Hepatitis Delta Virus (Current Topics in Microbiology and Immunology)

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307 Current Topics in Microbiology and Immunology

Editors R.W. Compans, Atlanta/Georgia M.D. Cooper, Birmingham/Alabama T. Honjo, Kyoto · H. Koprowski, Philadelphia/Pennsylvania F. Melchers, Basel · M.B.A. Oldstone, La Jolla/California S. Olsnes, Oslo · P.K. Vogt, La Jolla/California H. Wagner, Munich

J.L. Casey (Ed.)

Hepatitis Delta Virus

With 25 Figures and 12 Tables

123

John L. Casey, Ph.D. Department of Microbiology and Immunology Georgetown University 3900 Reservoir Road, NW Washington, DC 20007 USA e-mail: [email protected] The cover illustration is a simpliﬁed structure of hepatitis delta virus showing the internal ribonucleoprotein complex, which contains the circular RNA genome and the two forms of the hepatitis delta antigen; the envelope proteins of hepatitis B virus form the exterior of the virus. The inset is an electron micrograph of puriﬁed hepatitis delta virus particles, and was kindly provided by Dr. John Gerin. The background immunoﬂuorescence image is of transfected cells expressing hepatitis delta antigen, and was kindly provided by Dawn Defenbaugh.

Library of Congress Catalog Number 72-152360 ISSN 0070-217X ISBN-10 3-540-29801-0 Springer Berlin Heidelberg New York ISBN-13 978-3-540-29801-4 Springer Berlin Heidelberg New York This work is subject to copyright. All rights reserved, whether the whole or part of the material is concerned, speciﬁcally the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microﬁlm or in any other way, and storage in data banks. Duplication of this publication or parts thereof is permitted only under the provisions of the German Copyright Law of September, 9, 1965, in its current version, and permission for use must always be obtained from Springer-Verlag. Violations are liable for prosecution under the German Copyright Law. Springer is a part of Springer Science+Business Media springeronline.com © Springer-Verlag Berlin Heidelberg 2006 Printed in Germany The use of general descriptive names, registered names, trademarks, etc. in this publication does not imply, even in the absence of a speciﬁc statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. Product liability: The publisher cannot guarantee the accuracy of any information about dosage and application contained in this book. In every individual case the user must check such information by consulting the relevant literature. Editor: Simon Rallison, Heidelberg Desk editor: Anne Clauss, Heidelberg Production editor: Nadja Kroke, Leipzig Cover design: design & production GmbH, Heidelberg Typesetting: LE-TEX Jelonek, Schmidt & Vöckler GbR, Leipzig Printed on acid-free paper SPIN 11577317 27/3150/YL – 5 4 3 2 1 0

Preface

Since its discovery nearly 30 years ago, hepatitis delta virus (HDV) has continued to surprise and fascinate. Initially thought to be an antigenic variant of hepatitis B virus (HBV), HDV was soon found to be a defective virus that depends on an underlying or simultaneous hepatitis B infection. The clinical signiﬁcance of HDV infection is more severe acute and chronic liver disease than that caused by the HBV infection alone. The cloning and sequencing of the genome led to the realization that HDV is a unique RNA virus whose closest known relatives are plant viroids, but even that relationship is remote. In the current classiﬁcation scheme of the International Congress on the Taxonomy of Viruses, HDV remains the sole member of a ﬂoating genus, Deltavirus. The genome and its replication cycle bear no discernable resemblance to its helper virus, HBV, on which HDV depends for its envelope. At 1,680 nucleotides the HDV genome is the smallest known to infect man. The virus contains just one gene, which encodes an approximately 25-kDa protein, hepatitis delta antigen (HDAg, also sometimes referred to as delta protein or delta antigen). To compensate for this limited protein coding capacity, HDV relies heavily on host functions and on the structural dynamics of its circular RNA genome. Although HDV RNA is circular, it forms a characteristic unbranched rod structure in which over 70% of the nucleotides from Watson–Crick base pairs. One of the more remarkable aspects of HDV is that, unlike other RNA viruses, it does not produce a virally-encoded polymerase; rather, it somehow uses host DNA-dependent RNA polymerase to replicate its RNA genome and transcribe its mRNA. At a minimum, this process involves RNA polymerase II; HDAg also plays an as yet undeﬁned role. The potential involvement of another polymerase, such as polymerase I, or of other forms of RNA polymerase II remains an area of active investigation. RNA replication requires the unbranched rod structure of HDV RNA and occurs via a double rolling circle mechanism. Autocatalytic self-cleaving elements, termed ribozymes, in the genome and its complement, the antigenome, play essential roles in the processing of linear transcripts to circular forms. Ribozyme activity occurs via

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Preface

acid-base catalysis not unlike that accomplished by protein enzymes, and requires a complex double pseudoknot RNA structure. Ribozyme activity is also controlled by the structural dynamics of the RNA: formation of the unbranched rod structure interferes with ribozyme activity and likely prevents cleavage from occurring once the RNA circularized. HDV produces two forms of HDAg that have different roles in the replication cycle. The longer form has an additional 19 or 20 C-terminal amino acids that facilitate viral particle formation; the shorter form is required for RNA replication. The heterogeneity arises due to highly speciﬁc editing of an adenosine in the antigenome RNA by host RNA adenosine deaminase. This process requires particular secondary structure features in the RNA around the editing site. In some cases the unbranched rod structure competes with the conﬁguration required for editing; thus, structural dynamics of the RNA are important not only for HDV ribozyme activity, but for other processes as well. The functional activity of HDAg is affected by numerous post-translational modiﬁcations carried out by host enzymes. These modiﬁcations include farnesylation, phosphorylation, methylation and acetylation. Farnesylation is essential for interaction with the hepatitis B virus surface protein (HBsAg), and is thus required for viral particle formation. The speciﬁc signiﬁcance of the other modiﬁcations, as well as the nature of their effects on HDAg function, are not yet fully understood. Being derived from HBV proteins, the outside of the HDV particle is similar to that of HBV, only slightly smaller in size. Although the receptor for neither virus has been identiﬁed it is likely that attachment and entry occur by similar processes. Infectivity of both HBV and HDV involves elements of the preS1 and antigenic loop regions of HBsAg. Molecular genetic analysis of HDV isolates indicates geographical correlations that in some ways mirror those of its helper virus. That the greatest sequence diversity is found among isolates originating in Africa has led to the proposal that HDV might have radiated from that continent. One enigma is that the most distantly related sequences, for both HDV and HBV, come from South America. There is some evidence that infection with certain genotypes, or clades, can inﬂuence the severity of HDV disease. The mechanisms by which HDV thwarts the immune system to produce chronic infection are not yet understood. The woodchuck model of HDV has been the most accessible animal model of HDV infection and has been used both to analyze the natural history of HDV infection and to evaluate the efﬁcacy of vaccine strategies against the virus. Certainly, development of an effective vaccine strategy has been frustrating. Recent work suggests that HDAg may be poorly immunogenic, and may furthermore undergo genetic changes to avoid those limited immune responses that do occur.

Preface

VII

There are no effective licensed antiviral therapies for HDV, and although several therapies exist for combating its helper, HBV, none of these treatments affect HDV. This failure is due to the fact that HDV depends only on HBsAg production of the helper, and current anti-HBV therapies are not potent enough to signiﬁcantly diminish HBsAg levels, which are extraordinarily high. However, two potential therapeutic approaches have shown promise. One targets the host farnesyltransferase activity, which is required for virus production; the other approach advocates reducing HBsAg to levels that are too low to support continued HDV secretion. Both of these approaches are based to varying degrees on an understanding of the molecular virology of HDV, and it is likely that additional therapeutic avenues will be opened as our knowledge of HDV expands. The more we continue to learn about hepatitis delta virus the more fascinating it becomes. It is my hope that this book will stimulate additional interest in hepatitis delta virus among scientists, academic researchers and advanced students. I would like to thank the authors for their contributions, and the staff at Springer and members of my laboratory for their assistance in preparing this volume. Washington, DC, March 2006

John L. Casey

List of Contents

Structure and Replication of Hepatitis Delta Virus RNA . . . . . . . . . . . . . . . . . . J. M. Taylor

1

HDV RNA Replication: Ancient Relic or Primer? . . . . . . . . . . . . . . . . . . . . . . . 25 T. B. Macnaughton and M. M. C. Lai HDV Ribozymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 M. D. Been RNA Editing in Hepatitis Delta Virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 J. L. Casey Post-translational Modiﬁcation of Delta Antigen of Hepatitis D Virus . . . . . . . . 91 W.-H. Huang, C.-W. Chen, H.-L. Wu, and P.-J. Chen The Role of the HBV Envelope Proteins in the HDV Replication Cycle . . . . . . . . 113 C. Sureau Prenylation of HDAg and Antiviral Drug Development . . . . . . . . . . . . . . . . . . . 133 J. S. Glenn Hepatitis Delta Virus Genetic Variability: From Genotypes I, II, III to Eight Major Clades? . . . . . . . . . . . . . . . . . . . . . . . 151 P. Dény Functional and Clinical Signiﬁcance of Hepatitis D Virus Genotype II Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . 173 J.-C. Wu Immunology of HDV Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187 M. Fiedler and M. Roggendorf The Woodchuck Model of HDV Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211 J. L. Casey and J. L. Gerin Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226

List of Contributors (Addresses stated at the beginning of respective chapters)

Been, M. D. 47

Lai, M. M. C. 25

Casey, J. L. 67, 211 Chen, C.-W. 91 Chen, P.-J. 91

Macnaughton, T. B. 25

Dény, P. 151

Sureau, C. 113

Fiedler, M. 187

Taylor, J. M. 1

Gerin, J. L. 211 Glenn, J. S. 133

Wu, H.-L. 91 Wu, J.-C. 173

Huang, W.-H. 91

Roggendorf, M. 187

CTMI (2006) 307:1–23 c Springer-Verlag Berlin Heidelberg 2006

Structure and Replication of Hepatitis Delta Virus RNA J. M. Taylor (u) Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111-2497, USA [email protected]

1

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2

2 2.1 2.2 2.3 2.4 2.5

RNAs and Ribonucleoproteins . Genome and Antigenome . . . . mRNA . . . . . . . . . . . . . . . . . . Other HDV RNAs . . . . . . . . . . RNA Structure . . . . . . . . . . . . Ribonucleoproteins . . . . . . . .

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2 2 3 4 5 6

3 3.1 3.1.1 3.1.2 3.1.3 3.2 3.3 3.4 3.5 3.6 3.6.1 3.6.2 3.6.3 3.6.4 3.7

RNA Replication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Roles of Delta Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Essential Small Delta Protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . Other Forms of Delta Protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Replication with an Unchanging Separate Source of Small Delta Protein . Enzymology of RNA-Directed Transcription . . . . . . . . . . . . . . . . . . . . Initiation of Replication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Double Rolling-Circle Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Template-Switching, Reconstitution, and Recombination . . . . . . . . . . . Inhibition of Replication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Resistance to Interferons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sensitivity to Ribavirin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sensitivity to siRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Resistance to Dicer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cytopathic Effect of Replication . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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7 7 7 7 8 8 9 11 12 14 14 14 15 15 15

4 4.1 4.2 4.3

Evolution of the RNA Sequence Accumulation of Changes . . . . ADAR-Editing . . . . . . . . . . . . Origin . . . . . . . . . . . . . . . . . .

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16 16 17 17

5

Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Abstract While this volume covers many different aspects of hepatitis delta virus (HDV) replication, the focus in this chapter is on studies of the structure and replication of the HDV RNA genome. An evaluation of such studies is not only an integral part of our understanding of HDV infections but it also sheds new light on some

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important aspects of cell biology, such as the ﬁdelity of RNA transcription by a host RNA polymerase and on various forms of post-transcriptional RNA processing. Representations of the replication of the RNA genome are frequently simpliﬁed to a form of rolling-circle model, analogous to what have been described for plant viroids. One theme of this review is that such models, even after some revision, deceptively simplify the complexity of HDV replication and can fail to make clear major questions yet to be solved.

1 Introduction Other reviews on the topic of hepatitis delta virus (HDV) RNA structure and replication have been previously published (Cunha et al. 2003; Gerin et al. 2001; Taylor 2003, 2004). Moreover this volume contains current reviews on other aspects of HDV infection, in addition to one chapter on HDV replication (see chapter by T.B. Macnaughton and M.M.C. Lai, this volume). The objective of this chapter therefore will be to not only review information regarding HDV genome structure and replication, but also to consider what might be new insights and to point to questions yet to be solved. Over the years HDV has provoked interest because of the many unique features associated with its replication including RNA-directed transcription by a host enzyme, ribozyme domains, and essential RNA editing. It has also been associated with a deceptive simplicity: a very small genome encoding only one or two viral proteins to account for, and a rolling-circle model of replication that seems plausible. However, as described in this chapter, we are becoming aware of a greater complexity associated with the replication of this apparently simple virus.

2 RNAs and Ribonucleoproteins 2.1 Genome and Antigenome The genome of HDV is a small RNA of about 1,700 nucleotides in length with a circular conformation. We often refer to this RNA as single-stranded; however, based on predictions from the nucleotide sequence and certain experimental studies, we are convinced that this RNA can fold on itself via intra-molecular base pairing to form an unbranched rod-like structure. In this way, about 74% of all the nucleotides are involved in base pairing (Sect. 2.4).

Structure and Replication of Hepatitis Delta Virus RNA

3

The HDV genome, by deﬁnition, is the RNA species that is incorporated into new virus particles during an assembly process that depends upon envelope proteins provided by the helper hepadnavirus (see chapter by C. Sureau, this volume). However, inside a cell undergoing HDV genome replication, in addition to the genomic RNA, there are also many copies of an exactly complementary RNA, referred to as the antigenome (Chen et al. 1986). This antigenomic RNA contains an open reading frame for a 195-amino acid species, the small delta protein (S-HDAg), which is essential for genome replication, is apparently not translated from the circular antigenomic RNA, but from a less than genome-sized, polyadenylated RNA species that is present in the cytoplasm (Sect. 2.2). Both the genome and antigenome contain a domain that will act as a ribozyme. As discussed more fully in the chapter by M.D. Been (this volume), these domains are as short as 85 nucleotides in length (Ferre-D’Amare et al. 1998). They are sufﬁcient to allow RNA cleavage in vitro, in the presence of magnesium ions. The cleavage is a site-speciﬁc trans-esteriﬁcation reaction which produces a 5 -OH and a cyclic 2 -, 3 -monophosphate (Kuo et al. 1988b). This cleavage ability is needed for HDV replication (Macnaughton et al. 1993) and is considered to provide post-transcriptional cleavage of greater than unit-length HDV RNA multimers, thus releasing unit-length linear species that can be subsequently ligated to form new RNA circles (Taylor 1990). As considered in Sect. 2.4, the structures of the HDV ribozymes are different from the predicted rod-like folding. 2.2 mRNA The mRNA species for S-HDAg contains little more than the open reading frame. It is 5 -capped and 3 -polyadenylated, just as for a typical host mRNA. Recent studies indicate that S-HDAg mRNA can be bound with an antibody that can recognize 5 -cap structures (Nie et al. 2004). It is also bound by the poly(A) binding protein, PABP, a host protein that binds to the poly(A) sequences of host mRNAs (X. Nie, J. Chang, C. Tarn, C.-M. Chiang, J. Keene, L. Penalva and J. Taylor, unpublished results). For the mRNA there is evidence that the 5 -end has a preferred location at nucleotide 1630 (Gudima et al. 1999). At least for the majority of this mRNA the 5 -end has been modiﬁed to have a cap structure (Gudima et al. 2000; Nie et al. 2004). Therefore it is plausible but not directly proven, that this 5 -end corresponds to a preferred site for the initiation of RNA-directed transcription from a genomic RNA templates (Sect. 3.3).

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At the 3 -end of the mRNA is a poly(A) tail of about 150 adenosines, typical of cellular mRNA species. Consistent with this, the nascent RNA that is processed to become this mRNA, has a poly(A) signal, AAUAAA, and other features expected for an RNA transcript that undergoes polyadenylation. Mutation of this signal inhibits polyadenylation (Nie et al. 2004). 2.3 Other HDV RNAs It should be clear from the above that all three of the major HDV RNAs arise by post-transcriptional RNA processing. This means that for each, there are precursor RNAs, of relatively larger size, that act as the substrates for such processing. There is much more yet to be revealed about how such important precursor RNA species are initiated and how they are processed. 1. As discussed in Sect. 3.4, an unsubstantiated aspect of the rolling-circle model of replication is whether the RNA species that go on to be processed into antigenomic RNA circles are initiated from the same location as those which become mRNA species. 2. For the nascent genomic RNA transcripts we have as yet no clear data as to where they are initiated. One report has suggested, based on RNase protection assays, that there might be a preferred site, near one end of the rod-like RNA, and almost opposite to the site that corresponds to the 5 end of the mRNA (Beard et al. 1996). This result needs to be independently conﬁrmed. 3. Since the nascent transcripts of both the genomic and antigenomic RNA each contain a ribozyme domain per unit length, a transcript that is greater than unit length can contain two or more ribozyme domains, which will lead to ribozyme processing, to release RNA species that are of exactly unit length, and that can then be converted to unit length RNA circles. There is good evidence that the ribozyme domains are needed for the cleavage events to release the unit length linear RNAs (Macnaughton et al. 1993). There is also a report that the subsequent conversion of these to circles depends upon host factors, probably a host RNA ligase (Reid and Lazinski 2000). It will be important to identify this host ligase and determine how it is redirected to act on HDV RNAs. 4. During HDV replication additional processed RNAs that are relatively less abundant can be detected. Northern analyses can detect molecules that seem to be of twice or even three times unit length. Moreover, these species seem to exist in both circular and linear conformations (Chen et al. 1986). Apparently these species arise via alternative processing of multimeric nascent RNA transcripts.

Structure and Replication of Hepatitis Delta Virus RNA

5

2.4 RNA Structure It was promptly deduced from the ﬁrst full sequence of an HDV genome that this RNA could theoretically be folded into an unbranched rod-like structure (Wang et al. 1986). Further theoretical calculations predicted a high negative free energy of 805 kcal/mol, consistent with a stable structure (Kuo et al. 1988a). Experimental evidence also supports such a structure. In electron microscopic studies, the genomic RNA appears as a short double-stranded rod that upon progressive denaturation opens into a circle (Kos et al. 1986). In addition, by gel electrophoresis under nondenaturing conditions, both the genomic and antigenomic RNAs migrate as expected for double-stranded species (Lazinski and Taylor 1995). Moreover, upon prior denaturation, most of the RNAs migrate consistent with a circular conformation, the remainder behaving as linear species (Chen et al. 1986). Similar conclusions apply for the structure of the unit-length antigenome. While the rod-like folding is generally true, the details of the exact folding remain to be determined. One study attempted to use nuclease susceptibility assays to test the folding of a segment at one end of the rod-like structure of the genomic RNA in vitro. The detected folding was very close to that predicted (Beard et al. 1996). It is obvious that the structures of the two ribozyme domains are not compatible with the rod-like folding. Furthermore, folding of these domains into the rod-like structure should inhibit the ribozymes. Intuitively, if this inhibition were not the case the circular RNAs might undergo efﬁcient self-cleavage to form linear RNAs. The prediction that the rod-like folding overrides the ability to fold into the active ribozyme conformation has been proven using both in vivo and in vitro studies of natural and modiﬁed HDV RNAs (Lazinski and Taylor 1993, 1994a). Modiﬁed RNAs that cannot fold the ribozyme domain into a rod-like structure do not form stable circles in vivo. Conversely, unmodiﬁed HDV RNA circles are cleaved only inefﬁciently by ribozymes in vitro. And yet if these RNAs are ﬁrst hybridized with a separate oligonucleotide to stop the ribozyme domain from being inactivated by being drawn into the rod-like folding, then these RNAs are efﬁciently cleaved by ribozymes in vitro. Additional alternative foldings of HDV RNA sequences have also been reported. One such pairing is considered to produce a binding site for the protein PKR (Circle et al. 2003, 1997; Robertson et al. 1996). Another alternative folding, also based on in vitro data, has been proposed to explain a speciﬁc cross-linking induced by irradiation with ultraviolet light (Branch et al. 1989).

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In summary, it should not be surprising that HDV RNAs can, and do have, multiple ways in which they can fold. It is considered that most RNAs undergo a series of alternative foldings, that is, metastable states that can facilitate the molecule ultimately achieving a predominant ﬁnal more stable structure (Uhlenbeck 1995). On top of this, as will be considered in Sect. 2.5, the ﬁnal structure for HDV genomic and antigenomic RNAs probably involves the consequences of S-HDAg binding. 2.5 Ribonucleoproteins Inside a delta virus particle the genomic RNA is bound to molecules of S-HDAg (Bichko et al. 1996; Dingle et al. 1998; Ryu et al. 1993). As discussed later in Sect. 3.1, this protein that is the only one encoded by HDV, exists in two main size classes. The 195-amino acid S-HDAg is essential for HDV replication. During replication RNA editing leads to a change in the amber stop codon of S-HDAg to tryptophan (see chapter by J.L. Casey, this volume). This so-called amber/W mutation, leads to the translation of a protein form that is 19 amino acids longer at the C terminus. The resultant 214 amino acid large delta protein (L-HDAg) is both an inhibitor of replication and is essential, along with the envelope proteins of HBV, for the assembly of new virus particles (Chang et al. 1991; Chao et al. 1990). However, additional ﬁndings indicate that it is only when this L-HDAg is isoprenylated that it can function to assist assembly (Glenn et al. 1992) or to act as an inhibitor of replication (Sato et al. 2004). In natural infections, the assembled HDV particles contain both forms of HDAg in a ribonucleoprotein structure with the HDV genomic RNA (Ryu et al. 1992). In virions there are about 70 molecules of S-HDAg bound per molecule of genomic RNA (Ryu et al. 1993). These interactions are facilitated by an RNA-binding domain that is shared by both forms of HDAg (Lee et al. 1993). Within a cell undergoing HDV genome replication the majority of the accumulated genomic and antigenomic RNAs exist in complexes with S-HDAg. This has been demonstrated by immunoafﬁnity procedures using antibody speciﬁc for S-HDAg; in contrast, the mRNA for HDAg is not in such a complex (Nie, Chang, Taylor, unpublished). These ﬁndings are consistent with earlier reports that in vitro, S-HDAg can speciﬁcally recognize the rod-like folding of the genomic and antigenomic RNAs (Chao et al. 1991). A detailed comparison of the stoichiometry of S-HDAg per RNA has yet to be made for the intracellular ribonucleoprotein (RNP) relative to the virion RNP. Also, it will be important to determine the crystal structure of molecules of S-HDAg bound to a segment of HDV rod-like RNA.

Structure and Replication of Hepatitis Delta Virus RNA

7

3 RNA Replication 3.1 Roles of Delta Proteins 3.1.1 The Essential Small Delta Protein For some time it has been clear that the 195-amino acid S-HDAg is essential for HDV replication (Chao et al. 1990). Many activities of this protein have since been reported, and as summarized in Table 1, many speciﬁc roles in the HDV life cycle have been proposed. However, not yet solved is how many of these proposed roles actually contribute to the essential nature of this protein during HDV replication. It should not be unexpected that this protein will have several roles. 3.1.2 Other Forms of Delta Protein While S-HDAg is essential for replication other forms of the protein arise during replication. The best characterized other form is L-HDAg. It arises as a consequence of RNA-editing at a speciﬁc site, nucleotide 1012, in one genome numbering scheme (Kuo et al. 1988a). This location corresponds to the middle of the amber termination codon of S-HDAg. The RNA editing is carried out by ADAR-1, an adenosine deaminase acting on RNA (Sect. 4.2). The L-HDAg contains a single cysteine, located four amino acids from its novel C terminus. This cysteine is isoprenylated in vivo, and plays an essential role in the ability of this L-HDAg to facilitate virus assembly (Chang et al. 1991; Table 1 Proposed roles of S-HDAg in HDV replication 1. Form an RNP that stabilizes the genome and antigenome (Chao et al. 1991; Lazinski and Taylor 1995) 2. Form an RNP that protects HDV RNAs against ADAR editing (Cheng et al. 2003) 3. Form an RNP that facilitates transport of the HDV genome to the nucleus (Xia et al. 1992) 4. Act as an RNA chaperone to accelerate the HDV ribozyme activities (Huang and Wu 1998; Jeng et al. 1996) 5. Act as a facilitator of processivity during RNA-directed RNA transcription (Yamaguchi et al. 2001)

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Glenn et al. 1992). The L-HDAg does not support HDV genome replication and at least under certain conditions, acts as a dominant negative inhibitor of such replication (Chao et al. 1990; Sato et al. 2004). To consider HDV replication as being associated with just these two forms of HDAg is too simple. One has to factor in the consequences of posttranslational modiﬁcations, such as phosphorylation, acetylation, methylation, and isoprenylation (see chapter by W.-H Huang et al., this volume). In addition, it would seem that there are other RNA editing sites and there are certainly sites at which transcriptional errors occur (Sect. 4). Some of these sequence changes can lead to S-HDAg with altered sequence and functionality. Thus, once HDV replication is underway, there is a real heterogeneity in the amino acid sequence of those species, some of which will electrophoretically migrate the same as the prototypic S-HDAg or L-HDAg (Gudima et al. 2002). This heterogeneity is particularly true in situations where HDV replication is occurring in the absence of packaging followed by virus release and new rounds of infection; that is, when there is no selection for functional HDAg. 3.1.3 Replication with an Unchanging Separate Source of Small Delta Protein Because of some of the problems of delta protein heterogeneity described above in Sect. 3.1.2, a recent study has established a cell system in which HDV replication occurs in the presence of an unchanging DNA-directed source of S-HDAg (Chang et al. 2005a). In this system an integrated cDNA provides a single source, under tetracycline control, of S-HDAg. Added to these cells is an HDV RNA genome previously modiﬁed so that it can no longer express any S-HDAg. This new system provides a better approach to address important questions of HDV replication questions. Some applications will be described in subsequent Sections. 3.2 Enzymology of RNA-Directed Transcription Given that HDV genome replication involves RNA-directed RNA transcription and that S-HDAg is too small to have polymerase activity, it has been clear for some time that one or more host polymerases are required for HDV transcription. This being said, the characterization of such transcription leaves a lot to be desired. Much evidence invokes the interpretation that replication involves the redirection of host DNA-directed RNA polymerase II, Pol II (Macnaughton et al. 2002; Modahl et al. 2000; Moraleda and Taylor 2001). However, a complication is that it has been interpreted that a second polymerase, one that is more resistant to alpha-amanitin than Pol II, might be

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involved in the transcription of genomic RNA templates to produce antigenomic RNAs (Macnaughton et al. 2002; Modahl et al. 2000). In the absence of convincing experimental support it has been interpreted that RNA polymerase I is involved. This in turn has been incorporated into a highly speculative and complex rolling-circle model in which genomic RNA templates are sometimes transcribed by Pol II and other times by Pol I (Macnaughton et al. 2002). It is agreed that the transcription of antigenomic RNA templates into genomic RNA is sensitive to alpha-amanitin at levels consistent with the enzyme being Pol II. The accumulation of the HDV mRNA species is similarly sensitive. Furthermore, there is the circumstantial evidence for Pol II, in that accumulation of this mRNA is dependent upon poly(A) processing signals that in animal cells are only recognized by Pol II (Gudima et al. 2000; Hsieh and Taylor 1991; Nie et al. 2004). One would hope that stronger evidence obtained via robust experimental systems might be available for this important transcription question. The problem is that to date, no one has been able to obtain a reproducible and competent system for in vitro transcription of HDV RNA templates. One early in vitro study cannot be reproduced (Fu and Taylor 1993). Other in vitro studies achieve what is predominantly 3 -end addition to HDV RNAs, a process that might not be of biological relevance (Beard et al. 1996; Filipovska and Konarska 2000; Gudima et al. 2000). Why then has HDV transcription not been better characterized? The answer might be that no one has been able to use in vitro transcription reactions to achieve credible initiation of HDV RNA transcripts. In part, this may be because in vitro transcription will probably make use of ribonucleoprotein structures rather than naked HDV RNAs. Currently some progress is being made by the application of immunoafﬁnity procedures following disruption of cells undergoing a burst of HDV replication (Nie, Chang, Taylor, unpublished), just as others have done for other RNA viruses (Qanungo et al. 2004; Waris et al. 2004). Following such selections, both genomic and antigenomic unit-length HDV RNAs can be found bound to S-HDAg, and a fraction of these are also bound to RNA polymerase II (Nie, Chang, Taylor, unpublished). However, such complexes will have to be proven as competent for in vitro transcription and they will have to be carefully characterized for all the host proteins present. 3.3 Initiation of Replication In a natural infection, a receptor-mediated interaction of the virus with the host cell leads to the viral RNP reaching the nucleus and then initiating RNA-

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directed RNA transcription. Such infections have also been initiated using cultured primary hepatocytes but never with established cell lines. To initiate HDV replication in cell lines, many different strategies have been used. Some are listed in Table 2. Several important points need to be made about these systems: (1) S-HDAg has to be present at or soon after the transfection; (2) the in vitro RNAs and the in vivo DNA-directed transcripts are linear RNAs, not circular, as in the virions; (3) in some cases, even the total nucleic acid extracted from a cell undergoing replication, can be transfected into new cells and is sufﬁcient to initiate genome replication; (4) for the method described in Sect. 3.1.3, a low level of replication is initiated by transfecting HDV RNA into cells continuously expressing only a low level of S-HDAg. When these cells are induced by addition of TET to express large amounts of S-HDAg, there can be a rapid burst of replication; presumably this is because the RNA templates are already circular species resident within the cells. It has to be realized that in many transfection studies the HDV replication is initiated by nucleic acid species that are different from the circular genomic RNA present in the virions that initiate a natural infection. For some of these transfections the initial HDV RNA templates were greater than unit-length tandem multimers. It was considered that since each unit-length RNA, be it genomic or antigenomic, will contain a functional ribozyme, then initiation of replication might need an initial conversion of the RNA to unit-length linear and then to a circle, presuming that such circles might be the preferred template for RNA-directed transcription. While this may be true, recent studies show that linear RNAs, even unit-length RNAs, do not necessarily have to

Table 2 Components used for transfections that can initiate HDV genome replication in cell lines 1. 2. 3. 4.

HDV virions (Bichko et al. 1994) HDV RNP from virions (Bichko et al. 1994) HDV cDNA in expression vectors (Kuo et al. 1989) HDV RNA transcribed in vitro, into cells expressing S-HDAg (Glenn et al. 1990) 5. HDV RNA transcribed in vitro, pre-mixed with recombinant small S-HDAg (Dingle et al. 1998a) 6. HDV RNA transcribed in vitro, together with in vitro transcribed mRNA that can be translated into S-HDAg (Modahl and Lai 1998) 7. As in 4, but using total RNA extracted from cells in which replication was occurring (Gudima et al. 2004)

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be converted to circles before they can be transcribed to initiate replication (Chang and Taylor 2002). This result has made us aware that even after the initiation a natural infection, some of the nascent HDV RNA species that are noncircular and possibly of unit length or greater, might actually function as templates for transcription. To assess this contribution, a competition assay between RNA circles and linear HDV RNAs was able to show that unit-length circles are about 15 times better at initiation than linear RNAs. Also, genomic and antigenomic RNAs are of equal efﬁciency in initiation (Gudima et al. 2004). Many questions remain regarding the initiation of replication in a natural infection. Does S-HDAg function directly in this process and if so, how? What site(s) on the genomic RNA does the host polymerase recognize to facilitate such initiation? From what sites does the initiation actually take place? Are we correct in presuming that the 5 -end of the mRNA arose via sitespeciﬁc primer-independent initiation? Later, when new antigenomic RNAs are produced, again what does the host polymerase bind to and where is transcription initiated from? 3.4 Double Rolling-Circle Model For some time, attempts have been made to create models for the replication of HDV RNAs (Flint et al. 2004; Gerin et al. 2001; Macnaughton et al. 2002; Taylor 1990). Most of these attempts have borrowed from the concept of a ‘rolling-circle model of replication.’ This idea was applied previously to the replication of RNAs of the plant viroids (Branch and Robertson 1984), with which HDV has numerous similarities, as discussed in detail elsewhere (Taylor 1999). The viroid RNAs differ from HDV RNAs in that they are several times smaller and are noncoding. This, together with the fact that plant viroid RNAs are never assembled into virus-like particles, means that the concept of a viroid ‘genome’ has to be different for HDV. For some viroids RNA-directed RNA replication leads to the accumulation of unit-length RNA circles of both polarities. In contrast, for most viroids, circles of only one polarity can be found. For the complementary strand the RNA template is a linear RNA multimer of unit-length. It is considered that these linear RNAs act as templates for multimeric transcripts that can be processed to unit-length circular RNAs. Thus, for these two classes of viroids the replication models are described as double- and single-rolling circle models, respectively. For HDV, since both the genome and antigenome exist as unit-length circles, it was quickly extrapolated from the viroids, that the replication would

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Table 3 Some revisions to rolling-circle model of HDV genome replication 1. Nascent antigenomic transcripts can be ribozyme-cleaved independent of polyadenylation (Nie et al. 2004) 2. Nascent antigenomic transcripts can be polyadenylated independent of ribozyme cleavage (Nie et al. 2004) 3. Nascent antigenomic transcripts, undergo polyadenylation or ribozyme-cleavage as largely alternative processing events (Nie et al. 2004) 4. While circular forms of genomic and antigenomic RNA are preferred templates for RNA-directed transcription, linear forms can also act (Gudima et al. 2004)

be a double-rolling circle model. Furthermore, this basic model had to be adapted, to allow for the fact that a polyadenylated mRNA was also being produced. This model, with time, has required a number of additional modiﬁcations, some of which are listed in Table 3. Without resorting to a diagram, Table 4 attempts to describe the series of events that could be considered as essential steps in HDV genome replication. At the same time it should be clear from this review, and maybe from the other reviews in this volume, that for many of these steps we have yet to obtain actual experimental evidence. 3.5 Template-Switching, Reconstitution, and Recombination From studies of HDV replication as initiated by the transfection of linear HDV RNAs, it is clear that template-switching can occur during transcription of the transfected RNA. The best evidence for this is that when cells are transfected with linear RNAs that are one or two nucleotides less than unitlength, replication can be initiated but there are speciﬁc deletions and even nontemplated additions, on the RNAs that replicate (Chang and Taylor 2002). With this knowledge that template-switching can occur, experiments were undertaken in which the transfected RNA was replaced by two RNAs, each of which was signiﬁcantly less than unit-length, but which together provided representation of the whole genome. Following transfection with such RNAs, genome replication was detected, consistent with reconstitution of the HDV genome (Gudima et al. 2005). Such reconstitution was only achieved when the two RNA templates were pre-associated before the transfection. In fact, all of the available data concerning HDV template-switching is consistent with the role of the rod-like folding as a facilitator. That is, the inter-molecular association achieved prior to transcription depends on utilization of base-pairings normally considered to be part of the intra-molecular rod-like

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Table 4 Some key steps in HDV RNA-directed RNA transcription during a natural infection 1. Following virus attachment and entry the genomic RNA, as a ribonucleoprotein (RNP) complex with S-HDAg, is transported to the nucleus 2. This RNP is able to redirect either an inactive Pol II complex or one already active in DNA-directed RNA transcription 3. One of the sites for initiation of transcription on the genomic RNA corresponds to position 1630, the 5 -end of the mRNA 4. Following initiation, elongation using the circular RNA template, can produce antigenomic transcripts that are greater than unit length 5. Such nascent antigenomic RNA transcripts can be processed either to mRNA or to unit-length circular antigenomic RNA, both of which are relatively much more stable than the nascent transcript 6. The mRNA species are transported to the cytoplasm and the translation product, new S-HDAg, returns to the nucleus to support more RNA-directed RNA transcription. It may also alter the balance of processing in step 5 7. Transcription of new antigenomic RNA templates occurs, with the nascent transcripts being processed to form new unit-length genomic RNAs 8. ADAR editing can occur on all nascent genomic and antigenomic RNAs, and/or on processed unit-length RNAs. Essential to the life cycle is that some nascent antigenomic RNA and/or processed unit-length antigenomic RNAs, become a target for speciﬁc ADAR-editing at position 1012, leading to the production and translation of mRNAs encoding L-HDAg 9. After editing and maybe other sequence changes, translation produces altered forms of HDAg, especially of L-HDAg, that fail to support or even act as inhibitors of further RNA-directed transcription. In addition, L-HDAg, after isoprenylation, can complex with genomic RNA that in turn can interact with the envelope proteins of the helper virus HBV, if present, to achieve assembly and release of new virus particles

folding. Subsequently, it is considered that these new base-pairings within the RNA hybrid template force pauses in transcription at locations which allow template-switching to occur and achieve reconstitution of replication competent HDV RNA. It might be argued that the above examples of template-switching, although achieved within a cell rather than in vitro, are more relevant to the question of what an RNA polymerase can do, rather than to how an HDV genome is normally replicated. However, such studies are relevant to the question of whether there can be recombination between HDV RNA genomes. Some data from examination of patients infected with two different HDV genotypes have been interpreted as evidence for inter-molecular recombina-

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tion (Wang and Chao 2005; Wu et al. 1999). Also, it has been asserted that recombination can be achieved in cells transfected with two different HDV genotypes (Wang and Chao 2005). However, these data are not yet convincing and it is known that other attempts involving transfected RNAs have proven negative (Gudima et al. 2005). 3.6 Inhibition of Replication 3.6.1 Resistance to Interferons Interferons are often used as part of treatment therapies for HDV (as discussed in chapters by J.S. Glenn and J.L. Casey and J.L. Gerin, this volume). However, when applied to HDV replication as it occurs in cultured cells in the absence of HBV, no such inhibition has been detected with interferons α or γ (Chang et al. 2006; Ilan et al. 1992; McNair et al. 1993). In apparent contrast, a fraction of patient therapies are successful with high dose interferon treatments (Kleiner et al. 1993; Lau et al. 1999). However, it might be that these treatments are interfering with HDV indirectly, by inhibiting the helper virus HBV. 3.6.2 Sensitivity to Ribavirin Some years ago it was shown that ribavirin could block the replication of HDV in primary woodchuck hepatocytes (Choi et al. 1989; Rasshofer et al. 1991). Recent studies conﬁrm that this occurs in cell lines undergoing HDV replication (Chang et al. 2006). Moreover, this inhibition can be achieved with 30 µM ribavirin, a dose low enough to avoid cell toxicity. In contrast to this, others have cited that ribavirin treatments for HDV-infected patients are not effective (Hoofnagle 1998). However, given that ribavirin treatment is demonstrably selective for HDV replication in cultured cells, further studies in patients may be warranted. What might seem a possible hindrance to patient studies is that a side effect of ribavirin treatment can be anemia (GalbanGarcia et al. 2000). However, such side effects can be controlled, as judged by the current acceptance of ribavirin (combined with pegylated interferon) as part of a treatment for chronic hepatitis C virus infection. Also, ribavirin might be replaced with viramidine, an immediate precursor to ribavirin, a drug that more speciﬁcally targets the liver and should have fewer side effects (Lin et al. 2003). This drug, when applied to cultured cell lines at an appropriate concentration, can also speciﬁcally inhibit HDV genome replication (Chang et al. 2006).

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3.6.3 Sensitivity to siRNA Small interfering RNAs (siRNA) are short double-stranded RNAs of about 21 base pairs. They have offered great promise as ways to interfere with virus replication and therapies in humans are already underway. Many RNA viruses have been tested and found susceptible to siRNA attack (Bitko and Barik 2001; Coburn and Cullen 2002; Ge et al. 2003; Gitlin et al. 2002). Consistent with this, the replication of HDV in cultured cells can be inhibited by transfection of appropriate siRNA species (Chang and Taylor 2003). A caveat here is that only siRNA targeted against the HDV mRNA produce such inhibition. Those targeted against other regions on the genome or antigenome do not block replication. One possible reason for resistance is that the genomic and antigenomic RNAs are located in the nucleus, away from the RISC complex that is considered to mediate siRNA action. Another possibility is that the binding of S-HDAg to these RNAs confers resistance to siRNA-mediated degradation. 3.6.4 Resistance to Dicer Dicer is an enzyme present in animal cells that can act on RNA species that have 100% base pairing, to release siRNA species (He and Hannon 2004). Dicer also plays a role in the cleavage of microRNAs. These, like siRNA, are of about 21 nucleotides in length. They are derived from regions of RNA transcripts that have signiﬁcant levels of intra-molecular base pairing. Such precursors are ﬁrst cleaved in the nucleus by an enzyme known as drosha (Lee et al. 2003). The fragments produced are frequently RNA hairpins of about 70 nucleotides, with extensive but <100% base pairing. These precursors are cleaved further in the cytoplasm by dicer to release the microRNAs. The similarity between such precursors and the rod-like folding of HDV genomic and antigenomic RNAs is striking and leads to the question of whether HDV RNAs are also cleaved by dicer. Furthermore, there were reports that during the replication of two plant viroids, each with rod-like RNA genomes, that siRNA were detected (Itaya et al. 2001; Martinez De Alba et al. 2002). However, when an examination for siRNA was made for HDV RNAs under various conditions of replication, no siRNA could be detected nor could any siRNA be generated when in vitro transcribed HDV RNA species were subjected to recombinant dicer (Chang et al. 2003). 3.7 Cytopathic Effect of Replication From histological examinations of liver at the peak of natural and experimental HDV infections, whether fulminant or nonfulminant, it has been concluded

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that HDV replication can be cytopathic and that this can occur independently of inﬁltrating lymphocytes (Gowans and Bonino 1993). In contrast, for HDV replication in cultured cells the picture has been controversial (Lazinski and Taylor 1994b). In some cases, overexpression of no more than the delta protein has been sufﬁcient to cause cell death (Chang et al. 2000; Gowans et al. 1991; Liu et al. 2001). Also, transient replication of the HDV genome has been shown to interfere with cell colony forming ability (Wang et al. 2001). In other cases, neither expression of HDAg nor replication of the HDV genome has been sufﬁcient to induce detectable cell death (Lazinski and Taylor 1994b). In part, the variability of these results may be due to a combination of two factors. First, most replication initiated in cultured cells involves no cell-to-cell spread, and second, HDV replication in cultured cells becomes self-limiting within days because of the accumulation of mutated forms of S-HDAg which no longer support HDV genome replication. Consistent with this explanation are studies of replication in a cell system with an unchanging source of S-HDAg (Sect. 3.1.3). In such studies, low levels of replication are not detectably cytotoxic and replication can be maintained for more than a year. However, induction of an increased level of genome replication causes cytopathic effects detectable within 1–2 days. The cells stop growing, there is a speciﬁc accumulation of cells in G1 /G0 phase and within 6 days virtually all the cells have become nonadherent and proceed to cell death (Chang et al. 2005).

4 Evolution of the RNA Sequence 4.1 Accumulation of Changes As discussed in the chapter by P. Dény of this volume, from characterization of the many isolates of HDV from infected patients around the world, the length of the HDV genome can change by as much as 30 nucleotides, and the nucleotide sequence can change beyond 30% (Radjef et al. 2004). In the experimental situation in which an animal is infected with a single sequence of HDV, there can quickly accumulate a small number of single nucleotide changes and even nucleotide deletions (Gudima et al. 2002; Netter et al. 1995). Similarly, in the new model described in Sect. 3.1.2, when low amounts of functional S-HDAg are provided from a DNA master copy, the genome can continue replication for at least a year and accumulates many changes (Chang et al. 2005b). Most of the changes are single nucleotide substitutions although there are some single nucleotide deletions but no major changes in

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the genome size. This observed ability of HDV to continue replication in the absence of any (apparent) selective pressure and despite the accumulation of numerous sequence changes, demonstrates how successful and adaptive such a noncoding selﬁsh RNA can be. The similarities of this to the replication of the plant viroids are striking, with the exception that because of the natural intercellular communication that exists between plant cells, the viroids–unlike HDV–do not need any helper virus to spread from cell to cell. 4.2 ADAR-Editing After 1 year of HDV genome replication in culture in the presence of an exogenous source of S-HDAg, more than 97% of all genomes are edited at position 1012, the editing site responsible for the ability to switch from translation of S-HDAg protein to L-HDAg (Chang and Taylor 2005b). However, there are many other changes that accumulate in the HDV RNA. Moreover, 90% of these could be explained as ADAR-editing of either genomic or antigenomic strands. Thus, while it is tempting to see that change at position 1012 as site speciﬁc, we must admit that many other changes can occur and we detect whatever can be tolerated in the situation; that is, even within a few days of replication in culture, there are strong selective pressures for those HDV RNAs that can be replicated and are able to accumulate. 4.3 Origin For some time the similarities between HDV and the plant viroids have been apparent (Taylor 1999). The two major differences are that HDV has a genome at least four times bigger than that of viroids, and this genome encodes a protein, whereas the viroids encode no known proteins. Plants can also be infected by another class of agents that are known as viroid-like satellite RNAs. These also have small single-stranded RNA genomes that are frequently circular and sometimes encode a single small protein. These virusoids might seem more similar in that they, like HDV, need a helper virus. However, for virusoids the helper function is to provide the RNA polymerase activity. At one time it was suggested that HDV might have arisen via templateswitching between a putative human viroid and a host mRNA (Brazas and Ganem 1996; Robertson 1996). This model remains unlikely for several reasons, especially since we have yet to ﬁnd a single human viroid. Furthermore, it is relevant to note the following recent study (Gudima et al. 2005). A 5 -capped and 3 -polyadenylated mRNA for S-HDAg was pre-associated with a linear

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antigenomic RNA that lacked most of the open reading frame for S-HDAg. When these were pre-annealed and then transfected into a cell (expressing the S-HDAg) the reconstitution of replicating HDV RNA was detected. However, without this pre-annealing, there was no reconstitution. Furthermore, the linear antigenomic RNA was not able to achieve HDV reconstitution using abundant copies of DNA-directed HDV mRNA that were also present in the recipient cells.

5 Outlook It is good news that even before we can understand such things as the origin of HDV, we are seeing the demise of HDV as a clinically relevant infectious agent (Gaeta et al. 2000). The number of chronic HDV carriers is decreasing, maybe largely due to decreases in the number of HBV carriers as a result of increasing vaccination programs. However, HDV remains as one of the most interesting of animal viruses. HDV has so many unique features. It also serves as an important model for studies of RNA biology. In the future we expect to see a detailed understanding of HDV RNA-directed RNA transcription. What is it about this RNA, together with help from S-HDAg, that allows such successful redirection of a host polymerase? While it may currently be very difﬁcult to reconstitute RNA-directed transcription in vitro, a more accessible approach, already solved for other animal viruses (Qanungo et al. 2004), might be the isolation of transcription-competent replication complexes. Acknowledgements I especially thank the following members of the lab: Jinhong Chang, Severin Gudima, and Xingcao Nie, for discussions regarding this manuscript and for allowing citation of unpublished studies. Additional constructive comments on the manuscripts were provided by Glenn Rall and Richard Katz. Also, I thank collaborators Cheng-Ming Chiang, Jack Keene, and Luiz Penalva. Funding was provided by grants AI-26522 and CA-06927 from the N.I.H., and by an appropriation from the Commonwealth of Pennsylvania.

