner (Graziano et al. 1987). Over time, the “immortalized” T cells acquire the ability to grow without IL-2 and are said to be “transformed” (Miyoshi et al. 1981; Graziano et al. 1987; Ratner et al. 2000). The transition from IL-2-dependent to IL-2-independent growth in vitro is thought to resemble the progression of chronic ATL, which is characterized by indolent lymphocytosis, to acute ATL, an aggressive and rapidly fatal disease. In general, the IL-2-independent (transformed) T cells express Tax constitutively and, as a result,

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Table 25.1 HTLV-1-transformed T cells vs. ATL cells HTLV-1 transformed T cells ATL cells Constitutive Tax expression and IKK/NF-kB Frequent loss of Tax expression and activation NF-kB activated by Tax-independent cellular mechanisms Not tumorigenic in SCID mice due to host Tumorigenic in SCID mice immune response HBZ mRNA detectable HBZ mRNA invariably expressed Genomic and chromosomal alterations Damage to DNA repair mechanisms Frequent loss of p16INK4a, p15INK4b Downregulation of p27Kip1, inactivation of p21Cip1 Functional inactivation of p53 by Tax Mutations in p53 gene occur sometimes Jak3/Stat5 activation Jak3/Stat5 activation

their IKK/NF-kB pathway is chronically activated. By contrast, most ATL cells are Tax-negative. The loss of Tax expression from ATL cells may reflect a strong CTL response against Tax. The ability of Tax to induce cell cycle abnormalities, genomic instability (GIN), chromosome instability (CIN), and senescence (Majone et al. 1993; Marriott and Semmes 2005; Kuo and Giam 2006) likely also contribute to a negative selection against its continuous expression. Despite the strong evidence implicating Tax in cell immortalization/transformation, outside the context of viral infection, Tax alone is poorly capable of immortalizing/transforming primary T cells (Bellon et al. 2010). Whether other viral regulatory proteins, especially HBZ, may synergize with Tax in cell immortalization/transformation remains to be determined.

Signaling Pathways Involved in HTLV-1 Transformation in Cell Culture Earlier studies have found that transition of HTLV-1-immortalized cells to IL-2independence is associated with constitutive activation of the Jak3/Stat5 pathway (Migone et al. 1995). Altered expression of Src- and Syk-related tyrosine kinases has been observed in HTLV-1-transformed T cells (Weil et al. 1999). Downregulation of the cyclin-dependent kinase inhibitor (CKI), p27Kip1, has also been correlated with cell transformation (Cereseto et al. 1999). All HTLV-1-transformed T cells express another CKI, p21Cip1/Waf1, in great abundance (Cereseto et al. 1996; Kuo and Giam 2006). Interestingly, p21Cip1/Waf1 in these cells is localized to the cytoplasm and therefore functionally inactive (Liu et al. 2008). Downregulation of p27Kip1 and functional inactivation of p21Cip1/Waf1 appear to be integral parts of T cell transformation by HTLV-1 (Cereseto et al. 1996; Kuo and Giam 2006; Liu et al. 2008). It is possible that the altered activities of Src- and Syk-like tyrosine kinases, the activation of the Jak3/Stat5 pathway, and the impairment to the expression and function of p27Kip1 and p21Cip1/Waf1 are causally connected. Alternatively, they may evolve independently but act synergistically to transform T cells. Finally, alterations in p27Kip1 and p21Cip1/ Waf1 in HTLV-1-transformed T cells may be functionally equivalent to the loss of p16INK4a and p15INK4b in ATL cells.

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Aberrant Telomere Maintenance in ATL and Tax-Expressing Cells Constitutively high telomerase activity in neoplastic cells prevents the replicative senescence induced by telomere shortening that occurs during successive cell division cycles. ATL cells display constitutive telomerase activity (Uchida et al. 1999); however, since tax gene is often transcriptionally silenced in ATL cells, Tax is unlikely to play any role in the telomerase activity of ATL cells. Tax, however, has been found to promote hTERT expression in quiescent cells through NF-kB, but repress hTERT expression in cells stimulated to proliferate (Sinha-Datta et al. 2004; Gabet et al. 2003). Tax-mediated downregulation of hTERT was suggested to promote telomere shortening, fusion of chromosome ends, faulty segregation of the resulting dicentric chromosomes, and genetic instability (Gabet et al. 2003). Finally, HBZ has been reported to cooperate with with JunD to enhance hTERT expression (Kuhlmann et al. 2007).

Animal Models of HTLV-1 Infection Several animal models exist for the study of HTLV-1. Rabbits, nonhuman primates, and rats can all be infected by HTLV-1 and are useful for investigating virus spread and host immune responses. HTLV-1-infected rabbits, cynomolgus macaques, and squirrel monkeys do not show clinical diseases, however (see Zimmerman et al. 2010 for a recent review). The Wistar-King-Aptekman-Hokudai (WKAH) strain of rats develops spastic paraparesis with degenerative nerve lesions several months after HTLV-1 infection and has been used as a model for HAM/TSP (Ishiguro et al. 1992). A chimeric HTLV-1 with its envelope gene substituted by that of the ecotropic Moloney murine leukemia virus has also been used successfully to infect mice (Delebecque et al. 2002, 2005). The infected mice developed humoral and cellular immune responses against the chimeric virus and showed oligoclonal pattern of proviral integration, but no disease was reported (Delebecque et al. 2005). Finally, xenograft transplantation of cells from ATL patients or ATL cell lines into SCID mice has been useful for propagating tumor cells that are difficult to grow in cell culture (Feuer et al. 1993). The grafts exhibit many of the features of ATL cells in patients, including PTHrP expression and increased IL-2Ra levels (Feuer et al. 1993; Kondo et al. 1993; Liu et al. 2002). Interestingly, HTLV-1-transformed T cell lines derived in cell culture failed to engraft due to NK-cell-mediated lysis, but were tumorigenic in SCID/bg and NOD/SCID mice that have reduced natural killer (NK) cell activity (Liu et al. 2002). These results suggest that the lack of HTLV-1 gene expression in ATL cells likely allows them to evade immune surveillance. The nonobese diabetic/severe combined immunodeficient (NOD/SCID)/gc-null (NOG) mouse xenograft model has also been used to study HTLV-1 infection and treatments for ATL (Kondo et al. 1993; Dewan et al. 2003, 2006)

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Oncogenic Activities of Tax: IKK/NF-kB Activation, Cell Cycle, and Transgenic Mice Tax-Induced NF-k B Activation and Cell Transformation The oncogenic activity of Tax in cell culture and in transgenic mice is driven principally by NF-kB (Grossman et al. 1995; Matsumoto et al. 1997; Yamaoka et al. 1996, 1998; Hasegawa et al. 2006). In fact, NEMO was cloned as a gene whose loss prevented Tax-induced foci formation/cell transformation of rat fibroblasts (Yamaoka et al. 1998). The potent activation of NF-kB by Tax is known to upregulate many antiapoptotic proteins, including Bcl-xL (Tsukahara et al. 1999; Mori et al. 2001a; Nakashima et al. 2003) c-FLIP, (Krueger et al. 2006; Okamoto et al. 2006), survivin, (Mori et al. 2001b; Kawakami et al. 2005) HIAP-1, (Ng et al. 2001; Waldele et al. 2006), and Bcl-2 (Nicot et al. 1997; Akita et al. 2005). An earlier model of ATL development posits that Tax augments IL-2 and IL-2 receptor expression through NF-kB, thereby activating an autocrine stimulatory loop that leads to T cell proliferation and leukemia. Such a model, however, is not supported by further evidence. While IL-2 receptor a chain expression is upregulated by Tax, other essential IL-2 receptor subunits are not. Activation of human telomerase (hTERT) expression by Tax through an NF-kBdependent mechanism has been reported recently (Sinha-Datta et al. 2004). As mentioned above, increased hTERT expression in primary T cells as mediated by Tax is thought to prevent replicative senescence during the early stage of viral infection. Tax has been shown to inactivate p53 by facilitating the formation of a p65 RelA and p53 complex, which assembles on p53-responsive promoters to block p53-mediated transcription (Pise-Masison et al. 2000; Jeong et al. 2004).

Tax and Cell Cycle Entry Earlier studies have focused on demonstrating the functional resemblance of Tax to the oncoproteins of small DNA tumor viruses. It has been reported that Tax induces expression of genes encoding D-type cyclins, particularly cyclin D2, and CDK4 (Lemasson et al. 1998; Akagi et al. 1996; Santiago et al. 1999; Iwanaga et al. 2001; Ohtani et al. 2000) due, in part, to the potent NF-kB activation by Tax (Iwanaga et al. 2001). Tax also accelerates cell cycle progression through G1 apparently by binding to and stabilizing the enzymatic complexes formed by cyclin D family members and CDK4 and CDK6 (Neuveut et al. 1998; Schmitt et al. 1998; Haller et al. 2000; Li et al. 2003; Haller et al. 2002). Furthermore, Tax has also been demonstrated to increase E2F production (Kehn et al. 2005; Ohtani et al. 2000). Notably, Tax has been reported to reduce expression of p18INK4C and p19INK4D (Iwanaga et al. 2001) and inactivates p16INK4 (Low et al. 1997; Suzuki et al. 1996) and p15INK4B via direct binding (Suzuki et al. 1999). Some of these proposed activities of Tax clearly impact on G1–S transition.

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Tax and PDZ Domain-Containing Proteins Soon after the discovery of HTLV-1, a highly related human retrovirus known as HTLV-2 was isolated (Kalyanaraman et al. 1982). In contrast to HTLV-1, HTLV-2 is not associated with human malignancies. The HTLV-2 Tax is structurally and functionally similar to the HTLV-1 Tax, but lacks 22 amino acid residues in the carboxyl terminus. This domain in HTLV-1 Tax has been shown to constitute a PDZ domain-binding motif and interact with cellular PDZ domain-containing proteins including tumor suppressor hDlg (Ishioka et al. 2006), MAGI-3 (Ohashi et al. 2004), scaffolding protein hScrib (Arpin-Andre and Mesnard 2007), and the IL-16 precursor protein (Wilson et al. 2003). These interactions augment the transforming activity of HTLV-1 Tax in rat fibroblasts (Hirata et al. 2004) and possibly increase the pathogenicity of HTLV-1.

Tax in Transgenic Mice Transgenic mice expressing tax develop various tumors depending on the promoters used for Tax expression. Tax expressed by the HTLV-1 LTR caused neurofibroma, a tumor of mesenchymal tissue, after a long time (Nerenberg et al. 1987). Interestingly, one group of LTR-tax mice developed thymic atrophy and died soon after birth, consistent with the notion that Tax expression is detrimental to T cells (Nerenberg et al. 1987). Large granular lymphocytic leukemia has been found in mice transgenic for tax expressed from the T-cell specific granzyme B promoter (Grossman et al. 1995). More recently, tax under the control of the Lck proximal promoter has been found to induce large-cell lymphomas and leukemia with clinical, pathological, and immunological features characteristic of acute ATLL (Hasegawa et al. 2006). Here, again, the lymphoma/leukemia developed after prolonged latency periods. The Lck promoter-Tax transgenic mice were immunocompromised and prone to opportunistic infections (Hasegawa et al. 2006).

Oncogenic Activities of Tax: Tax and Genetic/Chromosomal Instabilities Tax is known to bring about both genetic instability (GIN) and chromosome instability (CIN) by causing defects in DNA damage repair and inappropriate chromosome segregation. Such genetic alterations are thought to increase the rate of mutations and chromosome aneuploidy that in turn facilitate leukemia development. While there is general agreement on the role of Tax in causing GIN and CIN, the exact mechanism(s) by which they are effected has (have) not been fully resolved. It is unclear whether the seemingly complex GIN and CIN caused by Tax share a common underlying mechanism and what role GIN and CIN may play in HTLV-1 replication.

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Tax and Defect in DNA Damage Repair With respect to GIN, Tax has been shown to downregulate the expression of DNA pol b, an enzyme involved in base-excision repair (Jeang et al. 1990). Tax also induces proliferating cell nuclear antigen (PCNA) expression, which likely contributes to continual DNA replication upon DNA damage and defect in nucleotide excision repair (Lemoine et al. 2000). Tax has also been reported to disrupt the ataxia telangiectasia-mutated (ATM) and ATR pathways by interacting with Chk1 and Chk2 (Park et al. 2004; Park et al. 2006; Gupta et al. 2007; Durkin et al. 2008). It was found to colocalize with and sequester Chk2 and DNA-PK to hamper cellular response to DNA damage (Gupta et al. 2007; Durkin et al. 2008), while the TaxChk1 interaction is thought to inhibit Chk1 activity, block phosphorylation-dependent degradation of Cdc25A, and prevent G(2) arrest in response to gamma-irradiation (Park et al. 2004). Tax was also found to reduce the association between MDC1 and DNA repair foci, which could potentially compromise cellular mechanism for DNA damage repair (Chandhasin et al. 2008). Insofar as there is consensus that Tax induces DNA damage, the multitude of models to explain its mechanisms of action suggests that additional work is needed to resolve the matter.

Tax and Double-Stranded DNA Breaks The association between Tax and micronuclei formation was first reported by Majones et al. (1993). Micronuclei consist of extranuclear chromosome fragments or whole chromosomes that form as a result of chromosome breaks (clastogenic events) or chromosome lagging during cell division. Tax-induced micronuclei formation is associated with increased free DNA 3¢ ends that are consistent with the increased occurrence of clastogenic DNA double-stranded breaks (DSBs) (Majone and Jeang 2000). In this vein, Lemoine and Marriott had used the PALA [N-(phosphonoacetyl)l-aspartate]-resistance assay, which measures increased copy number of the CAD (Carbamyl phosphate synthetase/Aspartate transcarbamylase/Dihydro-orotase) gene to demonstrate that Tax increase the rate of gene amplification (Lemoine and Marriott 2002), presumably caused by DNA recombination following DSBs. Finally, Ku80deficient cells are refractory to while loss of DNA-PKcs exacerbates Tax-induced micronuclei formation (Majone et al. 2005). Whether Tax directly induces DSBs, inhibits the repair of DSBs, or both has not been fully delineated.

Tax and Chromosome Instability Centrosomes, the microtubule organizing centers (MTOCs), are responsible for bipolar mitotic spindle formation and proper segregation of chromosomes during mitosis. Centrosome duplication occurs once in every cell division cycle (centrosome

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duplication “licensing”) and is tightly regulated. Centrosome amplification can lead to multipolar spindle formation and unequal chromosome segregation. Tax has been shown to cause centrosome amplification or fragmentation. This has been hypothesized as a contributing factor to the development of aneuploidy in ATL cells. The underlying mechanism for Tax-mediated centrosomal abnormality is not entirely clear. Interaction between Tax and RanBP1 that results in a disruption of Ran-RanBP1 regulation of centriole cohesion has been proposed (Peloponese et al. 2005). A role of NF-kB in Tax-mediated centrosomal amplification has also been suggested (Peloponese et al. 2005). Finally, Tax1BP2, an extensive coiled-coil protein identified in yeast 2-hybrid screen as a Tax-binding partner, has been shown to inhibit centrosome duplication (Ching et al. 2006). Tax1BP2 knockdown or putative inactivation through interaction with Tax, leads to centrosome hyperamplification. Another model for Tax-induced CIN posits that a direct interaction between Tax and HsMAD1, a critical component of the spindle checkpoint, causes spindle assembly checkpoint defect (Jin et al. 1998), which allows mitosis to proceed even though proper attachment of sister chromatids to the mitotic spindle is impaired, thus causing uneven distribution of chromosomes and aneuploidy. Tax-expressing HTLV-1transformed T cells arrest in metaphase after treatment with microtubule-disrupting agent, nocodazole, suggesting that the spindle checkpoint defect caused by Tax is likely to be subtle (Liu et al. 2005; Bellon et al. 2010).

Tax Induces Cellular Senescence Tax-Induced Senescence is a Precancerous Condition Despite the mitogenic and antiapoptotic activities of Tax outlined above, Tax is difficult to express in cultured cell lines (Liang et al. 2002). Few ATL cells consistently express Tax (Taniguchi et al. 2005), and cell transformation by HTLV-1 and especially by Tax in cell culture is inefficient (Graziano et al. 1987). Paradoxically, Tax expression also leads to drastic upregulation of p21Cip1/Waf1 and p27Kip1, and p53-/pRbindependent cellular senescence (Kuo and Giam 2006). The cellular senescence induced by Tax is most likely as a cellular protective measure against the potentially oncogenic activities of Tax such as GIN and CIN. Thus, impairment to this mechanism through downregulation of p27Kip1 and cytoplasmic mislocalization of p21Cip1/ Waf1 renders Tax expression permissible in HTLV-1-transformed cells and allows NF-kB to remain constitutively activated by Tax to promote cell survival and proliferation. The cellular senescence induced by Tax is not a result of overexpression of Tax. HTLV-1-infected lymphoid (SupT1) and nonlymphoid (HeLa) cells become senescent (Liu et al. 2008). Whether primary T cells infected by HTLV-1 also undergo senescence remains to be determined. It is worth noting that atypical lymphocytes that are binucleated or contain cleaved/cerebriform nuclei are readily seen in the blood smears of HTLV-I infected individuals (Taguchi and Miyoshi 1983; Kinoshita et al. 1985; Shimoyama 1991; Sacher et al. 1999). Such cells resemble senescent

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HeLa cells transduced by the Tax gene via a lentiviral vector (Kuo and Giam 2006). The said pathological findings are consistent with the notion that development of cellular senescence may reflect a precancerous condition due to oncogene activation or tumor suppressor inactivation by Tax (Adams 2009). Whether cellular senescence plays an active role in HTLV-1 replication is not clear. Senescent cells are known to secret chemoattractants for macrophages and NK cells (Adams 2009), which are responsible for their eventual elimination from affected tissues. Whether senescent HTLV-1-infected cells may use this mechanism for virus transmission and spread, especially to macrophages, can only be speculated upon at this point.

Upregulation of p21Cip1/Waf1 and p27Kip1 by Tax The p27Kip1 protein half-life in an actively dividing cell is short, but becomes greatly lengthened in the presence of Tax (Kuo and Giam 2006; Zhang et al. 2009). This appears to be a result of the unscheduled activation of the anaphase promoting complex/cyclosome (APC/C) by Tax (Kuo and Giam 2006), which leads to the premature polyubiquitylation and degradation of Skp2 and inactivation of SCFSkp2, the E3 ligase that mediates the destruction of p27Kip1. By constrast, the drastic rise in p21Cip1/Waf1 induced by Tax is due to p53-independent p21Cip1/Waf1 promoter activation (Chowdhury et al. 2003; Zhang et al. 2009) and p21Cip1/Waf1 mRNA stabilization (Zhang et al. 2009). The massive surge in p21Cip1/Waf1 and p27Kip1 in turn leads to p53and pRb-independent cellular senescence (Kuo and Giam 2006). Cell cycle arrest of CD34+ hematopoietic progenitor cells transduced by the tax gene has also been observed (Tripp et al. 2003, 2005), presumably mediated by the same mechanism. Because p21Cip1/Waf1 can serve as a chaperone for G1 cyclin D-CDK4/6 complex without inhibiting its kinase activity, it was hypothesized that p21Cip1/Waf1 may thereby facilitate cell-cycle transition of HTLV-1-infected cells (Kehn et al. 2004). However, in light of results showing that Tax causes cellular senescence, the increase in p21Cip1/ Waf1 brought on by Tax is most likely for senescence induction. Data showing that Tax causes mitotic aberrations and senescence support a model of ATL development in which the p21Cip1/Waf1- and p27Kip1-mediated senescence program has to be inactivated by cellular epigenetic or genetic changes to accommodate Tax expression and cell proliferation concurrently (Kuo and Giam 2006; Liu et al. 2008). Alternatively, the silencing of Tax and HTLV-1 expression by 5¢ LTR methylation (Taniguchi et al. 2005), or attenuation of Tax expression and activities by viral factors such as p30II and HBZ may allow virus-infected cells to continue to proliferate (Mesnard et al. 2006; Satou et al. 2006) and evolve into leukemia.

HBZ and HTLV-1 Leukemogenesis That HBZ antagonizes the activities of Tax at the levels of LTR transactivation and NF-kB activation immediately raises the possibility that it may attenuate or silence viral mRNA transcription and mitigate the various biological effects of Tax including

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senescence. It is conceivable that during HTLV-1 infection, if Tax expression is dominant over HBZ, then robust viral replication ensues and the senescence checkpoint becomes activated. Infected cells in this state likely reach a dead end. By contrast, when HBZ expression is favored, then Tax expression and activities together with HTLV-1 replication would be moderated and viral latency may ensue. In this vein, HBZ knockdown has been found recently to correlate with a reduction in T cell proliferation after HTLV-1 infection (Arnold et al. 2008). HBZ expression is determined by the abundance of the transcription factors that activate its promoter and by the sites of HTLV-1 proviral integration. SP1 binding sites in a TATA-less promoter located in the 3¢ LTR has been shown to mediate HBZ expression (Yoshida et al. 2008). Recent data have also indicated that Tax upregulates the expression of HBZ and that this upregulation is influenced by the HTLV-1 integration sites (Landry et al. 2009). Proviral DNA integrated near a strong cellular promoter that directs transcription in the opposite polarity of the major viral mRNA transcript is expected to express HBZ preferentially. Those infected cells that express HBZ in higher abundance are likely to continue to divide and evolve without triggering the senescence checkpoint because Tax expression and activities are downregulated. The reduction or silencing of viral replication in turn facilitates evasion from immune detection. Since ATL can only emerge from HTLV-1-infected cells that are capable of continuous cell division, and HBZ is likely important for lowering the Tax level and delaying or preventing the onset of cellular senescence induced by Tax, it is then logical that HBZ mRNA transcript is widely expressed in ATL cells. Finally, the ability of HBZ mRNA to stimulate cell proliferation suggests that it may actually be responsible for leukemia maintenance (Satou et al. 2006). The role of Tax in ATL development should not be underestimated, however. The ability of Tax to activate NF-kB remains a key factor in the proliferation, survival, and immortalization of HTLV-1-infected cells. The new adjustments to the role of Tax in leukemogenesis would be that (1) because of Tax’s ability to induce senescence, its expression needs to be moderated by HBZ, and perhaps p30II, to allow infected cells to proliferate and expand; and (2) the full oncogenic potential of Tax can only be realized when the senescence checkpoint is inactivated. The latter would favor elevated levels of Tax expression and increased NF-kB activation, and at the same time, exacerbate Tax-related genetic and chromosomal instabilities. In this sense, the eventual silencing of Tax expression would stabilize such preleukemic or leukemic cells genetically and prevent their killing by cytotoxic T lymphocytes (Fig. 25.4).

Conclusions and Perspectives The major obstacle to the study of HTLV-1 replication cycle remains the low infectivity of viral particles and the rather strict dependence on cell-to-cell contact for virus infection. This necessitates the coculture of virus-producing cells with target cells for only a modicum of virus transmission to occur. This technical difficulty is further compounded by the inability of the infected cells to spread the infection. Indeed, the low infectivity of HTLV-1 has made it difficult to study key events of

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Fig. 25.4 A model for HTLV-1 Leukemogenesis. HTLV-1 is transmitted by cell-to-cell contact. The expression levels of Tax and HBZ modulate the outcomes of infection. Robust viral replication stimulated by Tax is accompanied by cellular senescence. HBZ moderates transactivation by Tax, thereby downregulates viral replication and Tax expression to allow oligoclonal expansion of infected T cells. Cytotoxic T lymphocyte (CTL) killing can control virus replication in asymptomatic carriers and select for cells that carry latent proviral DNA. HTLV-1-infected cells develop chromosomal instability. Loss of p16INK4a, p15INK4b, and other tumor suppressors and constitutive Jak/Stat activation may contribute to the inactivation of the senescence checkpoint to allow persistent Tax expression and NF-kB activation. Loss of Tax expression is favored because Tax is a primary CTL target and has a propensity to induce genomic instability and cellular senescence. Inactivation of the senescence checkpoint can facilitate potent NF-kB activation by Tax at the early stage of leukemogenesis and aid the development of Tax-independent NF-kB activation later. The mitogenic activity of HBZ mRNA may help sustain the ATL tumor phenotype

viral life cycle such as the mechanisms of viral entry, assembly, and release, and the roles of viral regulatory proteins and their effects on infected cells during viral replication. Although recent data suggest that cell-free HTLV-1 particles can be transmitted to T cells via plasmocytoid dendritic cells (Jones et al. 2008), the efficiency of virus transmission remains quite modest. Thus, while T cells can be immortalized and transformed by HTLV-1 in cell culture, the simple question of what fraction of infected cells becomes so remains unanswered because of the low infectivity of HTLV-1 and the difficulty of tracking infected cells. Existing evidence suggests that immortalization and transformation of T cells by HTLV-1 is a rare occurrence and the notion that HTLV-1 infection invariably leads to cell proliferation requires critical reevaluation with better assay systems. Direct transfection of infectious HTLV-1 proviral DNA clones into cultured cells has been used to analyze the functions of viral accessory genes, but the in vivo roles of many accessory genes remain elusive. The existing animal models for HTLV-1 infection do not fully reflect the natural course of viral infection and disease development in human. Given that ATL develops

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over a course of several decades and only in a small fraction of infected persons, it would be unrealistic to expect an animal model to efficiently recapitulate all features of HTLV-1 pathogenesis. However, armed with the knowledge that inactivation of the senescence checkpoint or activation of the Jak/Stat and/or PI3K pathways can facilitate cell transformation and leukemogenesis, it may be possible to target these regulatory mechanisms in animal models to accelerate the progression to disease after HTLV-1 infection. The absence of Tax expression and the frequent presence of HBZ mRNA transcript in ATL cells now compel a reexamination of the roles of each gene, especially Tax, in HTLV-1 life cycle and leukemogenesis. While Tax expression is clearly not essential for the neoplastic phenotype of ATL cells in vivo, whether HBZ expression is necessary is not clear at present. Thus, the question remains whether Tax and HBZ are important respectively for the initiation and maintenance of ATL, or alternatively, both proteins may act complementarily at the beginning of viral infection up to the precancerous stage of ATL. The induction of cellular senescence by Tax is consistent with the difficulty in expressing it stably in cultured cell lines, and the loss of its expression from ATL cells. By moderating the level of expression of Tax and its activities, HBZ may delay or prevent Tax-induced senescence to allow HTLV-1-infected cells to proliferate and undergo oligoclonal expansion. Ultimately, the senescence mechanism triggered by Tax would have to be inactivated to allow the oncogenic potentials of Tax, such as potent NF-kB activation and induction of GIN and CIN, to become significant. In this sense, Tax may be considered as an opportunistic oncogene, and its leukemogenic potential requires specific collaborating cellular changes that prevent senescence induction. In vivo, Tax-specific cytotoxic T lymphocyte killing and Tax-induced cell cycle abnormalities eventually select for the loss of Tax expression from ATL cells. Since constitutive NF-kB activity is necessary for leukemic cell survival and proliferation, Tax-independent IKK/NF-kB activation likely evolves at a later stage of ATL development (see Fig. 25.4 for a depiction of this model). By contrast, since in vitro transformation of T cells by HTLV-1 takes place in the absence of host immune response, the loss of Tax and viral expression is not selected, and as such Tax expression and Tax-mediated IKK/ NF-kB activation is retained. This provides an explanation for the frequent inactivation of the senescence program in HTLV-1-transformed T cells through the downregulation of p27Kip1 and the functional inactivation of p21Cip1/Waf1. While alterations of p27Kip1 gene are relatively rare in ATL cases (Morosetti et al. 1995), p15INK4b and p16INK4a deletions are frequent occurrences (Hatta et al. 1995). Whether the latter loss can functionally substitute for the downregulation of p27Kip1 and inactivation of p21Cip1/ Waf1 is unclear. Recent progress on the HTLV-1 accessory genes, Tax and HBZ, has begun to shed light on how their interactions may regulate the proliferation and expansion of HTLV-1-infected cells and how HBZ may collude with Tax to cause cell transformation and cancer. It will be important in the future to investigate their functions in the context of viral infection in cell culture, in animal models, and in infected individuals, and to translate basic science findings into treatments for HTLV-1-related diseases.

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Notes In a recent paper (Zhi et al. 2011, PLoS Pathogens, 7(4): e1002025.doi:10.1371/ journal.ppat.1002025.), the persistent and potentially oncogenic activation of NF-kB by Tax was found to be responsible for triggering the cellular senescence response. Down-regulation of NF-kB activity by HBZ, by contrast, delayed or prevented the onset of Tax-induced senescence. These results support the model depicted in Fig. 25.4, showing that HTLV-1 executes two alternative genetic programs wherein robust HTLV-1 replication and elevated Tax expression drive IKK/NF-kB hyperactivation and trigger senescence. Moderation of Tax-mediated viral replication and NF-kB activation by HBZ, on the other hand, allows HTLV-1-infected cells to proliferate, persist, and evolve. Importantly, inactivation of the senescence checkpoint can facilitate persistent NF-kB activation and leukemogenesis.

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Chapter 26

Human T-Cell Leukemia Virus Type 2 (HTLV-2) Biology and Pathogenesis Rami Doueiri and Patrick L. Green

Discovery of HTLV-2 HTLV-2 was first identified in a T-cell line, termed MoT, derived from splenic tissue of a patient with a variant of hairy cell leukemia (Saxon et al. 1978). Serological data demonstrated that HTLV-2 was related to, but distinct from HTLV-1, and subsequent sequence analysis revealed that both viruses share approximately 70% nucleotide sequence homology (Kalyanaraman et al. 1982). HTLV-2 disease association has been less clear in comparison to HTLV-1, which is associated with adult T cell lymphoma (ATL) and HTLV-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Although HTLV-2 was discovered in a hairy cell leukemia patient, the limited number of individuals shown to harbor HTLV-2 in association with specific diseases has precluded convincing epidemiologic demonstration of a definitive etiologic role for HTLV-2 in human disease. However, several cases of HTLV2-associated neurological disease have been documented (Hjelle et al. 1992), and

R. Doueiri Department of Veterinary Biosciences, The Ohio State University, 1900 Coffey Road, Columbus, OH 43210, USA P.L. Green (*) Center for Retrovirus Research, The Ohio State University, 1900 Coffey Road, Columbus, OH 43210, USA Department of Veterinary Biosciences, The Ohio State University, 1900 Coffey Road, Columbus, OH 43210, USA Departments of Molecular Virology, Immunology and Medical Genetics, The Ohio State University, 1900 Coffey Road, Columbus, OH 43210, USA Comprehensive Cancer Center and Solove Research Institute, The Ohio State University, 1900 Coffey Road, Columbus, OH 43210, USA e-mail: [email protected] E.S. Robertson (ed.), Cancer Associated Viruses, Current Cancer Research, DOI 10.1007/978-1-4614-0016-5_26, © Springer Science+Business Media, LLC 2012

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more recent findings from an HTLV outcome study (HOST) provide strong support that HTLV-Z infection is associated with neurodegenerative disease, lymphocyte prolifecation, and may be a predisposition factor to cancer development and heart disease (Murphy et al. 2004; Biswas et al. 2009).

HTLV-2 Virion Structure HTLV-2 is a member of the deltaretrovirus family and, like all retroviruses, is enveloped and contains RNA as its genetic material (Fig. 26.1). Detailed description of the virion is beyond the scope of this chapter, but different components are briefly discussed. The components of the virion are not directly required for cellular transformation, but they are needed for viral attachment and entry, viral genome reverse transcription from RNA to double-stranded DNA, and integration into the cell chromosome, which eventually can lead to cellular transformation. Moving from outside to the inside of the virus particle, the virion envelope contains part of the cellular plasma membrane derived during virus exit or budding from the infected

Fig. 26.1 Schematic representation of the mature HTLV-2 virion highlighting the location of the structural components. The virion envelope is primarily from the host plasma membrane (formed on budding) and contains the viral Envelope proteins, surface unit (SU), and transmembrane (TM), alongside other host-encoded proteins. The inner envelope contains the matrix (MA), which is required for virion assembly at the inner cell membrane. The capsid (CA), which protects the viral RNA genome, the nucleocapsid (NC), integrase (IN), the protease (PRO), reverse transcriptase (RT), and tRNApro are shown

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cell, and two viral envelope proteins encoded by the env gene, the surface unit (SU) and transmembrane (TM) proteins. Beneath the envelope lies three viral proteins encoded by gag: the matrix (MA), the capsid (CA), and the nucleocapsid (NC) (which is associated with the RNA genome). Enzymatic viral proteins packaged in the virion include reverse transcriptase (RT), integrase (IN), and protease (PRO). Cellular factors also are found in the virion. Of critical importance is the cellular tRNA pro, which acts as the viral primer for the initiation of reverse transcription.

Genome Structure HTLV-2 is referred to as a complex retrovirus. In addition to the usual structural and enzymatic genes gag, pol, and env, there is a unique region at the 3¢end of the genome, not found in replication-competent simple retroviruses, which encodes regulatory and accessory genes. Historically, this region was termed X, which for HTLV-2 contains five (I–V) open reading frames (ORF) on the sense strand and one ORF on the antisense strand of the DNA proviral genome. ORFs III and IV encode the positive regulatory proteins, Rex and Tax, respectively. Tax acts as a transcriptional activator of the viral promoter located within the 5¢ long terminal repeat (LTR). Rex functions posttranscriptionally and is required for the efficient expression of structural and enzymatic proteins and thus viral progeny. ORFs I, II, V, and the antisense ORF encode accessory proteins p10, p28, p11, and the antisense protein HTLV-2 (APH-2), respectively. These proteins have been less well characterized to date, but emerging data suggest an important role early in the infectious process resulting in maintenance of proviral load and infected cell persistence in vivo. In the HTLV-2 infected cell, three major (high copy number) and five lower copy number messenger RNA (mRNA) species have been detected (Fig. 26.2). Like all retroviruses, the full-length (unspliced) RNA is utilized for synthesis of gag and pol-encoded gene products and also serves as the genomic RNA packaged into progeny virions. A singly spliced mRNA encodes the env gene products, while a doubly or completely spliced mRNA species encodes both the tax and rex gene products in partial overlapping reading frames: the start codons for Rex and the downstream Tax lie in the second exon, with Tax translation favored due to strong Kozac consensus sequences. Several lower copy number alternatively spliced mRNA species encode proteins from ORFs I (p10), II (p28), III (truncated Rex polypeptides p20/22), V (p11), and the antisense ORF (APH-2).

HTLV-2 Structural and Enzymatic Gene Products of the Virion Gag Gene The gag (group-specific antigen) region is translated into a polyprotein precursor (55 kDa) that subsequently is cleaved by the viral protease (Pro) into three mature

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Fig. 26.2 Structure and organization of the HTLV-2 genome based on the HTLV-2 MO isolate. The provirus genome is at the top of the figure. Location of the viral proteins, RxRE and CRS, are shown beneath the provirus. The structure of the structural, regulatory, and accessory proteins is shown at the bottom of the figure. (Oroszlan et al. 1984; Shimotohno et al. 1985; Halin et al. 2009)

proteins: 19-kDa matrix (MA), 24-kDa capsid (CA), and 15-kDa nucleocapsid (NC) proteins (Oroszlan et al. 1984). p19 is posttranslationally modified and contains a myristic acid at the NH2 terminus (Oroszlan et al. 1984). This modification targets the 55-kDa Gag precursor polypeptide to the inner surface of the plasma membrane (Hayakawa et al. 1992), which is required for virion assembly and budding of virus particles from infected cells.

Protease (Pro) Gene A reading frame that extends from the 3¢ end of gag to the 5¢ part of the pol region encodes the viral Pro. The synthesis of Pro requires ribosomal frameshifting of Gag (Nam et al. 1988). The protease activity has been elucidated in vitro, and studies have shown that Pro auto-catalytically processes itself into a mature form and is responsible for processing the Gag polyprotein into the mature products (Hatanaka and Nam 1989).

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Polymerase (Pol) Gene Similar to pro, a second frameshift is required to express the pol gene (Nam et al. 1988). HTLV-2 pol encodes a polyprotein containing 864 amino acids (Shimotohno et al. 1985). The 5¢end of pol encodes a RNA/DNA-dependent DNA polymerase termed reverse transcriptase (RT), which requires Mg2+ for optimal function (Rho et al. 1981). The downstream region of pol encodes the RNase H and integrase (IN). RNAse H is essential to the reverse transcription process by degrading the RNA of a RNA–DNA hybrid; IN is required to integrate the proviral DNA into the cellular genome.

Envelope (Env) Gene The env gene of the HTLV-2 produces an approximate 67-kDa glycoprotein (Lee et al. 1984), which then is cleaved into the 46-kDa surface glycoprotein (SU or gp46) and the 21-kDa transmembrane protein (TM or p21). The SU component is required for receptor recognition, whereas TM is required for fusion and entry into cells. Interestingly, human antibodies to HTLV-1 Env cross react with HTLV-2 Env, which led to the initial discovery of HTLV-2 (Kalyanaraman et al. 1982).

HTLV-2 Regulatory Genes Tax Gene HTLV-2 encodes two positive regulators of viral gene expression, Tax and Rex, which are required for efficient viral replication and cellular transformation. Tax-2 is a 37-kDa phosphoprotein that shares approximately 78% homology with HTLV-1 Tax (Tax-1). Tax is a trans-acting transcriptional activator and increases the rate of transcription initiation from the promoter in the 5¢ LTR of the provirus genome. In contrast to Tax-1, which is detected primarily in the nucleus, Tax-2 is located predominantly in the cytoplasm (Meertens et al. 2004a, Turci et al. 2006). However, Tax-2 also can be detected in nuclear bodies with RNA polymerase II, splicing complexes and specific transcription factors (Goh et al. 1985; Slamon et al. 1988; Semmes and Jeang 1996; Bex et al. 1997). Although Tax-2 and Tax-1 share a number of activities and associations with nuclear proteins, their distinct difference in localization has been proposed to contribute to the differences between HTLV-1 and HTLV-2 biology. Four functional domains have been described for Tax-2 (Fig. 26.3). The Tax-2 amino terminus contains an activation domain, a nuclear localization signal (NLS) that lies within the first 41 residues (Turci et al. 2006), and a zinc binding

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S130A/L131F (NFκB) Zinc finger

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100

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200

NES

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Activation domain

ΔPBM

Fig. 26.3 Domain structure of HTLV-2 Tax. The nuclear localization domain (NLS) is located within the first 41 amino acids in the N-terminus, in addition to an activation domain and a zinc finger domain. A cytoplasmic localization signal is found at region 90–100, and a leucine-rich region that contains a nuclear export signal (NES) is located at 189–202. A second activation domain is located between residues 189–322. Specific mutations affecting transcriptional activation pathways are indicated. The PDZ binding motif (PBM) domain, which is found in Tax-1 but not in Tax-2, has been added for illustrative purpose

domain between residues 23 and 49 that plays a role in protein–protein interactions. The central region of the polypeptide contains a cytoplasmic localization domain from residues 90–100 (Meertens et al. 2004a) and a leucine-rich nuclear export signal at residues 189–202 that appears to be indispensable for Tax-2 function (Chevalier et al. 2005). The carboxyl terminal 289–322 amino acids contain a second activation domain. One domain absent in Tax-2, but present at the carboxy terminus of Tax-1, is a four-amino-acid PDZ binding motif (PBM). The significance of this domain and other activities of Tax are discussed more detail in below.

Rex Gene In order to efficiently replicate, retroviruses need to override the nuclear retention of intron-containing mRNAs. The unspliced mRNA (genomic and gag/pol) and the singly spliced mRNA (env) are intron-containing viral mRNAs and the default pathway in the cell is to retain them in the nucleus until they are processed or targeted for degradation. At the molecular level, the most notable role of Rex is to regulate cytoplasmic levels of viral genomic unspliced and env singly spliced mRNAs, thus controlling the expression of the structural and enzymatic gene products that are essential for production of viral progeny (Hidaka et al. 1988; Kusuhara et al. 1999). Rex binds to the viral mRNAs via a cis-acting RNA Rex-response element (RxRE) and facilitates the export of these mRNA species from the nucleus to the cytoplasm (Ballaun et al. 1991; Black et al. 1991a; Bogerd et al. 1991). Previous studies revealed that HTLV-1 Rex (Rex-1), HTLV-2 Rex (Rex-2), and their RxREs are structurally similar and functionally interchangeable. The HTLV-2 RxRE is 226 nucleotides in length and is located in the R/U5 region of the LTR. A cis-acting repressive sequence (CRS) has been identified within the RxRE downstream of the splice donor site (Black et al. 1991a, b; Yip et al. 1991). Thus, a model consistent with the experimental data is that the CRS retains the unspliced mRNA in the nucleus to ensure the availability of sufficient amounts of Rex substrate (Ohta et al.

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S153A* S151A* T164A*

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57

71

81

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170

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Fig. 26.4 Domain structure of HTLV-2 Rex. The nuclear localization signal domain (NLS) and the RNA binding domain are located within the first 19 amino acids in the N-terminus. The activation domain and the nuclear export signal (NES) are located between positions 81–94, flanked by two multimerization domains between residues 57–71 and 124–132, respectively. A C-terminal regulatory domain from 144 to 164 that contains key phosphorylation sites (Serine 151, Serine 153, and Threonine 164) is required for efficient function and nucleocytoplasmic shuttling

1988; Black et al. 1991a, b), then Rex binds to the RxRE, overrides the repressive effects of the CRS, and exports the mRNA to the cytoplasm (Black et al. 1991a Younis and Green 2005). Mutational analyses of Rex-1 and Rex-2 identified important domains for their biological properties including (1) an arginine-rich sequence located at the N-terminus that serves both as an RNA binding domain and as a nuclear localization signal (NLS), (2) a central leucine-rich activation domain encompasses the nuclear export signal (NES), and (3) a multimerization domain composed of two regions flanking the NES (Siomi et al. 1988; Rimsky et al. 1989; Bogerd et al. 1991; Hope et al. 1991; Weichselbraun et al. 1992a, b; Bogerd and Greene 1993; Hammes and Green 1993; Palmeri and Malim 1996; Narayan et al. 2003) (Fig. 26.4). The Rex NLS interacts with importin-b, and is transported to the nucleus by a Ran-GTP-dependent mechanism (Palmeri and Malim 1999). Once Rex is in the nucleus, Ran-GTP binds to importin b releasing Rex and making it available to bind RxRE-containing mRNAs (Gorlich et al. 1994; Gorlich et al. 1996). Rex binding to its RxRE recruits additional Rex molecules, although this multimerization is not essential for Rex function (Weichselbraun et al. 1992b; Bogerd et al. 1993; Hataka et al. 1998; Narayan et al. 2003). Once a stable Rex/ RxRE/CRM1 complex is formed, CRM1 interacts directly with Rex and its cargo mRNA and facilitates nuclear transport through a series of protein–protein interactions with the FG repeat-containing nucleoporins. The Rex/RxRE/CRM1 complex then is recognized by Ran-GTP, which finally leads to the exit through the nuclear pore (Fornerod et al. 1997; Hataka et al. 1998; Heger et al. 1998; Otero et al. 1998; Askjaer et al. 1999; Fornerod and Ohno 2002; Younis and Green 2005). Both Rex-1 and Rex-2 are phosphoproteins, and phosphorylation has been shown to be critical for their function (Adachi et al. 1990, 1992; Green et al. 1992). In HTLV-2 infected cells, four different species of Rex are detected (20, 22, 24 and 26 Kd). Studies revealed that proteins p24/p26 are encoded by the tax/rex mRNA, and proteins p20/p22 are amino truncated forms of Rex-2 and are encoded from two alternatively spliced mRNAs. The function of p20/p22, which appear to be analogous to the amino terminal truncated p21 Rex of HTLV-1, is not well understood, but evidence

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suggests that they might interfere with full-length Rex function and localization (Ciminale et al. 1995, 1997; Kubota et al. 1996; Li and Green 2007). p24/p26 are the major Rex-2 protein species detected in HTLV-2 infected cells, which have the same amino-acid backbone, but differ by a conformational change that is induced by serine/threonine-specific phosphorylation (Green 1991; Narayan et al. 2001; Lairmore and Franchini 2007). This phosphorylation-induced conformational change is unique to Rex-2, as the Rex-1 phosphoprotein is detected as a single 27-kDa protein. Rex-2 p24 is localized primarily in the cytoplasm, whereas the p26 phosphorylated form, which is the active species, localizes predominantly to the nucleus and nucleolar speckles (Yip et al. 1991; Ciminale et al. 1997). Phosphorylation of Rex-2 correlates with its binding to RxRE-containing RNA and inhibition of mRNA splicing (Green et al. 1992; Bakker et al. 1996). Mutational analysis of Rex-2 revealed that the C-terminus contains a stability/inhibitory domain that is positively regulated through phosphorylation. Key residues (Ser 151, Ser 153, Thr 164) in the C-terminus appear to govern the switch between the p24 and p26 conformation and active function, supporting a model in which a phosphorylation continuum of Rex-2 at the C-terminus regulates its biological properties (Narayan et al. 2001, 2003; Kesic et al. 2009; Xie et al. 2009). Interestingly, Rex-2 can also negatively regulate levels of mRNA that contain LTR sequences (Watanabe et al. 1996) and thus might also play a role in viral latency. This negative regulation is observed in T-lymphocytes but not B-lymphocytes, does not require the RxRE, and therefore is a function distinct from Rex-2 positive posttranscriptional activity (Watanabe et al. 1996). In addition to Rex facilitating the export of HTLV-2-specific intron-containing mRNAs, there is evidence that Rex-2 also can inhibit the splicing of these mRNAs. Rex-2 binding to the mRNA may dislodge or prevent the binding of splicing factors (Black et al. 1991b; Bakker et al. 1996), thereby allowing the cellular machinery to recognize it as a processed mRNA and transport it to the cytoplasm. Interestingly, phosphorylation of Rex-2 is required for both efficient binding to mRNA and inhibition of splicing. In addition, there is evidence that Rex may increase the translational efficiency of Rex-responsive mRNAs possibly by enhancing binding to the translation initiation factor 5A (eIF-5A) (Katahira et al. 1994, 1995). Experimental data indicate that Rex-2 increases the level of incompletely spliced mRNA seven to ninefold in the cytoplasm, while Gag production increases 130-fold (Kusuhara et al. 1999). Moreover, the homologous HIV-1 Rev has been shown to facilitate translation (Arrigo and Chen 1991; D’agostino et al. 1992).

HTLV-2 Accessory Genes ORF I, II, and V The accessory proteins of HTLV-2 are encoded by several proximal X region ORFs between env and the 3¢ LTR. These proteins include p10 encoded by ORF I, p28 by ORF II, and p11 by ORF V (Ciminale et al. 1995). HTLV-2 p28, with the potential to

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be expressed from two distinct singly spliced mRNAs (both mRNAs also have the potential to produce p20/p22 truncated Rex), has been the best characterized and is, at least in part, functionally homologous to HTLV-1 p30. p28 functions to repress viral replication posttranscriptionally by retaining tax/rex mRNA in the nucleus (Younis et al. 2004). By repressing Tax and Rex positive regulatory functions, p28 downmodulates viral gene expression. Furthermore, it was shown that p28 was able to efficiently retain tax/rex mRNA in the nucleus only when the latter is expressed from a fulllength proviral clone and not from a cDNA, initially suggesting that the 5¢ untranslated region (UTR) of the target RNA, intron sequences, and/or splicing may play a role in mediating the inhibition. However, additional studies revealed that if the tax/ rex cDNA was expressed from the HTLV promoter, it was repressed by p28. The component of the HTLV-2 promoter, specifically Tax-2, was identified as the factor required for efficient recruitment of p28 to the newly transcribed mRNA. This finding suggested a complex interplay between the transcriptional machinery and the posttranscriptional regulation of tax/rex mRNA by p28, thereby coupling transcription with posttranscriptional inhibition. Yamamoto et al. investigated the functional significance of p28 in HTLV-2 infection, proliferation, and immortalization of primary T-cells in culture, and viral survival in an infectious rabbit animal model. They showed that p28 is dispensable for viral replication and cellular immortalization of primary T-lymphocytes. However, p28 function was critical for viral survival in vivo (Yamamoto et al. 2008). Together, the results are consistent with the hypothesis that p28 repression of Tax and Rex-mediated viral gene expression may facilitate survival of infected cells by downmodulating overall viral gene expression. p10 and p11 are expressed from the same doubly spliced mRNA in separate but overlapping reading frames. Although less is known about the role these two proteins play in the biology of HTLV-2, p10, like HTLV-1 p12, binds to the free chain of MHC class I but not to the IL-2R b and g chains (Johnson et al. 2000). p11 binds to MHC class I heavy chain (Johnson et al. 2001) and thus may facilitate escape from immune surveillance.

Aph-2 Antisense Gene The majority of retroviral gene products are encoded by the sense strand of the proviral genome. However, natural antisense viral transcripts have been recognized in retroviruses including HTLV, human immunodeficiency virus, and feline immunodeficiency virus (Larocca et al. 1989; Vanhee-Brossollet et al. 1995; Briquet et al. 2001). HTLV-1 encodes HBZ (HTLV-1 b-ZIP factor) (Gaudray et al. 2002; Matsuoka and Green 2009), and recently, APH-2 has been detected in HTLV-2 infected cells (Halin et al. 2009). APH-2 is a 183-amino-acid protein encoded by a singly spliced mRNA, and although APH-2 lacks a bZIP motif similar to HBZ, it can still interact with cyclic adenosine monophosphate-response element binding protein (CREB) and repress Tax-2 mediated transcription (Halin et al. 2009). Its role in HTLV-2 biology currently is not known, but drawing similarities from HTLV-1 HBZ, APH-2 may play a role in infectivity, viral persistence, and cellular proliferation (Arnold et al. 2006, 2008).

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Genetic Variability and Mode of Transmission of HTLV-2 The rate of genetic variability in the genome of HTLV-2 isolates is quite low, which is consistent with its mode of transmission. Infected individuals display relatively low levels of viral replication and virus propagation occurs primarily by mitotic clonal expansion of infected cells (Cimarelli et al. 1996). To date, there have been four HTLV-2 subtypes identified. Initially, analysis of env sequences identified subtypes a and b (Gessain et al. 1985; Hall et al. 1992). HTLV-2a is found mainly throughout North America and Europe in intravenous drug users (IDU), while HTLV-2b is predominantly found in the Amerindian population in North, South, and Central America (Switzer et al. 1995). Recently, a distinct variant of HTLV-2a has been identified in the Amazon basin that had a full tax gene similar, but distinct from HTLV-2b. This isolate was classified as HTLV-2c (Ishak et al. 1995; Eiraku et al. 1996). A new subtype found in the Efe Bambuti pygmy tribe in the African Congo has been designated HTLV-2d (Vandamme et al. 1998). Owing to the high incidence of HTLV-2 in Amerindians, the virus was thought to be of new world origin, contrary to HTLV-1, which is thought to have arisen from a zoonotic transmission of simian T-lymphotropic virus type 1 (STLV-1) from nonhuman primates to humans (Koralnik et al. 1994; Crandall 1996; Goubau et al. 1996; Voevodin et al. 1996; Liu et al. 1996; Song et al. 1994). However, the discovery of HTLV-2d along side STLV-2 in Africa supports an ancient African origin of HTLV-2 as well (Vandamme et al. 1996; Digilio et al. 1997; Van Brussel et al. 1998). HTLV-2 is transmitted via infected cellular blood products or intravenous drug use, mother-to-child and sexual contact (Roucoux and Murphy 2004). However, the route of transmission varies between different populations. Interestingly, vertical transmission from mother-to-child occurs mainly through breast-feeding, where the virus has been isolated from breast milk of infected mothers; children born to infected mothers have a higher prevalence of seropositivity (Heneine et al. 1992; Vitek et al. 1995).

HTLV Receptor and Infectivity The frequency of cell-free virus infection with HTLV is relatively low compared to cell-associated infection. Efficient infection of cells with HTLV in vitro occurs by cocultivating target cells with gamma irradiated or mitomycin C-treated virus producer cells. In vivo, HTLV-1 and HTLV-2 display distinct tropism for cellular transformation, transforming primarily CD4+ and CD8+ T-cells, respectively (Miyamoto et al. 1991; Ijichi et al. 1992; Lal et al. 1995). This in vivo tropism is recapitulated in tissue culture transformation assays (Robek and Ratner 1999; Wang et al. 2000; Ye et al. 2003). Cell tropism can be determined at the level of viral entry (receptormediated) as has been reported for poliovirus or HIV, or postentry (integration, transcription, or translation) as reported for murine leukemia virus (Chatis et al. 1984; Browning et al. 1997; Moore et al. 1997; Pleskoff et al. 1997). HTLV-1 and

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HTLV-2 efficiently enter CD4+ and CD8+ T-cells as well as B-cells, epithelial cells, and macrophages. However, the transformation tropism is restricted to CD4+ or CD8+ T-cells (Macatonia et al. 1992; Koyanagi et al. 1993; Jones et al. 2008), which suggests that the distinct HTLV-1 and HTLV-2 transformation tropism is not specifically determined by virus entry. Studies utilizing HTLV-1/HTLV-2 recombinant viruses revealed that the viral envelope is responsible for this preferential cellular tropism (Ye et al. 2003; Xie and Green 2005). The viral envelope has two glycoproteins – SU and TM. SU binds to the cellular receptor, while TM triggers the fusion of the viral and cellular membranes, facilitating viral entry. Binding studies have confirmed these findings and have revealed differential binding and entry properties of HTLV-1 and HTLV-2. HTLV-1 is highly dependent on an initial interaction with heparan sulfate proteoglycans (HSPG) found abundantly on CD4+ T-lymphocytes and subsequent GLUT-1 and/or neuropilin-1 (NRP-1) interactions. HTLV-2 binding and entry appears to be less dependent on HSPGs, which are limited on CD8+ T-cells, and highly dependent on interaction with GLUT-1 (Ghez et al. 2006; Jones et al. 2006).

HTLV-2 and Associated Diseases HTLV-2 and Neurologic Abnormality Although HTLV-2 was isolated from a patient with hairy cell leukemia (Kalyanaraman et al. 1982) and has the ability to transform T-cells in culture (Ross et al. 1996), clinical data associating HTLV-2 with malignancy have not been conclusive. On the contrary, an increased body of evidence correlates HTLV-2 infection with a neurological disease similar to HTLV-1 associated myelopathy/tropic spastic paraperesis (HAM/TSP) (Hjelle et al. 1992; Harrington Jr et al. 1993; Jacobson et al. 1993; Sheremata et al. 1993; Lehky et al. 1996; Murphy et al. 1997; Silva et al. 2002). The disease manifestation develops decades after infection, usually consists of neurologic disability and peripheral neuropathy (Murphy et al. 1997), and has a higher prevalence among women (Biglione et al. 1993; Harrington Jr et al. 1993; Jacobson et al. 1993; Sheremata et al. 1993; Black et al. 1996; Lehky et al. 1996; Murphy et al. 1997; Silva et al. 2002; Biglione et al. 2003; Orland et al. 2003; Araujo and Hall 2004; Biswas et al. 2009). Other reports of rare HTLV-2-associated disease include the development of a spinocerebellar syndrome (Castillo et al. 2000) and predominantly sensory polyneuropathy (PSP) (Zehender et al. 1995). (Fig. 26.5) A clear link between HTLV-2 and neurological disease has been difficult to conclude mainly due to the lower prevalence of identified HTLV-2 infection worldwide compared to HTLV-1, thus providing limited epidemiological data on disease association. Additional difficulties are due to concomitant infections with HIV-1, hepatitis B and C, and human herpes viruses (Berger et al. 1991; Silva et al. 2002; Araujo and Hall 2004; Biswas et al. 2009; Rosenblatt et al. 1992). Additionally, HTLV-2 neurologic disorders present with myelopathic and cerebellar features, similar to diseases such

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Rantes CCRs

HIV-1 infected CD4+ T-cell Cell death/viral spread

Fig. 26.5 List of neurologic signs and symptoms in HTLV-2 infected individuals

as multiple sclerosis and spinocerebellar degeneration (Oger and Dekaban 1995; Peters et al. 1999; Araujo and Hall 2004). Therefore, a thorough investigation and a careful interpretation of the symptoms and family history must be carried out before linking HTLV-2 with a particular disease manifestation.