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Branch AD, Benenfeld BJ, Baroudy BM, Wells FV, Gerin JL, Robertson HD (1989) An ultraviolet-sensitive RNA structural element in a viroid-like domain of the hepatitis delta virus. Science 243:649–652 Branch AD, Robertson HD (1984) A replication cycle for viroids and small infectious RNAs. Science 223:450–455 Brazas R, Ganem D (1996) A cellular homolog of hepatitis delta antigen: implications for viral replication and evolution. Science 274:90–94 Chang FL, Chen PJ, Tu SJ, Chiu MN, Wang CJ, Chen DS (1991) The large form of hepatitis δ antigen is crucial for the assembly of hepatitis δ virus. Proc Natl Acad Sci USA 88:8490–8494 Chang J, Gudima SO, Tarn C, Nie X, Taylor JM (2005a) Development of a novel system to study HDV genome replication. J Virol 79:8182–8188 Chang J, Gudima SO, Taylor JM (2005b) Evolution of hepatitis delta virus RNA genome following long-term replication in cell culture. J Virol 79:13310–13316 Chang J, Nie X, Gudima S, Taylor J (2006) Action of inhibitors on accumulation of processed hepatitis delta virus RNAs. J Virol (in press) Chang J, Moraleda G, Taylor J (2000) Limitations to the replication of hepatitis delta virus in avian cells. J Virol 74:8861–8866 Chang J, Provost P, Taylor JM (2003) Resistance of human hepatitis delta virus RNAs to dicer action. J Virol 77:11910–11917 Chang J, Taylor J (2002) In vivo RNA-directed transcription, with template switching, by a mammalian RNA polymerase. EMBO J 21:157–164 Chang J, Taylor JM (2003) Susceptibility of human hepatitis delta virus RNAs to small interfering RNA action. J Virol 77:9722–9731 Chao M, Hsieh S-Y, Taylor J (1990) Role of two forms of the hepatitis delta virus antigen: evidence for a mechanism of self-limiting genome replication. J Virol 64:5066–5069 Chao M, Hsieh S-Y, Taylor J (1991) The antigen of hepatitis delta virus: examination of in vitro RNA-binding speciﬁcity. J Virol 65:4057–4062 Chen P-J, Kalpana G, Goldberg J, Mason W, Werner B, Gerin J, Taylor J (1986) Structure and replication of the genome of hepatitis δ virus. Proc Natl Acad Sci USA 83:8774– 8778 Choi SS, Rasshofer R, Roggendorf M (1989) Inhibition of hepatitis delta virus RNA replication in primary woodchuck hepatocytes. Antiviral Res 12:213–222 Circle DA, Lyons AJ, Neel OD, Robertson HD (2003) Recurring features of local tertiary structure elements in RNA molecules exempliﬁed by hepatitis D virus RNA. RNA 9:280–286 Circle DA, Neel OD, Robertson HD, Clarke PA, Mathews MB (1997) Surprising speciﬁcity of PKR binding to delta agent genomic RNA. RNA 3:438–448 Coburn GA, Cullen BR (2002) Potent and speciﬁc inhibition of human immunodeﬁciency virus type 1 replication by RNA interference. J Virol 76:9225–9231 Cunha C, Freitas N, Mota S (2003) Developments in hepatitis delta research. The Internet Journal of Tropical Medicine Dingle K, Moraleda G, Bichko V, Taylor J (1998) Electrophoretic analysis of the ribonucleoproteins of hepatitis delta virus. J Virol Methods 75:199–204 Ferre-D’Amare AR, Zhou K, Doudna JA (1998) Crystal structure of a hepatitis delta virus ribozyme. Nature 395:567–574

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Ilan YM, Klein A, Taylor J, Tur-Kaspa R (1992) Resistance of hepatitis delta virus replication to alpha interferon treatment in transfected human cells. J Infect Dis 166:1164–1166 Itaya A, Folimonov A, Matsuda Y, Nelson RS, Ding B (2001) Potato spindle tuber viroid as inducer of RNA silencing in infected tomato. Mol Plant Microb Interact 14:1332–1334 Kleiner D, Di Bisceglie AM, Axiotis CA, Hoofnagle JH (1993) Prolonged alpha interferon therapy for chronic delta hepatitis: effect on liver histopathology. Prog Clin Biol Res 382:365–371 Kos A, Dijkema R, Arnberg AC, van der Meide PH, Schellekens H (1986) The hepatitis delta (δ) virus possesses a circular RNA. Nature 323:558–560 Kuo MY-P, Goldberg J, Coates L, Mason W, Gerin J, Taylor J (1988a) Molecular cloning of hepatitis delta virus RNA from an infected woodchuck liver: sequence, structure, and applications. J Virol 62:1855–1861 Kuo MYP, Sharmeen L, Dinter-Gottlieb G, Taylor J (1988b) Characterization of selfcleaving RNA sequences on the genome and antigenome of human hepatitis delta virus. J Virol 62:4439–4444 Lau DT, Kleiner DE, Park Y, Di Bisceglie AM, Hoofnagle JH (1999) Resolution of chronic delta hepatitis after 12 years of interferon alfa therapy. Gastroenterology 117:1229–1233. Lazinski DW, Taylor JM (1993) Relating structure to function in the hepatitis delta virus antigen. J Virol 67:2672–2680 Lazinski DW, Taylor JM (1994a) Expression of hepatitis delta virus RNA deletions: cis and trans requirements for self-cleavage, ligation, and RNA packaging. J Virol 68:2879–2888 Lazinski DW, Taylor JM (1994b) Recent developments in hepatitis delta virus research. Adv Virus Res 43:187–231 Lazinski DW, Taylor JM (1995) Intracellular cleavage and ligation of hepatitis delta virus genomic RNA: Regulation of ribozyme activity by cis-acting sequences and host factors. J Virol 69:1190–1200 Lee C-Z, Lin J-H, McKnight K, Lai MMC (1993) RNA-binding activity of hepatitis delta antigen involves two arginine-rich motifs and is required for hepatitis delta virus RNA replication. J Virol 67:2221–2227 Lee Y, Ahn C, Han J, Choi H, Kim J, Yim J, Lee J, Provost P, Radmark O, Kim S and others (2003) The nuclear RNase III Drosha initiates microRNA processing. Nature 425:415–419 Lin CC, Yeh LT, Vitarella D, Hong Z (2003) Viramidine, a prodrug of ribavirin, shows better liver-targeting properties and safety proﬁles than ribavirin in animals. Antivir Chem Chemother 14:145–152 Liu YT, Brazas R, Ganem D (2001) Efﬁcient hepatitis delta virus RNA replication in avian cells requires a permissive factor(s) from mammalian cells. J Virol 75:7489–7493 Macnaughton TB, Shi ST, Modahl LE, Lai MM (2002) Rolling circle replication of hepatitis delta virus RNA is carried out by two different cellular RNA polymerases. J Virol 76:3920–3927 Macnaughton TB, Wang Y-J, Lai MMC (1993) Replication of hepatitis delta virus RNA: effect of mutations of the autocatalytic cleavage sites. J Virol 67:2228–2234

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Martinez De Alba AE, Flores R, Hernandez C (2002) Two chloroplastic viroids induce the accumulation of small RNAs associated with posttranscriptional gene silencing. J Virol 76:13094–13096 McNair ANB, Monjardino J, Cheng D, Thomas HC, Kerr IM (1993) Hepatitis delta virus and the interferon system. Prog Clin Biol Res 382:161–164 Modahl LE, Macnaughton TB, Zhu N, Johnson DL, Lai MMC (2000) RNA-dependent replication and transcription of hepatitis delta virus RNA involve distinct cellular RNA polymerases. Mol Cell Biol 20:6030–6039 Moraleda G, Taylor J (2001) Host RNA polymerase requirements for transcription of the human hepatitis delta virus genome. J Virol 75:10161–10169 Netter HJ, Wu T-T, Bockol M, Cywinski A, Ryu W-S, Tennant BC, Taylor JM (1995) Nucleotide sequence stability of the genome of hepatitis delta virus. J Virol 69:1687–1692 Nie X, Chang J, Taylor JM (2004) Alternative processing of hepatitis delta virus antigenomic RNA transcripts. J Virol 78:4517–4524 Qanungo KR, Shaji D, Mathur M, Banerjee AK (2004) Two RNA polymerase complexes from vesicular stomatitis virus-infected cells that carry out transcription and replication of genome RNA. Proc Natl Acad Sci USA 101:5952–5957 Radjef N, Gordien E, Ivaniushina V, Gault E, Anais P, Drugan T, Trinchet JC, Roulot D, Tamby M, Milinkovitch MC and others (2004) Molecular phylogenetic analyses indicate a wide and ancient radiation of African hepatitis delta virus, suggesting a deltavirus genus of at least seven major clades. J Virol 78:2537–2544 Rasshofer R, Choi SS, Wolﬂ P, Roggendorf M (1991) Interference of antiviral substances with replication of hepatitis delta virus RNA in primary woodchuck hepatocytes. Prog Clin Biol Res 364:223–234 Reid CE, Lazinski DW (2000) A host-speciﬁc function is required for ligation of a wide variety of ribozyme-processed RNAs. Proc Natl Acad Sci USA 97:424–429 Robertson HD (1996) How did replicating and coding RNAs ﬁrst get together? Science 274:66–67 Robertson HD, Manche L, Mathews MB (1996) Paradoxical interactions between human hepatitis delta agent RNA and the cellular protein kinase PKR. J Virol 70:5611–5617 Ryu W-S, Bayer M, Taylor J (1992) Assembly of hepatitis delta virus particles. J Virol 66:2310–2315 Ryu WS, Netter HJ, Bayer M, Taylor J (1993) Ribonucleoprotein complexes of hepatitis delta virus. J Virol 67:3281–3287 Sato S, Cornillez-Ty C, Lazinski DW (2004) By inhibiting replication, the large hepatitis delta antigen can indirectly regulate amber/W editing and its own expression. J Virol 78:8120–8134 Taylor J (1990) Hepatitis delta virus: cis and trans functions needed for replication. Cell 61:371–373 Taylor JM (1999) Replication of human hepatitis delta virus: inﬂuence of studies on subviral plant pathogens. Adv Vir Res 54:45–60 Taylor JM (2003) Replication of human hepatitis delta virus: recent developments. Trends Microbiol 11:185–190 Taylor JM (2004) Structure and replication of hepatitis delta virus RNA. In: Yamaguchi Y, (ed) Hepatitis Delta Virus. Eurekah,

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Uhlenbeck O (1995) Keeping RNA happy. RNA 1:4–6 Wang D, Pearlberg J, Liu YT, Ganem D (2001) Deleterious effects of hepatitis delta virus replication on host cell proliferation. J Virol 75:3600–3604 Wang K-S, Choo Q-L, Weiner AJ, Ou J-H, Najarian C, Thayer RM, Mullenbach GT, Denniston KJ, Gerin JL, Houghton M (1986) Structure, sequence and expression of the hepatitis delta viral genome. Nature 323:508–513 Wang TC, Chao M (2005) RNA recombination of hepatitis delta virus in natural mixedgenotype infection and transfected cultured cells. J Virol 79:2221–2229 Waris G, Sarker S, Siddiqui A (2004) Two-step afﬁnity puriﬁcation of the hepatitis C virus ribonucleoprotein complex. RNA 10:321–329 Wu JC, Chiang TY, Shiue WK, Wang SY, Sheen IJ, Huang YH, Syu WJ (1999) Recombination of hepatitis D virus RNA sequences and its implications. Mol Biol Evol 16:1622–1632

CTMI (2006) 307:25–45 c Springer-Verlag Berlin Heidelberg 2006

HDV RNA Replication: Ancient Relic or Primer? T. B. Macnaughton1 · M. M. C. Lai1,2 (u) 1 Department of Molecular Microbiology and Immunology, Keck School of Medicine,

University of Southern California, Los Angeles, CA 90033, USA 2 Institute of Molecular Biology, Academia Sinica, Nankang, 115 Taipei, Taiwan

[email protected]

1

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

2 2.1 2.2

Hepatitis Delta Virus Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 Structure of HDV and HDV RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 HDAg . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

3 3.1 3.2 3.3 3.4

The HDV Replication Cycle . . . . . . . . . . . . . . Transcription and Replication . . . . . . . . . . . . The Role of HDAg in HDV RNA Replication . . Replication of Genomic vs. Antigenomic RNA: Differences and Similarities . . . . . . . . . . . . . . Mechanism of Transcription and Replication . .

4

Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

. . . . . . . . . . . . . . . . . . . 30 . . . . . . . . . . . . . . . . . . . 33 . . . . . . . . . . . . . . . . . . . 34 . . . . . . . . . . . . . . . . . . . 36 . . . . . . . . . . . . . . . . . . . 37

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 Abstract HDV replicates its circular RNA genome using a double rolling-circle mechanism and transcribes a hepatitis delta antigen-encodeing mRNA from the same RNA template during its life cycle. Both processes are carried out by RNA-dependent RNA synthesis despite the fact that HDV does not encode an RNA-dependent RNA polymerase (RdRP). Cellular RNA polymerase II has long been implicated in these processes. Recent ﬁndings, however, have shown that the syntheses of genomic and antigenomic RNA strands have different metabolic requirements, including sensitives to α-amanitin and the site of synthesis. Evidence is summarized here for the involvement of other cellular polymerases, probably pol I, in the synthesis of antigenomic RNA strand. The ability of mammalian cells to replicate HDV RNA implies that RNA-dependent RNA synthesis was preserved throughout evolution.

1 Introduction Early life is thought likely to have been RNA-based, a phase commonly referred to as ‘the RNA world’. Clearly, a means must have existed that permitted these

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primordial RNA molecules to be copied. Today, however, RNA-dependent RNA copying is regarded as a process reserved exclusively for RNA viruses, but not cellular RNAs. Virtually all RNA viruses (except retroviruses) undergo RNA-dependent RNA replication by a virus-encoded RNA-dependent RNA polymerase (RdRp), which speciﬁcally replicates the viral RNA genome but nothing else. Even satellite viral RNAs, which do not encode their own polymerase, rely on RdRp provided by the coexisting helper virus for their replication. The exceptions to this are hepatitis delta virus (HDV) and the small infectious agents of plants, viroids, neither of which encode an RdRp. Nevertheless, they undergo robust RNA replication once inside the cells. Increasing evidence is emerging to suggest that the ability of cells to copy RNA was not lost in antiquity after all. Most of this comes from plants and lower animal species that have been shown to encode RdRps. These RdRps could potentially be responsible for viroid replication and are also thought to be involved in gene silencing by amplifying the short pieces of RNA prepared by the dicer complex. However, very recently, results in arabidopsis suggest that these cellular RdRps may, in addition, be responsible for maintaining a novel extra-genomic cache of sequence information from generation to generation (Lolle et al. 2005). In contrast, mammalian cells have not been shown to encode any RdRps. Thus, the mechanism of HDV RNA replication is still a mystery although cellular enzymes must be responsible. In this article, evidence from our and other laboratories will be reviewed that indicate that HDV RNA replication likely occurs via a redirection of host cell DNA-dependent RNA polymerases. HDV RNA replication represents the ﬁrst example of RNA copying in mammalian cells; thus, the study of this system may provide a primer to the understanding of what may turn out to be a much more widespread phenomenon.

2 Hepatitis Delta Virus Background HDV was discovered following the detection of a novel antigen-antibody system in hepatitis B virus (HBV) carriers (Rizetto et al. 1977). Currently, HDV is classiﬁed as a subviral satellite of HBV due to an obligate relationship with HBV infections in nature. However, unlike other satellite viruses, the dependence of HDV on HBV is limited solely to the provision of an envelope of hepatitis B surface antigen for virus assembly. Nevertheless, this dependence requires that natural HDV infections occur as either a co-infection with HBV or as a super-infection of HBV carriers, with the resultant disease usually being more severe than that with HBV alone. Following HDV infection, the

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proportion of individuals that develops chronic disease is similar to that for HBV infections. Thus, approximately 5% of co-infected adults and almost all super-infected individuals will become carriers of both HBV and HDV. While there is currently still an estimated 15 million HDV carriers worldwide, public health initiatives established to control HBV have led to a very signiﬁcant and welcome decline in the rate of new HDV infections. 2.1 Structure of HDV and HDV RNA HDV is a small RNA virus consisting of spherical particles of about 36 nm in diameter. The virion itself is comprised of a short (1.7 kb) single-stranded, circular RNA that exists as a ribonucleoprotein complex with the only HDV-encoded protein, hepatitis delta antigen (HDAg). Together these form a roughly spherical core structure that is enveloped by hepatitis B surface antigen (HBsAg). There are approximately 70–200 HDAg molecules per RNA molecule (Ryu et al. 1993; Gudima et al. 2002). The genomic form of HDV RNA does not encode protein. However, the complementary strand (antigenomic HDV RNA), which is detected in HDV-infected cells, contains a single open reading frame (ORF) that is responsible for the synthesis of the HDAg (protein-coding domain, Fig. 1). Thus, by deﬁnition, HDV is a negative-strand RNA virus. Both genomic and antigenomic HDV RNA species exhibit a high degree of intramolecular self-complementarity that allows the respective molecules to fold into an unbranched rod structure under nondenaturing conditions (Kos et al. 1986; Wang et al. 1986; Fig. 1). These semi-double-stranded structures probably serve to stabilize the RNA. Interestingly, these structures are strikingly similar to that adopted by viroid RNAs under the same conditions, although HDV

Fig. 1 Genome organization of HDV RNA. Numbering is based on that of Wang et al. (1986). G, genomic HDV RNA; AG, antigenomic HDV RNA; S-HDAg, small hepatitis delta antigen; L-HDAg, large hepatitis delta antigen

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RNA is three to four times larger. Also like some viroids, HDV RNA possesses ribozyme activities on both genomic and antigenomic strands that catalyze cis-cleavage of their respective RNAs (Kuo et al. 1988; Branch et al. 1989; viroid-like domain, Fig. 1). Both the genomic and antigenomic ribozyme activities are essential for HDV RNA replication (Macnaughton et al. 1993a). These similarities suggest an evolutionary relationship between HDV and viroids. However, in addition to size differences, what sets these two infectious agents apart is that only HDV has the ability to encode protein. At one end of the rod structure, a short region has been identiﬁed that exhibits RNA promoter activity in nuclear extracts (Beard et al. 1996). This region comprises of a stem–loop structure with several bulges, all of which are essential for replication (Beard et al. 1996; Putative G to AG Promoter, Fig. 1). Exactly how this promoter operates is unclear. 2.2 HDAg The ORF encoding HDAg runs almost 75% of the length of the HDV rod RNA structure (Fig. 1) and is the reason why HDV RNA is so much larger than viroid RNA. This ORF exists in two sizes, a small and a large form, on different RNA molecules, with coding capacities for 195 and 214 amino acids, respectively. The difference between these two ORFs is base mutation at position 1015. This results from a speciﬁc editing event that is linked to HDV replication and catalyzed by the cellular enzyme ADAR-1 (adenosine deaminase that acts on RNA-1; Jayan and Casey 2002; Wong and Lazinski 2002). The nucleotide at position 1015 can be an A, which leads to a termination codon UAG, or G, giving UGG (Trp), which allows for the read-through of 19 additional amino acids. The resultant proteins are referred to as smalland large-HDAg, respectively (S-HDAg; L-HDAg), and are translated from an approximately 0.8 kb unspliced, antigenomic-sense, subgenomic transcript which is structurally identical to the conventional cellular mRNAs with both a 5 7-methylguanosine -cap structure and a poly A+ 3 -tail (Gudima et al. 2000; Fig. 1). Notably, only HDV genomes containing the smaller version of the ORF can initiate replication (Glen and White 1991; Macnaughton et al. 2003; see later) such that early in infection only S-HDAg is synthesized. With the exception of the19 aa C-terminal extension in the L-HDAg, the two HDAg species are identical in amino acid sequence and share many biochemical properties. Despite this, these proteins play very different roles in the HDV life cycle. S-HDAg is a crucial activator for the initiation and maintenance of HDV RNA replication in vivo. L-HDAg, which is only synthesized late in infection, is essential for virus packaging. Another function ascribed to L-HDAg

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is the suppression of HDV RNA replication (Chao et al. 1990). However, more recent studies in our laboratory indicated that L-HDAg-mediated suppression occurred only when this protein was expressed abnormally early in the HDV replication cycle (Macnaughton and Lai 2002b). Moreover, when expressed early, only suppression of synthesis of genomic RNA from the antigenomic RNA template, but not vice versa, was observed (Modahl and Lai 2000). When L-HDAg is expressed in the context of the natural replication cycle, it does not inﬂuence steady-state, cellular concentrations of either genomic or antigenomic HDV RNA (Macnaughton and Lai 2002b). These results were disputed recently (Sato et al. 2004), following observations using a mutant HDV genome designed to undergo rapid editing. This mutant expressed a higher level of L-HDAg early after initiation of replication, and HDV replication was rapidly terminated. While these results are consistent with the effects of early, unregulated L-HDAg expression, they do not establish that L-HDAg performs an inhibitory role in natural replication, in which L-HDAg is expressed only late in the infection. This point was noted by the authors, who concluded that at least ‘L-HDAg does not regulate the expression of wild type HDV’ (Sato et al. 2004). Despite the differences between S- and L-HDAg, these proteins also share some common functions, such as stabilization of HDV RNA (Lazinski and Taylor 1994), enhancement of ribozyme activity (Jeng et al. 1996) and RNA chaperon activity (Huang and Wu 1998). A number of functional domains have been identiﬁed within HDAg (Fig. 2), most of which are present on both S- and L-HDAg. Within the amino-terminal one-third there is a coiled-coil domain that promotes protein–protein interactions and is essential for the activator functions of S-HDAg (Xia and Lai 1992; Lazinski and Taylor 1993). Within the middle third are two domains, a nuclear localization sequence (NLS, Fig 2; Xia et al. 1992; Lazinski and Taylor 1993) and an RNA-binding

Fig. 2 Functional domain map of hepatitis delta antigen. NLS, nuclear localization signal; ARM, arginine rich motif; HLH, helix–loop–helix motif; S-HDAg, small hepatitis delta antigen; L-HDAg, large hepatitis delta antigen

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T. B. Macnaughton · M. M. C. Lai

motif that is semi-speciﬁc for HDV RNA (Lin et al. 1990; Chao et al. 1991). The latter domain is comprised of two arginine-rich motifs (ARM, Fig. 2), which are separated by a spacer region that includes a helix–loop–helix (HLH) motif (Lee et al. 1993; Chang et al. 1993). Both ARMs and the spacer region are required for RNA binding and the activation function of S-HDAg (Lee et al. 1993). The C-terminal third is characterized by a stretch of amino acids that is rich in proline and glycine residues, the function of which is not clear. Finally at the very C terminus of L-HDAg there is a four-amino-acid motif (CXXQ) that serves as a substrate for prenylation, with the modiﬁcation occurring at the cystine residue of this quartet (Glenn et al. 1992; Lee et al. 1994; Otto and Casey 1996; also see chapter by J.S. Glenn, this volume). Prenylation alters the conformation of L-HDAg, resulting in the masking of a C-terminal epitope (Hwang and Lai 1994) present on the native S-HDAg. This modiﬁcation also promotes membrane binding and is essential for the interaction between the L-HDAg and HBsAg (de Bruin et al. 1994; Hwang and Lai 1993), which is a critical step in HDV particle assembly. While the C-terminal 19 amino acids of L-HDAg are necessary and sufﬁcient for virus assembly (Lee et al. 1995), this process is enhanced by the presence of S-HDAg (Wang et al. 1994). This enhancement is likely due to a combination of direct effects such as protein–protein binding of S-HDAg with L-HDAg (Wang et al. 1994) and indirect effects such as the recently demonstrated enhancement of prenylation of L-HDAg by the presence of S-HDAg (O’Malley and Lazinski 2005). In addition to prenylation, HDAg undergoes a number of other posttranslational modiﬁcations, which likely play key regulatory roles during the HDV life cycle (see chapter by W.-H. Huang et al., this volume). These modiﬁcations are by phosphorylation (Chang et al. 1988; Mu et al., 1999,et al. 2001), acetylation (Mu et al. 2004) and methylation (Li et al. 2004). Speciﬁcally, Methylation of Arg13, acetylation of Lys72 and phosphorylation of Ser177 and Ser123 have been reported to affect the subcellular localization of HDAg and most of these modiﬁcations are important for antigenomic but not genomic HDV RNA replication (see later).

3 The HDV Replication Cycle Currently, there are no convenient cell culture model systems to study HDV infection. Thus, many of the details of the natural HDV replication cycle are still unclear. To date, most studies of the HDV replication cycle have been carried out using cultured cells transfected with HDV cDNA constructs longer than genome length (1.2× to 3× genomic length) under the control of strong

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foreign promoters. By this technique, the S-HDAg, essential for initiation of HDV RNA replication, is generated either from an mRNA transcribed directly from the transfected HDV cDNA or, alternatively, provided in trans from a co-transfected plasmid. In either case, this experimental approach introduces an artiﬁcial requirement of a DNA-dependent transcription step in order to generate a precursor HDV RNA, which, in turn, leads to subsequent RNA replication. Our laboratory has pioneered an alternative approach that circumvents the need for the cDNA step and involves the transfection of the 1.2× genomic-length HDV RNA together with an mRNA encoding HDAg (Modahl and Lai 1998). This method has led to very signiﬁcant revisions of the previous concepts of HDV RNA established by cDNA transfection. HDV presumably enters cells through a similar cellular receptor to that used by HBV as infection of both these viruses depends on the large form of HBsAg in the envelope (Sureau et al. 1993). The following steps, from uptake, uncoating to delivery of the viral RNA to the nucleus (where RNA replication takes place), are unknown although the latter is most likely reliant on the combined RNA-binding and nuclear localizing abilities of the HDAg present as part of the infecting ribonucleoprotein complex. The next step is HDV RNA replication, which is thought to proceed by a double rolling circle model similar to that proposed for viroids (Branch and Robertson 1984; Fig. 3). In this model, input circular genomic HDV RNA serves as a template for synthesis of the complementary antigenomic strand. As HDV RNA synthesis continues, monomers of antigenomic HDV RNA are cleaved from the growing transcript by the ribozyme activity intrinsic to both polarities of HDV RNA. The resultant antigenomic RNA species are then ligated into a circular form. The latter process was originally thought to be dependent on the self-ligating activity of HDV RNA (Sharmeen et al. 1989) but more recent evidence suggests that this may be carried out by a cellular RNA ligase (Reid and Lazinski 2000). The circularized antigenomic HDV RNA then serves as a template for genomic HDV RNA synthesis, with the subsequent replication steps proceeding by a similar mechanism (Fig. 3). Initially, evidence for this model came from the detection of greater-than-unit HDV RNA intermediates in infected or transfected cells (Chen et al. 1986; Kuo et al. 1989; Macnaughton et al. 1990) and from the observation that mutations interfering with ribozyme activity severely inhibited RNA replication (Macnaughton et al. 1993). More recently, metabolic labeling experiments have provided ﬁrm evidence that HDV RNA replication proceeds by this mechanism (Macnaughton et al. 2002) and that the replication intermediates are likely to be very long (at least 10× genome length). Nevertheless, the replication cycle must be asymmetrical as at least 20 times more genomic than antigenomic HDV RNA is synthesized in infected cells (Chen et al. 1986). The

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Fig. 3 HDV RNA double rolling circle replication. Adapted from the model for viroid replication originally put forward by Branch and Robertson (1984)

origins of replication on both genomic and antigenomic RNAs have not been unequivocally determined, although antigenomic-sense RNA synthesis may initiate from one end of the rod-like structure of HDV RNA downstream of a putative RNA promoter element (Beard et al. 1996; Fig. 1). The replication model above can account for the production of full-length genomic and antigenomic HDV RNA. However, since this model is based on viroid replication, not surprisingly, the synthesis of the antigenomic-sense 0.8-kb HDAg-encoding mRNA does not ﬁt easily into this scheme. The 5 end of this transcript starts at position 1631 just upstream of the initiation codon of the ORF for HDAg (Hsieh et al. 1990; Gudima et al. 2000). The 3 end of the transcript is 76 nucleotides downstream from the termination codon of HDAg and 15 nucleotides downstream of an AAUAAA polyadenylation signal (Hsieh et al. 1990; Fig. 1). The HDAg mRNA transcript is the least abundant of the three HDV RNA species, present in infected cells at approximately 1000 times lower frequency than the genomic species. As HDV RNA replication proceeds, RNA editing occurs at nucleotide 1015 of the genomic sense RNA (Casey et al. 1992) that ultimately leads to the pro-

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duction of L-HDAg. This event initiates the production of new virus particles by promoting the interaction of the HDV ribonuclearprotein complex and HBsAg. How the genomic RNA species is speciﬁcally selected for packaging was a puzzle for a long time, especially as HDV RNA replication is restricted to the nucleus and HBsAg occurs only in the cytoplasm. However, the likely mechanism behind this selection process was revealed recently when it was demonstrated that genomic HDV RNA species is speciﬁcally and rapidly exported to the cytoplasm soon after transcription (Macnaughton and Lai 2002a; see later). Nevertheless, many aspects of virus packaging and secretion are still unclear. The editing process is nonreversible; thus, the extent of RNA editing in the cells must be regulated, so that L-HDAg is not over-produced. Such a feedback inhibition is likely caused by the enhanced accumulation of deleterious mutations particularly near the putative promoter element that is triggered by the editing event (Macnaughton et al. 2003) and/or L-HDAg by itself (Cheng et al. 2003). This mechanism also explains why HDV RNA genomes encoding L-HDAg are noninfectious (Glen and White 1991; Macnaughton et al. 2003). 3.1 Transcription and Replication The ﬁrst event in the HDV replication cycle is likely to be the transcription of the 0.8-kb S-HDAg-encoding mRNA species since this protein is required for initiation of HDV RNA replication (Kuo et al. 1989). Originally, the mechanism for production of this transcript was thought to be an adjunct of rolling circle replication. This proposal, put forward from John Taylor’s laboratory (Hsieh and Taylor 1991), suggested that initiation of antigenomic HDV RNA synthesis from virion RNA occurs at position 1631, just upstream of the HDAg AUG codon. Transcription continues downstream until reaching the poly(A) addition signal, after which the nascent transcript is cleaved and a poly(A)+ tail attached. However, as is the case with eukaryotic mRNA transcription, the polymerase continues to transcribe downstream of the cleavage site. Normally, this downstream transcript is very unstable and is rapidly degraded. However, in the case of HDV, it was proposed that a ribozyme cleavage event occurring 34 nucleotides past the poly(A) cleavage/addition site stabilizes this downstream transcript to allow initiation of rolling circle replication of antigenomic HDV RNA (Hsieh and Taylor 1991). As the replication proceeds, the poly(A) addition signal is thereafter inhibited by a combination of HDAg and intramocular binding with nucleotides on the opposite side of the rod structure (Hsieh and Taylor 1991). Evidence for this model were based on observations using HDV cDNA constructs where transcription was under the control of foreign promoters, often in the absence of HDAg (Hsieh and

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Taylor 1991; Nie et al. 2004). Indeed, under these conditions, it was possible to produce both HDAg mRNA transcripts and full-length antigenomic HDV RNA. However, there are no cDNA intermediates in natural HDV replication. Thus, not surprisingly, this model cannot easily account for several observations made during natural HDV RNA-dependent RNA replication. The most signiﬁcant of these is the implication from this model that mRNA synthesis can occur only once per initiation of replication which, in turn, implies that mRNA would primarily be synthesized early in the RNA replication cycle. This is a very restrictive limitation because HDAg needs to be continually synthesized both for maintenance of replication and for packaging after the RNA editing has occurred. Work in our laboratory has suggested a different hypothesis. In particular, we demonstrated that the amount of the 0.8-kb mRNA initially showed a steady increase and was then maintained at the same level throughout the HDV replication cycle (Modahl and Lai 1998). These results suggested that the transcription of the mRNA and replication of the HDV genome are independent processes and occur concurrently. However, this model raises a different problem as it requires that mRNA transcription and RNA replication be synchronized on the same genomic sense HDV RNA template such that the poly(A) addition signal is recognized during transcription but not replication. A potential solution to this issue was suggested from recent observations in our laboratory indicating that transcription of mRNA and genomic to antigenomic RNA replication may be carried out by different polymerases and probably in different subnuclear domains (Modahl et al. 2000; Macnaughton et al. 2002; see below). Thus, these two processes may be both physically and biochemically separated in HDV-infected cells. There is a caveat to this, however, as infection requires only a single input genomic HDV RNA species. Thus, to serve as a template for both mRNA transcription and production of full-length antigenomic HDV RNA, this original infecting RNA species must be moved between different subnuclear domains. Since mRNA transcription is likely occur ﬁrst, this relocalization event probably depends on newly synthesized HDAg. If so, it might be expected that the de novo synthesized HDAg would be, in some way, different from the HDAg species present in the original infecting ribonucleoprotein complex, most likely due to different post-translational modiﬁcations (see next section). 3.2 The Role of HDAg in HDV RNA Replication S-HDAg is known to have multiple roles in HDV life cycle. Initially, HDAg is responsible for transport of HDV ribonucleoprotein complex to the site of RNA replication (Macnaughton et al. 1991). Subsequently, S-HDAg is required

HDV RNA Replication: Ancient Relic or Primer?

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for both the initiation and maintenance of HDV RNA replication, although how these roles are fulﬁlled is still not clear. Mutagenesis studies have shown that, with the exception of the C-terminal extension of L-HDAg, all of the known functional domains of HDAg (coiled-coil domain, nuclear localization signal and RNA-binding motifs) are essential for HDV RNA replication (Xia et al. 1992; Lazinski and Taylor 1993; Lee et al. 1993). Moreover, several posttranslational modiﬁcations (phosphorylation, acetylation, methylation) are also important, often having differential effects on genomic vs. antigenomic RNA syntheses (Chang et al. 1988; Mu et al. 1999, et al. 2001, et al.2004; Li et al. 2004; Table 1). An example of the latter came from the observation that unmodiﬁed recombinant S-HDAg from Escherichia coli, while allowing initiation of HDV RNA replication from a genomic template (antigenomic RNA synthesis), was defective for initiation of replication from an antigenomic template (genomic RNA synthesis) (Sheu and Lai 2000; Li et al. 2004). However, genomic RNA synthesis could be partially restored by ﬁrst methylating, in vitro, the E. coli-derived S-HDAg (Li et al. 2004). Results to date suggest that S-HDAg is a component of the HDV RNA transcriptional machinery. Indeed, some of the features of HDAg, such as

Table 1 Comparison of features and metabolic requirements for synthesis of the various HDV RNA species Feature

HDAg mRNA synthesis

G HDV RNA synthesis

AG HDV RNA synthesis

Relative ratios S-HDAg functional domains Protein–protein interaction Nuclear localization RNA Binding S-HDAg phosphorylation S-HDAg methylation S-HDAg acetylation Early L-HDAg expression Late L-HDAg expression Export to cytoplasm Site of synthesis

∼1

∼ 50

∼ 1000

? ? ? ? ? ? ? ? Yes Nucleoplasm

Required Required Required Sensitive Sensitive Sensitive Sensitive Resistant Yes Nucleoplasm near PML bodies Sensitive Pol II

Required Required Required Resistant Resistant Resistant Resistant Resistant No Nucleolus (in or near) Resistant Pol I?

α-Amanitin Polymerase

Sensitive Pol II

36

T. B. Macnaughton · M. M. C. Lai

nuclear localization and the presence of coiled-coil and HLH domains, are reminiscent of transcription factors. Moreover, like many DNA-dependent transcription factors, HDAg is both acetylated and methylated. HDAg binds directly to DNA-dependent RNA polymerase II (pol II; Yamaguchi et al. 2001) as well as several other cellular factors, including some pol II transcription factors, such as YY1 (Lee, personal communication), and nucleolar proteins, such as B23 and nucleolin (Huang et al. 2001; Lee et al. 1998). In a pol IImediated in vitro transcription system, S-HDAg was shown to promote RNA elongation by displacing NELF, a negative elongation factor, with which HDAg shares a limited sequence homology (Yamaguchi et al. 2001). HDAg has also been shown to have a low sequence homology to a novel cellular protein, DIPA, the expression of which inhibits HDV RNA replication (Brazas and Ganem 1996). However, the nature and signiﬁcance of DIPA remains to be established. In summary, S-HDAg may act as a transcription factor that interacts with both pol I and pol II transcription machineries. L-HDAg has been shown to inhibit HDV RNA replication when it is expressed at the beginning of HDV replication (Glen and White 1991; Macnaughton and Lai 2002b). Thus, it was regarded as a regulator of HDV RNA replication in the cell. As discussed above, this ﬁnding may be an artifact since L-HDAg normally is not expressed until late in the replication cycle. Late in replication, the presence or absence of L-HDAg did not affect the ﬁnal steadystate level of HDV RNA (Macnaughton and Lai 2002b). If L-HDAg indeed does not inhibit HDV RNA replication, its role in HDV RNA replication will be of considerable interest, as L-HDAg is usually colocalized with S-HDAg and has been found to be in the promyelocytic leukemia (PML) body of the nucleus, which is near the site of HDV RNA replication (unpublished observations). Curiously, L-HDAg has been shown to be able to activate a variety of transcription promoters (DNA-templated transcription) in a contransfection assay (Wei and Ganem 1998; Goto et al. 2000), although the functional signiﬁcance of this ﬁnding is currently unknown. 3.3 Replication of Genomic vs. Antigenomic RNA: Differences and Similarities RNA replication strategies of all single-stranded RNA viruses incorporate regulatory mechanisms to control the relative amounts and, in many cases, the temporal synthesis of genomic and antigenomic RNA species. Since genomic and antigenomic HDV RNA are also synthesized in signiﬁcantly different amounts, there must also be regulatory mechanisms operating during HDV replication. How this is achieved with just one virus-encoded protein is not clear. Certainly, both genomic and antigenomic RNA strands are synthe-

HDV RNA Replication: Ancient Relic or Primer?

37

sized continuously throughout the replication cycle. Moreover, genomic and antigenomic RNA intermediates of similar length are detected (dimer and longer multimers) and the ratios of circular to linear RNA molecules in both strands are also similar (Macnaughton and Lai 2002a), indicating that the both strands are made by an analogous rolling circle mechanism. There are, however, some different metabolic requirements for the synthesis of the two strands (Table 1). Genomic RNA synthesis (from the antigenomic template) is inhibited by very low concentrations of α-amanitin (1 µg/ml), whereas the antigenomic RNA synthesis (from the genomic template) is resistant to all concentrations tested (up to 100 µg/ml; Macnaughton et al. 2002). In contrast, transcription of the antigenomic-sense 0.8-kb HDAg mRNA is as sensitive to α-amanitin as genomic RNA synthesis (Modahl et al. 2000). Genomic RNA synthesis also requires some speciﬁc S-HDAg post-translational modiﬁcations (acetylation, phosphorylation and methylation), whereas antigenomic RNA synthesis can be mediated by an unmodiﬁed S-HDAg. For example: phosphorylation-defective mutant (S177A) and methylation-defective mutant (R13A) of HDAg can promote antigenomic RNA synthesis, but not the genomic RNA synthesis (Mu et al. 2001; Li et al. 2004); acetylation-defective mutant K72R (Mu et al. 2004) causes a dramatic reduction in genomic RNA accumulation while having no effect on antigenomic accumulation (Table 1). Genomic RNA synthesis is inhibited by early expression of L-HDAg, whereas the antigenomic RNA synthesis is not (Modahl and Lai 2000). Genomic RNA is rapidly exported to the cytoplasm after its synthesis and processing, whereas the antigenomic RNA is retained in the nucleus (Macnaughton and Lai 2002a). Furthermore, several site-speciﬁc mutations of HDV RNA sequence affect the synthesis of one RNA strand but not the other (Wang et al. 1997). Finally, genomic and antigenomic HDV RNA have different distribution patterns within the nucleus (Bell et al. 2000). Taken together, these results indicate that not only is the synthesis of genomic and antigenomic HDV RNA differentially regulated, but also that these two RNA strands are likely associated with different transcription machineries. 3.4 Mechanism of Transcription and Replication The enzymology of HDV RNA replication has commanded considerable interest over the last few years. Host cell RNA polymerase II, which normally uses a DNA template, has long been implicated as the likely replicase for HDV (Macnaughton et al. 1991). Recent studies based on α-amanitin resistance (see above) have conﬁrmed the major role this polymerase is likely to play (Macnaughton et al. 2002; Chang and Taylor, 2002). It is also clear that

38

T. B. Macnaughton · M. M. C. Lai

RNA polymerase II is likely responsible for transcription of the mRNA for HDAg (Modahl et al. 2000). In contrast, it seems likely that a different enzyme (probably pol I or a pol I-like enzyme) is responsible for the sysnthesis of fulllength antigenomic HDV RNA (Modahl et al. 2000; Macnaughton et al. 2002). However, this view is still somewhat controversial. Recently, a report from the Taylor laboratory (Nie et al. 2004), using various HDV cDNA constructs under the control of an SV40 promoter, concluded that poly(A) addition or ribozyme cleavage were alternative processing outcomes to generate the separate antigenomic HDV RNA transcripts. Signiﬁcantly, both processing events occurred on pol II-derived transcripts. This led the authors to conclude that only one polymerase (pol II) is required for HDV RNA replication. The system used in this study was designed to be replication-defective and thus overall bears little resemblance to natural HDV RNA-templated RNA replication. Moreover, a disproportionally high molar ratio of mRNA species to full-length antigenomes (3 : 1) was obtained as compared to that observed in HDV-infected livers (1 : 50), indicating that the system used for this study was prone to artifacts. In contrast, evidence for the multiple polymerase model is based primarily on RNA-only transfection studies, which more closely represents the natural situation. Moreover, using BrUTP labeling, we have recently observed that de novo genomic RNA synthesis was sensitive to α-amanitin and occurred in the vicinity of PML bodies whereas antigenomic synthesis was α-amanitin resistant and occurred either in or at the periphery of the nucleolus (Li et al. 2006). The involvement of different transcription machineries for genomic and antigenomic RNA synthesis is likely crucial for the HDV life cycle. For example, genomic RNA must be exported to the cytoplasm for packaging. Since pol II-mediated transcription is coupled to the nuclear export machinery (reviewed by Cullen 2003), this presents a convenient method for the export of genomic HDV RNA immediately following its synthesis. Interestingly, while the cellular mRNA export event is linked to splicing, only the completely or nearly completely processed forms of genomic HDV RNA (predominantly monomers) are exported. Thus, it is tempting to speculate that in the case of HDV, the ribozyme cleavage event, which is somewhat analogous to splicing, is linked to export. In contrast, the potential involvement of pol I in synthesis of antigenomic HDV RNA may be essential for the production of the full-length species. Speciﬁcally, pol I transcripts are never polyadenylated and production of full-length antigenomes of HDV requires that the polyadenylation signals (used during HDAg mRNA production) be silenced. How then can RNA pol II and other cellular polymerases use an RNA template when the normal template is DNA? There are several possibilities for this, none of which are mutually exclusive.

HDV RNA Replication: Ancient Relic or Primer?

39

1. HDAg shares some properties with transcription factors. Thus, direct or indirect binding of HDAg with the host polymerase (and/or other transcription factors) may lead to a relaxation of normal template requirements. Indeed, HDAg has been shown to bind to both HDV RNA and pol II (Lin et al. 1990; Chao et al. 1991; Yamaguchi et al. 2001). 2. The rod-shaped structure of HDV RNA resembles double-stranded DNA; therefore, it is conceivable that cellular RNA polymerases and transcription factors may recognize double-stranded RNA. In this regard, it has been shown that circular monomeric HDV cDNA contains endogenous promoters capable of directing HDV RNA synthesis (Macnaughton et al. 1993b). Interestingly, one of these, located near the transcription initiation site for HDAg mRNA, also exhibited promoter activity as an RNA molecule (Beard et al.; see Fig. 1: putative RNA promoter) The high GC content and extensive secondary structure would suggest that HDV RNA is a challenging template. Not surprisingly then, one of the functions attributed to S-HDAg is the promotion of elongation by pol II (Yamaguchi et al. 2001). Nevertheless, it is possible that RNA synthesis proceeds in a somewhat stop-start fashion with frequent polymerase pauses at regions of high secondary structure. In extreme cases, the polymerase may actually detach and subsequently re-anneal. Evidence for the latter comes from the likely intermolecular template switching required to reconstitute replicating HDV RNA from pairs of less than full-length RNAs (Gudima et al. 2005). Moreover, RNA recombination, which depends on template switching, has been demonstrated for HDV (Wang and Chao 2005). Examination of the ﬁne detail of the RNA secondary structure of the HDV rod (Fig. 1) reveals that the proteincoding domain consists of stretches of relatively intense intramolecular basepairing 30–40 nucleotides in length, interspaced with single-stranded regions of 5–12 bases. It is tempting to speculate that these single-stranded domains help the polymerase move along the template by providing regions that can be easily dissociated. In contrast, the most highly intramolecular base-paired region of HDV RNA occurs in the viroid-like domain (Fig. 1). Signiﬁcantly, for antigenomic HDV RNA synthesis, this region lies immediately downstream of the polyadenylation signal. Thus, polymerase pausing in this region may assist in the exonuclease-dependent termination of transcription demonstrated for cellular mRNA production (Kim et al. 2004; West et al. 2004). Finally, it remains a possibility that HDV RNA replication involves a cytoplasmic phase. While this is required for packaging of the genomic RNA into virions, it is curious that RNA export happens well before the virus assembly can take place. Moreover, HDV RNA has been shown to continually shuttle between the nucleus and cytoplasm (Tavanez et al. 2002). Thus, it is conceiv-

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T. B. Macnaughton · M. M. C. Lai

able that certain steps of HDV replication cycle, e.g. RNA ligation, take place in the cytoplasm. Thus, HDV RNA shuttling between the cytoplasm and the nucleus may be a critical step for successful RNA replication.

4 Perspectives HDV is a unique mammalian virus, containing the only RNA species that is known to be copied by host cell enzymes. It seems highly improbable that the cellular mechanism responsible for this activity is reserved solely for use by HDV. More likely, this is a previously unrecognized and yet innate ability of mammalian cellular polymrases. As such, the study of HDV replication should lay the initial ground rules that not only will assist in the identiﬁcation of other similar agents but may also open a new ﬁeld of cell research. An interesting question will be to determine the role that RNA replication has in normal cells. Simplistically, this ability could just be an artifact from the original RNA world that no longer serves any function. This view, however, is hard to sustain in light of evolutionary considerations and the likelihood that this property may be possessed by more than one mammalian RNA polymerase. In this regard, RNA-dependent RNA synthesis has been documented in certain mammalian cells (Volloch et al. 1996). What then are the possibilities: 1. An obvious candidate is in the operation of small interfering RNA (siRNA)-mediated inhibition of protein synthesis. In particular, RNA replication may be involved to amplify the small RNA fragments prepared by the dicer complex (reviewed Agrawal et al. 2003). However, while this RNA ampliﬁcation does take place in cells from simpler species, there is little indication that such ampliﬁcation occurs in mammalian cells. Hence, there is a need to repeatedly transfect siRNA in mammalian cells in order to maintain siRNA-mediated inhibition. 2. Regulation of protein synthesis and other cellular functions. The world of RNA-mediated regulation has been expanding steadily in recent years with the discovery of siRNA and micro RNAs. Perhaps a further mechanism exists that is dependent on RNA copying. Indeed, given the structural similarity of some micro RNAs to HDV and viroid, it is possible that an as yet undiscovered group of these RNAs are maintained by such a mechanism. 3. Perhaps the most interesting possibility of all is in the maintenance of an ancestral RNA-sequence cache such as that thought to be involved in

HDV RNA Replication: Ancient Relic or Primer?