HTLV-2 and Hematopoiesis Although the HTLVs infect T-cells in vivo (and to a lesser extent B-cells and monocytes), little has been done to evaluate the effect of the infection, particularly with HTLV-2, on hematopoiesis. Previously it was noted that HTLV-2 infection causes elevated lymphocyte counts (Itoyama et al. 1988; Prince et al. 1990, 1994). In the HTLV Outcome Study (HOST), Bartman et al. reported that HTLV-2 infected patients have an increase in absolute lymphocyte counts compared to HTLV-1 patients and seronegative individuals (Bartman et al. 2008). The mechanism for this significant increase is not well understood but several reasons have been postulated including (1) downstream effects of Tax-2 on T-cell proliferation and antiapoptotic factors, (2) inhibition of the immunologic responses to respiratory infections (Murphy et al. 2004; Asquith et al. 2007) and/or (3) an inflammatory response to the viral infection (Murphy et al. 1997; Oliveira et al. 2009). Interestingly, in infected individuals, antibodies are raised against all three Gag proteins p15, p24 and p19, and two envelope proteins gp46 and p21 (Clapham et al. 1983; Hoshino et al. 1983; Essex et al. 1984; Ratner 1996). Recently, an HTLV-2-specific CTL response against

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Tax-2 has been identified in infected individuals (Oliveira et al. 2009). The CTL response was described previously in HTLV-1 infected individuals and was found to play a pivotal role in controlling proviral load (Jacobson et al. 1990; Parker et al. 1992). In addition, high levels of CTL responses have been described in HIV infected individuals resulting in suppression of viral load and delayed AIDS progression (Goulder et al. 1997), as well as in healthy CMV seropositive individuals (Gillespie et al. 2000). The direct implication of a CTL response in HTLV-2 infected individuals is not yet clear but data suggest that it is a factor in limiting the number of infected cells as measured by proviral load, and might be important in preventing disease development in infected individuals.

HTLV-2 and HIV Coinfection In the past decade, there has been an increased incidence worldwide of HTLV coinfection with other viruses, but particularly with HIV-1. The rate of coinfections between HIV-1 and HTLV-1 is increasing among south Americans and Africans, while HIV-1 and HTLV-2 is increasing among north American and European intravenous drug users (IDVUs) (Khabbaz et al. 1992; Briggs et al. 1995; Salemi et al. 1995; Hershow et al. 1996; Egan et al. 1999; Soriano et al. 1999; Goedert et al. 2001; Guimaraes et al. 2001). The fact that HTLV-1 and HIV-1 infect CD4+ T-cells while HTLV-2 has a preferential tropism for CD8+ T-cells appears to influence HIV-1 pathogenesis (Casoli et al. 2007; Beilke et al. 2004). The outcome of HTLV-2– HIV-1 coinfection suggests that HTLV-2 has a protective role by maintaining CD8+ and CD4+ T-cell counts and lowering HIV-1 replication, resulting in delayed progression to AIDS. In vitro experiments investigating coinfection of peripheral blood mononuclear cells (PBMCs) with HTLV-2–HIV-1 provide valuable information on the mechanism by which HTLV-2 plays a protective role. Casoli et al. reported that in HTLV-2–HIV-1 coinfection, HTLV-2 expression occurs earlier than HIV-1 (Casoli et al. 2000). In addition, HTLV-2 infected CD8+ T-cells secrete increased concentrations of three HIV-1 suppressive chemokines including macrophage inflammatory protein 1a (MIP-1a or CCL3), MIP-1b (CCL4), and RANTES (CCL5) in vitro. Interestingly, the concentration of the chemokines (mainly MIP-1a) was inversely related to HIV-1 replication, and upon the addition of antichemokine monoclonal antibodies (Mabs), the protective HTLV-2 function was abrogated (Casoli et al. 2000). MIP-1a, MIP-1b and RANTES are CCR5 ligands, and CCR5 acts as a coreceptor for non-syncytium-inducing (NSI) macrophage-tropic strains of HIV-1, which dominate at the early onset of HIV-1 infection (Cocchi et al. 1995; Dragic et al. 1996; Oravecz et al. 1996; Moriuchi et al. 1998; Zagury et al. 1998). In addition, the HTLV-2 positive regulator Tax-2 has been shown to induce the expression of MIP-1b and RANTES (Lewis et al. 2000), but little is known about its effect on MIP-1a. Interestingly, Tax-1 positively regulates the expression level of MIP-1a (Baba et al. 1996). Tax-2 also can inhibit HIV-1 replication by interacting with the major histocompatibility complex class II transcriptional activator (CIITA) (Accolla

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Fig. 26.6 A model of the interplay between HTLV-2 and HIV-1 in coinfected individuals. HTLV-2 induces several C–C chemokines leading to inhibition of HIV-1 replication

et al. 2001; Casoli et al. 2004). CIITA can inhibit Tax-2 transactivation of the HTLV LTR, and similarly inhibit HIV replication by targeting the HIV positive regulator Tat (Accolla et al. 2002). HTLV-2 infected cells also secrete additional chemokines and cytokines that induce a protective Th1 response against invading pathogens (O’Garra 1998); a Th2 response seems to positively correlate with progression of HIV-1 infection (Clerici and Shearer 1994) (Fig. 26.6). Although the effect of HTLV-2 on HIV-1 appears more prominent than that of HIV-1 on HTLV, it has been demonstrated that HIV-1 Tat up-regulates both HTLV-1 and HTLV-2 gene expression in coinfected individuals (Beilke et al. 1998; Roy et al. 2008). The long-term implication of such interactions warrants further investigation for its importance on AIDS and HTLV-associated disorders. For example, it is poorly understood whether HTLV-2–HIV-1 coinfection can lead to additional complications, and epidemiological data suggest an increase in the incidence of neurologic disorders and liver dysfunction in HTLV-2–HIV-1 dually infected individuals (Beilke et al. 2004). On the contrary, special consideration should be given to HTLV-2–HIV-1 patients regarding highly active antiretroviral therapy (HAART).

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HAART can reduce HIV-1 morbidity and mortality but appears to increase HTLV-2 proviral load. In addition, the disorders affecting HAART treated HIV-1/HTLV-2 coinfected individuals are more of an inflammatory nature than of opportunistic pathogens. This is consistent with a greater incidence of neurological diseases in HIV-1/HTLV coinfected individuals (Casoli et al. 2007).

Tax Oncoprotein Mechanisms of Action Tax is a positive regulator of viral replication and displays oncogenic properties including the transformation of rodent fibroblasts and induction of tumors in transgenic animals. In addition, several studies using infectious molecular clones of HTLV-1 and HTLV-2 showed a direct role of Tax in transformation of human T-lymphocytes, and ample evidence points to a pivotal role of Tax in HTLV pathogenesis (Nerenberg et al. 1987; Tanaka et al. 1990; Grassmann et al. 1992; Yamaoka et al. 1992; Ross et al. 1996; Robek and Ratner 1999). Tax functions both at the transcriptional and posttranscriptional levels by modulating viral and cellular proteins required for viral replication and transformation. The different subtypes of HTLV-2 (A–D) encode slightly different Tax proteins. Tax-2B, 2C, and 2D are similar but not identical and vary slightly in length (356, 356, 344 amino acids), whereas Tax-2A is shorter and contains 331 amino acids (Feuer and Green 2005). However, it is not known if the different forms of Tax-2 contribute differently to viral replication, lymphocyte immortalization, and/or persistence.

Tax and Transcription Tax was first identified as a positive regulator of viral transcription. Interestingly, Tax is capable of transactivating several cellular genes important for cell proliferation and eventually transformation. Tax is expressed early after infection, and transactivates viral gene expression by recruiting activating transcription factor (ATF) and cyclic-AMP response element binding (CREB) proteins to the cyclicAMP response element (CRE) present at the viral promoter located at the 5¢LTR. Tax recruits these coactivators of transcription to its response element (TRE) located in the U3 region of the 5¢LTR (Zhao and Giam 1992; Wagner and Green 1993; Adya et al. 1994; Anderson and Dynan 1994; Yin et al. 1995; Bantignies et al. 1996). Recently, Tosi et al. have demonstrated that Tax-2 has the ability to recruit CBP/p300 to the viral LTR, while Tax-1 recruits CBP/p300-associated factor (PCAF), histone acetyl transferases (HATS) and CBP/p300 (Tosi et al. 2006). This difference might, in part, explain the differential profile of genes activated by Tax-1 compared to Tax-2. A mutational analysis was carried out to better understand the contribution of the different domains of Tax-2 to its transcriptional activity. Tax-2

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mutations that disrupted the zinc-finger domain and that affected the CREB/ATF domain failed to transactivate the HTLV-2 LTR (and HTLV-1 LTR) (Ross et al. 1997). However, it was found that HTLV-2-mediated T-cell transformation is dependent on Tax-2 activation of both NFkB and CREB/ATF, which then induces IL-2 independent T-cell transformation (Feuer and Green 2005; Ross et al. 1996). The activation of NFkB is absolutely essential at early stages of cellular transformation, while CREB/ATF activation by Tax is required for sustained cell growth (Ross et al. 2000). Recently, it has been shown that Tax-2 can interact with CIITA and NF-Y and this interaction was found to inhibit Tax-2-mediated activation of the viral LTR. CIITA is the master regulator of the expression of MHC class II genes that are important for the homeostasis and regulation of the immune system (Reith et al. 2005). CIITA is a non DNA binding transcriptional integrator recruited to the promoter by multiple transcription factors including nuclear factor Y (NF-Y) (Kretsovali et al. 1998; Spilianakis et al. 2000; Fontes et al. 1999a, b). One study showed that CIITA targets HIV-1 Tat and represses its transactivation of the HIV LTR (Accolla et al. 2002). Similarly, CIITA inhibits Tax-2 transactivation of the LTR promoter in HLA-II positive cells by recruiting NF-Y (Casoli et al. 2004; Tosi et al. 2006). The repression of Tax-2 transcriptional activity is the major, and probably the exclusive mechanism involved in reducing HTLV-2 replication in B-cells and T-cells (Tosi et al. 2009).

Tax Posttranscriptional Effects The transformation properties of Tax are a consequence of the ability of the protein to deregulate the transcription of genes and signaling pathways involved in cellular proliferation, cell cycle control and apoptosis (Hall and Fujii 2005). Interestingly, the Tax-1 carboxy terminus was shown to be responsible for increased transformation efficiency in rodent fibroblasts and increased micronuclei formation (Rousset et al. 1998; Hall and Fujii 2005). This region contains a PDZ binding motif (PBM), which is absent in Tax-2. The PDZ domain is a protein–protein interaction region, and is important for the interaction of Tax-1 with tumor suppressors such as hDLG, APC, Dlg-1, Scribbles, or precursor of interleukin-16 (proIL-16), and membraneassociated guanylate kinase (MAGUK) with inverted orientation (MAGI)-3 (Suzuki et al. 1999; Wu et al. 2000; Adamsky et al. 2003; Wilson et al. 2003; Ohashi et al. 2004; Yao et al. 2004; Ishioka et al. 2006; Okajima et al. 2008). In addition, this domain was found to contribute to HTLV-induced proliferation and immortalization of primary T-cells in vitro and viral survival in the rabbit animal model (Tsubata et al. 2005; Xie et al. 2006). A chimeric Tax-2 encoding the last 53 amino acids of Tax-1, which contains the PBM, resulted in increased transformation of Rat fibroblasts (Endo et al. 2002), increased micronuclei formation and increased T-cell proliferation in vitro (Xie et al. 2006). Data suggest that the interplay between the PDZ domain and interacting partners are major players in the differences in pathogenesis between HTLV-1 and HTLV-2.

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Tax/NFk B Interaction NFkB is a family of transcription factors that include RelA, RelB, c-Rel, NFkB1 (p105 catalyzed into p50), and NFkB2 (p100 catalyzed into p52). These transcription factors play an important role in immune function, innate and adaptive responses, development, and cell survival (Vallabhapurapu and Karin 2009). NFkb activity is tightly controlled in T-cells, but is constitutively active following HTLV infection. In the canonical pathway, NFkb is inhibited by inhibitor of kb (Ikb), which in turn is under the control of inhibitor of kb kinase (IKK). Tax ubiquitination is crucial for its interaction with the IKKg subunit (NEMO) and subsequent activation of NFkB (Shembade et al. 2007). One study reported that once Tax-2 is polysumoylated, it localizes to the nucleus (similar to Tax-1), thus bringing it in close proximity to RelA (Turci et al. 2009) and bypassing IKK and Ikb. On the contrary, the noncanonical pathway involves IKK phosphorylation of p100 in the NFkB2/p100 complex with subsequent processing into p52. In contrast to Tax-1, Tax-2 does not affect the noncanonical pathway (Higuchi et al. 2007; Higuchi and Fujii 2009). A region in Tax-1 located at 225–232 and not conserved in Tax-2 is responsible for the Tax1-p52 interaction (Shoji et al. 2009).

Tax-2 and Cell Cycle Dysregulation Cell cycle is a tightly regulated process, its perturbation is a hallmark of cellular transformation, and is a common target for viral oncoproteins (Feuer and Green 2005). The cell cycle is regulated by cyclin-dependent kinases (CDKs) and different D and E type cyclins. CDKs are under the control of CDK inhibitors (CDKI) such as p16ink and p21cip1/waf (Akagi et al. 1996; Mori et al. 2002). p21cip1/waf is part of the CDK2/ D1/PCNA complex and is required for G1 and G2 control; however, over-expression of p21 inhibits G1 and G2 control via p53-dependent and -independent mechanisms (Macleod et al. 1995). Stably transfected cell lines that produce Tax-2 (or Tax-1) have been useful in understanding the effect of Tax-2 on cellular proliferation. Tax-2 modulates the cell cycle at several steps. Tax-2 activates the expression of p21cip1/waf, but at a lower extent than Tax-1 (~25–35% less) (de La Fuente et al. 2000; Sieburg et al. 2004). In addition, Tax-2 has been shown to reduce cellular proliferation kinetics in Jurkat cells but to a lesser extent than Tax-1 (Sieburg et al. 2004; Feuer and Green 2005). Tax-2 induces the formation of multinucleated cells, suggesting that it might block progression or completion of mitosis or cytokinesis due to disruption of cell cycle checkpoints leading to unscheduled S phase entry and accumulation of DNA damage (Lemoine and Marriott 2001; Marriott et al. 2002). A great deal of knowledge about Tax comes from over-expression in transfected cell lines that do not represent the viral target cells in vivo, or are not likely to be encountered by the virus. Studies in primary hematopoietic CD34+ progenitor cells (HPC) failed to show arrest of the cell cycle, suppression hematopoiesis, or modulation of p21cip1/waf1 or p27kip1 gene expression via Tax-2 (contrary to Tax-1) (Feuer and Green 2005).

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Tax-2 Modulation of Apoptosis Apoptosis, a form of well controlled programmed cell death, is marked by a defined sequence of morphological changes. Apoptosis is required during embryonic development and in adult life to maintain proper homeostasis. It has been well documented that disruption of apoptosis can lead to cancer, neurodegenerative diseases, and immunological diseases. Expression of both Tax-1 and Tax-2 protects cells from apoptosis by modulating the activity of several pro-apoptotic genes. For example, inhibition of p53 function is associated with impaired function of Tax-2A. Interestingly, Tax-2B has been found to prevent apoptosis in infected cells by modulating p53 activity, an activity shared with Tax-1 (Mahieux et al. 2000; Van et al. 2001). Tax-2B represses p53 in T-cells, which is correlated with NFkB activation. However, in nonlymphoid cells, Tax-2B uses CBP binding to inhibit expression of p53, most likely through the interaction of CBP and the CR2 sequence of Tax-2 (Meertens et al. 2004b). Tax-2 also has been found to protect Jurkat T-cells as well as murine fibroblasts from apoptosis following serum deprivation (Saggioro et al. 2001; Sieburg et al. 2004). In addition, Tax-2 protects the cells from Fas-mediated apoptosis by up-regulating Bcl-XL expression (Zehender et al. 2001).

HTLV Experimental Models The development of culture and animal models to study HTLV-2 infection are important since they have the potential to yield valuable information about requirements for infectivity, persistence, prevention and treatment of the virus infection (Cockerell et al. 1991). HTLV experimental systems present a great challenge due to the poor replication of the virus in vitro and the inefficiency of cell-free infection; efficient infection requires the cocultivation of irradiated cells with PBMC. In addition, HTLV infects a wide variety of cells including B-cells, T-cells, endothelial cells, glial cells, and monocytes; but only primary T-cells are susceptible to transformation (Ho et al. 1984; Hoffman et al. 1984; Akagi et al. 1992; Koyanagi et al. 1993). Transformation of T-cells by HTLV is defined as IL-2-independent growth. Morphologically, transformed cells are evident microscopically as refractile cell clusters within 7–10 weeks of coculture; however, establishing transformed cells requires months in culture. Miyamoto et al. demonstrated that rabbit leukocytes can be transformed in vitro by HTLV-2 (Miyamoto et al. 1990). Rabbits then were assessed for their ability to be infected with HTLV-2. The in vivo infectivity was evident as early as 2 weeks and persisted until 24 weeks (the end of the experiment) as measured by anti-HTLV antibody response (against p24 and env gp46) and demonstration of viral antigens or detectable proviral sequences in tissues of inoculated rabbits (Cockerell et al. 1991). Interestingly, HTLV-2 was found to be less infectious, and to replicate less efficiently in the rabbit model than HTLV-1 (Cockerell et al. 1991; Lairmore et al. 1992). In addition, HTLV-2 infection does not seem to correlate with any clinicopathologic evidence of disease in rabbits (Cockerell et al. 1991; Yamamoto et al. 2008; Xie et al. 2009).

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Conclusion In recent years, there has been increased interest in HTLV-2 because it has been associated with several neurological abnormalities, increased viral spread due to IVDU and contaminated drug-related paraphernalia, and an effect on AIDS progression in HIV-1–HTLV-2 coinfected individuals. Furthermore, comparative studies between HTLV-1 and HTLV-2 have provided additional insight into the activity and function of viral regulatory and accessory proteins in the context of the host and during viral replication. Nevertheless, similarities and differences occur between HTLV-1 and HTLV-2 that account for the different pathological effects observed. It is becoming apparent that these differences cannot be attributed to any one single viral or host protein, but likely involves a contribution of Env (tropism), Tax, and accessory gene products and their differential interaction with host cellular factors. Despite advances made in understanding the molecular basis of viral infection, little is known about HTLV-2 pathogenesis, and research is warranted to better understand (1) the causes of the long latency period of HTLV-2 infection, (2) the reasons why HTLV-2 is associated with few cases of neurodegenerative diseases but no malignancies (unlike HTLV-1), (3) the effect of the viral infection on the immune system both short-term and long-term, and (4) the role of the host (in particular the restriction factors) in controlling HTLV-2 infection.

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Chapter 27

Mechanisms of Oncogenesis by Avian and Murine Retroviruses Karen Beemon and Naomi Rosenberg

Introduction The oncogenic retroviruses (formerly called RNA tumor viruses) have played an important role in cancer research since their discovery in chickens 100 years ago. The discovery of retroviral oncogenes established the central paradigm that cancer is a genetic disease. Since retroviral oncogenes are captured host genes, study of animal retroviruses is very relevant to understanding mechanisms of human cancer initiation and maintenance. Investigations of tumors induced by oncogene-containing retroviruses first demonstrated that altered or overexpressed cellular genes can provide a dominant signal initiating oncogenesis. The obligate integration step that characterizes retroviral replication allows these viruses to alter the expression of cellular genes by insertional mutagenesis. Our knowledge of the ways in which cellular genes can contribute to cancer is based on fundamental studies of retroviruses. Furthermore, many genes that participate in human tumor development were first isolated as retroviral genes or as sites of retroviral integration. Thus, studies directed at understanding viral mechanisms of oncogenesis revealed insights into more general cellular mechanisms of tumor induction. Novel ways to disrupt normal cell function and to regulate gene expression have all emerged from the study of these viruses. Several different mechanisms of oncogenesis have been associated with different classes of retroviruses (Table 27.1). First, the oncogene-containing retroviruses, such as the avian Rous sarcoma virus (RSV), the murine sarcoma viruses (MSV),

K. Beemon (*) Department of Biology, Johns Hopkins University, Baltimore, MD 21218, USA e-mail: [email protected] N. Rosenberg Sackler School of Graduate Biomedical Sciences, Tufts University School of Medicine, Boston, MA 02111, USA E.S. Robertson (ed.), Cancer Associated Viruses, Current Cancer Research, DOI 10.1007/978-1-4614-0016-5_27, © Springer Science+Business Media, LLC 2012

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Table 27.1 Mechanisms of retroviral oncogenesis Mechanism and representative viruses Oncogene(s) Oncogene capture Rous sarcoma virus v-src MC29 v-myc Fujinami sarcoma virus v-fps Avian myeloblastosis virus v-myb Reticuloendotheliosis virus-T v-rel Abelson murine leukemia virus v-abl Moloney murine sarcoma virus v-mos Ha/Ki murine sarcoma virus v-ras Feline sarcoma viruses v-fes, v-fms, v-fgr Simian sarcoma virus v-sis Walleye dermal sarcoma virus rv-cyclin Insertional mutagenesis Avian leukosis virus Murine leukemia virus Feline leukemia virus Mouse mammary tumor virus

myc, myb, erbB, TERT, bic, and others myc, myb, and others myc, myb, and others wnt-1, fgf, and others

microRNA induction Avian leukosis virus Reticuloendotheliosis virus-T Radiation leukemia virus Friend murine leukemia virus SL3-3 murine leukemia virus

miR-155 miR-155 miR-106-363 cluster miR-17-92 cluster miR-17-92, miR-106-363 clusters

Env signaling Friend spleen focus-forming virus Jaagsiekte sheep retrovirus Avian hemangioma virus

altered env product altered env product altered env product

Accessory genes Bovine leukemia virus Human T-cell leukemia virus

tax tax, HTLV-I basic leucine zipper factor (HBZ)

and Abelson murine leukemia virus, have all captured oncogenes from their hosts. Second, a large number of viruses lacking oncogenes, including avian leukosis virus (ALV), murine leukemia virus (MLV), feline leukemia virus (FeLV), and murine mammary tumor virus (MMTV), activate cellular oncogenes by insertional mutagenesis. A third type of oncogenesis involves activation of cellular microRNAs, either by insertional mutagenesis (ALV and MLV) or by transcriptional activation (reticuloendotheliosis virus). A fourth type of viral oncogenesis involves signaling by the viral env glycoprotein gene and is used by Friend spleen focus forming virus (SFFV) and Jaagsiekte Sheep Retrovirus (JSRV). Lastly, accessory (nonstructural) genes of human T-lymphotropic virus (HTLV) and bovine leukemia virus (BLV) such as tax and HBZ are key to cancer induction by these agents. HTLV is discussed in a separate chapter in this book. We discuss the first four mechanisms of oncogenesis here.

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Types of Oncogenic Retroviruses The prototypical oncogenic retroviruses were discovered over 100 years ago. RSV was discovered by Peyton Rous in 1910 (Rous 1910) and shown to be a filterable agent that causes solid tumors in chickens. Ellerman and Bang discovered ALV in 1908 (Ellerman and Bang 1908) and showed that the virus causes leukemia and lymphoma in chickens. Subsequent studies in mammals and other hosts led to discoveries of other tumor-inducing viruses, some of which contained oncogenes in their genomes and others that did not. The early discoveries of Bittner (Bittner 1942) and Gross (1951a, b) revealed that retroviruses were associated with mammary tumors and thymic lymphomas in mice. Subsequent studies revealed that many animals are infected by oncogenic retroviruses including cats, cows, rats, sheep, goats, koalas, several primates, and some fish (Rosenberg and Jolicoeur 1997; Hanger et al. 2000). Retroviral particles have also been found in tumors in vipers, but their role in the disease process has not been studied thoroughly (Andersen et al. 1979). The isolation of HTLV in 1980 marked the discovery of a retrovirus that caused malignant disease in humans (Poiesz et al. 1980). All of these viruses belong to the retrovirus subfamily called Orthoretrovirinae (Linial et al. 2005), a group that contains six different genera that are named for the first six letters in the Greek alphabet (e.g., Alpharetrovirus, Betaretrovirus, etc). The classification scheme is based on genome structure, virion morphology, and sequence relationships. All six of these genera contain members that have oncogenic potential. In addition to classification by genera, oncogenic retroviruses are often divided into two groups based on the mechanisms by which they induce tumors. One group of viruses contains oncogenes, while the other group does not. Viruses of the first type were derived from the second type by a process called oncogene capture. Retroviral oncogenes, called v-onc genes, were derived from cellular sequences called protooncogenes (c-onc genes) and incorporated into the viruses by recombination during virus integration and replication. Typically, these viruses are replication defective because one or more genes required for virus replication have been lost as a consequence of recombination with the c-onc gene. Viruses of this type typically induce tumors with a relatively short (several week) latent period and require a helper virus for replication. These viruses also typically transform cells in tissue culture, a property that advanced studies of oncogenic mechanisms used by these viruses. The second type of oncogenic retrovirus does not contain genes derived from cellular sequence and typically induces tumors by integrating into the host genome and altering the expression of a cellular gene. The majority of these viruses induce tumors after a much longer latent period than viruses that contain v-onc genes with tumors arising several months or more after infection. In addition, viruses of this type do not transform cells in tissue culture. ALV and MLV are prototypes of these viruses. A second type of virus that lacks an oncogene uses a modified env gene product to stimulate cell growth. Viruses of this type, such as JSRV and SFFV, are associated with tumors that arise in a relatively short latent period. They also transform some cell types in tissue culture. These properties reflect the fact that a virus-encoded product is responsible for causing altered growth.

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Fig. 27.1 Oncogene capture and the generation of v-onc-containing viruses. The process by which oncogenes are thought to be incorporated into retroviruses is illustrated. Each step is described in more detail in the text. Cellular exons: dark boxes; Cellular regulatory sequences: gray boxes; RNA: dashed lines with spliced information shown as solid lines

Oncogene Capture and the Generation of v-onc-Containing Viruses Oncogene capture is the process by which portions of c-onc genes are incorporated into retroviruses. Although over 100 different retroviruses have captured v-onc genes (Rosenberg and Jolicoeur 1997), the process is actually rare. Most of these viruses arose once and were identified because the animal from which the virus was isolated was part of a laboratory experiment or in close contact with people. Most commonly, these latter instances have involved pets, especially cats, and farm animals, especially chickens. The rarity with which v-onc gene capture occurs has made it difficult to study the process directly. Thus, the precise mechanisms involved have received limited experimental validation. Indeed, much of the information that has contributed to the model by which oncogene capture occurs has involved comparison of the viral and cellular sequences and a general knowledge of retrovirus replication. The integration of a replication-competent retrovirus into the host genome, an obligate step in the life cycle of all retroviruses, is the first step in oncogene capture (Fig. 27.1). Following integration, the viral genome is transcribed using cellular machinery and although most transcripts terminate in the 3¢ LTR, a fraction fail to do so and continue into flanking cellular sequences generating transcripts called readthrough transcripts. In one instance where this phenomenon has been studied (ALV), about 15% of the transcripts were shown to be of this type (Herman and Coffin 1986). In cases where the virus integration has occurred near a c-onc gene, the read-through transcript can contain these sequences. Because integration is largely random (Bushman et al. 2005), this event occurs rarely. However, if the

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readthrough transcript can lead to expression of the c-onc gene product, the cell is likely to be at a selective advantage. The second step in oncogene capture involves the packaging of the readthrough transcript in virions that are released from the cell. Retrovirus virions contain two copies of the virus genome, a feature that is necessary for successful replication following infection of a cell. Reverse transcription requires both copies to generate the cDNA copy that goes on to integrate into the genome. The final step in the process occurs after the virion containing one normal genome and one hybrid transcript infects a new cell. As part of the reverse transcription process, the template switching between the two RNAs can lead to recombination and incorporation of the cellular sequences within the viral genome (Fig. 27.1). The general aspects of this model are consistent with studies done using plasmids that mimic features of some of the intermediates that are postulated to play a key role in oncogene capture (Swain and Coffin 1992).

RSV and Oncogene-Containing Retroviruses The earliest characterized oncogene was the v-src oncogene of RSV (Martin 2001). The genomic RNA of RSV was found to be about 2 kb larger than that of ALV (Duesberg and Vogt 1970). Comparisons by RNase T1 fingerprinting between ALV and RSV genomic RNA showed them to be very closely related. However, RSV had an additional genomic sequence that was mapped near the 3¢ poly(A) sequence (Wang et al. 1975). Hybridization experiments, using a src-specific probe, showed that uninfected cells had related sequences; these were called c-src (Stehelin et al. 1976). In vitro translation experiments, together with generation of src-specific antibodies, defined the Src protein as a 60 kDa phosphoprotein called pp60src (Beemon and Hunter 1978; Brugge and Erikson 1977). The c-Src protein had a C-terminal amino-acid sequence, with repressive activity, that was replaced in the v-Src protein. Surprisingly, Src was found to be a tyrosine-specific kinase, a novel specificity at the time (Hunter and Sefton 1980). To date, approximately 100 different oncogene-containing retroviruses have been described. These viruses are derived from a variety of different hosts, with the majority coming from chickens, mice, cats, and rats. All of these animals can be readily infected with the replication-competent, non-oncogene-containing retroviruses that serve as the viral parent of these agents. Such infections are a prerequisite for generation of retroviruses that contain oncogenes. As noted earlier, most of these viruses are replication defective because the function of at least one viral gene required for replication has been lost during oncogene capture. RSV is a notable exception to this generalization and is the only replicationcompetent, v-onc gene-containing virus; it probably arose from more than one recombination event. The other viruses of this type exist naturally in combination with a replication-competent virus that provides the proteins required for replication. Viruses performing this function are typically referred to as “helper” viruses. Although helper viruses play a role in the spread of viruses containing v-onc genes,

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Fig. 27.2 Oncogene-containing retroviruses encode viral-onc fusion proteins. The structure of representative v-onc gene containing retroviruses is illustrated. The first five viruses were derived from ALV, and the last two were derived from MLV. Sequences derived from the replicationcompetent parent of the viruses are shown as open boxes; v-onc gene sequences are shown as shaded boxes. The proteins encoded by the v-onc genes are also illustrated but viral proteins that are expressed by some of the viruses are not shown. The drawing is not precisely to scale

study of helper virus free stocks, prepared using plasmids that provide the proteins required for replication, has revealed that the helper virus is not required for tumor induction or for transformation of cells in tissue culture (Green et al. 1987; Andersson et al. 1979; Dunbar et al. 1991; Fichelson et al. 1995). The genome structure of viruses that contain v-onc genes varies, but in all cases, the structure leads to a protein product that is directly capable of stimulating cell growth (Rosenberg and Jolicoeur 1997). In many instances, the v-onc gene exists as a fusion with part of a viral gene, particularly the gag gene that normally encodes virion structural proteins (Fig. 27.2). The product of such genes is typically a fusion

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Mechanisms of Oncogenesis by Avian and Murine Retroviruses Table 27.2 Functions of viral oncogenes Protein Function Gene Growth factor v-sis Receptor tyrosine kinase v-erbB c-erbB v-kit v-fms Nonreceptor tyrosine kinase v-src v-fps v-abl v-fgr v-fes G protein v-rasK v-rasH Serine/threonine kinase v-mos v-raf v-akt Adapter protein v-crk Transcription factor v-erbA v-myc v-myb v-ets v-fos v-rel

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Virus SSV AEV ALV HZ4-FeSV SM-FeSV RSV FuSV-ASV Ab-MLV GR-FeSV ST-FeSV Ki-MSV Ha-MSV Mo-MSV 3611-MSV Akt8 CT-10-ASV AEV MC29 BAI-AMV ALV E26-AMV FBR-MSV

protein containing sequences from both genes, and the determinants derived from the viral gene can influence the localization or function of the oncoprotein. Some v-onc fusions involve the viral env gene. Lastly, some v-onc genes are expressed without additional sequences derived from viral genes. In a few cases, viruses that contain two v-onc genes have been isolated. Examples include some isolates of avian erythroblastosis virus that contain both v-erbA and v-erbB (Engelbreth-Holm and Rothe-Meyer 1935), MH2, an avian virus that induces several types of tumors and contains both v-mil (v-raf) and v-myc, (Begg 1927) and E26, an avian virus that induces myeloblastosis and contains both v-myb and v-ets (Ivanov et al. 1962). Because the protooncogenes that were captured in these cases are not physically linked in the genome, these viruses appear to have arisen as a consequence of two independent capture events. Experimental evidence shows that both oncogenes contribute to the types of tumors induced by these viruses (Metz and Graf 1991; Beug and Graf 1989; Hartl et al. 2006). A survey of the different proteins encoded by retroviruses expressing v-onc genes reveals examples of molecules in many signaling pathways that modulate cell growth and differentiation (Table 27.2). For example, a number of oncoproteins, including Abl, Fes, Fps, and Yes, encode protein intracellular tyrosine kinases. Others, such as Fms, Fgr, Kit, ErbB, and Met, encode protein tyrosine kinase receptors, while Sis is a growth factor. Serine/threonine kinases including Akt, Raf, and Mos, the G protein Ras, and the adaptor protein Crk are expressed by one or more oncogenic retroviruses.

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Fig. 27.3 Activation of cellular oncogenes by insertional mutagenesis. Four different mechanisms by which cellular protooncogenes can be activated by insertional mutagenesis are illustrated: I. downstream transcriptional activation; II. enhancer mediated activation; III. read-through transcription; IV. alteration of 3¢ regulatory elements. Additional details are presented in the text

Transcription factors including Myc, Myb, Fos, Jun, ErbA, and the Cbl E3 ligase are other types of signaling molecules that are responsible for the oncogenic properties of rapidly transforming retroviruses.

Activation of Cellular Oncogenes by Insertional Mutagenesis Many oncogenic retroviruses do not contain oncogenes, and viruses of this type are common in many species and exist naturally. They are the predominant cause of retroviral induced tumors outside of laboratory settings (Rosenberg and Jolicoeur 1997). The long latency associated with oncogenesis by these viruses and their close relationship to endogenous retrovirus elements that exist in the genomes of many animals contributes to their maintenance in animal populations. The oncogenic properties of these viruses reflect the fact that integration is an obligatory part of the retroviral life cycle and thus these agents are insertional mutagens that disrupt the DNA structure at the site of integration (Fig. 27.3). These disruptions can separate exons of cellular genes, resulting in the production of nonfunctional proteins or proteins with altered function. They can also separate regulatory elements such as 3¢ untranslated sequences that control the stability of cellular mRNAs from coding sequences. As a consequence, altered expression of genes near integration sites can contribute to oncogenesis.

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A second consequence of integration relates to the structure of the integrated provirus, which has strong promoter and enhancer sequences within the long terminal repeats (LTRs) that are located at both ends of the genome. The LTRs also contain other regulatory sequences such as polyadenylation sequences that are required for proper expression of viral RNAs. These sequences allow the virus to integrate moreor-less randomly in the host genome and express the viral genes. However, these sequences can also affect the expression of genes in the vicinity of the integration. The promoter sequences found in the LTRs “substitute” for the promoter normally associated with a cellular gene in the vicinity of the integration. In addition, the strong enhancer sequences located in both LTRs can stimulate expression of cellular genes, including some that are located at considerable distance from the integration site and like all enhancers, can function independent of orientation. Examples of all of these mechanisms have been found in a wide range of retrovirus-induced tumors.

Promoter Insertion Study of ALV-induced bursal lymphoma led to the discovery of the promoter insertion mechanism of retroviral oncogenesis. This tumor arises in the Bursa of Fabricius, an organ that generates B lymphocytes in birds. The first clues to the mechanism were the presence of common integration sites in all cells, demonstrating a clonal relationship and indicating that the cells in the tumor arose from a single virus-infected cell. These early Southern blot analyses suggested that the integration site of the virus might be related in some fashion to tumor induction (Neel et al. 1981; Payne et al. 1981). Further study showed that the tumors contained novel fusion mRNAs with both viral and cellular sequences. Hayward and coworkers (1981) found that most ALV-induced tumors had sense integrations in the cellular myc locus, an oncogene previously observed in the oncogene-containing MC-29 virus that induces myelocytomatosis. In most tumors, the provirus had integrated into the first intron of myc, downstream of a transcriptional pause site. Surprisingly, the 3¢ LTR drove transcription of the downstream cellular myc gene by a mechanism called “Promoter Insertion” (Hayward et al. 1981). The 5¢ LTR was not used in these tumors because of mutations downstream of the 5¢ LTR which somehow inactivated it (Fung et al. 1982; Goodenow and Hayward 1987). Since the first c-myc exon is noncoding, this resulted in overexpression of the normal cellular Myc protein, a transcription factor that is normally expressed only briefly during the cell cycle. This was the first evidence of tumor initiation by disregulated expression of a normal cellular gene. It was predicted that nonviral agents might also induce elevated expression of c-onc genes. In fact, this was rapidly born out when translocations leading to myc overexpression were discovered in Burkitt’s lymphoma (Klein 1983; Taub et al. 1982). Myc has also been activated by insertional mutagenesis by MLV and FeLV in a large number of mouse and cat tumors (Neil and Stewart 2011). Retroviruses use promoter insertion to induce tumors involving activation of other protooncogenes. For example, in certain lines of chickens, the c-erbB gene is

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activated by ALV integration, an event that induces erythroblastosis (Nilsen et al. 1985). In these tumors, transcription initiates in the viral 5¢ LTR and reads through the poly(A) site into the downstream c-erbB gene, which encodes an EGF receptor protein family member. Alternative splicing generates both Gag-ErbB and GagEnv-ErbB products. v-erbB is the oncogene of avian erythroblastosis virus and is an amino-truncated form of the EGF receptor, lacking the EFG-binding domain. Remarkably, ALV integrations within the middle of the c-erbB gene result in generation of a similarly truncated form of the EGF receptor. Promoter insertion can also be associated with oncogenesis in other animals (Rosenberg and Jolicoeur 1997). For example, a variety of MLVs use this mechanism to induce thymic lymphoma, a T-cell tumor that is one of the most common types of retrovirus-induced tumor in mice and rats. Moloney MLV can activate the protooncogenes gfi-1 (Gilks et al. 1993), lck (Shin and Steffen 1993) and others via this mechanism. Several CasBr strains of MLV, viruses that induce several hematologic tumors, have been shown to cause tumors in the same fashion (Bergeron et al. 1993; Askew et al. 1991). Promoter insertion has also been documented in T cell lymphomas induced by FeLV in domestic cats (Neil et al. 1984; Forrest et al. 1987).

Insertions Affecting Regulatory Sequences While the majority of ALV integrations in classical lymphoid leukosis were in myc intron 1 or upstream of it, integrations were also observed in the 3¢ untranslated region (3’UTR) (Payne et al. 1982). The 3¢ UTR insertions most likely supplied a new poly(A) site, leading to a shorter 3¢UTR. While it was not clear why this event was oncogenic at the time, we now know that mRNAs are often downregulated by interactions of microRNAs or regulatory proteins with the 3¢UTR. Thus, a shorter 3¢UTR would likely stabilize the mRNA, leading to overexpression of Myc. Activating retroviral insertions have also been seen in the 3¢ UTRs of other oncogenes, including pim1 and gfi1 (Dabrowska et al. 2009; Selten et al. 1985). Recently, global shortening of 3¢UTRs of the majority of mRNAs has been observed in cancer cells (Mayr and Bartel 2009) and in proliferating cells (Sandberg et al. 2008), thus making the surprising observation of 30 years ago a more general phenomenon. A second example of regulatory sequence disruption has been documented in ALV-induced tumors that arise following infection of 10–14 day gestation chick embryos. In contrast to infection of newly hatched chicks where most ALV infection leads to bursal lymphoma after 4–6 months, about 10% of the chicks infected as embryos die from B-cell lymphomas in 1–2 months (Kanter et al. 1988; Pizer and Humphries 1989). These short latent period tumors often involve integration into the c-myb locus, a site that is not associated with the longer latency tumors that typically arise following neonatal infection. The incidence of short-latency lymphomas was much higher with a virus called EU8 that was found to have a 42-nt deletion in the gag gene that was important for the short latency (Jiang et al. 1997; Smith et al. 1997). This deletion occurred in an RNA regulatory sequence called the negative regulator of splicing (NRS), which inhibits splicing and promotes polyadenylation

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(McNally et al. 1991; O’Sullivan et al. 2002). A single point mutation in this element also led to rapid tumorigenesis (Polony et al. 2003). Most myb integrations were in intron 1, leading to a slightly truncated Myb protein which is highly oncogenic (Jiang et al. 1997). The truncated Myb protein lacks the N-terminal 20 amino acids, but their function is not known. The rapid-onset lymphomas required infection of embryos at mid-gestation and involved Myb overexpression, suggesting that the target cells are different than for the later occurring leukemias associated with myc integrations.

Enhancer-Mediated Insertional Mutagenesis During the same period when investigators were examining oncogenic mechanisms in the bursal lymphoma model, other scientists were focused on tumorigenesis in murine models using a variety of murine viruses including Moloney and Friend MLV, mouse mammary tumor virus (MMTV), and other viruses. Similar to the situation with ALV, tumors induced by these viruses were clonal with respect to virus integration. However, the initial analyses were complicated by the fact that the presence of large numbers of cross-reacting endogenous virus sequences made it difficult to identify integration patterns. Also, in most cases, no fusion RNAs similar to those detected in ALV-induced bursal lymphoma were found. Rather, enhancer sequences in the U3 region of the retrovirus LTRs were found to activate genes in the vicinity of the integration site. Early clues to the importance of the enhancer elements in insertional mutagenesis came from studies in which changes in the enhancer were shown to affect the type of tumor that was induced. For example, chimeric viruses constructed using F-MLV, which induces erythroleukemia and M-MLV, which induces T cell lymphoma, revealed that determinants in the LTR determined the type of tumor that was induced (Chatis et al. 1983). Repeats within the core sequence of the LTR enhancer or point mutations in these sequences also influence tumorigenicity by these viruses (Lenz et al. 1984; Morrison et al. 1995). Another example is variants of MMTV that induce T cell lymphoma rather than mammary tumors (Ball et al. 1988). Enhancer insertion involves activation of expression of the promoter of a cellular gene (or relief of repression) by complexes formed at the enhancer sequences in the retroviral LTR enhancer. In many cases, the integration site is upstream of the transcription start site of the cellular gene and in the opposite orientation. Since retroviral enhancers tend to be stronger than cellular enhancers, enhancer insertion leads to overexpression of the product of the affected gene, thereby leading to altered cell growth. In some cases, more than one cellular gene is affected and the products of all of the genes cooperate in tumor induction (see below). Enhancer insertion allows for considerable flexibility in the position of the integrated provirus with respect to the cellular gene that is affected. Indeed, insertions that affect genes located as much as 50–100 kb away have been reported and may affect higher order chromatin structure and looping. These include insertions into pvt1/ c-myc, evi1/mecom, and c-myb (Bartholomew and Ihle 1991; Hanlon et al. 2003; Lazo et al. 1990). In some cases the effects of long-range insertions have been reevaluated

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recently due to the discovery of microRNA gene activation nearer the integration site (Beck-Engeser et al. 2008). In the future, additional classes of noncoding RNA may be found to be oncogenic, possibly leading to alternatives for some of the longrange interactions proposed. Many protooncogenes have been identified as targets of enhancer-mediated insertion, including some that can also be targeted by other mechanisms of insertional mutagenesis or have been recovered in v-onc gene containing retroviruses. In addition, many of these genes have subsequently proven to be overexpressed in human tumors that are not associated with retrovirus infection. Among these are c-myc, bmi1, runx2, sox4, pim, ets, and many others (Neil and Stewart 2011). MMTV uses enhancer insertion to activate wnt1, fgf3, and several other genes in mammary tumorigenesis (Dickson et al. 1984; Fung et al. 1985). In some cases, a cryptic cellular internal promoter is activated, resulting in a truncated protein as in the case of integration into jdp2 (Stewart et al. 2007). Although most of the genes activated by these viruses in tumors have direct effects on cell signaling and growth, genes with other functions can also be affected. ALV activates telomerase reverse transcriptase (TERT) by enhancer insertion (Yang et al. 2007) in a fraction of short-latency B cell lymphomas that arise following infection of chick embryos. In these tumors, TERT is overexpressed and telomerase activity is increased. Because the integrations near TERT were clonal, the data suggest that these insertions occurred early in tumor development and that TERT activation may play a role in tumor initiation. This pattern is distinct from human tumors where telomerase activation is thought to be a late event in tumorigenesis (Shay and Bacchetti 1997). Because some of these tumors also overexpressed c-myb by a mechanism independent of proviral insertion, upregulation of Myb may also contribute to oncogenesis (Yang et al. 2007).

Insertional Mutagenesis in the Setting of Gene Therapy Until recently, examples of oncogenesis by proviral insertion were limited to naturally occurring examples in a few animals and laboratory experiments conducted using rodents, chickens and cats. In the past decade, an unintended and unfortunate example of insertional mutagenesis emerged from two gene therapy trials in which gammaretrovirus-based vectors were used in attempts to correct hematopoietic deficiencies in individuals with X-linked severe combined immunodeficiency (SCID-X1) (Hacein-Bey-Abina et al. 2003, 2002, 2008; Santilli et al. 2008). SCID-X1 results from mutations affecting the gene encoding the common g chain, a component of cytokine receptors that are critical for normal hematopoietic cell development. As a consequence, individuals affected with the condition are unable to mount normal immune responses and typically succumb to lethal infections early in life. In these trials, the study subjects were infused with stem cells that had been infected with a nonreplicating retrovirus vector that expressed the common g chain. Hematopoiesis was restored in all of the study subjects that became engrafted with the stem cells but five of the 20 individuals went on to develop leukemias that arose

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from the transplanted cells. Analyses revealed that the tumors in four of the five subjects had vector insertions in the vicinity of the LMO2 gene (Nam and Rabbitts 2006; Howe et al. 2008; Hacein-Bey-Abina et al. 2008) that activated expression probably via an enhancer-mediated mechanism. Insertions involving BMI1 and CCND2 were also found as were other somatic changes that likely contributed to tumor development. Although these genes are not prominent among those that are classically associated with insertional mutagenesis in murine models of tumor induction, insertions into these genes have been documented and changes in their expression are associated with oncogenesis (Dave et al. 2009). A second example of insertional mutagenesis in a gene therapy setting has been documented in a trial focused on X-linked chronic granulomatous disease (X-CGD) (Ott et al. 2006). This immunodeficiency stems from a defect in gp91phox, a critical component of the oxidative response that helps to control microbial infection. A protocol generally similar to that used in the SCID-X1 trial was followed. Two of the study subjects contained clonally-expanded populations of cells with proviral insertions in MDS-EVI1, PRDM16, or SETBP1. In this case, the original defect associated with X-CGD was not corrected, perhaps in part because of silencing of vector expression through methylation. Although leukemia did not develop in these cases, both subjects developed myelodysplastic syndrome, a preleukemic disorder, associated with the effects of proviral insertion (Stein et al. 2010). One of them succumbed to sepsis, and the second was treated with an autologous stem cell transplant. The experiences from these trials, coupled with the wealth of experience with models of leukemia induction by conventional murine and avian retroviruses, have led to renewed attempts to design retrovirus vectors that will not affect expression of host genes. A variety of approaches are being explored, including the use of lentivirus-based vectors that have been shown to have different integration patterns relative to cellular genes (Montini et al. 2006, 2009; Lewinski et al. 2006; Bushman et al. 2005). Nonetheless, clonal dominance has been noted in a study subject treated for b-thalassemia by stem cell transplant with a lentivirus vector (Cavazzana-Calvo et al. 2010). In this instance, a clear clinical benefit from the gene transfer is evident, and there is no evidence of tumor development or premalignant conditions.

Disruption of Gene Function and Insertion Into Tumor Suppressor Genes Most retrovirus integrations affect expression of cellular genes by upregulating genes in the vicinity of the integration site. However a fraction of all integrations disrupt expression of the gene product. Those that contribute to tumorigenesis likely disrupt tumor suppressor genes. The rarity with which this happens likely reflects two features. First, a more limited spectrum of integration sites is likely to block expression of a gene or gene product than those that can activate a gene. In addition, for the gene product to be lost both copies of the gene need to be compromised and retroviral integration into both copies is unlikely. Nonetheless, loss of the second allele by other mechanisms can occur, and there are multiple examples of this event in human cancers (Sherr and McCormick 2002; Fletcher and Houlston 2010).

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Fig. 27.4 Activation of micro-RNAs by insertional mutagenesis. Four examples of insertional mutagenesis events that activate miRNAs are illustrated. Additional details are presented in the text

Despite these limitations, retrovirus integration into tumor suppressor genes has rarely been observed in tumors induced by ALV or MLV. Nf1, a gene that encodes a Ras-GAP and is involved in neurofibromatosis in humans, is targeted in myeloid leukemias induced by MLV in BXH mice (Largaespada et al. 1995). In this instance, integrations into a large intron led to an mRNA that encodes a truncated and nonfunctional protein. In addition, Trp53, the gene that encodes the p53 tumor suppressor is frequently targeted in erythroleukemias induced by Friend MLV (Ben-David et al. 1988). However, even in this instance, inactivation of p53 in these tumors typically involves deletion and loss of the second allele independent of retroviral integration at the locus. Despite these examples, other studies using mice lacking a functional Trp53 reveal modest effects on Moloney MLV tumorigenesis, and other experiments have suggested that loss of heterozygosity is not a common feature of MLV-induced tumors (Lander and Fan 1997). Activation of microRNAs The discovery over 20 years ago of common ALV integration sites in the noncoding B-cell integration cluster (bic) in B-cell lymphomas (Clurman and Hayward 1989) was the first example of retroviral activation of a micro-RNA precursor (Fig. 27.4). The function of bic was puzzling for a long time, since it did not contain a long open reading frame but was replete with termination codons in all reading frames. Thus, it appeared to be an oncogenic noncoding RNA (Tam et al. 1997). Further, it had extensive secondary structure, which was conserved between chickens, mice and humans. Overexpression of myc and bic from retroviral vectors in chickens led to death from tumors in 30 days rather than 60 days with myc alone. While overexpression of bic alone did not lead to accelerated tumorigenesis over vectors alone, the tumors that appeared were of a higher grade (Tam et al. 2002). In addition, transgenic mice

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overexpressing bic in B cells led to death from tumors in 6 months (Costinean et al. 2006). The identification of microRNAs led to the observation that the largest bic hairpin is a precursor to miR-155. Thus, miR-155 was the first identified onco-miR. Its transcription is also activated by the rel oncogene in B cell lymphomas induced by avian reticuloendotheliosis virus T (REV-T) and was shown to promote cell survival (Bolisetty et al. 2009). Epstein–Barr virus (EBV) also activates miR-155 and this is needed for immortalization of B cell tumors (Yin et al. 2008; Linnstaedt et al. 2010). Furthermore, homologs of miR-155 with common targets are encoded by both human Kaposi’s sarcoma-associated herpes virus (KSHV) (Skalsky et al. 2007) and by an avian herpes virus, Mareks disease virus (Morgan et al. 2008). miR-155 is also overexpressed in a large variety of human tumors, including B-cell lymphomas, breast, colon, pancreatic, and lung tumors. When mir-155 was knocked out in mice, the immune system was severely compromised (Thai et al. 2007). miR-155 is also induced as an inflammatory response and has been claimed to be at the interface of cancer and inflammation (O’Connell et al. 2007). A number of other retroviruses integrate into microRNA loci and cause their overexpression (Fig. 27.4). One example is the Kis2 locus, which contains the miR 106-363 cluster and is targeted and activated by radiation leukemia virus (RadLV) and MLV SL3-3 in mouse T-cell leukemias (Landais et al. 2007). This microRNA cluster is also involved in human T-cell leukemias. Both Friend MLV and SL3-3 MLV commonly integrate into and activate the miR 17–92 cluster of seven microRNAs in erythroleukemias and T-cell lymphomas, respectively (Cui et al. 2007; Wang et al. 2006). This microRNA cluster is also amplified in human B cell lymphomas and other tumors. This microRNA cluster cooperates with myc to induce more rapid tumors in mice (He et al. 2005). In fact, some viral integrations far away from protein-coding genes have been reanalyzed and found to actually affect micro-RNA genes. The pvt1 locus encodes several microRNAs and is targeted by retroviral integration in mouse and rat T-cell lymphomas (Beck-Engeser et al. 2008). The pvt1 locus is about 50 kb upstream of the c-myc oncogene. Myc is also overexpressed in these tumors, possibly due to indirect effects of the Pvt1 microRNAs. Retroviral integration readily activates oncogenes, but it only rarely inactivates tumor suppressor genes (see above). Thus, discovery of onco-miRs led to the idea that perhaps they were suppressing the expression of tumor suppressor genes (Kent and Mendell 2006). Conversely, microRNAs that are downregulated in tumors may target oncogenes. A number of targets of miR-155 have been identified, some of them possible tumor suppressors, such as tumor protein 53-induced nuclear protein 1 (TP53INP1), which is reduced in pancreatic cancers (Gironella et al. 2007). Restoration of TP53INP1 decreased cell growth in culture and inhibited tumor formation in vivo. Similarly, JARID2 (Jumonji), which potentiates retinoblastoma protein (Jung et al. 2005) and negatively regulates cell growth is a target of miR-155 in chicken and human cells (Bolisetty et al. 2009). Overexpression of miR-155 has been shown to enhance cell survival (Bolisetty et al. 2009), which may explain its cooperation with apoptotic oncogenes.

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Oncogene Cooperativity Although oncogenic retroviruses have developed a variety of strategies to subvert cellular growth, even in the case of those that express v-onc genes, activation of additional oncogenes plays a central role in tumor development. These changes contribute both to tumor progression and to modulating the differentiation state of the cells that comprise the tumor. Indeed, like many other discoveries made with oncogenic murine and avian retroviruses, work with these agents contributed to our current understanding of tumorigenesis as a multistep process. Cooperativity between viral oncogenes was first noted as investigators became aware of retroviruses that had captured more than one oncogene. One focus of these investigations involved strains of avian erythroblastosis virus (AEV). Some strains such as AEV-H carry only the v-erbB gene, while others, such as AEV-ES4, have both v-erbB and v-erbA (Beug and Graf 1989; Hihara et al. 1983). The close relationship of these strains facilitated studies examining the pathogenesis of the two viruses and revealed that chickens infected with AEV-ES4 develop a more rapid disease than those infected with AEV-H. In addition, in vitro experiments using erythroid precursors revealed that expression of ErbA affects expression of genes that would normally stimulate erythroid development, thereby suppressing differentiation (Hayman and Beug 1992; Metz 1994). This suppression, in cooperation with the growth stimulatory signals transmitted by ErbB, contributes to the enhanced oncogenic potential of AEV-ES4 (Fuerstenberg et al. 1992). Similar findings have been observed when the E-26 strain of AMV was compared to the AMV-BAI strain. As noted earlier, E-26 expressed both v-myb and v-ets, whereas the BAI strain expresses only v-myb. In this instance, a broader range of hematopoietic cells can be transformed by the E-26 strain (Metz and Graf 1991). Cooperativity between oncogenes is not limited to situations where two v-onc genes have been captured or to retroviruses that have captured these genes. Analyses of integration patterns in tumors induced by ALVs and MLVs revealed that most tumors, despite their clonal nature, contained more than one integration site, raising the possibility that more than one gene was activated by proviral insertion in these tumors. For example, chickens with B-cell lymphomas frequently had integrations in both myc and bic loci (Clurman and Hayward 1989). Since bic encodes the precursor to microRNA-155 mentioned above, this is an instance of cooperativity between protein-coding and noncoding genes. There are also many examples of cooperation between protein-coding genes. Strong evidence in support of this idea emerged when investigators began to use mice carrying transgenes that encoded known oncogenes. For example, infection of mice expressing a c-myc transgene with Moloney MLV revealed that tumors developed with an accelerated kinetics. Analyses of common proviral insertion sites in the tumors led to the identification of bmi1 and pim1 as oncogenes that collaborate with c-myc to induce B-cell lymphomas (Moroy et al. 1991; Berns 1991; van Lohuizen et al. 1991). Other studies have identified additional gene networks that contribute to oncogenesis. With the advent of genomic technology and high-throughput methods to

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identify viral integration sites, several investigators have conducted large screens of tumor integration sites in different mouse backgrounds, including studies involving mice lacking functional tumor suppressor genes such as Trp53, Cdkn2a (Lund et al. 2002; Uren et al. 2008), or combinations of these genes (Kool et al. 2010). These data have been compiled in the readily accessible Mouse Retrovirus Tagged Cancer Gene Database (RTCGD), developed by Neal Copeland and collaborators to catalog retroviral insertion sites in many murine cancers (http://RTCGD.ncifcrf.gov/) (Akagi et al. 2004), and additional computational tools have been developed to assess the significance of integration patterns (de Ridder et al. 2006). These advances are helping to maximize the utility of information obtained in these screens and aid investigators in relating these patterns to discoveries in human cancer.

Endogenous Murine Retroviruses and Oncogenesis As noted earlier, integration is an obligate part of the retrovirus life cycle and is nonreversible. When integration occurs in a germ cell, all progeny of that cell will carry the integrated provirus. This phenomenon has led to the presence of integrated retroviruses that are a part of the genetic makeup of many species, including humans (Jern and Coffin 2008). Although the majority of these elements have become inactive as a consequence of mutations that have accumulated over the centuries that they have existed, some animals carry copies that are expressed. In certain strains of laboratory mice, these viruses play a special role in oncogenesis. The first clues to the role of endogenous retroviruses (ERV) in tumorigenesis emerged over 60 years ago in pioneering studies by Gross (Gross 1951a, b) using the AKR strain of laboratory mice. Almost all of these animals develop thymic lymphomas by 1 year of age. Long before the complexity of endogenous viruses was understood, Lilly and colleagues (1975) used classical genetic approaches to demonstrate that a single locus, now known as Akv1, was required for tumor induction. This locus corresponds to an endogenous provirus that is expressed from birth in AKR mice. Additional studies revealed that this virus, called AKR-MLV, undergoes recombination with other endogenous viruses expressed by the mice (Stoye et al. 1991; Hartley et al. 1977). The recombination events are central to the development of the tumors (Fig. 27.5). The newly formed viruses were initially recognized by virtue of an extended host range. AKV-MLV infection is limited to rodent cells that express the mCAT1 receptor (Albritton et al. 1989). However, viruses present in the tumor tissue could infect rodent cells and cells derived from other sources, including mink lung. Many of the viruses caused a specific type of cytopathic effect in these cells and were named MCFs or mink cell focus forming viruses (Hartley et al. 1977). The extended host range reflected recombination within the env gene that affects the amino terminal portion of the surface (SU) glycoprotein. A second recombination event alters the sequences within the U3 region of the LTR, a change that enhances transcription. Both of these events are important for generating the leukemogenic virus.

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Fig. 27.5 Endogenous murine retroviruses and oncogenesis. In AKR mice, the endogenous retrovirus, AKR-MLV is expressed from birth. It recombines with other ERVs in the genome to produce a leukemia virus, which causes thymic lymphomas by 1 year. The recombinant virus has an expanded host range due to changes in the SU glycoprotein and the LTR

The importance of ERV in tumorigenesis is not unique to AKR mice. Other strains of laboratory mice, including C58, HRS, and CWD, have a high frequency of spontaneous tumors and similar recombination events occur in these animals. Recombinants are also generated in the course of tumor induction by Moloney MLV and in MMTV-mediated induction of breast tumors. This phenomenon is not limited to mice; recombinations have been documented in FeLV-induced T cell tumors in cats (Rosenberg and Jolicoeur 1997).

Friend Virus and env Gene-Mediated Oncogenesis Although classical oncogenes are involved in tumor induction by most murine and avian retroviruses, Friend virus uses a distinct mechanism to induce tumors (Cmarik and Ruscetti 2010). Friend virus stocks contain two retroviruses, a replicationcompetent virus called Friend MLV and a replication-defective component called spleen focus forming virus (SFFV). The virus induces a rapid erythroid leukemia in mice, and the SFFV component is responsible for the disease. While these general features are reminiscent of v-onc gene-containing retroviruses, SFFV does not contain an oncogene but rather expresses an altered env gene product that contains an internal deletion and is not processed in the way functional Env gene proteins are processed (Kabat 1989). Other changes affect the carboxyl terminal sequences of the molecule. The SFFV-encoded Env protein, called gp55, exerts its leukemogenic effects by interacting with the erythropoietin receptor (Epo-R) that is expressed by developing red blood cells (Ferro et al. 1993; Nishigaki et al. 2001; Wang et al. 1993). Erythropoietin (Epo) typically stimulates these precursors by interacting with its receptor, initiating a cascade of signaling events that orchestrates the differentiation process. When gp55 interacts with the Epo-R, signaling cascades that are similar to those activated by Epo are triggered (Fig. 27.6), but in this case, constitutive expression of gp55 results in a sustained signal that drives cell proliferation (Cmarik and Ruscetti 2010). The effects of gp55 are not solely mediated via the Epo-R. A second cellular molecule called Stk also plays a role. This tyrosine kinase is expressed in two forms: a full length form and a truncated form called sf-Stk (Iwama et al. 1994).

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Fig. 27.6 Friend Virus and env Gene-Mediated Oncogenesis. The Friend SFFV altered env protein, gp55, interacts with the Epo Receptor and sf-Stk, initiating sustained signaling that drives proliferation

The latter form, highly expressed on erythroid precursors, also plays a key role in SFFV-mediated disease. The role of sf-Stk emerged from studies that identified Mstr1 as the gene responsible for Fv2, a locus associated with resistance to SFFVinduced disease (Persons et al. 1999). Only strains that express the truncated form are susceptible to the virus, a feature that reflects the ability of gp55 to interact and stimulate signal transduction via sf-Stk, but not the full-length form (Cmarik and Ruscetti 2010). Mechanisms similar to the one used by SFFV are likely important for tumors induced by the avian hemangioma retrovirus (Alian et al. 2000) and the sheep retrovirus JSRV (see Chap. 30).

Host Factors Influencing Oncogenicity A variety of host genes have been identified that affect tumorigenesis by oncogenic retroviruses. The majority of these can influence the process by affecting the outcome of infection and do not relate directly to the oncogenic effects of the virus. Other genes influence the growth and differentiation of the host cells that are the targets of the virus, while others, as described above for Mstr1, affect key signaling molecules that are important for stimulation of cell growth. Lastly, genes that influence the immune response to the virus can also play a key role in determining the outcome of infection. Genes that directly affect aspects of virus–cell interaction are highlighted here. One class of genes that play an important role is encoded by ERVs. Genes in this class, including Fv4, Rmcf, and Rmcf2, exert their effects by expressing env gene

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products that block infection by exogenous viruses (Stocking and Kozak 2008). Some chicken ERVs function in a similar fashion. These effects are mediated by a phenomenon called super-infection resistance whereby an Env protein expressed from an ERV prevents the interaction between the virus receptor and another virus. ERV gag gene products can also influence infection. A second type of ERV effect is exemplified by the Fv1 locus, which restricts infection at a stage after reverse transcription but before integration. This restriction, originally identified as affecting oncogenesis by Friend virus (Rosenberg and Jolicoeur 1997), affects virus replication and thereby reduces oncogenic potential. Although the precise way in which the Fv1 product exerts its effects are still not fully understood, a Gag-related protein similar to those encoded by the ERV-L family of ERVs is responsible (Best et al. 1996). Other host genes can affect virus replication and thereby influence the oncogenic potential of retroviruses. The clearest example illustrating this point is the APOBEC family of proteins. These molecules are cytidine deaminases that affect replication during reverse transcription through mutations caused by substitution of A residues for G residues. The protein also appears to be packaged in virions in at least some circumstances. Mice lacking the single gene that encodes APOBEC, mA3, have a higher virus load and develop thymic lymphomas more rapidly than wild-type mice when challenged with Moloney MLV (Ross 2009). Host genes that influence development of the cells that are transformed by retroviruses have been most thoroughly studied in the mouse. Examples of genes of this type include W and Sl, genes that influence the development of erythroid precursors. The W locus encodes the c-Kit receptor and the Sl locus encodes stem cell factor (SCF), the ligand for the receptor (Besmer 1991). Signaling mediated by SCF interaction with c-Kit is critical for appropriate development of hematopoietic precursors, and mutations affecting these pathways influence the frequency of cells that are susceptible to Friend virus-induced disease. A second host gene that plays a similar role is FoxN1, a gene that encodes a forkhead family transcription factor and is responsible for the nude mutation (Nehls et al. 1994). Nude mice do not develop a normal thymus because FoxN1 has undergone a spontaneous deletion in these animals and thus these animals lack the cells that give rise to thymic lymphomas. Acknowledgments We acknowledge the help of Mohan Bolisetty and Sarah Short with the preparation of the manuscript. KLB was supported by NIH grants R01CA048746-20 and R01CA124596-04.

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Chapter 28

Rous Sarcoma Virus: Contributions of a Chicken Virus to Tumor Biology, Human Cancer Therapeutics, and Retrovirology Leslie J. Parent

Discovery of A “Filterable Agent” That Causes Tumors in Chickens A century ago, Peyton Rous published a landmark article demonstrating that a spindle cell tumor could be transferred from one Plymouth Rock chicken to another (Rous 1910). Rous, a pathologist at Rockefeller Institute for Medical Research, had been given a hen with a large tumor protruding from its right breast. He removed the tumor and described its histological appearance as containing irregularly arranged cells, with frequent mitotic forms as well as multinucleated and giant cells. He inoculated pieces of the tumor into the peritoneal cavity of the original hen, which developed a large intraperitoneal tumor and died 35 days later. He also injected bits of the tumor into other animals and found that transplantation of the tumor was most successful in young birds and occurred between closely related birds of the same lineage, but not in more distant relatives or fowl purchased at market. In 1911, Rous reported that tumors could be transmitted using a small amount of liquid derived from the filtrate of macerated tumors. He noted that the filterable agent, or virus, caused tumors with the same histology as the original neoplasms, and metastatic disease occurred by dissemination through the bloodstream and lymphatics as well as by direct extension. Rous demonstrated that the agent in the filtrate fulfilled Koch’s postulates, proving that the tumor had an infectious origin (Rous 1911). Although Ellerman and Bang had reported in 1908 that filtrates could transmit leukemia and lymphoma in chickens, this disease was not recognized as a malignant process at the time (Vogt 1997; Javier and Butel 2008). Because of these circumstances, Rous sarcoma virus (RSV) became known as the first tumor virus identified, and it was at the forefront of the emerging fields of tumor virology and cancer biology.

L.J. Parent (*) Departments of Medicine and Microbiology and Immunology, Penn State College of Medicine, Hershey, PA 17033, USA e-mail: [email protected] E.S. Robertson (ed.), Cancer Associated Viruses, Current Cancer Research, DOI 10.1007/978-1-4614-0016-5_28, © Springer Science+Business Media, LLC 2012

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However, it was not until 1966, when Rous was 85 years old, that the Nobel committee formally recognized the value of his discovery and awarded him the Nobel Prize in Medicine or Physiology. In his Nobel Lecture, Rous described how his discovery of RSV was largely ignored for many years because the transmissible nature of the tumor was thought to be an anomaly unique to chickens (Rous 1972). In 1910 I described a malignant chicken sarcoma which could be propagated by transplanting its cells, these multiplying in their new hosts and forming new tumors of the same sort. In other ways the growth showed itself to be a neoplasm of a classical sort, yet, as reported in 1911, its cells yielded a causative virus… Hence the findings with the sarcoma were met with down-right disbelief, though soon several other, morphologically different, ‘spontaneous’ chicken tumors were propagated by transplantation and from each a virus was got causing growths of its kind. Not until after some 15 years of disputation amongst oncologists were the findings with chickens deemed valid, and then they were relegated to a category distinct from that of mammals because from them no viruses could be obtained (Rous 1972).

Although Peyton Rous turned his attention to other areas of research, RSV was distributed to scientists throughout the world. RSV was actively studied not only for its relationship to tumorigenesis, but also as a tool to explore the fundamentals of molecular biology and virus–host interactions (Martin 2004). The number and breadth of seminal discoveries that arose from studying RSV is quite remarkable: mechanism underlying cellular transformation; viral oncogenes and protooncogenes; signaling pathways that regulate cell growth; protein tyrosine kinases; retroviruses; virally encoded enzymes reverse transcriptase (RT), integrase (IN), and protease (PR); RNA packaging elements; retroviral gene therapy vectors; and host factors that contribute to the assembly of enveloped viruses. These discoveries have had far-reaching impacts on human health, leading to the development of tyrosine kinase inhibitors for cancer therapy and the inhibition of human retrovirus replication by interfering with the enzymatic activities of RT and PR. As a testament to the importance of RSV to the study of viral oncogenesis and cellular biology, two additional Nobel prizes in Medicine or Physiology were subsequently awarded: one to Harold Varmus and Michael Bishop for the discovery of the Src protooncogene and the other to David Baltimore and Howard Temin for their independent discoveries of reverse transcription and the RT enzyme. Remarkably, despite its humble beginnings, to this day RSV remains a powerful tool that continues to provide novel insights into tumor virology, retrovirus replication, and cellular transformation. Unfortunately, during his lifetime, Rous could not have imagined how his work in the early twentieth century would lay the foundation for treating human cancers, challenge the central dogma of molecular biology, or contribute to controlling the worldwide AIDS epidemic caused by a distantly related human retrovirus.

Pathogenicity of RSV in Birds and Mammals RSV readily establishes infection in chickens and other avian species, and it induces malignancies that ultimately lead to death of the animal. Inoculation of RSV into chicks younger than 1 month in age leads to the development of

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sarcomas in as little as 2–3 days. The tumors that arise are fibrosarcomas and histiocytic sarcomas that involve organs including liver, spleen, and lung (Rous 1911). Interestingly, although infection of adult birds also results in malignant transformation with formation of sarcomas, these tumors undergo spontaneous regression in immunocompetent animals (Fine and Sodroski 2000). Tumor induction by RSV is not strictly limited to birds, although the sarcomas formed in other species are not highly pathogenic. In rats and mice, injection of RSV in young animals rarely causes tumors locally, but malignant transformation at disseminated sites does not occur (Altaner and Temin 1970; Fine and Sodroski 2000; Maeda et al. 2008). In rodents, tumors at the inoculation site spontaneously undergo involution over time as the animals grow older. In cultured mammalian cells, cells do become infected, and integration of the viral genome occurs. These infected cells are transformed, although in most cases virus replication is abortive and virus particles are not released from tumor cells (Kotler 1971). The defect in virus release is complemented by fusion of chicken and mammalian cells, suggesting that a chickenspecific host factor required for RSV assembly is missing from mammalian cells (Coffin 1972). However, the block to budding is not absolute, because virus-like particles are released from mammalian cells upon expression of the RSV Gag protein from a nonretroviral promoter (Wills et al. 1989). To date, the avian factor needed for RSV to be released from mammalian cells following proviral integration has not been identified.

Rous Sarcoma Virus and the Biology of Transformation During the 1950s, more than 40 years after Rous’s initial discovery, many important developments set the stage for the major breakthroughs that would follow. Several groups of scientists reported that RSV-induced tumors were not isolated to avian species; rats, mice, hamsters, and rabbits also developed sarcomas when infected with certain strains of RSV (Martin 2004). The development of tumors in mammalian species legitimized the study of RSV and increased interest within the scientific community. The initial convincing evidence that the tumor-inducing activity associated with RSV was clearly linked to an actual virus was provided by electronmicroscopic examination of pelleted tumor extracts that revealed spherical virus particles 70–75 mm in diameter and enclosed by a double-layered membrane (Epstein 1956, 1958, Fig. 28.1). To ensure that the virus particles had biological activity, a sample of the pelleted fraction was divided into two portions, one that was subjected to microscopic examination and the other used to infect cells, which subsequently become became transformed (Epstein 1958). Around this time, quantitative methods for infectivity were developed that permitted careful investigation of the biological properties of the virus and the infected tumor cells (Rubin 1955). A particularly significant advance during this period was the invention of the “focus assay”, in which an agar overlay was applied to infected cells to isolate single infected cells into discrete foci that were all infected by a single virus (Temin and Rubin 1958).

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Fig. 28.1 (a) Diagram of immature (left) and mature (right) RSV virions depicting the arrangement of proteins, lipids, and RNA in the virus particles. vRNA viral RNA, CA capsid, MA matrix, NC nucleocapsid, PR protease, SU surface glycoprotein, TM envelope transmembrane domain, RT reverse transcriptase, IN integrase. Figure modified from (Garbitt 2004) with the author’s permission. (b) Electron-microscopic images of RSV virus-like particles produced by Gag expression that demonstrate immature morphology (left) and authentic RSV virions that have been released from the cell and have undergone maturation with arrangement of the core to form an electrondense center. Images obtained by L.Z. Scheifele and L.J. Parent with assistance from Roland Meyers, Penn State College of Medicine Imaging Core Facility (Scheifele 2004) and used with the author’s permission

This assay allowed for single virus variants, or genetic mutants, to be cloned and characterized in detail. The results of experiments based on this assay led Temin to propose the “provirus” hypothesis (described below) and contributed directly to the discovery of Src, the transforming protein encoded by RSV (Vogt 1997; Martin 2004).

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The biology of transformed cells was also studied using viruses isolated from foci of tumor cells in culture. RSV was a potent transforming agent and induced cancer in cultured cells more rapidly than any other virus, with initial changes of transformed morphology observed within 14 h following infection (Hanafusa 1969). Under optimal conditions, cellular transformation was produced in 90% of chicken embryo fibroblasts within 24 h. Transformation was most efficient if cells were infected within 4 h after subculture, to ensure that cells were actively dividing when exposed to virus. Incubation of cells with DEAE-dextran also increased the efficiency of transformation by enhancing virus-cell attachment. Cells transformed by RSV exhibited either rounded or fusiform shape with increased intracellular vacuoles, an ability to form colonies in agar suspension, increased growth rate, decreased dependence on serum factors for multiplication, changes in cell surface markers, and metabolic alterations including glucose utilization (Hanafusa 1969; Martin et al. 1971; Temin 1971; Bader 1972). Isolation of viral mutants with differing abilities to transform cells led to the hypothesis that the virus encoded a gene product that was responsible for maintaining the transformed state.

Discovery of a DNA Intermediate Required for RSV Replication The focus assay developed by Temin and Rubin allowed investigators to study the effects of single viruses on the properties of uniform populations of infected tumor cells (Temin and Rubin 1958). In the early 1960s, Temin observed two distinct phenotypes of tumor cells infected with the Bryan strain of RSV (Temin 1961). The wild-type virus, referred to as morphr, caused rounding of the cells, in contrast to the mutant strain morphf, which resulted in fusiform cellular morphology. Temin recognized that the morphological differences in the cells were maintained during long-term passage of the cells or by transfer of the virus to new cells, suggesting that the viral genetic material had become stably associated with the cell, likely through integration into the cellular DNA (Temin 1960, 1962; Temin and Kassner 1976). A major breakthrough in understanding the ability of RSV mutants to cause reproducible, transferrable cellular phenotypes came when Temin observed that actinomycin D, a DNA-dependent RNA synthesis inhibitor, interfered with virus production in infected cells (Temin 1963a). Inhibition of DNA synthesis prevented establishment of RSV infection, but only when cells were treated prior to formation of the provirus. Furthermore, Temin found that RSV infection was dependent on new DNA synthesis and cell mitosis, and RSV-infected chicken embryo fibroblasts contained DNA that was homologous to RSV RNA (Temin 1964a, b). Based on these experiments, he publically reported his “DNA provirus hypothesis,” which postulated that “infection of chicken cells by RSV leads to the formation of one or two copies of a regularly inherited structure with the information for progeny virus and for cellular morphology” (Temin 1976). In other words, the RSV RNA genome was converted to a DNA intermediate, which was integrated into the cell genome

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and used as a template to synthesize RSV RNA. Remarkably, Jan Svobda also proposed the existence of a provirus around the same time based on independent studies of rat cells infected with RSV (Svoboda et al. 1963; Temin 1976). However, data supporting the provirus hypothesis were considered by many scientists in the field to be too indirect and, therefore, inconclusive. Temin’s conviction that a DNA provirus was intrinsic to RSV replication was unwavering, and he and others continued to perform more rigorous experiments that were consistent with the theory. Then, in 1969, a key insight was made that greatly energized the field. In RSV-infected cells, RSV-derived DNA was synthesized in the absence of protein synthesis, suggesting that the enzyme that carried out viral DNA synthesis was present in incoming virus particles (Temin and Mizutani 1970; Temin 1976). These experiments prompted the development of an assay to look for RNAdependent DNA polymerase activity in RSV virions (Temin 1976; Weiss 2003). Purified virus particles were permeabilized with nonionic detergent, incubated with dideoxynucleotides dATP, dCTP, dGTP and radioactive dTTP in the presence of magnesium and dithiothreitol, and the incorporation of radioactively labeled TTP into DNA was measured (Temin and Mizutani 1970). There was a high degree of TTP incorporation under the standard assay conditions, but DNA synthesis was abrogated by pretreatment of the virions with ribonuclease A, suggesting that viral RNA was used as a template for production of DNA. Simultaneously, David Baltimore performed a similar polymerase assay using permeabilized Rauscher murine leukemia virus, discovering the same RNAdependent polymerase activity in virions, and concluding that all RNA tumor viruses shared the same enzyme (Baltimore 1970). Baltimore and Temin’s back-to-back manuscripts in Nature were groundbreaking, and the editors of the journal coined the term “reverse transcriptase” for this enzyme discovered in RNA tumor viruses (Baltimore 1995). Soon thereafter, RNA tumor viruses were called retroviruses based on their reverse transcriptase activity (Dalton et al. 1974). In 1975, only five years after their discovery, Temin and Baltimore shared the Nobel Prize in Physiology or Medicine with Renato Dulbecco. The relationship of reverse transcription to the study of cancer was eloquently described in Temin’s Nobel Lecture: “The Nobel Prize is an honor not only for me but also for all those who have been working with avian RNA tumor viruses… The genetic information in RNA is transferred to DNA during the replication of some viruses, including some that cause cancer. This transfer of information from the messenger molecule, RNA, to the genome molecule, DNA, apparently contradicted the ‘central dogma of molecular biology’, formulated in the late 1950s. This mode of information transfer was first postulated and established for the replication of Rous sarcoma virus, a strongly transforming avian C-type ribodeoxyvirus… In most of this discussion of virus replication and virus origins, I have not mentioned cancer. In fact, the absence of such discussion makes an important point: RNA tumor virus replication is not sufficient for cancer formation by RNA tumor viruses. Strongly transforming RNA tumor viruses like RSV cause cancer by introducing genes for cancer into cells… I do not believe that infectious viruses cause most human cancers, but I do believe that viruses provide models of the processes involved in the etiology of human cancer.” (Temin 1992).

The original RT assay was refined and used to delineate the molecular steps of the reverse transcription process in avian retroviruses (Boone and Skalka 1980, 1981).

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Fig. 28.2 Schematic diagram showing the integrated provirus of RSV, with the viral genes gag, pol, env, and src indicated. The unspliced viral RNA that serves as the viral genome and as the template for Gag and Gag-Pol fusion protein expression is shown below, in addition to the Gag and Gag-Pol proteins. The spliced viral RNAs that dictate Env and Src expression are illustrated with their respective protein products. Nucleotide numbering is based on the Prague C strain of RSV (Coffin et al. 1997). LTR long terminal repeat, U3 unique 3¢ region, R repeat, U5 unique 5¢ region, DR1 direct repeat 1, DR2 direct repeat 2, PBS primer binding site, PPT polypurine tract, RSE RNA stability element, AAAA poly(A) tail

These experiments demonstrated that everything needed for reverse transcription to proceed, except nucleotides, was contained within the virus particle (Boone and Skalka 1980). The molecular steps involved in the process of reverse transcription and the enzymatic activity of RT itself turned out to be more complex than originally imagined. The overall reaction involves transformation of the linear, single-stranded, positive-sense viral genomic RNA into the linear double-stranded proviral DNA. Importantly, the proviral DNA differs from the genomic RNA template at its 5¢ and 3¢ termini, which contain sequence known as the long terminal repeats (LTRs) (Fig. 28.2). Only portions of the LTR sequences are present in the genomic RNA. Oddly enough, the sequence that serves as the proviral promoter to drive viral transcription is located at the 3¢ end of the genomic RNA. Therefore, one of the primary goals of reverse transcription is to copy the promoter from the 3¢ end of the RNA and move it to the 5¢ end of the provirus to enable transcription of the viral genes by cellular RNA polymerase II. The polyadenylation signal at the 3¢ end of the provirus is also created through the process of reverse transcription. The molecular gymnastics required to properly synthesize the LTR-containing proviral DNA harnesses several different enzymatic activities of RT: RNA-dependent and DNA-dependent DNA synthetic capabilities and an RNA digestion activity (RNase H) that specifically degrades the RNA component of RNA–DNA hybrids (Telesnitsky and Goff 1997).

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The importance of the discovery of reverse transcription by Temin and Baltimore cannot be overstated. As stated in the press release from the Nobel Assembly at the Karolinska Institute announcing the 1975 Nobel Prize in Physiology or Medicine: “Temin postulated that the genetic information of an RNA virus capable of giving transformation could be copied into DNA, and that this DNA in a manner similar to that described for a DNA tumor virus could become integrated into the genetic material of cells. This proposal by the overall majority of scientists was considered as heresy since it was in conflict with the central dogma accepted in the field of molecular biology in those days. This dogma implied that information transfer in nature occurred only from DNA to RNA and not in the other direction. Temin accumulated certain indirect evidences supporting his theory but the major breakthrough occurred in 1970 when simultaneously Temin and also David Baltimore showed the occurrence of a specific enzyme in RNA tumor virus particles which could make a DNA copy from RNA. This enzyme was called reverse transcriptase”

(The Nobel Prize in Physiology or Medicine 1975 – Press Release 1975). Nobelprize.org. 1 Aug 2010 http://nobelprize.org/nobel_prizes/medicine/laureates/1975/press.html.

The Process of Reverse Transcription The steps involved in proviral DNA synthesis are depicted in Fig. 28.3, and several reviews present a more detailed description of the process (Goff 1990; Katz and Skalka 1990; Whitcomb and Hughes 1992; Telesnitsky and Goff 1997; Wilhelm and Wilhelm 2001; Hizi and Herschhorn 2008; Gotte et al. 2010; Herschhorn and Hizi 2010). Briefly, the primer tRNA-tryptophan is annealed to the primer-binding site (PBS) near the 5¢ end of the RNA genome. RT binds to the viral RNA and begins the synthesis of DNA using the RNA as a template. DNA synthesis continues until the 5¢ end of the RNA is reached, creating a short product called the minusstrand strong stop DNA. The RNAse H component of RT degrades the RNA strand of the RNA–DNA hybrid located upstream of the PBS. A molecular gymnastics event known as the first strand transfer occurs next, when the minus-strand strong stop DNA “jumps” to the opposite end of the RNA and hybridizes to a complementary repeated sequence, R. RNA-dependent DNA synthesis proceeds from R through a region known as the polypurine tract (PPT), then continues to the PBS at the end of the RNA strand. The PPT is not digested efficiently by RNase H and remains as the final vestige of the viral RNA template. The RNA PPT sequence forms a duplex with the DNA PPT sequence and primes DNA synthesis using the nascent minusstrand DNA as a template. DNA-dependent DNA synthesis continues through the end of the minus-strand DNA, a segment of the annealed tRNA primer, terminating at the PBS, and forms the plus-strand strong-stop DNA. The primer tRNA is degraded by RNase H, leaving the PBS intact. The next gymnastics event, the second strand transfer, involves translocation of the minus-strand PBS to the opposite end of the DNA duplex, annealing to the complementary plus-strand PBS sequence.

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Fig. 28.3 Diagram of the steps in the reverse transcription reaction. U3 unique 3¢ region, R repeat, U5 unique 5¢ region, PBS primer binding site, PPT polypurine tract, AAA poly(A) tail. Figure modified from (Spidel 2005) and used with the author’s permission

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DNA-directed DNA synthesis continues until both strands of the provirus are fully intact. In RSV-infected cells, synthesis of the double-stranded DNA provirus is not finished until after the reverse transcription complex enters the nucleus (Fig. 28.3) (Varmus et al. 1978; Lee and Coffin 1991). The completed products of reverse transcription may be integrated into the host genome or the blunt LTR ends may be joined by host ligases to the form dead-end products, 2-LTR circles and 1-LTR circles (Telesnitsky and Goff 1997) (Fig. 28.3). The consequences of reverse transcription have significant implications for retroviral pathogenesis, and carcinogenesis. The accurate execution of reverse transcription, including the precise construction of the LTRs, is required for the provirus to be integrated into the host’s cellular DNA. Integration is, by definition, a mutagenic event that permanently alters the genetic make-up of the host. The integration of proviral DNA may directly result in cancer by introducing the retroviral promoter upstream of a gene that regulates cell growth or other homeostatic functions, leading to deregulation of vital cellular activities and subsequent tumor formation. This process of inducing cellular gene expression driven by an integrated viral LTR promoter is known as insertional mutagenesis (Fig. 28.4). In addition, integration of the provirus near a growth-controlling gene may result in incorporation of a portion of the cellular gene into the virus particle, resulting in “oncogene capture” (Fine and Sodroski 2000). This latter mechanism accounts for the tumor-producing properties of RSV.

Genome Organization of RSV Early classification of RSV and its relatives was controversial and inconsistent. Throughout the mid-1900s, these viruses were called “Rousviruses,” for obvious reasons, “Leukoviruses,” because of their similarities to avian leukosis virus, or “Oncornaviruses” because they caused tumors in animals (Temin 1971). Members of this virus family are enveloped 80–100 nm diameter particles containing a nucleocapsid core that lacks apparent icosahedral symmetry. The discovery that ultimately unified this virus family occurred in 1961, when density gradient-purified RSV virions were shown to contain almost exclusively RNA and very little DNA (Crawford and Crawford 1961). This finding led to the classification of RSV and related viruses as RNA tumor viruses, to distinguish them from the families of DNA tumor viruses such as polyoma viruses. This terminology continued to be used until Temin and Baltimore discovered that RNA tumor viruses RSV and Rauscher murine leukemia virus contained a polymerase with reverse transcribing activity, which was encoded by the virus. Reverse transcription of the viral RNA genome into DNA was considered the hallmark of this class of viruses; hence, they were renamed “retroviruses,” and RSV was considered the prototype. RSV is presently classified as a member of the Retroviridae family, Orthoretrovirinae subfamily, and Alphoretrovirus genus, to denote it as belonging to the first group of retroviruses discovered. The RSV genome is a diploid, single stranded RNA molecule of approximately 9.3 kb in length (Schwartz et al. 1983). The genome is synthesized by the cellular

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enzyme RNA polymerase II using the integrated proviral DNA as a template (Rabson and Graves 1997). The viral RNA shares structural features with cellular mRNA, bearing a 5¢ methylguanine cap and a poly(A) tail at the 3¢ end (Vogt 1997) (Fig. 28.2). The two genomic RNAs in each virion are linked at their 5¢ ends through a noncovalent interaction that involves sequences in the dimer initiation site and additional nucleotides included in the dimer linkage site. Several regulatory elements are contained within the 5¢ and 3¢ untranslated regions (UTRs). The first nucleotide is at the start of the R region, a short sequence repeated at the 3¢end of the genome. The 5¢ R region is followed by a unique sequence denoted U5. The primer binding site (PBS), positioned between R and U5, serves as the binding site for the primer of reverse transcription. In RSV, this primer is the tryptophan-tRNA, which binds to a complementary sequence in the PBS (Harada et al. 1975). The psi sequence, a cis-acting element required for incorporation of the genomic RNA into virions, is located within the 5¢UTR, and extends seven codons into the gag region (Swanstrom and Wills 1997; Vogt 1997). Genetic separation of the viral psi packaging element from the genes encoding the viral structural proteins was a key discovery that ultimately led to the development of viral vectors for gene therapy (Linial et al. 1978; Linial 1981). The three genes absolutely required for replication are arranged in the same order in all retroviruses: 5¢-gag-pol-env-3¢ (Rabson and Graves 1997; Vogt 1997). In RSV, the PR-encoding sequence pro is located at the 3¢ end of the gag gene, although in other retroviruses, pro is contained within the pol gene or in a distinct pro reading frame (Swanstrom and Wills 1997). The src gene, unique to RSV, is dispensable for virus replication but is required for cellular transformation, and it resides downstream of env (Schwartz et al. 1983). The src coding region is flanked by short direct repeat sequences known as DR1 and DR2. The DR elements have pleiomorphic effects on virus replication; they have been implicated in viral RNA packaging, virus assembly, as a cis-acting signal for nuclear export of the unspliced viral RNA (Sorge et al. 1983; Ogert et al. 1996; Simpson et al. 1997, 1998). A regulatory element called the RSV stability element (RSE), recently discovered, lies just downstream of the src gene and prolongs the half-life of the unspliced mRNA (Weil and Beemon 2006; Weil et al. 2009). There are three polyadenylation signals in the 3’UTR, as well as the R region and a unique region called U3, which contains the viral promoter (Wang et al. 1975; Coffin and Billeter 1976). As discussed in the previous section, during reverse transcription, the U3 region is copied and translocated to the 5¢ end of the proviral DNA to recreate the promoter in its proper location, upstream of the viral genes (Hughes et al. 1978; Shank and Varmus 1978).

Identification of the src Viral Oncogene From studies of the cellular effects associated with RSV variants, Temin concluded that “viral genes controlled the morphology of transformed cells,” and this idea “led to the hypothesis that transformation is the result of the action of viral genes” (Temin 1976). While this idea might not seem radical or insightful today, the underlying cause of cancer was debated during the first part of the twentieth century.

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Thus, the study of RSV-induced tumors was the genesis for the hypothesis that alterations in cellular DNA caused transformation into the malignant state. One of the most intriguing observations made during this time was that different RSV mutants varied widely in their ability to promote cellular transformation. As discussed earlier, the morphf mutants described by Temin induced a different morphology in infected cells compared to the wild-type virus (Temin 1960). Based on these observations, Temin proposed that “…the virus becomes equivalent to a cellular gene controlling cell morphology (Temin 1960),” setting in motion the hunt for the transforming viral gene (Martin 2004). He proposed that viral oncogenes, or virogenes, were integrated into the host chromosome and caused cancer to arise in these cells. His work laid the foundation for the virogene–oncogene hypothesis, discussed in more detail below (Huebner and Todaro 1969). Another significant step forward was the description of the Bryan strain of RSV, which was replicationdefective but caused cellular transformation, highlighting the idea that virus replication and tumor formation were genetically separable (Bryan et al. 1955; Hanafusa et al. 1963; Temin 1963b). This idea was strengthened by the existence of irradiationinduced and naturally occurring mutants of RSV that were replication-competent but nontransforming (Vogt 1971; Martin and Duesberg 1972). Thus, it became apparent that the transformation gene of RSV was deleted or defective in these nontransforming variants. A series of experiments that followed in the late 1960s were instrumental in establishing the identity of the gene carried by RSV that was responsible for cellular transformation. The most critical advance was the description of an infectious, temperature-sensitive (ts) viral mutant that caused transformation when cells were grown at the usual temperature; remarkably, when shifted to a higher temperature, the cells reverted to normal morphology (Martin et al. 1971). Martin concluded that a protein encoded by the virus was required to maintain cellular transformation, and the loss of the transformed state was due to failure of the protein to function at the elevated temperature. The ts mutant reafffirmed the notion that virus replication and oncogenesis were genetically segregated. As a result of Martin’s findings, the quest for the oncogenic viral gene was undertaken by a number of investigators using the emerging molecular biology methods being developed at the time (Varmus 1990; Martin 2004). To identify the transforming gene associated with RSV, the genomes from transformation-competent and transformation-defective (td) strains were compared to determine whether there were any “extra” sequences present in the transforming genome (Vogt 1971). It had previously been shown that the RSV genomic RNA isolated from virus particles consisted of a 70 S RNA that could be reduced to two 35 S RNAs upon denaturation (Duesberg 1970). To determine whether the RNA isolated from nontransforming td viruses had a lower molecular weight than that of transforming viruses, Duesberg and Vogt metabolically labeled viral genomes with radioactive uridine or orthophosphate during their synthesis in infected cells and then compared the electrophoretic profiles of the 70S RNA species isolated from purified virions (Duesberg and Vogt 1970). They found a correlation between transformation activity and the presence of viral RNA of greater molecular weight.

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Subsequent analysis using RNA footprinting of RNAs from paired samples of different strains of RSV (Prague C and Schmidt-Ruppin A) and their transformationdefective counterparts revealed that the transforming genomes were approximately 10–15% larger than those lacking transformation capabilities (Lai et al. 1973). This key experiment led to the conclusion that the additional genetic information contained in the longer RNA genomes of the transforming viral strains was responsible for the transformed state of infected fibroblasts. It took four more years before three groups independently mapped the unique region of RNA linked to transformation activity to sequences near the 3¢ end of the viral genome by isolating polyadenylated RNA via oligo-dT chromatography (Wang et al. 1976). Electron-microscopic studies that examined heteroduplexes that formed upon hybridization of RSV Prague B RNA and complementary DNA synthesized from spontaneously arising td viruses determined that the defective genomes indeed contained deletions. These studies revealed that the src gene was approximately 2,000 nucleotides in length and was located 800–3,000 bases from the 3¢end of the genome (Junghans et al. 1977). This gene was referred to by many names, including “x”, “onc” and “sarc”, but ultimately the designation “src” was widely adopted to reflect the sarcomas in chickens caused by RSV strains harboring this gene. Because the td strains were replication competent and efficiently produced virus particles from infected cells, src was considered dispensable for infection. Instead, it was postulated that the viral src gene encoded a protein “whose sole function is the generation of the transformed phenotype.” (Kamine and Buchanan 1977).

Discovery of the Cellular Origin of v-Src: The Virogene–Oncogene Hypothesis At the time of the discovery of the src oncogene in RSV, there was active controversy about the mechanism of virus-induced oncogenesis. The “virogene–oncogene” hypothesis was proposed in 1969, based on observations that RNA tumor virus genetic material, and sometimes virus particles as well, had been detected in many different types of vertebrate animals (Huebner and Todaro 1969). Huebner and Todaro proposed that RNA tumor virus sequences (the virogene), which included information that caused malignant transformation (the oncogene), were ubiquitously present in cellular genomes and were transmitted vertically. In their view, the viral oncogenes were present in a dormant or covert form but became activated by environmental stimuli including irradiation, chemical carcinogens, DNA tumor viruses, or the normal aging process. This idea was bolstered by the existence of the DNA provirus of RNA tumor viruses, which meant that sequences derived from retroviruses were permanently inserted into the cellular genome. Harold Varmus and Michael Bishop became interested in testing the “virogene– oncogene” hypothesis. In support of the hypothesis, Varmus et al. (1972) reported the detection of RSV DNA sequences in uninfected chicken cells. A critical question to resolve was whether the RSV sequences they found in normal cells coded for

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only the structural proteins of the virus, or was the transforming gene src present as well? A major challenge was to develop single-stranded radioactively labeled DNA probes that were specific for src to use in hybridization experiments (Varmus 1990). Varmus and Bishop, along with colleagues Dominique Stehelin and Peter Vogt, took advantage of the td mutants, presuming that the src gene was deleted in its entirety in these transformation defective strains, to obtain a src-specific probe (Stehelin et al. 1976). They reasoned that they could apply subtraction hybridization to remove the genes that were present in both the td and transforming genomes, leaving only the src gene fragment since it had nothing to pair with in the td sequence. To accomplish this feat, RNA isolated from RSV was reverse transcribed to produce cDNA fragments that were used to anneal to viral RNA isolated from td RSV virions. The heteroduplexes were removed using hydroxylappetite chromatography, resulting in purification of a single-stranded DNA src fragment that hybridized selectively to RSV but not td sequences. The probe was approximately 1,600 nucleotides in length and corresponded to ~16% of the RSV genome, consistent with earlier studies that mapped the transforming activity of RSV (Duesberg and Vogt 1970; Lai et al. 1973; Junghans et al. 1977). This probe was the key reagent used to identify the cellular homologue of the viral src gene. To directly test the oncogene–virogene hypothesis, the src probe and the td-RSV probes were hybridized with DNA from chickens and other avian species (Stehelin et al. 1976). The src probe hybridized strongly to DNA from chicken, turkey, duck, and quail; unexpectedly, there was also evidence of a sequence with significant homology in DNA isolated from an Australian emu, which was genetically divergent from the other bird species. By contrast, only chicken DNA contained a match for the td sequences containing the remaining viral genes. The authors concluded that the src transforming gene originated in chickens or a closely related species. Their experiments confirmed the “oncogene” hypothesis, but refuted the “virogene” component of the theory. They also speculated, correctly, that the cellular gene corresponding to src was evolutionarily conserved in avian species and it served an important role, possibly involving the “normal regulation of cell growth” (Stehelin et al. 1976) based on the tumor activity of the viral src gene. It was proposed that the cellular src gene became incorporated into the RSV genome by mechanisms involving recombination or transduction (Stehelin et al. 1976). The slight differences in the nucleotide composition of the cellular src homolog and the viral src gene were proposed to have arisen during numerous rounds of virus replication during which mutations in the viral gene were introduced.

Confirmation of c-src in Vertebrates: The First Protooncogene Subsequent experiments showed that src was present in cells from virtually every vertebrate species (Spector et al. 1978a, b). Furthermore, the src sequence was not linked to the replicative genes of endogenous retroviral sequences (Padgett et al. 1977; Hughes et al. 1979). Thus, the origin of the viral src gene (known as

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v-src) was almost certainly derived from a sequence present in the cellular genome. Definitive proof was provided when the cellular src gene (referred to as “c-src”) was cloned from normal chicken DNA (Parker et al. 1981; Shalloway et al. 1981; Takeya et al. 1981). The gene was present in a single copy in the chromosomal DNA spanning 33 kb, and the coding sequence was interrupted by intervening sequences, or introns (Fig. 28.4a). By contrast, the v-src gene was a contiguous sequence of 1,590 bp (Czernilofsky et al. 1980b). What mechanism could explain how the spliced src gene became incorporated into the RSV coding sequence? Evidence supported the idea that incorporation of the c-src gene into the RSV genome occurred after splicing; therefore, a simple DNA recombination event could not have resulted in capture of the c-src oncogene. Instead, a more complex mechanism needed to be invoked (Fig. 28.4b). Comparison of the sequences of v-src with the cDNA encoding c-src revealed differences at both the 5¢ and 3¢ ends of the genes (Swanstrom et al. 1983; Takeya and Hanafusa 1983). In v-src, a region of the c-src coding sequence was deleted, intronic and untranslated sequences of the cellular gene were introduced, and codons for the 19 C-terminal residues of c-Src were replaced with a sequence coding for 12 different amino acids in v-Src (Swanstrom et al. 1983; Takeya and Hanafusa 1983). Additionally, there were some minor differences in how the v-src genes were arranged in different strains of RSV (Prague C and Schmidt-Ruppin A), suggesting that acquisition of src might have arisen from separate transduction events (Swanstrom et al. 1983). Even though capture of the c-src gene might have occurred independently in various strains of RSV, the transduction of cellular genes by retroviruses is a rare event that has not been faithfully recapitulated under experimental conditions. Sequencing of the RSV genome and subsequent experiments that probed the mechanism underlying retroviral transduction led to the development of two models that explain how c-src is incorporated into the RSV genome (Swanstrom et al. 1983; Takeya and Hanafusa 1983; Herman and Coffin 1987; Swain and Coffin 1992) (Fig. 28.4b). In each case, the initial event required is insertion of the provirus at or near the start of a cellular gene, just upstream of the coding sequence. One of two possible events likely occurred next. In the first, a region of the proviral sequence is deleted from the 3¢ long terminal repeat (LTR), bringing the beginning of the coding region of c-src just downstream of the viral env gene. Transcription by the host RNA polymerase II (polII) enzyme creates a premRNA that contained viral sequences linked to the introns and exons of the src mRNA. In the second potential mechanism, inefficient recognition of the proviral polyadenylation site by polII results in the synthesis of “readthrough” transcripts containing cellular DNA sequences linked to the provirus. The models converge at the next steps, in which the pre-mRNA undergoes splicing to remove the src introns. The spliced mRNA, which contains viral sequences merged with the src gene, is copackaged with wild-type unspliced viral RNA genomes into virions. During reverse transcription, the two viral RNAs recombine, even though they lack significant sequence homology at their 3¢ ends, through a process known as “illegitimate recombination.”

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Fig. 28.4 Potential mechanisms for transduction of the src oncogene into the genome of RSV. (a) The c-src gene is shown with 12 exons and the intervening intronic sequences. After transcription and splicing, the mRNA is generated, as indicated by the poly(A) tail shown as (AAA)n. (b) The proviral sequence of the retrovirus prior to acquisition of the src gene is shown. Integration of the proviral DNA into the chromosomal DNA of the cell occurs into a region near the 5¢ end of the src gene. Next, one of two possible mechanisms occurs (Swanstrom et al. 1983; Swain and Coffin 1992). In the first possible mechanism (1), there is spontaneous deletion of the LTR after integration; the fused DNA sequence undergoes transcription and splicing to generate a viral RNA product that contains RU5, gag, pol, env, and src. This viral RNA serves as a genome and is incorporated into a virus particle (oval) along with an unmodified viral genomic RNA consisting of RU5, gag, pol , and env. Through illegitimate recombination during reverse transcription, the src gene becomes incorporated into the viral RNA genome just upstream of the LTR sequence.

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The end result is incorporation of the coding region of c-src at the end of the env sequence, preceding the LTR (Schwartz et al. 1983, 1995; Swanstrom et al. 1983). Because the src gene is located between the LTRs, it is carried with the RSV genome, integrates into cells with the provirus, and is packaged into nascent virions with the viral genomic RNA in all subsequent replication cycles. After integration, the v-src gene is synthesized from a subgenomic, spliced mRNA to serve as the template for pp60 v-Src protein synthesis to promote tumor formation in the infected cell (Rabson and Graves 1997). RSV is unusual in that it acquired a cellular oncogene while maintaining all of the sequences needed for virus replication. In most other oncoretroviruses, cellular gene transduction was concurrent with deletion of an essential viral gene. In these cases, a helper virus was required to allow for transmission of the virally induced tumor from cell to cell (Fine and Sodroski 2000). The c-src gene became known as a “protooncogene’”, a term coined by Bishop to designate the cellular progenitor of the viral src oncogene (Bishop 1985, 1990). Bishop was not in favor of the term “cellular oncogene” because the cellular homologues of viral oncogenes do not cause malignant transformation in normal cells. Instead, the host genes encode proteins engaged in processes that regulate cell growth and differentiation. Examples of protooncogenes include certain growth factors and growth factor receptors, transcription factors, receptor and nonreceptor protein kinases involved in signal transduction, chromatin modifiers, and regulators of apoptosis (Croce 2008). A protooncogene can become oncogenic when its expression level or activity is deregulated. This disturbance in function can arise from a change in gene expression owing to insertion of a strong proviral LTR promoter upstream of the transcription start site of the protooncogene. The abnormal activation of protooncogene expression causes uncontrolled cell growth and malignant transformation. Other nonviral mechanisms also induce protooncogenes to become tumorigenic, including mutations that arise in sensitive coding regions that alter protein function, chromosomal rearrangements that place alternative promoters or enhancers nearby that stimulate gene expression, and gene duplications or amplifications that result in multiple copies of the protooncogene being expressed. Since the c-src gene was identified in 1976, over 80 protooncogenes have been discovered, providing critical tools needed to understanding how normal cell growth and differentiation are regulated and how cancer arises when these control mechanisms go awry (Cooper 1995; Vogelstein and Kinzler 2002).

Fig. 28.4 (continued) In the second possible mechanism (2), the cellular RNA polymerase II reads through the end of the LTR, ignoring the viral polyadenylation signal, resulting in the fudion of the src coding sequence at the 3¢ end of the viral RNA (Swain and Coffin 1992). This mutated viral RNA then serves as a viral genome and become encapsidated into a virus particle (oval) along with a wild-type genomic RNA. In the virus particle, reverse transcription occurs with illegitimate recombination generating the positioning of src within the 3¢ LTR

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Isolation of the Protein Product of the Viral src Gene Once the src gene was discovered, identification of the gene product of src quickly followed [reviewed in (Martin 2001, 2004)]. Several groups used the 35 S viral RNA isolated from td and nondefective (nd) variants of different strains of RSV to synthesize viral proteins using a reticulocyte lysate in vitro translation system (Purchio et al. 1977; Beemon and Hunter 1978; Kamine and Buchanan 1978; Kamine et al. 1978; Sefton et al. 1978). A series of proteins ranging in size from 13 to 180 kilodaltons (kDa) were detected, with a proteins of 60 kDa identified in the nd strains but not in the td strains. Brugge and Erikson (Brugge and Erikson 1977) had recently reported using serum from tumor-bearing rabbits infected with Schmidt-Ruppin subgroup D RSV to identify a 60 kDa transformation-specific antigen. The 60 kDa protein was detected in cells from two different species but not from cells infected with nontransforming variants. Ensuing studies of the viral Src oncoprotein determined that it possesses tyrosine kinase activity (pp60v-src), which was surprising, as there were no previously known tyrosine protein kinases (Collett and Erikson 1978; Levinson et al. 1978; Hunter and Sefton 1980; Hunter et al. 1980). The Src protein is synthesized on cytosolic ribosomes and transported rapidly to the plasma membrane by virtue of a short, myristylated membrane-targeting sequence at the N-terminus (Buss and Sefton 1985). The tyrosine kinase activation domain is located near the C terminus of the protein, and several residues within Src are autophosphorylated (Hunter and Cooper 1985). Tyrosine protein kinase activity is required for transforming activity (Bryant and Parsons 1984; Wilkerson et al. 1985), and cells transformed by v-src exhibit an approximately tenfold increase in total protein phosphorylation (Sefton et al. 1980). Analysis of the targets of Src phosphorylation yielded insights into mechanisms responsible for the transformed state (Martin 2001, 2004). Characterization of the c-Src protein revealed that it is also a protein tyrosine kinase, but it lacks robust transforming activity (Iba et al. 1984; Parker et al. 1984; Shalloway et al. 1984; Johnson et al. 1985; Wang 1987). The tyrosine kinase activity of c-src is tightly controlled, and in contrast to v-src, its overexpression does not result in a dramatic increase in tyrosine phosphorylation of cellular proteins (Iba et al. 1985). Furthermore, replacement of v-src with the c-src sequence in RSV results in a loss of tumor formation (Iba et al. 1984) and the differences in activity mapped to amino acid differences within the C-terminal sequence. Kinase activity is mediated by the catalytic domain, which is activated through autophosphorylation of a tyrosine residue, and mutants of v-Src that map to this region have temperaturesensitive phenotypes or lose transforming activity completely (Parsons and Weber 1989). The kinase domain of c-Src is functional, but its activity is regulated negatively by phosphorylation of its C-terminal tyrosine (Nada et al. 1991). Structural studies of the active and inactive forms of Src revealed the elegant mechanisms by which intermolecular interactions regulate phosphorylation and dephosphorylation that dictate its kinase activity (Gonfloni et al. 1997; Weijland et al. 1997; Williams

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et al. 1997; Xu et al. 1997, 1999; Roskoski 2004). By contrast, v-Src C-terminal tyrosine, and the kinase domain is, constitutively active (Parsons and Weber 1989), accounting for its potent transforming activity.