41

the extra-genomic inheritance of DNA sequence information observed in arabidopsis (Lolle et al. 2005). Could a system like this be present in human cells? Indeed, this has been suggested (Pearson 2005). In particular, rare cases of children who inherit disease-causing mutations but only show mild symptoms have been documented. Is it possible that in these individuals, the DNA of some cells have reverted to a nondisease state? There is much to do before we understand the mechanism(s) that drive HDV RNA replication. However, the future is very exciting. Thus, in spite of falling clinical relevance, HDV provides a novel window peering into a brave new world.

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CTMI (2006) 307:47–65 c Springer-Verlag Berlin Heidelberg 2006

HDV Ribozymes M. D. Been (u) Department of Biochemistry, Duke University Medical Center, Durham, NC, USA [email protected]

1

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

2 2.1 2.2

Sequence Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 Deﬁning a Ribozyme Sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 Two HDV Ribozymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50

3

Ribozyme Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50

4

Cleavage Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

5 5.1 5.2 5.3 5.4

Catalytic Strategies . . . . . . . . . . . . . In-line Orientation . . . . . . . . . . . . . . Divalent Metal Ion . . . . . . . . . . . . . . An Active Site Nucleobase . . . . . . . . . Site of Action of the Catalytic Cytosine

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 Abstract The self-cleaving RNA sequences, or ribozymes, in the genomic and antigenomic strands of hepatitis delta virus (HDV) RNA fold into structures that are similar to each other but distinct from those of small ribozymes associated with the RNA replicons that infect plants. HDV ribozymes have provided a tractable system for studying the mechanism of catalytic RNA, and results of biochemical and structural studies on the HDV ribozymes, from a number of labs, have enhanced our understanding and expanded our thinking about the potential for catalytic roles of RNA side chains. The results of these studies are consistent with models suggesting that both an activesite cytosine and a divalent metal ion have catalytic roles in facilitating the cleavage reaction in the HDV ribozymes. Despite recent advances, details about the catalytic mechanism of the HDV ribozyme continue to be debated, and these ribozymes should serve as a good system for further study.

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1 Introduction Self-cleavage sites in the RNA (ribozymes) of hepatitis delta virus (HDV) were hypothesized, identiﬁed, and initially characterized independently by Taylor and coworkers (Kuo et al. 1988; Sharmeen et al. 1988) and Wu et al. (1989). The ribozymes were proposed to process primary replication products, generated in rolling circle replication, to monomer size (Robertson 1992; MacNaughton et al. 1993; Lazinski and Taylor 1995a). Speciﬁc subfragments of each RNA strand were found to contain both the cleavage site and the sequences necessary and sufﬁcient for self-cleavage (Kuo et al. 1988; Wu et al. 1989). Although the sequences associated with the cleavage sites in HDV RNA differed from the sequence that formed the familiar hammerhead and hairpin ribozyme motifs, the reaction was the same. Self-cleavage of the RNA backbone involves a rearrangement of the 3 ,5 phosphodiester bond to generate a 2 ,3 -cyclic phosphate group and a 5 hydroxyl group, suggesting nucleophilic attack of the adjacent 2 hydroxyl on the scissile phosphate (Wu et al. 1989). The in vitro reaction required no protein or cellular factor but a divalent metal ion greatly stimulated cleavage rates (Wu et al. 1989). This chapter will review biochemical and structural data with emphasis on experiments and results that help us understand the catalytic mechanism used by the HDV ribozymes.

2 Sequence Requirements 2.1 Defining a Ribozyme Sequence With examination of the HDV sequences required for self-cleavage, a native sequence of about 85 nucleotides was found sufﬁcient for rapid cleavage and was deﬁned as a minimal or core ribozyme domain (Perrotta and Been 1990, 1991). These core sequences, both genomic and antigenomic, could fold into similar secondary structures (Fig. 1) (Perrotta and Been 1991; Rosenstein and Been 1991). A single nucleotide 5 to the cleavage site is sufﬁcient for cleavage. The 3 boundary is less precise in that the level of activity varied moderately— give or take a nucleotide or two at the 3 end. Internal deletions that shorten the P4 duplex can reduce the minimum size to about 65 nucleotides before the rate of self-cleavage begins to be signiﬁcantly reduced (Been et al. 1992; Thill et al. 1993). The possible involvement or contribution of HDV RNA sequences that ﬂank the core ribozyme remains of interest. In the process of elucidating

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Fig. 1 Secondary structures of the genomic and antigenomic HDV ribozymes. Numbering of the nucleotides in the ribozymes is 5 to 3 from the cleavage site with the nucleotide 5 of the cleavage site assigned −1. Due to differences in the sequences, corresponding positions in the genomic and antigenomic ribozymes deviate in the exact numbering. For this paper, numbering only impacts nomenclature for the catalytic cytosine; Cyt75 in the genomic ribozyme corresponds to Cyt76 in the antigenomic ribozyme

a core ribozyme that retains essential activity in vitro, important sequences could easily have been eliminated. Caution in deﬁning absolute boundaries is well advised, especially now that signiﬁcant contributions to cleavage activity from presumed ‘non-ribozyme’ ﬂanking sequences have been found for both the hairpin and hammerhead ribozymes. In both cases, it appears that a larger form of the ribozyme is either more stable or may fold more readily in ionic conditions approximating physiological conditions (Hampel and Tritz 1989; Murchie et al. 1998; Walter et al. 1998; Khvorova 2003; Canny et al. 2004). Studies of an HDV genomic ribozyme that included additional 5 viral sequence have revealed that those 5 ﬂanking sequences can inhibit activity by base pairing with ribozyme sequence but also participate in other interactions that facilitate folding of the active structure (Chadalavada et al. 2000, 2002; Brown et al. 2004). Similarly, a short sequence extension at the 3 end can alternatively stabilize either an active or inactive form of the antigenomic ribozyme (Perrotta et al. 1999a). Although these effects are different from what was seen with the hammerhead and hairpin ribozymes, the potential for additional interactions exists and deserves further investigation.

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2.2 Two HDV Ribozymes The ribozymes from the genomic and antigenomic strand of HDV RNA are similar in sequence, but are not identical. This sequence similarity reﬂects, in part, the fact that the HDV RNAs (both genomic and antigenomic) are about 70% self-complementary such that each single-stranded circle could form a partial-duplex rod-like structure (Wang et al. 1986). Each ribozyme sequence is transcribed from a region of the respective template that is partially complementary to the other ribozyme. Not surprisingly then, both ribozymes catalyze the same reaction to cleave the RNA backbone, and both appear to use a similar mechanism of catalysis. Although small differences in the activity of the two ribozymes are detectable under some conditions, at this point those differences appear to be idiosyncratic and relatively minor (Wadkins and Been 1997; Wadkins et al. 1999, 2001). It remains possible that there are less subtle and biologically important differences in the two ribozymes; however, for the major points addressed in this review, the two ribozymes appear to behave identically.

3 Ribozyme Structure Structures have been obtained from crystals of a modiﬁed form of the genomic ribozyme (Ferré-D’Amaré et al. 1998a; Ferré-D’Amaré and Doudna 2000; Ke et al. 2004). No physical structure is available for the antigenomic ribozyme, but because the secondary structures of the genomic and antigenomic ribozymes are so similar, it is likely that the three-dimensional structures will be similar as well. For an in-depth discussion of the structure and its significance, readers are directed to papers from the Doudna lab (Ferré-D’Amaré et al. 1998a; Ferré-D’Amaré and Doudna 1999, 2000; Doherty and Doudna 2000; Ke et al. 2004). A partial overview will be provided here and select aspects will be discussed later in context of the catalytic mechanism. Each ribozyme contains ﬁve duplexes or pairings labeled P1, P2, P3, P4 and P1.1. Extensive duplex formation and a nested pseudoknot arrangement of those duplex elements generates a compact structure. The importance of base pairing in all ﬁve duplex regions for in vitro cleavage activity was established by mutagenesis experiments in both ribozymes (Perrotta and Been 1991, 1993; Been et al. 1992; Thill et al. 1993; Wadkins et al. 1999; Nishikawa and Nishikawa 2000). An in vivo requirement for base pairing in P1 and P2 is also supported by the effect of mutations (Jeng et al. 1996). The lengths of P1, P3 and P1.1 (7 bp, 3 bp and 2 bp, respectively) are the same for both ribozymes

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and changes to their lengths might be expected to distort the structure of the active site. P1 and P3 show some sequence variation between the genomic and antigenomic ribozymes but those sequences do not vary amongst clinical isolates (Tanner 1995; Been and Wickham 1997; Wadkins and Been 2002). P1.1, an invariant and essential two base-pair duplex that forms a coaxial connector between P1 and P4 (Ferré-D’Amaré et al. 1998a), was only identiﬁed upon solving the crystal structure of the genomic ribozyme, and was not a feature of the secondary structures as originally proposed (Perrotta and Been 1991). P1.1 is always CC paired with GG, and that combination appears optimal for activity (Wadkins et al. 1999; Nishikawa and Nishikawa 2000). P4 is a long, imperfect duplex that extends away from the core ribozyme. It shows more sequence variation than the rest of the ribozyme domain, both between the two ribozymes and amongst the clinical isolates. The tolerance to sequence variation in P4 was exploited for the structural work. For the structures, the P4 hairpin was largely replaced by a U1A protein binding site and the crystals grown as the ribonuclear protein complex (Ferré-D’Amaré et al. 1998b). While P4 can be shortened without loss of in vitro ribozyme activity, the issue of whether the longer natural P4 hairpin sequence has a role in viral replication or ribozyme activity in vivo remains an interesting question. The 3 end of P2 deﬁnes the 3 boundary of the ribozyme domain. P2 would be predicted to play an important role in positioning Cyt75/76, the proposed catalytic cytosine, contained within the J4/2 joining segment. Alterations to the sequence and length of P2 can dramatically affect the extent of cleavage in vitro, which suggests that P2 may also function in directing the correct RNA folding (Perrotta and Been 1998; Perrotta et al. 1999a; Chadalavada et al. 2000, 2002). The cleavage site is located at the 5 end of P1, but in the structure (FerréD’Amaré et al. 1998a; Ke et al. 2004) this position is well buried in an activesite pocket between the two parallel coaxial stacks of P1–P1.1–P4 and P2–P3. The short sequence connecting P4 and P2 (J4/2) also forms part of that pocket and also contributes and positions Cyt75. No strict requirements for sequence 5 to the cleavage site have been identiﬁed (Perrotta and Been 1990, 1992; Shih and Been 1999). There are, however, sequence preferences. For example, a guanosine just 5 to the cleavage site slows cleavage activity roughly tenfold under our standard reaction conditions (Perrotta and Been 1992). In addition, sequence 5 to the cleavage site can interfere with ribozyme activity by participating in alternative pairings that disrupt the ribozyme structure (Perrotta and Been 1990, 1991; Chadalavada et al. 2000). Notably, cleavage site selection by the ribozyme does not appear to require the sequence 5 to the cleavage site to base pair with a particular sequence within the ribozyme domain. The most recent structures (Ke et al. 2004), which are of a precursor

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form of the genomic ribozyme, reveal a sharp bend in the backbone at the cleavage site that nearly reverses the direction of the RNA backbone. Two nucleotides just 5 of the cleavage site occupy a tight space between P1 and P3 without making speciﬁc base–base contacts. The structures of the 3 product form and the more recent precursor form of the ribozyme reveal changes that occur upon backbone cleavage and provide invaluable insights into the catalytic mechanism (Ferré-D’Amaré et al. 1998a; Ke et al. 2004). To prevent self-cleavage, the precursor structure was solved using an inactive ribozyme with a uracil replacing Cyt75. Near the position of the scissile phosphate, at the bend in the substrate strand mentioned above, are located a bound divalent metal ion and the pyrimidine base at position 75 (Ke et al. 2004). While there do not appear to be major changes in the general architecture of the ribozyme upon cleavage, there are local conformational changes in the vicinity of the cleavage site. It appears that, following cleavage, the 5 fragment bearing the newly generated 2 ,3 -cyclic phosphate is released along with the metal ion, and the space between P1 and P3 that had been occupied by the 5 fragment narrows slightly. The nucleotide sugar of G1, at the newly generated 5 end, moves further into the active site pocket. Thus, the 5 hydroxyl oxygen of the product is displaced relative to its position when it functioned as the 5 bridging oxygen of the precursor. Very importantly, the base at position 75 has moved in the product relative to its location in the precursor structures. In the product, Cyt75 can form a hydrogen bond between its N3 (or O4) and the 5 hydroxyl group oxygen of G1, while in the precursor, the uracil base at position 75 is positioned higher (~2 Å) in the active site and away from that same oxygen. As such, it is positioned closer to the predicted location of the 2 -OH group of the sugar at position −1, but only following a proposed rotation of that nucleotide that provides a more favorable geometry for the cleavage reaction. Knowing the site of action of Cyt75 is important for understanding details of the catalytic mechanism.

4 Cleavage Reaction The mechanism of the backbone cleavage reaction was ﬁrst inferred from the products (Wu et al. 1989) (Fig. 2a). A 5 hydroxyl group and 2 ,3 -cyclic phosphate group on the cleavage products are consistent with a mechanism in which the oxygen at the 2 position adjacent to the scissile phosphate attacks the phosphorus and the phosphorus to 5 -oxygen bond is broken. This reaction is common to the small self-cleaving ribozymes, but it is distinct from the reaction catalyzed by the large ribozymes where a 3 hydroxyl group is

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generated (RNase P RNA, group I and group II intron-derived ribozymes). For the small ribozymes, it is hypothesized that the 5 hydroxyl group displacement reaction occurs with inversion of conﬁguration through a pentavalent transition state where the attacking 2 oxygen and leaving 5 oxygen are at the apical positions of a trigonal bipyramidal arrangement of the phosphorane

Fig.2a–c Cleavage reaction and possible catalytic mechanisms for the HDV ribozymes. a Backbone cleavage results in a 2 ,3 -cyclic phosphate and a 5 hydroxyl group. Proposed catalytic groups include a base (B:) to facilitate deprotonation of the 2 hydroxyl group (the nucleophile) and an acid (B:H+) to donate a proton to the 5 bridging oxygen (the leaving group). Potential catalytic groups, as discussed in the text, include a Mg2+ ion-bound water (Mg2+ HO:H or Mg+ HO:) and an active site nucleobase (H+ :Cyt75/76 or :Cyt75/76). Two transition states (TS1 and TS2 ) are shown but in a concerted mechanism, concurrent proton transfer might occur. b :Cyt75/76 acting at the 2 hydroxyl group (a general base in the cleavage reaction). c H+ :Cyt75/76 acting at the 5 bridging oxygen (a general acid in the cleavage reaction)

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oxygens. Inversion of conﬁguration about the phosphorus, when a phosphorothioate stereoisomer is placed at the cleavage site, is strong support for this mechanism in the hammerhead and hairpin ribozymes (van Tol et al. 1990; Slim and Gait 1991); however, similar studies have not been reported for the HDV ribozymes. The reverse reaction, attack by the 5 hydroxyl on the cyclic phosphate center to restore the 3 ,5 linkage, has yet to be demonstrated with the HDV ribozymes. The lack of a detectable ligation reaction seems surprising given the chemistry of the reaction (Gerlt et al. 1975), the ability of both the hammerhead and hairpin ribozymes to catalyze ligation (Feldstein and Bruening 1993; Hegg and Fedor 1995; Hertel and Uhlenbeck 1995), and the biology in which RNA replication would appear to beneﬁt from ribozyme-catalyzed ligation. Nevertheless, two reports of in vitro ligation in the HDV ribozyme system have not been born out. The idea that a conformational change in the RNA may help drive the forward reaction is consistent with results from biophysical (Harris et al. 2002; Pereira et al. 2002) and structural studies (Ke et al. 2004) that reveal structural differences in the ribozyme domain between the precursor and product forms. In addition, a reaction that favors cleavage over ligation is consistent with the absence of binding interactions for both the 5 fragment and a catalytic metal ion in the 3 product following cleavage (Shih and Been 2001a; Ke et al. 2004).

5 Catalytic Strategies Among the various small, self-cleaving ribozymes, a variety of speciﬁc mechanisms may be used to achieve a similar set or subset of catalytic strategies. Those strategies include bringing the attacking group oxygen and phosphorus atoms into proximity and aligning those atoms with the leaving group oxygen atom, stabilizing the negative charge that develops in the transition state, and enhancing both the nucleophilicity of the 2 hydroxyl group and the leaving ability of the 5 oxygen. The HDV ribozymes have provided a good system to explore the potential for nucleobases or side chains of the RNA to act as general-acid-base catalysts (Fig. 2b,c). 5.1 In-line Orientation The structure of the hairpin ribozyme with a 2 O-methyl nonreactive substrate analog is a good example of a ribozyme facilitating the in-line orientation of the 2 and 5 oxygens with the phosphorus (Rupert and Ferré-D’Amaré

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2001, 2004; Ferré-D’Amaré and Rupert 2002; Ferré-D’Amaré 2004). Only small additional movement would be required to align the atoms to an in-line orientation for the reaction. In a recent structure of a prior-to-cleavage ‘tethered’ version of the hammerhead ribozyme, the 2 O–P5 O atoms are also approaching the in-line orientation (Murray et al. 2002; Dunham et al. 2003). In the precursor structures of the HDV ribozyme (Ke et al. 2004) the atoms involved in the reaction are not so favorably oriented. As noted above, the structure does reveal a dramatic sharp bend in the backbone at the scissile phosphate, and Ke et al. (2004) point out that there is room to rotate the nucleotide at the −1 position (5 to the cleavage-site phosphate) such that the necessary alignment could be attained. While an active site that positions the groups for the in-line arrangement will facilitate the reaction, the magnitude of the effect is difﬁcult to quantify. There is no HDV ribozyme-speciﬁc data that would address this issue. However, Soukup and Breaker (1999) in an insightful study, correlated the stability of phosphodiester bonds in RNA with the geometry of internucleotide linkage in RNAs of known structures. In addition to demonstrating a clear relationship of geometry to activity, they were able to estimate that the inline orientation could contribute as much as a 103 –104 -fold enhancement in cleavage rates relative to a linkage constrained in a non-inline orientation, or as little as a 10–20-fold enhancement relative to an unconstrained linkage. 5.2 Divalent Metal Ion Divalent metal ions, in addition to stabilizing overall structure, appear to have a more direct catalytic role in the cleavage reactions of the HDV ribozymes. Both HDV ribozymes are active in low (1–10 mM) concentrations of physiologically relevant divalent metal ions such as Mg2+ or Ca2+ (Wu et al. 1989; Perrotta and Been 1990; Rosenstein and Been 1990; Suh et al. 1993; Nakano et al. 2003). Altered metal ion speciﬁcity for cleavage of a 2 -5 linked phosphodiester bond suggested a possible intimate role for the divalent metal ion in the cleavage reaction (Shih and Been 1999). High concentrations (1–2 M) of NaCl will support cleavage activity, but it is much slower than that measured in 2–10 mM Mg2+ (Nakano et al. 2000; Wadkins et al. 2001). The exchange-inert cation complex, Co(III) hexamine, which structurally mimics a hexahydrated Mg2+ , does not support cleavage activity and inhibits the Mg2+ -dependent reaction (Nakano et al. 2000; Ke et al. 2004). More recently, structures of precursor forms of a mutant ribozyme revealed a metal ion in the active site, and that metal ion is lost when the ribozyme cleaves (Ke et al. 2004). Together, these data would be consistent with the involvement of a divalent metal ion in catalysis.

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Divalent metals tend to coordinate to the RNA through a water (outersphere coordination) but they also have the potential to coordinate directly to an oxygen or nitrogen in the RNA (inner-sphere coordination). An exhaustive kinetic and thermodynamic study of divalent-metal ion requirements of the genomic HDV ribozyme provided data that was evidence for both inner and outer-sphere binding at different metal sites (Nakano et al. 2003); a catalytic site that is strictly outer-sphere coordination but structural sites that also involve inner-sphere coordination. The absence of evidence for inner-sphere coordination of a catalytic metal ion was seen in other experiments as well. One approach to obtaining evidence for direct coordination of a metal ion to a scissile phosphate, nonbridging oxygen, is cleavage-rescue of thiophosphatesubstituted RNA with a soft metal ion (e.g., Mn2+ or Cd2+ ). Although, in Mg2+ , inhibition is seen with the pro-Rp thiophosphate, soft-metal ion rescue in the HDV ribozymes was not observed (Fauzi et al. 1997). Das and Piccirrili (2005) prepared a substrate oligonucleotide with a sulfur substituted for the 5 -bridging oxygen and found that a soft metal ion (Mn2+ ) did not stimulate the ribozyme-mediated cleavage even though it did stimulate the background cleavage of the same substrate. This result indicates that the metal ion does not directly coordinate to the phosphate 5 -bridging oxygen (the leaving group atom) in the ribozyme. Thus, so far at least, there is no evidence for innersphere coordination of a metal ion with catalytic activity. A catalytic role that involves outer sphere coordination is feasible because a water molecule coordinated to a metal ion has a lower pKa than bulk water, thus raising the possibility that the hydrated metal ion may function as a general acid-base catalyst. Nakano et al. (2001, 2003) propose that proton transfer catalyzed by a hydrated Mg2+ contributes about 25-fold to the overall rate in a reaction containing 1 M NaCl. Two speciﬁc models for the hydrated metal ion acting as a general acid-base catalyst in the HDV ribozyme reaction have now been proposed. In one model it is acting at the 2 hydroxyl group (Nakano et al. 2000), and in the other it is acting at the 5 bridging oxygen (Ke et al. 2004). In the structure of the precursor, a hydrated metal ion is well positioned to coordinate through a water to the 5 oxygen leaving group and thus may be a good candidate for a proton donor (Ke et al. 2004). On the other hand, in the earlier structure of the cleavage product, it appears that Cyt75 in the active site is well positioned for this same role (Ferré-D’Amaré et al. 1998a; Nakano et al. 2000). Kinetic data alone is unavoidably ambiguous with regard to assigning the position at which an acid-base catalyst might act in a reaction such as the one being studied here because the two mechanisms would be kinetically equivalent. However, Das & Piccirrili (2005) have recently shown that cobalt hexamine was effective in inhibiting the cleavage reaction even when the 5 bridging oxygen was replaced by sulfur, a much better leaving

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group. This result suggests that the inhibition by cobalt hexamine, which is a poor proton donor/acceptor, is not the result of it replacing a critical hydrated Mg2+ that acts at the 5 leaving group as a proton donor. Indeed they ﬁnd that Cyt76 likely fulﬁlls that role in the antigenomic form of the ribozyme they used (described below). 5.3 An Active Site Nucleobase Possible roles for a nucleobase in catalysis could range from the effective and ordinary (positioning reactive groups through hydrogen bond interactions) to the more exotic and elusive (general acid-base catalysts) to something in between (electrostatic shielding). Biochemical evidence and the crystal structures have led to the hypothesis that a cytosine located in the active site of the HDV ribozyme acts as a general acid-base catalyst (Tanner et al. 1994; Ferré-D’Amaré et al. 1998a; Perrotta et al. 1999b; Nakano et al. 2000; Shih and Been 2001b; Ke et al. 2004). Early mutagenesis results suggested that Cyt75 in the genomic HDV ribozyme and Cyt76 at the equivalent position in the antigenomic ribozyme may have an important role in the cleavage reaction (Tanner et al. 1994; Perrotta and Been 1996). Lower, but measurable, activity could be detected with the cytosine to adenine substitution (Perrotta and Been 1996; Perrotta et al. 1999b; Nakano et al. 2000). However, it was the structure of the cleaved genomic ribozyme that provided the strongest support for cytosine playing a speciﬁc catalytic role (Ferré-D’Amaré et al. 1998a). In the cleaved RNA, the cytosine is well positioned in the active site to act as a catalytic group, and both the O2 and N3 of Cyt75 are within hydrogenbonding distance to the 5 hydroxyl group oxygen generated following the cleavage reaction. A speciﬁc model with the nucleobase of Cyt75 acting as a general acid-base catalyst and accepting a proton from the 2 hydroxyl group was proposed (Fig. 2b) (Ferré-D’Amaré et al. 1998a). Shortly thereafter, a variation of that model was proposed (Nakano et al. 2000) with Cyt75 still acting as an general acid-base catalyst but donating a proton at the 5 bridging oxygen, the leaving group (Fig. 2c). The product structure would appear to be most consistent with Cyt75 acting at the 5 bridging oxygen leaving group but since the cleaved ribozyme lacked the 5 product, evidence for positioning of the 2 hydroxyl and phosphate groups was not available. Thus, both models propose a role for the Cyt75 side chain that is analogous to the catalytic role of the imidazole group of histidine in RNase A. However, unlike RNase A which has two histidines in the active site that can alternatively function as proton acceptors and proton donors in the two steps catalyzed by RNase A, the active site of the HDV ribozyme may contain only the single

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catalytic nucleobase. If we continue with the RNase A comparison, a third possible catalytic role for Cyt75 would be equivalent to the lysine side chain stabilizing charge on the cleavage-site phosphoryl group. In the ribozyme this might be through either the exocyclic amino group protons or, in the ionized form of the nucleobase, an N3 proton. Answers to the following questions would help distinguish between possible roles for Cyt75/76. First, is there evidence that the cleavage rate is linked to the ionization state (protonation) of Cyt75/76. If so, is there evidence that Cyt75/76 is actually acting as a generalacid-base catalyst. And, if it is, at what position is the cytosine acting to accept or donate a proton? For the latter, the possibilities are again the 2 hydroxyl group, the 5 leaving group, or maybe a phosphate oxygen. Results from several labs are consistent with the idea that the ionization state of Cyt75/76 is linked to cleavage activity. Differences in the pH–rate curves of the wild-type and Cyt75/76 to adenine mutants in both the antigenomic and genomic ribozymes are consistent with that hypothesis (Perrotta et al. 1999b; Nakano et al. 2000). More direct evidence came from nucleotide analog interference mapping (NAIM) experiments (Oyelere and Strobel 2000) in which the effect of analogs with different ionization constants were examined for their effect on cleavage activity. Recently, Das and Piccirilli (2005) showed a clear shift in the apparent pKa of the cleavage reaction when 6-azacytosine replaced C76 in an antigenomic ribozyme. Those data all revealed a correlation of the ionization of a base at position 75/76 and selfcleavage activity. The evidence that the cytosine is actually functioning as a general acid-base catalyst is indirect and requires correlating cleavage rates with the strength of the base or acid. Cleavage activity of antigenomic ribozyme variants in which Cyt76 was either changed to a U or deleted were partially rescued with the addition of free cytosine to the reaction (Perrotta et al. 1999b; Shih and Been 2001b). The rate of cleavage in the cytosine rescue reactions was both concentration and pH dependent. Base (buffer) rescue was also seen with imidazole and certain imidazole-like compounds with different pKa values (Shih and Been 2001b), and the shapes of the pH–rate curves for these rescue reactions were consistent with the idea that the cleavage rate reﬂected the ionization state of the base. More importantly, the reaction showed a dependence on the strength of the buffer which was consistent with general acid-base catalysis. A linear free-energy plot, the log of the second order rate constant for the base-rescued cleavage reaction versus pKa of the base, gave a line with a slope of ~0.5. This value is the Brønsted coefﬁcient (β) and, for this reaction, is consistent with an even distribution of charge between the proton donor and acceptor in the transition state; in other word the proton is ‘in-ﬂight’ and moved about half-way in the transition state and thus consistent with a mechanism

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of general acid-base catalysis (Jencks 1969; Fersht 1985). Strictly speaking, the conclusion from this analysis only applies to the buffer-rescue reactions; the assumption used here, to argue for general acid-base catalysis by Cyt75(76), is that free cytosine and the imidazole compounds fulﬁll a similar functional role in the mutants as Cyt75(76) does in the wild-type ribozyme. The base-rescue result, while providing strong support for a general acidbase mechanism, does not address the issue of where the general acid-base catalyst acts in the reaction. In this case, we cannot distinguish between the cytosine (or imidazole) acting to accept a proton from the 2 hydroxyl group (general base), or its conjugate acid donating a proton to the 5 oxygen leaving group (general acid). Ambiguity in assigning the site of action of a general acid-base catalyst from kinetic studies alone is unavoidable in this type of reaction (Jencks 1969). 5.4 Site of Action of the Catalytic Cytosine Although kinetic studies alone cannot identify the site at which the catalytic cytosine acts, structural studies and mechanistic studies with carefully chosen atom substitutions can. Structural data addressing this issue was discussed above. The recent mechanistic study by Das and Piccirrili (2005) speciﬁcally investigate the question of whether the active-site cytosine is acting at the 5 leaving group oxygen. To test that hypothesis, the 5 bridging oxygen was replaced with sulfur, which is a better leaving group, and the effect that substitution had on the reaction with and without the cytosine was examined. The logic behind this approach is that if the cytosine, acting as a general-acid catalyst, is donating a proton to the 5 bridging oxygen to make it a better leaving group in the cleavage reaction, then replacing the poor leaving group (oxygen) with a good leaving group (sulfur) will make the reaction less sensitive to the strength of the acid (Jencks 1969). The authors used an antigenomic form of the ribozyme with and without a C76U mutation and, in an impressive set of experiments, demonstrated that the 5 bridging sulfur ‘rescues’ cleavage activity in the C76U mutant background but had little effect in the wild-type background. Similar results were obtained with a 3-deazacytosine derivative at position 76 (C76c3C). The effect of 3-deazacytosine more precisely identiﬁes the suspected N3 atom of cytosine as the site of ionization. Chemogenetic suppression (Das and Piccirilli, 2005) aptly describes this approach because the deleterious effect of the cytosine to uracil (or 3-deazacytosine) mutation was suppressed by the oxygen to sulfur substitution. This was a beautiful test of the hypothesis that Cyt76 was acting as a proton donor in the cleavage reaction and provides strong support for that speciﬁc role.

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The interpretation of the most recent structural evidence (Ke et al. 2004) and the mechanistic data (Das and Piccirilli, 2005) thus provides two different models for the role of cytosine in the cleavage reaction. The structural data for the precursor form of the genomic ribozyme appears most consistent with the Cyt75 acting at the 2 hydroxyl group as a general base. The chemogenetic suppression with the antigenomic ribozyme is most consistent with Cyt76 acting at the 5 oxygen leaving group as a general acid. Although two different forms of the ribozymes (genomic and antigenomic) were studied, the ribozymes are so similar this is very unlikely to be an issue. Both studies relied heavily on an equivalent cytosine to uracil mutation in the ribozyme, so the mechanisms would not differ as a result of the mutation. The possibility that the cytosine could act at both positions, perhaps transferring a proton from the 2 oxygen nucleophile to the 5 bridging oxygen leaving group, would seem unlikely for this particular reaction given the distance involved. So can we say where the cytosine acts? Given the difﬁculties in obtaining accurate high-resolution structures of precursor forms of the HDV ribozymes, it may not be possible to solve a crystal structure of a precursor ribozyme that is not subject to criticism at some level. Modiﬁcations have to be made to the sequence or conditions to prevent it from cleaving. One might likewise argue that replacing an oxygen with sulfur alters the mechanism of the reaction but the internal consistency of the effect of substitutions in the chemogenetic suppression data appear very strong. Nevertheless, debate on this detail of the mechanism is likely to continue.

6 Conclusion HDV replication depends on small self-cleaving sequences in both the genomic and antigenomic strands of the replication intermediates. These ribozymes have provided a rich system for studying catalytic RNA and this review has focused on the chemical and catalytic mechanism of those ribozymes. However, it has ignored the equally interesting questions concerning the need for controlling cleavage/ligation activities and integrating ribozyme activity into the biology of the virus (MacNaughton et al. 1993; Lazinski and Taylor 1995b, 1995a). Ribozymes are typically thought to require metal ions or other cations for structural stability and usually, but not always, thought to use divalent metal ions for a direct catalytic role as well. Large ribozymes such at the self-splicing introns and RNase P RNA are probably correctly classiﬁed as obligate metallo-ribozymes. The picture is different for the small self-cleaving ribozymes; the hairpin, hammerhead, VS and HDV ribozyme all show vari-

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ous levels of activity in the absence of divalent metal ions (Murray et al. 1998; Curtis and Bartel 2001; Nakano et al. 2001; O’Rear et al. 2001; Wadkins et al. 2001). The hairpin ribozyme does not require a divalent metal ion (Hampel and Cowan 1997; Young et al. 1997) and the others might be considered metal-assisted since they cleave faster when Mg2+ is available. Of these, the HDV ribozymes have provided the most evidence for how the RNA may be directly involved in catalysis. The possibility that RNA nucleobases, like some familiar amino acid side chains, would be capable of acid-base catalysis had been discussed, but in the absence of a good example, it was beginning to look unlikely (Cech and Golden 1998). However, in providing what now appears to be strong evidence for cytosine (and adenine) catalyzed proton transfer, the ribozymes from HDV have made a unique contribution to our understanding of the RNA world hypothesis. Acknowledgements I thank A. Perrotta, S. Wilkinson, A. Brown and S. Chamberlin for comments on earlier version of the manuscript. I am especially grateful to Drs. S. Das and J. Piccirilli for providing their manuscript prior to publication. I also wish to acknowledge fun discussions of ideas over the years with Drs. J. Doudna, A. Ke, P. Bevilacqua and others working with the HDV ribozymes. This work was supported by a grant from the National Institutes of Health (GM047233).

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Curtis EA, Bartel DP (2001) The hammerhead cleavage reaction in monovalent cations. RNA 7:546–552 Das SR, Piccirilli JA (2005). Active site cytosine in the hepatitis delta virus ribozyme provides general acid catalysis. Nature Chem Biol 1:45–52 Doherty EA, Doudna JA (2000) Ribozyme structures and mechanisms. Annu Rev Biochem 69:597–615. 69:597–615 Dunham CM, Murray JB, Scott WG (2003) A helical twist-induced conformational switch activates cleavage in the hammerhead ribozyme. J Mol Biol 332:327–336 Fauzi H, Kawakami J, Nishikawa F, Nishikawa S (1997) Analysis of the cleavage reaction of a trans-acting human hepatitis delta virus ribozyme. Nucleic Acids Res 25:3124– 3130 Feldstein PA, Bruening G (1993) Catalytically active geometry in the reversible circularization of ‘mini-monomer’ RNAs derived from the complementary strand of tobacco ringspot virus satellite RNA. Nucl Acids Res 21:1991–1998 Ferré-D’Amaré AR (2004) The hairpin ribozyme. Biopolymers 73:71–78 Ferré-D’Amaré AR, Doudna JA (1999) RNA folds: insights from recent crystal structures. Annu Rev Biophys Biomol Struct 28:57–73 Ferré-D’Amaré AR, Doudna JA (2000) Crystallization and structure determination of a hepatitis delta virus ribozyme: use of the RNA-binding protein U1A as a crystallization module. J Mol Biol 295:541–556 Ferré-D’Amaré AR, Rupert PB (2002) The hairpin ribozyme: from crystal structure to function. Bioch Soc Trans 30:1105–1109 Ferré-D’Amaré AR, Zhou K, Doudna JA (1998a) Crystal structure of a hepatitis delta virus ribozyme. Nature 395:567–574 Ferré-D’Amaré AR, Zhou K, Doudna JA (1998b) A general module for RNA crystallization. J Mol Biol 279:621–631 Fersht A. (1985). Enzyme Structure and Mechanism. W.H. Freeman and Company, New York Gerlt JA, Westheimer FH, Sturtevant JM (1975) The enthalpies of hydrolysis of acyclic, monocyclic, and glycoside cyclic phosphate diesters. J Biol Chem 250:5059–5067 Hampel A, Cowan JA (1997) A unique mechanism for RNA catalysis: the role of metal cofactors in hairpin ribozyme cleavage. Chem Biol 4:513-517 Hampel A, Tritz R. 1989. RNA catalytic properties of the minimum (-)sTRSV sequence. Biochemistry 28:4929–4933 Harris DA, Rueda D, Walter NG (2002) Local conformational changes in the catalytic core of the trans-acting hepatitis delta virus ribozyme accompany catalysis. Biochemistry 41:12051–12061 Hegg LA, Fedor FJ (1995) Kinetics and thermodynamics of intermolecular catalysis by hairpin ribozymes. Biochemistry 34:15813–15828 Hertel KJ, Uhlenbeck OC (1995) The internal equilibrium of the hammerhead ribozyme reaction. Biochemistry 34:1744–1749 Jencks WP (1969) Catalysis in chemistry and enzymology. McGraw-Hill Book Company, New York Jeng K-S, Daniel A, Lai MMC (1996) A pseudoknot ribozyme structure is active in vivo and required for hepatitis delta virus RNA replication. J Virol 70:2403–2410 Ke A, Zhou K, Ding F, Cate JH, Doudna JA (2004) A conformational switch controls hepatitis delta virus ribozyme catalysis. Nature 429:201–205

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Khvorova A LA, Westhof E, Jayasena SD (2003) Sequence elements outside the hammerhead ribozyme catalytic core enable intracellular activity. Nature Struct Biol 10:708–712 Kuo MY-P, Sharmeen L, Dinter-Gottleib G, Taylor J (1988) Characterization of selfcleaving RNA sequences on the genome and antigenome of human hepatitis delta virus. J Virol 62:4439–4444 Lazinski DW, Taylor JM (1995a) Intracellular cleavage and ligation of hepatitis delta virus genomic RNA: Regulation of ribozyme activity by cis-acting sequences and host factors. J Virol 69:1190–1200 Lazinski DW, Taylor JM (1995b) Regulation of the hepatitis delta virus ribozymes: To cleave or not to cleave? RNA 1:225–233 MacNaughton TB, Wang Y-J, Lai MMC (1993) Replication of hepatitis delta virus RNA: effect of mutations of the autocatalytic cleavage sites. J Virol 67:2228–2234 Murchie AI, Thomson JB, Walter F, Lilley DM (1998) Folding of the hairpin ribozyme in its natural conformation achieves close physical proximity of the loops. Mol Cell 1:873–881 Murray JB, Dunham CM, Scott WG (2002) A pH-dependent conformational change, rather than the chemical step, appears to be rate-limiting in the hammerhead ribozyme cleavage reaction. J Mol Biol 315:121–130 Murray JB, Seyhan AA, Walter NG, Burke JM, Scott WG (1998) The hammerhead, hairpin and VS ribozymes are catalytically proﬁcient in monovalent cations alone. Chem Biol 5:587–595 Nakano S, Cerrone AL, Bevilacqua PC (2003) Mechanistic characterization of the HDV genomic ribozyme: classifying the catalytic and structural metal ion sites within a multichannel reaction mechanism. Biochemistry 42:2982–2994 Nakano S-I, Chadalavada DM, Bevilacqua PC (2000) General acid-base catalysis in the mechanism of a hepatitis delta virus ribozyme. Science 287:1493–1497 Nakano S-I, Proctor DJ, Bevilacqua PC (2001) Mechanistic characterization of the HDV genomic ribozyme: assessing the catalytic and structural contributions of divalent metal ions within a multi-channel reaction mechanism. Biochemistry 40:12022–12038 Nishikawa F, Nishikawa S (2000) Requirement for canonical base pairing in the short pseudoknot structure of genomic hepatitis delta virus ribozyme. Nucl Acids Res 28:925–931 O’Rear JL, Wang S, Feig AL, Beigelman L, Uhlenbeck OC, Herschlag D (2001) Comparison of the hammerhead cleavage reactions stimulated by monovalent and divalent cations. RNA 7:537–545 Oyelere AK, Strobel SA (2000) Biochemical detection of dytidine protonation within RNA. JACS 122:10259–10267 Pereira MJ, Harris DA, Rueda D, Walter NG (2002) Reaction pathway of the trans-acting hepatitis delta virus ribozyme: A conformational change accompanies catalysis. Biochemistry 41:730–740 Perrotta AT, Been MD (1990) The self-cleaving domain from the genomic RNA of hepatitis delta virus: sequence requirements and the effects of denaturant. Nucl Acids Res 18:6821–6827 Perrotta AT, Been MD (1991) A pseudoknot-like structure required for efﬁcient selfcleavage of hepatitis delta virus RNA. Nature 350:434–436

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Perrotta AT, Been MD (1992) Cleavage of oligoribonucleotides by a ribozyme derived from the hepatitis δ virus RNA sequence. Biochemistry 31:16–21 Perrotta AT, Been MD (1993) Assessment of disparate structural features in three models of the hepatitis delta virus ribozyme. Nucl Acids Res 21:3959–3965 Perrotta AT, Been MD (1996) Core sequences and a cleavage site wobble pair required for HDV antigenomic ribozyme self-cleavage. Nucl Acids Res 24:1314–1321 Perrotta AT, Been MD (1998) A toggle duplex in hepatitis delta virus self-cleaving RNA that stabilizes an inactive and a salt-dependent pro-active ribozyme conformation. J Mol Biol 279:361–373 Perrotta AT, Nikiforova O, Been MD (1999a) A conserved bulged adenosine in a peripheral duplex of the antigenomic HDV self-cleaving RNA reduces kinetic trapping of inactive conformations. Nucl Acids Res 27:795–802 Perrotta AT, Shih I-h, Been MD (1999b) Imidazole Rescue of a Cytosine Mutation in a Self-Cleaving Ribozyme. Science 286:123–126 Robertson HD (1992) Replication and evolution of viroid-like pathogens. Curr Top Microbiol Immunol 176:213–219 Rosenstein SP, Been MD (1990) Self-cleavage of hepatitis delta virus genomic strand RNA is enhanced under partially denaturing conditions. Biochemistry 29:8011– 8016 Rosenstein SP, Been MD (1991) Evidence that genomic and antigenomic RNA selfcleaving elements from hepatitis delta virus have similar secondary structures. Nucl Acids Res 19:5409–5416 Rupert PB, Ferré-D’Amaré AR (2004) Crystallization of the hairpin ribozyme: illustrative protocols. Methods Mol Biol 252:303–311 Rupert PB, Ferré-D’Amaré AR (2001) Crystal structure of a hairpin ribozyme-inhibitor complex with implications for catalysis. Nature 410:780–786 Sharmeen L, Kuo MY-P, Dinter-Gottlieb G, Taylor J (1988) Antigenomic RNA of human hepatitis delta virus can undergo self-cleavage. J Virol 62:2674-2679 Shih I-h, Been MD (1999) Ribozyme cleavage of a 2’,5’-phosphodiester linkage: mechanism and a restricted divalent metal ion requirement. RNA 5:1140–1148 Shih I-h, Been MD (2001a) Energetic contribution of non-essential 5’ sequence to catalysis in a hepatitis delta virus ribozyme. EMBO J 20:4884–4891 Shih I-h, Been MD (2001b) Involvement of a cytosine side chain in proton transfer in the rate- determining step of ribozyme self-cleavage. Proc Natl Acad Sci USA 98:1489–1494 Slim G, Gait MJ (1991) Conﬁgurationally deﬁned phosphorothioate-containing oligoribonucleotides in the study of the mechanism of cleavage of hammerhead ribozymes. Nucl Acids Res 19:1183–1188 Soukup GA, Breaker RR (1999) Relationship between internucleotide linkage geometry and the stability of RNA. RNA 5:1308–1325 Suh Y-A, Kumar PKR, Taira K, Nishikawa S (1993) Self-cleavage activity of the genomic HDV ribozyme in the presence of various divalent metal ions. Nucl Acids Res 21:3277–3280 Tanner NK (1995) The catalytic RNAs from hepatitis delta virus: structure, function, and application. Dinter-Gottlieb G. (ed) The Unique Hepatitis Delta Virus. pp 11–29. Springer-Verlag, New York

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Tanner NK, Schaff S, Thill G, Petit-Koskas E, Crain-Denoyelle A-M, Westhof E (1994) A three-dimensional model of hepatitis delta virus ribozyme based on biochemical and mutational analyses. Curr Biol 4:488–497 Thill G, Vasseur M, Tanner NK (1993) Structural and sequence elements required for the self-cleaving activity of the hepatitis delta virus ribozyme. Biochemistry 32:4254–4262 van Tol H, Buzayan JM, Feldstein PA, Eckstein F, Bruening G (1990) Two autolytic processing reactions of a satellite RNA proceed with inversion of conﬁguration. Nucl Acids Res 18:1971–1975 Wadkins TS, Been MD (1997) Core-associated non-duplex sequences distinguishing the genomic and antigenomic self-cleaving RNAs of hepatitis delta virus. Nucl Acids Res 25:4085–4092 Wadkins TS, Been MD (2002) Ribozyme activity in the genomic and antigenomic RNA strands of hepatitis delta virus. Cell Mol Life Sci 59:112–125 Wadkins TS, Perrotta AT, Ferré-D’Amaré AR, Doudna JA, Been MD (1999) A nested double-pseudoknot is required for self-cleavage activity of both the genomic and antigenomic HDV ribozymes. RNA 5:720–727 Wadkins TS, Shih I, Perrotta AT, Been MD (2001) A pH-sensitive RNA tertiary interaction affects self-cleavage activity of the HDV ribozymes in the absence of added divalent metal ion. J Mol Biol 305:1045–1055 Walter F, Murchie AI, Duckett DR, Lilley DM (1998) Global structure of four-way RNA junctions studied using ﬂuorescence resonance energy transfer. RNA 4:719–728 Wang K-S, Choo Q-L, Weiner AJ, Ou J-H, Najarian RC, Thayer RM, Mullenbach GT, Denniston KJ, Gerin JL, Houghton M (1986) Structure, sequence and expression of the hepatitis delta (δ) viral genome. Nature 323:508–514 Wu HN, Lin YJ, Lin FP, Makino S, Chang MF, Lai MM (1989) Human hepatitis delta virus RNA subfragments contain an autocleavage activity. Proc Nat Acad Sci USA 86:1831–1835 Young KJ, Gill F, Grasby JA (1997) Metal ions play a passive role in the hairpin ribozyme catalysed reaction. Nucl Acids Res 25:3760–3766

CTMI (2006) 307:67–89 c Springer-Verlag Berlin Heidelberg 2006

RNA Editing in Hepatitis Delta Virus J. L. Casey (u) Department of Microbiology and Immunology, Georgetown University Medical Center, Washington, DC, USA [email protected]

1 1.1 1.2 1.3 1.4

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . HDV Produces Two Forms of HDAg from the Same Gene . . . . . . . . . . What Is RNA Editing? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Adenosine Deamination at the Amber/W Site in the HDV Antigenome The Role of RNA Editing in the HDV Replication Cycle . . . . . . . . . . .