Role of c-Src in Human Cancers The cloning of the human c-src gene paved the way to explore the mechanism of tumorigenesis in human tumors (Gibbs et al. 1985). Initial insights into the potential signaling cascades controlled by c-Src were gleaned by studying the changes in cells that expressed v-Src. These transformed cells exhibited uncontrolled cellular proliferation mediated by mitogen signaling, anchorage and growth factor-independent growth, disruption of cell motility and cell–cell adhesion, deregulation of integrins, and stimulation of a host of signal transduction pathways, including MAPK (mitogen-activated protein kinase), PI3K (phosphatidylinositol 3-kinase), Ras, Stat3 and, FAK (focal adhesion kinase) (Martin 2001; Guarino 2010; Maslikowski et al. 2010). Even though many targets of v-Src were ultimately found to contribute directly to transformation and proliferation, the challenge remained to determine whether c-Src played a central role in the development of human cancers. Although beyond the scope of this chapter, a large collection of data supports the critical role of Src in tumor progression, angiogenesis, and metastasis in human cancers of the colon, breast and prostate (Mao et al. 1997; Irby and Yeatman 2000; Frame 2002; Mayer and Krop 2010). Inhibitors of Src and related tyrosine kinases have been used with great success to treat patients with hematologic malignancies (Krause and Van Etten 2005; Baselga 2006), and Src-specific antagonists are under development for clinical use for the treatment of various solid tumors and bone metastasis (Araujo and Logothetis 2009, 2010; Aleshin and Finn 2010; Lieu and Kopetz 2010; Mayer and Krop 2010; Rothschild et al. 2010). Many reviews of the Src oncoprotein and its relationship to human cancers are available to provide more in-depth information (Parsons and Weber 1989; Irby and Yeatman 2000; Martin 2001, 2004; Frame 2002; Alper and Bowden 2005; Guarino 2010). The importance of RSV as the stimulus for the discovery of protooncogenes was recognized by the Nobel Assembly in the press release that announced the winners of the Prize in Medicine or Physiology in 1989: “Michael Bishop and Harold Varmus used an oncogenic retrovirus to identify the growth-controlled oncogenes in normal cells. In 1976 they published the remarkable conclusion that the oncogene in the virus did not represent a true viral gene but instead was a normal cellular gene, which the virus had acquired during replication in the host cell and thereafter carried along. Bishop’s and Varmus’ discovery of the cellular origin of retroviral oncogenes has had an extensive influence on the development of our knowledge about mechanisms for tumor development.” (The Nobel Prize in Physiology or Medicine 1989 – Press Release 1989) Press Release, The Nobel Assembly at the Karolinska Institute, 9 October 1989, announcing the 1989 Nobel Prize in Physiology or Medicine. Nobelprize.org. 1 Aug 2010 http://nobelprize.org/nobel_prizes/medicine/ laureates/1989/press.html

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RSV Virion Assembly and Structure In addition to its critical role in establishing viruses as a cause of cancer, RSV was also the initial retroviruses identified. RSV serves as the prototype for the family of oncoretroviruses, leading the way in elucidating many fundamental features of the virus replication cycle. As the fields of cancer biology, oncogenesis, and retrovirology have diverged, RSV has remained a mainstay in pioneering studies of virus–cell interactions, retroviral enzymology and virus assembly. Many of the seminal discoveries arising from experiments using RSV have been applied directly to treatment strategies for human retroviral treatments and to the development of viral vectors for gene therapy. The virion structure of RSV shares many properties with all retroviruses, which are spherical, enveloped particles (Fig. 28.1) [reviewed in (Vogt 1997)]. Virus particles are composed of 65% protein, 35% lipid, and 1–2% RNA. Although the majority of the protein and RNA species in the virus particle are derived from the virus, many host proteins and RNAs are also incorporated into retrovirus particles. The envelope is derived from the host plasma membrane when the virus particle buds from the host cell [reviewed in (Wills and Craven 1991; Craven and Parent 1996; Swanstrom and Wills 1997; Demirov and Freed 2004)]. Envelope glycoproteins (made up of the SU, surface, and TM, transmembrane components) protrude as “spikes” from the virion envelope and attach to specific receptors on the target cell to permit virus entry [reviewed in (Hunter 1997)]. Virus particles are assembled within the cell as immature particles, which are not infectious. The morphology of immature particles is similar for all retroviruses, and consists of an electron dense layer of Gag and Gag–Pol proteins just under the lipid envelope (Swanstrom and Wills 1997). Gag and Gag-Pol proteins are arranged in a hexameric lattice with irregular defects to form a nearly spherical shell (de Marco et al. 2010). The arrangement of the viral RNA genome within the particle is not well understood. Concurrent with budding, the immature virus particle undergoes maturation, a process catalyzed by the retroviral PR (protease) enzyme, to generate the infectious virus particle (Swanstrom and Wills 1997) (Fig. 28.1). Although the sequence encoding PR is invariably located between the gag and pol genes in all retroviruses, the presence of PR as a fusion with the Gag polyprotein is unique to RSV. During processing, the viral protease dimerizes and cleaves the immature Gag polyprotein into the MA (matrix), CA (capsid), and NC (nucleocapsid) proteins, common to all Gag proteins, as well as the p2, p10, and SP (spacer) peptides, which are exclusively present in RSV. The crystal structure of RSV PR was the first high resolution PR structure obtained (Jaskolski et al. 1990). Importantly, the RSV PR structure was used as a model to design inhibitors of HIV-1 PR, which have become principal components of the anti-retroviral drug cocktail (Weber et al. 1989; Weber 1991; Wensing et al. 2010). With the processing of Gag and virus maturation, the appearance of the particle undergoes a dramatic transformation, with the appearance of an electron-dense irregular core structure near the center (Fig. 28.1) (Swanstrom and Wills 1997).

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Fig. 28.5 Current model of RSV replication cycle. Infection is initiated with binding of the viral glycoprotein (SU) to the cellular receptor (Tva), triggering fusion of the viral and cellular membranes. The viral core enters the cytoplasm and reverse transcription occurs within the preintegration complex (PIC) in the cytoplasm. The PIC is translocated to the nucleus, and nuclear entry occurs with nuclear envelope breakdown during mitosis and also through the intact nuclear pore in nondividing cells. Integration into the chromosome is mediated by the IN enzyme and facilitated by host factors. The proviral DNA is transcribed by polII and genome-length viral RNA are made. A subset of these mRNAs undergoes splicing and exit the nucleus for translation into Env and Src. Env is transported to the cell surface through the Golgi apparatus and secretory pathway. The remaining unspliced viral RNA has two fates. If it serves as the template for synthesis of Gag and Gag-Pol, it exits the nucleus, likely using the host cofactors Tap and Dbp5. If it is packaged into virus particles, there is evidence that the genomic viral RNA might interact with a fraction of the Gag protein that traffics back into the nucleus after its synthesis in the cytoplasm. Gag has a nuclear export signal that mediates exit of the viral ribonucleoprotein complex from the nucleus. In the cytoplasm, Gag-vRNA complexes are targeted to the plasma membrane using host factors, combine with additional Gag and Gag-Pol proteins, and deform the plasma membrane to release virus particle through budding. As budding occurs, the maturation process occurs to generate a fully infectious virion

The hexameric array of Gag polyproteins is reorganized into a polyhexagonal shell composed of the CA protein organized into hexamers and pentamers (Butan et al. 2008; Heymann et al. 2008; Cardone et al. 2009). The MA protein is arranged in an irregular fashion under the lipid envelope near the transmembrane (TM) domain of the envelope glycoprotein (Pepinsky and Vogt 1979). Presumably, the center of the particle contains the NC protein bound to the viral genomic RNA. The specific locations of other viral proteins, including p2, p10, SP, PR, RT, and IN are unknown. The RSV replication cycle begins with binding of the SU component of the envelope glycoprotein to its receptor, Tva, a relative of the low-density lipoprotein receptor (Bates et al. 1993, 1998; Young et al. 1993) (Fig. 28.5). Binding triggers

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fusion of the viral and cellular membranes, followed by entry of the viral core into the cytoplasm (Barnard et al. 2006). Reverse transcription of the viral genome takes place en route to the nucleus, but is not completed until after nuclear entry, when proviral synthesis is completed (Varmus et al. 1978; Lee and Coffin 1991). Although it was originally thought that nuclear entry of the RSV preintegration complex required breakdown on the nuclear envelope during mitosis, more recent analysis revealed that RSV integration does occur, albeit inefficiently, independently of cell division (Hatziioannou and Goff 2001; Katz et al. 2002). The proviral DNA is incorporated into the host chromosomal DNA by the viral IN enzyme, and integration occurs preferentially in regions of the genome that encode genes transcribed by RNA polymerase II (Brown 1997; Narezkina et al. 2004). Following integration, RNA polymerase II transcription of the proviral DNA results in the synthesis of a genome-length viral RNA, which is processed like cellular mRNAs to contain a 5¢ 7-methylguanine cap and a 3¢ poly(A) tail (Rabson and Graves 1997; Vogt 1997). A subset of the full-length viral mRNA undergoes splicing to produce mRNAs that encode the Env and Src proteins, and these mRNAs are exported from the nucleus, presumably by the usual route for spliced mRNAs that involves deposition of exon–exon junction machinery (Kim and Dreyfuss 2001). By contrast, the unspliced viral mRNA has two potential fates, one as the template for translation of the structural proteins Gag and Gag-Pol, and the other as the genome for encapsidation into assembling virions (Butsch and Boris-Lawrie 2002). The factors that govern whether the unspliced, genome-length viral mRNA is translated or packaged are not well understood. However, retroviruses must export their unspliced viral RNA from the nucleus to make Gag and Gag-Pol proteins to begin the process of assembling new virus particles (Rabson and Graves 1997; Vogt 1997). To do so, the normal quality-control measures that prevent unspliced cellular mRNAs from leaving the nucleus prematurely must be circumvented. For RSV, the cellular Tap/Dbp5 complex is reported to play a role in export of the unspliced viral RNA by binding to the cis-acting DR (direct repeat) elements that flank the src gene (Paca et al. 2000; Leblanc et al. 2007). Assembly of new virions is mediated by the Gag polyprotein, which is sufficient to drive particle production and release (Wills and Craven 1991; Craven and Parent 1996; Swanstrom and Wills 1997; Pincetic and Leis 2009) (Fig. 28.6). To initiate virus assembly, the Gag NC region selects the viral RNA for encapsidation by binding to the psi sequence in the RNA sequence (Jaskolski et al. 1990; Rein 1994; Swanstrom and Wills 1997; Jewell and Mansky 2000; D’souza and Summers 2005; Zhou et al. 2005, 2007). Recent evidence supports a model whereby the RSV Gag polyprotein, which undergoes transient nuclear trafficking, may capture its unspliced viral RNA genome in the nucleus, thereby circumventing cellular mechanisms that retain unspliced mRNAs in the nucleus (Scheifele et al. 2002; Garbitt-Hirst et al. 2009). The RSV Gag protein has a dynamic trafficking pathway throughout the cell and interacts with numerous host factors from its synthesis on ribosomes, through the nucleus, and to the plasma membrane (Craven and Parent 1996; Scheifele et al. 2002; Butterfield-Gerson et al. 2006; Pincetic and Leis 2009). The regions of Gag required for virus particle assembly have been identified as the M (membrane-binding),

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Fig. 28.6 Functional domains and host partners of the Gag protein. The RSV Gag protein is synthesized as a polyprotein, which is cleaved during virus maturation to generate the MA (matrix), p2a, p2b, p10, CA (capsid), SP (spacer), NC (nucleocapsid), and PR (protease) proteins. Gag contains two nuclear localization signals (NLS). The NLS in the N-terminal region of MA binds to host import factors importin-11 and transportin-SR/transportin 3. The NLS in NC interacts with the importin-alpha adaptor, which in turn binds to importin-beta to create a functional nuclear import complex. The NC region also contains a zinc-knuckle domain composed of Cis-His boxes, which are responsible for binding to the genomic vRNA for encapsidation. The CRM1 export factor binds to a nuclear export signal (NES) in the p10 region of Gag. The MHR, or major homology region, of Gag is indicated and plays an important structural role. The assembly domains are illustrated below the Gag molecule. The M (membrane-binding domain) folds as an alpha-helical domain that transports Gag to the plasma membrane through a poorly understood trafficking process. The I (interaction) domain in NC is present in two copies is made up of the Cis-His boxes (Zn2+ knuckle domains indicated) and basic residues (red + containing circles) that are important for NC–vRNA and NC–NC interactions. The L (late) domain is in the p2b region and interacts with a complex of host factors including the ubiquitin ligase Nedd4 and several members of the ESCRT-II and ESCRT-III families to facilitate the final pinching-off step of the budding process

I (interaction), and L (late) domains (Wills and Craven 1991). Analogous functional assembly domains were subsequently identified in other retroviral Gag proteins (Bennett et al. 1993; Weldon and Wills 1993; Wills et al. 1994; Zhou et al. 1994; Parent et al. 1995; Verderame et al. 1996; Swanstrom and Wills 1997; Bowzard et al. 1998; Sandefur et al. 2000; Pincetic and Leis 2009). The L domains of different retroviruses contain sequence motifs that interact with a multitude of host factors that belong to the ESCRT family of endosomal transport factors that mediate the final release of particles from the plasma membrane (Parent et al. 1995; Demirov and Freed 2004; Morita and Sundquist 2004; Pincetic and Leis 2009). Although initially discovered in RSV and HIV, L domains and the family of host proteins with which they interact are shared by many enveloped viruses (Freed 2002; Licata et al. 2003; Irie et al. 2004; Schmitt et al. 2005; Zhadina and Bieniasz 2010). Discovery of this common pathway usurped by many unrelated enveloped viruses may lead to the development of agents with broad antiviral activity.

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Conclusions RSV, “a humble chicken virus,” (Vogt 2009) has an important place in history for the breadth and magnitude of its contributions to biomedical sciences and molecular biology (Table 28.1). The scientists who studied RSV were rewarded for their insights, not only by the awards and prizes they received for their achievements but also with the excitement generated by the broad-reaching implications of their work. Table 28.1 Timeline of landmark discoveries derived from studies of RSV Year Discovery 1910 A spindle cell tumor could be transferred from one Plymouth Rock hen to another (Rous 1910) 1911 Tumor was transmitted by a filterable virus that fulfilled Koch’s postulates, proving infectious origin of the tumor (Rous 1911) 1938 Development of a method to measure the titer of infectious RSV (Keogh 1938) 1941 RSV-infected chicken embryo fibroblasts in culture demonstrate characteristic spindle-shape morphology (Halberstaedter et al. 1941) 1955 Each RSV-infected cell produces virus particles, and the virus perpetuates malignant state of the cell (Rubin 1955) 1956 Described the morphology of RSV using electron microscopy (Epstein 1956; Epstein and Holt 1958) 1958 Development of the focus assay, which isolated colonies of individual cells infected with RSV (Temin and Rubin 1958) 1959 RSV induces tumors in mammals (Schmidt-Ruppin 1964) 1960 “Provirus Hypothesis” proposed (Temin 1976) 1961 RSV virions contain an RNA genome (Crawford 1960) 1970 Discovery of reverse transcriptase activity in virions (Baltimore 1970; Temin and Mizutani 1970) 1976 Discovery of cellular src gene in avian species (Stehelin et al. 1976) 1977 Development of an antibody to detect v-Src protein (Brugge and Erikson 1977) 1980 Nucleotide sequence of the v-src gene (Czernilofsky et al. 1980a; Takeya and Hanafusa 1982; Czernilofsky et al. 1983) 1981 Cloning of chicken c-src gene (Shalloway et al. 1981) 1985 Cloning of human c-src gene (Gibbs et al. 1985) Src oncoprotein discovered to have tyrosine kinase activity (Collett and Erikson 1978; Levinson et al. 1978) 1978 Discovery of cis-acting packaging sequence sets stage for retroviral vectors 1985 Development of reverse transcriptase inhibitors for HIV treatment (Yarchoan et al. 1986) 1989 Crystal structure of RSV protease (Miller et al. 1989) 1991 Description of retroviral assembly domains (Wills and Craven 1991) 1995 Development of protease inhibitors for HIV treatment (Kitchen et al. 1995) 1997 Crystal structure of Src (Williams et al. 1997; Xu et al. 1997) 1999 Role of Src in human cancer demonstrated (Irby et al. 1999) 2004 Src-specific kinase inhibitors developed for human tumor treatment (Lombardo et al. 2004) 2009 Structure of the pentamer of mature polyhedral RSV capsids visualized (Cardone et al. 2009)

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Studies based on RSV launched entire new fields: tumor biology, cancer genetics, oncogenes, retrovirology, tyrosine kinases, reverse transcription, cDNA cloning, gene therapy vectors, and treatment of human cancers and AIDS. Ongoing research into Src and its activities in normal and malignant processes continues to make contributions to understanding the cell biology of cancer. Finally, the virus itself is the basis for contemporary novel findings regarding fundamental mechanisms in the retrovirus replication cycle. Acknowledgements The author is grateful for support from the national institutes of health great number ROI CA76534 and thanks members of her laboratory for eritical review of the manuscript.

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Chapter 29

Mouse Mammary Tumor Virus and Cancer Susan R. Ross

Introduction A key to understanding the transformation of normal cells is dissecting the various genetic changes that lead to abnormal cell division. Since their discovery at the beginning of the twentieth century, transforming retroviruses have been used to study both the mechanism and progression of tumor development (Rous 1911). Three general classes of transforming retroviruses have been described: acute transforming retroviruses, which encode oncogenes in their genome and thereby cause rapid, polyclonal tumors of virtually all infected cells; nonacute transforming retroviruses, which cause dysregulated expression of cellular oncogenes upon integration of the provirus into the host genome and usually cause monoclonal tumors with latencies longer than those seen with acute retroviruses; and transactivating, transforming retroviruses, which encode proteins that contribute to transformation but are not by themselves sufficient to induce full-blown cellular transformation (Coffin et al. 1997). Several other chapters in this book cover transforming retroviruses that cause cancer in humans as well as other mammals (see chapters on Jaagsiekte Sheep Retrovirus and Lung Cancer, Avian and Murine Retroviruses, Rous Sarcoma Virus, Human and Animal Retroviruses, HTLV-1 and HTLV-2). Here, I present a brief review of the betaretrovirus mouse mammary tumor virus (MMTV), what is known about how the virus induces tumors, and how this knowledge has been used to develop a large number of mouse models for human breast cancer. The recognition in the nineteenth century by “mouse fanciers” that mammary tumors occur in mice, coincided with the breeding of mice for particular genetic traits [for an excellent review of the history of mammary tumorigenesis in mice, see Cardiff and Kenney (2007)]. In 1933, a landmark paper from scientists at the Jackson

S.R. Ross (*) Department of Microbiology, Abramson Cancer Center, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA e-mail: [email protected] E.S. Robertson (ed.), Cancer Associated Viruses, Current Cancer Research, DOI 10.1007/978-1-4614-0016-5_29, © Springer Science+Business Media, LLC 2012

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Laboratory demonstrated that there was maternal inheritance of mammary tumor incidence; that is, the F1 female offspring of high tumor-incidence mothers mated with low tumor-incidence fathers developed mammary tumors while the F1 offspring of the reciprocal cross did not (Jackson and Little 1933). Shortly thereafter, J.J. Bittner showed that high mammary tumor-incidence mice could be freed of a milk-transmitted agent by foster nursing on low tumor-incidence mothers and suggested that this agent might be a virus, representing the first identified mammalian tumor virus (Bittner 1936). However, it was not until the 1960s that “Bittner agent” or MMTV was shown to have an RNA genome like Rous sarcoma virus and thus was definitively classified as a virus (Duesberg and Blair 1966). In the three intervening decades between Bittner’s observations and the definitive classification of MMTV as a virus, much was learned about its in vivo transmission and the genetics of susceptibility of different inbred strains of mice to infection (Nandi and McGrath 1973; Okeoma and Ross 2010). The use of recombinant DNA technology and the advent of transgenic and knockout mice in the 1980s led to the identification of the genes involved in susceptibility and resistance to virus infection and novel oncogenes associated with MMTV-induced mammary tumors [recently reviewed in Okeoma and Ross (2010) and Ross (2010)]. This in turn allowed the development of new mouse models of human breast cancer, as described below.

MMTV Infection and Germline Transmission Milk-transmitted MMTV is produced by the mammary epithelial cells of lactating mothers and is most likely transmitted as cell-free virus to suckling mice during the 1st week of life (Nandi and McGrath 1973). In nursing pups, the virus first infects dendritic cells in the gut and then spreads to B and T cells in the Peyer’s patches and mesenteric lymph nodes. During the first 3 weeks of life, MMTV infection amplifies throughout cells of the immune system, which then traffic to the mammary gland and deliver virus to puberty-driven dividing mammary epithelial cells (Ross 2000). MMTV infection of mammary epithelial cells also increases during pregnancyinduced cell division, but still depends on the presence of infected lymphocytes in this tissue (Fig. 29.1) (Golovkina et al. 1998). Since MMTV is a nonacute, transforming retrovirus and causes transformation by integrating near-cellular oncogenes and activating their expression, the more mammary cells infected, the more likely it is that such an integration event will occur. Thus, virgin mice which have lower levels of mammary epithelial cell infection have decreased tumor incidence compared to mice that have gone through multiple rounds of pregnancy and the average timeto-tumor formation is longer in virgin than in multiparous mice (Nandi and McGrath 1973). Tumor induction is close to 100% by 1 year of age in mouse strains that are susceptible to MMTV infection. In addition to acquiring exogenous virus through milk, mice can also inherit endogenous copies of the provirus, termed Mtv loci (Kozak et al. 1987). Indeed, most commonly used laboratory strains have from one to six copies of MMTV in their germline, the vast majority of which have one or more mutations in the viral genes

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Puberty and initial infection

Provirus integration into chromosome

PregnancyAdditional infection

Oncogene activation

Transformation

Partruition = apoptosis

Fig. 29.1 MMTV in vivo infection pathway. Lymphocytes in the small intestine become infected by MMTV and virus spreads in these cells. The infected lymphocytes traffic to the pubescent and lactating mammary gland and mammary epithelial cells get infected (Finke and Acha-Orbea 2001). This results in mammary epithelial cells that secrete virus into milk which is transmitted to the next generation. Proviral insertion occurs during puberty, but the higher virus load that occurs after multiple pregnancies increases the probability that activating insertions next to cellular oncogenes will occur, leading to transformation

and thus do not produce infectious virions. However, not all wild mice have endogenous MMTV proviruses and thus it has been estimated that MMTV first infected mice ~10 million years ago, after their speciation (Baillie et al. 2004). There are also several strains of inbred mice that inherit functional, fully infectious endogenous copies of MMTVs, most likely representing more recent germline integrations; such mice cannot be freed of mammary tumor induction by foster nursing (Nandi and McGrath 1973; Michalides et al. 1978). At least 10 different exogenous and more than 30 endogenous MMTVs have been identified (Kozak et al. 1987; Imai et al. 1994; Callahan and Smith 2000; Golovkina et al. et al. 1997).

MMTV Infection and Replication Enveloped viruses, like MMTV, enter cells when the viral glycoprotein (Env) on virions binds to receptors on the target cell surface. MMTV uses transferrin receptor 1 (TfR1) as its entry receptor (Ross et al. 2002). TfR1, whose normal function is to mediate uptake of iron-loaded transferrin, is highly expressed on actively dividing cells (Ponka and Lok 1999). Although virtually all cultured cells express large amounts of TfR1, expression in vivo is limited to activated lymphocytes and a few other tissues, including epithelial cells of the pregnant mammary gland (Brekelmans et al. 1994; Schulman et al. 1989). Thus, milk-transmitted virus infection is largely limited to these cell types in mice (Nandi and McGrath 1973). Upon binding transferrin, TfR1 is endocytosed to the recycling endosome, a pH 6 compartment, where iron is released; the receptor then recycles to the surface

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(Ponka and Lok 1999). However, MMTV requires both receptor binding and pH 5 to achieve entry into cells (Wang et al. 2008). MMTV binding to TfR1 redirects its endocytosis to a late endosomal compartment from which the virus enters cells. After entry into the cytoplasm, MMTV is reverse transcribed and the doublestranded DNA enters the nucleus and integrates into the chromosome; nuclear entry of the preintegration complex, which consists of viral DNA and proteins as well as cellular proteins, is believed to require cell division, similar to other retroviruses with the exception of the lentiviruses (Ross 1997b). However, these steps in the MMTV infection pathway have not been well-elucidated.

Transcriptional Control of MMTV Retroviral long terminal repeats (LTRs) contain the transcription start site as well as the regulatory sequences that determine proviral expression after integration into the host chromosomes. In the case of MMTV, which infects both lymphoid and mammary epithelial cells, there are sequences that specify expression in both cell types as well as hormone-regulated virus transcription (Ross 1997b). One of the earliest observations made about MMTV was that glucocorticoids and progesterone increase the amount of shed virus in both mice and cell lines derived from MMTVinduced mammary tumors (McGrath 1971). The increased virus production is due to enhanced transcription of MMTV, the result of direct interaction of the glucocorticoid or progesterone hormone receptors with sequences in the LTR (Payvar et al. 1983). More recently, it has been shown that other transcription factors, such as FoxA1 and NF1, participate in the hormone-regulated control of MMTV transcription (Fig. 29.2a) (Holmqvist et al. 2005; Vicent et al. 2010). Regulatory elements that determine high levels of viral transcription in mammary cells have also been identified in the LTR (Fig. 29.2a). These include recognition sites for the STAT5 transcription factor, which is a target of prolactin receptor signaling and is critical for the development of mammary alveolar epithelial cells, the cell type most often the target of MMTV-induced transformation (Qin et al. 1999; Cardiff and Kenney 2007). Indeed, MMTV transcription in mammary epithelial cells is induced by prolactin (Qin et al. 1999). While both MMTV proviruses acquired by infection and many endogenous Mtv loci are expressed in virgin mammary gland, pregnancy and lactation dramatically increase their transcription. This is due in part to increased prolactin, glucocorticoid, and progesterone levels and because another factor, the Cutl1/CCAAT displacement protein (CDP), which represses MMTV transcription in virgin mammary glands, declines in late pregnancy at the same time that there is an increase in the level of viral transcripts (Maitra et al. 2006). Additionally, there are other sequence elements that determine mammary epithelial cell transcription for which the transcription factors have not been identified (Mok et al. 1992; Mink et al. 1992; Qin et al. 1999). Because the MMTV LTR directs high levels of expression in mammary cells, it has been extensively used to create transgenic mice that develop breast cancer.

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b Wnt, Fgf (upstream/downstream; enhancer activation) Notch (exon; disrupt N terminus) eIF3e (intron; premature termination; truncated protein)

Fig. 29.2 The MMTV LTR contains transcriptional regulatory elements that control virus and oncogene expression. (a) Diagram of the MMTV LTR and the relative position of the transcription factor-binding sites that have been identified (see text). MCF mammary cell factor. (b) Possible mechanisms by which MMTV insertion near a cellular oncogene can result in dysregulated oncogene expression. Known examples of each mechanism are shown

Indeed, the MMTV-c-myc transgenic mouse was the first mouse model of breast cancer (Stewart et al. 1984). Since 1984, a large number of different genes have been placed under the control of the MMTV LTR (ras, neu, simian virus 40 large T antigen, polyomavirus middle T antigen, transforming growth factor b, etc.) and used to study mammary gland differentiation and transformation in transgenic mice (Cardiff 2001, 2003). In many of these models, the incidence and kinetics of mammary tumor induction are increased in multiparous versus virgin mice, most likely the result of the increase in LTR-driven transcription. MMTV-cre-recombinase transgenic mice have been crossed to mice with floxed genes to induce mammary epithelial cell-specific deletion of tumor-suppressor and other genes (Wagner et al. 2001). Although MMTV-transgenic mice have been an invaluable tool for researchers, in some models, high level transgene expression is dependent on pregnancy and thus this system is not always amenable to studying early events in mammary cell differentiation. MMTV also infects lymphocytes and other cells of the immune system and there are transcriptional regulatory elements in the virus that drive expression in these cells (Ross 1997a; Arroyo et al. 1997; Reuss and Coffin 2000). It is, therefore, not surprising that many MMTV-oncogene transgenic mice, including the original MMTV-myc mice, develop lymphomas in addition to mammary tumors (Stewart et al. 1984; Leder et al. 1986; Choi et al. 1987). MMTV expression levels are, however, much lower in lymphoid than mammary tissue, so MMTV LTR-driven transgenes generally have a much larger effect in the mammary gland. MMTV virus variants

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have also been found in some T-cell lymphomas (Ball et al. 1985; Michalides 1983). These T-cell tropic MMTVs have deletions/insertions in their LTRS that are believed to create novel transcription factor regulatory sites that drive higher expression of the variant viruses in T cells. Integration of the variant MMTVs in this cell type also activates oncogenes and transformation (see below) (Yanagawa et al. 1993; Bhadra et al. 2005).

MMTV Induction of Tumors MMTV is highly efficient at inducing mammary tumors in mice that are genetically susceptible to infection, with almost 100% of mice developing 1–2 independent tumors by the age of 1 year (Nandi and McGrath 1973). MMTV-induced mammary tumorigenesis occurs when the provirus integrates near-cellular oncogenes and activates their expression (Fig. 29.1). Because this integration appears to occur stochastically, the higher the level of infection, the more likely it is that such a transforming event will occur and as such, the kinetics and incidence of mammary tumor induction can be used as measures of infection levels (Golovkina et al. 1993). Retroviral insertion, which can occur >250 kb away from the cellular oncogene, alters oncogene expression through several mechanisms (Akagi et al. 2004). These include: enhancer activation, leading to de novo or increased expression of genes at the provirus integration site; promoter insertion, whereby the viral promoter is used in lieu of the normal promoter to initiate transcription; posttranscriptional dysregulation, which occurs when the virus integrates within the gene-coding region and either through read-through transcription or altered splicing results in an mRNA with an altered half-life; and gene disruption, where integration into the coding region results in the loss of gene expression or the production of a mutant or nonfunctional gene product [reviewed in Dudley (2003)]. Common integration sites (CISs) are defined by proviral integrations into the same genomic region in more than one independent tumor. Several groups have also defined CIS to include integrations into different genes of the same pathway; as an example, in MLVinduced leukemia, integrations into multiple members of the Notch pathway (notch1, notch2, jag1, lfng, dtx1, dtx2, tcfe2a, rbpsuh, hes1, hes2, hes5) have been detected (Suzuki et al. 2002). MMTV most frequently causes enhancer activation, although several examples of gene disruption/truncation have also been documented (Fig. 29.2b) (Callahan and Smith 2008). A large number of CISs (historically termed integration or int genes) have been implicated in MMTV-mediated mammary tumors (Table 29.1) (Callahan and Smith 2000, 2008). In particular, Wnt1 was the first oncogene cloned by insertion site analysis and Wnt family members and other genes in this pathway are frequently targeted by MMTV in tumors (Nusse and Varmus 1982; Callahan and Smith 2008). Numerous fibroblast growth factor genes, as well as their receptors, are also frequent sites of MMTV integration (Dickson et al. 1984; Theodorou et al. 2007; Callahan et al. submitted). Finally, members of the R-spondin (Rspo) family have

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Table 29.1 CIS in MMTV-induced mammary tumors in wild-type and transgenic mice Mouse strain Oncogene Associated with HBC a Wild type Multipleb Wnt1/Wnt10b − BALB/c Wnt3/Wnt9B/Nsf + Multiple Wnt3a/Wnt9a + Multiple Fgf3 + GR Tpl2/Cot + BALB/c Fgf4 − BALB/c Fgf10 + BALB/c, C3H Fgfr2 + Czech II Notch4 − BALB/c int-5/aromatase − Czech, BALB/c Rspo2 − BALB/c Rspo3 + Czech II eIF3e-p48 − C3H, Czech II Pdgfra − BALB/c Pdgfrb + Transgenic MMTV-Wnt1 Fgf4, Fgf8 −, + MMTV-neu Notch1 − WAP-TGFb Wnt1/Wnt3 −, + WAP-p53172H Pdgfra − Data is from Nusse et al. (1984), Theodorou et al. (2007), Callahan et al. (submitted), Lowther et al. (2005), Dickson et al. (1984), Erny et al. (1996), Theodorou et al. (2004), Gallahan and Callahan (1997), Durgam and Tekmal (1994), Gattelli et al. (2006), Marchetti et al. (1995), MacArthur et al. (1995), Shackleford et al. (1993), Dievart et al. (1999), Schroeder et al. (2000), Chatterjee et al. (2002), and Meyers and Dudley (1992) a Association with human breast cancer (HBC) analysis of the Oncomine database, the Cancer Genome Atlas, Cosmic (http://www.sanger.ac.uk/genetics/CGP/cosmic), and Broad Gene Ranker databases b More than two mouse strains, such as C3H/He, BALB/c, Czech II, and MMTV transgenics

also been identified as MMTV CIS (Gattelli et al. 2006; Lowther et al. 2005). These three gene families have been termed “core CIS” for MMTV. Several groups have recently also used high-throughput analysis insertion site analysis and uncovered genes not previously associated with MMTV-induced cancer (Theodorou et al. 2007; Callahan et al., submitted; Kim et al. unpublished observations). The variant MMTVs that induce T-cell lymphomas also integrate near-cellular oncogenes and activate their expression. Notch1, c-myc, and retinoic acid receptor g (RORg) have been identified as CISs in these tumors (Broussard et al. 2002, 2004; Yanagawa et al. 2000). MMTV-induced mammary tumors are thought to be the result of virus infection of a single initial stem cell, and mammary cell transplantation studies have indicated that oncogene integration events occur very early in transformation (Fig. 29.3) (Gattelli et al. 2006; Kordon and Smith 1998). Most MMTV-induced tumors proceed through at least two stages, the pregnancy-dependent hyperplastic alveolar nodule (HAN)

746

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env

Transcription/translation of Env(and other viral proteins)

Env expression on cell surface

ITAM-mediated signaling

CIS1 Oncogene activation CIS2

Activation of additional oncogenes Other mutations

TRANFORMATION Fig. 29.3 MMTV-mediated mammary tumorigenesis. MMTV infects mammary stem cells and after proviral integration, the viral genome is transcribed and translated. The Env protein then signals through its ITAM motif, leading to increased lobuloalveolar differentiation. After integration of the MMTV provirus into one or more CISs, the cell becomes fully transformed and develops into a tumor

followed by the hormone-independent mammary tumor (Callahan and Smith 2000). Some studies have suggested that progression of tumors from a pregnancy-dependent to a pregnancy-independent state depends on additional MMTV integration events and indeed, the former frequently present as polyclonal populations while the latter are generally monoclonal (Buggiano et al. 2002). Thus, integration events potentially affect both the early and late steps of transformation. Moreover, because MMTV infects stem cells, which can develop into ductal, alveolar, and epithelial–stromal cells within the mammary gland, insertional alteration of oncogene expression can occur in any of these cell types, which could affect the final tumor phenotype.

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Several other observations support the idea that the activation of multiple oncogenes is required for MMTV-induced mammary tumorigenesis. Most MMTV-induced tumors contain ten or more proviral integrations and a large percentage of mammary tumors derived from MMTV-infected mice have integrations at multiple CISs, such as Wnt1 and Fgf3 (Fig. 29.3) (Callahan and Smith 2000). Moreover, double-transgenic mice, such as MMTV-Wnt1/MMTV-Fgf3, exhibit accelerated mammary tumor induction in comparison to their single-transgenic MMTV-Wnt1 or MMTV-Fgf3 parental lines (Kwon and Weissman 1984). Similarly, MMTV infection of transgenic mice with a genetic predisposition to mammary tumorigenesis (e.g., MMTV-Wnt1, MMTVc-neu, or MMTV-Tgfb) accelerates tumorigenesis, and several novel oncogene insertion sites have been found in such mice (Dievart et al. 1999; Schroeder et al. 2000; Shackleford et al. 1993). There are rare MMTV-induced tumors with only one MMTV insertion site (Nusse and Varmus 1982); for these tumors, it has been suggested that genetic changes in cellular oncogenes that are not induced by virus could participate in transformation (Fig. 29.3) (Callahan et al. submitted). There is also evidence that MMTV infection of mammary epithelial cells leads to their dysregulated growth prior to the integration of the provirus into particular CIS. Early studies that examined the histopathology of mammary glands from uninfected and MMTV-infected mice indicated that lobuloalveolar differentiation, the result of increased cell division at terminal buds, was increased by viral infection (Squartini et al. 1983). This phenotype has been recapitulated in MMTV-transgenic mice (Ross et al. 2006a). More recent studies have indicated that this is due, at least in part, to expression of the MMTV Env protein in infected cells. The MMTV Env protein is found on the surface of infected cells and signals through an immunotyrosinebased activation motif (ITAM). ITAMs are highly conserved sequences found in receptors involved in the activation, proliferation, survival, and differentiation of hematopoietic cells (Grande et al. 2007). The tyrosine residues found in the canonical motif DxxYxx(L/I)x6-12Yxx(L/I) are necessary and sufficient for signaling and after phosphorylation by intracellular Src-family protein tyrosine kinases, the ITAMassociated tyrosines function as docking sites for SH2-containing signaling proteins, such as Syk, which link receptor-initiated signals to downstream cellular responses. Expression of the MMTV Env protein alone in cultured normal human or mouse mammary epithelial cells leads to morphological transformation (Katz et al. 2005). Moreover, infectious MMTV with mutations in the ITAM, while fully infectious in mice, shows slower kinetics and decreased incidence of mammary tumor induction (Ross et al. 2006b). Interestingly, a number of oncogenic viruses, some with tropism for nonhematopoietic cells, encode ITAM-containing plasma membrane-associated proteins that play a role in their ability to transform cells, including Epstein–Barr Virus (EBV) LMP2A, Kaposi’s Sarcoma Virus K1, and Bovine Leukemia Virus gp30 [reviewed in Grande et al. (2007)]. It has also been recently shown that the C35 protein, which is part of the HER2 amplicon and overexpressed in many invasive breast cancers, contains an ITAM and may be involved in transformation (Katz et al. 2010). These data suggest that ITAM-mediated signaling by the MMTV Env and other proteins in infected mammary epithelial cells participates in the transformation process (Fig. 29.3).

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MMTV and Human Breast Cancer MMTV-induced mammary tumors usually have different histopathological signatures when compared to human breast cancers. For example, the most commonly seen form of human breast tumors, invasive ductal carcinomas, are rarely found in MMTV-infected mice. Instead, MMTV tumors are usually characterized as acinar in origin (Cardiff and Wellings 1999). However, many, if not all, of the human mammary lesions are thought to originate in the terminal ductal lobular unit and atypical lobular type A (ALA) lesions are morphologically similar to the mouse mammary HAN lesions (Callahan and Smith 2000). Moreover, the tumors that develop in MMTV-transgenic strains can resemble more common forms of human breast cancer. For example, tumors in MMTV-ErbB2 transgenic mice are very similar to human ductal carcinoma in situ (Cardiff et al. 2000; Mikaelian et al. 2004; Rosner et al. 2002). The first identified MMTV CIS, Wnt1, has not been implicated in human breast cancer. However, other Wnt family members and the downstream targets of the Wnt signaling pathway (b-catenin, E-cadherin, cyclin D1) are mutated or deregulated in many human cancers, including breast cancer (Table 29.1) (Li et al. 2000). Additionally, several Fgf family members have been implicated in human breast cancer (Meyers and Dudley 1992; Theodorou et al. 2004). In silico comparison of genes identified in MMTV CIS screens with expression databases of human breast cancers have uncovered new potential markers that may be useful for molecular diagnosis (Theodorou et al. 2007; Callahan et al. submitted). Thus, MMTV target site analysis in both wild-type and transgenic mice strains with tumor morphology similar to that seen in humans may have potential for uncovering pathways relevant to the human disease. A long unresolved question is whether any virus similar to MMTV exists in humans. Very early studies indicated the presence of MMTV-like proteins in human breast cancer samples and milk, as well as antibodies to MMTV proteins in human sera (Levine et al. 1984; Keydar et al. 1984). However, it is now believed that these studies detected cross-reacting proteins and nonspecific antibodies (Dion et al. 1987; Goedert et al. 2006). More recently, several groups have also detected sequences with 85–95% homology to MMTV in human breast cancer biopsies using nested polymerase chain reaction (Wang et al. 1995; Ford et al. 2003), although an equivalent number of studies have failed to find such sequences (Mant et al. 2004; Park et al. 2011). Similarly, there have been some studies indicating that the mouse virus can infect human cells (Lasfargues et al. 1979; Indik et al. 2007), although our group has been unable to show that MMTV can use human TfR1 as an entry receptor (Ross et al. 2002; Wang et al. 2008). If MMTV-like viruses do exist in humans, it is unclear how such viruses are transmitted. In contrast to MMTV-infected mice that develop mammary tumors, where most if not all mammary cells are infected, a very low percentage of cells in human breast cancer biopsies appear to contain these sequences and additionally, normal breast tissue from the same individuals are negative for the viral DNA (Wang et al. 1995).

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That only breast cancer and not the normal mammary epithelial cells carry MMTVlike sequences would, thus, argue against a milk-borne mode of transmission. In mice, the major mode of transmission is through milk and horizontal spread in adult mice housed in the same cage is very rare (Nandi and McGrath 1973). Moreover, infection of adult mice with MMTV results in life-long, high-titer antibodies against the virus (Luther et al. 1997), so if the virus was transmitted from mice to humans, humans with breast cancer should have anti-MMTV antibodies, which, as discussed in the preceding paragraph, have not been detected. Thus, the mechanism by which the virus would make an efficient zoonotic leap from mice into humans is currently unclear.

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Stewart TA, Pattengale PK, Leder P (1984) Spontaneous mammary adenocarcinomas in transgenic mice that carry and express MMTV/myc fusion genes. Cell 38:627–637 Suzuki T, Shen H, Akagi K, Morse HC, Malley JD et al (2002) New genes involved in cancer identified by retroviral tagging. Nat Genet 32:166–174 Theodorou V, Boer M, Weigelt B, Jonkers J, van der Valk M et al (2004) Fgf10 is an oncogene activated by MMTV insertional mutagenesis in mouse mammary tumors and overexpressed in a subset of human breast carcinomas. Oncogene 23:6047–6055 Theodorou V, Kimm MA, Boer M, Wessels L, Theelen W et al (2007) MMTV insertional mutagenesis identifies genes, gene families and pathways involved in mammary cancer. Nat Genet 39:759–769 Vicent GP, Zaurin R, Nacht AS, Font-Mateu J, Le Dily F et al (2010) Nuclear factor 1 synergizes with progesterone receptor on the mouse mammary tumor virus promoter wrapped around a histone H3/H4 tetramer by facilitating access to the central hormone-responsive elements. J Biol Chem 285:2622–2631 Wagner KU, McAllister K, Ward T, Davis B, Wiseman R et al (2001) Spatial and temporal expression of the Cre gene under the control of the MMTV-LTR in different lines of transgenic mice. Transgenic Res 10:545–553 Wang Y, Holland JF, Bleiweiss IJ, Melana S, Liu X et al (1995) Detection of mammary tumor virus ENV gene-like sequences in human breast cancer. Cancer Res 35:5173–5179 Wang E, Obeng-Adjei N, Ying Q, Meertens L, Dragic T et al (2008) Mouse mammary tumor virus uses mouse but not human transferrin receptor 1 to reach a low pH compartment and infect cells. Virology 381:230–240 Yanagawa SI, Kakimi K, Tanaka H, Murakami A, Nakagawa Y et al (1993) Mouse mammary tumor virus with rearranged long terminal repeats causes murine lymphomas. J Virol 67:112–118 Yanagawa S, Lee JS, Kakimi K, Matsuda Y, Honjo T et al (2000) Identification of Notch1 as a frequent target for provirus insertional mutagenesis in T-cell lymphomas induced by leukemogenic mutants of mouse mammary tumor virus. J Virol 74:9786–9791

Chapter 30

Jaagsiekte Sheep Retrovirus and Lung Cancer Chassidy Johnson and Hung Fan

Introduction Jaagsiekte sheep retrovirus (JSRV) is the etiological agent of a contagious neoplasm in sheep termed ovine pulmonary adenocarcinoma (OPA) previously known as sheep pulmonary adenocarcinoma and ovine pulmonary carcinoma (Palmarini et al. 1997, 1999; Palmarini and Fan 2003). The first documentation of OPA was in the nineteenth century when a farmer from Cape of Good Hope, South Africa wrote to the magistrate stating that he was losing many of his sheep to a disease he called Jaagziekte. In Afrikaans, the word translated to Jaagsiekte (Jaag = chase; siekte = sickness) because the affected sheep had the appearance that they had been chased (Tustin 1969; York and Querat 2003). OPA has been reported on all continents with the notable exception of New Zealand and Australia. JSRV infection and OPA is a veterinary concern and economic burden in high endemic regions (i.e. Europe and Africa), and the possibility that it can act as a human pathogen has not been ruled out. OPA is derived from lung secretory epithelial cells, type II pneumocytes and Clara cells, and closely resembles bronchioloalviolar carcinoma (BAC) or adenocarcinoma of mixed type with BAC component in humans (Perk and Hod 1982; Palmarini et al. 1997). Compared to other types of cancers (i.e. leukemia, lymphoma and breast), model systems to study naturally occurring lung cancers are limited. The similarities between OPA and BAC make JSRV and OPA a useful model system to study human lung carcinogenesis. Furthermore, study of JSRV and enJSRV has proven to be an elegant model system to study retroviral/host evolution.

C. Johnson • H. Fan (*) Department of Molecular Biology and Biochemistry, Cancer Research Institute, University of California, Irvine, CA 92697, USA e-mail: [email protected] E.S. Robertson (ed.), Cancer Associated Viruses, Current Cancer Research, DOI 10.1007/978-1-4614-0016-5_30, © Springer Science+Business Media, LLC 2012

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Retroviruses The Retroviridae are a large family of enveloped and positive-stranded RNA viruses that infect a wide range of vertebrate species including humans, birds, cats, fish, sheep, and more. Retroviral infection can have pathogenic outcomes including malignant, degenerative, and immunodeficiency diseases. Retroviruses have provided useful tools to elucidate mechanisms of cancer. The fundamental genomic organization of all retroviruses is (5¢)-R-U5-gag-pol-env-R-U3 (3¢) (Fig. 30.1). Gag encodes the structural proteins that comprise the internal structure of the virions including the matrix (MA), capsid (CA), and the nucleocapsid (NC) proteins. The pol gene encodes the protease (PR), reverse transcriptase (RT), and integrase (IN) enzymes. The env gene encodes the envelope surface glycoprotein (SU) and transmembrane (TM) proteins. Gag, pol, and env are the genes required for the structure and replication of retroviruses. Simple retroviruses only encode these genes, whereas complex retroviruses encode additional genes. Some retroviruses have also captured cellular genes from the host that act as oncogenes. The virion structure of a simple retrovirus is depicted in Fig. 30.2. A hallmark feature of retroviruses is that they can convert the RNA genome into a DNA copy that becomes permanently integrated into the chromosomal DNA of the infected cell.

Viral genome 5’ CAP

gag R

pol

AAA 3’

env U3

U5

R

Provirus gag U3 R U5 LTR

pol

env U3

R U5 LTR

Fig. 30.1 Schematic organization of a simple retrovirus genome. The two forms of the genome are depicted. The RNA genome contains gag, pol, and env genes and is capped at the 5¢ end and polyadenylated at the 3¢ end. The viral RNA contains terminal repeats (R) and unique regulatory sequences (U3 and U5) at either end. The RNA genome is reverse transcribed into double stranded DNA by the viral reverse transcriptase. This process results in long terminal repeat elements (LTRs) at either end of the viral DNA. The viral DNA is integrated into the host DNA by viral integrase to form the provirus

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TM CA

RT

NC SU

MA IN

PR

Lipid bilayer

Fig. 30.2 Structure of a simple retrovirus. The virion of simple retroviruses contain two copies of the RNA genome that are coated with nucleocapsid (NC). The capsid (CA) forms the inner core that contains the genome, reverse transcriptase (RT), integrase (IN), and protease (PR) enzymes. The virion is enclosed in a lipid bilayer that contains envelope protein subunits, surface (SU) and transmembrane (TM). The matrix (MA) is located beneath the lipid bilayer

Retroviral Lifecycle The lifecycle of a simple retrovirus is shown in Fig. 30.3. Following SU binding to the cellular receptor, the virion is internalized and the nucleocapsid core is released into the cytoplasm of the host cell. The RT in the core particle is then activated. Reverse transcriptase is an RNA-dependent DNA polymerase that gives the virus the ability to transcribe the single-stranded RNA genome into a double-stranded DNA. The reverse transcribed copy of the viral RNA yields a slightly longer DNA copy because it is flanked by long terminal repeat elements (LTRs) that are formed during the duplication event (Fig. 30.1). The DNA copy is transported to the nucleus where the virus-encoded integrase (IN) catalyzes integration into the cellular DNA to form the provirus. The U3 region of the LTR contains the viral promoter and enhancer elements; the provirus is transcribed by cellular RNA polymerase II to give a primary transcript identical to virion RNA. Viral transcripts are exported to the cytoplasm (with and without mRNA splicing) and translated into viral polyproteins, or are used as genomes for newly packaged virions. Following packaging, virions are released at the cell surface by budding. After release, proteolytic processing of the virion polyproteins occurs and the mature virion can now infect new target cells. The fact that the virus does not kill the host cell is consistent with lifelong infection and in many cases development of malignancies. Details specific to JSRV will be discussed below.

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Receptor binding budding b Endocytosis Env localization to membrane Virion assembly g Uncoating

Viral proteins Genomic RNA

Reverse transcription Transcription Integration

Fig. 30.3 The retroviral lifecycle. The retroviral life cycle is shown as described in the text

Oncogenic Retroviruses Oncogenic retroviruses have been known for over 100 years. The first retroviral cancer was described in 1908 for avian myeloblastosis (Ellerman and Bang 1908; Rous 1911). Many other oncogenic retroviruses were later described such as Rous sarcoma virus (Rous 1911), murine mammary tumor virus (MMTV) (Bittner 1942), and several strains of murine leukemia virus (MuLV) (Gross 1951). The oncogenic retroviruses are largely animal viruses with human T cell lymphotropic virus (HTLV) being the exception. Retroviruses can be subdivided into two classes of retroviruses based on how fast they induce disease and the mechanism of transformation: acute transforming and non-acute retroviruses. Acute-transforming retroviruses induce disease rapidly and resulting malignancies are typically polyclonal. Additionally, they can often transform cells in culture. The oncogenic properties of acute transforming retroviruses are due to the presence of virally encoded oncogenes that were acquired from cellular genes (proto-oncogenes) by a process termed oncogene capture. Cellular proto-oncogenes encode proteins whose normal functions are to stimulate controlled cell growth or division. Oncogene capture typically renders the virus replication defective, so acutely transforming retroviruses usually replicate as a mixture with a related replication-competent “helper” virus. Retroviral oncogenes are typically altered from their cognate protooncogenes, expressing mutant proteins that can constitutively activate cellular growth pathways.

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Non-acute retroviruses have a long latency to tumor formation (months to years), do not transform cells in culture, and are replication competent. Non-acute retroviruses do not encode oncogenes or any other genes to which the malignant potential can be directly attributed. Rather tumor induction is due to insertional activation, where integration of the provirus into host DNA results in activated expression of an adjacent proto-oncogene (Fan et al. 1997). Proviral integration occurs at multiple sites (almost randomly) throughout the genome. However, when proviral integration occurs near a cellular oncogene, the viral promoter and/or enhancer elements can initiate over-expression of the proto-oncogene, ultimately leading to development of a tumor. The low probability of integration next to a proto-oncogene explains why tumors induced by non-acute retroviruses require relatively long times (multiple rounds of infection) to develop. In fact, non-acute retroviruses can be used to identify genes that can function as oncogenes (Li et al. 1999; Hansen et al. 2000; Mikkers et al. 2002). Although oncogene capture and insertional activation of proto-oncogenes are the two primary mechanisms that retroviruses use to induce tumors, some viral proteins can be directly transforming. For example, HTLV-1 and HTLV-2 express a viral protein Tax that can transform T cells (Nerenberg et al. 1987; Grassmann et al. 1992; Akagi and Shimotohno 1993; Grossman et al. 1995).

Biology of OPA and JSRV OPA Clinical Signs of OPA Animals that develop OPA show progressive signs of respiratory illness. The malignancy and fluid accumulation produced by the tumor cells make breathing difficult. The normal function of type II pneumocytes and Clara cells is to secrete surfactant and other fluids, and over-production of surfactant by the tumor cells leads to alveolar obstruction. Production of large amounts of lung fluid is considered a signature of OPA. Lung fluid can be collected by performing the “wheel barrow test” where the hind-quarters are held up, causing the lung fluid to drain through the nose. Four hundred milliliters per day have been collected by this method, although secretion of 10–40 ml per day is more common and in some cases no fluid secretion is observed (Cousens et al. 2009). Although OPA develops in sheep and goats, goats are less susceptible to OPA (Sharma et al. 1975a, b; Sharp et al. 1986; Tustin et al. 1988).

Gross Pathology OPA is classified as classical, atypical, or a mixture of the two types (Garcia-Goti et al. 2000), although no molecular differences in JSRVs have been attributed to the

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different OPA classifications. Classical OPA presents with frothy fluid that fills the trachea and exudes from the nares (Dungal 1938; Tustin 1969; Wandera 1971; Beytut et al. 2009). OPA can occur in all parts of the lung, but most frequently in the distal regions. Natural cases typically have one large tumor mass and experimental cases show numerous small nodules that are widely disseminated throughout the lung. When the tumor mass(es) are large, the normal architecture of the lung is distorted. Multiple lobes of the lung may be affected with a defined boundary between normal and neoplastic areas. Soft pockets of necrosis and abscessation are frequently observed. Bronchial and mediastinal lymph nodes are often enlarged and edematous and up to 10% contain metastase (Demartini et al. 1988; Rosadio et al. 1988b). Metastasis to liver, kidney, heart, skeletal muscle, or other extrathoracic tissues has been observed, although rarely (Mackay and Nisbet 1966; Nobel et al. 1969; Hunter and Munro 1983). Atypical OPA has only been reported in natural cases from Spain and Peru (Garcia-Goti et al. 2000; De Las Heras et al. 2003). Fluid is absent in the bronchial airway and the tumors are hard, pearly-white, and have a dry cut surface that can resemble scars (Garcia-Goti et al. 2000; De Las Heras et al. 2003). Atypical neoplasias are nodular in both early and advanced stages in contrast to classical tumors, which are diffuse. Atypical OPA is typically located in the diaphragmatic lobe.

Histological Descriptions Lung alveoli are comprised of two predominant epithelial cell types, type I and type II pneumocytes. Type II pneumocytes are surfactant secreting, cuboidal cells, and upon injury can act as progenitors for type I pneumocytes. Type I pneumocytes are squamous and are more abundant than type II pneumocytes, comprising the majority of the alveolar epithelia. Clara cells (found further up the airways in the bronchioles) and type II pneumocytes secrete surfactant, a mixture of glycerophospholipids, cholesterol, and proteins. Surfactant coats the surface of the alveoli where it has multiple functions: decreases the surface tension at the air–liquid interface to allow proper air–gas exchange, prevents alveolar collapse, and provides host defenses and pulmonary immune function (Schmitz and Muller 1991; Sakai et al. 1992; Rooney et al. 1994; Beytut et al. 2009). There are four surfactant proteins (A, B, C, and D) that have different functional and structural properties. SP-C is exclusively expressed in type II pneumocytes, whereas both type II pneumocytes and Clara cells can express SP-A and SP-B. Generally, surfactant protein expression is increased in OPA cells compared to normal lung cells, with SP-A levels being especially high. OPA tumors are well differentiated and consist of acinar or papillary forms, or a combination of the two. Acinar growth is most frequently observed in the lung parenchyma and consists of cuboidal cells, whereas papillary growth consists of columnar cells affecting intra-bronchiolar areas (Sharp et al. 1983; Rosadio et al. 1988b; Tustin et al. 1988; DeMartini and York 1997; Platt et al. 2002; Caporale et al. 2006; Beytut et al. 2009). OPAs are typically papillary in appearance and occasionally give the impression of solid growths. The neoplastic nodules compress

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the neighboring alveoli causing atelectasis (lack of gas exchange due to collapse) and/or merge to form a larger mass. OPA consists of malignantly transformed type II pneumocyte (82%), Clara (7%), and undifferentiated cells (11%) (Platt et al. 2002; Wootton et al. 2006b). JSRV infection has been detected in lung epithelial cells, CD4+ and CD8+ T cells, B cells, and cells of monocyte/macrophage lineage (Holland et al. 1999). Transformed stem cells have been reported to be the origin of many malignancies including breast, brain, colon, ovary, pancreas, prostate, and possibly the lung (Al-Hajj et al. 2003; Singh et al. 2003; Li et al. 2007; O’Brien et al. 2007; Maitland and Collins 2008; Zhang et al. 2008). A population of lung stem cells believed to be the origins of adenocarcinoma was recently identified, termed bronchioloalveolar stem cells (BASCs) (Kim et al. 2005). Despite the possibility that they are the targets for JSRV transformation in OPA, in transgenic mice where the JSRV LTR is driving expression of a reporter transgene, expression was not detected in BASCs (Dakessian and Fan 2008). OPA tumor cells in early stages of malignancy have a low mitotic index and at later stages, solid masses of pleomorphic cells with a high mitotic rate and scattered foci of necrosis are often observed. Although tumor cells generally have a low mitotic index, they express proliferating cellular nuclear antigen (PCNA), a marker for proliferation. JSRV capsid protein is detected in the cytoplasm of tumor cells with apical concentration in lung and lymph nodes (Palmarini et al. 1995; Palmarini et al. 1999; Garcia-Goti et al. 2000; DeMartini et al. 2001; Palmarini and Fan 2001; Palmarini and Fan 2003; Salvatori et al. 2004; Caporale et al. 2006). The fact that JSRV capsid protein is detected in OPA cells indicates that JSRV continues to replicate in these cells. Interestingly, capsid expression is not detected in all cells in OPA tumors, nor in normal cells adjacent to tumor areas. Additionally, not all nodules in multifocal OPA express capsid (Sharp et al. 1983; Palmarini et al. 1995; Platt et al. 2002). In contrast, JSRV SU has been detected in the cytoplasm of OPA cells and proliferating cells located in the bronchioles whereas expression was not detected in unaffected sheep (Payne and Verwoerd 1984; Salvatori et al. 2004). Atypcial OPA is commonly acinar with stroma that is more heavily infiltrated by inflammatory cells and connective fibers than classical OPA (De Las Heras et al. 1992; Garcia-Goti et al. 2000). Metastases in regional lymph nodes seem to be less frequent and less JSRV-positive cells are observed (Garcia-Goti et al. 2000). Early atypical lesions have numerous neoplastic polyps located in the terminal bronchioles and alveoli that develop into more elaborated intrabronchiolar polyps (Wandera 1970; Sharp et al. 1983).

Transmission Studies Suggest That the Causative Agent of OPA Is a Retrovirus The first suggestion that a retrovirus could be the etiological agent of OPA was the observation of particles resembling retroviruses in the lungs of sheep with clinical OPA (Malmquist et al. 1972; Payne et al. 1983; Perk et al. 1974). Biochemical analysis

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confirmed reverse transcriptase activity, retroviral RNA and retroviral antigens in tumor extracts (Perk et al. 1974; Verwoerd and de Villiers 1980; Sharp and Herring 1983). OPA was shown to be a transmissible malignancy by intratracheal inoculation of sheep with particles containing reverse transcriptase activity isolated from OPA (Martin et al. 1976), cytoplasmic fractions of tumor cells (Verwoerd and de Villiers 1980; Verwoerd et al. 1980) or pulmonary secretions (Sharp et al. 1983).

Molecular Biology of JSRV Deduction of JSRV Sequence and Identification of Endogenous JSRV Characterization of JSRV was a major focus of research during the early 1980s. JSRV could not be grown in culture, likely due to the fact that the LTR has highest activity in differentiated distal airway epithelial cells, which do not maintain the differentiated state in culture (Dobbs et al. 1985; Manzer et al. 2006; Wang et al. 2006). Therefore lung lavages of diseased sheep were used to purify the virus (Verwoerd et al. 1983; York 1987; York and Querat 2003). A major breakthrough was deduction of the sequence for a novel retrovirus (JSRV) in OPA lung fluids (York et al. 1991, 1992). This was accomplished by partially purifying virus from lung fluids, extracting RNA and using it as a template in in vitro reverse transcriptase reactions. The resulting cDNAs were cloned, and sequence analysis of overlapping clones allowed deduction of the JSRV sequence. The sequence analysis indicated that JSRV is a member of the betaretrovirus family (see below), and it contains an additional open reading frame termed orf-x, which is not present in other retroviruses. The assembled cDNA sequence was not infectious in vitro or in vivo likely for technical reasons. Hybridization experiments with the isolated DNA led to the discovery that in addition to the exogenous infectious JSRV in OPA, there are related endogenous retrovirus (enJSRVs) present in all sheep genomes (York et al. 1992; Hecht et al. 1994, 1996) (see below). Subsequently improved screening and cloning approaches allowed isolation of a molecular clone of a full-length integrated JSRV provirus from an OPA tumor from the UK, lJSRV21 (Palmarini et al. 1999). The JSRV sequences in lJSRV21 closely resembled the deduced JSRV sequence (Fig. 30.4a) (York et al. 1992; Palmarini et al. 1999).

JSRV Is a Betaretrovirus Early clues that JSRV is a betaretrovirus came from the fact that extracts from lung tumors and secretions from OPA-affected animals cross-reacted with antisera against capsid for betaretroviruses mouse mammary tumor virus (MMTV) and

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Fig. 30.4 The JSRV genome. (a) Schematic representation of the genomic organization of the JSRV21 genome. Typical of betaretroviruses, pro is in a different open reading frame from pol. Note the presence of an accessory open reading frame (orfx) overlapping pol. (b) pJSRV21 and pCMV2JS21 plasmid constructs. In pCMV2JS21, the U3 region of the proximal LTR was replaced by the human CMV promoter. (c) Western blot of 300-fold-concentrated supernatant from 293T cells transiently transfected with pCMV2JS21 and collected 24, 48, and 72 h post-transfection. The filters were probed with a rabbit polyclonal antiserum against the major capsid protein (CA) of JSRV (28). Lung secretions collected from an SPA-affected animal and concentrated in the same way as the 293T supernatant were used as a positive control (LF). Concentrated supernatant from mock-transfected 293T cells was used as a negative control (M). The 26-kDa CA protein is indicated. This figure is taken from Palmarini et al. (1999)

Mason-Pfizer monkey virus (MPMV) (Sharp and Herring 1983). Following cloning of JSRV, sequence analysis revealed genetic similarity of the RT protein to other betaretroviruses confirming that JSRV is a betaretrovirus. Based on Env and LTR sequence homology, two genotypes of JSRV have been isolated: type I from South Africa and France and type II from Scotland and Wyoming (Bai et al. 1996). It is not known if the sequence differences between the two subtypes result in functional differences. To date, three stains of JSRV have been isolated and sequenced. JSRV-SA is from South Africa (York et al. 1991, 1992) and JSRV21 (Palmarini et al. 1999)

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and JSRVJS7 (DeMartini et al. 2001) are from the UK. Other members of the betaretrovirus genus include mouse mammary tumor virus (MMTV), Mason–Pfizer monkey virus (MPMV) and squirrel monkey retrovirus (SMRV). JSRV is closely related to enzootic nasal tumor virus (ENTV), the etiological agent of a contagious nasal adenocarcinoma of the mucosal glands affecting sheep and goats, which will be discussed later. Assembly of betaretrovirus viral cores occur in the cytoplasm in contrast to other retroviruses where core assembly occurs during viral budding at the plasma membrane (Coffin 1992). Distinct JSRV core structures have been described for intracytoplasmic, budding, and extracellular virions. Intracytoplasmic virions, located below the apical membrane, are described as type-A particles that are doughnut or ring shaped with a small electron-lucent core, electron dense inner core, and double external membrane that range in size from 60 to 100 nm (Hod et al. 1977; Payne et al. 1983). Virions budding from the microvilli or into the intracytoplasmic vacuoles are complete round particles (not crescent shaped) containing a core that is more electron-dense than intracytoplasmic particles. JSRV particles observed outside the cell (type B/D) are round or pleomorphic, ranging in size from 90 to 120 nm in diameter (Verwoerd et al. 1980; Sharp et al. 1983).

JSRV Molecular Clones Are Infectious and Tumorigenic Transfection of a cloned full-length JSRV provirus into various cell lines did not result in production of virus, likely because the LTR has transcription specificity for differentiated airway cells (Palmarini et al. 2000a). To overcome this, the U3 region in the upstream LTR was replaced with the CMV immediate early enhancer/promoter to give pCMVJS21 (Fig. 30.4b). This plasmid would express native JSRV RNA from the CMV promoter that is highly active in many cells. Viral particles were efficiently produced in 293T cells transfected with pCMVJS21 (Fig. 30.4c). The resulting viral particles were concentrated and used to intratracheally inoculate newborn lambs. Two of four inoculated lambs developed OPA (Fig. 30.5), which was the first formal proof that JSRV was necessary and sufficient to induce OPA (Palmarini et al. 1999).

Transcription Specificity of the JSRV LTR Retroviral LTRs contain the viral promoter and enhancers that bind cellular transcription factors; therefore they dictate the cells in which the virus can be expressed. The cell-type specificity of the JSRV LTR was determined using LTR-luciferase reporter assays. The JSRV LTR had the highest activity in murine MLE-15 and mtCC1-2 cells (Palmarini et al. 2000a). These cells were derived from lung tumors of transgenic mice and they have retained differentiation characteristics of type II pneumocytes and Clara cells, respectively (Malkinson et al. 1997). JSRV LTR activity was low in human lung epithelial lines (A549, H358 and H441) and the sheep BAC

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Fig. 30.5 Induction of SPA in JSRV21infected lambs. (a–c) Lung tumor tissues from lambs inoculated with JSRV21 produced from transfected 293T cells. Hematoxylin and eosin-stained lung tumor sections are shown. (a) Low-magnification micrograph (bar, 380 mm) showing many neoplastic foci in the microscopic field (some are indicated by arrows). (b) High-magnification micrograph (bar, 40 mm) of a neoplastic nodule with a clear papillary pattern (asterisk). Myxoid tissue-containing cells with elongated or round nuclei are present in the interstitium of the neoplastic tissue. (c) Papillary proliferation (asterisk) occluding the lumen of a bronchiolus (bar, 40 mm). (d–e) Immunohistochemistry for JSRV CA antigen (brownish stain). (d) A neoplastic focus (asterisk) is shown. No staining is present in the cells infiltrating the tumor or in adjacent normal cells. (e) Rabbit preimmune serum staining; no staining in the tumor nodule (asterisk). (f) A lung section from an uninoculated lamb stained for JSRV CA antigen. This figure was taken from Palmarini et al. (1999)

cell line (JS7). The latter cell lines have not retained epithelial differentiation, indicating that maintenance of differentiation of lung cells may be required for JSRV LTR activity. JSRV LTR sequences important for activity in MLE-15 have been determined by mutational analysis and in vivo footprinting of an M-MLV driven by an LTR containing JSRV enhancers (McGee-Estrada and Fan 2006). A map of putative and confirmed transcription factor binding sites is shown in Fig. 30.6. The NF-1, HNF3b, and C/EBP binding sites in the JSRV LTR are critical for activity in MLE-15 cells although not for the low level activity in other cell lines examined (McGee-Estrada et al. 2002; McGee-Estrada and Fan 2006).

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Fig. 30.6 In vivo footprinting of the JSRV enhancer. A composite of in vivo DMS footprinting for both strands of the JSRV enhancers in the LTR of DMo+JS Mo-MuLV-infected MLE-15 cells is shown. Factor-binding sites where strong footprints were observed are shown in bold and italics. Protected bases are indicated by arrows pointing away from the sequence, and hypermethylated bases are indicated by arrows pointing toward the sequence. Filled and open arrowheads indicate strong protection and hypersensitivity, respectively. Canonical binding sites that have been analyzed by EMSA are shown in bold. A shaded rectangle indicates boundaries for the putative Gfi-I binding site. This figure was taken from McGee-Estrada and Fan (2006)

LTR Binding Sites HNF3s (Hepatocyte Nuclear Factor 3, aka FOXA2) are a family of nuclear proteins expressed in the liver and lungs and are critical in development of these tissues (Overdier et al. 1994; Costa et al. 2001). HNF-3a and -b are highly expressed in both type II pneumocytes and Clara cells (Clevidence et al. 1994; Kaestner et al. 1994) where they are important for expression of SP-B, CC-10, and other lungspecific genes (Sawaya et al. 1993; Bohinski et al. 1994; Clevidence et al. 1994; Bingle et al. 1995; Bruno et al. 1995; Margana and Boggaram 1997). Two putative HNF-3 binding sites have been identified in the JSRV LTR. The upstream HNF-3 binding site, which binds HNF-3b, is critical for LTR activity in MLE-15 cells and the downstream site is not important. Results were different in NIH-3T3 and mtCC1-2 cells where the HNF-3 sites were not required. These results indicate that there are cell-type specificities for LTR activity and sites other than HNF-3 can mediate expression of the JSRV LTR. However, binding sites identified in cell lines such as NIH-3T3 where the LTR only shows low basal activity may not be as important as those identified in differentiated type II pneumocytes (e.g. MLE-15).

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C/EBP (CCAAT/Enhancer binding protein) has three isoforms (a, b and d) that are expressed in many tissues (Birkenmeier et al. 1989; Sugahara et al. 1999). Isoforms a and d are highly expressed in the bronchiolar epithelium, while b is mostly found in alveolar macrophages (Sugahara et al. 1999). C/EBPa and b bind to a C/EBP binding site in the JSRV LTR, which is critical for activity (McGee-Estrada and Fan 2006). NF-1 (nuclear factor protein-1) controls expression of many target genes, including the surfactant protein C promoter (Bachurski et al. 1997). There are at least four mammalian NF-1 genes (A, B, C and X), with multiple isoforms as a result of splicing (Rupp et al. 1990; Chaudhry et al. 1997). NF-1A has been shown to transactivate the SP-C promoter in HeLA cells (Bachurski et al. 1997). The NF-1 binding site in the JSRV LTR is important for LTR activity, although not as critical as other sites such as those for C/EBP and HNF-3b. The core binding sequences of the two HNF-3b and C/EBPab binding sites are conserved in type I and type II JSRV LTRs (McGee-Estrada and Fan 2007), illustrating the importance of these sites for JSRV LTR activity.

LTR Elements Are Determinants of Disease Outcome Env and the LTR are the main determinants of disease for many retroviruses. Env determines cell specificity at the level of viral entry and the LTR dictates where the virus can be expressed transcriptionally. For JSRV, several findings indicate that the LTR plays a more important role in disease specificity than Env. First, the JSRV receptor (hyaluronidase 2; Hyal2) is expressed in many cell types throughout the body and viral DNA is found in many different cell types including PBMCs, macrophages, B lymphocytes, CD4+, and CD8+T lymphocytes in lymph nodes (Holland et al. 1999; Miller 2003). Despite the presence of viral DNA in the different cell types, expression is highly restricted to OPA tumor cells in vivo and differentiated type II pneumocytes and Clara cells in vitro (Palmarini et al. 1995). Indeed lung epithelial cell lines that have not maintained the differentiation state do not support activity of the LTR (Palmarini et al. 2000a). Second, when mice are infected intranasally with adeno-associated virus expressing JSRV Env in the absence of the LTRs, lung adenocarcinomas develop even though the vector transduced multiple cells and tissues in the airways (Wootton et al. 2005). Third, JSRV, ENTV, and enJSRV are closely related betaretroviruses with high sequence similarity, with the most differences in the LTR (McGee-Estrada and Fan 2007). All three JSRV isolates contain two conserved HNF-3 binding sites in U3, while the ENTV and enJSRV U3 regions do not. The C/EBP binding site of JSRV is interrupted in enJSRV but conserved in ENTV LTRs. Although several other binding sites are conserved between the JSRV and ENTV LTRs, the ENTV-1 LTR is less active than the JSRV LTR in MLE-15 cells. The transcriptional specificities of the enJSRV and exogenous JSRV LTRs differ because enJSRVs respond to progesterone and they do not respond to lung-specific factors (Palmarini et al. 2000b). Indeed enJSRV is highly expressed in the reproductive tract of ewes and at low levels in lungs.

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Rej Retroviral RNA is transcribed as genome-length RNA. Nuclear viral RNA can be spliced in the nucleus to form mRNA for envelope protein. Spliced viral mRNA is exported to the cytoplasm for translation. Alternatively, unspliced viral RNA is transported to the cytoplasm where it is packaged into virions or translated as the mRNA for Gag and Gag-pol polyproteins. For cellular mRNAs, splicing is typically a prerequisite for RNA export to the cytoplasm. Retroviruses have evolved two mechanisms to export unspliced RNA: virally encoded trans-acting proteins or cisacting RNA elements (Cullen 2003). For example, human immunodeficiency viruses (HIVs) encode Rev that facilitates export of unspliced RNA to the cytoplasm (Cullen 2003). Rev binds to a Rev response element (RRE) on the viral RNA, and it facilitates nuclear export via the cellular protein Crm1 (Cullen 2003). Simple retroviruses do not encode accessory proteins; rather they export RNA via cis-acting constitutive transport elements (CTEs), secondary stem-loop structures that directly bind cellular RNA export proteins (e.g. TAP/NXF-1 for MPMV). Recently, the mechanism for export and translation of unspliced RNA for JSRV has been described. A trans-acting factor Rej or JSE-SP is encoded in the 5¢ end of env (Caporale et al. 2009; Hofacre et al. 2009). The protein is generated by translation of a multiply spliced mRNA and/or by cleavage of the signal peptide from the full-length Env precursor polyprotein (Hofacre et al. 2009). JSRV Rej is analogous to regulatory proteins of other betaretroviruses, mouse mammary tumor virus (MMTV), and human endogenous retrovirus K (HERV-K) (Rem and Rec; respectively) (Lower et al. 1995; Indik et al. 2005; Mertz et al. 2005). In human 293T cells, Rej is required for Gag expression and it appears to act by facilitating export of unspliced RNA, mRNA translation and post-translational modifications (Caporale et al. 2009; Hofacre et al. 2009). However, in other cells, Rej is not required for export of unspliced viral RNA but it is still essential for expression of Gag protein; it apparently facilitates translation of the cytoplasmic unspliced viral RNA (Hofacre et al. 2009) – an unusual mechanism for the Rev-like proteins. A cis-acting Rej response element (RejRE) is contained in the 3¢ of Env, and this is embedded in a larger JSRV RNA expression and export element (JREE) that also contains a CTE for export of unspliced viral RNA in cell lines other than 293T (Nitta et al. 2009). Export of unspliced viral RNA is CRM1 and B23-dependent but not TAP-dependent (Caporale et al. 2009; Nitta et al. 2009). A core stem-bulge-stem structure in the RejRE is required for activity and secondary structure of the RejRE core is important (Nitta et al. 2009). While Rej plays a role in viral RNA export only in 293T cells, Rej binds to the RejRE in all cell lines tested (Nitta et al. 2009). Thus Rej binding to the RejRE may also be important for translation of unspliced viral RNA.

Orf-X The JSRV genome contains an additional open reading frame termed orf-x. Orf-x overlaps with the 3¢ end of the pol gene and the codon usage differs from the rest of

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the virus (York et al. 1992). Based on sequence analysis, orf-X shares sequence similarity to the adenosine 3A receptor and is predicted to encode a very hydrophobic accessory protein of 166 amino acids containing four putative transmembrane domains (Bai et al. 1999). ORFs Homologous to orf-x are not found in other betaretroviruses and it has been debated if it truly represents an accessory gene for JSRV (York et al. 1992; Bai et al. 1999; Rosati et al. 2000). Orf-x is not required for Envmediated transformation in vitro (Maeda et al. 2001) or in vivo (Wootton et al. 2005; Caporale et al. 2006; Cousens et al. 2007). Evidence that orf-x plays a role in the JSRV lifecycle is based on the fact that the sequence is strongly conserved among different JSRV isolates and endogenous JSRV, suggesting a selective pressure to continue carrying it (York et al. 1992; Bai et al. 1996, 1999; Rosati et al. 2000; York and Querat 2003). Additionally, orf-x mRNA has been detected in JSRV-infected cells and lung tumors (Palmarini et al. 2002). However, a mutant of JSRV in which the orf-X reading frame was mutated by insertion of two stop codons induced OPA with the same efficiency as wild-type virus (Cousens et al. 2007). Thus while OrfX might be important for some aspect of JSRV infection in vivo, it is not required for oncogenesis by JSRV.

The JSRV Receptor: Hyal2 The cellular receptor for JSRV was mapped and cloned from human cells by a combination of somatic cell hybrid, molecular cloning, and gene transfer experiments (Rai et al. 2000, 2001). The JSRV receptor was found to be hyaluronidase 2 (Hyal2) (Rai et al. 2001). Initial studies reported that Hyal2 is a lysosomal hyaluronidase (Lepperdinger et al. 1998), but it was later found that it is a glycosylphosphatidylinositol (GPI)-anchored cell surface receptor with very little hyaluronidase activity (Rai et al. 2001; Liu et al. 2003a; Duh et al. 2005; Van Hoeven and Miller 2005; Miller et al. 2006). Hyal2 is located on human chromosome 3p21.3, and it is interesting that this chromosomal region may encode a tumor suppressor gene, because it is in a region of loss of heterozygosity (LOH) in human lung cancer (Zabarovsky et al. 2002). Hyal2 is present on the surface of many cell types, which explains how JSRV DNA can be detected not only in lung cells but also lymphocytes, macrophage, and other cell types of sheep with OPA (Holland et al. 1999; Miller 2008; Rai et al. 2001). Mouse cells cannot be infected by retroviral vectors pseudotyped with JSRV Env because murine Hyal2 does not bind JSRV Env (Rai et al. 2001). Likewise rat cells are only slightly permissive for infection (Liu et al. 2003a). The relevance of these results to the potential role of Hyal2 in JSRV-induced malignancies will be discussed later.

Mechanisms of JSRV Oncogenesis The mechanism by which JSRV induces tumors has been of considerable interest. Considering the existing paradigms for oncogenic retroviruses, it was important to determine if JSRV is an acute transforming retrovirus or a non-acute retrovirus.

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The rapid time course to disease and multifocal neoplasia were consistent with JSRV being an acutely transforming retrovirus, i.e., that it carries an oncogene. However, the JSRV genome does not contain any open reading frames with high homology to cellular proto-oncogenes – the hallmark of acute transforming retroviruses. On the other hand, the viral orf-x reading frame of JSRV and related viruses does not have an obvious role in viral replication, so it might potentially be an oncogene – although it is contained in non-oncogenic enJSRVs as well (York et al. 1992; Bai et al. 1999; Rosati et al. 2000). However, a JSRV mutant in which orf-x was mutated shows the same rapid disease induction as wild-type virus (Cousens et al. 2007). At the same time, there is no concrete evidence that JSRV uses insertional activation in induction of malignancy. In an OPA-derived cell line containing only one copy of JSRV DNA, the provirus is integrated into the structural gene for pulmonary surfactant protein A (SPA), as opposed to a proto-oncogene (DeMartini et al. 2001). Another study found only two out of 70 OPA tumors with insertions in the same locus (chromosome 16) (Cousens et al. 2004). Identification of more OPA tumors with common insertion sites will be needed before a role for insertional activation in JSRV oncogenesis could be established.

The JSRV Genome Can Transform Cells Given the rapid rate of JSRV oncogenesis, we tested if the JSRV genome might contain an oncogene using a functional assay. NIH-3T3 cells are contact-inhibited, but they are highly susceptible to morphologic transformation and they have been used to detect both viral oncogenes and activated cellular proto-oncogenes (Shih and Weinberg 1982). Transfection of pCMVJS21 DNA into NIH-3T3 cells resulted in formation of transformed foci; cells in the foci could grow in soft agar while control NIH-3T3 cells cannot (Maeda et al. 2001). Thus, the JSRV genome contains a gene that is capable of transformation, i.e. an oncogene. This observation has been confirmed by other laboratories (Rai et al. 2001).

Env Is the Oncogene Further experiments indicated that expression of JSRV Env was sufficient to transform NIH-3T3 cells and in some studies env alone was more efficient at transformation than the entire JSRV molecular clone (Maeda et al. 2001; Rai et al. 2001). This was the first report that a native retroviral envelope protein could transform cells. JSRV Env has been shown to transform various cell lines including murine NIH-3T3 fibroblasts (Maeda et al. 2001), rat 208F fibroblasts (Rai et al. 2001; Maeda et al. 2005), avian DEF and DF-1 fibroblasts (Allen et al. 2002; Zavala et al. 2003), human bronchial BEAS-2B epithelial cells (Danilkovitch-Miagkova et al. 2003), MDCK canine kidney epithelial cells (Liu and Miller 2005), and RK3E rat kidney epithelial cells (Maeda et al. 2005).

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JSRV Env not only can transform cell lines in culture, but it can also induce lung (and other) tumors in animals. This has been shown in transgenic mice expressing env transgenes (Dakessian et al. 2007; Chitra et al. 2009), and in mice infected with an adeno-associated viral vector (Wootton et al. 2005). A JSRV-based retroviral vector expressing Env also induces lung tumors in sheep (Caporale et al. 2006). Together, these results indicate that Env is an oncogene and it alone is sufficient to induce OPA. Additional viral genes and/or viral spread are not required for oncogenesis.

Domains of Env Required for Transformation The domains of JSRV Env involved in transformation have been studied extensively (Palmarini et al. 2001b; Chow et al. 2003; Hofacre and Fan 2004; Hull and Fan 2006). Initial attention was focused on the cytoplasmic tail (Lander et al. 2001) of the TM protein, since sequence comparisons between exogenous (oncogenic) JSRV and endogenous (non-oncogenic) enJSRV Env proteins indicated relative conservation between the proteins except for the TM CT (Palmarini et al. 2001b). Chimeras exchanging the CTs between these two Env proteins indicated that the CT of exogenous JSRV is essential for transformation (Palmarini et al. 2001b). There is a tyrosine residue in the JSRV CT at position 590 while the CT of enJSRV lacks tyrosines, suggesting that this residue is important for Env transformation. This was confirmed when mutation of the tyrosine to phenylalanine (Y590F) or aspartic acid (Y590D) abolished transformation in NIH-3T3 cells (Palmarini et al. 2001b). Additionally, when JSRV virions containing the Y590D mutation were inoculated into sheep, the virus could not establish infection or induce disease (Cousens et al. 2007). Thus Y590 in the CT is important for JSRV Env transformation. The amino acid sequence surrounding Y590 is YRNM, and if Y590 is phosphorylated it could potentially bind cellular proteins with SH2 domains. YXXM and YXN are putative binding motifs for the SH2 domains of the PI3K regulatory subunit (p85) and growth factor receptor binding protein-2 (Grb-2), respectively (Songyang et al. 1993). Mutation of the YXXM motif (e.g. M593T) abolished or decreased transformation in cellular transformation assays (Palmarini et al. 2001b; Allen et al. 2002; Liu et al. 2003a, b). On the other hand, mutation of the YXN (e.g. N592T) did not abolish transformation, and in fact this increased transformation potential (Palmarini et al. 2001b). Taken together these results suggested that the YXXM motif in the JSRV CT might bind PI3K, leading to downstream signaling. Indeed, JSRVtransformed cells show constitutive phosphorylation (activation) of the downstream kinase Akt (Palmarini et al. 2001b). Alanine scanning mutagenesis on the JSRV CT has been conducted (Hull and Fan 2006). The CT is 44 amino acids in length, and the N-terminal 14 residues form an amphipathic helix that is likely embedded in the lipid bilayer of the cell membrane. The alanine scanning mutations indicated that the C-terminal 10–12 residues are not essential for transformation. In contrast, mutations in the amphipathic helix and downstream amino acids generally affected transformation. For some residues,

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alanine substitution resulted in complete inhibition of transformation, for others there was partial inhibition, and for four residues there was enhancement. These mutations could affect either the global structure of the CT, or binding of cellular proteins involved in transformation to the CT. They will be useful in future studies on the mechanism of JSRV transformation. While the TM CT is essential for JSRV transformation, other regions of Env protein are also important. Deletions within SU (from the signal peptide to the junction between SU and TM) also abolished transformation (Hofacre and Fan 2004), indicating that SU is also important for Env transformation. The role of SU in transformation is independent from that of the TM CT, since co-transfection with two transformation-defective JSRV env mutants with mutations in SU and in the TM CT results in transformation (Hofacre and Fan 2004). The mechanism by which SU acts in JSRV transformation has not been determined. The potential role of the extracellular portion of the TM protein (the ectodomain) has also been investigated. The ectodomains of endogenous and exogenous JSRV TMs are highly conserved, with only four amino acid differences (Palmarini et al. 2000b). Sequential mutation of the exogenous JSRV TM ectodomain amino acids to those of enJSRV TM partially reduced transformation efficiency (S. Hull and H. Fan, unpublished). Thus the ectodomain of TM may also contribute to JSRV transformation, although in a limited fashion.

Signaling in JSRV Transformation PI3K-Akt-mTor Pathway Class I phosphatidylinositol-3-kinases (PI3K) are cellular lipid kinases consisting of a regulatory and catalytic subunit. They are further subdivided into class IA and class IB. Activation of cellular receptor tyrosine kinases and/or Ras proteins mediate recruitment to the plasma membrane and activation of class IA PI3Ks, whereas class IB PI3Ks are activated by G protein-coupled receptors (Stephens et al. 1994, 1997). At the plasma membrane activated PI3K phosphorylates phosphatidylinositol 4,5 bisphosphate (PIP2) forming the secondary messenger, phosphatidylinositol 3,4,5-trisphosphate (PIP3), which in turn recruits proteins with pleckstrin homology (PH) domains such as the serine-threonine kinases Akt and 3-phosphoinositide-dependent kinase 1 (PDK) by binding pleckstrin homology (PH) domains in those proteins (Corvera and Czech 1998). Once at the plasma membrane, PDK1 activates Akt by phosphorylation at threonine 308 (Alessi et al. 1997). Activated Akt can phosphorylate numerous downstream targets that regulate many cellular pathways, including those leading to oncogenic transformation. mTor is one of the major downstream targets of Akt and is a master regulator of cell growth, size, and protein synthesis (Lawlor and Alessi 2001; Abraham 2002; McManus and Alessi 2002); dysregulation of the PI3K-Akt-mTor pathway has been described for many cancers (Bellacosa et al. 2004).

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As discussed above, the requirement of the YXXM motif for transformation suggested that JSRV Env protein directly binds the PI3K regulatory subunit (p85) to activate signaling through the PI3K-Akt-mTOR pathway. Indeed, Akt is activated in many different Env-transformed cell lines including rodent fibroblasts (NIH-3T3 and 208F) (Palmarini et al. 2001b; Alberti et al. 2002; Chow et al. 2003; Liu et al. 2003b; Maeda et al. 2003; Hofacre and Fan 2004; Maeda et al. 2005; Hull and Fan 2006), chicken embryonic fibroblasts (CEF and DF-1) (Allen et al. 2002; Zavala et al. 2003), and kidney epithelial cells (MDCK and RK3E) (Liu and Miller 2005; Maeda et al. 2005). Akt activation was found to be PI3K dependent because PI3Kspecific inhibitors (LY294002 and/or wortmannin) reverted a transformed phenotype, inhibited transformation, or inhibited kinase activity (Palmarini et al. 2001a, b; Maeda et al. 2001; Liu et al. 2003b; Maeda et al. 2003; Zavala et al. 2003). A positive correlation between the levels of Akt activation and degree of Env transformation has also been reported (Liu et al. 2003b). Thus PI3K appears to play a role in Akt activation during Env transformation. At the same time, increasing evidence indicates that Env can also activate Akt independently of PI3K. First, Env efficiently transforms cells and activates Akt in cells where the PI3K regulatory subunit (p85) was inactivated by a dominant negative p85 protein or in cells derived from p85a/b double knockout mice (Maeda et al. 2003). However, other regulatory subunits for class IA PI3Ks potentially could be compensating for the loss of p85. Second, PI3K inhibitors (LY294002 or wortmannin) did not inhibit Env-mediated transformation or revert the transformed phenotype of NIH-3T3 cells (Maeda et al. 2003). Third, phosphorylation of Env at Y590 would be required for p85 binding, and this has not been detected in JSRV-transformed cells. Moreover, in vivo binding of p85 and JSRV Env has not been observed (Liu et al. 2003b; Liu and Miller 2005) although the lack of detection could have been technical. Fourth, in some studies cells could be transformed by an Env with a mutant YXXM motif, and the resulting transformed cells still showed Akt phosphorylation (Liu et al. 2003b; Zavala et al. 2003; Liu and Miller 2005). Finally, Akt activation is not always observed in Env transformed cells or OPA tumor cells. One study found that activated Akt was not detected in Env-transformed (DF-1) cells or lung sections from OPA (Zavala et al. 2003). In another study, only 37% of late-stage OPA tumors showed activated Akt (Suau et al. 2006). Thus Akt activation may not always be required for Env transformation and other pathways may compensate. The role of the PI3K-Akt-mTOR pathway in JSRV transformation has also been assessed by performing transformation assays in the presence of the mTOR inhibitor rapamycin (Maeda et al. 2005). Rapamycin generally inhibits JSRV transformation, although inhibition is partial. Moreover, depending on the cell line, the effects differ. For instance, rapamycin has a modest effect on JSRV transformation in NIH-3T3 fibroblasts (30–40% reduction), while it has a stronger effect in RK3E epithelial cells (~70% reduction). Cell-type specific signaling also has been reported for other viral oncogenes (Aftab et al. 1997). In summary, signaling through Akt and mTOR is observed in JSRV-transformed cells, and its relative importance for transformation varies among cell lines. In JSRV-transformed cells activation of Akt appears to result from both PI3K-dependent

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and -independent mechanisms. Despite the importance of the YXXM motif in Env for transformation, it is probably not directly binding and recruiting PI3K to the plasma membrane. Other as-yet-unidentified mechanisms are likely responsible for activation of this signaling pathway.

Ras-Raf-MEK-MAPK Pathway MAP kinases are serine/threonine protein kinases that respond to extracellular stimuli, and they regulate many cellular functions including mitosis, differentiation, proliferation, and cell survival vs. apoptosis (Pearson et al. 2001). There are five (and likely more) MAPK protein families that are each activated by a cascade of upstream regulatory kinases. Each cascade includes two activating kinases: MAPKKK, MAPKK and MAPK. The extracellular signal-related kinase I and 2 (ERK1/2, aka p44/42) are prototypical MAPKs. They connect extracellular signals at the cell membrane (e.g. activated receptor tyrosine kinases) to activation (phosphorylation) of transcription factors in the nucleus, thus regulating gene expression (Ballif and Blenis 2001). Dysregulated signaling to ERK1/2 is one of the most important pathways in cancer. In MAPK signaling, extracellular mitogen stimulation indirectly leads to activation of intracellular Ras proteins – small G-proteins. One effect of Ras protein activation is binding effector proteins, such as Raf (a MAPKKK), which assists in activation at the plasma membrane. Activated Raf phosphorylates (activates) MEK-1/2 (a MAPKK), which in turn phosphorylates (activates) ERK1/2 (a MAPK). Activated ERK1/2 migrates to the nucleus where it phosphorylates and activates transcription factors such as ELK-1. Other MAPK families include c-Jun amino terminal kinase (JNKs 1, 2 and 3), p38 (isoforms a, b, g and d), ERKs 3/4, and ERK-5. The Ras-Raf-MEK-MAPK pathway has also been implicated in JSRV Env-mediated transformation, although again the relative importance may be cell type and/or species specific. In transformation of NIH-3T3 and RK3E cells, the Ras-Raf-MEKMAPK pathway is of major importance, since treatment with MEK-1 inhibitors (e.g. PD98059) abolishes transformation (Maeda et al. 2005). In NIH-3T3 cells, the predominant signaling is initiated through H or N-Ras, since the H/N-Ras inhibitor FTI-277 also abolishes JSRV transformation (Maeda et al. 2005). FTI-277 can also revert the transformed phenotype of JSRV-transformed NIH-3T3 cells (Maeda et al. 2005). On the other hand, in RK3E cells, FTI-277 only partially inhibits transformation, indicating that stimulation of Raf and MEK-1 in these cells is occurring through alternate proteins; K-Ras is at least partially involved (Maeda et al. 2005). Despite the evidence for Ras-Raf-MEK-ERK signaling being important for JSRV transformation, constitutive phosphorylation of ERK1/2 was not observed in NIH-3T3 and 208F cells (Liu et al. 2003b; Maeda et al. 2005), although it was in RK3E cells (Maeda et al. 2005). The p38 MAPK negatively regulates JSRV transformation in NIH-3T3 and RK3E cells since the p38 inhibitor SB203580 substantially increases JSRV transformation (Maeda et al. 2005). p38 appears to be inhibiting phosphorylation of MEK1/2 in

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these cells, since treatment of JSRV-transformed cells with SB203580 results in enhanced MEK1/2 and ERK-1/2 phosphorylation. The Ras-Raf-MEK-MAPK pathway has been implicated in vivo because phosphorylated ERK1/2 has been consistently detected by immunohistochemistry in naturally and experimentally induced OPA tissues (De Las Heras et al. 2000, 2003, 2006; Maeda et al. 2003). On the other hand, phosphorylated p38 was not consistently detected in OPA tumors (Maeda et al. 2005). In summary, signaling through the Ras-Raf-MEK-MAPK pathway is important for JSRV transformation in the cell lines studied, and this is consistent with activation of this pathway in OPA tumors. p38 is activated in JSRV-transformed cells, and it may negatively modulate signaling through Ras-Raf-MEK-MAPK, at least in tissue culture. The details of which isoforms in this pathway are involved in signaling may differ depending on the cell line, type or species of origin. Finding the cellular protein(s) that interact with Env would provide insight into how Env activates the PI3K-Akt-mTOR and Ras-Raf-MEK-MAPK pathways. A diagram of the signaling pathways involved in JSRV transformation is shown in Fig. 30.7.

Other Pathways Src is an intracellular non-receptor tyrosine-specific protein kinase that was the first proto-oncogene discovered. Studies on JSRV CT mutants suggested that Env may signal to Src, and treatment with the Src inhibitor PP2 inhibited transformation (Hull and Fan 2006). Treatment of 208F cells with Src inhibitors also reverted or inhibited Env transformation as did a dominant negative Src protein (Varela et al. 2008). Exactly how Src is functioning in Env transformation or how it is activated is unknown. Mutations in the epidermal growth factor receptor (EGFR) are frequently observed in human BAC tumors, and treatment with EGFR inhibitors (gefitinib) has significantly improved survival rates (Marchetti et al. 2005). Although OPA resembles BAC, EGFR inhibitors do not affect Env transformation in vitro (Varela et al. 2008). Primary type II pneumocytes derived from primary OPA tissues had constitutively active Akt and increased levels of telomerase activity compared to normal type II pneumocytes (Suau et al. 2006). This has suggested that Akt activation could lead to telomerase activation and inhibition of senescence. Inhibition of telomere shortening is believed to be required for malignancy. It has also been suggested that Env could be regulating total Akt levels through Hsp90, a molecular chaperone that functions in folding, assembly, maturation, and stabilization of proteins (Varela et al. 2008). Hsp90 can affect several signaling proteins including Akt, c-Src, and p53 (Varela et al. 2008). Inhibition of Hsp90 reverted and/or inhibited JSRV Env transformation of rodent fibroblasts at least partially by inducing Akt degradation. Furthermore, Hsp90 was found to be expressed in naturally occurring OPA and inhibition of Hsp90 reduced proliferation of primary and immortalized cells from OPA tumors (Varela et al. 2008).

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?

SU

Hyal2 RON

TM

Src

RAS

?

PI3K Rac1 Akt

R Raf

MEK 1/2

p38

?

TSC1-TSC2

RheB ERK1/2 ERK mTOR

TRANSFORMATION Fig. 30.7 Signaling Pathways for transformation by JSRV Env. Pathways and intermediates that have been established in JSRV Env transformation are shown in solid arrows. Dashed arrows indicate where the mechanisms of signaling to the pathway are not yet understood. Question marks are where signaling has been established in other biological systems, but it is unclear if it is important for JSRV Env transformation. The two predominant pathways in Env transformation are the Ras-Raf-MEK1/2-ERK1/2 and PI3K-Akt-mTOR pathways. Signaling from Hyal2-RON, Src and Rac1 signaling has also been found to be important Env transformation in at least some cases. Interaction between Env and the proteins in the indicated pathways has not been detected. It is likely that additional intermediates are involved in some of these signaling pathways

Env Transformation in 3-D Culture The JSRV Env transformation studies discussed above were performed in monolayer culture. While these experiments have proven informative, there are limitations because monolayer cultures do not recapitulate the polarized epithelial structures or the cell–cell and cell–environment interactions that occur in the lung. An in vitro model has been to suspend epithelial cells in extracellular matrix (e.g. Matrigel). Under these conditions, MDCK cells readily form well-differentiated hollow spheres termed acini, consisting of a single layer of polarized epithelial cells with

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many of the properties of normal epithelia (O’Brien et al. 2001, 2002; Lubarsky and Krasnow 2003; Debnath and Brugge 2005). MDCK cells expressing Env that were cultured in 3-D formed aberrant structures with multiple lumens (Johnson et al. 2010). The disruption was largely mediated by an increase in proliferation and also by resistance to the proliferative suppression signal associated with lumen clearing. On the other hand, JSRV Env did not disrupt establishment of cell polarity, tight junctions, or apoptosis associated with lumen clearing. The PI3K-Akt-mTor and Ras-Raf-MEK-MAPK pathways are important in MDCK transformation but differences were observed in monolayer vs. 3-D culture conditions. In monolayer culture, inhibition of PI3K reverted the transformed phenotype more efficiently than did inhibiting mTOR. In contrast, in 3-D culture, inhibition of mTor (rapamycin) more efficiently reverted transformation than did inhibition of PI3K (LY294002). Thus in 3-D culture, Env signals to mTor in both PI3K-dependent and -independent manners, but the PI3K-independent pathway may be more important. This is reminiscent of the previous finding in monolayer that JSRV Env can signal to Akt (upstream of mTOR) through PI3K-dependent and -independent mechanisms (Maeda et al. 2005; Hull and Fan 2006). Similar to other cell lines, inhibition of MEK (PD98059) inhibited Env transformation of MDCK cells in monolayer, indicating a positive role for MEK in this process (Johnson et al. 2010). In contrast, inhibition of H/N-Ras (FTI-277) enhanced transformation of Env-expressing MDCK cells, indicating that H/N-Ras has a negative role in MDCK transformation in monolayer. FTI-277 also enhanced transformation of parental MDCK cells, indicating that H/N-Ras may have a general negative effect on growth of MDCK cells in monolayer. In contrast, in 3-D culture inhibition of both MEK and H/N-Ras enhanced transformation and the size of Env structures; this was also true for control acini. These results indicate a general inhibition of MDCK cell growth in 3-D culture by the H/N-Ras-MEK pathway, in contrast to a positive role for MEK in transformation in a monolayer. The studies on MDCK 3-D cultures emphasize the fact that the differentiation state of the cells can influence signaling pathways involved in JSRV transformation. In particular, conditions that maintain differentiation of lung epithelial cells (e.g. 3-D culture) will be most informative about the mechanisms of JSRV lung carcinogenesis.

The Hyal2/RON Pathway As described above, the cell surface receptor for JSRV Env is Hyal2. Studies on the minimally transformed human lung epithelial cell line BEAS-2B have indicated that in these cells Hyal2 is complexed with a membrane-spanning growth factor receptor RON (Danilkovitch-Miagkova et al. 2003). RON (also known as STK) is a receptor tyrosine kinase that belongs to the Met proto-oncogene family. It is widely expressed in human tissues, in particular those of epithelial origin and immune cells (Comoglio and Boccaccio 1996). Interestingly, RON is overexpressed in a variety

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of human tumors, and certain RON mutants induce tumorigenic transformation in vitro; RON may also promote tumor metastasis (Santoro et al. 1996; Wang et al. 2003). Additionally, transgenic mice overexpressing RON develop lung tumors (Wang et al. 2003), which supports a role for RON in lung cancer. Indeed RON is overexpressed and constitutively activated in some human lung cancer cell lines, and in particular those derived from BAC (Danilkovitch-Miagkova et al. 2003). The facts that RON can associate with Hyal2, and that over-expression of RON can lead to lung cancer, have suggested that JSRV transformation may involve the Ron/ Hyal2 axis in lung epithelial cells. RON is phosphorylated in Env transformed BEAS-2B cells and expression of a dominant- negative kinase-dead RON mutant blocked Env-mediated transformation (Danilkovitch-Miagkova et al. 2003). In normal BEAS-2B cells, Hyal2 is constitutively associated with RON, inhibiting its ability to bind its growth factor ligand (macrophage-stimulating protein; MSP) and activate its tyrosine kinase. When JSRV Env is introduced into BEAS-2B cells, Env binds to Hyal2, resulting in its degradation. This in turn frees RON to respond to MSP and become activated; activation ultimately leads to phosphorylation of downstream targets such as Akt (Danilkovitch-Miagkova et al. 2003). Thus, in effect, Hyal2 functions as a tumor suppressor in BEAS-2B cells by binding and blocking the mitogenic activity of RON; Env counteracts Hyal2 by binding it and stimulating its degradation. Although the Hyal2/RON interaction is likely involved in JSRV transformation in BEAS-2B cells, it is not required for transformation in several other cell lines and mouse models. First, JSRV Env does not bind murine Hyal2, yet Env transforms murine cell lines and induces tumors in mice (Wang et al. 1995; Liu et al. 2003a, b; Miller et al. 2004; Maeda et al. 2005; Wootton et al. 2005, 2006a, b). Additionally, over-expression of Hyal2 or RON did not affect Env transformation or activation of RON in any cell lines tested (Liu et al. 2003a; Miller et al. 2004). When the receptor binding domain (RBD) of Env (that binds Hyal2) is deleted, the resulting Env can still induce transformation of rodent cell lines (Rai et al. 2001; Liu et al. 2003a; Miller 2003; Miller et al. 2004). Moreover, MDCK cells do not express RON (Wang et al. 1994, 2004; Danilkovitch-Miagkova et al. 2001). Thus while the RON-Hyal2 pathway may contribute to Env transformation in human lung airway cells or BEAS-2B cells, it is probably not absolutely required. It will be interesting to explore the role of RON-Hyal2 in ovine lung epithelial cells that would express RON as well as a Hyal2 that is a functional Env receptor.

Pathogenesis by JSRV Natural History of OPA in Flocks OPA was shown to be a transmissible malignant disease by intratracheal inoculation of sheep with: tumor cells (Coetzee et al. 1976), tumor homogenate (Wandera 1970; Verwoerd et al. 1980; Salvatori et al. 2004), OPA lung fluid, and finally JSRV produced in culture (Palmarini et al. 1999; Cousens et al. 2007). In nature, a major route of

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JSRV transmission appears to be by aerosols (Dungal 1938, 1946; Tustin 1969) because lung fluid from OPA-affected sheep contain high concentrations of JSRV (107–1010 JSRV particles per ml) (Cousens et al. 2009), making spread through aerosolized lung secretions plausible. The environment of the lung is rich in surfactant proteins, proteases, and denaturing agents that inactivate viruses as well as other pathogens. However, JSRV can survive in this environment as well as outside the host for several weeks (Cousens et al. 2009). The stability of the virus is largely attributed to the Env protein. MuLV-based vectors pseudotyped with JSRV Env are remarkably stable at room temperature and relatively resistant to the effects of surfactant (Zsengeller et al. 1999; Coil et al. 2001). JSRV infection can also be spread vertically (intra-uterine) and by suckling of colostrum from infected animals (Salvatori et al. 2004; Grego et al. 2008).

Latency to Disease Incubation time to development of OPA is 6–8 months when JSRV is introduced into flocks of sheep where infection is not endemic (Dungal 1938). Although sheep of all ages are susceptible, cases of OPA from natural infection rarely occur in animals younger than 7–9 months of age (Dungal 1938; Tustin 1969; Hunter and Munro 1983; Gonzalez et al. 1993). The disease latency following intratracheal inoculation of lambs that are several months old is 5–12 months (Tustin 1969; Wandera 1970; Martin et al. 1976). When very young lambs are inoculated, clinical signs appear at 3–6 weeks or even more rapidly (4–6 days) (Sharp et al. 1983). It has been suggested that younger lambs are more susceptible to OPA because they have more proliferating type II pneumocytes and Clara cells (Salvatori et al. 2004; Caporale et al. 2005). Not all animals infected with JSRV develop OPA, (Salvatori et al. 2004; Caporale et al. 2005), and some breeds may be more resistant to OPA than others. In 1933, an outbreak of OPA in Iceland resulted from introduction of a JSRV-infected breeding stock into the island (York and Querat 2003); up to 90% of Gottorp sheep died from OPA compared to 10% of Adalbol sheep (Dungal 1938). Additionally, flock immunity might theoretically provide protection to animals. Development of OPA was high (20% or more) during the first few years following introduction of infection into a flock and it decreased (less than 5%/year) after a few years (Dungal 1938; Shirlaw 1959; Wandera 1967; Tustin 1969; Sharp and DeMartini 2003). However, an immune response to JSRV has not been detected in infected animals. In natural cases, co-infection with the ovine lentivirus maedi-visna virus (MVV) is frequently observed in OPA tumors (Markson et al. 1983; Snyder et al. 1983). Animals also frequently present with secondary bacterial infections. In most studies, OPA sheep co-infected with maedi-visna virus (MVV) present with more severe clinical signs than those only infected with JSRV (Markson et al. 1983; Snyder et al. 1983; Rosadio et al. 1988b). Development of OPA was also studied in animals experimentally co-infected with JSRV and MVV (Hudachek et al. 2010).

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OPA was observed along with the spontaneous regression of the tumors and regression of OPA was more common than progression; CD3+ T cells and antibodies against JSRV were detected, which could have been responsible for the regression. Thus under these conditions, MVV might stimulate the immune response, leading to protection against progression of JSRV-induced OPA.

Immune Responses JSRV-infected sheep rarely develop antibodies to JSRV. The general lack of detectable cellular and/or humoral immunity in infected animals is a unique property of JSRV infection (Holland et al. 1999; Summers et al. 2002; Sharp and DeMartini 2003; Ortin et al. 2007). Expression of closely related endogenous JSRV-related proviruses (enJSRVs, see below) in the fetal thymus during T cell development is believed to cause immunological tolerance to exogenous JSRV (Palmarini et al. 2001a; Spencer et al. 2003; Palmarini et al. 2004). Despite this, OPA tumors generally show a large influx of macrophages and downregulation of MHC I and II complex expression (Hunter and Munro 1983; Summers et al. 2005). Downregulation of MHC 1 in OPA may prevent elimination of virally infected cells by virus-specific CD8+ T cells (Summers et al. 2005). The effects of the infiltrating macrophages are unknown, but it is possible that they secrete IFN-g, which could modulate MHC I and II levels. A recent study of natural OPA cases reported numerous CD3+ T cells located in pulmonary tissue and surrounding the neoplastic foci (Beytut et al. 2009). However, sheep with natural OPA have reduced CD4+ T cells in the peripheral blood, increased circulating neutrophils or increased CD3+ T cells at neoplastic foci (Holland et al. 1999; Summers et al. 2002; Sharp and DeMartini 2003). These changes are not always observed in experimentally infected animals. Therefore, it is believed that the presence of neutrophils, macrophage, lymphocytes, and plasma cells in naturally infected cases are due to secondary infections (Rosadio et al. 1988a; Garcia-Goti et al. 2000; Summers et al. 2005).

Endogenous JSRV ERVs Retroviral integration is an essential step in the lifecycle resulting in integration of proviral DNA into the host genome. If a retrovirus infects a germ cell progenitor, this results in inheritance of the proviral DNA as a Mendelian element in resulting progeny. Vertically transmitted retroviruses are termed endogenous retroviruses (ERVs) (Boeke and Stoye 1997; Coffin 2004). The process of accumulating copies of retroviruses into the germ line is termed endogenization. While endogenization of retroviruses is relatively infrequent, over evolutionary time scales this has resulted

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in accumulation of numerous “ancient” and “modern” (recently acquired) ERVs in most eukaryotes. Since there is no mechanism for elimination of ERV DNAs from cells, ERVs accumulate over time. For instance, approximately 8% of the human genome consists of ERV proviruses. Most ERVs are replication defective, likely reflecting selection against replication competent ERVs. However, some modern ERVs can encode viral proteins, or in some cases complete viruses. When expressed, ERVs can have various biological effects and in some cases expression is selected. Biological effects of ERVs include immune suppression (Golovkina et al. 1992; Gifford and Tristem 2003), resistance to exogenous retroviral infection (Best et al. 1997), and facilitating early embryonic development (Villarreal 1997; Mi et al. 2000; Dupressoir et al. 2005). ERVs have also been implicated in diseases such as autoimmunity (Nakagawa and Harrison 1996; Perron et al. 1997), multiple sclerosis (Perron et al. 1997), and cancer (Lower et al. 1996) in humans, although causal roles have not been definitively established. Sheep are interesting because they carry copies of active ERVs that are highly similar to the exogenous betaretroviruses JSRV and ENTV – the enJSRVs. Indeed endogenization of enJSRVs appears to be ongoing (Arnaud et al. 2007, 2008). Studies in sheep by Palmarini and collaborators provide strong evidence of co-adaptation between exogenous betaretroviruses, enJSRVs, and their hosts.

enJSRV enJSRVs have been integrating into the sheep genome for the last 5–7 million years. Currently, there are at least 27 enJSRV proviruses in the sheep germline DNA (Palmarini et al. 2000b; Arnaud et al. 2007), and five have intact proviruses with uninterrupted ORFs (Arnaud et al. 2007). These five enJSRVs have 85–89% sequence identity to exogenous JSRV (Palmarini and Fan 2003). Regions of sequence divergence reside in the U3 region of the LTR and three regions in Gag and Env termed variable regions 1, 2, and 3 (VR1-3) (Palmarini et al. 2000b). None of the EnJSRV Envs contain the YXXM motif in the CT region of the Env TM domain and are unable to induce transformation (Palmarini et al. 2001b; Arnaud et al. 2007). enJSRV and exogenous JSRV LTRs differ because enJSRV LTRs are not preferentially active in lung epithelial cells (McGee-Estrada and Fan 2007). They also may respond to progesterone, consistent with their high expression levels in the female reproductive tract tissue (Palmarini et al. 2000b).

EnJSRV Restriction Factor Certain EnJSRVs function as restriction factors for exogenous JSRV infection. EnJSRV and JSRV use the same receptor, Hyal2, and expression of EnJSRV Env in some tissues can block JSRV entry through receptor competition. In addition, two

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recently acquired enJSRVs (56A1 and −20) express Gag protein that transdominantly inhibit exogenous JSRV viral particle release through a mechanism termed JSRV late restriction (JLR) (Mura et al. 2004). JSRV Gag normally coalesces into viral cores in the pericentrosomal area, after which the cores engage the intracellular trafficking machinery for transport to the cell membrane. enJS56A1 Gag behaves as a mis-folded protein, likely due to tryphophan at position 21 (W21); however, it can associate with exogenous JSRV Gag forming multimers and/or chimeric viral particles that are unable to traffic to the cell membrane. It has been suggested that the inhibitory effects of enJSRV on exogenous JSRV infection may have driven a switch in tissue tropism for the exogenous virus from the genital tract (where enJSRV is expressed) to the alveolar epithelium of the lung (no enJSRV expression) (Palmarini et al. 2000b; Palmarini et al. 2004). Indeed, LTRs of the restrictive enJSRVs are not active in differentiated lung cells (Palmarini et al. 2000b).