2

Host Enzymes Required for HDV RNA Editing . . . . . . . . . . . . . . . . . . . 72

3 3.1 3.2

Factors Affecting Substrate Selection . . . . . . . . . . . . . . . . . . . . . . . . . . 72 RNA Sequence and Structural Requirements for Editing . . . . . . . . . . . . . 73 Variations in Amber/W Site Structures Among HDV Genotypes . . . . . . . 75

4 4.1 4.2

Effects of Variations in Editing on HDV RNA Replication and Virus Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 Effects of Excessive Editing at the Amber/W Site . . . . . . . . . . . . . . . . . . 77 Effects of Diminished Editing at the Amber/W Site . . . . . . . . . . . . . . . . 78

5 5.1 5.2 5.2.1 5.2.2 5.2.3

Control of HDV RNA Editing . . . . . . . . . . Restriction of Editing to the Amber/W Site Regulation of Editing Levels . . . . . . . . . . Effects of HDAg . . . . . . . . . . . . . . . . . . . Effects of RNA Structural Dynamics . . . . . Negative Feedback Regulation . . . . . . . . .

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Abstract Hepatitis delta virus (HDV) relies heavily on host functions and on structural features of the viral RNA. A good example of this reliance is found in the process known as HDV RNA editing, which requires particular structural features in the HDV antigenome, and a host RNA editing enzyme, ADAR1. During replication, the adenosine at the amber/W site in the HDV antigenome is edited to inosine. As a result, the amber stop codon in the hepatitis delta antigen (HDAg) open reading frame is changed to a tryptophan codon and the reading frame is extended by 19 or 20 codons. Because these extra amino acids alter the functional properties of HDAg, this change serves a critical purpose in the HDV replication cycle. Analysis of the RNA secondary

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structures and regulation of editing in HDV genotypes I and III has indicated that although editing is essential for both genotypes, there are substantial differences. This review covers the mechanisms of RNA editing in the HDV replication cycle and the regulatory mechanisms by which HDV controls editing.

1 Introduction 1.1 HDV Produces Two Forms of HDAg from the Same Gene Hepatitis delta virus (HDV) is often compared to viroids because of the characteristic unbranched rod secondary structure formed by its RNA and the relatively small size of its genome. However, unlike viroids, HDV does contain one gene that encodes the sole viral protein, HDAg. Early analyses showed two electrophoretic forms of HDAg in liver and viral particles isolated from serum (Bergmann and Gerin 1986; Bonino et al. 1981, 1984, 1986). (These forms were sometimes referred to by their apparent molecular weights, p-24 and p-27; they are denoted here as S-HDAg and L-HDAg for short and long, respectively.) Following the cloning of HDV cDNAs (Makino et al. 1987; Wang et al. 1986), a series of studies illuminated the functional roles of S-HDAg and L-HDAg in HDV replication: S-HDAg is required for replication of HDV RNA, and LHDAg is required for the formation of HDV particles (Chang et al. 1991; Glenn et al. 1992; Hwang et al. 1992). Early studies found that L-HDAg also inhibits HDV RNA replication (Chao et al. 1990; Kuo et al. 1989), but more recent analyses suggest that this might not always be the case, particularly for antigenome RNA synthesis (Macnaughton and Lai 2002; Modahl and Lai 2000). Cloning and sequencing of the genome in 1986 indicated heterogeneity at several positions in the 1679 nucleotide (nt) genome (Wang et al. 1986). This variability affected the predicted length of HDAg: some clones contained a UAG (amber) stop as the 196th codon and encoded a 195 amino acid protein, other clones had UGG at this location and encoded a protein 214 amino acids in length (Wang et al. 1986; Xia et al. 1990). Expression of protein from clones that contained either the UAG or UGG sequence showed that the former encoded S-HDAg and the latter L-HDAg (Weiner et al. 1988; Xia et al. 1990). Subsequently, a series of studies in cultured cells and in a chimpanzee infected by injection of an HDV cDNA clone led to the remarkable discovery that the heterogeneity at this position arose during the course of HDV replication. Although transfected cDNAs encoded only S-HDAg, both S-HDAg and L-HDAg were detected (Luo et al. 1990; Sureau et al. 1989). No L-HDAg was detected

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when cells were transfected with an expression construct for S-HDAg that did not produce replicating HDV RNA. Thus, the appearance of L-HDAg was linked to HDV replication. Because of the different functions of S-HDAg and L-HDAg, the synthesis of L-HDAg late in the replication cycle is an example of a classic switch from viral RNA replication to genome packaging. Analysis of HDV RNA isolated from the serum of the transfected chimpanzee and from transfected cultured cells showed that heterogeneity appeared at the position corresponding to the adenosine in the UAG stop codon for S-HDAg (Luo et al. 1990). Subsequent studies in transfected cells showed that the appearance of L-HDAg and sequence heterogeneity at this site are temporally correlated; moreover, mutations that abolished the appearance of heterogeneity also prevented L-HDAg production (Casey et al. 1992). These studies indicated that some genomes encoding S-HDAg are converted, or edited, to encode L-HDAg during the course of HDV replication. The site at which editing occurs has been termed amber/W in accord with the codon change that accompanies the sequence modiﬁcation. Because of the essential functions of S-HDAg and L-HDAg editing plays a central role in the HDV replication cycle. 1.2 What Is RNA Editing? The term RNA editing was ﬁrst used in the late 1980s to describe an unusual process in which multiple Us are inserted and deleted in trypanosome mitochondrial mRNAs (Benne et al. 1986). The usage of the term was subsequently expanded as it was applied to other, less drastic, examples of nucleotide changes in RNAs, including deamination of C to U in apoB100 mRNA in small intestine (Scott 1989), deamination of glutamate receptor subunit B (gluRB) pre-mRNA in brain (Higuchi et al. 1993), and insertion of nontemplated Gs in the P gene of paramyxoviruses (Curran and Kolakofsky 1990). Thus, broadly deﬁned, the term RNA editing describes processes other than splicing that result in the modiﬁcation of an RNA sequence from that of its template. While collectively referred to as RNA editing, these sequence revisions involve a wide range of mechanisms. In the two types of editing used by mammalian cells, C to U and A to I, the modiﬁed base within the RNA molecule is deaminated; there is no evidence that the phosphate backbone is broken during the editing process. 1.3 Adenosine Deamination at the Amber/W Site in the HDV Antigenome One difﬁculty encountered in establishing the mechanism of editing at the amber/W site was identifying the RNA substrate: assays performed on repli-

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cating RNAs could not deﬁnitively determine whether the substrate for editing was the genome or the antigenome, or even whether editing was the result of co-transcriptional misincorporation. Although initial attempts led to the erroneous suggestion that the genomic RNA might be the substrate, in which case editing would occur as a U to C transition (Casey et al. 1992; Zheng et al. 1992), the use of nonreplicating RNA expression constructs that could exclusively produce either genomic or antigenomic RNA in transfected cells led to the unambiguous conclusion that editing occurs on the antigenome RNA (Casey and Gerin 1995). This result was further supported by analysis of editing on in vitro transcribed RNAs mixed with nuclear extracts: only antigenomic RNA was edited at the amber/W site (Casey and Gerin 1995). This observation indicated that HDV editing occurs post-transcriptionally, and is not the result of transcriptional misincorporation. Subsequently, it was shown that RNA adenosine deaminase (ADAR) from Xenopus laevis can edit the amber/W site in the HDV antigenome with considerable speciﬁcity in vitro (Polson et al. 1996). Accordingly, the type of RNA editing used by HDV is adenosine deamination. In this process, the amino group of adenosine is removed and replaced with a keto oxygen. Because this position of the base is changed from a hydrogen bond donor to an acceptor, the Watson–Crick base-pairing preference of this nucleotide is changed from pairing with U to pairing with C. Therefore, in any subsequent functions that involve base pairing (such as translation, RNA-templated transcription, and splice site identiﬁcation) the edited position will behave as G rather than the original A. Adenosine deamination has the potential to produce as many as 15 different recodings of an RNA transcript, including the creation of a methionine start codon and the abolition of stop codons. Thus, for example, when the adenosine at the amber/W site in the HDV RNA is edited, a UAG amber stop codon is changed to UIG, which behaves like UGG, and encodes tryptophan. As indicated by this example, sites in RNAs that undergo adenosine deamination have been named according to the coding change brought about by editing. 1.4 The Role of RNA Editing in the HDV Replication Cycle In the HDV replication scheme editing occurs at the amber/W site on the antigenomic RNA (see Fig. 1). The cycle begins with genomes encoding S-HDAg, the form required for RNA synthesis. Three RNA species are produced: the mRNA encoding S-HDAg, the antigenome, which serves as replication intermediate, and the genome. During replication, in some antigenome RNAs the adenosine at the amber/W site is deaminated to inosine. Because inosine forms base pairs with C rather than U, subsequent genome RNA synthe-

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Fig. 1 The role of RNA editing in the HDV replication cycle. Dark heavy lines represent antigenomic sense RNAs; gray lines indicate genome RNAs. 1, Synthesis of mRNA encoding S-HDAg; 2, replication of full-length antigenomic and genomic RNA; 3, translation of S-HDAg, which is required for RNA replication; 4, during replication some of the antigenomic RNA is edited at the amber/W site by the host RNA adenosine deaminase ADAR1; 5, antigenomic RNA containing I at the editing site serves as template for the synthesis of genomic RNA containing C at the complementary position; 6, synthesis of mRNA encoding L-HDAg; 7, replication of genomic and antigenomic RNA encoding L-HDAg; 8, translation of L-HDAg, which inhibits RNA replication, and is required for virion packaging

sis results in the appearance of C at the corresponding position in the genome. Transcription from such genomes leads to the production of mRNA encoding L-HDAg, which can limit further RNA synthesis and initiates the packaging process. It is important to note that, unlike cellular mRNA substrates for RNA adenosine deamination, HDV mRNA is not edited directly. Rather, editing occurs on the full-length antigenome, which is a replication intermediate. Consistent with this model, some of the sequences that form the structure required for editing (see below) are more than 300 nt downstream of the polyadenylation and ribozyme sites, and are not included in the mRNA sequence. Furthermore, analysis of RNA in viral particles indicates that genome RNAs contain the expected C at the position complementary to the amber/W site. From the scheme depicted in Fig. 1 it is clear that editing plays a central role in the HDV replication cycle. Because L-HDAg is a limiting factor for virus production, insufﬁcient editing reduces virus output and is likely to limit propagation in the host (Jayan and Casey 2002b, 2005). Conversely, excessive editing strongly diminishes viral RNA accumulation (Jayan and Casey 2002a, 2005; Sato et al. 2004). Moreover, because edited genomes are

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also packaged into virions, excessive editing reduces the infectivity of viral progeny. The remainder of this chapter will cover recent efforts to identify the speciﬁc host enzymes involved in HDV RNA editing, the RNA secondary structures involved, and the factors that determine where and how much editing occurs.

2 Host Enzymes Required for HDV RNA Editing ADAR edits adenosines in double-stranded RNA (dsRNA); this activity is present in nuclear extracts from numerous metazoan species (Bass). As mentioned above, it was shown that ADAR from Xenopus laevis can edit the amber/W site in the HDV antigenome with considerable speciﬁcity in vitro (Polson et al. 1996). While only one ADAR has been identiﬁed in Xenopus, mammalian cells contain two related genes, ADAR1 and ADAR2, that encode proteins capable of editing adenosine in dsRNA (Melcher et al. 1996; O’Connell et al. 1995; Patterson and Samuel 1995; Yang et al. 1997). These proteins contain a catalytic deaminase domain along with three or two, respectively, copies of dsRNA binding motifs (DRBMs). Both genes are essential for viability in mice (Brusa et al. 1995; Wang et al. 2000). Both ADAR1 and ADAR2 can edit HDV RNA at the amber/W site in transfected cultured cells (Jayan and Casey 2002a; Sato et al. 2001; Wong et al. 2001). However, because the level of ADAR1 mRNA expression is considerably higher than ADAR2 in liver, it seems likely that ADAR1 is responsible for editing during HDV infection of hepatocytes. Consistent with this idea, knockdown experiments using small interfering RNA (siRNA) have shown that the short form of ADAR1, which is localized in the nucleus, is responsible for amber/W site editing during HDV replication in Huh-7 cells (Jayan and Casey 2002b; Wong and Lazinski 2002). Because the HDV amber/W site was edited with high speciﬁcity in vitro using just puriﬁed Xenopus ADAR, no additional factors aside from HDV RNA and ADAR are required for amber/W site editing to occur (Polson et al. 1996). Nevertheless, it is possible that additional factors, such as HDAg, can contribute to the efﬁciency and speciﬁcity of editing (see Sect. 3).

3 Factors Affecting Substrate Selection ADAR activity was ﬁrst identiﬁed due to its ability to extensively modify adenosines in dsRNA. Indeed, the deamination of up to 50% of adenosines

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in dsRNA destabilized base pairing to such an extent that the activity was initially described as an ‘RNA unwindase’ (Bass and Weintraub 1987, 1988; Wagner et al. 1989). In such dsRNAs, the likelihood of editing at individual adenosines is determined largely by: (1) the identity of the 5 nucleotide neighbor—G is strongly disfavored; and (2) the distance from the 3 end of the RNA—adenosines less than 20 nt from the 3 end are not deaminated (Polson and Bass 1994). Despite the role of its activity on dsRNAs in the initial characterization of ADAR, it is not yet clear to what extent editing on dsRNAs is an important cellular function. However, it is clear that ADARs edit several RNAs with high speciﬁcity and that some of these editing events are highly important (Bass 2002; Gott and Emeson 2000; Seeburg 1998, 2002). 3.1 RNA Sequence and Structural Requirements for Editing Inspection of the predicted structures of known sites for speciﬁc editing reveals several common features. All include at least six contiguous base pairs around the editing site, and many substrates contain more. In most cases the target adenosine occurs as either an A–U pair or an A–C mismatch pair. Mutational analyses of some substrates, including the HDV genotype I amber/W site, have indicated that editing levels are higher when the adenosine occurs as an A–C mismatch rather than an A–U pair (Casey et al. 1992; Herb et al. 1996; Lomeli et al. 1994; Polson et al. 1996; Wong et al. 2001). Moreover, at least for HDV genotype I, any change in the position opposite the amber/W site (deletion, or substitution by A or G), led to markedly reduced editing levels. (Casey et al. 1992) None of the known substrates for speciﬁc editing contain the disfavored G as the 5 neighbor of the editing site. Outside the immediate vicinity of editing sites the requirements for base pairing are distributed asymmetrically along the RNA (Lehmann and Bass 1999; Polson and Bass 1994). Base-pairing on the 5 side of sites varies between two and ﬁve base-pairs. On the 3 side base-pairing is greater, in most cases extending for at least about 20 base-pairs. Analysis of editing on dsRNA templates in vitro has led to a model in which ADAR1 interacts with a base-paired region extending about 20 nt to the 3 side of edited adenosines (Lehmann and Bass 1999; Polson and Bass 1994); this interaction most likely occurs via the three DRBMs of ADAR1 (Liu et al. 1998; Liu and Samuel 1996). In dsRNA substrates, editing sites could tolerate small disruptions of base pairing in the region downstream, but the presence of a 6-nt internal loop strongly diminished editing (Lehmann and Bass 1999; Polson and Bass 1994). In most cases base pairing 3 of sites that are substrates for highly speciﬁc editing are

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disrupted by mismatches, bulges and internal loops, but these disruptions are generally not as large as a 6-nt internal loop. The role of these disruptions may be to orient the ADAR protein via the double-stranded RNA binding domains such that the deaminase domain is positioned correctly at the editing site (Bass 2002; Lehmann and Bass 1999; Ohman et al. 2000; Polson and Bass 1994).

Fig. 2 Comparison of amber/W sites in genotypes I and III. Upper: schematic diagram of HDV genome, indicating the unbranched rod and the location of the HDAg coding region. The ﬁlled bar above the genome indicates the S-HDAg coding region; the open bar, including the W indicates the additional amino acids added to make L-HDAg. Dashed boxes indicate sequences from the coding and noncoding regions that make up the amber/W editing site. Middle: the amber stop codon (UAG) is edited by ADAR1in the unbranched rod conformation of genotype I RNA; for type III, however, editing occurs in the double hairpin structure, not the unbranched rod. Lower: the predicted secondary structures around the genotype I and III amber/W sites are shown. Vertical lines indicate A–U and G–C base-pairs; dots indicate G–U pairs. Positions that have been shown to be critical for editing are shaded in the genotype I structure. Arrows indicate internal loops and bulges; improved base-pairing at these locations increased editing (Jayan and Casey 2005)

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The RNA secondary structure downstream of the HDV amber/W site in HDV genotype I contains base-paired segments, but is more frequently disrupted by bulges and mismatches than other editing substrates. Mutations that improved base-pairing, particularly in the region 15–25 nt 3 of the editing site (see arrows in Fig. 2, left panel), increased editing signiﬁcantly (Jayan and Casey 2005; Sato et al. 2004). However, the increased editing resulted in dramatically lower levels of HDV RNA replication, principally due to excessive L-HDAg production (Jayan and Casey 2005; Sato et al. 2004). These results led to the suggestion that the HDV editing site (at least for genotype I) may have been selected to be suboptimal in order to prevent the rapid accumulation of too much L-HDAg, which could inhibit viral RNA replication. In light of the model for ADAR1 substrate activity, the bulges and mismatches 3 of the HDV genotype I amber/W site raise questions about the role of this region in editing at the amber/W site. Some studies have suggested that extensive base-pairing 3 of editing sites may not be essential for efﬁcient editing. Sato and Lazinski (Sato et al. 2001) found that a minimal substrate that was derived from the HDV amber/W site and that contained only eight base-pairs could be efﬁciently edited when ADAR1 was overexpressed. Herbert and Rich (2001) showed that ADAR1 could efﬁciently edit even when the three DRBMs were removed. Perhaps consistent with these ﬁndings, more extensive disruption of the base-pairing 3 of the HDV genotype I amber/W site had no apparent effect on editing due to endogenous ADAR1 in Huh-7 cells (Jayan and Casey 2005). These results could indicate that the deaminase domain itself possesses some RNA binding activity that can be effective under certain conditions. Alternatively, the role of the ADAR1 DRBMs in editing at the HDV amber/W site may be more complex than previously thought; perhaps these domains can also recognize and bind RNA segments that do not exhibit signiﬁcant dsRNA character. Further analyses of editing under more controlled conditions, such as in vitro, and using variant forms of ADAR1 and amber/W substrates may be necessary to resolve the role of dsRNA segments and the ADAR1 DRBMs in editing at the HDV amber/W site. 3.2 Variations in Amber/W Site Structures Among HDV Genotypes It is interesting to note that both the RNA secondary structure around the amber/W site and the C-terminal sequences speciﬁc to L-HDAg are distinguishing features of some HDV genotypes (Casey 2002; Casey et al. 1993; Hsu et al. 2002; Ivaniushina et al. 2001; Niro et al. 1997; Shakil et al. 1997) (see the chapter by P. Dény, this volume). The genotypic differences in the RNA secondary structure required for editing fall into two categories: (1) the dif-

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ferent portions of the HDV antigenome that form the structure; and (2) the speciﬁc RNA secondary structure formed around the amber/W site. Despite these variations, all three of the genotypes analyzed (I, II and III) are edited by the same enzyme—ADAR1 (Jayan and Casey 2002b). In HDV genotype I the structure required for amber/W site editing is part of the unbranched rod structure characteristic of HDV RNA (Fig. 2; Casey et al. 1992). The eight Watson–Crick base-pairs ﬂanking the amber/W site and the A–C mismatch pair involving the amber/W site are highly conserved among over 50 genotype I sequences (Niro et al. 1997; Yang et al. 1995). The role of this structure in editing has been conﬁrmed by site-directed mutagenesis studies (Casey et al. 1992; Casey and Gerin 1995; Polson et al. 1996; Wong et al. 2001). HDV genotype III RNA also forms an unbranched rod structure, which is required for RNA replication; however, the base-pairing in the immediate vicinity of the amber/W site is disrupted such that this structure does not function as a substrate for amber/W editing (Casey 2002). Rather, editing in genotype III requires an alternative, ‘double hairpin’ structure that creates better base-pairing in the immediate vicinity of the amber/W site (Fig. 2; Casey 2002). This structure, which differs from the unbranched rod structure by about 80 base-pairs, contains two stem–loops that essentially shift the positions of the noncoding side of the HDV antigenome that are base-paired with the amber/W site region. In the unbranched rod structure the amber/W site is opposite position 580; in the double hairpin structure, the paired position is 509. The structure required for editing in the other HDV genotypes has not yet been determined. Comparative analysis of the predicted secondary structure in the vicinity of the amber/W site in the unbranched rod reveals structures similar to the genotype I structure but more disrupted (Hsu et al. 2002; Ivaniushina et al. 2001; Radjef et al. 2004). Inspection of the predicted RNA secondary structures around the amber/W sites of genotypes I and III indicates that the genotype III amber/W site differs from the type I site. In some cases the differences occur at positions that have been shown to be essential for efﬁcient editing in genotype I (Fig. 2). For example, the A–C mismatch pair that involves the amber/W adenosine and which is highly conserved among genotype I isolates, occurs as an A– U pair in genotype III; when introduced by site-speciﬁc mutagenesis into a genotype I genome, this speciﬁc change substantially reduces both editing and virus production (Casey et al. 1992; Jayan and Casey 2005). The significance and effect of these variations on editing, RNA replication and virus production in genotype III remains to be determined. Perhaps the variations at the genotype III site can be explained by compensatory effects, such as changes elsewhere in the editing site region, including sequences/structures 3 of the editing site, or differences in the mechanisms by which HDV regulates

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editing during replication. Another (but not mutually exclusive) possibility is that the differences described are responsible for variations in the levels of editing among genotypes (Hsu et al. 2002). Deﬁning the structural determinants for editing remains an important goal. Despite recognition of the common features among editing sites noted in Sect. 3.1, the sequence and structural determinants for highly speciﬁc editing are still not well understood. Only a handful of substrates for highly speciﬁc editing have been identiﬁed to date in mammals, and it is anticipated that many more remain to be found (Paul and Bass 1998). Knowledge of sequence and structural requirements for editing will likely facilitate the prediction of potential adenosine deamination editing sites from analysis of genomic sequences. Moreover, it is reasonable to expect that differences in editing levels among different substrate adenosines are due in part to variations in the structure of the RNA in the vicinity of the editing sites. Understanding the effects of structural variations will contribute to our understanding of how this important post-transcriptional regulatory mechanism is modulated. Variations among the amber/W sites in the HDV genotypes may provide a valuable opportunity to evaluate the effects of different sequences and RNA secondary structures on editing at the HDV amber/W site.

4 Effects of Variations in Editing on HDV RNA Replication and Virus Production Examination of the role of editing in the HDV replication cycle (Fig. 1) suggests that varying the efﬁciency of editing at the amber/W site, either by altering levels of ADAR expression or by the introduction of mutations near the amber/W site, is likely to affect HDV replication, virus production, or both. Premature editing at the amber/W site could reduce levels of RNA replication because edited antigenomes encode L-HDAg, which is a trans-dominant inhibitor of HDV RNA replication (Chao et al. 1990; Glenn and White 1991). Insufﬁcient editing could inhibit virion production because L-HDAg is required for virus production (Ryu et al. 1992; Wang et al. 1992). Several studies have indicated just how sensitive HDV replication and virus production are to variations in editing (Casey 2002; Jayan and Casey 2002a, 2005; Sato et al. 2004). 4.1 Effects of Excessive Editing at the Amber/W Site Overexpression of ADAR1 by cotransfection of ADAR1 expression constructs resulted in increased editing at the amber/W site, and increased production

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of L-HDAg (Jayan and Casey 2002a; Sato et al. 2004). Concomitantly, levels of HDV RNA synthesis were strongly inhibited. Another approach gave similar results: increasing the base-pairing 3 of the amber/W site led to higher levels of editing and L-HDAg synthesis, and dramatic inhibition of viral RNA synthesis (Jayan and Casey 2005; Sato et al. 2004). In all cases, overproduction of LHDAg accounted for a signiﬁcant fraction of the inhibition. The sensitivity of replication to editing (via L-HDAg) is remarkable; in one study mutations that increased editing by approximately threefold led to a 50-fold decrease in RNA replication (Jayan and Casey 2005). It might be expected that inhibition of HDV RNA synthesis due to increased editing and subsequent L-HDAg overproduction would automatically inhibit viral particle production (because of decreased viral RNA levels within the cell). Indeed, inhibition of virus production was observed 6–12 days post-transfection with a site-directed mutant that exhibited increased editing (Jayan and Casey 2005). However, there was no inhibition of virus production before day 6, even though intracellular RNA levels were signiﬁcantly decreased. One explanation of these results is that intracellular viral RNA is not normally the limiting factor for particle production. Virus secretion was closely correlated with levels of L-HDAg, consistent with the interpretation that L-HDAg is the limiting factor for virus particle production. ADAR1 has several isoforms, one of which is induced by interferon. Although siRNA knockdown studies have shown that the shorter form of ADAR1, which is not induced by interferon, is primarily responsible for editing at the amber/W site (Wong and Lazinski 2002), treatment of Huh-7 cells with interferon has been shown to increase ADAR1 p150 expression and increase editing (Hartwig et al. 2004). Levels of HDV RNA were not analyzed in this study; based on the inhibition of replication associated with modest increases in editing due to ADAR co-transfection or editing site mutations (Jayan and Casey 2002a, 2005, Sato et al. 2004), it might be expected that HDV RNA levels would decrease. Conversely, previous studies indicated that interferon treatment of cultured cells did not affect HDV RNA replication (Ilan et al. 1992; McNair et al. 1994); however, the effects of interferon treatment on ADAR1 and amber/W editing were not assessed. Analysis of the effects of interferon on both editing and replication in the same study is required to clarify this issue. 4.2 Effects of Diminished Editing at the Amber/W Site The requirement of L-HDAg for production of HDV particles is clear (Chang et al. 1991; Ryu et al. 1992). Not surprisingly, inhibition of editing, either by site-directed mutagenesis (Casey 2002; Jayan and Casey 2005) or by siRNA-

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mediated knockdown of ADAR1 expression (Jayan and Casey 2002b), inhibited viral particle production in transfected cells. Early studies on the effect of L-HDAg indicated that L-HDAg expression strongly inhibited HDV RNA replication (Chao et al. 1990; Glenn and White 1991). However, editing site mutations that prevent L-HDAg production do not result in increased levels of HDV RNA replication, at least for genotype I constructs (Macnaughton and Lai 2002; Sato et al. 2004; Wu et al. 1995). Partly based on this result, it has been suggested that L-HDAg might not actually inhibit HDV RNA accumulation, at least under normal circumstances in Huh-7 cells (Macnaughton and Lai 2002). However, the results discussed in Sect. 4.1 indicate that inhibition of replication does occur when L-HDAg is overproduced by excessive editing during the course of HDV RNA replication. In contrast to the observations with genotype I, genotype III RNA replication is increased at least ﬁvefold by mutations that abolish editing (Casey 2002). One interpretation of these results is that, at least in Huh-7 cells, maximum levels of HDV RNA replication are limited by factors other than L-HDAg production; thus, decreased L-HDAg production does not increase replication. However, when L-HDAg is overproduced, replication becomes sensitive. Genotype III RNA replication may either be less sensitive to these as yet undeﬁned limitations, or more sensitive to L-HDAg.

5 Control of HDV RNA Editing Control of HDV RNA editing occurs on several levels. First, the HDV antigenome contains about 337 adenosines, but editing is highly speciﬁc for the amber/W site. Second, both the rate and extent of editing appear to be carefully controlled. Some host substrates for editing exhibit modiﬁcation rates approaching 100%, and this editing likely occurs rapidly; most known host substrates are pre-mRNAs that are edited prior to splicing. In contrast, for HDV, edited viral RNAs accumulate slowly and levels typically plateau at less than 30% edited after 12 days in transfected cells in culture. 5.1 Restriction of Editing to the Amber/W Site ADAR1 and ADAR2 can extensively edit long (≥ 50 base-pairs) doublestranded RNAs, in which up to 50% of adenosines may be deaminated. Clearly, promiscuous editing such as occurs on dsRNA could be deleterious to virus replication. Indeed, spurious editing on HDV RNA by overexpressed ADAR1

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and ADAR2 led to the production of protein variants that inhibited replication (Jayan and Casey 2002a). However, even though HDV RNA exhibits signiﬁcant base-pairing in the unbranched rod structure, promiscuous editing does not typically occur during HDV infection; the amber/W site is edited 600-fold more efﬁciently than the other 337 adenosines in the RNA (Polson et al. 1998). It is worth noting here that, although editing at nonamber/W sites does not appear to occur at levels important for the replication cycle, the genetic evolution of the virus may nevertheless be affected by ADAR editing during the course of infection (see the chapter by J.L. Casey and J.L. Gerin, this volume). It is likely that the primary and secondary structure of the HDV RNA have evolved to avoid undesirable (for the virus) editing at sites other than amber/W. As noted in Sect. 3, analysis of editing on dsRNAs has indicated that adenosines with a 5 guanosine neighbor are much less likely to be deaminated than other adenosines (Polson and Bass 1994). In both the HDV genome and antigenome, guanosine is by far the most common 5 neighbor for adenosine, and the ratios of observed to expected occurrences for the dinucleotides GA and UC (which would be GA in the complementary strand) are higher than for any other dinucleotides. This bias may be due, in part, to selection for sequences that place nonamber/W adenosines in contexts that are less likely to be edited. As for secondary structure, base-pairing in the HDV RNA unbranched rod structure is interrupted by frequent bulges, internal loops and mismatches, which have been shown to restrict editing on artiﬁcial dsRNA substrates (Aruscavage and Bass 2000; Lehmann and Bass 1999; Ohman et al. 2000). 5.2 Regulation of Editing Levels HDV must regulate both the rate and the extent of editing at the amber/W site because, as shown in Sect. 4, levels of viral RNA replication and virion production are sensitive to the kinetics and amount of L-HDAg produced. Moreover, as shown in Fig. 1, editing occurs not on the mRNA, but on the antigenome, which is a replication intermediate. Hence, HDV RNA editing levels within an infected cell at any given time are the result of the accumulation of all editing events within that cell up to that time, and the percentage of antigenomes containing the UGG codon (and genomes with ACC at the corresponding positions) increases with time. The cost of this mechanism to the virus is that a fraction of viral particles contain genomes that encode L-HDAg; such genomes cannot replicate (Glenn and White 1991). Thus, HDV must control the level of editing in order to ensure viability. HDV does not appear to regulate editing by affecting ADAR1 expression because ADAR1

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levels are unaffected by HDV replication (Wong and Lazinski 2002). Control mechanisms for editing rely on several viral components and functions, including: RNA structure, HDAg, and viral RNA replication. These mechanisms vary among genotypes, at least for genotypes I and III. Some of the control mechanisms may be described as passive, in that they are not affected by (or responsive to) the level of editing. This category includes the secondary structure of the RNA around the amber/W site. As mentioned in Sect. 3.1, the disruptions in base-pairing 3 of the amber/W site in HDV genotype I create a suboptimal substrate for editing. Mutations that increase base-pairing in this region increase editing, but severely reduce replication and virion production (Jayan and Casey 2005; Sato et al. 2004). It is not yet known whether the structures in the vicinity of the amber/W sites of other genotypes are also suboptimal. One potential dilemma for the virus that is posed by using a suboptimal structure to limit editing efﬁciency is that the speciﬁcity of editing is likely to be compromised because the speciﬁcity is determined by the ratio of the efﬁciency of editing at the amber/W site to the efﬁciency of editing at other ‘non-speciﬁc’ sites. The danger for the virus of nonspeciﬁc editing is the production of additional genomes defective for replication, or even the creation of dominant negative S-HDAg mutants (Jayan and Casey 2002a). Thus, there may be limits as to how much amber/W editing can be restricted by using suboptimal structures. HDV does appear to have a mechanism for minimizing the effects of editing at nonamber/W sites: in one study of HDV replicating in transfected cells, all nonamber/W changes that occurred during replication were found on genomes that were also edited at the amber/W site (Polson et al. 1998). 5.2.1 Effects of HDAg HDV genotype I uses an additional mechanism to slow down editing early in the replication cycle. For this genotype, S-HDAg, which is known to bind HDV RNA (Chang et al. 1988), is a strong inhibitor of editing at the amber/W site. While editing on replicating RNA 2–3 days post-transfection is nearly undetectable, up to 40% of nonreplicating RNAs produced in transfected cells in the absence of HDAg are edited. However, co-transfection of an S-HDAg expression construct leads to markedly reduced levels of editing on nonreplicating RNAs (Polson et al. 1998). The levels of S-HDAg required for this inhibition are similar to those seen in cells replicating HDV RNA. Thus, it appears that S-HDAg prevents the rapid accumulation of editing early in the HDV genotype I replication cycle. It has been suggested that inhibition occurs by HDAg binding to HDV RNA (Polson et al. 1998); however, it is not clear whether

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the inhibition is due to steric effects–such as blocking of an ADAR1 binding site by HDAg–or HDAg-mediated sequestration of HDV RNA in a cellular compartment in which ADAR1 is not active. Whether the ability of HDAg to inhibit editing varies during the course of viral RNA replication remains to be determined. In contrast to the sensitivity of genotype I amber/W site editing to S-HDAg, editing at the amber/W site in the genotype III double hairpin structure is not inhibited by S-HDAg (Cheng et al. 2003). The hairpin denoted SL2, on the 3 side of the amber/W site, plays an essential role (Cheng et al. 2003), and might somehow interfere with S-HDAg binding near the amber/W site. 5.2.2 Effects of RNA Structural Dynamics HDV genotype III uses the distribution of the RNA between at least two conformations to restrict editing (Casey 2002; Cheng et al. 2003). Only RNA molecules that adopt the double hairpin structure can be edited (Fig. 2). However, the majority of genotype III RNA appears to assume the unbranched rod conformation, which is not a substrate for editing (Casey 2002; Cheng et al. 2003). The introduction of mutations in the genotype III RNA that shift the distribution of the RNA to the double hairpin structure increases editing to levels comparable with those seen with nonreplicating genotype I RNA (Casey 2002). Thus, while the amber/W site itself in genotype III RNA can be edited with efﬁciency similar to the genotype I site, editing levels in nonreplicating genotype III RNAs are much lower because most of the RNA assumes the unbranched rod conformation, which is not a substrate for editing (Casey 2002). Preliminary data from our laboratory (Linnstaedt and Casey, unpublished results) indicates that the double hairpin structure is less stable than the unbranched rod and can only be formed co-transcriptionally. Thus, the structural dynamics of the RNA determine the amount of the double hairpin structure formed, which in turn determines how much RNA is available to be edited at the amber/W site. S-HDAg is not an effective inhibitor of editing for this genotype and likely does not play a direct role in limiting editing levels (Cheng et al. 2003). Possibly because editing in genotype III is downmodulated by the distribution of the RNA between two structures, further control by S-HDAg is not necessary. 5.2.3 Negative Feedback Regulation Two recent studies have indicated that editing can be regulated by negative feedback (Cheng et al. 2003; Sato et al. 2004). In HDV genotype I mutants that overproduce L-HDAg, levels of L-HDAg plateau as replication is shut down—

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by L-HDAg (Sato et al. 2004). This inhibition of L-HDAg production occurs because of the location of editing in the HDV replication scheme (Fig. 1). Editing occurs on the antigenome–the replication intermediate–and does not result in L-HDAg synthesis until the edited antigenome ﬁrst serves as template for the synthesis of genomes, which then serve as templates for transcription of mRNAs encoding L-HDAg. Thus, L-HDAg can, under these circumstances, limit its own production. In addition to the plateau in L-HDAg production, Sato et al. observed a plateau in editing at the amber/W site, which is not explained by the above model (Sato et al. 2004). They hypothesize that editing occurs only on newly transcribed antigenomic HDV RNA, perhaps before HDAg has a chance to bind and form a ‘mature’ RNP, and that L-HDAg indirectly inhibits editing by blocking new RNA synthesis. One important consideration of this model is the mechanism whereby L-HDAg prevents further antigenome synthesis. Other reports have indicated that L-HDAg does not inhibit antigenome RNA synthesis (Macnaughton and Lai 2002; Modahl and Lai 2000). Does inhibition of antigenome synthesis occur indirectly via shutdown of genomic RNA synthesis, or does L-HDAg produced as a result of editing inhibit antigenome RNA synthesis in a manner that is not obvious when L-HDAg is produced in trans? Another consideration is that the observed control of editing by L-HDAg was only observed when editing levels were artiﬁcially elevated—either by mutation or by ADAR overexpression. As mentioned above in Sect. 4, L-HDAg does not appear to limit replication of HDV genotype I RNA in transfected Huh-7 cells; rather, as yet undetermined factors limit RNA levels. Perhaps, editing in HDV genotype I is calibrated to these limitations, such that appropriate levels of editing are achieved just before replication is restricted; alternatively, L-HDAg may play a more important role limiting replication in infected hepatocytes than in Huh-7 cells. Cheng et al. recently showed that L-HDAg is a much better inhibitor of editing in HDV genotype III than is S-HDAg, which is a very poor inhibitor (see Sect. 5.2.1). This observation led to the suggestion that genotype III L-HDAg might directly inhibit its own production by directly inhibiting amber/W site editing. However, preliminary data from our laboratory (R. Chen and J.L. Casey, unpublished results) suggest that this inhibitory activity, which is unrelated to replication, might not be the predominant factor involved in a negative feedback loop to limit editing levels (Cheng et al. 2003). Although L-HDAg is a potent inhibitor of genotype III editing, mixtures of genotype III S-HDAg and L-HDAg at ratios similar to those found in cells replicating genotype III RNA exhibit inhibitory activities similar to S-HDAg. Hence, it appears that levels of L-HDAg achieved during replication might not be sufﬁcient to directly affect editing.

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In contrast to the behavior of HDV genotype I, wild-type HDV genotype III replication is affected by levels of L-HDAg production. Genotype III mutants that do not produce L-HDAg replicate at signiﬁcantly higher levels, and accumulate higher levels of editing (Casey 2002; Cheng et al. 2003). Thus, it seems likely that the predominant factor in the control of maximal editing levels in HDV genotype III is the ability of L-HDAg to inhibit replication. As discussed in Sect. 5.2.1, in the current model for genotype III, editing occurs on a metastable structure that is formed co-transcriptionally; cessation of transcription would prevent further editing because the structure required for editing would not be formed. Thus, while it remains to be determined whether L-HDAg functions to control editing of wild-type HDV genotype I RNA, it seems likely that it does in genotype III. A remaining question for HDV genotype III is why do S-HDAg and L-HDAg inhibit editing at such different levels (Cheng et al. 2003)? One possibility is that the more important aspect for the virus is that S-HDAg is not a good inhibitor. Because editing is already modulated by the conformational dynamics of the RNA, further inhibition by S-HDAg could lead to insufﬁcient levels of editing.

6 Perspective Editing at the amber/W site plays a critical role in the HDV replication cycle. Analysis of how the virus controls editing has led to valuable contributions to the ﬁeld of RNA adenosine deamination. Thus far, amber/W site editing is the only example of speciﬁc editing that occurs in an organ other than the brain in mammals, but it is highly likely that more examples will be identiﬁed. The differences in editing sites, structures, and regulatory mechanisms between HDV genotypes I and III is remarkable, and emphasizes just how different these two genotypes are. Understanding how editing is regulated during the course of HDV replication remains an important goal. There is still much to learn in this area, which is largely undeveloped for host targets of speciﬁc editing. One of the more exciting recent developments has been the identiﬁcation of the role of RNA structural dynamics in controlling amber/W site editing in HDV genotype III. Further analysis of editing at the amber/W site will advance our understanding of the determinants of viral replication and is likely to continue to contribute to the ﬁeld of RNA editing. Acknowledgements The work in the author’s laboratory is supported by NIH grant R01-AI42324. I thank Dr. Renxiang Chen and Sarah Linnstaedt in my laboratory for sharing unpublished results and for comments on the manuscript.

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References Aruscavage PJ, Bass BL (2000) A phylogenetic analysis reveals an unusual sequence conservation within introns involved in RNA editing. RNA 6:257–269 Bass BL (2002) RNA editing by adenosine deaminases that act on RNA. Annu Rev Biochem 71:817–846 Bass BL, Weintraub H (1987) A developmentally regulated activity that unwinds RNA duplexes. Cell 48:607–613 Bass BL, Weintraub H (1988) An unwinding activity that covalently modiﬁes its doublestranded RNA substrate. Cell 55:1089–1098 Benne R, Van den Burg J, Brakenhoff JP, Sloof P, Van Boom JH, Tromp MC (1986) Major transcript of the frameshifted coxII gene from trypanosome mitochondria contains four nucleotides that are not encoded in the DNA. Cell 46:819–826 Bergmann KF, Gerin JL (1986) Antigens of hepatitis delta virus in the liver and serum of humans and animals. J Infect Dis 154:702–706 Bonino F, Heermann KH, Rizzetto M, Gerlich WH (1986) Hepatitis delta virus: protein composition of delta antigen and its hepatitis B virus-derived envelope. J Virol 58:945–950 Bonino F, Hoyer B, Ford E, Shih JW, Purcell RH, Gerin JL (1981) The delta agent: HBsAg particles with delta antigen and RNA in the serum of an HBV carrier. Hepatology 1:127–131 Bonino F, Hoyer B, Shih JW, Rizzetto M, Purcell RH, Gerin JL (1984) Delta hepatitis agent: structural and antigenic properties of the delta- associated particle. Infect Immun 43:1000–1005 Brusa R, Zimmermann F, Koh DS, Feldmeyer D, Gass P, Seeburg PH, Sprengel R (1995) Early-onset epilepsy and postnatal lethality associated with an editing- deﬁcient GluR-B allele in mice. Science 270:1677–1680 Casey JL (2002) RNA Editing in Hepatitis Delta Virus Genotype III Requires a Branched Double-Hairpin RNA Structure. J Virol 76:7385–7397 Casey JL, Bergmann KF, Brown TL, Gerin JL (1992) Structural requirements for RNA editing in hepatitis delta virus: evidence for a uridine-to-cytidine editing mechanism. Proc Natl Acad Sci USA 89:7149–7153 Casey JL, Brown TL, Colan EJ, Wignall FS, Gerin JL (1993) A genotype of hepatitis D virus that occurs in northern South America. Proc Natl Acad Sci USA 90:9016– 9020 Casey JL, Gerin JL (1995) Hepatitis D virus RNA editing: speciﬁc modiﬁcation of adenosine in the antigenomic RNA. J Virol 69:7593–7600 Chang FL, Chen PJ, Tu SJ, Wang CJ, Chen DS (1991) The large form of hepatitis delta antigen is crucial for assembly of hepatitis delta virus. Proc Natl Acad Sci USA 88:8490–8494 Chang MF, Baker SC, Soe LH, Kamahora T, Keck JG, Makino S, Govindarajan S, Lai MM (1988) Human hepatitis delta antigen is a nuclear phosphoprotein with RNA- binding activity. J Virol 62:2403–2410 Chao M, Hsieh SY, Taylor J (1990) Role of two forms of hepatitis delta virus antigen: evidence for a mechanism of self-limiting genome replication. J Virol 64:5066– 5069

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Cheng Q, Jayan GC, Casey JL (2003) Differential inhibition of RNA editing in hepatitis delta virus genotype III by the short and long forms of hepatitis delta antigen. J Virol 77:7786–7795 Curran J, Kolakofsky D (1990) Sendai virus P gene produces multiple proteins from overlapping open reading frames. Enzyme 44:244–249 Glenn JS, Watson JA, Havel CM, White JM (1992) Identiﬁcation of a prenylation site in delta virus large antigen. Science 256:1331–1333 Glenn JS, White JM (1991) trans-dominant inhibition of human hepatitis delta virus genome replication. J Virol 65:2357–2361 Gott JM, Emeson RB (2000) Functions and mechanisms of RNA editing. Annu Rev Genet 34:499–531 Hartwig D, Schoeneich L, Greeve J, Schutte C, Dorn I, Kirchner H, Hennig H (2004) Interferon-alpha stimulation of liver cells enhances hepatitis delta virus RNA editing in early infection. J Hepatol 41:667–672 Herb A, Higuchi M, Sprengel R, Seeburg PH (1996) Q/R site editing in kainate receptor GluR5 and GluR6 pre-mRNAs requires distant intronic sequences. Proc Natl Acad Sci USA 93:1875–1880 Herbert A, Rich A (2001) The role of binding domains for dsRNA and Z-DNA in the in vivo editing of minimal substrates by ADAR1. Proc Natl Acad Sci USA 98:12132–12137 Higuchi M, Single FN, Kohler M, Sommer B, Sprengel R, Seeburg PH (1993) RNA editing of AMPA receptor subunit GluR-B: a base-paired intron-exon structure determines position and efﬁciency. Cell 75:1361–1370 Hsu SC, Syu WJ, Sheen IJ, Liu HT, Jeng KS, Wu JC (2002) Varied assembly and RNA editing efﬁciencies between genotypes I and II hepatitis D virus and their implications. Hepatology 35:665–672 Hwang SB, Lee CZ, Lai MM (1992) Hepatitis delta antigen expressed by recombinant baculoviruses: comparison of biochemical properties and post-translational modiﬁcations between the large and small forms. Virology 190:413–422 Ilan Y, Klein A, Taylor J, Tur-Kaspa R (1992) Resistance of hepatitis delta virus replication to interferon-alpha treatment in transfected human cells. J Infect Dis 166:1164–1166 Ivaniushina V, Radjef N, Alexeeva M, Gault E, Semenov S, Salhi M, Kiselev O, Deny P (2001) Hepatitis delta virus genotypes I and II cocirculate in an endemic area of Yakutia, Russia. J Gen Virol 82:2709–2718 Jayan GC, Casey JL (2002a) Increased RNA editing and inhibition of hepatitis delta virus replication by high-level expression of ADAR1 and ADAR2. J Virol 76:3819– 3827 Jayan GC, Casey JL (2002b) Inhibition of hepatitis delta virus RNA editing by short inhibitory RNA-mediated knockdown of Adar1 but not Adar2 expression. J Virol 76:12399–12404 Jayan GC, Casey JL (2005) Effects of conserved RNA secondary structures on hepatitis delta virus genotype I RNA editing, replication, and virus production. J Virol 79:11187–11193 Kuo MY, Chao M, Taylor J (1989) Initiation of replication of the human hepatitis delta virus genome from cloned DNA: role of delta antigen. J Virol 63:1945–1950