The Role of enJSRV in Placentation The fusigenic properties of retroviral Env proteins have led to the proposal that ERVs play a role in placental morphogenesis. Indeed, retroviral particles are observed in the reproductive tract of many different species (Kalter et al. 1975; Harris 1991), and the human proteins syncitin-1 and -2 that promote syncitial trophoblast fusion are HERV env proteins (Blond et al. 1999; Mi et al. 2000). Studies in sheep are the first to provide in vivo evidence that ERVs play a physiological role in conceptus and placental development (Dunlap et al. 2006a, b). EnJSRV expression is most abundant in the female reproductive tract of sheep, including the vagina, cervix, uterus, and oviduct (Spencer et al. 1999; Palmarini et al. 2000b; Palmarini et al. 2001a; Dunlap et al. 2005). Env expression is temporally regulated during conceptus development and placental morphogenesis (Dunlap et al. 2005). It has been proposed that co-expression of Env and Hyal2 mediate cell fusion leading to formation of multinucleated syncytial plaques that are critical for formation of the placenta (Dunlap et al. 2006a, b). Indeed, it was shown that targeted downregulation by siRNA of EnJSRV Env expression in the oviduct leads to failure of the sheep conceptus to implant (Dunlap et al. 2006a, b).

ENTV ENTV is the causative agent of contagious ovine and caprine nasal adenocarcinoma (ONA and CNA), which arises from the secretory cells of the ethmoid turbinate (Cousens et al. 1999; De Las Heras et al. 2003; Ortin et al. 2003). ENTV shares ~95% homology with JSRV. Two strains of ENTV, ENTV-1, and ENTV-2 have been identified and are largely distinguished by their host tropism and dissemination in infected animals. ENTV-1 infects sheep and is mainly confined to the tumor.

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ENTV-2 infects goats and also establishes lymphoid infection (Cousens et al. 1996; Ortin et al. 2003). As expected from the high degree of sequence homology, ENTV shares many properties with JSRV. Specifically, Hyal2 is the cellular receptor for entry, Env acts as an oncogene in in vitro and in vivo experiments (Alberti et al. 2002; Dirks et al. 2002; Wootton et al. 2006a) and the same signaling pathways are involved in ENTV transformation (PI3K-Akt-mTor and Raf-MEK-MAPK pathways). Although ENTV-1 employs Hyal2 for entry, additional factors are required because Hyal2 is not sufficient for entry in some cell lines (Dirks et al. 2002). JSRV and ENTV sequence are divergent in three regions: 3¢ of Env, the U3 region of the LTR and Orf-x (Cousens et al. 1999). ENTV-1 has two stop codons within the orf-x open reading frame, making it unlikely that orf-x protein is important for ENTV-1 replication (Cousens et al. 1999). While ENTV-1 Env has been shown to transform rodent fibroblast and epithelial cell lines (Alberti et al. 2002; Dirks et al. 2002; Liu et al. 2003a, b; Liu and Miller 2005) and induce lung tumors in mice (Wootton et al. 2006a), there are significant sequence differences in the TM CT region between JSRV and ENTV. However, utilization of the same pathways for transformation suggests that the minimally essential amino acids and/or structure are maintained. Specifically, residue Y590 in CT of JSRV Env is critical for transformation. This residue is conserved in ENTV-1 and shown to be required because mutations ablate transformation (Alberti et al. 2002; Liu et al. 2003b). Although JSRV and ENTV show an overall high sequence similarity, the U3 regions of the LTR are only 62% homologous. Most differences are observed in the mid- and promoter proximal enhancer regions. A site critical for activity of the JSRV LTR in lung epithelial cells, the upstream HNF-3b binding site, is absent in the ENTV LTR and is believed to contribute to its lower activity compared to the JSRV LTR in those cells (McGee-Estrada and Fan 2007). When expressed in an AAV vector, ENTV-1 Env induces adenocarcinoma of the lung that closely resembles that induced by JSRV Env. Thus ENTV Env is not the major determinant of disease specificity. Rather, similar to JSRV, the ENTV LTR is likely the major determinant.

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Markson LM, Spence JB et al (1983) Investigations of a flock heavily infected with maedi-visna virus. Vet Rec 112(12):267–271 Martin WB, Scott FM et al (1976) Experimental production of sheep pulmonary adenomatosis (Jaagsiekte). Nature 264(5582):183–185 McGee-Estrada K, Fan H (2006) In vivo and in vitro analysis of factor binding sites in Jaagsiekte sheep retrovirus long terminal repeat enhancer sequences: roles of HNF-3, NF-I, and C/EBP for activity in lung epithelial cells. J Virol 80(1):332–341 McGee-Estrada K, Fan H (2007) Comparison of LTR enhancer elements in sheep beta retroviruses: insights into the basis for tissue-specific expression. Virus Genes 35(2):303–312 McGee-Estrada K, Palmarini M et al (2002) HNF-3beta is a critical factor for the expression of the Jaagsiekte sheep retrovirus long terminal repeat in type II pneumocytes but not in Clara cells. Virology 292(1):87–97 McManus EJ, Alessi DR (2002) TSC1-TSC2: a complex tale of PKB-mediated S6K regulation. Nat Cell Biol 4(9):E214–E216 Mertz JA, Simper MS et al (2005) Mouse mammary tumor virus encodes a self-regulatory RNA export protein and is a complex retrovirus. J Virol 79(23):14737–14747 Mi S, Lee X et al (2000) Syncytin is a captive retroviral envelope protein involved in human placental morphogenesis. Nature 403(6771):785–789 Mikkers H, Allen J et al (2002) High-throughput retroviral tagging to identify components of specific signaling pathways in cancer. Nat Genet 32(1):153–159 Miller AD, Van Hoeven NS et al (2004) Transformation and scattering activities of the receptor tyrosine kinase RON/Stk in rodent fibroblasts and lack of regulation by the jaagsiekte sheep retrovirus receptor, Hyal2. BMC Cancer 4:64 Miller AD, Vigdorovich V et al (2006) Hyal2, where are you? Osteoarthritis Cartilage 14(12):1315–1317 Mura M, Murcia P et al (2004) Late viral interference induced by transdominant Gag of an endogenous retrovirus. Proc Natl Acad Sci USA 101(30):11117–11122 Nakagawa K, Harrison LC (1996) The potential roles of endogenous retroviruses in autoimmunity. Immunol Rev 152:193–236 Nerenberg M, Hinrichs SH et al (1987) The tat gene of human T-lymphotropic virus type 1 induces mesenchymal tumors in transgenic mice. Science 237(4820):1324–1329 Nitta T, Hofacre A et al (2009) Identification and mutational analysis of a Rej response element in Jaagsiekte sheep retrovirus RNA. J Virol 83(23):12499–12511 Nobel TA, Neumann F et al (1969) Histological patterns of the metastases in pulmonary adenomatosis of sheep (jaagsiekte). J Comp Pathol 79(4):537–540 O’Brien LE, Jou TS et al (2001) Rac1 orientates epithelial apical polarity through effects on basolateral laminin assembly. Nat Cell Biol 3(9):831–838 O’Brien LE, Zegers MM et al (2002) Opinion: building epithelial architecture: insights from threedimensional culture models. Nat Rev Mol Cell Biol 3(7):531–537 O’Brien CA, Pollett A et al (2007) A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature 445(7123):106–110 Ortin A, Cousens C et al (2003) Characterization of enzootic nasal tumour virus of goats: complete sequence and tissue distribution. J Gen Virol 84(Pt 8):2245–2252 Ortin A, Benito AA et al (2007) Bronchioloalveolar carcinoma not related to jaagsiekte sheep retrovirus in a goat. Vet Pathol 44(5):710–712 Overdier DG, Porcella A et al (1994) The DNA-binding specificity of the hepatocyte nuclear factor 3/forkhead domain is influenced by amino-acid residues adjacent to the recognition helix. Mol Cell Biol 14(4):2755–2766 Palmarini M, Fan H (2001) Retrovirus-induced ovine pulmonary adenocarcinoma, an animal model for lung cancer. J Natl Cancer Inst 93(21):1603–1614 Palmarini M, Fan H (2003) Molecular biology of jaagsiekte sheep retrovirus. Curr Top Microbiol Immunol 275:81–115

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Chapter 31

Small RNAs and Their Role in Herpesvirus-Mediated Cancers Sankar Swaminathan and Rolf Renne

EBV EBER RNAs Structure and Expression of EBERs The EBV-encoded RNAs (EBERs) are 166 and 172 nucleotide single-stranded RNAs encoded by two genes separated by 161 bp in the EBV genome (Arrand and Rymo 1982; Lerner et al. 1981; Rosa et al. 1981). They are the most abundant RNA species in most EBV-infected cells, and are present at more than 5 × 106 copies per cell (Lerner et al. 1981). EBERs are transcribed by RNA polIII and contain intragenic control sequences typical of A and B boxes found in polIII transcripts (Jat and Arrand 1982; Rosa et al. 1981; Howe and Shu 1989). However, both EBER 1 and 2 also contain upstream elements and TATA-like sequences typical of polII promoters (Howe and Shu 1989). Although EBER transcription is primarily polIII dependent, the upstream sequences are required for efficient transcription (Howe and Shu 1989, 1993). These upstream sequences are highly conserved in Herpesvirus papio, the baboon herpesvirus homologous to EBV, suggesting that they are important in regulation of EBER expression (Howe and Shu 1988). EBERs are expressed in nasopharyngeal carcinoma (NPC) and Burkitt lymphoma (BL) biopsies and in BL cell lines and lymphoblastoid cell lines (LCLs) passaged in vitro (Jat and Arrand 1982; Minarovits et al. 1992). Where transcription initiation

S. Swaminathan (*) Division of Infectious Diseases, Department of Medicine, University of Utah School of Medicine, Salt Lake City, UT 84132, USA e-mail: [email protected] R. Renne Department of Molecular Genetics and Microbiology, UF Shands Cancer Center, University of Florida, Gainesville, FL 32610-3633, USA E.S. Robertson (ed.), Cancer Associated Viruses, Current Cancer Research, DOI 10.1007/978-1-4614-0016-5_31, © Springer Science+Business Media, LLC 2012

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rate has been directly measured in LCLs by nuclear run-on assay, EBERs are transcribed at a rate at least ten times higher than that of the most highly transcribed latency-associated EBV mRNA (Sample and Kieff 1990). EBERs are expressed in all BL cell lines, but the relative level of EBER 1 and 2 varies among isolates (Minarovits et al. 1992). In most cases, EBER1 is the predominant species, although there are instances when EBER2 is expressed at higher levels. There appear to be type-specific sequence differences in the EBER region among different EBV isolates, although these variations are mostly in noncoding regions (Arrand et al. 1989). The entire EBER locus is hypomethylated in BL cells in comparison to other regions of the EBV genome (Banati et al. 2008; Minarovits et al. 1992), suggesting that hypomethylation of the EBER locus may allow persistent EBER expression even during restricted latency, when most latent gene expression is curtailed. Although EBERs are detectable in cells in which EBV is undergoing lytic replication, they do not appear to play an essential role in the process. The steady-state level of EBER RNAs does not decline significantly upon induction of lytic replication in latently infected cells in culture (Weigel et al. 1985). Nevertheless, based on nuclear run-on assays performed in BL cells in vitro, it is likely that EBER transcription declines during lytic replication (Greifenegger et al. 1998). In lesions of oral hairy leukoplakia, an EBVassociated proliferative epithelial disease, lytic EBV replication predominates and EBERs are not expressed at significant levels (Gilligan et al. 1990). These findings and the ability of EBER-deleted EBV recombinants to replicate lytically (Swaminathan et al. 1991) demonstrate that EBERs are dispensable for lytic replication. EBERs form stable protein complexes with two cellular proteins, La and L22. Based on folding algorithms and experimental evidence from chemical modification and enzymatic cleavage, EBER RNAs are predicted to form stable secondary structures with several stem-loops (Glickman et al. 1988; Rosa et al. 1981). Both EBERs associate with the abundant cellular cytoplasmic autoantigen La and are immunoprecipitable with anti-La antibody (Lerner et al. 1981; Rosa et al. 1981). La binds to many polIII transcripts but associates only transiently with such transcripts during 3¢ end maturation rather than forming stable complexes as it does with EBERs. EBER1 also forms complexes with the ribosomal protein L22 (Toczyski and Steitz 1991). Each EBER1 molecule binds three molecules of L22 simultaneously at three distinct stem-loops (Fok et al. 2006b; Toczyski and Steitz 1993). This binding leads to a partial relocalization of L22 to the nucleoplasm from its usual nucleolar and cytoplasmic locations (Toczyski et al. 1994). This interaction of EBERs with L22 may have important functional consequences (see below).

Functions of EBERs During Latent Infection Several cellular functions have been proposed for EBERs but almost all remain controversial. These functions may be broadly categorized into the following categories: preventing host cell translational shutoff, preventing apoptosis, enhancing specific gene expression and facilitating B cell transformation (Fig. 31.1).

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Nucleus

c

IRF-3

RIG-I Viral Episome

?

Human Genome

IFN, cytokines EBER 2

?

EBER 1 Nucleolus

?

a PKR

b

?

Apoptosis

Apoptosis Translation blockade

L22

Promoting Oncogenesis

(d)

L22

Fig. 31.1 Interaction of EBV EBER RNAs with cellular proteins and proposed mechanisms of action. (a) EBER1 is shown binding and inhibiting cellular kinase PKR, blocking PKR-mediated apoptosis and PKR-mediated protein translation shutoff. (b) A PKR-independent role for EBERs in preventing apoptosis. (c) EBERs are shown binding and activating RIG-1, leading to downstream activation of IRF-3 and transcription of IFN and cytokines, such as IL-10. Question marks denote controversies or uncharacterized mechanisms: In (a) and (c), the localization of EBERs to the cytoplasm is debated. The alternative mechanism of EBERs in preventing apoptosis is uncharacterized (b). How L22 sequestration may promote oncogenesis remains to be defined (d)

Preventing Protein Translation Shutoff A potential role for EBER RNAs in protecting EBV-infected cells from translational arrest was suggested by analogy to the small adenovirus RNAs, VAI and II, which bear structural similarities to EBERs. During adenovirus replication, VA RNAs rescue cells from inhibition of protein translation mediated by the cellular kinase PKR, which is induced by interferon and activated by double-stranded RNAs produced during replication of many viruses (Ghadge et al. 1994; Hovanessian 1989). Activated PKR phosphorylates eukaryotic initiation factor eIF2-a, thereby inhibiting initiation of peptide synthesis (Hershey 1991). Adenovirus mutants in which VA RNAs were replaced by EBER1 could functionally substitute for VA RNAs (Bhat and Thimmappaya 1983, 1985). Although rescue of adenovirus replication by EBER1 was only partial, it suggested that EBERs might play a role in counteracting the antiviral effects of interferon and PKR activation in EBV-infected cells. Consistent with this model, several in vitro studies demonstrated that EBERs could directly bind

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PKR and inhibit its activity. Both EBERs inhibit PKR kinase activity in peptide phosphorylation assays and bind to PKR in vitro (Clarke et al. 1991; Sharp et al. 1993). When added to reticulocyte lysates at high concentrations, EBER1 prevented inhibition of translation by double-stranded RNA (Clarke et al. 1990). When transfected into cells in culture, EBER plasmids enhanced overall protein synthesis; however, PKR-negative cells from PKR knockout mice still exhibited the EBER effect (Laing et al. 1995, 2002). Similarly, although EBER-transfected 3T3 cells exhibited higher soft agar clonogenicity, there was little correlation with EBER expression and EBER expression did not reliably predict tumorigenicity in nude mice (Laing et al. 2002). Overall these data suggest that while EBER expression may have effects on cell physiology, they are not due to PKR inhibition. Whether EBERs actually interact with PKR and inhibit its activation during EBV infection remains controversial. One of the main objections to EBERs acting as a barrier to translational inhibition is their nuclear location. In situ hybridization studies have demonstrated nuclear localization of EBERs (Barletta et al. 1993; Howe and Steitz 1986). A subsequent report described EBERs in a perinuclear location, consistent with endoplasmic reticulum and Golgi localization, by confocal microscopy, but this is the only such report (Schwemmle et al. 1992). A recent study demonstrates that under conditions where UI small nuclear RNAs undergo nucleocytoplasmic shuttling in heterokaryons, the EBERs do not shuttle (Fok et al. 2006a). In addition, EBERs injected into Xenopus oocyte nuclei remained confined to the nucleus whereas tRNAs underwent rapid cytoplasmic export. EBER1 was shown to have a half-life of 25–30 h and was more stable than RNAs that did undergo shuttling, indicating that rapid cytoplasmic degradation was not responsible for the inability to detect shuttling. A nuclear location for the EBERs is clearly difficult to reconcile with a role in modulating the cytoplasmic function of PKR in blocking translation initiation. Another line of evidence suggesting that EBER RNAs are not critical in protection from the effects of interferon comes from studies performed using LCLs transformed by EBER-deleted recombinant EBV (Swaminathan et al. 1991). Replication of VSV, which is highly sensitive to inhibition by interferon, is not quantitatively impaired in EBER-negative LCLs treated with interferon, nor is the growth of EBER-negative LCLs inhibited by interferon (Swaminathan et al. 1992).

Lymphomagenesis and Protection from Apoptosis EBERs are able to enhance survival of Burkitt lymphoma cells under certain experimental conditions. However the mechanism of this effect remains to be fully explained. EBV-infected Burkitt lymphoma cells examined shortly after biopsy usually express EBER RNAs and EBNA1, a nuclear protein required for episomal EBV maintenance. Several cell lines derived from Burkitt lymphoma cells maintain this restricted pattern of EBV gene expression in vitro. One such cell line, Akata, spontaneously loses EBV genomes in culture (Shimizu et al. 1994). The resulting EBV-negative cells are noticeably less robust, undergo spontaneous apoptosis, are more serum-dependent, and are less able to form colonies in soft agar or tumors in

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nude mice (Komano et al. 1998; Ruf et al. 1999). Reinfection of the EBV-negative clones with EBV restores the parental phenotype, indicating that EBV gene expression contributes to the oncogenic phenotype (Komano et al. 1998; Ruf et al. 1999). Transfection of EBERs partially restores resistance to both spontaneous and interferon-induced apoptosis (Komano et al. 1999; Ruf et al. 2000). Interferon treatment and PKR activation can induce apoptosis by multiple mechanisms, including eIF2-phosphorylation. Whether PKR inhibition is actually the mechanism by which EBERs protect against apoptosis has remained controversial. Studies from independent laboratories measuring the effect of EBERs on PKR kinase activity, on either PKR itself or eIF2-a as a substrate, have yielded conflicting results (Nanbo et al. 2002; Ruf et al. 2005; Wang et al. 2005). When considered in combination with the data on nuclear EBER location, it appears that while EBERs may inhibit apoptosis, it is unlikely that inhibition of PKR is the primary mechanism for this effect.

EBERs and Cellular Gene Expression Several cell signaling pathways have been implicated in EBER effects in protection from apoptosis and enhancement of B cell survival. Expression of a variety of cytokines and growth factors is enhanced in several types of EBER-expressing cells. In EBV-negative B lymphoma, T cell lymphoma, or gastric carcinoma cell lines, stable expression of EBERs is associated with IL-10, IL-9, or insulin-like growth factor 1 (IGF1) induction, respectively (Iwakiri et al. 2003, 2005; Kitagawa et al. 2000; Yang et al. 2004). In the case of gastric carcinoma, 5–15% of cases are reported to be EBV-positive. Infection of EBV-negative gastric carcinoma cell lines with EBV led to expression of a limited number of EBV genes including EBERs and was correlated with increased IGF1 production, as was transfection of EBER genes (Iwakiri et al. 2003). Similarly, infection of EBV-negative NPC-derived cell lines with EBV led to increased IGF1 production and enhanced growth at low serum concentrations. Increased IGF1 levels were associated with increased IGF1 mRNA, implying induction of transcription or transcript stabilization by the EBERs. EBV infection and EBER expression in EBV-negative subclones of two BL cell lines, Akata and Mutu, have been correlated with increased expression of human IL-10 (Kitagawa et al. 2000). A model for induction of IL-10 transcription by EBERs has been proposed based on findings that transfected EBERs interact physically and functionally with RIG-I, a cytoplasmic protein important in interferon induction by double-stranded RNA [for review, see Bird (2007)]. According to this model, EBERs may bind and induce the downstream activity of RIG-I, thereby affecting transcription of a number of IFN target genes. Several lines of evidence support the interaction of RIG-I with EBERs, including the use of dominant negative forms of RIG-I and the ability of RIG-I to coimmunoprecipitate with EBER RNA (Samanta et al. 2006, 2008). Transfection of RIG-I led to production of type I IFNs in EBV-positive BL cells but not in EBVnegative cells (Samanta et al. 2006). Transfection of RIG-I siRNA also decreased

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IL-10 expression in BL cells but this effect was not observed in EBV-negative cells or BL cells infected with the EBER-negative recombinant (Samanta et al. 2008). Silencing IRF-3 expression also led to decreased IL-10 expression, suggesting that RIG-I may activate the IL-10 promoter through IRF-3 induction. There is, thus, a large body of experimental evidence that EBERs induce expression of several cytokines that are capable of enhancing cell growth. This model, although attractive, still must contend with the preponderance of evidence that EBERs are confined to the nucleus whereas the RIG-I interaction must occur in the cytoplasm.

EBERs Role in B Cell Transformation and Lymphomagenesis The ability of EBV to efficiently transform primary B lymphocytes in vitro is dependent on several EBV genes expressed during latent infection. Because of the virtually ubiquitous expression of EBER RNAs in EBV-associated tumors, and different types of latent infection and their conserved expression among different primate lymphocryptoviruses, it was thought that they might be important for the process of transformation. The generation of EBER-deleted EBV recombinants in the early 1990s demonstrated that they were not essential for transformation of B lymphocytes in vitro (Swaminathan et al. 1991). EBER-negative recombinants were generated by transfection of cosmid DNA deleted for EBERs into a BL cell line (P3HR-1) harboring an EBV genome which has lost the essential transforming gene EBNA2. Virus replication was induced and recombinants which had acquired both EBNA2 and the EBER deletion were selected by virtue of their ability to transform and immortalize primary B lymphocytes. EBER-deleted recombinants were isolated and arose at a frequency (>15%) that suggested no intrinsic defect in their transforming capacity. Furthermore, outgrowth of EBER-negative LCLs was not slower than that of EBERpositive LCLs, again suggesting that EBERs did not provide a significant growth advantage. Nevertheless, it remained possible that the LCLs were initially coinfected with P3HR-1 virus, which provided EBERs in trans and were subsequently lost prior to analysis. However, pure EBER-negative EBV was passaged from these EBER-negative LCLs and was capable of immortalizing primary B lymphocytes de novo. Immortalized cell lines derived from EBV deleted for EBERs also maintained latent infection and a growth phenotype typical of EBER-positive LCLs, indicating that in vitro, EBERs are not essential for maintenance of the transformed phenotype (Swaminathan et al. 1991). EBER-deleted recombinants were also unimpaired in their ability to enter the lytic phase of replication (Swaminathan et al. 1991). By contrast, EBER-negative recombinants generated by another method suggest that EBERs may provide a quantitative advantage in transforming ability (Yajima et al. 2005). Using the Akata BL cell line, recombinant EBER-negative viruses were derived by using drug selection and cre recombinase to remove the EBER genes from the resident EBV genome. EBER-positive revertant knock-ins were also generated by transfection of wild-type EBER plasmids and reselection of a stable

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transfectant carrying EBER-positive recombinants. Transformation assays performed with high titers of recombinant EBV generated from the EBER knockout and revertant strains revealed that the EBER revertant possessed approximately 20-fold more transforming ability than the EBER-negative recombinant. Further, growth of the EBER-negative LCLs was impaired compared to that of the revertants under low serum conditions. Although these data suggest that EBERs play a role in B lymphocyte growth and transformation, the results were based on comparisons of recombinants ultimately derived from single clones of serially selected transfectants, which may vary considerably in growth rates and phenotype. Most recently, EBER-negative recombinant EBV and revertant EBER-positive viruses derived from B95-8 EBV have been generated using bacmid technology. When a panel of these viruses was tested for their ability to transform primary B lymphocytes in vitro, and the transforming efficiency was quantitatively measured, no differences were observed between EBER-negative recombinants and revertants (Gregorovic G, et al. 2011). These data overall indicate that while strain-specific differences may account for the varying reports, EBERs are not required for the in vitro transforming ability of EBV. The dispensability of EBERs for transformation in vitro suggests that they may play an important role latent infection in vivo and possibly in tumorigenesis. Some clues to their in vivo function may come from known cellular EBER-binding proteins. EBERs interact with two cellular RNA-binding proteins, La and L22; the latter is a component of the large ribosomal subunit (Fok et al. 2006b; Toczyski et al. 1994). La is known to be important in the biogenesis and maturation of polIII transcripts, and is possibly involved in enhancing the stability or translation of some mRNAs (Wolin and Cedervall 2002). Although EBERs are present at high copy numbers, the levels of La in the cell are high enough that sequestration of La does not seem to be a likely mechanism for EBER effects (Wolin and Cedervall 2002). L22 is also an RNA binding protein and each EBER1 RNA binds three L22 molecules (Fok et al. 2006b). EBER binding causes relocalization of L22 to the nucleoplasm from the nucleolus and cytoplasm (Toczyski et al. 1994). Thus, a significant amount of L22 may be sequestered by EBERs although depletion of L22 from ribosomes has not been observed to occur. A recent report on the association of a Marek’s disease virus (MDV) small RNA that also associates with L22 suggests that relocalization and/or RNA binding of L22 may have oncogenic effects in vivo (Kaufer et al. 2010). MDV expresses two copies of a ~450 nt RNA highly homologous to the telomerase-associated RNA (TR) that is an essential component of the telomerase complex (Fragnet et al. 2003). MDV TR (vTR) is fully functional as a telomerase component and is essential for efficient lymphomagenesis in chickens as deletion of the vTR genes leads to decreased and delayed tumor formation (Trapp et al. 2006). Importantly, MDV with a mutation of MDV vTR that abrogates telomerase binding is still lymphomagenic, indicating that the vTR has other functions essential for lymphomagenesis (Kaufer et al. 2010). EBER-1, vTR and the host chicken TR have similar effects on L22 localization as does the vTR mutant which does not bind telomerase. Significantly, in another recent study, when EBER1 was mutated so that it no longer bound L22, the ability of EBER1 to enhance BL cell

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growth and colony formation in soft agar was significantly impaired (Houmani et al. 2009). Both EBV and MDV small RNAs, although very different in their structure and transcriptional regulation appear to target the same cellular protein. How the interaction with L22 might mediate growth effects remains to be determined.

Herpesvirus saimiri HSURs H. saimiri infection leads to aggressive T cell leukemia and lymphoma in a variety of New World primates and transforms T lymphocytes from marmosets in vitro into continuously proliferating cell lines expressing T and NK activation markers [for review, see Ensser and Fleckenstein (2005)]. H. saimiri infected tumor cells and cell lines express seven small RNAs similar to human snRNAs that form snRNPs containing Sm proteins (Biesinger et al. 1990; Lee and Steitz 1990). They are assembled into snRNPs in the cytoplasm by the SMN complex which performs this function in cellular U snRNP assembly (Golembe et al. 2005). During latent infection, HSURs are found in the nucleus and colocalize with cellular snRNPs (Golembe et al. 2005). HSUR1 and HSUR2 are the most highly conserved HSURs among HVS isolates and are the only HSURs found in the closely related H. ateles (Albrecht 2000). HSUR1 is the most abundant HSUR and the most abundant latent H. saimiri transcript, expressed at approximately 20,000 copies per cell (Lee et al. 1988; Murthy et al. 1986). The level of other HSURs is about 2,000 copies per cell. Despite their abundance, conservation, and expression in H. saimiri-related tumors, HSURs are dispensable for H. saimiri lytic infection and transformation in vitro (Ensser et al. 1999; Murthy et al. 1989). They are thus similar to EBERs and likely to be important in vivo to ensure viral persistence and infected cell growth and survival. Their mechanism of action is similarly incompletely characterized although recent studies have provided tantalizing clues to their function and interactions with other small RNAs. Three of the HSURs, namely, HSUR1, HSUR2, and HSUR5, contain AU-rich elements (AREs) in their 5¢ region, similar to those present in the 3¢ UTRs of many cytokine mRNAs and growth factors. Several cellular proteins (HuR, hnRNPD (AUF1), TTP, and BRF1), which either stabilize or destabilize ARE-containing mRNAs, bind specifically to the ARE. HSUR1 and HSUR2 have been shown to bind HuR and hnRNPD in transformed cells and HSUR1 binds to TTP (Cook et al. 2004). Although the presence of AREs in HSURs had led to the hypothesis that HSURs might regulate the levels of host ARE-containing mRNAs, extensive and thorough analysis of cellular mRNAs in cells transformed by HSUR1- and HSUR2deleted HVS failed to demonstrate changes in the levels of mRNAs with AREs (Cook et al. 2004). However, the levels of HSUR1 itself appear to undergo regulation by the ARE-mediated pathway as mutation of the ARE in HSUR1 increased its steady-state levels (Cook et al. 2004; Fan et al. 1997). Despite the lack of any discernible effect on ARE-containing mRNAs, HSUR1 and HSUR2 appear to enhance expression of a specific set of genes important in NK and T cell activation (Cook et al. 2005). When the transcriptional profile of marmoset T lymphocytes transformed by mutant HVS deleted for HSUR1 and

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HSUR2 was compared to cells transformed by wild-type HVS, HSUR expression correlated with significantly increased levels of T cell receptor b and g chains, the T cell and natural killer (NK) cell-surface receptors CD52 and DAP10, and intracellular proteins SKAP55, granulysin, and NKG7. Expression of these genes could be rescued by exogenous HSUR1 and HSUR2 expression. The mechanism by which HSUR1 and HSUR2 exert these effects remains to be defined. Recently, another intriguing mechanism by which HSUR1 and HSUR2 may modulate host cell gene expression has been identified. These two HVS small RNAs were found to contain sequences complementary to miR-142-3p (HSUR1 and HSUR2), miR-27 (HSUR1), and miR-16 (HSUR2) (Cazalla et al. 2010). This suggested that HSUR 1 and HSUR2 may sequester or inactivate these miRNAs, leading to enhanced accumulation of the miRNA target transcripts. Both HSURs were found in Ago2 complexes from HVS-transformed marmoset cells, whereas other cellular snRNAs and other HSURs were not, indicating that HSUR1 and HSUR2 are selectively incorporated into miRNPs. Cell lines generated by transforming primary cells with either wt HVS or HVS specifically deleted for HSUR1 and HSUR2 were then used to assess the effect of HSUR1 and HSUR2 on the levels of these miRNAs and to investigate their association with each other. All three miRNAs, miR-142-3p, miR-27, and miR-16, were immunoprecipitated with anti-Sm serum, but only in wt HVS-transformed cells, demonstrating that they interact specifically with HSUR1 and HSUR2 snRNPs. Furthermore, the level of miR-27 was markedly higher in HSUR-deleted HVS transformed cells, suggesting that HSUR1 leads to downregulation of miR-27. Knockdown of HSUR1 confirmed the negative effect of HSUR1 on miR-27 accumulation and mutation of the miR-27 complementary sequence in HSUR1 abolished the interaction and the negative effect on miR-27 levels. Significantly, levels of FOXO1, a miR-27 target protein, were reduced in cells transformed by HVS lacking HSUR1 and HSUR2, indicating that the effect of HSUR1 on miR-27 has functional consequences on miR-27 target gene expression. Although these findings suggest a mechanism of action for HSUR1, they raise several questions. First, the implications of interactions with miR-16 and miR-142-3p are unclear, as steady state levels of these miRNAs do not appear to be affected by HSURs, nor do the levels of some of their potential target mRNAs. Second, miR-27 targets were not identified among the set of activation genes described earlier that were identified by microarray comparison of HSUR-deleted and wt HVS-transformed cell lines. Thus, there are clearly additional mechanisms by which HSURs selectively enhance gene expression that remain to be identified. Finally, as for EBV EBERs, the in vivo role of HSURs in lymphomagenesis remains to be fully elucidated in animal models.

Viral MicroRNAs In 2004, Tuschl and colleagues discovered the first viral encoded miRNAs in EBV infected Burkitt’s lymphoma cells (Pfeffer et al. 2004). To date, the miRNA registry miRBase (http://www.mirbase.org/) (Griffiths-Jones 2004; Griffiths-Jones et al. 2006) contains 173 herpesvirus-miRNAs, encoded by members of all three herpesvirus

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subfamilies [Alphaherpesvirinae, Betaherpesvirinae, and Gammaherpesvirinae, recently reviewed in Boss and Renne (2010) and Umbach and Cullen (2009)]. Here, we summarize the current knowledge on miRNAs encoded by the two g-herpesviruses EBV and KSHV that are associated with human cancer.

MicroRNA Biogenesis and Function There is no inherent difference between expression and maturation of viral and cellular miRNAs. EBV and KSHV miRNAs, are expressed from polII transcripts (Ambros 2004; Bartel 2004; Bogerd et al. 2010; Diebel et al. 2010) and processing begins with formation of an imperfect stem loop with a hairpin bulge that forms within a miRNA precursor termed the pri-miRNA. The dsRNA region of the pri-miRNA is recognized by DGCR8, which recruits the endonuclease Drosha to cleave and release a 60–80 nt long hairpin. This pre-miRNA is then exported into the cytoplasm via the Exportin 5/RAN-GTPase pathway, where it is recognized by Dicer and cleaved at the bulge. One strand of this dsRNA product is loaded into the RNA-induced silencing complex (RISC). The remaining strand, known as the star (*) strand, is degraded but in many cases can also be loaded into RISC with variable efficiency [for review see: Ambros (2004) and Bartel (2004)]. RISC functions by guiding miRNAs to semicomplementary sites within the 3¢UTRs of target transcripts and induces translational silencing and/or degradation. The 5¢ end of the miRNA, specifically nucleotides 2–8 termed the seed sequence, is critical for determining mRNA target binding, but there are rare cases of miRNA binding sites that have little seed binding, but significant 3¢ compensatory complementarity instead. Transcript 3¢UTRs have multiple binding sites for a specific miRNA, and it is also common that one gene is targeted by multiple miRNAs. Due to the flexible requirements for target recognition, a single miRNA can regulate many targets and as a result miRNAs form large posttranscriptional regulatory networks (Grimson et al. 2007). After binding of RISC to the 3¢UTR of the target transcript, silencing is accomplished through an incompletely deciphered mechanism(s). Current evidence suggests several different mechanisms: inhibition of translational initiation by interfering with the interaction of eIF4E, eIF6E, and the poly A binding protein, premature termination of translation by inducing ribosomal drop-off after initiation, and messenger RNA degradation by relocation of the RISC to cytoplasmic processing (P)-bodies, which contain the RNA degradation machinery (Filipowicz et al. 2008).

EBV- and KSHV-Encoded miRNAs After the initial identification of five EBV miRNAs (Pfeffer et al. 2004), a combination of tiled arrays, cloning, and bioinformatic approaches identified 18 additional EBV miRNAs, located within the 12 kb deletion specific to the B95-8 strain analyzed

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B95-8 deletion

BHRF1 BFLF2

BILF2

LF3

BALF5

LF2

EBV BHRF1- 1

23

BART- 1 5 16 17

Cluster 1 ORF69

2

Cluster 2

K12

v-Flip

v-Cyclin

LANA

KSHV KSHV-miR-K12-12 10

9 8 7 11 6 5 4 3 2 1

Fig. 31.2 Schematic representation of EBV and KSHV miRNAs. EBV and KSHV genomes are represented with black arrows for ORFs, and black bars or rectangles for repeat sequences. MiRNA locations are indicated with red arrows. Genomes are not drawn to scale. Figure was compiled from Burnside et al. (2006), Cai and Cullen (2006), Cai et al. (2005), Cui et al. (2006), Dunn et al. (2005), Grey et al. (2005), Grundhoff et al. (2006), Pfeffer et al. (2004, 2005), Samols et al. (2005), and Yao et al. (2007)

in the original report (Pfeffer et al. 2004) and three more within the BART region outside of the B95-8 deletion (Cai et al. 2006; Grundhoff et al. 2006). Recently, two additional BART miRNA genes have been identified in EBV-positive NPC tissue samples (Zhu et al. 2009). This brings the total of EBV miRNA genes to 25. Since B95-8 immortalizes human B cells, none of the 18 miRNAs within the deletion are required for immortalization in vitro – however, as discussed below this does not rule out a contributing role to pathogenesis and/or tumorigenesis in vivo. Like their metazoan counterparts, EBV miRNAs are expressed in a tissue specific fashion. BHRF1 and BART miRNAs are differentially expressed in lymphoid and epithelial cells and are latency program specific. BART miRNAs are predominantly expressed in epithelial cells, whereas BHRF1 miRNAs are only expressed during latency program III (when most latency-associated genes are expressed). A subset of EBV miRNAs is induced during reactivation and some EBV miRNAs are expressed early after de novo infection of B cells suggesting a role in the establishment of latency (Cai et al. 2006; Cosmopoulos et al. 2009; Edwards et al. 2008; Xing and Kieff 2007). These temporal and spatial expression differences may point to cell-type-specific regulation of host genes in EBV-associated NPCs versus lymphomas. In 2005, four laboratories cloned miRNAs from Kaposi’s sarcoma-associated herpesvirus (KSHV)-infected primary effusion lymphoma cells (PEL) and identified a total of 12 miRNA genes giving rise to 18 mature miRNAs (Cai et al. 2005; Grundhoff et al. 2006; Pfeffer et al. 2005; Samols et al. 2005; Umbach and Cullen 2010). All KSHV miRNAs are located within the major latency-associated region of the genome with 10 of the 12 miRNAs organized in a cluster in the intragenic region between v-Flip and the K12/Kaposin gene. Two additional miRNAs were found to be located within the K12/Kaposin gene (Fig. 31.2). In PEL cells all KSHV miRNAs are highly expressed and induction of lytic replication leads only to a moderate increase of some KSHV miRNAs (Boss and Renne 2010; Cai and Cullen 2006; Pearce et al. 2005). PCR-based miRNA profiling showed that all KSHVinfected tumor cells of both lymphoid and endothelial origin express KSHV miRNAs, albeit at different levels. KSHV miRNAs are also detectable early after de novo

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infection of endothelial cells (O’Hara et al. 2009; Karlie Plaisance and Rolf Renne, unpublished results). The fact that all KSHV miRNAs are expressed in both KS and PEL suggests a role in pathogenesis and or tumorigenesis for this novel class of latency-associated posttranscriptional regulators. Additional support for this notion came from phylogenetic studies. Marshall et al. (2007) analyzed clinical samples from AIDS-KS and KS patients of different geographical origin and found that most miRNAs were highly conserved, suggesting in vivo selection for functional miRNA genes. However, this study also identified a number of miRNA polymorphisms, and a recent study suggests a linkage of such polymorphisms with pathogenesis (Whitby, in press, Journal of Infectious Disease). Molecular studies revealed that KSHV miRNA polymorphisms can affect both miRNA maturation and targeting in infected cells (Gottwein et al. 2006; Han and Renne, unpublished results).

Identifying Targets and Functions of Viral miRNAs Understanding the function of viral miRNAs, and more specifically their contribution to tumorigenesis, will ultimately require comprehensive and cell-type specific target gene identification. Conceptually, viral miRNAs can target cellular transcripts to modulate the host environment, and/or target viral transcripts to regulate viral gene expression. Examples of both types of regulation have been identified for at least one viral miRNA from each human herpesvirus. This field is relatively new, and technologies for miRNA target identification are rapidly evolving from bioinformatic predictions and individual target gene analysis to genomic and proteomic scale analysis (Chi et al. 2009; Hafner et al. 2010). However, studies over the past 6 years strongly suggest that these novel viral posttranscriptional regulators modulate important biological processes including oncogenesis, proliferation and cell survival, innate and adaptive immunity, and control of latent/lytic infection, all of which clearly have implications for viral persistence and pathogenesis (Table 31.1 and Fig. 31.3).

KSHV and EBV miRNAs Targeting Host Cellular Genes To date, most efforts on identifying cellular genes targeted by viral miRNAs have been focused on KSHV and EBV. The first cellular target genes for KSHV viral miRNAs were identified by gene expression profiling of HEK 293 cells stably expressing a miRNA cluster containing ten miRNAs (Samols et al. 2007). A total of 65 genes were downregulated in miRNA expressing cells. Among these, thromobospondin 1 (THBS1) was verified as an miRNA target using luciferase reporter constructs containing THBS1 3¢UTRs. Moreover, protein levels of THBS1 were decreased greater than tenfold in KSHV miRNA-expressing cells. THBS1, a strong tumor suppressor and anti-angiogenic factor, had previously been reported to be downregulated in KS lesions (Taraboletti et al. 1999). The 3¢UTR of THBS1 contained

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Table 31.1 Experimentally verified KSHV and EBV miRNA targets Viral targets miRNA Gene target Gene function miR-K12-9* RTA Replication and KSHV transcriptional activator EBV miR-BART2 BALF5 DNA polymerase miR-BART22 LMP2A Viral oncogene in NPC miR-BART 1-5p LMP1 Viral oncogene miR-BART16 miR-BART17-5p Cellular targets

References Bellare and Ganem (2009) Barth et al. (2008) Lung et al. (2009) Lo et al. (2007) Lo et al. (2007) Lo et al. (2007) References

HCMV

miR-UL112-1

MICB

NK cell ligand

KSHV

miR Cluster

THBS1 EXOC6 ZNF684 CDK5RAP1

miR-K12-1

IkBa p21

miR-K12-3

LRRC8D NHP2L1

miR-K12-3 miR-K12-7 miR-K12-4-3p miR-K12-5

C/EBPb (LIP) GEMIN8 BCLAF1

Angiogenesis inhibitor SEC15 gene family Zinc finger protein Regulation of neuronal differentiation NF-kB inhibitor Inducer of cell cycle arrest Immune cell activator U4 snRNA nuclear binding protein Transcriptional activator Required for splicing Proapoptotic factor

EBV

805

miR-K12-6 miR-K12-11 miR-K12-7

Rbl-2 MAF

Rb-like protein Transcription factor

MICB

NK cell ligand

miR-K12-11a

BACH1

Transcriptional suppressor

miR-BHRF1-3

CXCL11

miR-BART2

MICB

Chemokine, T-cell attractant NK cell ligand

miR-BART3 miR-BART5 miR-BART16

IPO7 PUMA TOMM22

Nuclear import protein Proapoptotic factor Mitochondrial membrane protein a Shown to have seed sequence homology with human miR-155

Stern-Ginossar et al. (2007) Samols et al. (2007) Dolken et al. (2010) Dolken et al. (2010) Dolken et al. (2010) Lei et al. (2010) Gottwein and Cullen (2010) Dolken et al. (2010) Dolken et al. (2010) Qin et al. (2010b) Dolken et al. (2010) Ziegelbauer et al. (2009) Lu et al. (2010) Hansen et al. (2010) Nachmani et al. (2009) Gottwein et al. (2007) and Skalsky et al. (2007) Xia et al. (2008) Nachmani et al. (2009) Dolken et al. (2010) Choy et al. (2008) Dolken et al. (2010)

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nucleus

Viral Episome

Human Genome

Host Cellular Targets

Viral Targets

a

e Latent/ Lytic

Promoting Oncogenesis

Control BALF5 LMP1 LMP2A RTA (BCLAF1) (IkBa) (Rbl-2)

C/EBP beta MAF p21

b

Proliferation & Cell Survival BACH1 THBS1 BCLAF1 PUMA

c

Innate & Adaptive Immunity MICB CXCL11 IkBa

d

Host miRNA Function

Fig. 31.3 Themes of viral miRNA gene regulation. Virus infected cells can produce miRNAs which target both viral and cellular genes. (a) Inhibition of genes that may promote viral oncogenesis. (b) Targets involved in promoting proliferation and cell survival. (c) Gene targets important in innate and adaptive immunity. (d) Modulating host cell miRNA expression and function. Experiments in MCMV show that viral miRNA synthesis completely overtakes host cell miRNA production early after infection (Dolken et al. 2007). Viral miRNAs may hijack Drosha processing or RISC loading leading to impaired function of host miRNA, leading to global de-repression of cellular miRNA targets. (e) The maintenance of latency is a common function of viral miRNAs that target immediate-early or early lytic viral genes. BCLAF1, IkBa, and Rbl-2 are host genes that contribute to the latent/lytic switch by either sensitizing cells to reactivate or functioning to maintain latency. Known viral miRNAs and target genes, including all references, are listed in Table 31.1

seed sequence binding sites for multiple KSHV miRNAs, suggesting that viral miRNAs in clusters coordinately regulate host cellular target genes. Additionally, osteopontin (SPP1) and PRG1, genes involved in cell-mediated immunity and apoptosis, were identified as KSHV miRNA targets by similar techniques. These initial findings demonstrated that KSHV-encoded miRNAs contribute to viral pathogenesis by promoting angiogenesis (a hallmark of KS tumors) and by inhibiting cellular immunity and apoptosis (Samols et al. 2007). Using an elegant tandem-array approach, The Ganem group identified several KSHV miRNA targets that were either induced by miRNA knockdown in latently infected PEL cells or inhibited in uninfected B cells ectopically expressing KSHV miRNAs (Ziegelbauer et al. 2009). This analysis revealed that three KSHV miRNAs

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(miR-K12-5, K12-9, and miR-K12-10b), target Bcl-2-associated factor (BCLAF1). BCLAF1 is a transcriptional repressor that can promote apoptosis. However, BCLAF1 expression in latently infected PEL cells can also inhibit KSHV replication. Antagomir-based inhibition of KSHV miRNAs miR-K12-5, K12-9 and miR-K12-10b resulted in sensitizing latently infected endothelial cells for lytic reactivation. These data suggest that KSHV miRNAs can contribute to latency control by targeting both the viral RTA gene (as discussed below) and cellular genes such as BCLAF1 (Ziegelbauer et al. 2009). Viral miRNAs can also mimic cellular miRNA function. Two groups showed that miR-K12-11 and human miR-155 shared complete seed sequence identity (Gottwein et al. 2007; Skalsky et al. 2007). Mir-155 is aberrantly expressed in many human malignancies and when overexpressed in mice causes lymphoproliferative disease (Garzon and Croce 2008). This led to the question of whether miR-K12-11 and miR-155 target a common set of genes. Prediction programs revealed that the BACH1 gene contains four binding sites for both miR-K12-11 and miR-155 within its 3¢UTR. BACH1 is a transcriptional repressor affecting expression of hemeoxygenase 1 (HMOX1), a protein that promotes cell survival and proliferation (Igarashi and Sun 2006). Luciferase reporter assays confirmed miRNA regulation of BACH1 and BACH1 protein levels were decreased in miR-K12-11 and miR-155 expressing cells. Gene expression profiling also revealed that miR-K12-11 and miR-155 can regulate a common set of genes (Gottwein et al. 2007; Skalsky et al. 2007). In addition, Qin et al. (2010a) showed that miR-K12-11-dependent regulation of BACH-1 not only affected oxidative stress responses but also led to increased expression of xCT, an amino-acid transporter, which has previously been shown to function as a fusion receptor for KSHV. Qin et al. (2010b) also showed that KSHV-encoded miRNAs induce IL-6 and IL-10 secretion in murine macrophages and human myelomonocytic cells. C/EBPb, a known regulator of IL-6 and IL-10 transcription, was shown to be targeted by the KSHV miRNA cluster. Specifically, miR-K12-3 and miR-K12-7 inhibited the LIP isoform of C/EBPb, which functions as transcriptional suppressor. These data suggest that KSHV-encoded miRNAs directly regulate cytokine secretion of latently infected cells (Qin et al. 2010b). KSHV infected endothelial cells undergo transcriptional reprogramming, expressing markers for both lymphatic and blood endothelial cells (Carroll et al. 2004; Wang et al. 2004). Hansen et al. have recently demonstrated that KSHV miRNAs regulate this reprogramming by targeting the cellular transcription factor musculoaponeurotic fibrosarcoma oncogene homolog (MAF). MiR-K12-6 and miR-K12-11 together target the 3¢UTR of MAF, thereby inducing endothelial cell differentiation, and possibly contribute to KSHV oncogenesis (Hansen et al. 2010). Interestingly, miR-K12-11, the ortholog of miR-155 is also involved in B cell differentiation and proliferation in vivo (Boss and Renne, unpublished). Two recent studies addressed the role of KSHV miRNA within the context of the viral genome by generating a recombinant KSHV miRNA virus (Lei et al. 2010; Lu et al. 2010). Lei et al. reported inhibition of NF-kB in 293 cells infected with the mutant virus which was accompanied by a moderate increase in lytic replication.

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IkBa, the NF-kB repressor, was subsequently shown to be targeted by miR-K12-1 (Lei et al. 2010). Gottwein and colleagues reported another target for miR-K12-1, which like NF-kB is crucial for cell survival and proliferation. Several lines of evidence demonstrate that miR-K12-1 directly targets p21, a key inducer of cell cycle arrest and tumor suppressor (Gottwein and Cullen 2010). Using a similar miRNA knockout virus, Lu et al. also observed a moderate increase in lytic replication but identified entirely different mechanisms. In addition to a moderate inhibition of RTA by miR-K12-5 Lu et al. observed a drastic genome wide inhibition of DNA methylation after deleting the miRNA cluster. Retinoblastoma (Rb)-like protein 2 (Rbl-2), a potent inhibitor of DNA (cytosine-5-)-methyltransferases (DnmT1, 3a, and 3b) was shown to be targeted by several KSHV miRNAs. These data showed for the first time a role of viral miRNAs in epigenetic regulation of latency (Lu et al. 2010). The latest technique to identify miRNA targets utilizes cross-linking and immunoprecipitation of RISCs followed by microarray or high-throughput sequencing analysis of the RISC-bound miRNA targets (Chi et al. 2009; Hafner et al. 2010). Using this technique, Dolken et al. (2010) were able to confirm a significant number of the above discussed targets and in addition determined six novel targets of KSHV miRNAs and two targets of EBV miRNAs. KSHV miR-K12-3 was shown to target LRRC8D, thought to be involved in proliferation and activation of lymphocytes and macrophages, and NHP2L1, a nuclear protein that binds to U4 snRNA. MiR-K12-4-3p targets GEMIN8, which is required for spliceosomal snRNP assembly in the cytoplasm and pre-mRNA splicing in the nucleus. The KSHV miR-cluster was also found to target EXOC6, ZNF684, and CDK5RAP1; however, no functional studies have been presented on these novel target genes (Dolken et al. 2010). For the more than 25 EBV-encoded miRNAs, few cellular targets have been identified. Dolken et al. showed that EBV miR-BART16 target TOMM22 and miRBART3 targets IP07, both involved in cellular transport processes. However, their role in EBV biology has not been determined (Dolken et al. 2010). Previously, EBV miRNAs were shown to regulate PUMA and CXCL11. PUMA modulates apoptosis through p53 upregulation and is targeted by miR-BART5 (Choy et al. 2008). CXCL11 is an IFN-inducible T-cell chemoattractant and is targeted by miRBHRF1-3, thereby inhibiting T-cell recognition (Xia et al. 2008). In this context, EBV miR-BART2 also suppresses the major histocompatibility complex 1-related chain B (MICB) (Nachmani et al. 2009). The first viral miRNA targeting a host gene involved in immune response was reported by Stern-Ginossar et al. (2007) who identified MICB targeted by miR-UL112-1 in HCMV-infected cells. MICB is a stressed-induced ligand that is essential for natural killer (NK) cell recognition of virus-infected cells. Using elegant genetics approaches, they demonstrated that targeting MICB significantly reduced NK cell killing of HCMV infected cells (Stern-Ginossar et al. 2007). Later, two more g-herpesvirus-encoded miRNAs were found to target MICB. Nachmani et al. (2009) showed that KSHV miR-K12-7 and EBV miR-BART2 both directly target MICB mRNA and reduce its expression by utilizing three different sites within the MICB 3¢UTR. Interestingly, MICB is also targeted by the HCMV encoded UL16 protein and the KSHV MIR3 and MIR5 proteins

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(Areste and Blackbourn 2009).This coevolution suggests that targeting MICB to inhibit NK cell function is a critical step for herpesviral persistence in vivo.

Viral miRNAs Targeting Viral Genes MiR-BART2 is encoded antisense to BALF5, a transcript encoding the EBV DNA polymerase, and was initially hypothesized to function as siRNA (Pfeffer et al. 2005). Barth et al. (2008) later confirmed that miR-BART2 does cleave BALF5 in a siRNAlike manner in EBV-infected cells. The first example of a viral gene targeted by viral miRNAs through seed sequence binding was latent membrane protein 1 (LMP1) (Lo et al. 2007). LMP1 is a viral oncoprotein required for immortalization of human B-cells. However, overexpression of LMP1 leads to induction of apoptosis (Izumi and Kieff 1997). The 3¢UTR of latent LMP1 contains potential binding sites for several BART miRNAs. The BART cluster 1 miRNAs miR-BART16, miR-BART17-5p, and miR-BART1-5p were shown to decrease LMP1 protein expression. MiRNA-dependent targeting of LMP1 in latently infected cells provides a mechanism of fine tuning a balance between proliferation and apoptosis (Lo et al. 2007). Recently, novel miRNAs including miR-BART22 have been identified in EBV-associated NPCs. MiR-BART22, which is expressed at high copy numbers in NPCs was shown to target the latent membrane protein 2A (LMP2A) and reduce its expression in NPC-derived cell lines, which may facilitate NPC carcinogenesis (Lung et al. 2009). To investigate whether KSHV miRNAs target KSHV immediate early transactivators, Bellare and colleagues utilized luciferase reporter assays in which the 3¢UTR of the KSHV reactivation and transcriptional activator gene (RTA) was cotransfected with individual KSHV miRNA mimics. Further analysis of miRNA knockdown using antagomirs (sequence-specific miRNA inhibitors) in latently infected PEL cells showed that miR-K12-9* modulates RTA expression at the protein level (Bellare and Ganem 2009). Lu et al. also found that KSHV miR-K12-5 can inhibit RTA expression. However, this may reflect an indirect effect rather than direct targeting, since the 3¢UTR of RTA does not contain a favorable miR-K12-5 seed sequence (Lu et al. 2010). In summary, while the mechanisms by which herpesvirus-encoded miRNA function may vary (miRNA-like versus siRNA-like for antisense control), it becomes clear that all herpesviruses have evolved to utilize miRNAs as a means to regulate both lytic and latent gene expression. Future work using appropriate in vivo models will reveal how important these regulatory pathways are in the context of viral latency and persistence.

Open Questions and Future Perspectives The noncoding small RNAs and microRNAs expressed by EBV and KSHV are likely to play important roles in the pathogenesis of both viruses for the reasons

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outlined above. These RNAs are expressed in large amounts, are evolutionarily conserved in many cases, and are expressed in a wide variety of cell types and human cancers. The dispensability of the noncoding small RNAs of EBV and primate rhadinoviruses for in vitro transformation despite these characteristics strongly suggests important roles in either tumorigenesis or infected cell survival or immune evasion in vivo. The molecular mechanisms of action of these small RNAs remain elusive despite intensive investigation. The likelihood of EBERs playing a role in cell proliferation and apoptosis resistance makes identification of their cellular target pathways potentially important for devising novel therapeutic approaches for EBVrelated malignancies. Another important question is whether KSHV and EBV miRNAs directly contribute to viral tumorigenesis. A strong candidate for such a role in KSHV is miR-K12-11, a functional mimic of human miR-155, which was shown to have oncogene activity (Garzon and Croce 2008). This mechanism is conserved among viruses, since MDV-1, an avian tumorigenic virus, also encodes a miR-155 homolog (Morgan et al. 2008; Zhao et al. 2009) and EBV strongly upregulates miR-155 (Cameron et al. 2008). Very recently, Linnstaedt et al. (2010) have demonstrated that inhibition of miR-155 during immortalization can prevent the outgrowth of LCLs and even reduce proliferation of some EBV+ cell lines. Together, these data strongly suggest that modulation of this central pathway may be a central mechanism in g-herpesviral lymphomagenesis. Additionally, it has recently been discovered that miRNAs and EBERs can be secreted by exosomes; hence, the regulatory effects of viral-encoded small RNAs may not be limited to infected cells, but may also affect the tumor microenvironment (Iwakiri et al. 2009; Pegtel et al. 2010; Valadi et al. 2007). In addition to targeting pathways involved in apoptosis, proliferation, angiogenesis, and differentiation, KSHV and EBV miRNAs modulate innate immunity by targeting MICB (Stern-Ginossar et al. 2007) and the control of viral latent and lytic replication by targeting viral genes. While these additional functions are not directly related to tumorigenesis, they contribute to a hallmark of herpesvirus infection, which is lifelong persistence, a prerequisite of viral oncogenesis. Acknowledgments We thank Karlie Plaisance-Bonstaff for reading and editing the manuscript and generating the figures.

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Wang HW, Trotter MW, Lagos D, Bourboulia D, Henderson S, Makinen T, Elliman S, Flanagan AM, Alitalo K, Boshoff C (2004) Kaposi sarcoma herpesvirus-induced cellular reprogramming contributes to the lymphatic endothelial gene expression in Kaposi sarcoma. Nat Genet 36:687–693 Wang Y, Xue SA, Hallden G, Francis J, Yuan M, Griffin BE, Lemoine NR (2005) Virus-associated RNA I-deleted adenovirus, a potential oncolytic agent targeting EBV-associated tumors. Cancer Res 65:1523–1531 Weigel R, Fischer DK, Heston L, Miller G (1985) Constitutive expression of Epstein-Barr virusencoded RNAs and nuclear antigen during latency and after induction of Epstein-Barr virus replication. J Virol 53:254–259 Wolin SL, Cedervall T (2002) The La protein. Annu Rev Biochem 71:375–403 Xia T, O’Hara A, Araujo I, Barreto J, Carvalho E, Sapucaia JB, Ramos JC, Luz E, Pedroso C, Manrique M, Toomey NL, Brites C, Dittmer DP, Harrington WJ Jr (2008) EBV microRNAs in primary lymphomas and targeting of CXCL-11 by ebv-mir-BHRF1-3. Cancer Res 68:1436–1442 Xing L, Kieff E (2007) Epstein-Barr virus BHRF1 micro- and stable RNAs during latency III and after induction of replication. J Virol 81:9967–9975 Yajima M, Kanda T, Takada K (2005) Critical role of Epstein-Barr Virus (EBV)-encoded RNA in efficient EBV-induced B-lymphocyte growth transformation. J Virol 79:4298–4307 Yang L, Aozasa K, Oshimi K, Takada K (2004) Epstein-Barr virus (EBV)-encoded RNA promotes growth of EBV-infected T cells through interleukin-9 induction. Cancer Res 64:5332–5337 Yao Y, Zhao Y, Xu H, Smith LP, Lawrie CH, Sewer A, Zavolan M, Nair V (2007) Marek’s disease virus type 2 (MDV-2)-encoded microRNAs show no sequence conservation with those encoded by MDV-1. J Virol 81:7164–7170 Zhao Y, Yao Y, Xu H, Lambeth L, Smith LP, Kgosana L, Wang X, Nair V (2009) A functional MicroRNA-155 ortholog encoded by the oncogenic Marek’s disease virus. J Virol 83:489–492 Zhu JY, Pfuhl T, Motsch N, Barth S, Nicholls J, Grasser F, Meister G (2009) Identification of novel Epstein-Barr virus microRNA genes from nasopharyngeal carcinomas. J Virol 83:3333–3341 Ziegelbauer JM, Sullivan CS, Ganem D (2009) Tandem array-based expression screens identify host mRNA targets of virus-encoded microRNAs. Nat Genet 41:130–134

Chapter 32

Viral Malignancies in HIV-Associated Immune Deficiency Pankaj Kumar, Veenu Minhas, and Charles Wood

Introduction There has been a large volume of experimental evidence that supports the idea that immune system is capable of suppressing the growth of tumors. In fact, this concept of immune surveillance mechanism against cancer was proposed a long time ago by Paul Ehrlich. Clinical data that supports the importance of immune surveillance as a mechanism for tumor prevention has been generated largely by two sets of epidemiologic studies. First, studies that reported that long-term usage of immunosuppressive drugs following organ transplant is associated with higher risk for developing malignant tumors. Second, with the advent of human immunodeficiency virus (HIV) epidemic in the early 1980s, it was reported that HIV-positive patients had an increased risk for developing certain malignancies. As early as 1982, the US Center for Disease Control and Prevention (CDC) included Kaposi Sarcoma (KS) and primary central nervous system lymphoma (PCNSL) as AIDS defining malignancies (CDC 1982; Anonymous 1992). Non-Hodgkin lymphoma (NHL) and invasive cervical carcinoma were subsequently added as AIDS-defining conditions in 1987 (CDC 1987) and 1992 respectively (Anonymous 1992). The development of these AIDS defining malignancies was considered sufficient to signify the progression of HIV-infected patients to AIDS. Interestingly, malignancies found in organ transplant patients have a common unifying feature with malignancies found in the HIV patients; both generally have a viral etiology. Co-infection with other oncogenic viruses is considered a major risk factor associated with the development of malignancies in HIV-infected individuals. Although it is being debated whether HIV acts directly as an oncogenic agent or not, it is generally accepted that HIV provides a dysfunctional immunologic background

P. Kumar • V. Minhas • C. Wood (*) Nebraska Center for Virology, School of Biological Sciences, University of Nebraska-Lincoln, 4240 Fair Street, Morrison Center, Lincoln, NE 68583, USA e-mail: [email protected] E.S. Robertson (ed.), Cancer Associated Viruses, Current Cancer Research, DOI 10.1007/978-1-4614-0016-5_32, © Springer Science+Business Media, LLC 2012

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for other oncogenic viruses to escape immune surveillance and induce tumors. Other risk factors that are likely to play a role in the development of cancers in HIV-infected individuals include the duration of HIV immunosuppression, longevity, especially in those patients who are undergoing anti-retroviral therapy and therefore survive longer without the development of AIDS, family history of cancers, lifestyle, such as smoking and exposure to sunlight. In addition to AIDS-defining malignancies listed above, HIV-infected individuals have a higher incidence of non-AIDS defining tumors compared to the general population. These non-AIDS defining tumors include Hodgkin’s lymphoma, lung cancer, hepatocellular carcinoma, hematopoietic malignancies, cutaneous cancer, anal cancer, and various sarcomas other than Kaposi’s sarcoma. The focus of this chapter is to give an overview on major malignancies associated with viral etiology in the setting of HIV-associated immune deficiency.

AIDS-Defining and AIDS-Associated Malignancies AIDS-Associated Lymphomas AIDS-Associated Lymphomas (ARLs) are usually derived from B-cells as demonstrated by B-cell specific markers (such as CD19 and CD20) and rearrangement of immunoglobulin (Ig) heavy-chain gene (Carbone et al. 2001). They present an aggressive clinical course with widespread involvement of one or more extranodal sites. ARL is generally a late event in the course of HIV infection and prognosis is associated with the stage or extent of the disease, bone marrow involvement, age, severity of the underlying immunodeficiency (measured by CD4 lymphocyte count) and the history of opportunistic infections prior to AIDS diagnosis (Levine et al. 2000). The pathogenesis of ARL is probably multifactorial but ARL are very frequently associated with either Epstein-Barr virus (EBV) or human herpesvirus 8 (HHV-8) or both. These viruses are closely related members of the gamma herpes virus family. Abnormally high levels of IL-6 and IL-10 have been demonstrated in ARL. Both cytokines function as autocrine growth factors. It is noteworthy that all three viruses namely HIV, HHV8, and EBV have the ability to deregulate the cytokine network. The World Health Organization (WHO) has classified ARLs into three categories (Raphael et al. 2001); (a) lymphomas that also occur in immunocompetent patients and includes Burkitt’s lymphoma, diffuse large B-cell lymphomas (DLBCL) and Hodgkin’s lymphoma (HL); (b) lymphomas that occur more specifically in HIV+ patients such as primary effusion lymphoma (PEL) and plasmablastic lymphoma of the oral cavity and; (c) lymphomas that occur in other immunodeficiency states such as polymorphic B-cell lymphoma or post-transplant lymphoproliferative disorder (PTLD)- like B-cell lymphoma.

Burkitt’s Lymphoma Burkitt’s Lymphoma (BL) was first described as an obscure tumor in African children by Denis Burkitt almost 50 years ago (Burkitt 1958). Since its first description, BL

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has generated a lot of scientific interest because of its unique epidemiological features and has led to seminal discoveries including the discovery of Epstein–Barr virus (EBV), the first virus to be associated with a human cancer. BL is genetically characterized by a chromosomal translocation of the Myc gene to one of the three immunoglobulin loci leading to high level of c-Myc expression (Ballerini et al. 1993; Pelicci et al. 1986). Myc family of proteins are important transcriptional factors that globally regulate multiple cellular processes including proliferation, differentiation, and apoptosis. Three epidemiologically distinct forms are now recognized that differ in their geographic distribution, sites of involvement, and frequency of association with EBV (Diebold et al. 2001). The endemic form is present in areas of equatorial Africa and Papua New Guinea where malaria is very common. It occurs mainly in children and clinically involves the jaw or other facial bone although other organs can also be affected. Nearly all cases of endemic BL are positive for EBV. Elsewhere, BL occurs as a sporadic form affecting mostly the young adults. It usually presents as an abdominal mass in the ileocecal region but can involve other organs such as the endemic form. EBV is associated with about 20% of the sporadic cases. HIVassociated BL typically involves lymph nodes and bone marrow and has a high association with EBV (~30–40%) (Brady et al. 2007). Histological picture of BL consists of homogenous infiltration of medium-sized cells with round nuclei containing several nucleoli and very little basophilic cytoplasm. Mixed within the tumor cells are numerous benign macrophages with engulfed cell debris which confer a histologic pattern referred to as “starry sky” appearance. BL cells express pan-B markers (CD19, CD20 and CD22), are positive for surface IgM and Ig light chain and BCL6, but are negative for BCL2 and Terminal deoxynucleotidyl transferase (TdT), and have a high Ki67 proliferation index (Bellan et al. 2010). Microarray-based gene expression profiling studies have been employed to establish the molecular signature of BL that would discern BL from diffuse large B-cell lymphomas (DLBCL) (Hummel et al. 2006). Distinguishing BL from DLBCL is critical as both tumors require different treatment strategies. BL is rapidly fatal if untreated, but is curable with intensive chemotherapy that includes high doses of cyclophosphamide and antimetabolites, as well as intrathecal chemotherapy. On the other hand, treatment of DLBCL employs a less aggressive approach which typically consists of cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP), with the monoclonal anti-B-cell antibody, rituximab. Accurate diagnosis helps the clinicians to follow appropriate therapeutic regimen for treatment. Although considerable progress has been made in understanding the role of EBV in the transformation process, deciphering its exact role in the pathogenesis of BL is still a work in progress. Apart from the endemic form where the virus can be detected in virtually all cases; its association with other forms remains variable. This observation gives a strong indication that EBV may be a cofactor and not the initiating factor for tumorigenesis. Southern blot analysis of both: the c-Myc translocation and the fused terminal repeats of the EBV genome shows that the tumor cells are monoclonal and probably result from expansion of a single infected progenitor cell (Raab-Traub and Flynn 1986). Monoclonality of the tumors presents

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two possible scenarios; (1) EBV infection facilitates tumor initiation and argues for EBV being the etiologic agent or; (2) EBV infection merely facilitates the malignant progression by supporting unrestricted clonal expansion of B-cells that harbor a Myc/Ig translocation and EBV clonality is a consequence of selective growth advantage conferred by viral episomes. The second scenario is particularly interesting as it may explain that EBV’s role can be replaced by other factors in tumors where EBV is not detected, such as in sporadic BL (Moody et al. 2003). Primary infection with EBV can have diverse outcomes ranging from asymptomatic infection, self-resolving infectious mononucleosis, to the development of EBV-associated malignancies including Burkitt’s lymphoma, Hodgkin’s disease, nasopharyngeal carcinomas, and B cell lymphomas in transplant recipients or AIDS patients. EBV establishes lifelong latency in memory B cells by mimicking the normal behavior of B-cells in response to an antigen. The current accepted model for the establishment of EBV latency suggests that latent proteins expressed by EBV during primary infection provide signals to the infected B cell to become proliferating B-lymphoblasts (Young and Rickinson 2004). These activated lymphoblasts later differentiate into resting memory cells through a mechanism analogous to the germinal center reaction. Germinal centers are the sites within lymph nodes where antigen-activated B-lymphocytes undergo isotype switching and somatic mutation in their Ig genes to become memory B-cell. Isotype switching enables the B cell to replace the class of antibody expressed while somatic hypermutation increases the affinity of B cell receptor for an antigen. The latently infected memory cells express little or no genetic information and are therefore not recognized by the immune response. EBV displays distinct latency programs in various malignancies or EBV-derived cell lines which are defined by the specificity of latent gene expression (Young and Rickinson 2004). EBV positive cases of BL have a highly restricted pattern of latent gene expression, only expressing EBNA-1 and EBV-encoded small non-polyadenylated RNAs or EBERs (called latency-1). EBNA-1 plays a crucial role in the maintenance and replication of the viral genome (Rawlins et al. 1985) while EBERs possess anti-apoptotic activity and have been shown to promote the tumorigenic phenotype (Nanbo et al. 2002). However, recent studies report that some endemic cases of BL display alternate patterns of viral latency characterized by the expression of EBNA3 proteins in addition to EBNA-1 and EBERs (Kelly et al. 2006). It was suggested that tumor evolution ultimately selects these different forms of EBV latency that are compatible with high levels of pro-apoptotic c-Myc expression provides a more detailed discussion on EBV encoded latency genes and their contribution to B-cell transformation.

Diffuse Large B-Cell Lymphomas Diffuse Large B-Cell Lymphomas (DLBCL) is one of the most common lymphomas among ARLs accounting for about 70% cases. DLBCL are a heterogeneous group of non-Hodgkin lymphomas that typically present as a nodal or extranodal mass with fast tumor growth. Tumor cells are large with a nuclear size exceeding that of

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normal lymphocyte and display diffuse growth pattern that effaces the normal lymph node architecture. Based on their histologic features, DLBCL are classified into various subtypes that include centroblastic, immunoblastic, T-cell/histiocyterich, and anaplastic variants (Raphael et al. 2001). AIDS-related DLBCL is reported to occur in younger patients and display immunoblastic morphology more often than non-AIDS cases (Madan et al. 2006). Certain subtypes of DLBCL defy clear-cut classification based on histopathology reflecting the underlying clinical heterogeneity. In recent years, gene expression profiling (GEP) studies have contributed significantly toward classifying tumors based on differential patterns of gene expression. In a seminal study, Alizadeh et al. classified DLBCL into two major molecular subtypes that seem to have derived from distinct stages of B-cell differentiation: the germinal center B-cell (GCB) like and the activated B-cell (ABC) like (Alizadeh et al. 2000). The gene expression profile of GCB-DLBCL resembles to that of normal germinal center B-cells, whereas the gene expression profile of ABC-DLBCL shares similarity to mitogenactivated B-cells. The genes overexpressed in GCB-DLBCL include GC markers such as CD10, BCL-6, LMO2, A-MYB, and OGG1 while representative genes in the ABC-DLBCL include IRF4/MUM1, FLIP, cyclinD2, FoX-P1, and BCL-2. Interestingly, gene expression profile for GC or ABC subtypes of DLBCL does not correlate with any of the previously described histopathological subtype. EBV has been found in both subtypes of DLBCL, although less frequently in the GC subtype (Chadburn et al. 2009). These distinct gene expression profiles have a prognostic implication with some studies suggesting that patients of GCB subtype have a significantly better overall survival than those of ABC-DLBCL (Alizadeh et al. 2000; Rosenwald et al. 2002). Since AIDS-DLBCL have an aggressive course and poor clinical outcome compared to DLBCL, few studies have sought to identify the differentially expressed genes in AIDS-DLBCL versus DLBCL. Patrone et al. reported that gene expression patterns in AIDS-related DLBCL was very similar to non-AIDS related DLBCL with the exception of TCL1 (T-cell leukemia-1), a proto-oncogene that was highly expressed in AIDS-DLBCL (Patrone et al. 2003). However, findings from a recent report from Madan et al. suggest otherwise. They performed hierarchical cluster analysis using scores of immunohistochemical stains for GC differentiation and ABC markers on DLBCL from HIV-positive and HIV-negative patients and found that the immunophenotypic profile of AIDS-related DLBCL was different as it overlapped between the immunophenotypic profile of GC and ABC subtype of nonAIDS DLBCL suggesting a unique pathophysiology (Madan et al. 2006). Biological mechanisms underlying DLBCL pathogenesis are complex and probably involve multiple factors such as chronic antigen stimulation, immunosuppression, cytokine deregulation, and several genetic alterations. Using comparative genomic hybridization (CGH), several groups have identified distinct genetic alterations in different subgroups of DLBCL (Tagawa et al. 2005; Bea et al. 2005). EBV has been found to be associated more commonly with the immunoblastic variants (~90%) of AIDS related DLBCL than the centroblastic variants (~30%). EBV-encoded transforming protein LMP-1 is found in 90% of the immunoblastic cases but is not usually

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detected in the centroblastic subtype. LMP1 is an integral membrane protein that resembles constitutively active CD40, a glycoprotein belonging to tumor necrosis factor receptor (TNFR) (Uchida et al. 1999). LMP-1 has been found to be transforming in rodent fibroblasts (Wang et al. 1985). Transgenic mice expressing LMP-1 in B-cells have increased propensity to develop B-cell lymphomas (Thornburg et al. 2006). LMP-1 activates a number of signaling pathways including NFkB, JNK, and p38 pathways in a ligand-independent manner. LMP-1 also inhibits apoptosis in latently EBV-infected cells by upregulating the expression of antiapoptotic proteins BCL-2, BFL-1, and A20 while inhibiting the expression of pro-apoptotic BAX protein (Soni et al. 2007). Immunophenotyping using markers, BCL-6 and syn-1 have been used to define the histogenesis of AIDS-related DLBCL. BCL-6 is a zinc finger transcription repressor that is selectively expressed in B-cells located within GC while syn-1 is an integral transmembrane glycoprotein and is a marker of post-GC B-cell differentiation. The differential expression of BCL-6 and syn-1 segregate AIDS-related DLBCL into two major phenotypic subsets: BCL-6+/syn-1− and BCL-6−/syn-1+. These different patterns of expression basically correspond to two physiologic stages of B-cell development with B-cells within the GC displaying BCL-6+/syn-1 phenotype while terminally differentiated post-GC B-cells showing BCL-6−/syn-1+ phenotype. Interestingly, among EBV-infected AIDS-related DLBCL, the expression of EBVencoded LMP-1 antigen can be detected only in BCL-6−/syn-1+ phenotype which correspond to post GC state and these tumors display immunoblastic morphology. These findings suggested that the post-GC phenotype is the sole condition permissive for LMP-1 expression in the context of AIDS-NHL or alternately, that LMP-1 expression forces post-GC maturation of these lymphomas (Carbone et al. 1998a). Primary Central Nervous System Lymphomas (PCNSL) is a rare extranodal variant of DLBCL that is associated with the advanced stages of HIV infection. PCNSL was the most frequent brain tumor in AIDS patients till the introduction of highly active antiretroviral therapy (HAART) in mid 1990s (Sacktor et al. 2001). The confinement of the lymphoma within the CNS during the course of the disease despite the fact that CNS lacks lymphatic system adds an intriguing feature to the pathogenesis of PCNSL. It has been hypothesized that PCNSL originate from B-cells derived from systemic lymphoid tissues that normally traffic in and out of CNS. These B-cells are exposed to a germinal center environment outside the brain and later, a malignant clone expressing specific adhesion molecule metastasize to the brain (Uccelli et al. 2005). Experimental evidence seems to support this view as mutations in BCL-6 5¢ noncoding regions, which are regarded as a marker for B-cell transition through the GC, are frequently found in PCNSL (Larocca et al. 1998). Gene expression studies demonstrate that PCNSL exhibit a distinct expression profile compared to nodal lymphomas with an overlapping state of differentiation that is characterized by an expression of both GCB and ABC genes (Rubenstein et al. 2006). AIDS-related PCNSL is universally associated with EBV and frequently express EBV-encoded LMP-1 protein. PCNSL is found in patients with very low peripheral blood CD4 count. However, a recent study suggests that it is not the absolute CD4+ T-cell count but the lack of EBV-specific CD4+ T-cells that account

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for rapid progression in PCNS lymphomas in AIDS patients (Gasser et al. 2007). Detection of high levels of EBV DNA from the cerebrospinal fluid (CSF) of HIV-associated PCNSL has been shown to be a highly sensitive and specific marker for the diagnosis and monitoring of the disease (De Luca et al. 1995). Plasmablastic lymphoma of the oral cavity (PBL) is another variant of DLBCL that is predominantly found in HIV-positive patients. It was first described in the jaws and oral cavity of HIV-infected patients (Delecluse et al. 1997); although later it was also reported from other extra-oral sites including gastrointestinal tract, soft tissues, skin and heart of HIV-negative patients (Dong et al. 2005). The histopathological picture is characterized by the presence of large round/oval cells with abundant eosinophilic cytoplasm. The tumor cells have an eccentrically placed nucleus with a single prominent nucleolus that is located in the center. The tumor cells have a terminally differentiated B-cell immunophenotype and are typically positive for plasma cell markers such as CD38 and CD138. Although PBL is considered to be of differentiated B-cell lineage, they are negative for other common B-cell markers such as CD20, CD79a, and PAX-5 (Vega et al. 2005). Monoclonal rearrangement of immunoglobulin heavy chain and positivity for post GC B-cell marker multiple myeloma-1 (MUM1)/IRF4 are indicative of their origin from post-GC (Gaidano et al. 2002). An array-based comparative genomic hybridization of PBL indicates that these tumors are more similar to DLBCL than the plasmacytomas (Chang et al. 2009). While most PBL are positive for EBV, the prevalence of HHV8 remains somewhat unresolved.

Hodgkin’s Lymphoma Although not classified as an AIDS-defining malignancy, HIV-related HL is one of the most common non-AIDS defining condition. Several studies have reported that HIV-infected patients have a 3–18-fold increased risk of developing HL compared to the general population (Biggar et al. 2006; Engels et al. 2008). The presence of large mononucleated (Hodgkin) and multinucleated (Reed Sternberg) neoplastic cells (HRS) within a reactive inflammatory cell background are the defining characteristic of HL. HL is unique among lymphomas because the neoplastic component i.e., the HRS cells constitute <1% of the tumor mass (Brauninger et al. 2006). HL tends to manifest more obvious clinical symptoms than non-Hodgkin’s lymphoma (NHL) which often leads to early detection and favorable treatment. HL is divided into two major entities that differ in their clinical and histopathologic features: more commonly found (about 95%) classical HL (cHL) and not so frequent, nodular lymphocyte-predominant HL (NLPHL). CHL has been classified into four morphological subtypes: nodular sclerosis, mixed cellularity, lymphocyte rich, and lymphocyte depleted (Stein 2001). There is considerable geographical and age group variation within different subtypes of HD, e.g., young adults in the United States and Europe are likely to have the nodular sclerosis variant, while mixed cellularity or the lymphocyte-depleted subtype is more frequent in the developing world (Cartwright and Watkins 2004). Most cases of HL in HIV patients are either

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of mixed cellularity or the lymphocyte-depleted forms. In the United States, HL follows a bimodal age distribution showing a peak at young age and second peak in late life. Also white young adults with a history of infectious mononucleosis have a higher incidence of HL. The exact origin of the HRS cells has long been a controversial topic as these cells display a phenotype that is not consistent with a defined hematopoietic cell type. The HRS cells express CD15 and CD30 but are negative for CD45 and T-cell markers (Schmid et al. 1991). CD30 is a membrane glycoprotein of the TNF receptor family that is expressed in activated T and B cells. The B-cell markers, CD20, CD79A, and B-cell-specific activator protein (BSAP) are variably expressed in a subset of cases (Schmid et al. 1991). HRS cells show clonal immunoglobulin gene rearrangement and thus support the idea that they represent clonal populations of GC-derived tumor B cells. In an elegant study, Cossman et al. performed high throughput gene expression studies from individual Reed–Sternberg cells isolated from HL. This expression profile, closely resembled to that of GC B-cells and not with dendritic cells, thus corroborated the generally held view that HRS cell is of B-cell lineage (Cossman et al. 1999). A hallmark of HRS cells is the lack of a functional B-cell receptor (BCR) (Marafioti et al. 2000). In a subset of cHLs, this lack of a functional BCR has been attributed to “crippling” somatic mutations of the rearranged immunoglobulin gene (Kanzler et al. 1996). However, in other cases of cHL with functional gene rearrangements, the absence of Ig has been ascribed to downregulation of B-cell specific transcription factors (OCT2, BOB1 and PU1) (Theil et al. 2001) or epigenetic silencing of immunoglobulin gene transcription by promoter methylation (Ushmorov et al. 2004). Normally, antigen-activated B-cells undergo somatic hypermutation of the BCR in GC to generate B cells with higher affinity BCRs. These high affinity B-cells are preferentially selected for survival and undergo clonal expansion and differentiation into antibody-producing plasma cells. B cells in GC that lack B cell-receptor expression are eliminated by apoptosis. The HRS cells are an exception to this general rule as they continue to proliferate in spite of having deleterious mutations which suggests that somehow they are rescued from apoptosis by a transforming event. Several different mechanisms have been proposed to explain how HRS cells avoid apoptosis including genetic alterations of the bcl-2 family proteins, constitutive activation of the NF-kB pathway and expression of EBV latent genes (Brauninger et al. 2006). Aberrant signaling of NF-kB pathway has generated considerable interest. NF-kB family of transcription factors plays a critical role in the regulation of apoptosis by upregulating anti-apoptotic members of the Bcl-2 family and caspase inhibitors such as XIAP and FLIP. NF-kB is constitutively active in HRS cells independent of the EBV status and several factors have been found to contribute toward deregulated NF-kB pathway (Krappmann et al. 1999). These factors include frequently found mutations that inactivate NF-kB inhibitor kappa beta (IkB) alpha and epsilon subunits, gene amplification of c-rel, signaling through tumor necrosis factor receptors (CD30 and receptor activator of NF-kB (RANK), and expression of LMP1 and LMP2A in EBV-positive cases (Kuppers 2009). In addition, NF-kB-dependent upregulation of cellular

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FLICE-inhibitory protein (c-FLIP) has been suggested to mediate resistance of HRS cells to death receptors (CD95/TRAIL) induced death (Mathas et al. 2004). Although EBV is not detected in all cases of HL, several lines of evidence favor the idea that EBV has some role in the pathogenesis of HL. In the US, EBV can be frequently detected in mixed cellularity variant of cHL but rarely in other subtypes. Strikingly, all cases of HIV-related HLs are positive for EBV (Bellas et al. 1996). The association between EBV and HL has been demonstrated by numerous seroepidemiological studies in which antibody titers against viral capsid antigens (VCAs) have been consistently found to be higher in HL cases compared to the general population (Besson et al. 2006). The definitive proof revealing an etiologic association of EBV with HL was the detection of the viral genome and virally encoded proteins in the HRS cells (Weiss et al. 1989). EBV has been found to transform primary human B lymphocytes including GC B cells that are incapable of expressing a functional immunoglobulin heavy chain due to crippling mutations introduced during somatic hypermutation (Mancao et al. 2005). HRS cells positive for EBV display a type II form of latency with viral antigen expression limited to EBNA1, LMP1, LMP2, as well as the EBER1, EBER2, and BamHI A transcripts (Deacon et al. 1993). The transforming potential of LMP-1 has been described earlier. Unlike LMP-1, LMP2 is not required for transformation in B-cells. It is an integral membrane protein with two isoforms, LMP2A and LMP2B. LMP2A can constitutively induce several signaling cascades. The LMP2A amino-terminal cytoplasmic domain contains an immunoreceptor tyrosine-based activation motif (ITAM) which is a conserved 6-amino acid sequence present in a number of proteins that serve as signal transducers for immunoreceptors. LMP2A functionally acts as a BCR mimic in vivo by providing constitutive signaling required for B-cell development and survival (Mancao and Hammerschmidt 2007). This was supported by studies using transgenic mice that expressed LMP2A in cells of B cell lineage. The study reported that B-cells with nonfunctional BCRs that are normally eliminated by apoptosis were rescued by the expression of LMP2A (Caldwell et al. 1998). A follow-up study showed that oncogene Ras is constitutively active in peripheral; BCR-negative B cells from LMP2A transgenic mice (Portis and Longnecker 2004). By contrast, the function of LMP2B, a variant of LMP2A, is still not resolved in the pathogenesis of EBV infections.

Primary Effusion Lymphoma Primary Effusion Lymphoma (PEL) is one of the least common of the ARLs, accounting for less than 1–4% of cases (Simonelli et al. 2003). PELs are almost universally associated with KSHV infection and are found in patients with advanced stages of AIDS. The prognosis of PEL is poor with a median survival of less than 6 months. PELs are recognized as clinical subtype of DLBCL where neoplastic cells proliferate within major body cavities (pleural, peritoneal and pericardial), typically without the formation of a grossly visible mass. However, a solid variant that does not involve body cavities has also been recognized (Carbone et al. 2005). Mouse model studies have shown some differences in the gene expression profile of both variants with the solid tumors more likely to express adhesion molecules and structural

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proteins (Yanagisawa et al. 2006). In most cases, EBV coinfection is very frequent. Although the pathogenetic role of EBV in PELs is not known, microarray studies have shown differences in gene expression patterns between the EBV-positive and EBV-negative cases (Fan et al. 2005). Morphologically, PEL cells are large with abundant basophilic cytoplasm and exhibit either immunoblastic (round nuclei with central prominent nucleoli) or plasmablastic (eccentric nuclei with abundant cytoplasm) or anaplastic (very large round cell with pleomorphic nuclei) features. PELs have aberrant immunophenotypic characteristics with positive reactivity for CD45 (Leukocyte common antigen) but reactivity for B-cell lineage markers like immunoglobulin (Ig), CD19, CD20, and CD79a or T-cell specific markers like CD3, CD4, CD8 is absent (Nador et al. 1996). Activation (CD30, CD38) and plasma cell markers (CD138, MUM-1/IRF4) are typically present. PELs display rearrangements in immunoglobulin genes at the genetic level suggesting that they are derived from the B-cell lineage (Matolcsy et al. 1998). Gene expression profile analysis shows that PEL cells exhibit a gene expression pattern that shares features of both immunoblasts and plasma cells, i.e., a plasmablastic profile (Klein et al. 2003). Nuclei of PEL cells stain positive for KSHV LANA protein by immunohistochemistry, which is the standard assay to establish diagnosis for KSHV infection. Although PEL cells are positive for EBV (as detected by in situ hybridization for EBERs) in vast majority of cases, they are negative for LMP1 expression (Horenstein et al. 1997). The exact mechanism by which KSHV and EBV promote tumorigenesis in PEL is not clear. KSHV is present in PEL cells as episomes and majority of the infected cells express a latent pattern of gene expression; however, a small percentage (<3%) of cell populations spontaneously undergo lytic reactivation (Renne et al. 1996). PEL cell lines derived from PEL have been used extensively as a model to study KSHV biology. Unlike KS tumor explants, PEL cell lines stably maintain viral episomes at high copy numbers (50–150 copies per cell) (Ballestas et al. 1999). Among the latently expressed genes, LANA-1, viral cyclin (v-Cyc), viral FLICE inhibitory protein (v-FLIP), and LANA-2/viral interferon regulatory factor 3 (vIRF3) are consistently expressed (Luppi et al. 1999). Another KSHV protein that is expressed in a minority population of PEL tumor cells is viral interleukin-6 (vIL-6), a cytokine that shares 24.7% amino acid identity with human IL-6. Studies have shown that vIL-6 contributes some of the autocrine growth factor activity in PEL cell lines suggesting that vIL-6 may help KSHV persist and spread in the human host (Jones et al. 1999). Inoculation of NIH3T3 cells expressing vIL-6 into nude mice resulted in the development of tumors more rapidly than the control cells. Moreover, vIL-6-positive tumors were more vascularized than controls and expressed increasing levels of vascular endothelial growth factor (VEGF) expression (Aoki et al. 1999). Chromosomal translocations seen in other non-Hodgkin’s lymphomas like those involving BCL2, BCL6, and Myc loci are consistently absent in PEL (Carbone et al. 1998b). A recent study investigated the genomic profiles of 28 PELs and ten PEL cell lines using comparative genomic hybridization (CGH) and identified selectin-P ligand (SELPLG) and coronin-1C (CORO1C) as the targets of amplification at chromosome 12 (Luan et al. 2010). SELPLG is critical for cell migration and chemotaxis, while CORO1C regulates actin-dependent processes and is important

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for cell motility. Overexpression of these two genes in PELs may play an important role in its pathogenesis.

Kaposi’s Sarcoma Kaposi’s sarcoma (KS) is a complex, angiogenic tumor characterized by angiogenesis, infiltration of inflammatory cells, and the proliferation of spindle-shaped cells, which are the hallmark cells of this neoplasm. KS lesions appearing in young homosexual men in New York and San Francisco were one of the first indications of what later became the AIDS epidemic. KS is one of the three AIDS-defining malignancies. It was originally described by Moritz Kaposi in 1872 as an idiopathic, multipigmented sarcoma of the skin (Kaposi 1872). Since then, four clinical variants of KS (classic, endemic, iatrogenic, and AIDS-associated), which most likely represent different manifestations of the same pathologic process, have been described. The histological features of all these variants are indistinguishable and after the discovery of Kaposi’s sarcoma-associated herpesvirus (KSHV) in 1994, it became clear that this virus is associated with all variants of KS. Since then, various diagnostic assays have been employed to understand the epidemiology of KSHV. It has been found that seroprevalence of KSHV is uneven and mirrors the incidence of KS. Conclusive data on transmission of KSHV are still lacking but both horizontal (sexual and non-sexual) and vertical modes of transmission have been reported. Saliva is emerging as one of the important routes of transmission because the virus is shed frequently from the oropharynx (Brayfield et al. 2004; Duus et al. 2004). Classic KS is a rare disease and the geographic location, ethnicity, age, and gender heavily influence the incidence rate (Iscovich et al. 2000). This is the least aggressive form of KS. It manifests as a slow or non-progressing skin condition of the limbs and occurs primarily among older men of Mediterranean and Jewish descent (Iscovich et al. 2000; Reynolds et al. 1965). The male to female ratio has been reported to be 15:1 with age of onset usually greater than 50 years of age and rarely (4–8%) prior to 30 years of age (Biggar et al. 1984; Dictor and Attewell 1988; Geddes et al. 1994). The incidence of classic KS in North America, Northern Europe, and Asia is low (Iscovich et al. 1998a, b, 2000; DiGiovanna and Safai 1981; Guttman-Yassky et al. 2006). Endemic KS was identified in parts of Central Africa, where the highest incidence is observed in a belt-like path stretching from Cameroon, the former Zaire, to Uganda and downward through Tanzania, north and eastern Zambia, Zimbabwe, and northern South Africa (Cook-Mozaffari et al. 1998). It is recognized to be one of the most common neoplastic diseases in equatorial Africa (Lothe and Murray 1962; Oettle 1962; Olweny 1984). The male to female ratio is similar to that of classic KS and a mean age of onset of 48 years but the lymphadenopathic form is seen mostly in prepubertal children with no sex predilection. Rapid visceral involvement can cause early death and skin lesions are sparse (Friedman-Kien and Saltzman 1990).

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Both the iatrogenic and AIDS-associated forms are linked to overt immune suppression. The iatrogenic form of KS was recognized in 1960s and 1970s following advances in transplantation medicine. With the introduction of immunosuppressive regimens used to prevent graft rejection, post-transplant patients developed disseminated KS as early as 2 months and up to 8 years after initiation of therapy (Friedman-Kien and Saltzman 1990). This form of KS often resolves when immune-suppressive therapy is stopped, calling attention to immune deficiency as an etiologic cofactor. Among transplant recipients, KS develops mostly in those with preexisting HHV-8 infection. Seropositive recipients have been reported to have a 28–75 fold higher KS risk than seronegative recipients (Mbulaiteye et al. 2006a; Parravicini et al. 1997). This type of KS is aggressive and involves lymph nodes, mucosa, and visceral organs in about half of the patients, sometimes even in the absence of skin lesions (Antman and Chang 2000). At the beginning of the AIDS epidemic, the risk of developing KS among homosexual men with HIV infection approached 50%, and approximately 40% of the patients who were diagnosed positive for AIDS also presented with KS lesions (Biggar and Rabkin 1996). AIDS-associated KS is a very aggressive form that often disseminates to visceral organs (Beral et al. 1990). It has been noted that among HHV-8/HIV coinfected males, the 10-year risk of developing KS is 39–50% (Martin et al. 1998; O’Brien et al. 1999). In Africa, the ubiquitous presence of the virus in the pre-HIV era, combined with the HIV epidemic has led to a dramatic increase in KS incidence (Feller et al. 2010). In developed countries, AIDS-KS incidence decreased after HAART introduction in 1996, probably due to enhanced immune reconstitution and perhaps a direct effect of protease inhibitors as anti-angiogenic factors. There are three distinct histologic stages of KS; the patch, plaque, and nodular forms. Though KS is most commonly localized to the skin, organ involvement can occur and is common in AIDS-associated KS (Wong and Damania 2005). KS is not a conventional neoplasm. In KS lesions, proliferation, inflammation, and angiogenesis seem to occur in parallel. This is markedly different from other cancers where proliferation precedes inflammation and neoangiogenesis (Ganem 2010). Also, KS lesions are comprised of several cell types including spindle cells, infiltrating inflammatory cells as well as extravasated erythrocytes. The percentage of spindle cells in KS lesions increases with the stage of the disease. Most researchers believe that the spindle cells originate from endothelial lineage as they express many markers of the endothelial lineage including CD31 and CD34; however, several reports show that spindle cells also express markers for smooth muscle cells, macrophages, and dendritic cells (Huang et al. 1993; Nickoloff and Griffiths 1989; Weich et al. 1991). A report published by Dupin et al. (1999) showed clearly that VEGFR-3, which is restricted to lymphatic endothelium of adult tissues, was extensively expressed in early KS. Additionally, spindle cells cannot be stained for VW/PalE and eNOS (specific markers for blood vessels). Together, these results suggested that spindle cells are lymphatic in origin. Recent large-scale gene expression studies have revealed that KSHV infection induces dramatic reprogramming of the endothelial cell transcriptome with induction of lymphatic lineage-specific genes, including

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PROX1, a master regulator of lymphatic development; and downregulation of blood vascular genes (Carroll et al. 2004; Hong et al. 2004; Wang et al. 2004). Like other herpes viruses, KSHV replication alternates between two phases: latent and lytic. The latent phase is characterized by a restricted pattern of viral gene expression that facilitates the evasion of immune surveillance. In the late stages of KS, essentially all spindle cells are latently infected by KSHV and express latent genes. The latency locus is comprised of three open reading frames (ORFs) which includes latency-associated nuclear antigen (LANA), viral cyclin (v-cyclin), and viral FLICE inhibitory protein (v-FLIP), all controlled by a single promoter. LANA is a chromatin-interacting transcriptional factor that has been found to be essential for viral episome maintenance in proliferating cells. It tethers the viral genome to the host chromosomes assuring their segregation to daughter cells during mitosis. LANA modulates the cellular transcription program by altering the functions of various transcription factors. It can also inhibit tumor suppressor genes such as Rb, p53, and VHL leading to impaired apoptosis and activation of genes involved in angiogenesis, cell proliferation, and survival. v-Cyclin, a viral homolog of the cellular cyclin D associates with cyclin-dependent kinases (CDKs) and promotes cell-cycle progression by phosphorylating specific target proteins such as pRb and Histone H1 (Godden-Kent et al. 1997). v-FLIP is a potent anti-apoptotic effector due to its ability to activate NF-kB and therefore, has been associated with cell survival, morphologic changes, and inflammatory activation. As mentioned earlier, majority of the tumor cells in KS are latently infected; however, a small number of KS tumor cells also undergo lytic infection. The lytic phase drives the replication cycle and majority of viral genes are expressed in this phase. This phase mainly allows for the spread of the virus in the infected individual. Since the cells with lytic viral replication will ultimately die, it was safely assumed that lytic genes are not likely to play a significant role in oncogenesis. However, emerging evidence from several laboratories supports a key role for many of these lytic proteins which have key regulatory functions. Among the lytic genes, the G-protein-coupled receptor (vGPCR) is considered an important candidate responsible for the initiation of KS. vGPCR is a constitutively active member of the family of CXC chemokine G-protein-linked receptors and its expression has been found sufficient to induce cell transformation and VEGF-mediated angiogenesis in NIH3T3 cells (Bais et al. 1998). Transgenic mice expressing vGPCR within hematopoietic cells develop angioproliferative lesions in multiple organs that morphologically resemble KS lesions (Yang et al. 2000). Another lytic gene that may contribute to KSHV tumorigenesis is viral homologue of human IL6 (vIL6) which has been described earlier. Immune suppression is a major force in the development of KS as observed during HIV infection and in transplant patients. But the mechanism associated with this phenomenon is still being debated. HIV-associated KS patients present a more aggressive clinical course than that of other immunosuppressed patients, suggesting that HIV contributes more than just providing the immune-deficient state. Several studies support a role of HIV-encoded tat (Trans Activating Factor) protein as a cofactor in the pathogenesis of AIDS-associated KS (Aoki and Tosato 2004; Ensoli et al. 1990). HIV tat is a small polypeptide comprising of 86–101 amino acids that

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functions as a potent transactivator (Romani et al. 2010). Tat has been shown to induce the expression of several cytokines and angiogenic factors including IL-6, IL-10, and vascular endothelial growth factor receptors 1 and 2 (VEGFR1 and 2), which play an important role in KS progression. Tat is released from HIV-1 infected cells and Tat-containing supernatants promote the growth of KS infected cells (Ensoli et al. 1990). Overexpression of tat gene in transgenic mice results in the formation of skin lesions that closely resemble KS (Vogel et al. 1988). Tat can induce KSHV replication in lymphoma cells that are infected with KSHV (Harrington et al. 1997) and recent studies demonstrate that Tat activates KSHV lytic replication in part by modulating Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling (Zeng et al. 2007). Tat has also been implicated in promoting KSHV transmission by enhancing the entry of KSHV into endothelial and other cells (Aoki and Tosato 2004). These studies highlight the importance of cross talk between HIV-1 and KSHV in the pathogenesis of KS-associated malignancies.

Cervical Cancer Cervical cancer is the second most common cancer among women worldwide, with an estimated 529,409 new cases and 274,883 deaths in 2008 (IARC, GLOBOCAN 2008). Cervical cancer kills more women in the developing world than any other cancer. More than 80% of these cases occur in developing countries with highest incidence rates recorded in Sub-Saharan-Africa. In developed countries, the incidence and mortality rates due to cervical cancer have been declining over the years due to widespread screening and intervention. Over the past 10 years, it has been clearly established that persistent infections with oncogenic human papilloma virus (HPV) types is causally associated with almost all cases of cervical cancer. This association is supported by epidemiological studies and the detection of viral DNA from nearly all cases of cervical cancers worldwide (Bosch et al. 2002). However, HPV is not a sufficient cause of cervical cancer; other cofactors including smoking, multiple pregnancies, and immunosuppression may also contribute to the development of cervical cancer (Baseman and Koutsky 2005). Sexual intercourse is the primary route of transmission for HPV and sexual promiscuity is the most important risk factor associated with acquiring genital HPV infections (Kataja et al. 1993). HPV is most common in young women in their late teens and early 20s. Dunne et al. reported that in United States, the prevalence of HPV infection in women increases from 14 years through 24 years, and then decreases at older ages (Dunne et al. 2007). HPV is a member of the Papillomaviridae family of non-enveloped DNA viruses. These viruses are associated with a variety of cutaneous and mucosal conditions ranging from benign warts to several oropharyngeal and genital neoplasms. The virus specifically infects the basal cell layer of the squamous epithelium that is exposed as a result of minor abrasions. HPV infections are often subclinical and transient, resolving spontaneously over time, 90% cases clearing the infection within 2 years (Ho et al. 1998). Long-term viral persistence is required for the malignant progression of cervical cancer. Most cervical cancers originate at the transformation zone,

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an area of the cervix where columnar epithelium changes to squamous epithelium by a process called squamous metaplasia. Cervical Intraepithelial Neoplasia (CIN)/ Squamous Intraepithelial Lesions (SIL) are commonly used clinical terms that describe precancerous lesions or the abnormal growth of squamous cells observed in the cervix. Histologically, 90–95% of the cervical cancers are squamous cell carcinomas, while adenocarcinoma constitutes less than 5% of cervical cancers. HPV genome consists of approximately 8 Kb of double-stranded, circular DNA. More than 100 different HPV types have been classified on the basis of their nucleotide sequence, of which about 40 infect the genital tract. Based on their oncogenic potential, these viruses are grouped into high risk, intermediate risk, and low risk types. Types 16 and 18 are considered high-risk (oncogenic) types and are associated with aggressive forms of cervical cancers throughout the world (Clifford et al. 2003; Munoz et al. 2003). HPV 16 is detected more often in squamous cell carcinomas whereas HPV 18 is frequently detected in adenocarcinomas. Infection with low-risk HPV subtypes such as 6, 11, and 42 is usually associated with benign warts (Lorincz et al. 1992). Some studies have shown that there are geographical variations in HPV type distribution, e.g., in Sub-Saharan Africa, the high risk HPV types associated with high grade SIL (HSIL) in HIV-infected women include HPV 52, 58, 53, 35, and 45 in addition to types 16 and 18 (Blossom et al. 2007; Sahasrabuddhe et al. 2007). For high risk HPV types, early viral gene products, E6 and E7, play critical roles in the transformation of epithelial cells (Hawley-Nelson et al. 1989). These proteins bind to and inactivate a number of cellular proteins involved in regulating cell cycle progression and apoptosis. One of the best characterized function of E6 protein is its ability to promote the degradation of p53, an important tumor suppressor that regulates G1/S and G2/M cell cycle check point in response to DNA damage. E6 does so by interacting with a cellular protein E6-AP to form a ubiquitin ligase that targets p53 for degradation via the proteosome (Scheffner et al. 1993). Another major function of E6 important for immortalization is to activate telomerase via up regulation of hTERT expression (McMurray and McCance 2003). Furthermore, the high-risk E6 protein have been shown to interact with a number of other cellular proteins, such as the focal adhesion protein paxillin, p300/CBP, BAK12, IRF-3, and PDZ domain proteins such as hDLG, MUPP-1, MAGI-1, and hScrib. These interactions contribute to HPV-induced cellular transformation (Narisawa-Saito and Kiyono 2007). E7 binds to members of the Rb tumor suppressor protein family and inhibit their ability to modulate the function of E2F transcription factors, leading to constitutive activation of S phase genes (Dyson et al. 1989). The HPV-16 E7 protein has been shown to interact with HDACs, and this interaction occurs through an intermediary protein called Mi2ß. This binding is independent of E7’s interaction with Rb and has been found to be important for the maintenance of HPV episomes in undifferentiated keratinocytes (Brehm et al. 1999). The E6 and E7 protein derived from low-risk types have been shown to have impaired ability to interact with cellular proteins thus partially explaining their non-transforming nature (Heck et al. 1992). The productive life-cycle of HPV is intimately tied to epithelial cell differentiation (Longworth and Laimins 2004). On primary infection, the virus maintains its genome as a low copy episomal DNA within the nuclei of the infected basal cells.

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As basal cells divide, daughter cells migrate to the upper layers of the epithelium and differentiate. Normally the cells exit the cell cycle as differentiation progresses; however, HPV infected cells fail to exit the cell cycle and continue to support DNA synthesis. The contribution of viral oncogenes E6 and E7 in creating a cellular environment favorable for viral DNA replication has briefly been discussed before. During carcinogenic progression of cervical lesions, HPV DNA often integrates into cellular chromosomes; an event that has been shown to positively correlate with the severity of the lesions (Jeon et al. 1995). Whether the integration event is essential for carcinogenesis is a matter of debate. However, it has been noticed that viral genome integration occurs downstream of the early genes E6 and E7, often resulting in the disruption of E2 gene, a transcription factor that regulates viral transcription. Loss of E2 is thought to relieve repression of early promoter resulting in increased expression of transforming E6 and E7 proteins (Pett and Coleman 2007). HPV integration sites are randomly distributed over the whole genome with preferences for relatively few loci (Wentzensen et al. 2004). There is no evidence to support the targeted disruption of a critical cellular oncogene by integrated viral genome. Although cervical cancer has been listed as an AIDS-defining condition by CDC since 1993, numerous studies have failed to consistently demonstrate high incidence of cervical cancer among HIV-infected women. The incidence of cervical cancer never reached epidemic proportions in the pre-HAART era like other AIDS-associated malignancies (KS and NHL) in women with AIDS, even though both HIV and HPV share the same epidemiological risk factors (being sexually transmitted infections). The association between HIV and cervical cancer varies considerably from country to country. Studies conducted in Africa, where both HIV and cervical cancer are endemic, have found a weaker association between the two than the data reported from Europe or America (La Ruche et al. 1998; Mbulaiteye et al. 2006b; Sitas et al. 1997). It is likely that progression of HPV infection to cervical cancer, which can take years, might exceed the mean survival time in developing countries. Also patients in developing countries have higher background rates of cervical cancer in general population as they usually do not have access to routine screening and timely treatment thus making an inconclusive link between cervical cancer in HIVpositive and HIV-negative patients. Data from several AIDS-cancer registries match studies in the United States have reported fivefold or greater risk of cervical cancer in patients with AIDS compared to the general population (Frisch et al. 2000, 2001). However, it is not entirely clear whether HIV increases the oncogenicity of HPV types or how HIV alters the natural history of HPV infection. It seems plausible that diminished HPV-specific immune response due to HIV infection may allow for viral persistence and subsequent progression to cervical cancer. The contribution of HIV-induced immunosuppression to the increased risk of HPV infection and CIN is poorly understood. Recent studies by Bigger et al. and Chaturvedi et al. have reported that the relative risk for cervical cancer was not significantly associated with low CD4 T-cell count at the time of AIDS onset and did not increase with increasing time after AIDS onset suggesting that there is no relationship between the incidence of cervical cancer and HIV-induced immunosuppression (Biggar et al. 2007; Chaturvedi et al. 2009). Although increasing risk for

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in situ carcinoma, a precursor for cervical cancer was found to be associated with increasing time after AIDS onset. This is consistent with several studies that have documented that HIV-seropositive women are at increased risk of low-grade cytological abnormalities e.g., CIN1 (Harris et al. 2005; Strickler et al. 2005). It has been suggested that lower CD4 count (reflective of immunosuppression) levels may promote early stages of cervical dysplasia through poorly controlled HPV infection while later events that are involved in the progression of malignant state seem not to be related to immune status (Biggar et al. 2007). Strickler et al. (2003) investigated the prevalence of high-risk HPV16 in HIV-seropositive women and found that its detection was not greatly affected by lower CD4 counts suggesting that HPV16 may be more efficient at evading immune surveillance than other HPV types, contributing to its strong association with cervical cancer (Strickler et al. 2003). Some studies have addressed the possibility of a direct interaction between HIV and HPV which in theory could result in enhanced HPV-induced pathology. The HIV-encoded tat protein has been shown to directly increase gene expression of HPV 16 E6 and E7 genes in infected keratinocytes (Vernon et al. 1993). Similar experiments showed that tat protein could increase HPV-18 E1 and L1 protein expression (Dolei et al. 1999).There is little knowledge about the immune factors that influence the progression of CIN to cancer in HIV-infected women. Nicol et al. analyzed the local cytokines milieu from cervical biopsies of HIV/HPV co-infected women and found that there was overexpression of the cytokine, RANTES, member of the interleukin-8 super family of cytokines suggesting that cytokines may have an important role in the pathogenesis of HIV/HPV co-infection (Nicol et al. 2008).