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Lehmann KA, Bass BL (1999) The importance of internal loops within RNA substrates of ADAR1. J Mol Biol 291:1–13 Liu Y, Herbert A, Rich A, Samuel CE (1998) Double-stranded RNA-speciﬁc adenosine deaminase: nucleic acid binding properties. Methods 15:199–205 Liu Y, Samuel CE (1996) Mechanism of interferon action: functionally distinct RNAbinding and catalytic domains in the interferon-inducible, double-stranded RNAspeciﬁc adenosine deaminase. J Virol 70:1961–1968 Lomeli H, Mosbacher J, Melcher T, Hoger T, Geiger JR, Kuner T, Monyer H, Higuchi M, Bach A, Seeburg PH (1994) Control of kinetic properties of AMPA receptor channels by nuclear RNA editing. Science 266:1709–1713 Luo GX, Chao M, Hsieh SY, Sureau C, Nishikura K, Taylor J (1990) A speciﬁc base transition occurs on replicating hepatitis delta virus RNA. J Virol 64:1021–1027 Macnaughton TB, Lai MM (2002) Large hepatitis delta antigen is not a suppressor of hepatitis delta virus RNA synthesis once RNA replication is established. J Virol 76:9910–9919 Makino S, Chang MF, Shieh CK, Kamahora T, Vannier DM, Govindarajan S, Lai MM (1987) Molecular cloning and sequencing of a human hepatitis delta virus RNA. Nature 329:343–346 McNair AN, Cheng D, Monjardino J, Thomas HC, Kerr IM (1994) Hepatitis delta virus replication in vitro is not affected by interferon-alpha or -gamma despite intact cellular responses to interferon and dsRNA. J Gen Virol 75:1371–1378 Melcher T, Maas S, Herb A, Sprengel R, Seeburg PH, Higuchi M (1996) A mammalian RNA editing enzyme. Nature 379:460–464 Modahl LE, Lai MM (2000) The large delta antigen of hepatitis delta virus potently inhibits genomic but not antigenomic RNA synthesis: a mechanism enabling initiation of viral replication. J Virol 74:7375–7380 Niro GA, Smedile A, Andriulli A, Rizzetto M, Gerin JL, Casey JL (1997) The predominance of hepatitis delta virus genotype I among chronically infected Italian patients. Hepatology 25:728–734 O’Connell MA, Krause S, Higuchi M, Hsuan JJ, Totty NF, Jenny A, Keller W (1995) Cloning of cDNAs encoding mammalian double-stranded RNA-speciﬁc adenosine deaminase. Mol Cell Biol 15:1389–1397 Ohman M, Kallman AM, Bass BL (2000) In vitro analysis of the binding of ADAR2 to the pre-mRNA encoding the GluR-B R/G site. RNA 6:687–697 Patterson JB, Samuel CE (1995) Expression and regulation by interferon of a doublestranded-RNA-speciﬁc adenosine deaminase from human cells: evidence for two forms of the deaminase. Mol Cell Biol 15:5376–5388 Paul MS, Bass BL (1998) Inosine exists in mRNA at tissue-speciﬁc levels and is most abundant in brain mRNA. EMBO J 17:1120–1127 Polson AG, Bass BL (1994) Preferential selection of adenosines for modiﬁcation by double-stranded RNA adenosine deaminase. EMBO J 13:5701–5711 Polson AG, Bass BL, Casey JL (1996) RNA editing of hepatitis delta virus antigenome by dsRNA-adenosine deaminase. Nature 380:454–456 Polson AG, Ley HL, 3rd, Bass BL, Casey JL (1998) Hepatitis delta virus RNA editing is highly speciﬁc for the amber/W site and is suppressed by hepatitis delta antigen. Mol Cell Biol 18:1919–1926

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Radjef N, Gordien E, Ivaniushina V, Gault E, Anais P, Drugan T, Trinchet JC, Roulot D, Tamby M, Milinkovitch MC and others (2004) Molecular phylogenetic analyses indicate a wide and ancient radiation of African hepatitis delta virus, suggesting a deltavirus genus of at least seven major clades. J Virol 78:2537–2544 Ryu WS, Bayer M, Taylor J (1992) Assembly of hepatitis delta virus particles. J Virol 66:2310–2315 Sato S, Cornillez-Ty C, Lazinski DW (2004) By inhibiting replication, the large hepatitis delta antigen can indirectly regulate amber/W editing and its own expression. J Virol 78:8120–8134 Sato S, Wong SK, Lazinski DW (2001) Hepatitis delta virus minimal substrates competent for editing by adar1 and adar2. J Virol 75:8547–8555 Scott J (1989) Messenger RNA editing and modiﬁcation. Curr Opin Cell Biol 1:1141– 1147 Seeburg PH (2002) A-to-I editing: new and old sites, functions and speculations. Neuron 35:17–20 Seeburg PH, Higuchi M, Sprengel R (1998) RNA editing of brain glutamate receptor channels: mechanism and physiology. Brain Res Brain Res Rev 26:217–229. Shakil AO, Hadziyannis S, Hoofnagle JH, Di Bisceglie AM, Gerin JL, Casey JL (1997) Geographic distribution and genetic variability of hepatitis delta virus genotype I. Virology 234:160–167 Sureau C, Taylor J, Chao M, Eichberg JW, Lanford RE (1989) Cloned hepatitis delta virus cDNA is infectious in the chimpanzee. J Virol 63:4292–4297 Wagner RW, Smith JE, Cooperman BS, Nishikura K (1989) A double-stranded RNA unwinding activity introduces structural alterations by means of adenosine to inosine conversions in mammalian cells and Xenopus eggs. Proc Natl Acad Sci USA 86:2647–2651 Wang JG, Cullen J, Lemon SM (1992) Immunoblot analysis demonstrates that the large and small forms of hepatitis delta virus antigen have different C-terminal amino acid sequences. J Gen Virol 73:183–188 Wang KS, Choo QL, Weiner AJ, Ou JH, Najarian RC, Thayer RM, Mullenbach GT, Denniston KJ, Gerin JL, Houghton M (1986) Structure, sequence and expression of the hepatitis delta viral genome. Nature 323:508–514 Wang Q, Khillan J, Gadue P, Nishikura K (2000) Requirement of the RNA editing deaminase ADAR1 gene for embryonic erythropoiesis. Science 290:1765–1768 Weiner AJ, Choo QL, Wang KS, Govindarajan S, Redeker AG, Gerin JL, Houghton M (1988) A single antigenomic open reading frame of the hepatitis delta virus encodes the epitope(s) of both hepatitis delta antigen polypeptides p24 delta and p27 delta. J Virol 62:594–599 Wong SK, Lazinski DW (2002) Replicating hepatitis delta virus RNA is edited in the nucleus by the small form of ADAR1. Proc Natl Acad Sci USA 99:15118–15123 Wong SK, Sato S, Lazinski DW (2001) Substrate recognition by ADAR1 and ADAR2. RNA 7:846–858 Wu TT, Bichko VV, Ryu WS, Lemon SM, Taylor JM (1995) Hepatitis delta virus mutant: effect on RNA editing. J Virol 69:7226–7231 Xia YP, Chang MF, Wei D, Govindarajan S, Lai MM (1990) Heterogeneity of hepatitis delta antigen. Virology 178:331–336

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Yang A, Papaioannou C, Hadzyannis S, Thomas H, Monjardino J (1995) Base changes at positions 1014 and 578 of delta virus RNA in Greek isolates maintain base pair in rod conformation with efﬁcient RNA editing. J Med Virol 47:113–119 Yang JH, Sklar P, Axel R, Maniatis T (1997) Puriﬁcation and characterization of a human RNA adenosine deaminase for glutamate receptor B pre-mRNA editing. Proc Natl Acad Sci USA 94:4354–4359 Zheng H, Fu TB, Lazinski D, Taylor J (1992) Editing on the genomic RNA of human hepatitis delta virus. J Virol 66:4693–4697

CTMI (2006) 307:91–112 c Springer-Verlag Berlin Heidelberg 2006

Post-translational Modification of Delta Antigen of Hepatitis D Virus W.-H. Huang · C.-W. Chen · H.-L. Wu · P.-J. Chen (u) Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, and Hepatitis Research Center, National Taiwan University Hospital, Taipei, Taiwan [email protected]

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92

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The Hypothesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92

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Isoprenylation of L-HDAg . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role of L-HDAg Farnesylation in the HDV Replication Cycle . . . . . . Isoprenylation Enhances the trans-Suppression Activity of L-HDAg . Other Viral Prenylated Proteins . . . . . . . . . . . . . . . . . . . . . . . . . .

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Phosphorylation of Delta Antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 Phosphorylated Delta Antigens in the HDV Life Cycle . . . . . . . . . . . . . . 98 Putative Delta Antigen Kinase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100

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Acetylation of Delta Antigen . . . . . . . . Enzyme for Delta Antigen Acetylation . Acetylation Site of Delta Antigen . . . . . Functions of Delta Antigen Acetylation .

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Abstract The hepatitis delta virus (HDV) genome has only one open reading frame, which encodes the viral small delta antigen. After RNA editing, the same open reading frame is extended 19 amino acids at the carboxyl terminus and encodes the large delta antigen. These two viral proteins escort the HDV genome through different cellular compartments for the complicated phases of replication, transcription and, eventually, the formation of progeny virions. To orchestrate these events, the delta antigens have to take distinct cues to trafﬁc to the right compartments and make

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correct molecular contacts. In eukaryotes, post-translational modiﬁcation (PTM) is a major mechanism of dictating the multiple functions of a single protein. Multiple PTMs, including phosphorylation, isoprenylation, acetylation, and methylation, have been identiﬁed on hepatitis delta antigens. In this chapter we review these PTMs and discuss their functions in regulating and coordinating the life cycle of HDV.

1 Introduction Among the animal viruses, hepatitis D virus (HDV) is currently the smallest known. The length of its single-stranded, negative polarity genome, which contains a single open reading frame encoding for delta antigen, contains only 1678 nucleotides. Despite the extreme simplicity of the HDV genome, it replicates and produces progeny even more actively than many RNA viruses. Therefore, many of the fundamental features of the life cycles of viruses can probably be embodied in the simple HDV. In this review, we focus on the role of delta antigen (HDAg). Viral delta antigen has been shown to be essential for viral replication, and its variant, the large delta antigen (L-HDAg), is required for viral assembly. The replication and assembly processes, however, take place in different compartments of infected cells. For example, HDV RNA enters the nucleus and even the nucleolus for replication and transcription. Recent studies have indicated that newly synthesized viral genomic RNA further moves to the cytoplasm. This scenario suggested that different stages of viral RNA replication might take place in several distinct subcellular compartments. As these stages of the HDV life cycle require the presence of viral delta antigen, the protein must be able to trafﬁc to different but appropriate subcellular compartments to synchronize viral replication and assembly. The mechanisms by which the delta antigen orchestrates these complicated steps are the topics of this chapter.

2 The Hypothesis We propose that the delta antigens are modiﬁed post-translationally to produce many different isoforms. Although the amino acid backbone remains unchanged, the modiﬁed delta antigens can exert different functions in the viral replication cycle in different subcelluar compartments. Perhaps the posttranslational modiﬁcations (PTMs) of proteins can draw a similarity with dressing codes for humans. A person has to perform multiple social functions

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on different occasions, for which the person has to put on different but appropriate costumes. In this way, the same person enriches the life and expands one’s social capacity. In a similar fashion, for delta antigen, different PTMs produce uniquely modiﬁed delta isoforms that recognizes the right cellular compartment and interacting partners to conduct the requested activity. There are already many precedents for PTMs altering the functional capacity of delta antigens. Many proteins interacting with nucleic acids frequently adopt this strategy. Eukaryotic chromosomal packing or modeling proteins, such as histones, rely on PTM to reversibly modulate their interaction with the genome. Transcriptional factors, such as NFκB, trafﬁc between the cytoplasm and the nucleus by differential acetylation. Drawing from these cases, delta antigens may undergo different PTMs in order to transform into distinct isoforms to conduct the required functions at successive stages of the HDV life cycle.

3 Isoprenylation of L-HDAg Protein isoprenylation is the post-translational addition of an isoprenoid lipid to the cysteine residue. The protein isoprenylation mediates protein– protein and protein–membrane interactions. Current evidence suggests that isoprenylated proteins are involved in cell cycle regulation, signal transduction, cytoskeleton reconstruction and protein trafﬁcking (Crowell 2000). A 15-carbon farnesyl or 20-carbon geranylgeranyl isoprenoid lipid is bound to the carboxyl terminus of a protein (Clarke 1992). The metabolic enzyme was classiﬁed into three kinds of isoprenyltransferase. Farnesyltransferase (Ftase) transfers a farnesyl to a protein substrate that has a tetrapeptide CaaX motif at the C terminus. The ‘X’ is generally methionine, cysteine, serine, glutamine or alanine. Geranylgeranyltransferase type I (GGTase I) transfers geranylgeranyl to proteins containing a CaaX motif, where the ‘X’ commonly refers to leucine. After adding the isoprenoid lipid to the cysteine residue, the ’-aaXmotif is removed by carboxyl peptidase and a new COOH-terminal prenylcysteine synthesized by carboxyl methyltransferase (Zhang and Casey 1996). The third isoprenyltransferase is geranylgeranyltransferase type II, which catalyzes the geranylgeranylation of proteins having XCXC, XXCC or CCXX at the C terminus. Through RNA editing, L-HDAg contains an additional 19 amino acids at its C terminus. A tetrapeptide Cys-Arg/Thr-Pro/Gln-Gln (C-R/T-P/Q-Q) was identiﬁed in this region and can be prenylated by FTase or GGTase I (Table 1). Previously, it was suggested that L-HDAg is modiﬁed by geranylgeranyl

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Table 1 Post-translational modiﬁcations of delta antigens and their putative biological activities Antigen modiﬁcation

Modiﬁcation residue

Consensus Candidate sequence enzyme

Biological functions

Prenylation (L-HDAg)

Cysteine 211

CXXQ

trans-dominant inhibition Virus assembly Replication

Phosphorylation Serine 2 MSX4 R/K (S-HDAg) Serine 4, 6, 22a ? Serine 177 PESPF

Threonine 95a Phosphorylation Serine 123 (L-HDAg) Acetylation Lysine 72

Methylation

Arginine 13

RXXR

Farnesyltransferase Casein kinase II ?

Protein kinase C ? Replication ? AG RNA replication Editing PKR PKR pseudosubstrate Casein kinase II ? Subcellular localization P300 Replication Earlier appearance of LHDAg SHDAg subcellular localization PRMT1 Replication AG RNA nuclear transport

a Found in only some HDV isolates

isoprenoid lipid (Glenn et al. 1992; Koff 1992). It could be farnesylated and geranylgeranylated when incubated with bovine brain cytosolic extract. However, a subsequent experiment using puriﬁed prenyltransferase and recombinant L-HDAg indicated that L-HDAg is a better substrate for farnesyltransferase in vitro (Otto and Casey 1996). Conﬁrming this result, reverse-phase HPLC analysis found a 15-carbon isoprenoid residue on L-HDAg expressed in COS cells. HDV isolates are classiﬁed into three genotypes I, II and III based on the differences in their nucleotide sequences. The CXXQ motif in genotype III is CTQQ and is less efﬁciently farnesylated than other genotypes (O’Malley and Lazinski 2005). During the early phase of HDV replication, the small form of hepatitis delta antigen (S-HDAg) is more abundant than L-HDAg, and the L-HDAg/S-HDAg ratio increases when replication progresses. The level

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of farnesylated L-HDAg can be increased in the presence of S-HDAg. It was proposed that L-HDAg is farnesylated optimally and thus functions optimally only when S-HDAg is also present. (O’Malley and Lazinski 2005). 3.1 Role of L-HDAg Farnesylation in the HDV Replication Cycle L-HDAg shares an identical 195 amino acid sequence with S-HDAg, but their biological effect(s) are completely different. L-HDAg is essential for virus packaging. Previous studies demonstrated that both of extra 19 amino acids at the C terminus and farnesylation at the CXXQ motif are required for HDV packaging (Lee et al. 1994). In the HDV packaging process, hepatitis B surface antigen (HBsAg) interacts with L-HDAg to form particles; this process can occur even in the absence of HDV RNA (Hwang and Lai 1993). HBsAg is mainly localized in the endoplasmic reticulum (ER) membrane, while the localization of newly synthesized L-HDAg is very dynamic (Hourioux et al. 1998). Analyses of transiently transfected cells revealed the sequential appearance of L-HDAg in the nucleoplasm, then in the nucleolus, and ﬁnally in nuclear speckles (Shih and Lo 2001). Prenylated L-HDAg is concentrated in the nuclear speckles. Far-Western protein blotting analysis indicated a direct protein–protein interaction between HBsAg and L-HDAg (Hwang and Lai 1993). The L-HDAg isoprenylation also mediates this protein–protein interaction. When HDV particles assemble, L-HDAg must interact with HBsAg in the ER membrane. From where the prenylated ER-associated L-HDAg comes remains unknown. The protein is probably either transported from the nucleus or comes directly from the newly synthesized L-HDAg. Prenylation is not sufﬁcient for the interaction between delta antigen and HBsAg; the primary amino acid sequence upstream the CXXQ motif is also critical for HDV packaging (Chang et al. 1994). Attachment of the CXXQ isoprenylation motif to S-HDAg did not enable it to be packaged with HBsAg. Furthermore, deletions of any ﬁve amino acids in the last 15 amino acids unique to the L-HDAg abolished HDV packaging ability (Lee et al. 1994). A prolinerich sequence, spanning amino acids 198–210 on L-HDAg, was identiﬁed as a nuclear export signal (NES) that can direct nuclear export of L-HDAg to the cytoplasm via a chromosome region maintenance 1 (CRM-1) independent pathway. Within this sequence, proline 205 is critical for the NES function and L-HDAg packaging activity (Lee et al. 2001). Results from these experiments seem to imply that the newly synthesized L-HDAg is isoprenylated by farnesyltransferase in the cytoplasm, then translocated to the nucleolus. The prenylated L-HDAg inhibits HDV RNA replication and shifts the HDV life

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Fig. 1 Illustration of the farnesylation pattern, subcellular localization and biological function of L-HDAg in the HDV replication cycle. The farnesylated L-HDAg sequentially translocates from the cytoplasm, nucleolus, and nuclear speckles to the ER membrane. Farnesylation enhances the inhibitory function of L-HDAg in the nucleolus, and is required for virus packaging; thus prenylation inhibitors can block viral assembly (also see chapter by J.S. Glenn, this volume)

cycle to the late packaging phase. In the late stage, through a CRM-1 independent pathway, prenylated L-HDAg is transported to the ER membrane where it can interact with HBsAg and HDV RNA to form the intact virion (Fig. 1). 3.2 Isoprenylation Enhances the trans-Suppression Activity of L-HDAg L-HDAg exerts inhibitory effects on HDV replication. This inhibitory activity was suggested to be due to conformational differences between S-HDAg and LHDAg. A prenylation-defective L-HDAg mutant, in which the cysteine 211 was changed to serine, has a lower trans-dominant inhibitory activity than wildtype L-HDAg (Hwang and Lai 1994). As most of the nonprenylated L-HDAg was retained in the cytoplasm, the effect of prenylation on the inhibitory activity of L-HDAg for HDV replication is probably not direct. Rather, it seems that isoprenylation enhances the inhibitory activity of L-HDAg by facilitating translocation of L-HDAg to the nucleus where HDV replicates (Lin et al. 1999; Tan et al. 2004).

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3.3 Other Viral Prenylated Proteins Pseudorabies virus Us2 is another prenylated viral protein (Clase et al. 2003). Although Us2 protein is also packaged as part of the tegument of mature virions, prenylation is not required for Us2 incorporation into virions. The Us2 protein isolated from puriﬁed virions is not prenylated. Lovastatin (a prenylation inhibitor) treatment caused a dramatic relocalization of Us2 to microtubules, but the biological signiﬁcance of Us2 prenylation is still unknown.

4 Phosphorylation of Delta Antigens The two forms of HDAg, S-HDAg and L-HDAg, perform opposite biological functions in the HDV replication cycle even though they share 195 identical amino acids. As regulatory proteins in the HDV life cycle, both forms of HDAg are phosphorylated when they are expressed in insect cells, mammalian cells and infected hosts (Chen et al. 1997; Hwang et al. 1992). Two-dimensional phosphoamino acid analysis indicated that the S-HDAg is phosphorylated at both serine and threonine residues while L-HDAg is phosphorylated only at serine (Mu et al. 1999). Nevertheless, athough L-HDAg is only phosphorylated at serine, the phosphorylation level of L-HDAg is approximately sixfold higher than that of S-HDAg, and the modiﬁcation proﬁle is also more complicated (Choi et al. 2002; Hwang et al. 1992). The actual phosphorylation sites of HDAgs have not been completely identiﬁed. Putative phosphorylated residues on HDAgs were predicted based on either the conserved serine/threonine phosphorylation domains of known kinases or conserved serine/threonine residues among different HDV isolates. Sequence alignment among three genotypes of HDV indicates that there are only three conserved serine residues on S-HDAg, serine 2, 123 and 177 (Fig. 2). Among these putative phosphorylated residues, serine 177 is the only one that has been identiﬁed in vivo by mass spectrometry analysis so far (Chen et al. 2002). Serine 2 and 123 are located in a potential casein kinase II (CK II) recognition domain (Yeh et al. 1996). However, phosphorylation of these two residues has not yet been proven. Replacing these three conserved serine residues with alanine did not completely abolish S-HDAg phosphorylation (Mu et al. 1999), indicating that there are other phosphorylated residues. The threonine phosphorylation site of S-HDAg is still unknown. Threonine 95 is highly conserved among different genotypes, but it can be serine in some isolates.

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Fig. 2 Alignment of S-HDAg sequences from different hepatitis delta virus isolates

Within the extra C terminal 19 amino acid of L-HDAg, there are two candidate serine residues for phosphorylation: amino acids 207 and 210. Serine 210 is a potential protein kinase C recognition site. Mutations at these two serine residues had no signiﬁcant effect on L-HDAg phosphorylation, indicating that phosphorylation probably does not occur within the 19-aa extension unique of L-HDAg. Surprisingly, it was found that mutation at the acceptor site for farnesylation, cysteine 211, completely inhibited phosphorylation, which suggested that farnesylation may play a signiﬁcant role in L-HDAg phosphorylation. (Bichko et al. 1997). It is still not known why the L-HDAg is more heavily phosphorylated than S-HDAg. 4.1 Phosphorylated Delta Antigens in the HDV Life Cycle Protein conformation is directly correlated with its biological function. One must therefore be very careful when using mutant proteins to investigate function, because mutations can interfere with function indirectly by affecting protein conformation. More reliable approaches to investigate the biological function of a phosphorylated protein include abolishing kinase activity by using dominant negative mutants or RNA interference (RNAi) technology. However, because most of the phosphorylation sites and corresponding kinases for HDAg phosphorylation have still not been identiﬁed, site-directed mutagenesis and kinase inhibitor(s) were used to investigate the inﬂuence of HDAg phosphorylation on HDV replication. Some of the serine and threonine residues have been mutated in S-HDAg (Table 1). Mutation at Serine 2 diminished the activity of SHDAg in assisting HDV replication. Mutations at Threonine/Serine 95 of SHDAg also inﬂuenced HDV RNA replication. Serine 177 has been identiﬁed as a phosphorylation site in vivo and is located within a conserved motif, Pro-Glu-Ser-Pro-Phe (PESPF). Mutation of serine 177 to alanine interfered with synthesis of HDV genomic RNA from the antigenomic template (Mu et al. 2001). Furthermore, mutation at serine 177 also reduced the phosphorylation level of S-HDAg,

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the degree of RNA editing and production of L-HDAg. Serine to alanine mutations at positions 4 and 123 seemed not to exert any effect on the HDV life cycle. DRB (5,6-dichloro-1-β-D-ribofuranosylbenzimidazole), an inhibitor of the CK II, diminished phosphorylation levels of both HDAgs and suppressed HDV replication. This observation is in accordance with a S-HDAg mutant assay that implies serine 2 is phosphorylated by CK II (Yeh et al. 1996). Nevertheless, as CK II also regulates the phosphorylation of other cellular proteins, the effect of kinase inhibitors on cells is global rather than HDAg-speciﬁc. Therefore, it was not clear whether the effect of kinase inhibitor on HDV replication was due to the direct inhibition of HDAg phosphorylation or to the modulation of cellular protein phosphorylation. For example, the activity of a transcription factor, CHOP, could also be inﬂuenced by CK II (Ubeda and Habener 2003). As HDV replication depends on cellular transcription machinery, (Lai 1995, also see the chapters by J.M. Taylor, and T.B. Macnaughton and M.M.C. Lai, this volume), it is possible that blocking the phosphorylation of these transcription factors by kinase inhibitors indirectly impaired HDV replication. More experiments will be required to clarify this issue. Protein kinase C (PKC) inhibitor suppressed HDV replication more signiﬁcantly. Mutation of S-HDAg at the putative PKC phosphorylation site did not have any effect on HDV replication, implying that the inﬂuence of PKC inhibitor on HDV is an indirect consequence of modulating the activity of cellular proteins. The nucelolar protein, B23 probably is the communicator. It is a substrate of PKC and its interaction with S-HDAg enhances HDV replication (Beckmann et al. 1992; Huang et al. 2001). Histone and serine/threonine phosphatase can also be phosphorylated by PKC (Jakes et al. 1988). The effects of PKC on these proteins and their roles in the HDV life cycle require more detailed studies. Furthermore, the phosphatase responsible for HDAg dephosphorylation is still unknown. Although L-HDAg is more heavily phosphorylated, phosphorylation seems not to affect the biological functions of L-HDAg signiﬁcantly. The only effect found so far is that L-HDAg with mutation at serine 123 was translocated from nucleolus to nuclear speckle SC35 (Tan et al. 2004). The same phenomenon was observed when the cells expressing wild-type L-HDAg were treated with the CK II inhibitor, dichlororibofuranosyl benzimidazole. Compared with wild type L-HDAg and serine 2-mutated L-HDAg, L-HDAg mutated at serine 123 was transported to the cytoplasm less efﬁciently and resulted in a lower level of HDV particle secretion.

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4.2 Putative Delta Antigen Kinase HDAg presents a very complicated phosphorylation pattern. No single serine/threonine mutation can abolish its phosphorylation completely. The conserved serine 2, threonine/serine 95, serine 123 and serine 177 are most likely residues for phosphorylation in HDAg. If the phosphorylation is critical for HDV replication, phosphorylation at the remaining nonconserved serine/threonine residues may not have signiﬁcant effect for HDV. Kinase inhibition assay suggested that the CK II phosphorylates serine 2 and serine 123 (Yeh et al. 1996). However, there is still no direct evidence to prove serine 2 and 123 are phosphorylated in vivo by CK II. Ion trap tandem mass spectrometry conﬁrmed that serine 177 is phosphorylated in an S-HDAg-expressing stable cell line (Chen et al. 2002). An in-gel kinase assay indicated that double-stranded RNA activated kinase (PKR) could phophorylate S-HDAg. Furthermore, immunoprecipitation-puriﬁed endogenous PKR could phosphorylate recombinant SHDAg at serine 177, 180, and threonine 182 (Chen et al. 2002). Among the residues phosphorylated in vitro by PKR, serine 177 is the only one that has also been identiﬁed to be phosphorylated in vivo. Apart from the phosphorylated residues on S-HDAg, another critical question for HDAg phosphorylation is the nature and number of kinase(s). Previous studies have shown that HDV replication was not reduced in cells expressing dominant negative PKR mutants. Therefore, PKR was considered to be a regulatory kinase but not the essential kinase for HDAg phosphorylation. In HDV cDNA-transfected cells, HDV replication was not suppressed by interferon treatment even though the level of PKR was increased. This result implies that part of the function of S-HDAg may resemble the trans-acting protein (Tat) of HIV-1 and vaccinia virus K3L protein that behave as a decoy substrate to inhibit PKR activity. To more completely understand the role of phosphorylated S-HDAg in the HDV life cycle, it will be essential to further identify the HDAg modiﬁcation sites and the responsible kinase(s).

5 Acetylation of Delta Antigen HDV replication takes place within the nucleus (Rizzetto et al. 1977; Wu et al. 1992). However, both HDAg and genomic HDV RNA can be found in the cytoplasm (Macnaughton and Lai 2002). Kinetic studies of newly synthesized HDV RNA in heterokaryons indicated a transient phase of shuttling viral genomic RNA into the cytoplasm and then re-importing into the nucleus

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(Tavanez et al. 2002), indicating the importance of nucleocytoplasmic shuttling in the HDV life cycle. We have demonstrated that acetylation of S-HDAg may be a signal responsible for modulating such shuttling. Both isoforms of HDAg are acetylated, and lysine 72 (K72) of S-HDAg was identiﬁed as one of the acetylation sites (Mu et al. 2004). Substitution of K72 by arginine (K72R) caused the S-HDAg mutant to redistribute from the nucleus to the cytoplasm. The K72R mutant also reduced genomic RNA synthesis from antigenomic RNA template and resulted in the earlier appearance of L-HDAg. These results demonstrated that HDAg is an acetylated protein and acetylation on K72 of S-HDAg may modulate the subcellular localization of S-HDAg and participate in viral RNA nucleocytoplasmic shuttling and replication. 5.1 Enzyme for Delta Antigen Acetylation Histone acetyltransferases (HATs) modify a wide spectrum of cellular factors, including histones, coactivators, nuclear transport proteins, structural proteins, cell cycle components, transcription factors, and nuclear receptors (for reviews see Fu et al. 2004; Quivy and Van Lint 2004; and references therein). A-type HATs, which are generally localized in the nucleus and are thereby linked with transcriptional regulation, are divided into ﬁve families, including Gcn5-related acetyltranferases (GNATs), the MYST-related HATs, p300/CBP HATs, TAFII250, and nuclear hormone-related HATs (Carrozza et al. 2003). We demonstrated that S-HDAg could be acetylated in vitro by p300 (Mu et al. 2004). However, it is still possible that other acetyltransferases also participate in catalyzing S-HDAg acetylation in vivo. The cellular deacetylases (HDACs) responsible for the deacetylation of acetylated HDAg are currently under investigation. 5.2 Acetylation Site of Delta Antigen We have demonstrated that HDAg was acetylated in vivo and in vitro (Mu et al. 2004). Notably, S-HDAg acetylated in vitro by p300 was used for MASS analysis, which identiﬁed K72 as the acetylation site. Nevertheless, we cannot rule out the possibility that, in addition to K72, S-HDAg might also contain other sites that could be acetylated by p300 or other acetyltransferases. This phenomenon occurs on some cellular proteins such as p53 (Brooks and Gu 2003) and YY1 (Yao et al. 2001), or viral proteins such as HIV-1 Tat (Kiernan et al. 1999). These proteins were found to possess multiple acetylation sites and were acetylated by several HATs. Furthermore, the common acetylation

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motif of nuclear receptors, KXKK (Fu et al. 2004), is also present within amino acids 36–43 of HDAg. Thus, further study is required to verify the candidate K72 and other possible unidentiﬁed acetylation sites. 5.3 Functions of Delta Antigen Acetylation HDAg is an acetylated protein and mutation at lysine 72 of S-HDAg affects its subcellular localization as well as viral RNA replication. How does acetylation of S-HDAg modulate the HDV life cycle? Acetylation has been demonstrated to occur on a wide spectrum of cellular proteins as well as viral proteins and affect these factors’ functions and activities (for reviews see Chan and La Thangue 2001; Goodman and Smolik 2000; Greene and Chen 2004; Yang 2004; and references therein). The K72R mutant of S-HDAg displayed a different subcellular distribution pattern from that of the wild-type. Considering the cases that acetylation is crucial for nuclear accumulation of HNF4 (hepatocyte nuclear factor-4) (Soutoglou et al. 2000), MHC II (major histocompatibility class II) trans-activator CIITA (Spilianakis et al. 2000), NF-κB (Chen et al. 2001), and Signal transducers and activators of transcription 3 (Stat 3) (Wang et al. 2005), acetylation on K72 of HDAg is likely to be the signal to modulate its nuclear localization. Moreover, S-HDAg could stimulate the transcription elongation of pol II (Yamaguchi et al. 2001), which is believed to be responsible for the replication of HDV genomic RNA from antigenomic RNA (Macnaughton et al. 2002; Moraleda and Taylor 2001). It may explain the mechanism that non-nucleoplasmic localization of K72R mutant impairs the accumulation of genomic RNA when antigenomic RNA was introduced to cells as template for replication. Although amino acids 68–88 of HDAg had been identiﬁed as a bipartite nuclear localization signal (NLS) (Chang et al. 1992; Xia et al. 1992), it is unlikely that K72R mutant of S-HDAg impairs the NLS since treatment with leptomycin B, an inhibitor for CRM-1 dependent nuclear export pathway, could retain K72R mutant of S-HDAg in the nucleus (Mu et al. 2004). S-HDAg has been demonstrated to interact with nuclear proteins such as RNA polymerase II (Yamaguchi et al. 2001), B23 (Huang et al. 2001), nucleolin (Lee et al. 1998) and SC35 (Bichko and Taylor 1996). Since lysine acetylation has also been reported to affect protein–protein interactions (Kiernan et al. 1999; Zhang et al. 2001), the poor nuclear retention ability of the K72R mutant may suggest that acetylation is required for the interaction of S-HDAg with these proteins. Furthermore, acetylation of K72 may also be able to modulate the interaction of S-HDAg and HDV RNA, just like the cases that acetylation of lysine residues present on the tails of core histones by CBP/p300 weakens internucle-

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osomal interactions (Bannister and Kouzarides 1996; Ogryzko et al. 1996) and acetylation of HIV Tat promotes its dissociation from TAR (Tat transactivation response region) RNA (Kiernan et al. 1999). Acetylation of lysine residues of several proteins, such as histones, p53, HIV Tat, and MyoD, can form speciﬁc sites to interact with bromodomain of HATs such as Gcn5, PCAF, and CBP so as to exert speciﬁc activities (for reviews see Yang 2004; and references therein). In contrast, acetylation of the lysine residue(s) adjacent to the binding motif of some proteins, such as activator of retinoid receptor (ACTR) (Chen et al. 1999b) and adenovirus E1A (Zhang et al. 2000), can hinder the access of its binding partners. The acetylation of S-HDAg may thus either recruit a transcriptional coactivator or block a negative regulator to facilitate HDV replication. Protein lysine acetylation can modulate several functions of cellular and viral proteins. Further studies are required to verify the function(s) of acetylation on S-HDAg and HDV life cycle.

6 Methylation of Delta Antigen S-HDAg was shown to be methylated in vitro and in vivo (Li et al. 2004). The major methylation site is at arginine 13 (R13) in the RGGR motif of S-HDAg. The methylation of S-HDAg is crucial for HDV RNA replication, especially for replication of the genomic RNA from the antigenomic RNA. A substitution at R13 of S-HDAg, or treatment with methylation inhibitors, reduced HDV RNA replication. The methylation status of S-HDAg affected its subcellular localization. Both the R13A mutant of S-HDAg, and wild-type S-HDAg in cells treated with a methylation inhibitor, were localized mainly in the cytoplasm, whereas wild-type S-HDAg formed speckled structures in the nucleus. Furthermore, the methylation of S-HDAg is involved in the transportation of antigenomic RNA, but not genomic RNA. Moreover, when introduced as a ribonucleoprotein (RNP) complex, antigenomic RNA, but not genomic RNA, failed to be targeted to the nucleus with unmethylated S-HDAg. These results indicate that methylation of S-HDAg plays an important role in the replication of HDV RNA and methylated HDAg is required for the initiation of replication of genomic RNA from the antigenomic strand. 6.1 Enzyme for Delta Antigen Methylation Arginine methylation involves the addition of one or two methyl groups to the guanidine nitrogen atoms of arginine (Gary and Clarke 1998; McBride and

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Silver 2001). Arginine may be dimethylated asymmetrically or symmetrically. At least three types of protein arginine methyltransferase (PRMT) activities have been reported (Lee et al. 1977). PRMT1 (Tang et al. 2000a), PRMT3 (Tang et al. 1998), PRMT6 (Frankel et al. 2002), and coactivator-associated arginine methyltransferase 1 (CARM1, also known as PRMT4) (Chen et al. 1999a) are type I enzymes. PRMT5 is so far the only known type II enzyme (Branscombe et al. 2001). Another class of PRMTs, including PRMT7, only catalyzes the formation of monomethylarginines (Miranda et al. 2004). No clear consensus sequences/motifs are known for CARM1 substrates or for the newly identiﬁed PRMT6 and PRMT7. In vitro analysis demonstrated that S-HDAg was methylated by PRMT1, but not other type I protein PRMTs including PRMT3 and CARM1. PRMT1 is the predominant type I protein arginine methyltransferase in mammalian cells (Tang et al. 2000a). It is localized in the nucleus and catalyzes methylation on arginine residues in RGG, RXR or GAR motifs of several proteins (Boisvert et al. 2003; Liu and Dreyfuss 1995; Wada et al. 2002). The substrates of PRMT1 involve several proteins, including many RNA-binding proteins, transporting proteins, transcription factors, and nuclear matrix proteins (Abramovich et al. 1997; Mowen et al. 2001; Nichols et al. 2000; Smith et al. 1999; Smith et al. 2004; Tang et al. 2000b). It is noteworthy that PRMT5 (type II) also preferentially catalyzes arginine methylation located in RG-rich clusters (Boisvert et al. 2003), and cellular proteins, such as DRB-sensitive inducing factor (DSIF), p160 or Spt5, were identiﬁed to be arginine methylated by PRMT1 as well as PRMT5 (Kwak et al. 2003). PRMT6, which is also a type I PRMT, catalyzed methylation on arginine-rich motif (ARM) of HIV-1 Tat protein (Boulanger et al. 2005). Notably, the RNA binding domain of HDAg contains two ARMs (Lee et al. 1993). Hence, the possible involvement of PRMTs in addition to PRMT1 (i.e. PRMT5, PRMT6) in arginine methylation of S-HDAg still requires further exploration. Moreover, methylation of lysine residue(s) also occurs in HDAg, as Li et al. mentioned that antibodies detecting methylated lysine residues could recognize cellular HDAg. This result indicates the possible involvement of lysine methyl transferases such as the SET domain-containing methyltransferase family (Nishioka et al. 2002; Strahl et al. 2002), and suggests the possibility that, similar to histones, S-HDAg methylation might be catalyzed by two families of proteins—one that methylates arginine residues, the other lysine residues.

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6.2 Methylation Site of Delta Antigen Li et al. demonstrated that the methylation of S-HDAg could be catalyzed speciﬁcally by PRMT1 and the residue R13 of S-HDAg is essential for methylation in vitro. PRMT1 has been found to catalyze methylation on arginine residues in RGG, RXR and GAR motifs of RNA-binding proteins (Boisvert et al. 2003; Wada et al. 2002). SHDAg contains two RGG motifs, 10-RGG-12 and 91-RGG93. S-HDAg containing alanine substitution mutations of arginine 10 and arginine 91 could still be methylated in vitro, although mutation of arginine 10 reduced the intensity of S-HDAg methylation. However, arginine 13, which is adjacent to the 10-RGG-12 motif, appeared to be important for methylation to occur. Mutation of arginine 13 to alanine abolished methylation. Further study such as MASS analysis of puriﬁed cellular SHDAg might be necessary to verify methylation on the candidate R13 and other unidentiﬁed sites including arginine as well as lysine residues. 6.3 Functions of Delta Antigen Methylation Methylation of S-HDAg plays very important roles in HDV RNA replication. Methylated HDAg is likely to be essential for the initiation of replication of genomic RNA from the antigenomic strand. How does methylation of S-HDAg affect HDV replication? Protein arginine methylation has been demonstrated to modulate transcription (Chevillard-Briet et al. 2002; Davie and Dent 2002), nucleic acidbinding afﬁnity (Boulanger et al. 2005), protein–protein interaction (Bedford et al. 2000; Kwak et al. 2003), and nuclear targeting (Shen et al. 1998). The most likely role for methylation in HDAg function is that methylation regulates its subcellular distribution, as has been frequently observed for the methylation of other RNA-binding proteins, such as hnRNP A2 (Nichols et al. 2000) and RNA helicase A (Smith et al. 2004). Wild-type S-HDAg generally formed speckles in the nucleus, but the R13A mutant protein was localized predominantly in the cytoplasm. Treatment with methylation inhibitors also showed similar cytoplasmic localization of wild-type S-HDAg. Considering that HDV RNA replication was suggested to occur in the nuclear speckle structures by RNA polymerase II machinery (which is believed to be responsible for the replication of HDV genomic RNA from antigenomic RNA template) (Bichko and Taylor 1996; Macnaughton et al. 2002; Modahl et al. 2000; Moraleda and Taylor 2001), unmethylated HDAg might thus affect HDV replication by dislocating HDV from the antigenomic RNA template. This model is further

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supported by the observation that introduction of RNP complexes containing HDV antigenomic RNA and unmethylated HDAg could not target to the nucleus while RNP containing genomic RNA with unmethylated HDAg did. However, although arginine methylation of some RNA binding proteins has been reported to affect RNA binding, analysis of RNA binding of in vitromethylated and unmethylated S-HDAg by UV cross-linking or gel mobility shift did not show signiﬁcant differences (Li et al. 2004). Methylation on arginine residues of transcriptional factors, such as DSIF or Spt5, were shown to inhibit association with RNA polymerase II and transcriptional activity (Kwak et al. 2003). HDAg has been demonstrated to interact with polymerase II and affect Pol II activity (Yamaguchi et al. 2001). Perhaps methylation of S-HDAg affects its interaction with the cellular transcription machinery responsible for HDV RNA replication. That the methylation of SHDAg is required for the replication of genomic RNA from antigenomic RNA and for the formation of the speckled structures in the nucleus further suggests that methylation on arginine may modulate the interaction of S-HDAg and the RNA polymerase II transcription machinery. Lysine methylation was also identiﬁed on S-HDAg, but the effects have not yet been pursued. HIV Tat and histone 3 also undergo methylation and acetylation on lysine, and thus may offer some clues. Arginine methylation diminishes the transactivation capacity of Tat, in contrast to the positive effect of lysine acetylation on Tat activity (Boulanger et al. 2005). Meanwhile, lysine acetylation of some proteins at a site that also serves as a substrate for another modiﬁcation (such as methylation on lysine 9 of histone 3 and sumoylation on lysine 539 of sp3) blocked such modiﬁcation. If it is the case for S-HDAg, the balance of methylation and acetylation on lysine residues will offer more delicate control of the HDV life cycle.

7 Prospect Phosphorylation of S177, acetylation of K72 and methylation of R13 of S-HDAg could all modulate the replication of HDV genomic RNA from antigenomic RNA but not replication of HDV genomic RNA from antigenomic RNA. These results suggest that PTMs of S-HDAg play critical roles for its functions to facilitate the replication of HDV genomic RNA. The different requirements of S-HDAg for the replication of genomic and antigenomic RNA are also consistent with the previous suggestion that rolling circle replication of hepatitis delta virus RNA is carried out by different cellular RNA polymerase machineries (Macnaughton et al. 2002; see also the chapter by T.B. Macnaughton

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and M.M.C. Lai, this volume). The crosstalk between these different PTMs of S-HDAg and their roles in the HDV life cycle require further evaluation. Other PTMs such as sumoylation, ubiquitination, and ADP-ribosylation, occur on several cellular proteins as well as viral proteins and modulate their biological functions. Whether these modiﬁcations are present on HDAg have not yet been identiﬁed. In conclusion, the PTM of HDAg may make them more versatile. Dissecting the exact function of individual isoforms will become an important area of HDV virology. Useful state-of-art tools, such as LC/MS/MS and PTM-speciﬁc antibodies, may help to relate the modiﬁed HDAg to functions.

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CTMI (2006) 307:113–131 c Springer-Verlag Berlin Heidelberg 2006

The Role of the HBV Envelope Proteins in the HDV Replication Cycle C. Sureau (u) Laboratoire de Virologie Moléculaire, Institut National de la Transfusion Sanguine, 6 Rue Alexandre Cabanel, 75739 Paris, France [email protected]

1 1.1 1.2

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114 The Virion Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114 The Helper HBV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118

2

The Interaction Between the HDV RNP and the HBV Envelope Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120

3

The S-HBsAg Protein and the Assembly of HDV Particles . . . . . . . . . . . 121

4

The L-HBsAg Protein and the Infectivity of the HDV Particles . . . . . . . . 124

5

The Effect of HDV Infection on the HBV Life Cycle . . . . . . . . . . . . . . . . 125

6

Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127 Abstract The hepatitis delta virus (HDV) is a subviral agent that utilizes the envelope proteins of the hepatitis B virus (HBV) for propagation. When introduced into permissive cells, the HDV RNA genome replicates and associates with multiple copies of the HDV-encoded proteins to assemble a ribonucleoprotein (RNP) complex. The mechanism necessary to export the RNP from the cell is provided by the HBV envelope proteins, which have the capacity to assemble lipoprotein vesicles that bud into the lumen of a pre-Golgi compartment before being secreted. In addition to allowing the release of the HDV RNP, the HBV envelope proteins also provide a means for its targeting to an uninfected cell, thereby ensuring the spread of HDV. This chapter covers the molecular aspects of the HBV envelope protein functions in the HDV replication cycle, in particular the activity of the small envelope protein in RNP export and the function of the large envelope protein at viral entry.

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1 Introduction In 1977, the initial description of the hepatitis delta antigen (HDAg) by M. Rizzetto, was made from the examination of liver biopsies of hepatitis B virus (HBV) chronic carriers, and it was logically thought to constitute a new HBV antigen (Rizzetto et al. 1977). After its characterization as a nuclear antigen, the immunoreactive material was found to reside in particles coated with the HBV envelope proteins, and was consequently referred to as a virus-like agent that could be transmitted to chimpanzee only in the presence of HBV (Bonino et al. 1984, 1986; Rizzetto et al. 1980a). Because of this absolute requirement for HBV coinfection, it has been considered as a defective virus. The cloning of the hepatitis D virus (HDV)-associated RNA was achieved in 1986 (Chen et al. 1986; Wang et al. 1986), and the nucleotide sequence analysis revealed a genome structure that was unique among animal viruses: it was a circular, single-stranded RNA of negative polarity, with an open reading frame coding for the HDAg-associated protein, the only protein that HDV RNA is known to encode, but it lacked the coding capacity for envelope proteins. Thus, since the very early phase of its discovery, HDV has been closely associated with HBV although its genome sequence presents no homology to that of HBV. 1.1 The Virion Structure The HDV virions are heterogeneous in size with an average diameter of 36 nm and a buoyant density of 1.25 g/cm3 in CsCl (Fig. 1), and they display a chimerical structure consisting of an outer lipid membrane in which the HBV envelope proteins are anchored, and an inner ribonucleoprotein (RNP) made of HDV-speciﬁc elements (Bonino et al. 1984; He et al. 1989; Rizzetto et al. 1980b). The RNP includes a 1,700-nucleotide single-stranded RNA genome associated with approximately 200 copies of the HDAg protein (Gudima et al. 2002). This protein appears as two isoforms: the small form (S-HDAg) of 195 amino acid residues and the large form (L-HDAg), which is 19 amino acids longer. The difference in size arises as a consequence of an RNA editing event that occurs on a replication intermediate of the viral genome and is copied onto the HDAg mRNA (see the chapter by J.L. Casey, this volume). The examination of the RNP by electron microscopy reveals a spherical, core-like structure, with no apparent icosahedral symmetry and a diameter of approximately 19 nm. The HDV envelope appears undistinguishable from the one of HBV. It consists of a lipid membrane in which the three HBV coat proteins, bearing the hepatitis B surface antigen (HBsAg) and designated small (S-HBsAg),

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Fig. 1 Schematic representation of the HDV virion. The particle comprises two types of components: (a) the viral envelope of HBV origin, including the HBV envelope proteins S-HBsAg, M-HBsAg and L-HBsAg; and (b) the ribonucleoprotein (RNP) that comprises the circular genomic RNA associated with multiple copies of the HDVencoded delta proteins, S-HDAg and L-HDAg

middle (M-HBsAg) and large (L-HBsAg), are inserted (Bruss 2004; Ganem and Schneider 2001). It was initially reported that S-HBsAg, M-HBsAg and L-HBsAg were present at a ratio estimated at 95 : 5 : 1, respectively, at the surface of HDV, as opposed to a 4 : 1 : 1 ratio in the envelope of HBV virions also called Dane particles (Bonino et al. 1986; Gerlich et al. 1987; Heermann et al. 1984). However, these numbers may not be accurate, considering that experimentally, HDV particles can be assembled with the S-HBsAg protein only (Wang et al. 1991). It is therefore expected that serum-derived HDV particles would be very heterogeneous in the relative amounts of each type of envelope proteins. In the serum of the infected host, HDV particles are present at titers as high as 1011 infectious units/ml, along with the different forms of HBV particles: the infectious Dane particles and the empty subviral particles (SVPs) (Ganem and Schneider 2001; Lai 1995). All have in common the HBV surface proteins (Fig. 2A). HDV is directly dependent on HBV to acquire its envelope, and for that reason it should be considered as a defective virus or a subviral agent, and a satellite of HBV. It does not fulﬁll the criteria for the deﬁnition of a virus; nonetheless, it is referred to as Hepatitis delta virus

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Fig. 2 A,B Model for the budding of HBV and HDV particles into the lumen of a cellular intermediate compartment (IC lumen) between ER and Golgi. HBV envelope protein aggregates are thought to bud spontaneously at the IC membrane. Aggregates of S-HBsAg proteins only, or S-HBsAg + M-HBsAg, lead to the formation of spherical subviral particles (SVP). When L-HBsAg is present in the aggregates and the HBV nucleocapsid is absent, it leads to the secretion of ﬁlamentous SVP. When aggregates include L-HBsAg in the presence of HBV nucleocapsid, budding leads to the secretion of HBV virion. When HDV RNPs are present, they can be included in aggregates, irrespective of the presence of L-HBsAg for budding of HDV virions. Incorporation of the L-HBsAg protein in the HDV envelope confers infectivity. HDV virions devoid of L-HBsAg are noninfectious (non-inf HDV)

(HDV), and it constitutes the only species of the Deltavirus genus (ICTVdB: The Universal Virus Database of the International Committee on Taxonomy of Viruses, http://www.ictvdb.iacr.ac.uk). Based on the structure of the HDV virion, it would appear that HBV is just a provider of coat proteins. In fact, transfection of cultured mammalian cells with a cloned HDV cDNA, in the absence of HBV, led to the replication of HDV RNA and to the formation of RNPs, thereby demonstrating that, in vitro, HBV is not required at this stage of the replication cycle (Kuo et al. 1989). The accumulation in transfected cells of large amounts of replicating viral RNA and RNPs, combined with the absence of detectable RNPs in the culture medium,

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were evidence of a defect in the export function (Chen et al. 1986). The RNPs were not able to induce cell membrane lysis for egress or to bud on their own at a cellular membrane for secretion. When transfections were conducted in the presence of a plasmid driving the expression of the HBV envelope proteins, RNPs were included in lipoprotein vesicles and were released from the cells as mature virions. The conclusion of these experiments was that HBV assists in assembly and secretion of HDV by providing the export system. Moreover, it

Fig. 3 The S-HBsAg and L-HBsAg HBV envelope proteins are key elements of the HDV replication cycle. S-HBsAg and L-HBsAg are integral membrane proteins. S-HBsAg comprises TMD1 (I) between amino acid residues 4 and 24; the cytosolic loop (c.l.) 29–79; TMD2 (II) 80–100; the antigenic loop (a.l.) 101–164; two predicted carboxyl terminal TMDs, 173–193 and 202–222. L-HBsAg is identical to S-HBsAg except for the additional pre-S (pS) domain (pre-S1 + pre-S2) at its amino terminus. The pre-S1 domain contains a putative receptor binding site (thick line). For HDV morphogenesis, S-HBsAg proteins interact with each other and with L-HBsAg (step 1) to form aggregates that bud in the lumen of the IC. The HDV RNP is probably recruited by the envelope proteins through an interaction between S-HBsAg and L-HDAg (step 2). Viral entry is mediated by the L-HBsAg protein through an interaction between the pre-S1 domain and a receptor on the hepatocyte membrane (step 3). The glycosylation site (N146) in the antigenic loop is indicated. The broken line indicates the myristate group linked to the amino terminus of L-HBsAg. Elements important for interaction with L-HDAg are indicated: W196, W199, W201

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was proven that the S-HBsAg protein could fulﬁll these functions on its own (Sureau et al. 1992; Wang et al. 1991). Hence, all the information necessary for assembly and secretion of HDV resides in the S-HBsAg polypeptide (Fig. 3). 1.2 The Helper HBV HBV belongs to the Hepadnaviridae family. It is an enveloped virus with an icosahedral nucleocapsid that contains a circular, partially double-stranded DNA, and it replicates its genome through a reverse transcription mechanism (Ganem and Schneider 2001). The prominent characteristic of HBV is its budding mechanism, which is nucleocapsid independent and driven solely by the viral coat proteins at the membrane of a cellular intermediate compartment (IC) between the endoplasmic reticulum (ER) and the Golgi complex (Huovila et al. 1992; Patzer et al. 1984, 1986). The three envelope proteins, S-HBsAg, M-HBsAg and L-HBsAg are found at the surface of HBV virions but in reality, the driving force of the budding process is provided by S-HBsAg (Nassal 1996). As this protein is produced in enormous amounts in the host cell, the vast majority of HBV particles are S-HBsAg-coated SVPs (Heermann and Gerlich 1992). A single open reading frame on the HBV genome encodes the three HBV envelope proteins, but translation is initiated from three distinct in-frame AUG codons (Heermann et al. 1984) (Fig. 4A). The S-HBsAg protein is 226 amino acid residues in length, and the M-HBsAg protein has 55 additional residues (the pre-S2 domain) at the amino terminus. L-HBsAg comprises the entire M polypeptide with an additional amino terminal polypeptide (pre-S1) of 108 or 119 residues (Fig. 4B). S-HBsAg is an integral membrane glycoprotein, synthesized at the ER membrane (Simon et al. 1988) (Fig. 4C). It is anchored in the lipid bilayer through the amino terminal transmembrane domain 1 (TMD1) between residues 4 and 24 (Eble et al. 1986, 1990). It comprises the downstream cytosolic loop (residues 25– 79), the second TMD (TMD2) between residues 80 and 100, the antigenic loop (residues 101–164) that contains the immunodominant epitopes facing the ER lumen (or the surface of extracellular particles), and the hydrophobic carboxyl terminus (residues 165–226), whose structure is predicted to include two alpha helices (Persson and Argos 1994) (Fig. 4C). The topology of MHBsAg is similar to that of S-HBsAg, with the amino terminal pre-S2 facing the ER lumen. L-HBsAg, whose carboxyl terminal half consists of the entire S polypeptide, has been described as having two topologies: the amino terminal pre-S domain (pre-S1 + pre-S2) is either cytosolic at the ER membrane (internal on secreted virions) or luminal (exposed at the virion surface) (Bruss et al. 1994; Lambert and Prange 2001; Ostapchuk et al. 1994; Prange and Streeck

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1995). The internal conformation (Li-HBsAg, Fig. 4C) is involved in recruiting the nucleocapsid for virion assembly, whereas the external form (Le-HBsAg, Fig. 4C) corresponds to a receptor binding function at viral entry (Bruss and Vieluf 1995; Le Seyec et al. 1999). S-HBsAg proteins can dimerize and form multimers at the ER membrane through lateral protein–protein interactions, and the resulting aggregates are thought to bud spontaneously into the lumen of the IC (Fig. 2B). The budding mechanism is very efﬁcient and unique to the Hepadnaviridae family. Although S-HBsAg protein provides the driving force in the budding process, it cannot direct HBV virion assembly if the L-HBsAg protein is absent from the budding aggregates because the incorporation of the HBV nucleocapsid is mediated by a short linear sequence (residues 92 to 113) in pre-S1 (Fig. 4C). Owing to the overproduction of S-HBsAg and its capacity for autoassembly, virion formation is a very rare event in comparison with SVP production. It is estimated that more than 99% of HBV-related par-

Fig. 4 A–C A Domains of the HBV envelope proteins open reading frame (upper line). B The HBV envelope proteins. L-HBsAg, M-HBsAg and S-HBsAg proteins are translated from three in-frame initiation sites. C Membrane topology of the HBV envelope proteins. M-HBsAg and S-HBsAg adopt a similar topology at the ER membrane. The two transmembrane topologies of L-HBsAg are represented: the pre-S domain (pre-S1 + pre-S2) can reside on the cytoplasmic side of the ER membrane (Li-HBsAg), or it can be translocated through the membrane as found on the secreted particles (LeHBsAg). The broken line indicates the myristate group linked to the amino terminus of L-HBsAg. Open rectangles represent TMDs. Glycosylation site (G); antigenic loop (a.l.); cytosolic loop (c.l.); pre-S domain (pS)

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ticles are SVPs, and this translates into an average infectious serum containing approximately 1012 –1013 SVPs and only 108 –109 virions. HBV thus appears to have developed an overactive budding mechanism, which is quite a puzzling characteristic of the Hepadnaviruses, because there is no obvious advantage for the production of such an enormous amount of export vesicles that are devoid of any cargo. Fortuitously, it constitutes the export system that HDV is in need.

2 The Interaction Between the HDV RNP and the HBV Envelope Proteins In general, the assembly of enveloped viruses occurs in a speciﬁc subcellular compartment where all the structural components colocalize (Garoff et al. 1998). Newly synthesized structural proteins are prevented from initiating budding reactions until all virion components are present at the site of assembly. This rule, however, does not apply to HBV because for this virus, budding is driven by the envelope proteins, irrespective of the presence of the nucleocapsid. This is obviously an important consequence for HDV, which can utilize the budding machinery to its proﬁt. Overall, the production of progeny HDV virions by an infected cell involves two processes, which are independent of each other: the formation of the RNP and that of the viral envelope. They are directed by distinct viral species, and they are spatially separated, occurring in the nucleus and at the ER membrane, respectively. Therefore, a critical step of the HDV life cycle is the encounter of the RNP with the HBV budding system. The RNP comprises both the SHDAg and L-HDAg proteins associated with the viral RNA, and it is thought to shuttle between the cell nucleus and the cytoplasm (Lee et al. 2001; Tavanez et al. 2002). Export from the nucleus has been shown to rely on a speciﬁc nuclear export signal (NES) located in the 19 amino acid carboxyl terminus of L-HDAg (Lee et al. 2001), which also contains a carboxyl terminal CXXQ motif (where C = cysteine, Q = glutamine and X = any amino acid) for farnesylation (Glenn et al. 1992). The farnesyl group is covalently bound to the cysteine residue at position 211, and probably serves to anchor the RNP in the ER membrane where the envelope proteins assemble (Otto and Casey 1996). As an indirect proof of this phenomenon, treatment of HDV-producing cells with a farnesyl transferase inhibitor prevents assembly of RNPs into enveloped particles (Bordier et al. 2002; see also the chapter by J.S. Glenn, this volume). Interestingly, when expressed with the HBV envelope proteins in the absence of HDV RNA and S-HDAg, L-HDAg protein can be packaged and secreted in the SVPs (Chang et al. 1991; Chen et al. 1992; Ryu et al. 1992). This

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strongly suggests that L-HDAg mediates the incorporation of the RNP in the HBV envelope. Since deletions in the L-HDAg polypeptide can be performed between amino acids 2 and 195 without preventing packaging with S-HBsAg (Chang et al. 1994; Chen et al. 1992), the 19 amino acid carboxyl terminus is likely to constitute the packaging signal. Support for such a role comes also from the observation that its appending to the carboxyl terminus of a foreign protein, namely cHRas, leads to the co-secretion of the latter with SVPs (Lee et al. 1995). Ras is similar in size to L-HDAg and naturally farnesylated at its carboxyl terminus but cannot be packaged as such by S-HBsAg. In addition, a farnesylated S-HDAg could not be assembled with S-HBsAg indicating that the farnesyl group, per se, is not sufﬁcient (Lee et al. 1994). The carboxyl terminus of L-HDAg is likely to mediate an interaction with S-HBsAg but, surprisingly, its amino acid sequence is not well conserved among the different HDV genotypes, except for the farnesylation signal CXXQ, for a tryptophan residue at position 196 and for the presence of at least ﬁve proline residues (Radjef et al. 2004). A mutational analysis of this domain has shown that substitution of Ala for Trp196 had no effect on packaging, whereas mutation of the proline residues at positions 201, 204, 205 and 208 (positions in L-HDAg genotype I) were detrimental (O’Malley and Lazinski 2005). Although Pro-201 and Pro-205 reside in the NES, it was demonstrated that the lack of packaging with S-HBsAg was not due to a deﬁcient NES in the corresponding mutants but to a probable defect in S-HBsAg interaction. Whether L-HDAg binds directly to S-HBsAg or not, whether binding occurs during budding or beforehand, and whether the free form of L-HDAg binds to the envelope proteins in addition to the RNP-associated form, remain unknown (Sheu et al. 1996).

3 The S-HBsAg Protein and the Assembly of HDV Particles It is worthy of note that the members of the Hepadnaviridae family closest to HBV, namely the Woodchuck hepatitis virus (WHV) and the Woolly monkey hepatitis B virus (WMHBV), can assist in HDV propagation because their small envelope proteins (S-WHsAg and S-WMHBsAg, respectively) are competent for HDV RNP envelopment (Barrera et al. 2004; Ponzetto et al. 1984; Ryu et al. 1992). Experimental transmission of HDV has been achieved in woodchucks, and this animal model has been useful to study the interactions between HDV and the helper Hepadnavirus. In contrast, the envelope protein of a more distantly related Hepadnavirus, namely the Duck hepatitis B virus (DHBV), is unable to package the HDV RNP (O’Malley and Lazinski 2005). Therefore, determinants that are speciﬁc for HDV maturation on the S-HBsAg

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protein should be present on S-WHsAg and absent on the small DHBV envelope protein (S-DHBsAg). Compared to S-HBsAg or S-WHsAg, the S-DHBsAg polypeptide appears to lack the region corresponding to the antigenic loop between TMD2 and the carboxyl terminal hydrophobic domain (Fig. 3). When part of this domain (from residues 107 to 147) was experimentally deleted on S-HBsAg, it led to a drastic reduction in the capacity of the mutant for HDV maturation (O’Malley and Lazinski 2002). Interestingly, this deletion mutant was competent for the envelopment of the singly expressed L-HDAg protein, suggesting that the hindrance observed in RNP envelopment may rather reﬂect a lesser ﬂexibility of the envelope, which could no longer accommodate an RNP, than a lack of binding. On one hand, S-DHBsAg cannot package the RNP and cannot interact with L-HDAg, and on the other hand, a S-HBsAg mutant that mimics S-DHBsAg in lacking the antigenic loop, is also deﬁcient for packaging the RNP, while competent for L-HDAg interaction. Hence, it would be interesting to swap subdomains between the two proteins to precisely identify determinants of L-HDAg interaction. The deﬁciency in HDV maturation observed with the antigenic loop-deleted S-HBsAg, could also be explained, at least in part, by its lack of N-linked carbohydrates, since the removal of the glycosylation site (Asn146) was shown to prevent glycosylation, and partially inhibit HDV assembly (Sureau et al. 2003; Wang et al. 1996). Considering the topology of the S-HBsAg protein at the ER membrane, it was expected that regions most likely to contain an RNP binding site will be exposed to the cytosolic face of the membrane (Fig. 3). The S-HBsAg loop, from residues 24 to 80, thus appeared as a good candidate because its disposal to the cytosol had been experimentally established. A genetic analysis of residues 24 to 59 revealed that a S-HBsAg mutant carrying a deletion of residues 24–28 was not affected for SVP secretion or L-HDAg packaging, but was partially deﬁcient for HDV virion assembly (Jenna and Sureau 1998). It thus might reﬂect a hindrance in the envelope ﬂexibility to accommodate an RNP. The same study also revealed that the 28–59 domain does not contain motifs essential for HDV maturation. Interestingly, a determinant of HBV virion assembly was shown to reside in a region that overlaps with the 28–59 sequence (Löfﬂer-Mary et al. 2000). When mutational analysis was conducted on the carboxyl terminus of S-HBsAg, it was initially found that the tryptophan residue at position 196 was a determinant of HDV assembly (Jenna and Sureau 1999). When reexamined in a recent study, it was observed that the carboxyl domain of S-HBsAg contained a conserved tryptophan-rich domain of which Trp196, Trp199 and Trp201 were demonstrated to be critical for HDV assembly and for interaction with L-HDAg (Komla-Soukha and Sureau 2006). The entire carboxyl terminus of S-HBsAg (residues 164–226) is highly hydrophobic; it includes eight tryptophan residues, and it is predicted to con-

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tain two TMDs located at positions 173–193 and 202–222 (Persson and Argos 1994). They bracket a short sequence (194–201), including Trp196, Trp199 and Trp201, which presents a low degree of ﬂexibility (Fig. 3). Hydrophobicity and secondary structure predictions are compatible with the orientation of the tryptophan residues at the cytosolic side of the ER membrane in a position potentially adequate for interaction with the RNP. However, a topological model of S-HBsAg obtained by epitope mapping of monoclonal antibodies raised against HBV particles, proposed that the 187–207 region would not be buried inside the S-HBsAg particles, but would, instead, lie on the surface (Chen et al. 1996; Paulij et al. 1999). One could speculate that after synthesis at the ER membrane, the loop is initially disposed to the cytosolic face, and is translocated to the outside of the viral membrane after budding. The two topologies may also coexist at the virion surface. The fact that motifs identiﬁed as essential to HDV assembly, such as Trp196, Trp199 and Trp201, are dispensable for subviral particle secretion and yet strictly conserved among HBV, WHV and WMHBV isolates, suggests that the selection pressure that has led to their conservation concerns functions other than those involved in subviral particle assembly, for instance the processes of HBV maturation or entry. But they can also be conserved on S-HBsAg because the corresponding DNA coding sequence also encodes critical domains of the HBV polymerase. Interestingly, the Trp196 codon is included in the DNA sequence that codes for the YMDD motif of the polymerase catalytic domain. This motif is crucial to the activity of the enzyme and only in lamivudine-resistant virus is YMDD converted to YVDD, YSDD or YIDD (Torresi 2002; Torresi et al. 2002). The latter mutation results in a W196S mutation in S-HBsAg. As a consequence, patients infected with a YIDD mutant, are expected to be resistant to HDV superinfection because the W196S mutation in the S-HBsAg prevents RNP packaging (Komla-Soukha and Sureau 2006). For a better understanding of the HDV maturation process, the determinants of incorporation of L-HDAg proteins into SVPs need to be sorted from those involved in RNP envelopment. In the former case, assembly should proceed through colocalization of L-HDAg and S-HBsAg followed by a speciﬁc interaction between these two partners, whereas in the latter case, assembly is likely to depend also on the constraints exerted on the envelope to accommodate a 19 nm RNP. The capacity of S-HBsAg to modulate its intrinsic membrane bending force is suggested by the fact that in nature HBV manages to assemble three types of particles, namely the 22 nm spheres, the ﬁlaments that are 22 nm in diameter and up to several hundred nanometers in length, and the 42 nm Dane particles. Therefore, ﬂexibility of the viral envelope is another characteristic of HBV that is beneﬁcial to HDV (Fig. 3). In the light of a recent study that measured the concentration of HDAg proteins at up to six million copies per

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infected cell, it seems that the encounter between L-HDAg and S-HBsAg, or any cellular factor involved in this process, should be facilitated (Gudima et al. 2002). However, the possibility of obtaining S-HBsAg mutants that are deﬁcient for both HDV maturation and L-HDAg packaging, while being permissive for SVP secretion, strongly suggests that the RNPs cannot be passively incorporated in the budding vesicles. In favor of a direct interaction between S-HBsAg and HDV RNPs are the following observations: (a) S-HBsAg and LHDAg have been reported to bind to each other in a far Western binding assay (Hwang and Lai 1993); and (b) synthetic peptides speciﬁc for HBV envelope proteins have been shown to bind both L- and S-HDAg proteins in an in vitro assay (de Bruin et al. 1994; Hourioux et al. 1998). At present, we are left with a tryptophan-rich domain as a binding motif candidate on S-HBsAg, and a carboxyl terminal proline-rich domain as a possible ligand on the partner L-HDAg. Since examples of protein interaction mediated by a proline-rich sequence binding to a tryptophan-rich motif have been reported (Kay et al. 2000; Simon et al. 1998), it would be interesting to investigate this hypothesis in the process of HDV maturation.

4 The L-HBsAg Protein and the Infectivity of the HDV Particles For the HDV replication cycle to be completed, secreted virions must be targeted to uninfected cells. Therefore it was expected for the L-HBsAg protein, which mediates infectivity of the HBV virion (Gripon et al. 2005; Le Seyec et al. 1999), to be required as an integral component in the envelope of an infectious HDV particle. This was demonstrated in an in vitro culture system: particles coated with the S-HBsAg protein, or S-HBsAg and M-HBsAg, were not infectious when tested on primary hepatocyte cultures. But when L-HBsAg was coexpressed with S-HBsAg, infectivity was restored (Sureau et al. 1993). We can presume that in using the same glycoprotein, namely L-HBsAg, as that used by HBV to mediate viral entry, HDV increases its chance of propagation because only HBV-susceptible cells could become infected with HDV. However this is not proven, and abortive infection may occur if HDV is driven to a hepatocyte that is not already infected by HBV, or is not to become infected. While it is clear that HBV does not make an efﬁcient use of its budding system, it ensures infectivity to its virion because maturation and infectivity are linked to the same molecule, namely the L-HBsAg protein. This dual function of L-HBsAg has been mapped to two regions in the pre-S1 domain: (a) a receptor binding site at the amino terminal end (Le Seyec et al. 1999); and (b) a HBV nucleocapsid binding site at the carboxyl terminus, which should

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be dispensable for HDV (Bruss 1997). In contrast, taking full advantage of the HBV budding machinery, HDV uses all available S-HBsAg aggregates at the IC membrane, regardless of the presence of the L-HBsAg, to coat its RNP and bud. As a result, a signiﬁcant amount of exported HDV virions is likely to be enveloped with S-HBsAg proteins only (or S-HBsAg and M-HBsAg), and thereby, to be noninfectious (Fig. 2). The minimum amount of L-HBsAg proteins in the HDV envelope to confer infectivity is unknown. Overall, the HDV life cycle depends on two HBV elements only: the S-HBsAg protein for the export of the RNP, and the L-HBsAg protein for entry into an uninfected hepatocyte (Fig. 3). With regard to the M-HBsAg protein, its role, which is still enigmatic in the HBV replication cycle, is not essential for in vitro assembly and infectivity of HDV (Bruss and Ganem 1991; Fernholz et al. 1993; Sureau et al. 1994; Wang et al. 1991). As for the details of viral entry into the host cell, it seems reasonable to assume that HBV and HDV use the same cellular receptor on the human hepatocyte, but at present, its identity remains unknown. At a post-binding step, intracellular uptake is likely proceeding via receptormediated endocytosis (Kock et al. 1996), and internalization of HDV RNP and that of HBV nucleocapsid, most likely follow separate pathways in which S-HBsAg and L-HBsAg may participate. For both viruses, however, the outer shell has to be dismantled, and the interactions between envelope proteins and the inner cargo must be abolished.

5 The Effect of HDV Infection on the HBV Life Cycle Since HDV is directly dependent on HBV for propagation, it can be transmitted concomitantly with HBV to an individual who has no history of prior HBV infection, this is referred to as a coinfection pattern – or it can be transmitted to an HBV chronic carrier, this is referred to as superinfection. Coinfections are often acute and self-limiting, and they result in a concomitant replication of both HBV and HDV, whereas superinfections cause severe acute and chronic type D hepatitis in 70% of cases. They also lead to the inhibition of HBV replication during the acute phase of HDV infection. This phenomenon has been described in both humans and experimentally infected chimpanzees, but it remains poorly understood (Chen et al. 1988; Sureau et al. 1989; Wu et al. 1991). It could result from a direct suppression of HBV replication exerted by the coexpressed HDV proteins, RNA or RNPs, or could be the consequence of an indirect interfering mechanism driven by inﬂammatory cytokines. The suppressive effect could also result from a hijacking of the envelope proteins by the RNPs in host cells when HDV RNA replication reaches maximum levels

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upon superinfection. Considering that an infected cell might contain as many as 6,000,000 copies of HDAg proteins and 60,000 copies of HDV RNA (Gudima et al. 2002), it is likely that the helper nucleocapsids be heavily outnumbered by the HDV RNPs in their access to the HBV budding machinery. In addition, faced with these huge amounts of RNPs ready for export, the budding system, though oversized for HBV, may reach saturation. There are at least seven HDV genotypes with various geographical distributions and a sequence divergence as high as 38% (Radjef et al. 2004; also the chapter by P. Dény, this volume), and eight HBV genotypes (designated A to H), also presenting different geographic distributions and a degree of sequence divergence greater than 8%. This raises the possibility for a broad range of HBV/HDV coinfection characteristics. A speciﬁc pathogenicity could result, in part, from the capacity of the HBV envelope proteins to export the RNP and/or to drive a progeny virion to uninfected hepatocytes. The most severe form of HBV/HDV coinfection has been recorded in South America where genotype III HDV was associated with genotype F HBV (Casey et al. 1996), the most divergent types in their respective family (Casey et al. 1993; Norder et al. 2004; Radjef et al. 2004). It would thus be interesting to characterize the envelope proteins of each HBV genotype for their ability to complement a given HDV genotype.

6 Conclusion Among enveloped viruses that achieve maturation through a nucleocapsidindependent assembly mechanism, HBV has developed the most active budding process that is known. It is carried out by the S-HBsAg envelope protein, and it leads to the formation of a large excess of empty subviral particles over mature virions. For that reason, HBV appears to be the best-suited virus to supply HDV with transport vesicles. For cell egress, the HDV RNP must be embedded in the HBV envelope proteins, and it is likely mediated by a speciﬁc interaction between L-HDAg and S-HBsAg. Recent experiments based on genetic analysis have identiﬁed a short sequence at the carboxyl terminus of S-HBsAg as a candidate for interaction with L-HDAg. At viral entry, assuming that HBV and HDV use a single primary receptor, the recent advance made in the identiﬁcation of a HBV receptor binding domain in the L-HBsAg protein is likely to be relevant for HDV. Overall, there are still many important and interesting questions about the HBV envelope protein functions in the HDV life cycle, but to better understand morphogenesis and infectivity of HDV, we would need to obtain ultrastructural data that reveal both the architecture of the envelope and the precise organization of the RNP.

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Prenylation of HDAg and Antiviral Drug Development J. S. Glenn (u) Division of Gastroenterology and Hepatology, Stanford University School of Medicine, CCSR 3115, 269 Campus Drive, Palo Alto, CA 94305–5187, USA [email protected]

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 Abstract Hepatitis delta virus (HDV) is an important cause of acute and chronic liver disease. Current medical therapies are unable to effectively eradicate HDV infections. Research into the molecular virology of the HDV life cycle has revealed a fascinating collection of biology. These insights are now beginning to be translated into new potential treatment strategies. For example, an essential step in the virus assembly process involves the post-translational lipid modiﬁcation of a speciﬁc HDV protein, namely prenylation of large delta antigen. Preventing prenylation abolishes virus particle formation. Drugs capable of speciﬁcally inhibiting prenylation have been developed for use in humans. These agents represent a new class of antiviral agents, with HDV as a ﬁrst target. Here, a brief review of the HDV life cycle emphasizing the role of prenylation is presented along with implications for drug development and therapy.

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1 Introduction 1.1 HDV Disease Burden It has been estimated that at least 15 million of the over 300 million people infected with hepatitis B virus (HBV) also harbor a hepatitis delta virus (HDV) infection and such infections can be found throughout the world (Gerin et al. 2001; Rizzetto et al. 1991). The clinical course associated with HDV is typically more severe than for HBV infection alone. Unfortunately, current therapies are largely ineffective against HDV. The study of HDV molecular virology, however, has revealed exciting new avenues for potential therapeutic intervention. After a brief review of the basic HDV virion composition and life cycle (covered in more detail in other chapters in this volume), this chapter will focus on a special post-translational modiﬁcation of a key HDV protein. This modiﬁcation reaction, termed prenylation, turns out to be both a mechanism exploited by the virus to mediate its assembly, and the basis for an exciting new form of antiviral therapy. HDV can be viewed as a ‘parasite virus’ of HBV. HDV has its own genome and encodes its own core-like protein, but it requires HBV to provide a source of envelope protein. This provides a molecular explanation for why natural HDV infections are always found in association with hepatitis B. There are two major clinical scenarios: (1) coinfection–acute simultaneous infection of HDV with HBV in a previously uninfected patient; and (2) superinfection– HDV infection of a chronically infected HBV patient (Hoofnagle 1989). This is often manifested by a sudden worsening or ‘ﬂare’ of previously stable chronic HBV disease. 1.2 The HDV Virion The HDV particle is composed of a single-stranded circular 1.7 kb RNA genome, small and large delta antigen (S- and L-HDAg), which together are surrounded by a lipid envelope containing the HBV surface antigen proteins (HBsAgs). The fully assembled particle diameter is about 36 nm. Sequencing of isolates from around the world has led to a classiﬁcation into three genotypes–I, II, and III–based on sequence variation (Casey 1996), and recent data suggests the existence of up to four additional genotypes (Radjef et al. 2004) (see the chapter by P. Dény, this volume). The two major isoforms of delta antigen, termed small and large, are identical in sequence except that L-HDAg has an extra 19 amino acids at its carboxyl (C-) terminus. As detailed

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by W.-H. Huang et al. (this volume), this addition is the result of a speciﬁc RNA editing event which occurs during replication of the HDV genome and converts the S-HDAg stop codon into a tryptophan codon which then allows translation to proceed to the next downstream stop codon. The presence of these extra C-terminal amino acids dramatically changes the function of delta antigen. For example, while S-HDAg promotes HDV genome replication, the L-HDAg can act as a potent transdominant inhibitor (Chao et al. 1990; Glenn and White 1991; Sato et al. 2004). The two isoforms also have differences in their ability to transactivate heterologous genes (Wei and Ganem 1998). As discussed in the next section, perhaps the most striking functional difference between these isoforms has emerged from studies of HDV assembly. 1.3 The HDV Life Cycle Figure 1 diagrams the major steps in the HDV life cycle. Infection of a target cell begins with attachment and entry. This step depends on the HBV L (or preS1) surface antigen which is required for infectivity (Sureau et al. 1993). The speciﬁc host receptor(s) and how the incoming virion enters the cell await further clariﬁcation. The second stage involves translocation to the nucleus where most viral nucleic acids and encoded proteins are observed in liver biopsy specimens from infected patients and animals. Transport of HDV RNA into (and out of) the nucleus appears to be mediated by delta antigen in the form of a ribonucleoprotein particle, exploiting in part the nuclear localizing signals contained in delta antigen (Chang et al. 1992; Chou et al. 1998; Xia et al. 1992). The third stage, genome replication occurs in the nucleus. Based on analyses of the detected intermediates a ‘rolling circle’ mechanism, similar to that of plant viroids, has been proposed. Only RNA to RNA transcription is involved in both HDV genome replication and transcription of the mRNA encoding delta antigen. Interestingly, HDV does not encode a known polymerase and the host cell hepatocyte is not classically thought of as having RNA to RNA transcriptional activity. There is evidence suggesting RNA polymerase II, perhaps modulated in some fashion by delta antigen, is redirected for this purpose (see the chapter T.B. Macnaughton and M.M.C. Lai in this volume for a detailed review). In the ﬁnal stage of the HDV life cycle, the complex of newly replicated RNA genome and associated delta antigens are incorporated into an enveloped particle for release. The precise intracellular location of this assembly is not known but several key molecular determinants have been deﬁned. Speciﬁc mutations in HBsAg can impair HDV genome packaging (see the chapter

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Fig. 1 Schematic of the HDV life cycle. (1) Attachment and entry. (2) Translocation to the nucleus. (3) Genome replication. (4) Virion assembly and release. (Note: diagram is simpliﬁed for clarity. For example, genome replication intermediates are omitted and nuclear HDV RNAs depicted without associated protein. No speciﬁc assembly site is intended. See text for further details). (From Glenn, JS 1999)

by W.-H. Huang et al., this volume). With respect to delta antigen, only the large form is capable of producing particles with HBsAg (Chang et al. 1991; Ryu et al. 1992; Shih and Lo 2001). The S-HDAg does not, although it can be co-packaged by L-HDAg into particles (Chen et al. 1992; Lazinski and Taylor 1993). Thus, the aforementioned RNA editing event which changes the form of delta antigen produced from small to large can be viewed as a molecular switch in the viral life cycle which favors the initiation of virus assembly. This also suggests that the extra 19 amino acids unique to L-HDAg isoform contain a critical signal for assembly.

2 Prenylation and HDV Assembly Within the 19 amino acids unique to L-HDAg lies a four-amino acid sequence motif conserved across HDV isolates. This motif, consisting of a cysteine

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situated exactly three amino acids from the C terminus, is termed a ‘CXXX box’ (where C = cysteine, and X = any amino acid) (Maltese 1990). CXXX boxes are found in a variety of proteins and are signiﬁcant because they represent the substrates recognized by a family of enzymes called prenyltransferases. Prenyltransferases catalyze the covalent attachment of a prenyl lipid to the CXXX box cysteine, a process termed prenylation (Zhang and Casey 1996). Farnesyltransferase (FTase) adds a 15-carbon prenyl lipid (farnesyl) and geranylgeranyltransferase-I (GGTase I) adds a 20-carbon prenyl lipid (geranylgeranyl). These prenyl lipids are both derived from mevalonic acid. Of note, a second class of GGTases, GGTase type II, has a more complex substrate recognition and catalyzes the transfer of geranylgeranyl to cysteine residues contained in C-terminal motifs such as CC or CXC (Pereira-Leal and Seabra 2001). Examples of farnesylated proteins include lamin B, Ras, and the Batten disease CLN3 protein, whereas the γ-subunit of G proteins and the Rab proteins are examples of geranylgeranylated proteins (Farnsworth et al. 1989; Hancock et al. 1989; Novick and Brennwald 1993; Pullarkat and Morris 1997; Yamane et al. 1990). One effect of prenylation is to promote membrane association of the modiﬁed protein (Casey 1995). Prenylation can also play a major role in protein–protein interactions (Hoffman et al. 2000; Pfeffer and Aivazian 2004). It was hypothesized that the conserved CXXX box in large delta antigen was a substrate for prenylation and that such a lipid modiﬁcation could help mediate interaction with the membrane-associated HBsAg required for HDV morphogenesis. Labeling studies with [3 H]-mevalonate – the metabolic precursor of prenyl lipids – have demonstrated both in in vitro translation reactions (Glenn et al. 1992) and in intact cells (Glenn et al. 1992; Hwang et al. 1992) that large delta antigen is indeed subject to prenylation. The speciﬁc type of prenyl lipid added is farnesyl (Otto and Casey 1996). That such modiﬁcation is critical to the HDV assembly process was indicated by site-directed mutagenesis studies. Indeed, genetic disruption of the delta antigen CXXX box–such as by substitution of the CXXX box cysteine with serine–abolishes prenylation of large delta antigen. The same mutation also abrogates large delta antigen’s ability to interact with, and form secreted particles with, HBsAg (Glenn et al. 1992; Hwang and Lai 1993). This was the ﬁrst demonstration that a viral protein could be modiﬁed by prenylation, and highlighted a novel mechanism of virion assembly. Although genetically disrupting the delta antigen CXXX box would not be practical in natural infections, achieving a similar end result – namely prevention of prenylation – pharmacologically might be translated into a practical clinical strategy. As detailed below, this approach has been progressively eval-

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uated, ﬁrst with virus-like particles (VLPs), then complete infectious virions, and most recently in an in vivo mouse model of HDV.

3 Targeting HDV Assembly by Prenylation Inhibition 3.1 Inadequacy of Current Therapy for HDV Current therapy for HDV infections is suboptimal. Although efﬁcacy of interferon in obtaining a biochemical and virologic response has been demonstrated by randomized controlled trial, even with prolonged treatment, most patients who respond initially relapse after cessation of therapy (Farci et al. 1994). Even among patients followed for more than 12 years, only those in whom HBV surface antigen could be eradicated were able to be cleared of their delta infection (Farci et al. 2004). A more drastic approach such as orthotopic liver transplantation of HDV patients can, like for HBV, yield good long term results when appropriate immunoprophylaxis with anti-HBsAg antibodies (anti-Hbs) is used (Samuel et al. 1995). Moreover, HDV has been associated with decreased rates of graft re-infection compared to HBV alone (Zignego et al. 1993). Because of HDV’s dependence on HBV for providing a source of HBsAg, anti-HBV agents which completely eradicate HBV would be expected to eventually lead to clearance of HDV as well. Unfortunately, if the drug is only effective at decreasing HBV DNA levels, but leaves HBsAg largely unaffected, it would be predicted to have little effect on HDV. Indeed, when used as a single agent or in combination with high dose interferon, lamivudine does not improve disease activity or lower HDV-RNA levels in HDV patients (Lau et al. 1999; Wolters et al. 2000). Similarly, famciclovir which can inhibit HBV (Cirelli et al. 1996), was shown to be ineffective in the setting of HDV infection. In particular, no therapeutic effect on serum HDV RNA, alanine aminotransferase, or liver histology could be observed (Yurdaydin et al. 2002). 3.2 Prenylation Inhibition—Effect on HDV VLPs To begin to test the hypothesis that prenylation inhibition-based antiviral therapy might be effective against HDV, a cell line which produces HDV VLPs was created (Glenn et al. 1998). This is the simplest model of HDV assembly and relies on the fact that the minimal requirements for particle assembly are

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the L-HDAg and the small HBV surface antigen (Chang et al. 1991). Next, a candidate inhibitor was identiﬁed based on the fact that prenylation of L-HDAg involves the addition of the prenyl lipid farnesyl via a reaction catalyzed by farnesyltransferase. BZA-5B, a farnesyltransferase inhibitor (FTI) originally developed to prevent prenylation of the farnesylated oncogenic form of Ras (James et al. 1994) was chosen for this purpose. BZA-5B could indeed inhibit L-HDAg prenylation, and the drug was shown to speciﬁcally inhibit the prenylation-dependent production of HDV VLPs in a dose-dependent manner (Glenn et al. 1998). Beginning at a BZA-5B concentration of 50 µM, no particles could be detected. Controls for nonspeciﬁc inhibition of protein synthesis and secretion showed essentially no effect of BZA-5B. 3.3 Prenylation Inhibition–Effect on Infectious HDV Particles These results provided the ﬁrst demonstration that HDV assembly might be susceptible to pharmacologic inhibition. Next this strategy was extended to a system that produces complete, infectious HDV virions containing an intact genome and another farnesyltransferase inhibitor, FTI-277 (Bordier et al. 2002). This system was based on co-transfecting Huh7 cells with plasmids encoding the complete HDV and HBV genomes (Sureau et al. 1992). Midnanomolar concentrations of FTI-277 dramatically inhibited virion production, and low micromolar drug concentrations decreased virion production to below the limit of detection (Bordier et al. 2002). Again, there was no signiﬁcant effect on general protein synthesis and secretion or cell metabolism. Because the molecular structures of BZA-5B and FTI-277 are very different, these results strongly suggested that their mechanism of action against HDV was indeed a reﬂection of their common FTI activity rather than some other feature of the inhibiting drugs. Thus even with the added complexity and assembly determinants of infectious HDV virions compared to VLPs, the former are also sensitive to pharmacological inhibition of prenylation. Moreover, using a similar virion production model, it was demonstrated that HDV genotype III virions–which are associated with particularly severe clinical disease (Casey et al. 1993)–are as sensitive to prenylation inhibition as were HDV genotype I virions (Bordier et al. 2002). 3.4 Prenylation Inhibition—In Vivo Efficacy Most recently, the antiviral efﬁcacy of FTIs was evaluated further in vivo using a new mouse model of HDV (Bordier et al. 2003). The latter was established

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by hydrodynamically transfecting HBV-transgenic mice with HDV-encoding plasmids, and was an extension of previous experiments performed with HDV alone (Chang et al. 2001). Immunohistochemistry reveals that up to 20%–30% of the hepatocytes in the thus treated HBV-transgenic mice express delta antigen, and replicated genomic RNA is readily detectable in the liver, as are HDV virions in the serum (Bordier et al. 2003). This was the ﬁrst demonstration of HDV viremia in an immunocompetent mouse model, and the percentage of mouse hepatocytes expressing delta antigen is 10–50-fold greater than reported for mouse models using other techniques. Cohorts of mice in which HDV viremia had been established in this manner were treated with single daily doses of the prenylation inhibitors FTI-277 or FTI-2153, or vehicle controls. Both drugs were very effective at clearing HDV from the serum (Bordier et al. 2003). Because a pool of prenylated L-HDAg can accumulate before the initiation of treatment with FTIs, it might be predicted that the efﬁciency of clearing HDV from the serum would be proportional to the length of treatment with prenylation inhibitor. This indeed appears to be the case. About an 85% reduction in serum HDV titer was observed after 2 days of therapy. By 4 days of treatment, the serum levels of HDV were reduced ~95%, and they became undetectable by 7 days of therapy (Bordier et al. 2003). In addition, similar levels of alanine transaminase – a marker of liver toxicity – were observed among all treatment groups. This argues against the possibility that a nonspeciﬁc hepatotoxic effect of the FTIs was responsible for their potent clearing of HDV viremia. Together these results represent a dramatic and clear ﬁrst in vivo conﬁrmation of the potential of FTIs as a novel class of antiviral agents. Moreover, the clinical relevance and importance for human HDV infections of these results is obvious.

4 Prenylation Inhibition – Rationale for Human HDV Infections Because mouse hepatocytes do not have the natural receptors for HDV infection, in the above mouse model newly produced virions cannot go on to infect additional rounds of hepatocytes, and therefore the effect of FTIs on virus-related liver injury cannot be fully evaluated. In human HDV infections, however, it would be expected that inhibition of crucial steps in the virus life cycle, such as virion assembly and release, would have a major impact on the course of HDV infection and its associated liver disease. In addition to the above preclinical data in mice, one of the more compelling reasons to pursue FTI therapy for HDV is that precisely the types of

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drugs needed have already been developed and used in humans, albeit for a completely different purpose. This fortuitous development is because the requirement for prenylation in transformation by the Ras oncogene was found to be dependent on the same farnesyl modiﬁcation as occurs on L-HDAg (James et al. 1993; Liu et al. 1999: Sun et al. 1995). Thus, the enzymes responsible for this modiﬁcation have been important targets for anti-cancer drug design. A variety of prenylation inhibitors has been developed and undergone evaluation for clinical use (Rowinsky et al. 1999; Sebti and Hamilton 2000). These efforts include one of the best examples of successful rational drug design, although it turns out that a wide array of chemical entities can have potent inhibitory activity against FTase. Drugs employing both benzodiazepine and tricyclic scaffolds, as well as compounds isolated from natural product screens have yielded effective FTIs. The most clinically advanced FTIs have been developed into oral formulations and used in several phase I/II and III trials (Mazieres et al. 2004). Interestingly, their mechanism of action may turn out to involve inhibition of other farnesylated proteins beyond Ras. Except for certain hematological malignancies (Jabbour et al. 2004), however, to date the anti-tumor efﬁcacy of FTIs has proved somewhat disappointing for many oncologic indications. This may well reﬂect the fact that the targeted form of Ras turns out not to play a primary role in many of these cancers, and that the targeted tumors involve a variety of other important factors. Fortunately, these same considerations do not apply for HDV. In addition, the evaluated FTIs have been shown to be potent and effective inhibitors of FTase and their use was found to generally be quite safe with collectively the main reported side effects being reversible, dose-dependent myelosuppression, fatigue, reversible neuro-cortical toxicity, prolongation of QT interval, and mild gastrointestinal toxicity. Moreover, side effects differed among individual FTIs, suggesting that many side effects might be more related to each speciﬁc compound rather than a result of their common inhibition of farnesylation.

5 Attractive Features of Prenylation Inhibition-Based Antiviral Therapy Prenylation inhibition-based antiviral therapy differs from more classical approaches to antiviral treatment, in that it seeks to deprive the virus access to a host function. By this targeting of a host cell enzyme rather than a virusspeciﬁc target, such a strategy may actually impose some difﬁcult challenges for a virus attempting to develop resistance. This is because the targeted locus is not under genetic control of the virus. Moreover, because the farnesyl moi-

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ety of L-HDAg may serve more of a speciﬁc ligand role (Hoffman et al. 2000), rather than simply being a mediator of transient interactions with membranes, simple substitution of geranylgeranyl under conditions of FTI-mediated inhibition of farnesylation may be insufﬁcient for restoring HDV assembly. Certainly to date, no such mechanism of resistance has been observed. At ﬁrst glance, it might be predicted that inhibiting host cell mediated prenylation would cause intolerable side effects. Surprisingly, however, this does not appear to be the case as FTIs are remarkably well tolerated by host cells in vitro (Dalton et al. 1995), and more importantly in human in vivo trials (Sharma et al. 2002). This might reﬂect that more host cell prenylated proteins are modiﬁed by geranylgeranyl rather than farnesyl (Farnsworth et al. 1990), although recent data suggest this may not be the case (Winter-Vann and Casey 2005). Alternatively, because there is a family of prenyltransferase enzymes, ‘cross prenylation’ by nontargeted prenyltransferases may occur. It is worth emphasizing that prenylation inhibition-based antiviral therapy has implications for other viruses besides HDV which are found to have similarly prenylated proteins. Indeed a CXXX box motif is present in proteins of numerous other medically important viruses, as well as in agents with a potential for bioterrorism (Elazar and Glenn 2005). The precise role played by prenylation, however, may differ in each case and need not be restricted to mediating assembly as in HDV. For example, the polymerase proteins of hepatitis A virus and foot and mouth disease virus have a conserved CXXX box. Because the replication of these positive single-strand RNA viruses is thought to occur in intimate association with intracellular membranes, prenylation of these proteins may provide a membrane anchoring function for the catalytic subunit of the respective replication complexes. On the other hand, the UL32 gene product of herpes simplex virus (HSV), which is thought to be involved in virus particle formation, also contains a CXXX box (McGeoch et al. 1988). This suggests that HSV can be considered for targeting by prenylation inhibition and indeed HSV sensitivity to FTI-I treatment has been demonstrated. Interestingly, the interpretation of these latter experiments performed in a Ras-transformed cell line infected with HSV-1 was that the virus exploits the host-cell Ras signaling pathway for infection (Farassati et al. 2001). Thus it is possible to consider that, in addition to direct antiviral effects brought about by inhibiting the prenylation of speciﬁc viral proteins, antiviral activity may also be observed as the result of prenylation inhibition-mediated perturbation of host cell pathways dependent on host prenylated proteins. Another example of this has been described for hepatitis C virus where it was proposed that the effect of geranylgeranyltransferase type I inhibitor on HCV RNA replication reﬂects the virus’ dependence on the prenylation of a host cell protein (Ye et al. 2003). This dependence of HCV on host cell geranylgeranylation has

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recently been suggested by others as well (Kapadia and Chisari 2005). Finally, the mechanism of lovastatin antiviral activity against respiratory syncytial virus was suggested to reﬂect dependence on prenylation of the host protein RhoA (Gower and Graham 2001).