HIV Pathogenesis and Immune Suppression Depletion of CD4+ T cells represents a fundamental event in HIV pathogenesis and is generally associated with HIV-induced immunodeficiency. However, in recent years, it has been increasingly recognized that HIV-associated immune suppression is not primarily due to inactivation of the immune system, but rather due to chronic activation, rapid turnover, and activation-induced cell death. At first, immune activation may seem like a compensatory mechanism to achieve T cell homeostasis after virusmediated CD4+ T cell destruction; however, several studies demonstrate that immune activation is directly responsible for increased proliferation and death of CD4+ and CD8+ T cells (Hazenberg et al. 2000; Sousa et al. 2002). In order to understand how immune activation leads to defects in immune surveillance and the reactivation of other co-infecting oncogenic viruses, one must first have a better understanding of the natural history of HIV infection and the underlying immunopathology. The natural history of HIV infection has been well studied and reviewed extensively (Cadogan and Dalgleish 2008; McMichael et al. 2010; Mogensen et al. 2010) (Fig. 32.1). Exposure to infected bodily fluids through sexual contact or sharing needles is the primary route HIV enters the body. The initial exposure of the virus is followed by an acute phase of infection, which is defined as the period from the

836

P. Kumar et al. Acute HIV syndrome Dissemination of virus to secondary lymphoid organs

Clinical latency

Development of AIDS associated malignancies and opportunistic infections

Immune activation Circulating CD4+ T cells

HIV load Immune response Mucosal CD4+ T cells

Acute (5-10 Wks)

Chronic (1-15 years)

AIDS

Fig. 32.1 The course of natural HIV-1 infection and kinetics of immune activation

initial detection of viral RNA until the presence of HIV-specific antibodies. Acute phase usually range from days to several weeks after infection. Prior to the acute phase and shortly after initial exposure of the virus via sexual route, there is a brief eclipse phase where HIV first replicates locally in the vaginal or rectal mucosa before any viral RNA can be detected in the plasma. It has been demonstrated in the SIV model that the cells initially infected are resident memory T cells in the mucosa. As a result of innate immune response, granulocytes, macrophages, and lymphocytes are recruited to the initial site of infection. Macrophages are among the first cells infected by HIV. In contrast to T cells, they are more resistant to the cytopathic effects of HIV and thus are believed to be the principal reservoirs for long-term infection (Salazar-Gonzalez et al. 2009; Veazey et al. 2003). Dendritic cells (DC) located beneath the mucosal surfaces are also involved in HIV transmission. HIV exploits the physiological migration properties of DCs to gain access to CD4+ T cells in lymph nodes. Some subsets of mucosal DCs expressing DC-SIGN lectin receptor bind gp120, the viral envelope, with high affinity. These DCs facilitate the initial establishment and spread by carrying the virus from mucosal sites of exposure to the lymphoid tissues and transferring infection to CD4+ T cells in the lymph node. This further allows the virus to replicate and disseminate to secondary lymphoid tissues such as the gut-associated lymphoid tissue (GALT). Acute phase of infection is followed by a massive depletion of CD4+ memory T cells in the mucosa, the lymph nodes, and the GALT within a few days after infection, accompanied by production of high levels of circulating virus. However, acute infection does not seem to affect the naïve and resting central memory T cells that

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do not express CCR5 co-receptor, enabling these cells to replenish the T-cell population after the acute phase of infection. As these cells are short-lived, they only partially substitute for the CD4+ effector memory T cells deleted during the acute phase of infection (Grossman et al. 2006). Active viral replication in the infected individuals subsequently leads to viremia, which usually peaks, between 2 and 3 weeks after initial infection. During this period, patients suffer from influenza-like symptoms like fever, sore throat, and lymphadenopathy. T cell levels in the peripheral blood gradually return close to normal but T cells in the GALT remain severely suppressed. There is an establishment of latent viral reservoirs in resting memory CD4+ T cells, which are generated when activated CD4+ T cells return to a quiescent state. Latent HIV persists as a stably integrated but transcriptionally silent provirus. In this state, the virus is unaffected by immune responses or antiretroviral drugs. Eventually, the viremia levels decrease as the initial infection is partially contained by the immune response and reach a steady viral set point, which marks the initiation of the chronic phase of infection. The chronic phase of infection can last for years even in the absence of any antiretroviral treatment but vary from individual to individual. During the chronic phase, there is continuous immune activation and an accelerated cell turnover even though the circulating peripheral CD4+ T cells are still near the normal level (Ford et al. 2009) HIV-infected individuals display elevated markers of activation (e.g. CD38, Ki67) and apoptosis of CD8+ and CD4+ T-cells. Although the precise origin of generalized immune activation is still not fully understood but several mechanisms may be involved, including antigenic stimulation by the virus itself or its encoded protein like envelope protein gp120 that has been shown to activate B cells via the stimulation of various cytokines (He et al. 2006). The accessory protein Nef can also promote lymphocyte activation either directly (Wang et al. 2000) or through infection of macrophages (Swingler et al. 1999). Immune activation may also be induced by other viruses, like EBV, KSHV or CMV that reactivate as a result of suboptimal immune control. Last but not the least, substantial damages in the mucosal barriers occurred during both the acute and chronic phase of infection, allow the translocation of bacterial products such as lipopolysaccharide (LPS) into the circulation which may lead to systemic activation of immune system (McMichael et al. 2010). Immune activation causes considerable cellular turnover, senescence, and apoptosis. Repeated stimulation gradually drives HIV-specific T cells toward an irreversible exhaustion of their replicative capacities. In other words, immune activation results in consumption of naïve and memory cell pools with a concomitant expansion of short lived effector CD4+ cells which ultimately become the new targets for viral replication. Over time, this vicious cycle of immune activation and HIV replication results in a constant decline in CD+ T cell counts, a gradual increase in viral loads, followed by the development of opportunistic infections and AIDSassociated malignancies. There are a number of possible mechanisms that can contribute to tumorigenesis in HIV-infected individuals. The most discernible one being the depletion of CD4+ T cells during the course of infection, which subsequently leads to a lack of immune surveillance against various co-infecting oncogenic viruses like EBV, HPV, and

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KSHV. These viruses can be reactivated during HIV-induced immunosuppression. The role and mechanism of how these viruses can lead to oncogenic transformation has been described earlier. Another possible mechanism for the development of AIDS-associated malignancies, especially AIDS-associated B cell lymphomas is the chronic activation of B cells. AIDS-associated B cell lymphomas are often characterized by the presence of genetic abnormalities like the chromosomal translocation of c-Myc proto-oncogene next to the immunoglobulin heavy chain gene locus, a seminal event in the genesis of BL, or mutations involving other oncogenes, such as BCL2 or BCL6, commonly observed in DLBCL. These chromosomal alterations may occur due to defects in either of the two germinal center reactions following B-cell activation namely somatic hypermutation or class switch recombination. Chronic activation of B cells increases the likelihood of chromosomal translocations and somatic mutations in cellular oncogenes. One factor that can potentially contribute toward defects in germinal center reactions following chronic B cell hyper activation is an aberrant expression of activation-induced cytidine deaminase (AID), the DNA-mutating enzyme that plays a central role in somatic hypermutation and class switch recombination. A recent study found that indeed AID expression was markedly elevated in those patients who developed AIDS-NHL, when compared to AIDS and HIV-negative controls (Epeldegui et al. 2007). Dysregulation of the cytokine network may be another independent risk factor for the development of B-cell malignancies in HIV patients. Cytokines play important roles in B-cell activation, proliferation, and apoptosis. In fact, numerous cytokines associated with B cell lymphomagenesis have been found to be elevated during HIV infection. Notably levels of IL-10 (Sasson et al. 2010), IL-6, TNF alpha, and CXCL13, which is a homeostatic B cell chemokine (Patke and Shearer 2000); were found to be elevated prior to the development of AIDS-NHL (Widney et al. 2005). It is noteworthy that IL-6 and IL-10 are potent B cell-stimulatory cytokines. Earlier studies proposed that HIV induced a TH1 to TH2 shift which would result in an inability to mount a successful cell-mediated immune response (Clerici and Shearer 1993) but later studies did not support this idea (Graziosi et al. 1994). We do not fully understand whether cytokine dysregulation is the cause or outcome of lymphomagenesis. However, it is important to understand the role of cytokines during HIV-induced immunosuppression as it may allow new therapeutic approaches based on the use of either recombinant cytokines or specific antagonists.

Evolving Spectrum of AIDS Associated Malignancies in Post-HAART Era The introduction of highly active antiretroviral therapy (HAART) in the mid-nineties significantly changed the natural history of AIDS, its prognosis and the mortality rates for people infected with HIV. Since then, many studies have been conducted to determine the effect of HAART on the incidence of AIDS defining and non-AIDS defining malignancies. The results of these studies are not very consistent and some

KS and NHLs

San Francisco City Clinic cohort, USA

North America, Europe, and Australia

52 European HIV clinics

Chelsea and Westminster Cohort, Great Britain

Buchbinder et al. (1999)

International collaboration on HIV and cancer (2000)

Mocroft et al. (2000)

Matthews et al. (2000)

NHLs

NHLs

KS, NHLs, Cervical cancer and 20 other cancers

1992–1994 vs. 1997–1998

KS and NHLs

1988–1999

1994–1998

1977–1999

1993–1996

1988–1997

NHLs

17 Western European countries Ledergerber et al. (1999) Swiss HIV cohort study

Franceschi et al. (1999)

Table 32.1 Studies reporting the impact of HAART on the incidence of malignancies Author/group Study site/cohort Malignancies studied Study period Jacobson et al. (1999) Multicenter AIDS cohort KS and NHLs 1984–1997 study, USA

(continued)

Major findings Decline in KS (25.6–7.5 per 1,000 person-years) Increase in NHLs (21% per year) but recent possible decrease Increase in NHLs (3.6% in 1994 to 4.9% in 1997) Decline in KS (hazard ratio – 0.08) No change in NHL (hazard ratio – 0.61) Decline in KS incidence (3.5–0 per 100 person-years) No change in NHL incidence (remained between 1.4–1.8) Decline in KS and NHL incidence (IRR, 0.32 and 0.58, respectively) No change in other cancers (IRR, 0.96) Percentage of NHLs as ADI have increased (from 4 to 16%) The incidence of AIDS-related lymphomas has not changed over time (P = .933), but contributes to a greater percentage of first ADI in the HAART era (P < .0001).

32 Viral Malignancies in HIV-Associated Immune Deficiency 839

Study site/cohort

Los Angeles, USA

Australia

London, Great Britain

Euro-SIDA study, Europe

29 centers in France

SEER program, USA

Australia

France

Univ of Alabama at Birmingham HIV clinic

Table 32.1 (continued) Author/group

Levine et al. (2000)

Grulich et al. (2001)

Ives et al. (2001)

Kirk et al. (2001)

Besson et al. (2001)

Eltom et al. (2002)

Dore et al. (2002)

Herida et al. (2003)

Bedimo et al. (2004)

AIDS defining and nonAIDS defining

Non-AIDS defining

NHLs

KS and NHLs

Non-AIDS defining

NHLs

KS and NHLs

KS and NHLs

Lymphomas

Malignancies studied

1989–2002

1992–1999

1993–2000

1973–1998

1993–1994 vs. 1997–1998

1994–1995 vs. 1997–1999

1990–1998

1980–1998

1982–1998

Study period

Decrease in prevalence of small noncleaved lymphoma (P = 0.005) Increase in diffuse large cell lymphoma (P < 0.0001) Decline in KS and NHL incidence (P trend – 0.045) 34% decline in KS, non-significant increase (51%) in NHLs Significant decline in NHLs (1.99–0.30 cases/100 person years of follow-up) Decline in incidence of AIDSrelated lymphomas (86.0–42.9 per 10,000 person-years) Decline in KS and NHL incidence Increase in non-AIDS defining cancers Percentage of NHLs as ADI have increased (from 4.4 to 6.3%) No change in overall incidence, Lung cancer increased Decrease in AIDS-defining malignancies (IRR, 0.31) Increase in non-AIDS defining malignances (IRR, 10.87)

Major findings

840 P. Kumar et al.

Millitary Clinics, Multicenter study, USA San Diego County, USA

Mocroft et al. (2004)

Burgi et al. (2005)

Tri-service AIDS Clinical Consortium and Multicenter AIDS Cohort Study Swiss HIV cohort study

Shiels et al. (2008) 1990–2006

1984–2006

KS

1991–2002

1988–2000

1988–2003

1994–2003

Study period

AIDS defining

AIDS defining and AIDS associated

NHLs

Non-AIDS defining

KS

Malignancies studied Annual reduction of 39% in KS incidence Decline in non-AIDS defining cancers (OR – 0.21) Decline in NHL incidence (29.6–6.5 per 1,000 person-years) Decline in KS (SIR – 2,800–790 per 100,000 person years) and NHL incidence (SIR – 9.8–6.5 per 100,000 person years) Increase in non-AIDS defining cancers (from 31.4 to 58% of cancers) Decrease in AIDS-defining malignancies (IRR, 0.29)

Major findings

Franceschi et al. (2008) Powles et al. (2009)

Decline in KS incidence (33.3–5.1 per 1,000 person-years) Chelsea and Westminster Non-AIDS defining 1985–2007 Increase in non-AIDS defining Cohort, Great Britain cancers (SIR, 1.96) Millitary beneficiaries, AIDS defining and non1984–2006 Decrease in AIDS-defining in late Crum-Cianflone et al. Multicenter study, USA AIDS defining HAART era (P < 0.0001) (2009) Increase in non-AIDS defining cancers (P < 0.0004) Seaberg et al. (2010) Multicenter study, USA AIDS defining and non1984–2007 KS and NHL declined (IRR, 0.13 AIDS defining and 0.23, respectively) Anal cancer increased (IRR, 5.84) No chance in HL (IRR, 0.75) NHLs Non-Hodgkins lymphomas, ADI AIDS-defining illnesses, KS Kaposi’s sarcoma, IRR Incidence rate ratio, SIR Standardized incidence ratios, OR Odds ratio

Colorado, Florida, and New Jersey

Engels et al. (2008)

Diamond et al. (2006)

Study site/cohort

Euro-SIDA study, Europe

Author/group

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recent and prominent studies have been summarized in Table 32.1 (Levine et al. 2000; Engels et al. 2008; International Collaboration on HIV and Cancer 2000; Bedimo et al. 2004; Besson et al. 2001; Buchbinder et al. 1999; Burgi et al. 2005; Crum-Cianflone et al. 2009; Diamond et al. 2006; Dore et al. 2002; Eltom et al. 2002; Franceschi et al. 1999, 2008; Grulich et al. 2001; Herida et al. 2003; Ives et al. 2001; Jacobson et al. 1999; Kirk et al. 2001; Ledergerber et al. 1999; Matthews et al. 2000; Mocroft et al. 2000, 2004; Powles et al. 2009; Seaberg et al. 2010; Shiels et al. 2008). In recent years, the longer survival rates associated with HAART have led to a steady increase in diseases with a longer latency period, especially non-AIDSdefining malignancies. Decreased incidence for certain malignancies may be due to a better immune surveillance for malignancies. These studies were mostly conducted in HIV-infected individuals in Western countries, where one-third of deaths in these individuals are due to cancer. One point that needs to be emphasized from this table is the lack of any report from sub-Saharan Africa, despite this region bearing the brunt of the HIV epidemic. One of the reasons has been the slower roll out of HAART in this region and poor availability of registries and records in order to evaluate a change in incidence. Incidence of cancer in the pre- and post-HAART era in Africa has also been difficult to measure due to the considerably shorter life-span of people living with HIV as compared to Western countries. Another limitation of many of these studies is that the length of time the participants have been on anti-HIV therapy has been short. Considerably longer follow-up is required to gather more comprehensive data on this topic. As evident from Table 32.1, the incidence of non-AIDS-defining malignancies have increased in the post-HAART era. In contrast, most studies conclude that KS and NHLs have declined in incidence. The data on the impact of anti-HIV therapy on the development and persistence of HPV-related disease is conflicting. Some studies have found a regression in HPV-related disease, while others have found limited or no regression (De Vuyst et al. 2008). More recent studies are now evaluating the factors associated with the risk of developing malignancies including the use of specific antiretroviral agents, ART drug class, the duration of ART, and the level of immunosuppression.

Concluding Remarks The incidence of AIDS-related KS and NHL has decreased dramatically since the introduction of HAART, especially in the developed countries where HAART is widely available and drug failures are being monitored. Similarly, in the developing countries with the recent scale up of the treatment, it is expected that these AIDSdefining cancers will also experience a decrease. However, higher risk in developing other non-AIDS-associated malignancies in HIV-infected and treated individuals are already being observed. The impact of HAART on these cancers remains unclear but it is expected there will be an increase in the overall risk in developing nonAIDS-defining cancers, and that there will a shift in the spectrum of these malignancies because of the increasing size of the HIV-infected and treated population.

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The development of these cancers could be due to a number of reasons but the exact mechanisms are not known. It could be due to chronic immune suppression or due to co-infection or reactivation of other viruses associated with cancers such as EBV or KSHV. For HIV-infected individuals in developing countries, there could be other additional factors which include poor nutritional status, other co-morbid diseases, drug–drug interactions, and cytotoxicity of HAART therapy. In conclusion, the evolving spectrum of AIDS-defining malignancies along with increasing incidence of non-AIDS associated cancers remains a challenge for the diagnosis, care, and treatment of HIV-infected patients and warrants further research.

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Index

A Acute-transforming retroviruses, 758 Adenovirus type 12 E1A, tumorigenesis E1A and E1B genes, 489 MHC class I COUP-TFII/HDAC recruiting, transcription repression, 495–496 CTL avoidance, shutoff, 490–491 transcription level, shutoff, 492, 493 neuronal and tumor-related genes, expression of E1A–12 oncoprotein, 500 Spacer region, 499, 500 neuronal gene induction NRSF degradation, 502–504 NRSF, schematic representation of, 501, 502 NF-kB binding and COUP-TFII/HDAC binding, MHC class I repression, 496–497 p50 and p65 phosphorylation, blocking of, 492–494 NK cell lysis activating ligands, reducing expression of, 497–499 resistance to, 497 Adult T-cell leukemia (ATL), 613 genomic instability, 621–623 vs. HTLV–1-transformed cells, 623–624 oligoclonal expansion, 620–621 telomerase activity, 625 tumor suppressors, 621 AEV. See Avian erythroblastosis virus (AEV) AIDS-associated lymphoma (ARL) Burkitt’s lymphoma (BL), 820–822

diffuse large B-cell lymphomas (DLBCL), 822–825 Hodgkin’s lymphoma, 825–827 primary effusion lymphoma (PEL), 827–829 Alpha fetoprotein (AFP), 522 ALV. See Avian leukosis virus (ALV) Antiviral therapy and HCC, 572–573 ARL. See AIDS-associated lymphoma (ARL) Ataxia telangiectasia mutated (ATM), 473, 474 ATL. See Adult T-cell leukemia (ATL) Avian erythroblastosis virus (AEV), 692 Avian leukosis virus (ALV), 83, 681–684 Avian reticuloendotheliosis virus T (REV-T), 691

B BAC. See Bronchioloalviolar carcinoma (BAC) BART miRNA, 803 BHRF1 miRNA, 803 Bittner agent. See Mouse mammary tumor virus (MMTV) BK polyomavirus and transformation cytopathic disease, 422–423 discovery, 419 genome and life cycle agnoprotein, 422 infection, replication/transformation, 422 large (LTag) and small tumor antigen (sTag), 421 noncoding control region (NCCR), 421 primary host cell specificity, 420 regions, 420 secondary host cell specificity, 421

E.S. Robertson (ed.), Cancer Associated Viruses, Current Cancer Research, DOI 10.1007/978-1-4614-0016-5, © Springer Science+Business Media, LLC 2012

853

854 BK polyomavirus and transformation (cont.) in human cancers kidney-pancreas transplant patient, 425 prostate cancer, 426, 427 urothelial cancers, 426 oncogenic transformation, in experimental systems, 424 BL. See Burkitt’s lymphoma (BL) Bone marrow-derived nonhematopoietic cells transformation, JC virus, 441–442 Bone marrow reconstitution, 591 Bronchioloalviolar carcinoma (BAC), 755 Burkitt’s lymphoma (BL), 820–822

C Cervical cancer HIV-associated immune deficiency AIDS-defining condition, 834 HIV/HPV co-infection, 835 HPV infections, 832–833 immunosuppression, 834–835 and HPV integration of, 470–471 oncogenes, contribution of, 471–472 c-onc genes, 679 Consensus degenerate hybrid oligonucleotide primers (CODEHOP), 254 CTL epitopes, T-antigen, 394–396 Cytochrome P450 (CYP) pathway, 590–591

D Decoy cells, 426 Diffuse large B-cell lymphomas (DLBCL), 822–825 DNA viruses human cytomegalovirus, 120 PI3K-Akt-TSC-mTOR signaling pathway cap-dependent translation, 122–124 4E-BP1 phosphorylation, 122 eIF4F translation initiation complex, 120 intersections, 124, 125 oncoprotein, 121 PKB, 121 RAFT1/FRAP, 121 SV40 large T (SVLT), 123 TSC1 and TSC2, 122 replication, 119 tumor, oncogenic viruses and cancer transmission, 106 Warburg effect citrate diversion, 127 glucose and glutamin metabolism, 126

Index HCMV-infected cells, 126 tricarboxylic acid cycle, 125

E EBER. See EBV-encoded RNA (EBER) EBV. See Epstein-Barr virus (EBV) EBV-encoded RNA (EBER) B cell transformation and lymphomagenesis, 798–800 cellular gene expression, 797–798 lymphomagenesis and apoptosis protection, 796–797 protein translation shutoff prevention, 795–796 structure and expression, 793–794 EBV nuclear antigen 2 (EBNA–2), 89 EBV nuclear antigen leader protein (EBNALP), 180–181 Endogenous retroviruses (ERV), 693–694 env gene, 756 Enzootic nasal tumor virus (ENTV), 782–783 Epidermodysplasia verruciformis (EV), 111 Epstein-Barr virus (EBV). See also Lymphocryptovirus (LCV) animal models, 142 antiviral agents, 190 associated tumors, oncogenic viruses and cancer transmission, 110–111 BILF1, 55 biology, 134–137 and Burkitt’s lymphoma, 137–139 EBER (see EBV-encoded RNA (EBER)) EBNA–1, 59 EBNA–3C, 60 ENBA–5, 61 encoded oncogenes, 89–90 encoded proteins, 174–175 gastric carcinoma, 141 genome, 172 HHV–8 and Kaposi’s sarcoma, 142–143 HIV–1, coinfection with, 596–597 and Hodgkin’s disease, 140 immunotherapy, 188–189 infection diagnosis and malignancies, 188 KS lesions, 144–145 LMP1, 59, 64–65 LMP2A, 50 lymphomas, immunosuppression, 141 lytic and latent genes, 173 microRNA host cellular genes targeting, 804–809 structure, 802–804 and nasopharyngeal carcinoma, 139–140

Index NK-cell lymphomas, 141–142 and notch signaling, 66–68 structure, 172–173 T-cell lymphomas, 141–142 tumorigenesis cell-cycle progression, 185–188 cell signaling, 182–183 chromatin remodeling, 183–185 vaccination, 190–191 virus-mediated cell proliferation, 46–48 and Wnt Signaling, 70

F Flexner, S., 13–15 FoxN1, 696 Fv1, 696

G Gag gene, 756 g 1-herpesvirus. See Lymphocryptovirus (LCV)

H HAART. See Highly active antiretroviral therapy (HAART) HCV. See Hepatitis C virus (HCV) Head and neck cancer (HNC), HPV role in, 475–476 Hepadnaviruses and hepatocellular carcinoma chronic HBV infection biology of, 533–536 hepatocyte evolution and liver cancer, 544–553 virus and liver, changes in, 541–543 ground squirrel models, 531, 532 integration of, 532 origin of, 536–537 orthohepadnavirus and avihepadnavirus, 531 persistent injury, to liver, 532 progression of, 533 replication double-strand, linear virus DNA (DSL DNA), structure of, 540 HBeAg and HBx, 537, 538 HBV life cycle, in hepatocytes, 538, 539 HBV, rcDNA genome of, 537, 538 polymerase/pregenome complex, 538 therapy, 554 woodchuck models, 531, 532, 553 Hepatitis B virus (HBV). See also Viruses and cancer

855 biology of endothelial cell layer and hepatocytes, 534, 535 immune response, 535, 536 perinatal infection, stages of, 533, 534 conjectures, 37–39 discovery of, 28–31 DNA integration, into host chromosomes, 513 encoded oncogenes, 88 genetics of, 36–37 and hepatocellular carcinoma, 110 hepatocyte evolution and liver cancer HBx and HCC, 547–550 oncogenes, 544 regeneration and HCC, 545–547 transgenic mice, 544 viral envelope proteins and HCC, 550–553 life cycle, in hepatocytes, 538, 539 pre-S2/S proteins and large envelope protein, 514 protein-primed reverse transcription, 512 rcDNA genome of, 537 viral load, 513 virus and liver, changes in genetic basis, 542 host immune response, 541–542 resistance to infection, 542, 543 titer production of, 541 X protein, 513 Hepatitis C virus (HCV) clinical perspective, and HCC antiviral therapy, 572–573 epidemiological link, 571–572 etiology of, 573 core protein, 515–516 encoded oncogenes, 88 genome, 514–515 HIV–1, coinfection with, 600 NS5A protein, 516 replication, 515 steatosis and oxidative stress, 516 virological perspective, and HCC core protein, 576–577 experimental systems, 574 genome and polyprotein product, 575, 576 molecular biology of, 575–578 NS3–4A enzyme complex, 577 NS5A phosphoprotein, 578 NS5B RNA polymerase, 578 proteins and carcinogenesis, 575–576 tropism, 574–575

856 Hepatitis viruses causes, of cancer death, 509, 510 epidemiology, 511–512 HBV DNA integration, into host chromosomes, 513 pre-S2/S proteins and large envelope protein, 514 protein-primed reverse transcription, 512 viral load, 513 X protein, 513 HCC (see Hepatocellular carcinoma (HCC)) HCV core protein, 515–516 genome, 514–515 NS5A protein, 516 replication, 515 steatosis and oxidative stress, 516 Hepatocellular carcinoma (HCC) age, incidence distribution, 511, 518 detection and management of alpha fetoprotein (AFP), 522 chronic hepatitis virus effect, 523 molecular and biomarker information, subclassification, 523 poor prognosis and, 521, 522 serum markers for, 524 etiologies of, 509, 510 HBV and HCV, 509, 511 HBx and DDB1, 547, 549 indirect effect, 550 nucleotide excision repair, 549 reported activities of, 547, 548 transformed state, of tumor cells, 549, 550 HCV (see Hepatitis C virus (HCV)) hepatocyte regeneration and cirrhotic nodules and non-cirrhotic livers, 546 clonal hepatocyte repopulation, 545, 546 molecular pathogenesis of chronic hepatitis virus, immune response, 519 chronic infection, molecular and cellular events, 520 clinical conditions, progression of, 518 inherited and acquired mutations, 519–521 microRNA role, in hepaocarcinogenesis, 521 molecular targets and pathways, 520 pathways of, 517–519

Index origin of, 536–537 viral envelope proteins and ground glass hepatocytes (GGH), 551 HBx, 553 hepatocyte death, 551 as oncogenes, 551 PreS2 mutation, 552 S, M, and L protein, 550 spheres and rods, 550–551 Hepatocyte evolution and liver cancer HBx and HCC, 547–550 oncogenes, 544 regeneration and HCC, 545–547 transgenic mice, 544 viral envelope proteins and HCC, 550–553 regeneration and cirrhotic nodules and non-cirrhotic livers, 546 clonal hepatocyte repopulation, 545, 546 Herpesviruses EBV (see also Epstein-Barr virus (EBV)) animal models, 142 biology, 134–137 and Burkitt’s lymphoma, 137–139 gastric carcinoma, 141 HHV–8 and Kaposi’s sarcoma, 142–143 and Hodgkin’s disease, 140 KS lesions, 144–145 lymphomas, immunosuppression, 141 and nasopharyngeal carcinoma, 139–140 NK-cell lymphomas, 141–142 T-cell lymphomas, 141–142 KS (see Kaposi’s sarcoma (KS)) KSVH (see also Kaposi’s sarcoma herpes virus (KSVH)) animal models, 155–156 biology, 146 latent proteins and oncogenesis, 146–151 multicentric Castleman’s disease, 145–146 primary effusion lymphoma, 145 MDV, 156–157 (see also Marek’s disease virus (MDV)) Herpesvirus-mediated cancers EBV EBER RNAs B cell transformation and lymphomagenesis, 798–800 cellular gene expression, 797–798 lymphomagenesis and apoptosis protection, 796–797

Index protein translation shutoff prevention, 795–796 structure and expression, 793–794 HSUR, 800–801 microRNA biogenesis and function, 802 EBV-encoded, 802–803 host cellular genes targeting, 804–809 identifying targets and functions, 804 KSHV-encoded, 803–804 viral genes targeting, 809 Herpesvirus saimiri (HVS) classification, 203 HSURs, 800–801 Saimiri sciureus, 202 Saimiri transformation-associated protein (Stp), 203 tyrosine kinase-interacting protein (Tip), 204 HHV–8. See Human herpesvirus 8 (HHV–8) Highly active antiretroviral therapy (HAART) drug interactions demand management, 590–591 HIV–1/HTLV–2 coinfection, 660, 661 HIV–1, treatment, 589 with chemotherapy, 590 HIV–1. See Human immunodeficiency virus type 1 (HIV–1) HIV-associated immune deficiency AIDS-associated lymphoma Burkitt’s lymphoma, 820–822 diffuse large B-cell lymphomas, 822–825 Hodgkin’s lymphoma, 825–827 primary effusion lymphoma, 827–829 cervical cancer AIDS-defining condition, 834 HIV/HPV co-infection, 835 HPV infections, 832–833 immunosuppression, 834–835 Kaposi’s sarcoma AIDS-associated KS, 830 classic KS, 829 endemic KS, 829 histological features, 829, 830 iatrogenic KS, 830 immune suppression, 831–832 KSHV replication, 831 pathogenesis and immune suppression acute phase, 836–837 CD4+ T cell destruction, 835 chronic phase, 837 immunosuppression, 838 infection and immune activation, 836 macrophage, 836

857 tumorigenesis, 837–838 viral entry, 835–836 post-HAART era, AIDS, 838–842 Hodgkin’s lymphoma, 825–827 HPV. See Human papillomavirus (HPV) HSUR, 800–801 HTLV–1. See Human T-lymphotropic virus type 1 (HTLV–1) HTLV–2. See Human T-cell leukemia virus type 2 (HTLV–2) HTLV–1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), 613 HTLV–1 uveitis, 613 Human breast cancer, 748–749 Human cytomegalovirus (HCMV), 120 Human herpesvirus 8 (HHV–8) epidemiology, 592 Kaposi’s sarcoma, coinfection, 592–595 Human herpes viruses (HHV), 46–48. See also Herpesviruses Human immunodeficiency virus type 1 (HIV–1) associated malignancies, 586–588 immunodepletion heightens, 588–589 Kaposi’s sarcoma, 592–595 treatment bone marrow reconstitution, 591 drug interactions demand management, 590–591 HAART with chemotherapy, 590 highly active antiretroviral therapy, 589 viral coinfection, malignancy anti-EBV cytotoxic T lymphocytes, 598 EBV, 596–597 HCV, 600 HHV–8, 592–595 HPV, 598–599 HTLV–1, 600–601 IL–10 role, 597 multicentric Castleman’s disease, 596 rare lymphoma, 595–596 Human infections, polyomavirus SV40 contemporary human infections predictions, 401–403 evidence of, 403–404 SV40 association, human cancer, 405–408 SV40-contaminated vaccines, 400–401 Human papillomaviruses (HPVs) abortive viral infection, 467 cervical cancer, 832–834 encoded oncogenes, 88 in head and neck cancer (HNC), 475–476 high-risk, 463 HIV–1, coinfection with, 598–599

858 Human papillomaviruses (HPVs) (cont.) molecular events, malignant progression integration and cervical cancers, 470–471 oncogenes contribution, to human cervical cancers, 471–472 oncogenes repression, E2 protein, 471 oncogenic activities, host DNA damage response, and genomic instability, 473–475 persistent latent infection bovine papillomavirus type 1 (BPV1), 468 Brd4, 468, 469 E6 and E7 oncoproteins, 468 mitotic chromosomes, tethering mechanism, 469 productive viral life cycle basal cells, 465 E4 protein, 467 E5 protein, 466, 467 E2 proteins, 465, 466 HPV16 genome organization and functions, of gene products, 464 suprabasal cells, 466 therapeutic approaches, 477 Human T-cell leukemia virus type 2 (HTLV–2) accessory genes Aph–2 antisense gene, 655 ORF I, II, and V, 654–655 culture and animal models, 664 discovery, 647–648 genetic variability, 656 genome structure, 649, 650 hematopoiesis, 658–659 HIV coinfection, 659–661 neurologic abnormality, 657–658 receptor and infectivity, 656–657 regulatory genes Rex gene, 652–654 Tax gene, 651–652 structural and enzymatic genes envelope (env) gene, 651 gag gene, 649–650 polymerase (pol) gene, 651 protease (Pro) gene, 650 Tax oncoprotein mechanisms apoptosis, 664 cell cycle dysregulation, 663 NFkB interaction, 663 posttranscriptional effects, 662 viral transcription, 661–662 virion structure, 648–649 Human T-cell lymphotropic virus (HTLV), 45

Index Human T-lymphotropic virus type 1 (HTLV–1) animal models, 625 ATL cells (see Adult T-cell leukemia (ATL)) cell-associated transmission, 619–620 cell transformation, oncoproteins, 586 encoded oncogenes, 87 epidemiological studies, 613 genome and replication, 614–615 genome structure, 586 HBZ functions, 619, 630–632 history of, 620 HIV–1, coinfection with, 600–601 p12I, p13II, and p30II functions, 618 Rex functions, 618 Tax functions IKK/NF-kB, 616–618 viral mRNA transcription, 615–616 viral origins, human cancers, 27 in vitro T-cell transformation vs. ATL cells, 623–624 signaling pathways, 624 telomerase activity, ATL cells, 625

I Interferon treatment, HCC, 572–573 International Committee on Taxonomy of Viruses (ICTV), 449

J Jaagsiekte sheep retrovirus (JSRV) endogenous enJSRV, 781–782 ERVs, 780–781 molecular biology betaretrovirus, 762–764 disease determinants, 767 Hyal2, 769 LTR binding sites, 766–767 molecular clones, 764 Orf-X, 768–769 Rej, 768 sequence deduction and endogenous identification, 762 transcription specificity, 764–766 oncogenesis mechanisms cell transformation, 770 Env, 770–771 transformation, env domains, 771–772 pathogenesis disease latency, 779–780

Index immune responses, 780 OPA, flocks, 778–779 transformation, signal EGFR inhibitors, 775 env transformation, 776–777 Hyal2/RON pathway, 777–778 PI3K-Akt-mTor pathway, 772–774 Ras-Raf-MEK-MAPK pathway, 774–775 Src inhibitors, 775 JC virus (JCV) bone marrow-derived nonhematopoietic cells transformation, T-Ag, 441–442 cell culture and animal models, of tumorigenesis, 435–436 and human cancer, association of, 433, 434, 436–438 latency, reactivation, and cellular tropism, 434–435 neural stem cells transformation, 443, 444 and oncogenic potential, molecular mechanism studies, 435 pathogenesis of considerations, 443 and dissemination, from bone marrow, 440, 441 stem cells in, 439, 440 T-Ag-induced, 440 PML-type configuration, 442, 443 and stem cells, brain and bone marrow hematopoietic cells, 438 nonhematopoietic cells, 439 T-Ag expression, 442 JSRV. See Jaagsiekte sheep retrovirus (JSRV)

K Kaposi’s sarcoma (KS) classical, 219 clinical features and treatment, 221–222 co-factors, 220–221 cytokines and angiogenic factors, 151–155 endemic, 219 epidemic/AIDS, 220 epidemiology, 251 etiological agent, 252–253 HIV–1 and HHV–8 coinfection, 592–595 HIV-associated immune deficiency AIDS-associated KS, 830 classic KS, 829 endemic KS, 829 histological features, 829, 830 iatrogenic KS, 830 immune suppression, 831–832 KSHV replication, 831

859 iatrogenic, 220 KSHV latency, 253 molecular characteristics, 252–253 morphology, 251–252 proliferative disorder, 223 Kaposi’s sarcoma herpes virus (KSHV). See also Rhadinoviruses animal models, 155–156 associated diseases, 216 biology, 146 epidemiology, 252 encoded oncogenes, 90–92 etiological agent, KS, 252–253 gene expression, 252–253 genomic conservation, 255–257 host, 258 K1 gene, 51 K15 gene, 51 LANA–1, 62 latency-associated nuclear antigen (LANA) functions, 253 vs. RFHV LANA sequence analysis, 256–257 latent proteins and oncogenesis, 146–151 microRNA host cellular genes targeting, 804–809 structure, 802–804 modulator of immune recognition, 256 molecular biology, 252 multicentric Castleman’s disease, 145–146 multicentric Castleman’s disease (MCD) clinical features and treatment, 227–228 IL–6 role, 227 incidence, 226–227 and notch signaling, 68 origin and evolution, 215–216 primary effusion lymphoma, 145 primary effusion lymphoma (PEL) clinical features and therapy, 224–225 KSHV-associated lymphomas, 223–224 pathogenesis, role of, 225–226 solid, 224 primary infection, 216–218 vs. RFHV genome, 255–257 vs. rhadinoviruses genome, 258–260 RRV, 208, 209, 211 transmission and risk factors, 216 tumorigenesis mechanisms, 228–233 viral proteins involvement, 233–236 vCyclin, 61 vGPCR, 53–54 vIL–6, 56

860 Kaposi’s sarcoma herpes virus (KSHV). See also Rhadinoviruses (cont.) viral chemokines, 53 vIRFs, 62–63 virus-mediated cell proliferation, 47–48 and Wnt signaling, 70 Kidney transplantation, BK polyomavirus, 425 KS. See Kaposi’s sarcoma (KS) KSHV. See Kaposi’s sarcoma herpes virus (KSHV)

L Latency-associated nuclear antigen–1 (LANA) Kaposi sarcoma, 222 viral protein involvement, tumorigenesis, 233 Liver, digestive system, 517 Lung alveoli, epithelial cell, 760 Lymphocryptovirus (LCV) BARTs, 181–182 and cancer, 170–171 chemotherapy, 190 cytotoxic T cells use EBV-specific T cells, 189 unmanipulated donor T cells, 189 EBERs, 181 EBNA1, 177–178 EBNA2, 178–179 EBNA3 family, 179–180 EBNALP, 180–181 EBV antiviral agents, 190 cell-cycle progression, 185–188 cell signaling, 182–183 chromatin remodeling, 183–185 encoded proteins, 174–175 genome, 172 immunotherapy, 188–189 infection diagnosis and malignancies, 188 structure, 172–173 vaccination, 190–191 in human, 171 LMP1, 175 LMP 2A and 2B, 176–177 radiation therapy and surgery, 190 targeting B-cells, antibody therapy, 189–190 viral species, 170 Lymphomagenesis, MDV initiating events, 325–326 lymphoma progression, 326–327 Lymphoproliferative disorder (LPD), 208

Index M Macaca nemestrina rhadinovirus 2 (MneRV2), 258 Major histocompatibility complex (MHC) class I, adenovirus type 12 E1A COUP-TFII/HDAC recruiting, transcription repression, 495–496 NF-kB and COUP-TFII/HDAC binding, repression of, 496–497 shutoff CTL avoidance, 490–491 transcription level, 492, 493 Mardivirus. See Marek’s disease virus (MDV) Marek’s disease virus (MDV) encoded oncogenes, 309–311 gene products, oncogenesis MDV-encoded microRNAs, 320–324 phosphoprotein 14 and RLORF9, 319–320 RLORF4, 319 herpesviruses, 156–157 history of, 308 lymphomagenesis initiating events, 325–326 lymphoma progression, 326–327 Meq binding proteins, 313–314 deletion mutant, phenotype, 316–317 dimerization partners, 312–313 localization and dynamics, 311–312 splice variants, 315–316 target genes, 315 viral interleukin 8, 317–318 viral telomerase RNA, 317 viral ubiquitin-specific protease, 318–319 pathogenesis, 308–309 RNA association, 799 MDV. See Marek’s disease virus (MDV) Meq, MDV Binding Proteins, 313–314 deletion mutant, phenotype, 316–317 dimerization partners, 312–313 localization and dynamics, 311–312 splice variants, 315–316 target genes, 315 viral interleukin 8, 317–318 viral telomerase RNA, 317 viral ubiquitin-specific protease, 318–319 Merkel cell polyomavirus (MCV) genome, appearance and structure of, 450 Merkel cell carcinoma (MCC) and viral detection digital transcriptome subtraction (DTS), 451, 452

Index in immunosuppressed populations, 451 integration of, 452, 453 origin of, 451 prevalence of, 452 viral carcinogenesis of, 453, 454 polyomaviruses, 339 small and large T antigens conserved region 1 (CR1), 456 DnaJ domain, 456 domains, 455 indirect evidence for, 458 p53, 456–458 PP2A, 458 replication, 453 retinoblastoma (RB) family-binding motif LXCXE, 456 T antigens, 456–458 transformation of, 455 MicroRNAs (miRNAs) role, in hepaocarcinogenesis, 521 Epstein–Barr virus biogenesis and function, 802 host cellular genes targeting, 804–809 identifying targets and functions, 804 and KSHV-encoded, 802–804 viral genes targeting, 809 mLANA, 278–279 MLV. See Murine leukemia virus (MLV) Mouse mammary tumor virus (MMTV) genome transformation, 740 germline transmission, 740–741 human breast cancer, 748–749 replication, 741–742 transcriptional control mammary gland differentiation and transformation, 743 proviral expression, 742 regulatory elements, 742 virus production, 742 tumor induction common integration sites, 744–745 Env protein, 747 oncogene integration, 745–746 oncogenes activation, 747 pregnancy state, 746 proviral integrations, 744 retroviral insertion, 744 wild-type and transgenic mice, 745 Mtv loci, 740 Multicentric Castleman’s disease (MCD), 596 Murine gammaherpesvirus 68 (MHV68). See also Murine gammaherpesvirusassociated tumorigenesis

861 immune response antibody response, 289–293 malignant disease control, 293–296 immunomodulation M1, 286–287 M2, 282–284 mK3, 285–286 Murine gammaherpesvirus-associated tumorigenesis immune system role, 287 implications and application, 296–297 MHV68 immune response antibody response, 289–293 malignant disease control, 293–296 MHV68 immunomodulation M1, 286–287 M2, 282–284 mK3, 285–286 mLANA, 278–279 prooncogenic alterations, cell signaling NF-kB, 279–280 PI3K/Akt, 280 twist/snail, 281 v-bcl2 characteristics, 274 gammaherpesvirus Bcl–2 orthologs, 275 oncogene, 276 v-cyclin characteristics, 271 classification, 271 effects, 273 oncogene, 272 role, 272 and tumorigenesis, 273–274 vGPCR, 276–277 Murine leukemia virus (MLV), 685–686

N Natural killer (NK) cell lysis, adenovirus type 12 E1A NKG2D activating ligands, reducing expression of, 497–499 resistance to, 497 Neural stem cells transformation, JC virus, 443, 444 Neuronal gene induction, adenovirus type 12 E1A, 501–504 Neuron-restrictive silencer factor (NRSF) Ad5-and Ad12-, 502, 503 degradation, 502–504 schematic representation of, 501, 502 ubiquitination, 501–502

862 NF-kB binding, adenovirus type 12 E1A and COUP-TFII/HDAC binding, MHC class I repression, 496–497 p50 and p65 phosphorylation, blocking of, 492–494 murine gammaherpesvirus-associated tumorigenesis, 279–280 NHL. See Non-Hodgkin lymphoma (NHL) Non-acute retroviruses, 758 Non-Hodgkin lymphoma (NHL) anti-EBV cytotoxic T lymphocytes, 598 IL–10 role, 597 lymphocryptoviruses, 170 Nonhuman primate gamma-herpesviruses herpesvirus saimiri (HVS) classification, 203 Saimiri sciureus, 202 Saimiri transformation-associated protein (Stp), 203 tyrosine kinase-interacting protein (Tip), 204 RhLCV Cercopithecine herpesvirus 15, 204 EBV infection, 206 miRNAs, 207 retroperitoneal fibromatosis (RF), 205 RRV G protein-coupled receptor (GPCR), 209 KSHV, 208, 209, 211 lymphoproliferative disorder (LPD), 208 vIL–6 detection, 210 Nonobese diabetic/severe combined immunodeficient (NOD/SCID), 625 NS3–4A enzyme complex, HCV, 577 NS5A phosphoprotein, HCV, 578 NS5B RNA polymerase, HCV, 578

O Oncogenesis mechanisms endogenous murine retroviruses, 693–694 Env signaling friend virus and env gene-mediated, 694–695 gene cooperativity, 692–693 host factors influences, 695–696 insertional mutagenesis enhancer-mediated, 687–688 gene function disruption and tumor suppressor genes, 689–690 gene therapy, 688–689 microRNA activation, 690–691 promoter insertion, 685–686 regulatory sequence disruption, 686–687

Index oncogene capture ALV and RSV, 681–684 v-onc-containing viruses generation, 680–681 retroviral classification, 679 Oncogenic viruses and cancer transmission control and prevention, 105–106 epidermodysplasia verruciformis (EV), 111 Epstein–Barr virus-associated tumors, 110–111 hepatitis B virus and hepatocellular carcinoma, 110 infection and human cancer, 101–104 in molecular biology, 104–105 rumor viruses, 112–113 transmissible cancer cells, 113–114 transmission and virulence, 108–109 transmission dynamics DNA tumor viruses, 106 iatrogenic infections, 107–108 retroviruses, 106–107 viral cancers, multifactorial facets, 109–110 OPA. See Ovine pulmonary adenocarcinoma (OPA) Open reading frame (ORF), 255–256 Orthoretrovirinae, 679 Ovine pulmonary adenocarcinoma (OPA) clinical signs, 759 gross pathology, 759–760 histological feature acinar growth, 760 mitotic index, 761 papillary form, 760 retrovirus transmission, 761–762 transformed cells, 761 history, 755 Oxidative stress, HCV, 516

P p53 in human cervical cancers, 472 Merkel cell polyomavirus, 456–458 Papillomaviruses (PVs). See Human papillomaviruses (HPVs) PEL. See Primary effusion lymphoma (PEL) Phosphoinositide 3 kinase-like kinase (PIKK), 473 PI3K-Akt-TSC-mTOR signaling pathway cap-dependent translation, 122–124 4E-BP1 phosphorylation, 122 eIF4F translation initiation complex, 120 intersections, 124, 125 oncoprotein, 121

Index PKB, 121 RAFT1/FRAP, 121 SV40 large T (SVLT), 123 TSC1 and TSC2, 122 pol gene, 756 Polyomaviruses (PyV). See also Merkel cell polyomavirus BK virus, 419 cell cycle progression, LT, 345 DNA damage responses, 359–360 genome destabilization, 357–359 genome organization, 340–343 history and discovery, 338–339 icosahedral capsid and MCV genome, appearance and structure of, 450 MPyV middle T antigen, 352–355 non-neoplastic disease association, 345–346 phylogenetic comparison, 339–340 properties, 344 small T antigen, 355–356 species of, 449 transformation assays immortalization, 346 non-SV40 LT proteins, 351–352 SV40 large T antigen, 347–351 transformation, 347 tumors, in animals, 356–357 viral life cycle, 343–344 Polyomavirus SV40 hamster model, infection and oncogenesis, 390–393 human infections contemporary human infections predictions, 401–403 evidence of, 403–404 SV40 association, human cancer, 405–408 SV40-contaminated vaccines, 400–401 large T-antigen early protein cell cycle dysregulation, 382 cytoplasmic T-antigen, 382–385 transformation, maintenance, 382 mouse model, cellular immune responses CTL epitopes, T-antigen, 394–396 SV40 microRNA and CTL evasion, 396 SV40 T-antigen, cellular proteins DnaJ domain and hsc70, 387–388 p53 tumor suppressor protein, 388–389 T-ag, 389–390 tumor suppressor proteins, Rb family, 388 T-antigen variable domain, 385–387

863 transgenic mouse models loss of dependence, T-antigen, 398 Rb and p53 Pathways, 397–398 T/t-antigen gene signature, 399 viral genome and virus-encoded proteins, 377–379 viral late proteins, 380–381 viral regulatory region, 379–380 Posttransplant lymphoproliferative disorder (PTLD), 171 Primary effusion lymphoma (PEL), 827–829 Progressive multifocal leukoencephalopathy (PML), 433–435, 437 Prostate cancer, BK polyomavirus, 426 Protein kinase A catalytic subunit (PKAc), 494 Protooncogene, 721

R Retroperitoneal fibromatosis-associated herpesvirus (RFHV) etiological assessment, RF tumors immunohistochemical analysis, 262–263 spindle tumor cells detection, 262 viral load, 260–261 genomic conservation, 255–257 host, 258 vs. KSHV genome, 255–257 LANA vs. KSHV LANA sequence analysis, 256–257 prevalence, macaque, 260 Retroperitoneal fibromatosis (RF), macaques epidemiology, 253 etiology, 254 phylogenetic analysis, 254 Retroviral transformation, 739 Retroviruses cell transformation, oncoproteins, 586 cellular proto-oncogenes activation, 585 genome structure, 756 lifecycle, 757–758 oncogenesis mechanisms (see Oncogenesis mechanisms) oncogenic properties, 758–759 oncogenic types, 679 oncogenic viruses and cancer transmission, 106–107 structure, 757 viral-encoded genes and cancer, 82–83 RF. See Retroperitoneal fibromatosis (RF), macaques RFHV. See Retroperitoneal fibromatosisassociated herpesvirus (RFHV)

864 Rhadinoviruses (RV) associated diseases, 216 discovery, 258 Kaposi sarcoma classical, 219 clinical features and treatment, 221–222 co-factors, 220–221 endemic, 219 epidemic/AIDS, 220 iatrogenic, 220 proliferative disorder, 223 molecular characteristics, 258–260 multicentric Castleman’s disease (MCD) clinical features and treatment, 227–228 IL–6 role, 227 incidence, 226–227 origin and evolution, 215–216 primary effusion lymphoma (PEL) clinical features and therapy, 224–225 KSHV-associated lymphomas, 223–224 pathogenesis, role of, 225–226 solid, 224 primary infection, 216–218 transmission and risk factors, 216 tumorigenesis mechanisms, 228–233 viral proteins involvement, 233–236 Rhesus macaque lymphocryptovirus (RhLCV) Cercopithecine herpesvirus 15, 204 EBV infection, 206 miRNAs, 207 retroperitoneal fibromatosis (RF), 205 Rhesus macaque rhadinovirus (RRV) G protein-coupled receptor (GPCR), 209 KSHV, 208, 209, 211 lymphoproliferative disorder (LPD), 208 vIL–6 detection, 210 Rhesus rhadinovirus (RRV) discovery, 258 prevalence, macaque, 260 RLORF4, 319 RNA-dependent DNA polymerase, 757 Rous, P., 1, 2, 9–12, 15–19 Rous sarcoma virus (RSV) in biomedical sciences, 728 c-src confirmation in vertebrates mRNA splicing, 719 protooncogene, 721 provirus insertion, 719 role, human cancers, 723 transduction mechanism, 720 virion recombination, 719, 721 DNA discovery and replication, 709–712

Index filterable agent discovery, 705–706 genome organization, 714–715 pathogenicity, birds and mammals, 706–707 reverse transcription circular DNA formation, 712–714 DNA synthesis, 712 integration, host chromosome, 713–714 src viral oncogene identification, 715–717 protein product isolation, 722–723 transformation biology, 707–709 virion structure, 708, 724 virion assembly attachment, 724 Gag protein, 727 maturation, 724 replication, 725–726 transformation, 724–725 viral RNA genome arrangement, 724 virogene-oncogene hypothesis, 717–718 Rumor viruses, 112–113 RV. See Rhadinoviruses (RV)

S Schmorl, G., 6–9 SFFV. See Spleen focus forming virus (SFFV) Sheep pulmonary adenocarcinoma. See Ovine pulmonary adenocarcinoma (OPA) Simian vacuolating virus 40 (SV40), 449 Solid tumors, JC virus, 436 Spindle cells, 251 Spleen focus forming virus (SFFV), 694–695 Squamous cell carcinomas (SCCs), 475 Steatosis and oxidative stress, HCV, 516 Stem cells and JC virus (JCV), brain and bone marrow, 438–439 SV40 T-antigen, cellular proteins DnaJ domain and hsc70, 387–388 p53 tumor suppressor protein, 388–389 T-ag, 389–390 tumor suppressor proteins, Rb family, 388

T T-antigen variable domain, 385–387 Tax (transactivator encoded by the X region), HTLV–1 cellular senescence p21Cip1/Waf1 and p27Kip1 upregulation, 630 precancerous condition, 629–630 oncogenic activities cell cycle progression, 626

Index chromosome instability, 628–629 DNA damage, 628 double-stranded DNA breaks, 628 genetic/chromosome instability, 627 NF-kB activation and cell transformation, 626 PDZ domain-containing proteins, 627 in transgenic mice, 627 Telomerase reverse transcriptase (TERT), 688 Thromobospondin 1 (THBS1), 804 Transferrin receptor 1 (TfR1), 741 Transgenic mouse models, polyomavirus SV40 loss of dependence, T-antigen, 398 Rb and p53 Pathways, 397–398 T/t-antigen gene signature, 399 Tumorigenesis. See also Murine gammaherpesvirus-associated tumorigenesis EBV cell-cycle progression, 185–188 cell signaling, 182–183 chromatin remodeling, 183–185 rhadinoviruses mechanisms, 228–233 viral proteins involvement, 233–236 viral proteins involvement K1/VIP, 235–236 LANA, 233 miRNAs, 234 vFLIP, 234 vGPCR, 235 vIL–6, 234–235 viral cyclin, 233–234

V v-bcl2 characteristics, 274 gammaherpesvirus Bcl–2 orthologs, 275 oncogene, 276 vGPCR, 276–277 Viral cyclin (vcyclin) characteristics, 271 classification, 271 effects, 273 oncogene, 272 role, 272 and tumorigenesis, 273–274 viral proteins, in tumorigenesis, 233–234 Viral-encoded genes and cancer human cancer EBV-encoded oncogenes, 89–90 HBV-encoded oncogenes, 88

865 HCV-encoded oncogenes, 88 HPV-encoded oncogenes, 88 HTLV–1-encoded oncogenes, 87 KSHV-encoded oncogenes, 90–92 large DNA tumor viruses, 92 retroviruses, 82–83 small DNA tumor viruses, 84–85 tumor viruses, 81–82 Virogene-oncogene hypothesis Rous sarcoma virus, 717–718 Viruses and cancer first cancer vaccine, HBV, 35–36 (see also Hepatitis B virus (HBV)) global vaccination programs, 34–35 origins of, 27–28 prevention, 26 secondary antiviral prevention, 36 vaccine field trials, 32–33 vaccine invention, 31–32 Virus-mediated cell proliferation cell cycle machinery modulation EBV EBNA–1, 59 EBV EBNA–5, 61 EBV EBNA–3C, 60 EBV ENBA–5, 61 EBV LMP1, 59 KSHV LANA–1, 62 KSHV vCyclin, 61–62 KSHV vIRFs, 62–63 p53-mediated cell cycle arrest, 58–59 Rb-mediated cell cycle arrest, 57–58 cellular signaling pathways EBV and notch signaling, 66–68 EBV and Wnt Signaling, 70 EBV LMP1, 64–65 KSHV and notch signaling, 68 KSHV and Wnt signaling, 70 KSHV K13 (vFLIP), 65–66 NF-kB signaling pathway, 63–64 notch signaling pathway, 66 wingless-type (Wnt) signaling pathway, 68–70 chemokine/cytokine system, modulation biological effects, 52 EBV BILF1, 55–56 KSHV vGPCR, 53–54 KSHV vIL–6, 56 KSHV viral chemokines, 53 signaling pathways, 54–55 oncogenic human herpesviruses, 46–48 self-proliferation, viral strategy, 48 viral proteins mimicking growth receptor, 48–52 v-onc genes, 679

866 W Warburg effect citrate diversion, 127 glucose and glutamin metabolism, 126 HCMV-infected cells, 126 tricarboxylic acid cycle, 125 Warthin, A.S., 4–6 Welch, W.H., 3–4

Index Wistar-King-Aptekman-Hokudai (WKAH), 625 Wnt, 744

X X-linked severe combined immunodeficiency (SCID-X1), 688