6 Other Targets for Anti-HDV Therapy Because the production of mevalonate by the enzyme HMG-CoA reductase is a committed step in both the cholesterol and prenyl lipid biosynthetic pathways (Zhang and Casey 1996), it has been suggested that HMG-CoA reductase inhibitors–which are in widespread clinical use to treat hypercholesterolemia– might be used to inhibit prenylation. Prenylation can indeed be inhibited in vitro by an HMG-CoA reductase inhibitor, but the doses required are cytotoxic (Sinensky et al. 1990). Because cellular needs for cholesterol can be exogenously supplied via the low density lipoproteins receptor system, synergy between HMG-CoA reductase inhibitors and FTIs can be considered. Similar potential synergistic effects might also be achieved by combining FTIs with other classes of drugs which inhibit additional steps in the pathway of prenyl protein synthesis. For example, most prenylated eukaryotic proteins are further processed by the sequential activity of the products of the Rce1 (CXXX-box endoprotease) and ICMT (isoprenylcysteine carboxylmethyl transferase) genes, respectively (see Fig. 2), which result in the removal of the CXXX box-XXX tripeptide, and methylation of the resulting C-terminal carboxyl group (Winter-Vann and Casey 2005). Both of these reactions increase the membrane association of the processed protein (Hancock et al. 1991; Parish and Rando 1996; Silvius and l’Heureux 1994) and their inhibition can interfere with the protein’s function (Bergo et al. 2004; Kim et al. 1999; Winter-Vann et al. 2005). To the extent that prenylated large delta antigen and other viral proteins are indeed further processed by these mechanisms, drugs which inhibit these reactions may also prove useful as antiviral agents alone or in combination with FTIs. Additional therapies based on knowledge of the HDV life cycle can also be considered. For example, targeting the RNA editing process, ribozyme activities, or RNA-dependent RNA transcription offer the prospect of HDV-speciﬁc therapeutic strategies. Finally, small interfering RNA and immune-based therapies are being increasingly considered for a variety of viral diseases, and HDV might also be targeted by such approaches. Taken together, the study of HDV molecular virology has revealed an exciting array of potential new antiviral strategies. Perhaps one of the most

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Fig. 2A–C Prenylation-induced processing reactions. Proteins harboring a ‘CXXXbox,’ where C = cysteine and X = one of the last three amino acids at the carboxyl terminus of the protein, are the substrate for covalent attachment of the prenyl lipids farnesyl or geranylgeranyl. A This reaction is catalyzed by prenyltransferase enzymes (farnesyltransferase or geranylgeranyltransferase I). B The prenylated CXXX-box is recognized by CXXX-box endoprotease (the product of the Rce1 gene) which removes the ‘-XXX’ tripeptide. C The now C-terminal prenylated cysteine undergoes methylation of its carboxyl group in a reaction catalyzed by isoprenylcysteine carboxylmethyltransferase (the ICMT gene product). See text for details

practical and pre-clinically validated approaches involves the inhibition of L-HDAg by FTI treatment. Because such inhibitors have been developed and used in phase I and II trials, the ﬁrst human evaluation of their antiviral efﬁcacy against HDV should hopefully soon be forthcoming. Acknowledgements This work was supported by a Burroughs Welcome Fund Career Award and Veterans Administration Merit Review Award.

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Kim E, Ambroziak P, Otto JC, Taylor B, Ashby M, Shannon K, Casey PJ, Young SG (1999) Disruption of the mouse Rce1 gene results in defective Ras processing and mislocalization of Ras within cells. J Biol Chem 8383–8390 Lau D, Doo E, Park Y, Kleiner D, Schmid P, Kuhns M, Hoofnagle J (1999) Lamivudine for chronic delta hepatitis. Hepatology 546–549 Lazinski DW, Taylor JM (1993) Relating structure to function in the hepatitis delta virus antigen. J Virol 2672–2680 Liu M, Bryant MS, Chen J, Lee S, Yaremko B, al. e (1999) Effects of SCH 59228, an orally bioavailable farnesyl protein transferase inhibitor, on the growth of oncogenetransformed ﬁbroblasts and a human colon carcinoma xenograft in nude mice. Cancer Chemother Pharmacol 50–58 Maltese WA (1990) Posttranslational modiﬁcation of proteins by isoprenoids in mammalian cells. FASEB J 3319–3328 Mazieres J, Pradines A, Favre G (2004) Perspectives on farnesyl transferase inhibitors in cancer therapy. Cancer Lett 159–167 McGeoch DJ, Dalrymple MA, Davison AJ, Dolan A, Frame MC, McNab D, Perry LJ, Scott JE, Taylor P (1988) The complete DNA sequence of the long unique region in the genome of herpes simplex virus type 1. J Gen Virol 1531–1574 Novick P, Brennwald P (1993) Friends and family: The role of the Rab GTPases in vesicular trafﬁc. Cell 597–601 Otto JC, Casey PJ (1996) The hepatitis delta virus large antigen is farnesylated both in vitro and in animal cells. J Biol Chem 4569–4572 Parish CA, Rando RR (1996) Isoprenylation/methylation of proteins enhances membrane association by a hydrophobic mechanism. Biochemistry 8473–8477 Pereira-Leal JB, Seabra MC (2001) Evolution of the Rab family of small GTP-binding proteins. J Mol Biol 889–901 Pfeffer S, Aivazian D (2004) Targeting Rab GTPases to distinct membrane compartments. Nat Rev Mol Cell Biol 886–896 Pullarkat RK, Morris GN (1997) Farnesylation of Batten disease CLN3 protein. Neuropediatrics 42–44 Radjef N, Gordien E, Ivaniushina V, Gault E, Anais P, Drugan T, Trinchet JC, Roulot D, Tamby M, Milinkovitch MC and others (2004) Molecular phylogenetic analyses indicate a wide and ancient radiation of African hepatitis delta virus, suggesting a deltavirus genus of at least seven major clades. J Virol 2537–2544 Rizzetto M, Ponzetto A, Forzani I (1991) Epidemiology of hepatitis delta virus: Overview. In: Rizetto M, editor. The hepatitis delta virus. New York: Wiley-Liss Inc. pp 1–20 Rowinsky EK, Windle JJ, Von Hoff DD (1999) Ras protein farnesyltransferase: A strategic target for anticancer therapeutic development. J Clin Oncol 3631–3652 Ryu W-S, Bayer M, Taylor J (1992) Assembly of hepatitis delta virus particles. J Virol 2310–2315 Samuel D, Zignego A-L, Reynes M, Feray C, Arulnaden JL, David M-F, Gigou M, Bismuth A, Mathieu D, Gentilini P and others (1995) Long-term clinical and virological outcome after liver transplantation for cirrhosis caused by chronic delta hepatitis. Hepatology pp 333–339

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Sato S, Cornillez-Ty C, Lazinski DW (2004) By inhibiting replication, the large hepatitis delta antigen can indirectly regulate amber/W editing and its own expression. J Virol 8120–8134 Sebti SM, Hamilton AD (2000) Farnesyltransferase and geranylgeranyltransferase I inhibitors and cancer therapy: lessons from mechanism and bench-to-bedside translational studies. Oncogene 6584–6593 Sharma S, Kemeny N, Kelsen DP, Ilson D, O’Reilly E, Zaknoen S, Baum C, Statkevich P, Hollywood E, Zhu Y, et al. (2002) A phase II trial of farnesyl protein transferase inhibitor SCH 66336, given by twice-daily oral administration, in patients with metastatic colorectal cancer refractory to 5-ﬂuorouracil and irinotecan. Ann Oncol 1067–1071 Shih KN, Lo SJ (2001) The HDV large-delta antigen fused with GFP remains functional and provides for studying its dynamic distribution. Virology 138–152 Silvius JR, l’Heureux F (1994) Fluorimetric evaluation of the afﬁnities of isoprenylated peptides for lipid bilayers. Biochemistry 3014–3022 Sinensky M, Beck LA, Leonard S, Evans R (1990) Differential inhibitory effects of lovastatin on protein isoprenylation and sterol synthesis. J Biol Chem 19937– 19941 Sun J, Qian Y, Hamilton AD, Sebti SM (1995) Ras CAAX peptidomimetic FTI 276 selectively blocks tumor growth in nude mice of a human lung carcinoma with K-Ras mutation and p53 deletion. Cancer Res 4243–4247 Sureau C, Guerra B, Lanford RE (1993) Role of the large hepatitis B virus envelope protein in infectivity of the hepatitis delta virion. J Virol 366–372 Sureau C, Moriarty AM, Thornton GB, Lanford RE (1992) Production of infectious hepatitis delta virus in vitro and neutralization with antibodies directed against hepatitis B virus pre-S antigens. J Virol 1241–1245 Wei Y, Ganem D (1998) Activation of heterologous gene expression by the large isoform of hepatitis delta virus. J Virol 2089–2096 Winter-Vann AM, Baron RA, Wong W, dela Cruz J, York JD, Gooden DM, Bergo MO, Young SG, Toone EJ, Casey PJ (2005) A small-molecule inhibitor of isoprenylcysteine carboxyl methyltransferase with antitumor activity in cancer cells. Proc Natl Acad Sci USA 4336–4341 Winter-Vann AM, Casey PJ (2005) Opinion: Post-prenylation-processing enzymes as new targets in oncogenesis. Nat Rev Cancer 405–412 Wolters LM, van Nunen AB, Honkoop P, Vossen AC, Niesters HG, Zondervan PE, de Man RA (2000) Lamivudine-high dose interferon combination therapy for chronic hepatitis B patients co-infected with the hepatitis D virus. J Viral Hepat 428–434 Xia Y-P, Yey C, Ou J-S, Lai MMC (1992) Characterization of nuclear targeting signal of hepatitis delta antigen: Nuclear transport as a protein complex. J Virol 914–921 Yamane HK, Farnsworth CC, Xie H, Howald W, Fung BK-K, Clarke S, Gelb MH, Glomset JA (1990) Brain G protein g subunits contain an all-trans-geranylgeranylcysteine methyl ester at their carboxyl termini. Proc Natl Acad Sci USA 5868–5872 Ye J, Wang C, Sumpter R, Jr., Brown MS, Goldstein JL, Gale M, Jr. (2003) Disruption of hepatitis C virus RNA replication through inhibition of host protein geranylgeranylation. Proc Natl Acad Sci USA 15865–15870

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CTMI (2006) 307:151–171 c Springer-Verlag Berlin Heidelberg 2006

Hepatitis Delta Virus Genetic Variability: From Genotypes I, II, III to Eight Major Clades? P. Dény (u) Service de Bactériologie, Virologie, Hygiène, Laboratoire Associé au Centre National de Référence des hépatites B et C, Hôpital Avicenne, Assistance Publique – Hôpitaux de Paris, Université Paris 13, EA3406, 93009 Bobigny Cedex, France [email protected]

1

Viral Genetics of HBV and HDV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152

2

Nucleotide Similarity Approach: The Notion of Genotypes . . . . . . . . . . . 155

3 3.1 3.2 3.3 3.4

Phylogenetic Analyses of African HDVs . . . . . . . . . . Patients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Analysis of the R0 Region . . . . . . . . . . . . . . . . . . . . Full-Length Genome Analysis . . . . . . . . . . . . . . . . . Use of sHD Gene Sequence for Phylogenetic Analyses

4

HD Protein and RNA Secondary Structure . . . . . . . . . . . . . . . . . . . . . . 162

5 5.1 5.2 5.3

HDV Phylogeography . . . . . . . . . . . . . . . . . . . . HDV Genetic Distances and Geography . . . . . . . Co-epidemiological Speciation of HBV and HDV . HDV Genetic Variability and Clinical Patterns . . .

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Proposal for the Deltavirus Genus Classiﬁcation . . . . . . . . . . . . . . . . . . 165

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Abstract Hepatitis D virus (HDV) is a satellite of hepatitis B virus (HBV) for transmission and propagation, and infects nearly 20 million people worldwide. The HDV genome is composed of a compact circular single-stranded negative RNA genome with extensive intramolecular complementarity. Along with epidemiological, geographic distribution and pathological patterns, the variability of HDV has been limited to three genotypes and two subtypes that have been characterized to date. Recently, extensive phylogenetic reconstructions based on the delta antigen gene and full-length genome sequence data, have shown a wide and probably ancient radiation of African lineages, suggesting that the genetic variability of HDV is much more complex than previously thought. Indeed, sequences previously afﬁliated with genotype IIb should now be considered as belonging to clade 4 (HDV-4) and African HDV sequences segregate within four additional clades: HDV-5, HDV-6, HDV-7 and HDV-8. These results bring the geographic distribution of HDV closer to the genetic variability of its helper HBV.

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1 Viral Genetics of HBV and HDV Despite an effective vaccine, infection with hepatitis B virus (HBV) is mainly transmitted through the mother-to-neonate route in endemic countries. In most cases, chronic infection results and the transmission will therefore occur from generation to generation. HBV could be considered as an indirect marker of population migration. Transmitted from the mother, it might be considered as an alternate to mitochondrial DNA (mtDNA) (Ingman et al. 2000). MtDNA analyses indicate that around 59,000–100,000 years before present, earth colonization might have occurred from Africa to the Middle East, Asia then to Australia, Europe and to Americas. Several hypothesis have been proposed to link the HBV to human evolution and dispersal around the globe (reviewed in Simmonds 2001). (1) The existence of Hepadnaviridae in other primates, mammals and birds makes possible a co-speciation of HBVs during evolution. (2) The higher divergence of the South American HBV genotype F strains (HBV/F) could have given rise to HBV spreading from this area to the rest of the world during the slave trade. (3) The existence of primate-speciﬁc strains might also explain a cross species contamination such as in HIV. These hypotheses are not necessarily mutually exclusive, and each could contribute, in part, to the present day distribution of HBV among humans around the world. Regardless of the origin and evolution of HBV, nucleotide similarity approaches and evolutionary reconstructions indicate the existence of at least eight HBV genotypes (labelled HBV/A to HBV/H), the existence of many subtypes and speciﬁc gene phylogenies identify recombinant forms (Norder et al. 2004). Hepatitis delta virus (HDV) was identiﬁed in 1977 as a foreign antigen in the serum and the liver of Italian patients infected with HBV (Rizzetto et al. 1977). The origin of HDV remains difﬁcult to understand and the age of the HDV–HBV association needs to be clariﬁed. This viral-like agent has been classiﬁed as a satellite of HBV, being dependant on HBV for virion assembly and propagation (Sureau et al. 1993), but not for HDV–RNA replication. The HDV genome is a circular single-stranded RNA of 1672–1697 bases with extensive intramolecular complementarity (Wang et al. 1986; Radjef et al. 2004). Part of the HDV genome might share historical homology with viroids or plant virus satellite RNA sequences (Elena et al. 1991). Interestingly, a pseudoknot ribozyme is evidenced in both genomic and antigenomic RNA strands, corresponding to the best conserved parts of the genome. However, due to a low degree of similarity between viroids and the HDV genome, this ancestral viroid afﬁliation is disputed (Jenkins et al. 2000). Furthermore, viroids are not known to code any protein, while HDV does. A double rolling circle model

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is involved for viral RNA replication (reviewed in the chapters by J.M. Taylor and by T.B. Macnaughton and M.M.C. Lai, this volume). The lack of ﬁdelity and the absence of a proofreading activity of RNA polymerases give rise to heterogeneous molecules that can be considered as quasispecies, as has been described for the RNA virus world (Eigen and Biebricher 1988). Indeed, the original paper (Wang et al. 1986) describing a full-length HDV genome sequence derived following experimental transmission to chimpanzees (Chimp A20), indicates that the RNA sequence from an HDV strain, derived from a chronically infected Italian patient-isolate, was heterogeneous. In fact, the 1679 nucleotide long sequence showed 17 ambiguous positions, mostly consisting of transitions (n=16). From the same strain successfully transmitted to the woodchuck model (where delta ribonucleoprotein is budding through the woodchuck hepatitis virus surface antigen envelope), six positions were heterogeneous in the W5 isolate (Dény et al. 1991). All the viruses characterized from this lineage show a nucleotide sequence with global similarity higher than 98% during these experimental transmissions (Wang et al. 1986; Kuo et al. 1988; Dény et al. 1991; Kos et al. 1991). This heterogeneity and evolution rate has been particularly well studied on the HDV coding gene (Imazeki et al. 1990), 1068 bases (Zhang and Hansson 1996) and on the complete genome (Lee et al. 1992; Chao et al. 1994). Lee et al. described the long-term evolution of a Lebanese HDV strain. The evidence of an acute delta

Table 1 Similarity percentage (%) for the sHD gene within (bold) and between the eight HDV major clades: mean (range) HDV-1 HDV-1 HDV-2 HDV-3 HDV-4 HDV-5 HDV-6 HDV-7 HDV-8

HDV-2

HDV-3

HDV-4

HDV-5

HDV-6

HDV-7

HDV-8

89.5

(84.2–99.4)

81.6

91.5

73.3

73.3

92.3

80.1

81.8

73.8

92.8

78.9

84.0

72.4

80.9

92.1

77.8

79.3

72.4

77.8

80.5

91.4

77.7

79.0

72.1

78.1

81.6

77.3

89.9

78.9

82.0

71.3

80.0

83.4

80.6

81.3

(78.3–84.0) (87.7–95.6) (71.7–75.4) (72.1–74.4) (88.5–97.0) (76.9–83.0) (79.6–84.5) (72.9–75.1) (90.4–97.5) (76.7–80.5) (82.6–85.4) (71.1–73.5) (79.8–82.1) (90.6–93.9) (75.0–80.9) (77.7–80.9) (70.2–74.1) (76.5–78.7) (79.4–82.2) (91.2–91.7) (74.5–80.8) (76.7–80.7) (70.7–73.5) (75.7–80.4) (79.4–83.5) (75.8–79.5) (87.5–91.6)

93.2

(76.2–81.3) (80.7–83.2) (69.9–72.5) (77.9–81.8) (82.1–84.5) (79.1–81.7) (78.8–83.8) NA

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Fig. 1 Detection of deleted HDV genome in the serum by the use of ampliﬁcation (1) and primer extension (2). 2a: Successive samples obtained from a patient before (lanes 1–4) and after (lane 5) Interferon treatment. 2b: Successive follow-up (lanes 1 and 2) of HDV in a patient harbouring two forms of deleted HDV molecules. 3: Sequence analyses indicate the 13 to 14 base deletions from the respective strains

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hepatitis followed by a chronic course of the infection with ﬂares, gave the opportunity to compare the evolutionary rates of the genome during the different phases. The acute/ﬂare phase was associated with a rapidly evolving rate of 3.0×10−2 substitutions/nucleotide/year, whereas the chronic phase seemed to be less dynamic–3.0×10−3 substitutions/nucleotide/year. Obviously, such different rates of evolution could make the hypothesis of a ‘molecular clock’ not as easily suitable for HDV as for other viruses (Simmonds 2001). Furthermore, treatment-induced environmental constraints also accelerate such evolutionary rates. For example, in the case of a chronically infected patient by HDV type 1, a substantial modiﬁcation of the viral genome occurred with the appearance of naturally deleted HDV-RNA molecules that were detected in the plasma (Fig. 1) (Dény 1994). Under high dose alpha-interferon, both defective and natural molecules disappeared, but a resistant strain emerged (without the defective molecule) (Fig. 1) indicating that a relapse occurred under interferon treatment. In such an environment the mutation rate of the HDV coding sequence before and after treatment was 1.09×10−2 substitutions per site per year, which was higher than observed in natural infection for the delta protein coding gene: i.e., 1.14×10−3 to 1.28×10−3 mutations/ nucleotide/year (Imazeki et al. 1990; corrigendum in Lee et al. 1992) or 2.60×10−3 mutations/nucleotide/year (Chao et al. 1994). Interestingly, a recent paper from J.C. Wu et al., conﬁrmed the existence of such deletions in four out of ﬁve patients chronically infected with HDV-1 or HDV-2 (Wu et al. 2005). The evidence of mixed HDV infection has been described in Taiwan (Wu et al. 1999) and HDV genome recombination is another evolutionary pathway recently observed (Wang and Chao 2005). T.C. Wang and M. Chao described a patient who was infected with both a genotype I (HDV-1) strain and a genotype IIb (HDV-4) strain. Using a wide range of techniques, they found evidence that HDV homologous recombination occurred in 6% of clones and mapped the recombination events within homologous regions from those two strains.

2 Nucleotide Similarity Approach: The Notion of Genotypes Although HDV was expected, like many RNA viruses, to exhibit considerable genetic variability, only three HDV genotypes (labelled genotype I, II and III) have been characterized on the basis of a small number of complete genome sequences (Casey et al. 1993; Imazeki et al. 1991; Wang et al. 1986). In 1993, J. Casey and coworkers identiﬁed strains from Columbia and Peru that have the lowest similarity score when compared with all identiﬁed HDV strains of other lineages (range, 67.5%–69.4%) (Casey et al. 1993).

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Therefore, the deﬁnition of ‘genotype’ is based on the comparison of nucleotide sequence similarity between pairs of sequences that have been discovered and characterized at the time. A nucleotide similarity percentage corresponds directly to the number of positions bearing identical nucleotides between pairs of sequences, divided by the length of the alignment. For HDV genomes that are known at present, with regards to the region studied, the divergence in nucleotide sequence (= 100% – similarity score) is less than 14%–15.7% among different isolates of the same genotype and ranges from 19% to 38% between sequences from different genotypes (Casey et al. 1993; Imazeki et al. 1991; Shakil et al. 1997; Wu et al. 1998). Table 1 indicates the nucleotide similarity of the sHD coding gene. Genotype I includes the European, Mediterranean, North American, and some Russian, African and Asian HDV isolates (Makino et al. 1987; Chao et al. 1990; Shakil et al. 1997); it was also described in the Paciﬁc island of Nauru (Chao et al. 1990). Genotype II was initially found in Japan and Taiwan (Imazeki et al. 1990; Lee et al. 1996); some sequences from Taiwan and Okinawa islands were tentatively assigned to a subtype of genotype II (i.e., genotype IIb, see below) (Wu et al. 1998; Sakugawa et al. 1999). However, addition to the data set of sequences from Yakutia (Sakha republic, Russia), which were clearly identiﬁed as a subclade of genotype II strains, suggested that the IIb strain TW-2b (genotype IIb) might not be afﬁliated with HDV-IIa (HDV-2) (Ivaniushina et al. 2001). Therefore Ivaniushina et al. suggested that TW-2b might be the prototype of a new clade. Genotype III has been exclusively found in the Amazonian part of South America (Peru, Colombia, Equator, Brazil and Venezuela) (Casey et al. 1993; Nakano et al. 2001; A Kay, personal communication). These studies on HDV genome variability have been performed in nonAfrican countries except for the description of two sequences (assigned to genotype I) from Ethiopia and Somalia (Zhang et al. 1996). The existence of different subclades among genotype I sequences have been proposed but dismissed (Zhang et al. 1996; Shakil et al. 1997). On studying African HDV sequences (Radjef et al. 2004 and unpublished data from our laboratory), our phylogenetic analyses indicated that, in addition to genotype I-like sequences, approximately 70% of the characterized African isolates (mostly from West and Central Africa) form highly divergent groups (see the score of similarity in Table 1). These results (1) suggest a possible ancient African radiation, and (2) extend the known HDV genetic variability to probably eight major monophyletic groups or ‘clades’, thus bringing this satellite variability closer to human HBV genetic variability. In the following section of this review, we shall focus on these different HDV clades and shall make a proposal to suggest an updating of the classiﬁcation among the Deltavirus genus.

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3 Phylogenetic Analyses of African HDVs 3.1 Patients Between January 1999 and February 2005, 391 samples, collected from HDV infected patients that were positive in a routine search of HDV RNA detection in serum, were analysed. In the ﬁrst part of the study (Radjef et al. 2004), we selected 25 patients whose preliminary examination suggested that the HDV viral strains varied from previously described HDV genotypes. Indeed, we selected the 25 samples for which (1) HDV cDNA could not be ampliﬁed using previously described primers 6A and 6S (Dény 1994) even though HDV serology was positive or (2)) the R0-DNA amplicon restriction pattern was atypical (Gordien et al., unpublished results, see Fig. 2). Interestingly, 22 samples were obtained from patients from Africa or who had travelled to Africa. The male to female ratio was 0.8 and the mean age was 35 years (range, 15–53 years). Most patients had chronic active hepatitis or cirrhosis and only one patient, aged 33 years, had acute HDV superinfection. 3.2 Analysis of the R0 Region We ﬁrst used a 329-nucleotide (nt) HDV cDNA fragment (here called R0: position 907–1235; sequence numbering according to Wang et al. 1986) to characterize a large number of clinical samples. Brieﬂy, HDV RNA was extracted from 250µl of serum and the R0 region was ampliﬁed as described (Ivaniushina et al. 2001). For all samples, two short RT–PCR-generated DNA fragments were directly sequenced. This region was chosen because it encompasses the 3’-end of the large delta protein (L-HDAg) coding sequence, as proposed by J. Casey, and is widely used in the literature to differentiate genotype I, II and III. The new 25 R0 sequences were aligned with 16 sequences from Russia (Ivaniushina et al. 2001) and 36 R0 sequences from public databases. Alignment of HDV R0 sequences was generated with ClustalW 1.8 using a gap opening penalty (GOP) of 15 and a gap extension penalty (GEP) of 6.66 with minimum manual corrections. Analyses using maximum parsimony (MP), distance and neighbour joining reconstruction (NJ) and maximum likelihood (ML) methods yielded results compatible with the phylogenetic tree shown in Fig. 3a. Of the 22 newlycharacterized African sequences, 15 form at least three lineages (boxed in grey in Fig. 3a) spanning a range of variability much larger than those within type I or type III. The other seven African sequences are scattered within the type I clade, conﬁrming the presence of HDV-1 in the African continent.

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Fig. 2 Rapid algorithm for HDV genome characterization using the ampliﬁed R0 restriction fragment proﬁle. The use of SmaI and XhoI gave at least ten different proﬁles on 275 samples studied. SacII, which is used in secondary analysis, can differentiate some HDV-2 and HDV-5 or HDV-6 isolates. In all, at least 13 different restriction proﬁle patterns have been observed using the three different restriction enzymes (E. Gordien and N. Radjef, unpublished results)

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Fig. 3 Phylogenetic trees inferred from delta-virus nucleotide sequences. a NJ phylogram obtained from the R0 data set; bootstrap values obtained for NJ/MP are indicated above the branches, whereas ML posterior probabilities inferred from 400 metaGA samplings are indicated below the branches. b ML cladogram obtained from the full genome data set (uppercase taxon); numbers at the branches indicate ML posterior probabilities inferred from 400 metaGA samplings. Fifteen of the 25 viral HDV sequences reported here form new clades (boxed in grey) characterizing a range of variability larger than that among type-I or type-III sequences. The remaining ten new sequences cluster with type-I. Asterisks indicate newly characterized HDV sequences. Scale is in percent-expected substitution per position

3.3 Full-Length Genome Analysis To test the validity of our results, we sequenced the full genome of isolates (Fig. 3b) spanning the range of the newly-characterized clades (accession

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numbers AJ584844–AJ584849). Alignments of these sequences showed a level of similarity of approximately 75% (see Table 3 in Radjef et al. 2004), in the same range as that observed between type I and type II sequences, suggesting the existence of additional genotypes. Because different values of GOP and GEP gave different results, full-genome alignments were performed using the SOAP program (Loytynoja and Milinkovitch 2001). Using this program, positions with different alignments can be excluded, or a proportion of GOP and GEP combinations yielding the same alignment can be studied. We also used ProAlign (Loytynoja and Milinkovitch 2003), a program that provides a statistical approach to multiple sequence alignment, such that a posterior probability is assigned to each aligned position. Positions with a posterior probability below a user-deﬁned threshold (here <90%) can be excluded before phylogeny inference. MP (using branch-and-bound) and NJ (ML distances) phylogenetic analyses were performed with PAUP*4.0b10 (Swofford 1998). ML analyses were carried out both with PAUP*4.0b10 (full-length and sHD data sets) and the metapopulation genetic algorithm (metaGA) (Lemmon and Milinkovitch 2002) implemented in the program MetaPIGA (www.ulb.ac.be/sciences/ueg). Bootstrap analyses (103 , 104 and 102 replicates for PAUP MP, NJ and ML, respectively) and 10000 metaGA samples (2500 replicates with four populations, ML analyses) were performed to assess the robustness and posterior probabilities of nodes, respectively. We used the HKY model and estimated transition:transversion ratio, proportion of invariable sites, and rate heterogeneity (gamma distribution with four categories) parameters from the data. These analyses yielded results (Fig. 1b) similar to those described above (Fig. 1a): the genetic variability of the HDV genus has more major monophyletic groups (i.e., clades) than previously thought. 3.4 Use of sHD Gene Sequence for Phylogenetic Analyses The HDV RNA secondary structure clearly violates the assumption of character independence. Furthermore, there is functional evidence for the sHD protein to trans-complement more efﬁciently the corresponding HDV-type (Casey and Gerin 1998; Lin et al. 2003). In the absence of recombination, coevolution of the sHD coding gene and the corresponding viral genome likely occurred. We therefore phylogenetically analysed sHD coding sequences from 44 isolates including newly characterized strains from Okinawa island (Ma et al. 2003) and Taiwan (Wang and Chao 2003) labelled as genotype IIb strains, three Peruvian HDV sequences kindly provided by J. Casey and 12 sHD gene sequences from African strains from our laboratory. Alignment of this part

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of the genome was unambiguous. The phylogenetic results obtained again conﬁrmed that the African HDV clades account for a large proportion of HDV variability worldwide (Fig. 4). These analyses, including the African HDV sequences, also conﬁrm that sequences from the ‘genotype IIb’ complex (Ma et al. 2003; Wu et al. 1998; Watanabe et al. 2003; Wang and Chao 2003) form a dis-

Fig. 4 ML analyses of 44 sHD cDNA sequences. Analyses were conducted using PAUP4b10. Numbers indicate bootstrap values (BV) (1000 NJ replicates with Paup*, parameters constrained to those obtained from the ML analysis of the original data set). BV of 50 or greater are indicated. Note that BV of 100 and 98 are also present within clade 1 for (ITALY,(W5,W15)) and (US1,TH7), respectively. Scale is in percentexpected substitution per position. Suggested clade names are indicated (see Table 2)

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tinct clade (proposed as ‘HDV-4’ in Fig. 4), which is not more closely related to the genotype IIa sequences (cf. ‘HDV-2’ in Fig. 4) than others. In all, at least probably eight major clades, including the well deﬁned genotype I (HDV-1) and genotype III (HDV-3) were identiﬁed; four of them (HDV-5, HDV-6, HDV-7 and HDV-8) correspond exclusively to African HDV sequences (Fig. 4).

4 HD Protein and RNA Secondary Structure All the newly-characterized complete HDV sequences exhibited the two expected overlapping open reading frames (s-HDAg and L-HDAg), and most of the conserved motifs were located in the central and carboxy-terminal regions of the S-HDAg (Fig. 5). As most patients studied had a clear antibody response to recombinant type-I HDAg, one might expect some immunogenic epitopes to correspond to these conserved regions. Analysis of the carboxy-terminal part of L-HDAg proteins reveals a highly-variable proline-rich domain except for three conserved residues (Fig. 5): a tryptophan (W) at position 195, a cystein (C) involved in the farnesylation signal in position 211 and a glutamine (Q) at the very end carboxy-terminal of L-HDAg (see the chapter by J.S. Glenn, this volume). Predictions of HDV-RNA antigenomic secondary structure indicate that the characterized African isolates exhibit slightly different patterns in the vicinity of the RNA editing site (at the amber/tryptophan codon; see the chapter by J.L. Casey, this volume). Each prototype sequence from each clade had a speciﬁc antigenomic RNA pseudo double-strand secondary structure. Alternative branched secondary structure were also observed for some sequences (Radjef et al. 2004). Fig. 5 Alignment of HD amino-acid sequences from African (dFr-910, dFr-47, dFr73, dFr-48, dFr-45, and dFr-644,), Italian (A20, genotype I), Japanese (-S, genotype IIA), Peruvian (-1, genotype III), and Taiwanese- (TW-2b, genotype IIB) isolates. Dots indicate identities with the dFr-910 sequence whereas asterisks indicate positions conserved across all aligned sequences. Bold characters indicate polymorphisms. Motifs corresponding to functional properties are boxed (NLS, nuclear localization signal; ARM, arginin-rich motifs; PKR, protein kinase R). The carboxy-terminal part of LHD constitutes a highly-variable proline-rich domain corresponding to the packaging signal except for three conserved residues: a tryptophan, a cystein, and a glutamine corresponding, respectively, to the UAG-to-UGG editing site, the farnesylation box CXXX, and the C-terminal residue. Characters on the bottom line indicate when all (uppercase) or 50% to 99% (lower case) of the ten aligned sequences yielded identical secondary structure consensus predictions (H,h, helix; C,c, coiled; E,e, beta-sheet)

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5 HDV Phylogeography 5.1 HDV Genetic Distances and Geography Similar to HCV (Morice et al. 2001) and HBV (Ganne-Carrié et al., 2006), the delta viruses characterized in Paris (France) showed a wide African distribution. For example, the six full-length viral RNA sequences were obtained from ﬁve patients from Western or Central sub-Saharan African countries (Cameroon, Guinea, Ivory Coast, Mali, and Republic of Congo) and from an adult Polish woman who had lived in Cameroon for 3 years. To determine whether the geographic distribution of the HDV isolates was correlated with their levels of sequence divergence, we compared the Kimura-2-parameter pairwise distances between full-genome sequences with the relative geographic distances matrix of the capitals of the countries where the patients had been infected. To compare two quantitative continuous variables, we decided to calculate the correlation coefﬁcient to evaluate the degree of proportionality of data sets. When the ubiquitous type-I sequences were removed, a signiﬁcant statistical correlation was observed between the two matrices (Pearson correlation coefﬁcient: r = 0.791; P < 0.0001). Because r = 0.791, we can estimate that in this data set the Kimura-2 genetic distances and the corresponding relative geographical distances were directly proportional, with a minimal risk of error (0.0001). 5.2 Co-epidemiological Speciation of HBV and HDV One of the main difﬁculties to characterize both HBV and HDV in the same sample is the inhibition of the HBV replication in the case of HDV co- or superinfection. However, using sensitive approaches it is now possible to obtain such information. An alternative would be to extract both RNA and DNA from the liver tissue when available. Analyses of Amazonian isolates clearly identify a strong association between HBV/F and HDV genotype III (Casey et al. 1996; Nakano et al. 2001; Quintero et al. 2001). HDV-1, which is ubiquitous, is mainly associated with HBV/D around the Mediterranean region (Saudy et al. 2003; Bozdayi et al. 2004), Russia (Flodgren et al. 2000) and has recently been described in Mongolia (Inoue et al. 2005). However, HDV-1 has also been found together with HBV/A and, in Taiwan, with HBV/B and HBV/C (Kao et al. 2002). HDV-2, which corresponds to the ﬁrst Japanese prototype lineage and the Yakutian lineage, is also linked with Asian HBV genotypes (HBV/B and HBV/C) (Kao et al. 2002). Interestingly, HDV-4 (previously genotype IIb)

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is associated with HBV/B (Moriyama et al. 2003) and might be comprised of two subgroups of Taiwan and Okinawa HDV strains (Watanabe et al. 2003). HDV clades 5 to 8 are associated with HBV African strains mainly HBV/E and HBV/A (P. Dény, unpublished results). 5.3 HDV Genetic Variability and Clinical Patterns The wide radiation of HDV we describe might contribute to the spectrum of pathologies associated with HDV. For example, speciﬁc liver lesions, including morula cells, have been observed in severe hepatitis in African and Amazonian patients (Casey et al. 1993; Parana et al. 1995). It is therefore considered that the association of HDV-3–HBV/F leads to severe acute hepatitis. By contrast HDV-2 and HDV-4 have been typically associated with less severe hepatitis disease than Type I-associated infections (Wu et al. 1995). However, a recent study among the Miyako island strains suggests that the HDV-4 Okinawa subgroup (labelled IIb-M in the original paper) induces a greater progression to chronic hepatitis and to cirrhosis (Watanabe et al. 2003). Type-I viruses have a wide spectrum of pathologies, ranging from severe fulminant hepatitis in Sweden (Hansson et al. 1982; Zhang and Hansson 1996), Russia (Flodgren et al. 2000) and Taiwan (Wu et al. 1995) to very mild disease in the town of Archangelos in the island of Rhodes (Hadziyannis et al. 1991) and in Mongolia (Inoue et al. 2005). All African samples studied in our work came from the screening of HDV replication markers in patients with liver disease who were immigrating into France. Most patients suffered from active chronic hepatitis or cirrhosis and some of them underwent liver transplantation. Although the delta viruses corresponding to the African radiation in this study are associated with severe liver-speciﬁc HDV histological lesions, it should be emphasized that these virus lineages might not necessarily be as pathogenic in the general population. Obviously, other factors such as the time and duration of infection, the viral load (Le Gal et al. 2005), the appearance of defective HDV RNA molecules (Wu et al. 2005), the HBV helper strain and the genetic background of the patient (Dieye et al. 1999) may contribute to the pathogenicity.

6 Proposal for the Deltavirus Genus Classification Clearly, the classiﬁcation of HDV into only three ‘genotypes’ does not reﬂect the actual range of variability of the Deltavirus genus. Indeed, using the

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delta antigen gene and full genome sequence data, we identiﬁed a wide and probably ancient radiation of African lineages (as suggested by several clades branched inside the deepest part of the unrooted tree, see Figs. 3 and 4), making the genetic variability of HDV much wider than previously thought. Nevertheless, the South American sequences (HDV clade 3) remain the most divergent group (Table 1). Interestingly, this is also the case for HBV/F when compared to all other human HBV available all over the World. Furthermore, the strains Taiwan-TW-2b, Miyako, L215, and AF209859 and many other available sequences from the work of Watanabe et al. (2003) should be considered as a speciﬁc major clade, distinct from those including genotypes I, II and III. We suggest that the sequence TW-2b (previously labelled TWD62; Figs. 3 and 4) be considered the HDV-4 clade prototype due to the anteriority of the study by J.C. Wu et al. (Wu et al. 1998) (Table 2). Finally, the additional African lineages described here suggest that there are at least four other major clades. In our geographical area (near Paris, France), among 75 African non-HDV-I strains, characterized between 1999–2005, clades HDV-5, HDV6, HDV-7, and HDV-8 represent 74.7%, 9.3%, 13.3% and 2.7%, respectively. All our results would mean that the Deltavirus genus includes at least eight major clades (Table 2), which, interestingly, is very similar to the human HBV genetic variability which includes eight distinct genotypes (A–H; reviewed in Norder et al. 2004) To assume a correct labelling and classiﬁcation of HDV, we would suggest that after an analysis of the R0 region (or by RFLP or by nucleotide similarity approaches), the sequence of the S-HDAg coding gene (or the complete

Table 2 Proposed nomenclature for the eight HDV clades (1–8) in the Deltavirus genus Clade

Genotype

Prototype reference isolate

Accession number

Length (nt)

Reference

HDV-1 HDV-2 HDV-3 HDV-4 HDV-5 HDV-6 HDV-7 HDV-8

I IIa III IIb

Italy-A20 Japan-S Peru-1 TW-2b dFr-910 dFr-48 dFr-45 dFr-644

X04451 X60193 L22063 AF018077 AJ584848 AJ584847 AJ584844 AJ584849

1679 1683 1676 1676 1697 1687 1672 1680

Wang et al. 1986 Imazeki et al. 1991 Casey et al. 1993 Wu et al. 1998 Radjef et al. 2004 Radjef et al. 2004 Radjef et al. 2004 Radjef et al. 2004

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genome) would be obtained. Phylogenetic reconstruction from nucleotide alignment including the proposed FASTA ﬁle of sHD reference strains (available on request) could help to identify the afﬁliation of each HDV isolate. An international network of HDV could also help to identify the HDV–HBV co-epidemiological evolution. Among many questions that remain to be assessed, some can be listed: – Is there any clinical evidence of a severe prognosis occurring with a speciﬁc HBV-HDV association? – Could the introduction of an HDV clade to a speciﬁc HBV background with which it is not usually linked enhance liver disease? – Why are HBV/F and HDV-3, which are linked together in South America, the most divergent isolates? – Are the different HDV S-HDAg and L-HDAg proteins functionally clade speciﬁc for replication and assembly, respectively? Acknowledgements I am particularly grateful for the help given by all physicians to collect clinical data from patients. I thank many people from the lab and others who make this work possible. In particularly, I am grateful to Nadjia Radjef, Patricia Anaïs, Valeria Ivaniushina, Maïté Rico-Garcia, Emmanuel Gordien, Elyanne Gault, Sibel Oymac, Selym Badur and Frédéric Le Gal for allowing me to present their unpublished results. I thank all the technicians and students of the lab for the care in sample conservation and handling. I am especially grateful to Michel Milinkovitch from the Université Libre de Bruxelles for discussions and help in many phylogenetic aspects and concepts. I thank John Casey for giving unpublished genotype III sequences from Peru. I thank Camille Sureau, Patrick Mardulyn, John Casey, Emmanuel Gordien and Elyanne Gault for helpful comments. This work is part of the program of the ‘Laboratoire Associé au Centre National de Référence des Hépatites B et C pour le virus delta’ supported by the French Ministry of Health and the ‘Institut de Veille Sanitaire’.

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Casey JL, Niro GA, Engle RE, Vega A, Gomez H, McCarthy M, Watts DM, et al. (1996) Hepatitis B virus (HBV)/hepatitis D virus (HDV) coinfection in outbreaks of acute hepatitis in the Peruvian Amazon basin: the roles of HDV genotype III and HBV genotype F. J Infect Dis 174:920–926 Chao YC, Chang MF, Gust I, Lai MM (1990) Sequence conservation and divergence of hepatitis delta virus RNA. Virology 178:384–392 Chao YC, Tang HS, Hsu CT (1994) Evolution rate of hepatitis delta virus RNA isolated in Taiwan. J Med Virol 43:397–403 Dény P (1994) Le virus de l’hépatite D. Etude de l’ARN viral lors de l’infection humaine et expérimentale, analyse des variations génétiques. pp 1–191 Université Paris 7 Deny P, Zignego AL, Rascalou N, Ponzetto A, Tiollais P, Brechot C (1991) Nucleotide sequence analysis of three different hepatitis delta viruses isolated from a woodchuck and humans. J Gen Virol 72:735–739 Dieye A, Obami-Itou V, Barry MF, Raphenon G, Thiam A, Ndiaye R, Ndiaye M, et al. (1999) Association between Class I HLA alleles and HBs antigen carrier status among blood donors in Senegal. Dakar Med 44:166–170 Eigen M, Biebricher CK (1988) Sequence space and quasispecies distribution. In: Domingo E, Holland J, Ahlquist P (eds) RNA genetics, Variability of RNA genomes. Vol. III. CRC press, Boca Raton, pp 211–245 Elena SF, Dopazo J, Flores R, Diener TO, Moya A (1991) Phylogeny of viroids, viroidlike satellite RNAs, and the viroidlike domain of hepatitis delta virus RNA. Proc Natl Acad Sci USA 88:5631–5634 Flodgren E, Bengtsson S, Knutsson M, Strebkova EA, Kidd AH, Alexeyev OA, KiddLjunggren K (2000) Recent high incidence of fulminant hepatitis in samara, russia: molecular analysis of prevailing hepatitis B and D virus strains. J Clin Microbiol 38:3311–3316 Hadziyannis SJ, Papaioannou C, Alexopoulou A (1991) The role of the hepatitis delta virus in acute hepatitis and in chronic liver disease in Greece. Prog Clin Biol Res 364:51–62 Hansson BG, Moestrup T, Widell A, Nordenfelt E (1982) Infection with delta agent in Sweden: introduction of a new hepatitis agent. J Infect Dis 146:472–478 Ganne-Carrié N, Williams V, Thar T, Kaddouri H, Jean-Trinchet JC, Dziri-Mendil S, Alloui C, Al Hawjri N, Dény P, Beaugrand M, Gordien E (2006) Signiﬁcance of Hepatitis B Virus Genotypes A, B, C, D, E in a Cohort of Patients with Chronic Hepatitis B in the Seine Saint Denis District of Paris (France). J Med Virol (in press) Imazeki F, Omata M, Ohto M (1990) Heterogeneity and evolution rates of delta virus RNA sequences. J Virol 64:5594–5599 Imazeki F, Omata M, Ohto M (1991) Complete nucleotide sequence of hepatitis delta virus RNA in Japan. Nucl Acid Res 19:5439–5440 Ingman M, Kaessmann H, Paabo S, Gyllensten U (2000) Mitochondrial genome variation and the origin of modern humans. Nature 408:708–713 Inoue J, Takahashi M, Nishizawa T, Narantuya L, Sakuma M, Kagawa Y, Shimosegawa T, et al (2005) High prevalence of hepatitis delta virus infection detectable by enzyme immunoassay among apparently healthy individuals in Mongolia. J Med Virol 76:333–340

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Moriyama M, Taira M, Matsumura H, Aoki H, Mikuni M, Kaneko M, Shioda A, et al (2003) Genotype analysis, using PCR with type-speciﬁc primers, of hepatitis B virus isolates from patients coinfected with hepatitis delta virus genotype II from Miyako Island, Japan. Intervirology 46:114–120 Nakano T, Shapiro CN, Hadler SC, Casey JL, Mizokami M, Orito E, Robertson BH (2001) Characterization of hepatitis D virus genotype III among Yucpa Indians in Venezuela. J Gen Virol 82:2183–2189 Norder H, Courouce AM, Coursaget P, Echevarria JM, Lee SD, Mushahwar IK, Robertson BH, et al. (2004) Genetic diversity of hepatitis B virus strains derived worldwide: genotypes, subgenotypes, and HBsAg subtypes. Intervirology 47:289–309 Quintero A, Uzcategui N, Loureiro CL, Villegas L, Illarramendi X, Guevara ME, Ludert JE, et al. (2001) Hepatitis delta virus genotypes I and III circulate associated with hepatitis B virus genotype F In Venezuela. J Med Virol 64:356–359 Radjef N, Gordien E, Ivaniushina V, Gault E, Anais P, Drugan T, Trinchet JC, et al. (2004) Molecular phylogenetic analyses indicate a wide and ancient radiation of African hepatitis delta virus, suggesting a deltavirus genus of at least seven major clades. J Virol 78:2537–2544 Rizzetto M, Canese M, Arico S (1977) Immunoﬂuorescence detection of a new antigen antibody system (Delta/ anti-Delta) associated to hepatitis B virus in liver and serum of HBs Ag carriers. Gut 18:997–1003 Sakugawa H, Nakasone H, Nakayoshi T, Kawakami Y, Miyazato S, Kinjo F, Saito A, et al. (1999) Hepatitis delta virus genotype IIb predominates in an endemic area, Okinawa, Japan. J Med Virol 58:366–372 Shakil AO, Hadziyannis S, Hoofnagle JH, Di Bisceglie AM, Gerin JL, Casey JL (1997) Geographic distribution and genetic variability of hepatitis delta virus genotype I. Virology 234:160–167 Saudy N, Sugauchi F, Tanaka Y, Suzuki S, Aal AA, Zaid MA, Agha S, et al. (2003) Genotypes and phylogenetic characterization of hepatitis B and delta viruses in Egypt. J Med Virol 70:529–536 Simmonds P (2001) The origin and evolution of hepatitis viruses in humans. J Gen Virol 82:693–712 Sureau C, Guerra B, Lanford RE (1993) Role of the large hepatitis B virus envelope protein in infectivity of the hepatitis delta virion. J Virol 67:366–372 Swofford D (1998) PAUP*: Phylogenetic Analysis Using Parsimony (and other methods), version 4.0b10 (in progress). Sinauer Associates, Sunderland, MA Wang K-S, Choo Q-L, Weiner AJ, Ou J-H, Najarian RC, Thayer RM, Mullenbach GT, et al (1986) Structure, sequence and expression of the hepatitis delta (∂) viral genome. Nature 323:508–514 Wang TC and Chao M (2003) Molecular cloning and expression of the hepatitis delta virus genotype IIb genome. Biochem Biophys Res Commun 303:357–363 Wang TC and Chao M (2005) RNA recombination of hepatitis delta virus in natural mixed-genotype infection and transfected cultured cells. J Virol 79:2221–2229 Watanabe H, Nagayama K, Enomoto N, Chinzei R, Yamashiro T, Izumi N, Yatsuhashi H, et al (2003) Chronic hepatitis delta virus infection with genotype IIb variant is correlated with progressive liver disease. J Gen Virol 84:3275–3289

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Wu JC, Chiang TY, Sheen IJ (1998) Characterization and phylogenetic analysis of a novel hepatitis D virus strain discovered by restriction fragment length polymorphism analysis. J Gen Virol79:1105–1113 Wu JC, Choo KB, Chen CM, Chen TZ, Huo TI, Lee SD (1995) Genotyping of hepatitis D virus by restriction-fragment length polymorphism and relation to outcome of hepatitis D. Lancet 346:939–941 Wu JC, Hsu SC, Wang SY, Huang YH, Sheen IJ, Shih HH, Syu WJ (2005) “Defective” mutations of hepatitis D viruses in chronic hepatitis D patients. World J Gastroenterol 11:1658–1662 Wu JC, Huang IA, Huang YH, Chen JY, Sheen IJ (1999) Mixed genotypes infection with hepatitis D virus. J Med Virol 57:64–67 Zhang YY, Hansson BG (1996) Introduction of a new hepatitis agent in retrospect: genetic studies of Swedish hepatitis D virus strains. J Clin Microbiol 34:2713– 2717 Zhang YY, Tsega E, Hansson BG (1996) Phylogenetic analysis of Hepatitis D Viruses indicating a new genotype I subgroup among africain isolates. J Clin Microbiol 1996:2713–2717

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Functional and Clinical Significance of Hepatitis D Virus Genotype II Infection J.-C. Wu (u) Department of Medical Research and Education, Institute of Clinical Medicine, Taipei Veterans General Hospital, National Yang-Ming University, 201 Shih-Pai Road, Sec. 2, 112 Taipei, Taiwan [email protected]

1

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174

2 2.1

Functional Signiﬁcance of Genotype II HDV . . . . . . . . . . . . . . . . . . . Comparisons of Genomic Sequence Between Genotype II and Other Genotypes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Comparisons of Amino Acid Sequences of HDAg Between Genotype II and Other Genotypes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Interactions of HDAgs of Genotypes I and II . . . . . . . . . . . . . . . . . . . Transactivation of HDV RNA Replication by the S-HDAgs of Genotypes I and II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Differences in Viral Editing, Packaging and Replication Efﬁciencies Between Genotypes I and II HDV . . . . . . . . . . . . . . . . . . . . . . . . . . .

2.2 2.3 2.4 2.5

3 3.1 3.2

. . 176 . . 176 . . 177 . . 177 . . 178 . . 179

Clinical Signiﬁcance of Genotype II HDV . . . . . . . . . . . . . . . . . . . . . . . 180 Comparison Between Genotypes I and II HDV . . . . . . . . . . . . . . . . . . . 180 Inﬂuence of Replication and Genotypes of HBV . . . . . . . . . . . . . . . . . . . 182

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182

Abstract Hepatitis D virus (HDV) infection is one of the important etiologies of fulminant hepatitis and may aggravate the clinical course of chronic HBV infection to cirrhosis and liver failure. HDV was classiﬁed into three genotypes. Recent molecular phylogenetic analysis of HDV suggests at least seven major clades. The genotype I HDV is widely spread, genotype II is found in East Asia and genotype III HDV is prevalent in South America. The genomic size is 1682–1685 nucleotides (nt) for genotype II, and 1676 nt for genotype IV (IIb). The divergence in HDV nucleic acid sequences between genotype II and other genotypes varies from 13.8% to 35.3%. The divergences in the HDAg-coding region may range from 17.8% to 29.8% between genotype II and other genotypes. There is no genotypic or size restriction on the interactions of either the small or the large hepatitis delta antigens (HDAgs) between genotypes I and II, and there is also no genotypic incompatibility during co-package of HDAgs

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of different genotypes into virus like particles. There appears no apparent universal genotypic restriction of the transactivation of genotype I HDV RNA replication by small HDAg of genotype II. In contrast, there appears more genotypic restriction for genotype I small HDAgs to transactivate genotype II HDV RNA replication. Of the functional domains of HDAg, the 19 amino acids at the carboxyl-end of the large HDAg show the greatest divergences (70%–80%) between genotypes I and II. The viral packaging efﬁciencies of genotype I HDV isolates are usually higher than those of genotype II. The 19 amino acids at the carboxyl-end seem to be the most important determinant for viral packaging efﬁciencies. The editing efﬁciencies of the genotype I HDV are also higher than those of the genotype II. Genotype II HDV infection is relatively less frequently associated with fulminant hepatitis at the acute stage and less unfavorable outcomes [cirrhosis or hepatocellular carcinoma (HCC)] at the chronic stage as compared to genotype I. It appears that the clinical manifestations and outcomes of patients with genotype IV (IIb) HDV infection are more like those of patients with genotype II HDV infection. Persistent replication of HBV or HDV was associated with higher adverse outcomes (cirrhosis, HCC or mortality) compared to those who cleared both viruses from the sera. HBV of the genotype C is also a signiﬁcant factor associated with adverse outcomes (cirrhosis, HCC or mortality) in patients with chronic hepatitis D in addition to genotype I HDV and age. However, most patients with chronic HDV infection have low or undetectable hepatitis B virus DNA levels. During longitudinal follow-up, genotype I HDV is the most important determinant associated with survival.

1 Introduction The hepatitis D virus (HDV) is composed of an envelope of hepatitis B surface antigen (HBsAg), a genome of 1.7 kb and the only encoded protein, hepatitis delta antigen (HDAg) [1–4]. HDV is of negative polarity and the HDAg is encoded by the antigenomic strand of the virus. There are two molecular weight forms of HDAg. The large HDAg (L-HDAg) that has an additional 19-amino acid (aa) extension at the C terminus after editing of the antigenomic HDV RNA plays a key role in the assembly of HDV virions [2–4]. However; it inhibits HDV replication in a transdominant negative manner. The small HDAg (S-HDAg) transactivates the replication of HDV RNA [2, 3]. The HDV is a defective virus. It can replicate by itself, through a double-rolling circle mechanism, but it needs the supply of HBsAg from its helper hepatitis B virus (HBV) to complete the assembly of HDV particles and the subsequent secretion and transmission [5, 6]. Although HDV infection is not a common cause of infection except in high risk groups and certain areas, it is one of the important etiologies of fulminant

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hepatitis and may aggravate the clinical course of chronic HBV infection to cirrhosis and liver failure [7–12]. However, a subclinical course or relatively slow progression have been reported in some HDV infections [13–15]. The causes of varied clinical manifestations and outcomes of HDV infection are not yet completely clear. Host and viral factors may both play roles in the wide spectrum of HDV infection. Of viral factors, persistent replication of HDV and/or HBV is related to elevated alanine transaminase levels and progression of liver disease [14, 16]. HDV genotypes may also inﬂuence viral behaviors and subsequent disease courses [17–19]. Based on sequence comparison and phylogenetic analysis, HDV is classiﬁed into three genotypes by Casey [17]. HDV genotype I is the most widely spread and is found in patients with chronic active hepatitis or fulminant hepatitis in Europe, North America, Asia and Africa [17, 18, 20–24]. Genotype II HDV is found in East Asia including Japan, Taiwan, and Yukutia [18, 25–26]; this genotype is less often associated with fulminant hepatitis or rapid progression to cirrhosis or hepatocellular carcinoma (HCC) as compared to genotype I [18]. Genotype III HDV is prevalent in South America (Peru, Colombia and Venezuela), and is often found in patients with severe acute hepatitis [17, 27]. A novel HDV isolate discovered by Wu et al. was originally named genotype IIB because of its relatively close association with genotype II sequences [28]. This novel subtype may be the result of recombination between genotype I and II [28, 29]. Genotype IIB was later found to be a dominant genotype in Okinawa [30–32]. Recently, a molecular phylogenetic analysis of HDV sequences that included novel divergent isolates from Africa suggested at least seven major clades [32], and the original genotype IIb was re-classiﬁed as a new clade, HDV-4 [33]. The novel divergent isolates were further classiﬁed into additional new clades, HDV-5, -6, and 7, and 8 [33] (see also the chapter by P. Dény, this volume). Taiwan is an area of endemic HBV infection. The prevalence of HBV carriers is around 15% in adults. Both genotype I and II HDV have been discovered in this area. In addition, genotype IIb (HDV-4) was also discovered in Taiwan. In this setting, the clinical manifestations and virological characteristics of both HDV genotypes could be compared in patients with similar ethnic backgrounds. In the following sections we will report on the epidemiology and transmission of genotype II HDV, and the functional and clinical significance of genotype II HDV infection will be compared to that of genotype I. Although a recent report reclassiﬁed genotype IIb as belonging to a separate phylogenetic clade [33], it is also included in this review because of its initial association with genotype II and because of its prevalence in parts of east Asia.

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2 Functional Significance of Genotype II HDV 2.1 Comparisons of Genomic Sequence Between Genotype II and Other Genotypes Two isolates from each genotype are obtained from GenBank to show the identities in nucleic acid sequences within and between genotypes. The genomic size is 1682–1685 nucleotides (nt) for genotype II, and 1676 nt for genotype IIb. As shown in Table 1, the divergence in HDV nucleic acid sequences within genotype II isolates may be up to 14.3%. The divergence between genotype II and other genotypes varies from 13.8% to 35.3%. Consistent with previous reports [21], the autocleavage region (see the chapter by M.D. Been, this volume) appears to be the most conserved in the HDV genome, The sequence divergence in this region within the same genotype is usually less than 9%, although divergence as high as 15.4% is seen for HDV-7. Between genotype II and other genotypes the divergence in this region varies from 6.6% to 24.3% (Table 1). This region is responsible for autocleavage during HDV replication; the variation in sequences in this region may suggest possible functional differences in this region among genotypes. The divergence in the HDAgcoding region within each genotype ranges from 7% within genotype V to Table 1 Comparison of nucleic acid identities in various regions of HDV among HDV genotypes/clades Identities (%) in HDV nucleic sequences between genotype II and other genotypes/clades Regions

I

II

III

Complete HDV Autocleavage HDAg-coding Hypervariable

74.9–75.6 90–92 77–79.2 66.3–67.1

_85.7 _95.3 _89 _79.3

64.7–65.9 75.7–78.7 70.2–70.7 55.6–57.3

IIb (HDV-4) 75.5–79 85.9–89.7 80–82.8 66.4–72

HDV-5

HDV-6

HDV-7

77.1–86.2 90–93.3 82.2–83.6 65–69.8

75.4–76 89.4–90.1 79.4–82.6 66.4–67.5

71.2–75.1 80.9–84.6 77.8–81.4 60–66.8

Autocleavage region: nt 659–959; HDAg-coding region: nt 960–1601; Hypervariable region: nt 1602-end/1–658 Isolates (accession number, number of nucleotides) used for comparison: genotype I, Italy (AJ307077, 1679 nt), TW2667–66 (AF425644, 1674 nt); genotype II, TW2476–38 (AF425645, 1682 nt), Yakutia (AJ309879, 1685 nt); genotype III, Peru-1 (L22063, 1677 nt), VNZD8375 (AB037947, 1672 nt); genotype IV (IIb), TWD62, (AF01877, 1676 nt). Miyako (AF309420, 1676 nt); genotype V, dFr 47 (AJ584845, 1697 nt), dFr910 (AJ584848, 1697 nt); genotype VI, dFr48 (AJ584847, 1687 nt); genotype VII, dFr45 (AJ583868, 1672 nt), dFr644 (AJ583882, 1680 nt)

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11% within genotype II. The divergence in this region may be up to 17.5% within HDV-7. The divergence in this region may be further increased from 17.8% to 29.8% between genotype II and other genotypes. The variations in HDAg coding sequences are expected to result in heterogeneity in amino acid sequences of HDAg. The variations in various functional domains of HDAg may subsequently affect replication or packaging of HDV [3, 4, 19]. Sequence divergence in the hypervariable noncoding region is the largest, being 20.7% within genotype II, and up to 28% within HDV-7. The divergence in the hypervariable region between genotype II and other genotypes ranges from 33.6% to 44.4% (Table 1). 2.2 Comparisons of Amino Acid Sequences of HDAg Between Genotype II and Other Genotypes The HDAg has the following functional domains: the cryptic RNA-binding domain (CRBD; aa 2–27); the coiled-coil structure (CCS; aa 31–52) essential for the oligomerization between the small and the large HDAg; the nuclear localization signal (aa 68–88) essential for the nuclear translocalization of HDAg; the RNA-binding domain (RBD; aa 97-146) essential for HDV RNA binding and replication; the packing signal of the large HDAg (PAS; aa 196–214) for the packaging of HDV virions [2–4]. Variations in amino acid sequences in these domains may result in differences in HDV replication or packaging. The predicted amino acid sequence of genotype II HDAg is compared with those other genotypes and shown in Table 2. Of the various domains of HDAg, the RNA-binding domain appears the most conserved within and between genotypes. The packaging signal at the carboxyl end of L-HDAg is genotype speciﬁc; it is usually highly conserved within the same genotype, but highly divergent between different genotypes. The divergence in PAS within genotype II is up to 15%, and 25%–80% between genotype II and other genotypes (Table 2). Interestingly, the amino acid sequences of some domains of a HDV genotype may be closer to a different genotype as compared to those of the same genotype, suggesting possible genetic recombination during evolution. 2.3 Interactions of HDAgs of Genotypes I and II The coil–coil sequence is a segment of amino acids responsible for the oligomerization of S-HDAg and L-HDAg. The oligomerization of S-HDAgs is essential for the transactivation of HDV RNA replication, while the oligomerization between S-HDAg and L-HDAgs may result in the suppression of the

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Table2 Comparison of amino acid sequences in various domains of the HDAgs among HDV genotypes/clades Identities(%) in HDAg sequences between genotype II and other genotypes

HDAg CRBD CCS NLS RBD HLH RBD PAS

I

II

III

72.1–75.4 46.2–65.4 72.7 85.7–95.2 90.9 75–92.9 72.7–100 20–30

_86.9 _80.8 _90.9 _81 _100 _85.7 _81.8 _85

62.3–64.4 34.6–50 63.6–86.4 52.4–66.7 81.8 75–82.1 63.6–72.7 33.3–40.9

IIb (HDV-4) 74.8–80.4 53.8–65.4 63.6–81.8 81–95.2 81.8 85.7–92.9 81.8–100 75–90

HDV-5

HDV-6

HDV-7

76.6–81.3 65.4–73.1 72.7–81.8 76.2–95.2 81.8–90.9 78.6–85.7 81.8–100 75

73.8–77.1 50–53.8 68.2 91.9–81 81.8 85.7–89.3 72.7 65–70

70.2–74.8 38.5–57.7 72.7–77.3 81–90.5 72.7–81.8 75–82.1 63.6–81.8 70–71.4

Cryptic RNA-binding domain (CRBD): aa 2–27; coil–coil sequence (CCS): aa 31–52; Nuclear localization sequence (NLS): aa 68–88; RNA-binding domain (RBD) (aa 97–146); Packaging signal (PAS) (aa 195–214) Isolates (accession number) used for comparison: genotype I, Italy (AJ307077), TW2667–66 (AF425644); Genotype II, TW2476–38 (AF425645), Yakutia (AJ309879); genotype III, Peru-1 (L22063), VNZD8375 (AB037947); genotype IV (IIb), TWD62 (AF01877); Miyako (AF309420); genotype V, dFr 47 (AJ584845), dFr910 (AJ584848); genotype VI, dFr48 (AJ584847); genotype VII, dFr45 (AJ583868), dFr644 (AJ583882)

synthesis of genomic HDV RNA from the antigenomic template [34, 35]. S-HDAg needs the help of L-HDAg through oligomerization at the CCS for co-packaging into the HDV virion [36, 37]. The difference in the amino acid sequence at the CCS between different genotypes may be up to 45%–50%. However, there is no genotypic or size restriction on the interactions of either S-HDAg or L-HDAgs between genotypes I and II, and there is also no genotypic incompatibility during co-packaging of HDAgs of different genotypes into virus-like particles [38]. The ﬁndings that variations in HDAg amino acid sequences do not change the critical hydrophobic residues in the heptad repeats may explain the lack of genotypic restriction of oligomerization of HDAgs between different genotypes. 2.4 Transactivation of HDV RNA Replication by the S-HDAgs of Genotypes I and II Casey reported that there is genotype-speciﬁc complementation of HDV RNA replication by HDAg [39]. The S-HDAg of the genotype III is unable to transactivate viral RNA replication of genotype I HDV, and vice versa [39]. However,

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the transactivation activity of the S-HDAgs from different genotype I and II isolates on a HDAg synthesis-defective genotype I mutant varied from 6% to 172% as compared to that of the cognate S-HDAg, and there appears to be no apparent universal genotypic restriction on the transactivation of genotype I HDV RNA replication by S-HDAg of genotype II [38]. In general, genotype I S-HDAgs are more likely to be stronger supporters of HDV RNA replication of the same genotype [38]. With regard to HDAg synthesis-defective genotype II HDV, the transactivation activities of S-HDAgs from various isolates of the same genotype range from 22% to 250% as compared to that of the cognate S-HDAg. In contrast, there appears to be more genotypic restriction for genotype I S-HDAgs to transactivate genotype II HDV RNA replication. The greater divergence between genotypes I and III HDV sequences than between genotypes I and II may account for the stricter genotype-speciﬁc complementation between genotypes I and III. However, genotype per se could only partly predict the degree of the S-HDAgs of different isolates of the same or different genotypes in transactivating the replication of a second HDV isolate [38]. A minimum of 56 residues from the N-terminal portion of the S-HDAg, which covers the CCS responsible for oligomerization, determines the strength of transactivation of HDV RNA replication [38]. 2.5 Differences in Viral Editing, Packaging and Replication Efficiencies Between Genotypes I and II HDV There are several functional domains of HDAg that are closely associated with viral replication or packaging. Mutations or alteration of amino acids within these domains may inﬂuence functions associated with viral replication or packaging. Of these functional domains, the 19 amino acids at the carboxyl-end of the L-HDAg show the greatest divergences (70%–80%) between genotypes I and II (Table 2) [2–4, 17–19]. It is reasonable to consider that viral packaging efﬁciencies may vary greatly between these two genotypes. Hsu et al. reported that viral packaging efﬁciencies of genotype I HDV isolates are usually higher than those of the genotype II. The difference in packaging efﬁciencies may be 50-fold or greater. Viral packaging efﬁciencies vary not only between genotypes I and II, but also among HDV isolates of the same genotype [38]. Isoprenylation is essential for the L-HDAg to interact with HBsAg and the subsequent packaging of HDV virions [40]. Isoprenylation increases hydrophobicity of L-HDAg and facilitates the interaction between L-large HDAg and the envelope proteins composed of HBsAg and membrane lipids [41]. The 11 hydrophobic residues (including proline) of the 19 aa of the genotype I HDV may further increase the interactions compared

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to only nine hydrophobic residues in the same region of the genotype II. Segment swapping experiments of the L-HDAg indicate that the 19 aa at the carboxyl-end seem to be the most important determinant for viral packaging efﬁciencies [38]. However, amino acids outside the carboxyl-end 19 residues may also inﬂuence packaging efﬁciency. Synthesis of L-HDAg requires editing at adenosine 1012 (Amber/W site) of the antigenomic HDV RNA [42–44]. The editing efﬁciencies of the genotype I HDV are also higher than those of genotype II [38]. Genotype I HDV isolates usually have a unique base-pairing structure required for maximal editing [44] in which nucleotides 1008–1016 are paired with nucleotides 576– 584 in the predicted rod structure. The pairing is relatively stronger, because there are base pairs on each side of the editing site [26, 44]. Disruption of the 1009A/583U pairing markedly reduces the editing efﬁciency of 1012A. However, the nucleotide sequences surrounding the editing area are different in genotype II HDV isolates which result in no pairings of 1009A/583U and 1008U/584U and a lower RNA editing efﬁciency [26, 38]. Recently, Jayan and Casey reported that the conserved RNA secondary structure around the HDV genotype I amber/W site has been selected not for the highest editing efﬁciency but for optimal viral replication and secretion [45]. In a short report the replication of a genotype I HDV isolate from Italy is 100 fold-higher than that a genotype II HDV from Taiwan [46]. More isolates from each genotype are needed to conﬁrm the difference in viral replication efﬁciencies between genotypes I and II.

3 Clinical Significance of Genotype II HDV 3.1 Comparison Between Genotypes I and II HDV It has been reported that genotype II HDV infection is relatively less frequently associated with fulminant hepatitis at the acute stage and less unfavorable outcomes (cirrhosis or HCC) at the chronic stage as compared to genotype I of the same area [18]. This study was composed of symptomatic inpatients and asymptomatic outpatients for regular check up. In a longer follow-up for more than 15 years, about 45% of patients with chronic genotype I HDV infection survived, while more than 75% of patients with chronic genotype II HDV infection remained alive [63]. The difference is statistically signiﬁcant. The long-term prognosis of patients with chronic genotype II HDV infection seems better than that reported previously in western countries where only

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genotype I HDV is currently found [10, 11, 17]. All the patients in the study by Ivaniushina et al. had a history of chronic liver disease, and all except two presented with grave liver disease or cirrhosis [26]. It is not surprising that there was no difference in the severity of infection between genotype I and II in this cross-sectional study of a selected group of patients. Moreover, the genotype II isolates from Yakutia clustered into a clade different from those from Taiwan and Japan (see the chapter by P. Dény, this volume); it is possible that the genotype II variant in Yakutia may be associated with different virological characteristics and clinical manifestations. In addition, different genotypes of HBV may also inﬂuence outcomes of patients with chronic hepatitis D. The impact of viral replication, mutations and genotypes of HBV on clinical manifestations were not analyzed in that study [26]. The inﬂuence of HBV will be further discussed in the following section. Genotype IIb (HDV-4 by Dény’s classiﬁcation) infection is not as common in Taiwan as in Okinawa [18, 19, 30–32]. It appears that the clinical manifestations and outcomes of patients with genotype IIb infection are more like those of patients with genotype II HDV infection in Taiwan (J.C. Wu, unpublished results) with relatively less unfavorable outcomes compared to genotype I [18, 19, 30–32]. In the study by Watanabe et al. genotype IIb infection is associated with milder liver diseases as compared to the genotype IIb variant, type IIbM, in patients of the same ethnic background living in Miyako [32]. Genetic variations in nucleotide and amino acid sequences of HDV may account for the heterogeneity in disease manifestations (Table 3).

Table 3 Comparison of virological and clinical behaviors between genotype I and II HDV Genotype I

Genotype II

Distribution Genomic size Oligomerization of HDAg Transactivation by heterotypic small HDAg

Worldwide 1674–1679 nt No genotype-restriction Transactivated by both genotypes I and II

Replication Editing Assembly Unfavorable outcomes Acute infection Chronic infection

Comparable or higher Higher efﬁciency Higher efﬁciency More More fulminant hepatitis More cirrhosis and HCC

Japan, Taiwan, Yakutia 1682–1685 nt No genotype-restriction Not signiﬁcantly transactivated by genotype II Lower Lower efﬁciency Lower efﬁciency Less Fewer fulminant hepatitis Fewer cirrhosis and HCC

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3.2 Influence of Replication and Genotypes of HBV Smedile et al. reported that HBV replication modulates pathogenesis of HDV in chronic hepatitis D [16]. Wu et al. also reported that persistent replication of HBV or HDV are associated with elevated serum transaminase levels [14]. Based on an intergroup divergence of 8% or more in the complete nucleotide sequence, HBV can be classiﬁed into eight genotypes A–H [47]. Genotypes and core promoter mutations of HBV have been reported to be associated with time of HBeAg seroconversion, HBV DNA levels, treatment response to interferon and long-term outcomes [47–62]. Because chronic hepatitis D patients still have underlying chronic hepatitis B, replication status, genotypes and mutations of HBV may also inﬂuence clinical course and outcomes of chronic HDV infection. In a recent study in our laboratory, persistent replication of HBV or HDV was associated with higher adverse outcomes (cirrhosis, HCC or mortality) compared to those who cleared both viruses from sera [63]. HBV genotype C is also a signiﬁcant factor associated with adverse outcomes (cirrhosis, HCC or mortality) in patients with chronic hepatitis D, in addition to genotype I HDV and age. However, most of patients with chronic HDV infection have low or undetectable HBV DNA levels [12, 14]. During longitudinal follow-up, genotype I HDV is the most important determinant associated with survival.

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39. Casey JL, Gerin JL (1998) Genotype-speciﬁc compentation of hepatitis delta virus RNA replication by hepatitis delta antigen. J Virol 72:2806–2814 40. Glenn JS, Watson JA, Havel JM, White JM (1992) Identiﬁcation of a prenylation site in delta virus large antigen. Science 256:1331–1333 41. Moraleda G, Seeholzer S, Richko V, Dunbrack R, Otto J, Taylor J (1999) Unique properties of the large antigen of hepatitis delta virus. J Virol 73:7147–7152 42. Casey JL, Gerin JL (1995) Hepatitis D virus RNA editing: speciﬁc modiﬁcation of adenosine in the antigenomic RNA. J Virol 69:7593–7600 43. Polson AG, Bass BL, Casey JL (1996) RNA editing of hepatitis delta virus antigenomr by dsRNA-adenosine deaminase. Nature 380:454–456 44. Casey JL, Bergmann KF, Brown TL. Gerin JL (1992) Structural requirements for RNA editing in hepatitis delta virus: evidence for a uridine-to-cytidine editing mechanism. Proc Natal Acad Sci USA 89:7149–7153 45. Jayan GC, Casey JL (2005) Effects of conserved RNA secondary structures on hepatitis delta virus genotype I RNA editing, replication, and virus production. J Virol 79:1187–1193 46. Lin FM, Lee CM, Wang TC, Chao M (2003) Initiation of RNA replication of cloned Taiwan-3 isolate of hepatitis delta virus genotype II in cultured cells. Biochem Biophys Res Commun 306:966–972 47. Kao JH (2002) Hepatitis B viral genotypes: clinical relevance and molecular characteristics. J Gastroenterol Hepatology 17:643–650 48. Okamoto H, Tsuda F, Sakugawa H et al. (1998) Typing hepatitis B virus by homology in nucleotide sequence: Comparison of surface antigen subtypes. J Gen Virol 69:2575–2583 49. Magnius LO, Norder H (1995) Subtypes, genotypes and molecular epidemiology of the hepatitis B virus as reﬂected by sequence variability of the S-gene. Intervirology 38:24–34 50. Kao JH, Chen PJ, Lai MY, Chen DS (2000) Hepatitis B genotypes correlate with clinical outcomes in patients with chronic hepatitis B. Gastroenterology 118:554– 559 51. Fujie H, Moriya J, Shintani Y et al. (2001) Hepatitis B virus genotypes and hepatocellular carcinoma in Japan. Gastroenterology 120:1564–1565 52. Orito E, Mizokami M, Sakugawa H et al. (2001) A case-control study for clinical and molecular biological differences between hepatitis B viruses of genotypes B and C. Hepatology 33:218–223 53. Orito E, Ichida T, Sakugawa H et al. (2001) Geographic distribution of hepatitis B virus (HBV) genotype in patients with chronic HBV infection in Japan. Hepatology 34:590–594 54. Chan HLY, Tsang SWC, Liaw CT et al. (2002) Viral genotypes and hepatitis B virus DNA levels are correlated with histological liver damage in HBeAg-negative chronic hepatitis B virus infection. Am. J. Gastroenterol 97:406–412 55. Wai CT, Chu CJ, Hussain M, Lok ASF (2002) HBV genotype B is associated with better response to interferon therapy in HBeAg (+) chronic hepatitis than genotype C. Hepatology 36:1425–1430 56. Chu CJ, Hussain M, Lok ASF (2002) Hepatitis B virus genotype B is associated with earlier HBeAg seroconversion compared with hepatitis B virus genotype C. Gastroenterology 122:1756–1762

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57. Sumi H, Yokosuka O, Seki N et al. (2003) Inﬂuence of hepatitis B virus genotypes on the progression of chronic type B liver disease. Hepatology 37:19–26 58. Yuen MF, Sablon E, Yuan HJ et al. (2003) Signiﬁcance of hepatitis B genotype in acute exacerbation, HBeAg seroconversion, cirrhosis-related complications, and hepatocellular carcinoma. Hepatology 37:562–567 59. Tanaka Y, Hasegawa I, Kato T et al. (2004) A case-control study for differences among hepatitis B virus infections of genotypes A (subtypes Aa and Ae) and D. Hepatology 40:747–755 60. Lindh M, Hannmoun C, Dhillon AP, Norkrans G, Horal P (1999) Core promoter mutations and genotypes in relation to viral replication and liver damage in East Asian hepatitis B virus carriers. J Infect Dis 179:775–782 61. Hunt CM, McGill JM, Allen MI, et al. (2000) Clinical relevance of hepatitis B virus mutations. Hepatology 31:1037–1044 62. Kao JH, Chen PJ, Lai MY, Chen DS (2003) Basal core promoter mutations of hepatitis B virus increase the risk of hepatocellular carcinoma in hepatitis B carriers. Gastroenterology 124:327–334 63. Su CW, Huang YH, Huo TI, Shih HH, Shen IJ, Chen SW, Lee PC, Lee SD, Wu JC (2006) Genotypes and viremia of hepatitis B and D viruses are associated with outcomes of chronic hepatitis D patients. Gastroenterology 130 (in press)

CTMI (2006) 307:187–209 c Springer-Verlag Berlin Heidelberg 2006

Immunology of HDV Infection M. Fiedler · M. Roggendorf (u) Institute of Virology, University Clinic Essen, Hufelandstrasse 55, 45122 Essen, Germany [email protected]

1

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188

2

Natural History of the Clinical Course of HDV Infection . . . . . . . . . . . . 189

3 3.1 3.2 3.2.1 3.3 3.3.1 3.3.2 3.3.3

Immunopathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Is HDV a Cytopathic Virus? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-Cell Immune Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role of IgM Anti-HDV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T-Cell Immune Responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T-Helper Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cytotoxic T Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T-Cell Immune Responses After Immunization of Mice or Woodchucks .

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190 190 191 191 192 192 193 194

4 4.1 4.2 4.3 4.4 4.5 4.6

Vaccination Studies . . . . . . . . . . . . . . . . . . . . . . . Immunization with HDV Protein . . . . . . . . . . . . . . Immunization with Synthetic Peptides . . . . . . . . . . DNA Immunization . . . . . . . . . . . . . . . . . . . . . . . Immunization with Vaccinia Virus Expressing HDAg HDV Is a Poor Immunogenic Protein . . . . . . . . . . . Conclusions on Vaccination Studies . . . . . . . . . . . .

. . . . . . .

195 198 200 200 201 202 202

5

Immunogenic Domains of HDAg . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203

6

Closing Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205

Abstract Hepatitis delta virus (HDV) infection may occur as coinfection with hepatitis B virus (HBV) or as superinfection of a chronically HBV-infected patient. A strong antibody response is mounted, which persists for many years; however, it is not able to modulate the course of infection. In most cases the superinfection takes a chronic course. In patients with inactive disease (HDV PCR negative) an oligospeciﬁc T-helper cell immune response and a cytotoxic T-cell response were found, which were absent in patients with persistent viremia. The role of the cellular immune response in liver injury during acute infection has not been investigated. Vaccination strategies tested in the woodchuck model induced speciﬁc B- and T-cell responses but failed to protect from HDV infection.

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1 Introduction Hepatitis delta virus (HDV) has been recognized to be an important cause of acute or chronic hepatitis in humans. HDV infection may occur as coinfection with hepatitis B virus (HBV) or as superinfection of a chronically HBV-infected patient (Rizetto et al. 1984). The course of simultaneous infection is similar to HBV infection alone and clearance of HBV is accompanied by elimination of HDV. HDV superinfection of chronically HBV-infected patients results in chronic HDV infection in more than 80% of cases. This often progresses rapidly to liver cirrhosis and hepatocellular carcinoma (Fattovich et al. 1987). The immunopathogenesis of HDV infection has not been investigated in detail so far. Antibodies recognizing both HDV proteins are detected at low titers during acute infection and reach high levels during chronic infection, but are not able to modulate the course of infection. Therefore, antibodies are probably not able to neutralize the virus (Rizetto 1981, 1984). The predictive role of antibodies of the IgM-class for the course of HDV infection has been studied extensively and is somewhat controversial (Aragona et al. 1987; Borghesio et al. 1998; Farci et al. 1986; Govindarajan et al. 1989). Several groups investigated the ﬁne speciﬁcity of the antibody response and deﬁned immunogenic epitopes (Bergmann et al. 1989; Seizer et al. 2005; Wang et al. 1990). Knowledge of the cellular immune response in HDV infection is still incomplete. A polyspeciﬁc, but weak T helper (Th) cell response is observed in patients with acute self-limiting HDV infection, but is absent in chronically HDV-infected patients (Nisini et al. 1997). The liver damage results in serum alanine and aspartate aminotransferase (ALT and AST) elevation. This observation, as well as the fact that HDV itself is not cytopathic (Guilhot et al. 1994), may indicate that cytotoxic T cells (CTL) are responsible for destruction of hepatocytes; however, little is known about the CTL response in HDV infection. Recently, Huang et al. identiﬁed two HLA-A*0201-restricted CD8+ T-cell epitopes on HDV (Huang et al. 2004). Vaccination studies might also give insight into the role of the immune response in HDV infection. Woodchucks chronically infected with woodchuck hepatitis virus (WHV) can be superinfected with HDV and therefore can be a good model to test vaccine candidates. Different strategies have been investigated to establish a protective vaccine against HDV superinfection. Synthetic peptides, HDAg expressed in Escherichia coli, yeast, or baculovirus, infection with vaccinia virus expressing the small or the large hepatitis delta antigen (S-HDAg, L-HDAg), and DNA immunization by gene gun have been studied. So far, none of these protocols has been able to protect woodchucks

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from HDV superinfection, but in some studies a modulation of the course of infection was observed (see Sects. 4.1–4.6). The current knowledge about the B- and T-cell immune responses in HDV infection and an update of vaccine trials will be covered in this review.

2 Natural History of the Clinical Course of HDV Infection HDV infection in patients shows two courses of disease. HBV and HDV coinfection induces a disease similar to the classical acute HBV hepatitis with a cellular immune response to HBV resulting in a downregulation of HBV replication by cytokines and an elimination of infected cells by cytotoxic cells (Chisari and Ferrari 1995; Guidotti et al. 1996b). Neutralizing antibodies against HBsAg prevent hepatocytes from reinfection with HBV and HDV. The humoral immune response to HDV in this mode of infection is characterized by low titers of anti-HDV, which disappear soon after infection (Rizetto 1981, 1984). A speciﬁc immune response against HDV may not be required in the context of HBV clearance because HDV, as a helper-dependent virus, essentially needs HBV envelopes to form complete particles. HDV superinfection of chronically HBV-infected patients results in most cases in chronic infection. The course of HDV superinfection may be divided into the following phases: (1) an acute phase with active HDV replication and suppression of HBV with high ALT levels; (2) a chronic phase characterized by decreasing HDV replication and increasing HBV replication with moderate or ﬂuctuating ALT levels followed by development of cirrhosis and hepatocellular carcinoma; or (3) elimination of HDV, or HBV and HDV (Wu et al. 1995). Woodchucks chronically infected with WHV can also be superinfected with HDV. In our hands, all superinfected animals developed chronic HDV infection. The correlation between high levels of HDV viremia and a decrease of the HBV load is also observed in woodchucks. Figure 1 shows the typical course of HDV superinfection in a WHV carrier woodchuck. We have observed this kind of ﬂuctuation of HDV replication followed by a ﬂuctuation of HBV replication in a couple of animals. So far, the inverse correlation between HDV and HBV viremia is not understood. It has been previously shown that the intrahepatic induction of gamma interferon (IFNγ), tumor necrosis factor alpha (TNFα), and IFNα/β downregulates HBV replication noncytopathically in the livers of HBV transgenic mice. This antiviral effect can be achieved by injecting these mice with an unrelated hepatotropic virus, such as lymphocytic choriomeningitis virus (LCMV) or adenovirus (Cavanaugh et al. 1998; Guidotti et al. 1996a). The downregulation of the HBV replication in the set-

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Fig. 1 Natural history of the course of HDV superinfection in a WHV carrier woodchuck (kinetics of HDV RNA and WHV DNA measured by spot blot hybridization)

ting of HDV superinfection could also be mediated by HDV speciﬁc T-cells secreting these cytokines. On the other hand, a direct interference between HDV and HBV replication has not been ruled out up to now.

3 Immunopathogenesis In most viral infections B- and T-cell immune responses contribute in a speciﬁc manner to antiviral defense. A successfully mounted immune response leads to elimination of the virus or persistence at low levels. For HDV infection the contribution of speciﬁc B- and T-cells for immunopathogenesis (e.g. liver damage) or elimination of HDV has not been clariﬁed. Approximately 80% of HBV carriers superinfected with HDV develop chronic infection. It is not known whether elimination of HDV is correlated to a speciﬁc T-cell response. As we will discuss below speciﬁc T-cell responses to HDV have been described in patients who recovered from HDV superinfection. However, the timing of the occurrence of this response in acute infection is not known. The speciﬁc CTL response identiﬁed so far is obviously not effective in eliminating the virus from the liver in the majority of cases. 3.1 Is HDV a Cytopathic Virus? The existence of a cytotoxic immune response to HDV has been assumed, as HDV itself is probably not directly cytopathic. Mice made transgenic for

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both small and large HDAg and expressing these proteins in the liver do not develop any form of liver disease. Hence, the hepatitis in humans may be induced rather by the immune response than by HDV itself (Guilhot et al. 1994). The issue of the cytotoxicity of HDV itself has been investigated in different in vitro systems and yielded conﬂicting results. More recently, Wang et al. studied this issue extensively (Wang et al. 2001). When cells transfected with replication-competent HDV-cDNA were followed, a progressive decline in viral RNA replication and a steady decrease in the cells expressing HDAg were found. However, in transient transfection assays, no evidence was found to link HDV replication to apoptosis or cell cycle arrest. Thus, HDV does not appear to be acutely cytotoxic. In dividing cells, however, HDV replication was associated with a slight growth disadvantage. The authors discuss that this may not cause hepatitis in vivo but might contribute to impaired liver regeneration in the setting of ongoing hepatocellular injury. In short, liver injury caused by HDV infection may be mediated both by the immune system and the virus itself. 3.2 B-Cell Immune Response Antibodies recognizing both HDV proteins are detected at low titers during acute infection and reach high levels during chronic infection (Rizetto 1981, 1984). Analogous to the immune response to HBV core antigen, antibodies to HDV may not able to modulate the course of infection and, therefore, may not have neutralizing activities. 3.2.1 Role of IgM Anti-HDV The serologic proﬁle of acute HDV infection consists of a prodromic phase of viremia followed by an early IgM and a delayed IgG anti-HDV response (Aragona et al. 1987). Whereas IgG anti-HDV may persist for a lifetime, regardless of the clinical outcome, IgM anti-HDV disappears rapidly in some patients and persists in others. A correlation between chronicity of HDV infection and the persistence of high titer IgM anti-HDV was found in early studies (Aragona et al. 1987; Farci et al. 1986). After the introduction of sensitive techniques to detect HDV RNA some authors could not conﬁrm this relationship between chronic infection and IgM anti-HDV detection (Govindarajan et al. 1989). A more recently conducted long term study in patients with HBV and HDV infection under interferon therapy revealed again a complete concurrence

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between the activity of liver disease and the IgM course (Borghesio et al. 1998). The discrepancies in the different studies may be explained by the use of techniques of different sensitivity and space of time that was investigated. 3.3 T-Cell Immune Responses Little data exist about the HDV-speciﬁc cellular immune response. Two studies in humans identiﬁed CD4+ and CD8+ T cells that, interestingly, were only identiﬁed in patients with inactive HDV disease (HDV RNA negative). The number of investigated patients, however, was very low and no study has explored the kinetics of the HDV-speciﬁc immune response up to now. Negro et al. demonstrated that rechallenge with HDV of chimpanzees, which had apparently recovered from a ﬁrst HDV infection, resulted in the reappearance of HDV replication, sometimes associated with hepatitis (Negro et al. 1989). However, only low levels of viremia were detected and ALT elevations, when present, were mild. This ﬁnding suggests that at least a partial immunity against HDV can be raised. 3.3.1 T-Helper Cells A Th-cell immune response has been demonstrated in HDV-infected patients who eliminated HDV after acute hepatitis due to superinfection. Immunization studies (protein and DNA, see Sects. 4.1 and 4.3) in mice and woodchucks also revealed a multispeciﬁc Th response. Nisini et al. analyzed the proliferative response to recombinant HDAg and synthetic peptides in HDV-infected patients (Nisini et al. 1997). Eight of 30 patients speciﬁcally responded to HDAg. Interestingly, all responders had an inactive HDV infection (HDV PCR negative), while none of the patients with active disease showed any signiﬁcant proliferation. The use of the synthetic peptides revealed that the T-cell recognition was directed against different epitopes, with patient-to-patient variation. The response to peptides was generally oligospeciﬁc: all together 15 different peptides induced proliferation. T-cell clones generated from the cells of three patients produced large amounts of IFN-γ belonging to either Th1 or Th0 subsets, and some of them exerted cytotoxic activity in an antigen speciﬁc manner. The authors speculate that these T cells could play a pivotal role either in the defense against HDV infection or for the immunopathogenesis of liver disease. A further characterization of the clones using the overlapping synthetic peptides allowed the identiﬁcation of four Th epitopes [amino acids (aa) 26–41, 50–65, 66–81, and 106–121;

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Fig. 2 Localization of CTL, Th, and B-cell epitopes on HDAg (aa 1–214). The corresponding studies are indicated

see Fig. 2]. HDAg and these four peptides were recognized in the context of different class II gene products (either DR, DP, or DQ alleles). In particular, peptide amino acids 50–65 and 106–121 were presented in association with two or more different HLA-DR molecules (promiscuous peptides). The authors discuss the possibility that these regions could be relevant for further vaccine development. One of the identiﬁed HDV epitopes (aa 106–121) may be generated by extracellular processing for presentation to speciﬁc CD4+ T cells, a rare form of processing (Accapezzato et al. 1998). The authors discuss that this 106–121 peptide could enhance the presentation to speciﬁc CD4+ T cells for mounting of a protective immune response against HDV after extracellular processing. Alternatively, this peptide may play a crucial role in the pathogenesis of HDV-mediated liver disease: the 106–121 peptide could bind to MHC class II molecules that are expressed on hepatocytes activated by inﬂammatory cytokines and cause extensive lysis of bystander class II hepatocytes by liver-inﬁltrating HDV-speciﬁc cytotoxic CD4+ T cells. Either of these proposed mechanisms could be relevant in a large cohort of patients, because the 106–121 peptide is recognized by T cells in the context of multiple HLA-DR molecules (Nisini et al. 1997). 3.3.2 Cytotoxic T Cells Little is known about the CTL response during acute or chronic HDV infection and its contribution to elimination of HDV in a subset of patients. Staining of stimulated peripheral blood mononuclear cells (PBMC) of patients with

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tetramer complexes loaded with peptides aa 26–34 and 43–51 showed signiﬁcant binding (Fig. 2) (Huang et al. 2004). Interestingly, these patients presented with inactive disease (HDV RNA negative), whereas the patients with PBMCs without binding to these peptides had active disease (HDV RNA positive). For one patient the frequencies of peptide-speciﬁc CD8+ T cells increased significantly after peptide stimulation. ELISPOT assays also conﬁrmed that these cells were functional to HDV. These two HLA-A*0201-restricted CD8+ T-cell epitopes (genotype 1) were identiﬁed in HLA-A*0201 transgenic mice that were immunized with plasmid DNA coding for the large HDAg. Splenocytes were screened directly ex vivo with tetramers presenting potential HLAA*0201-restricted HDV peptides. Two of six tetramers loaded with epitopes aa 26–34 and 43–51, respectively, showed signiﬁcantly enhanced responses in HLA-A*0201 transgenic mice after immunization. After stimulation in vitro, both CTL lines of mice were able to trigger speciﬁc CTL responses to peptides 26–34 or 43–51. 3.3.3 T-Cell Immune Responses After Immunization of Mice or Woodchucks Immunization studies in mice and woodchucks support the hypothesis that Th-cell and CTL responses can be generated, that may contribute to a protective immunity to HDV. Different DNA immunization protocols using either plasmids expressing the small or the large HDAg induced signiﬁcant CD4+ Th-cell proliferative responses in mice of different strains (Mauch et al. 2002). Huang et al. further characterized the cytokine proﬁle after immunization in mice and found a prominent increase in Th1 cytokine production, especially in IFN-γ and interleukin (IL)-2, but not in IL-4 (Huang et al. 2000). A cytolytic activity of an HDV-speciﬁc CTL response was demonstrated by Mauch et al. in mice (Mauch et al. 2002). After immunization with DNA coding for the small or large HDAg speciﬁc CD4+ Th and CD8+ CTL responses were detected in different mouse strains. Both CD4+ and CD8+ T cells were required for the antitumor activity in a syngenic tumor model as determined by in vivo T-cell depletion experiments. In woodchucks a proliferative Th-cell immune response was detected after immunization and after challenge, and was further characterized using a panel of overlapping synthetic peptides spanning the whole HDAg (D’Ugo et al. 2004; Fiedler et al. 2001). In both studies the small HDAg expressed in yeast was used. In our study HDAg/CpG immunization induced a Th-cell response in WHV carrier woodchucks equivalent to that induced in WHV negative ones by immunization with protein and complete Freund’s adjuvant (CFA). Both protocols are known to induce a strong Th1 cell immune response

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(Chu et al. 1997; Forsthuber et al. 1996; Schirmbeck et al. 1999). A polyspeciﬁc, but weak lymphoproliferative response was seen. Overall, 10 peptides were able to induce a Th-cell immune response (Fig. 2). The recognized epitopes grouped into two clusters, one near the N terminus and one closer to the C terminus of HDAg. We did not investigate the proliferative response after challenge. D’Ugo et al. detected a Th-cell immune response against HDAg in two of four woodchucks immunized with HDAg/MF59 and in three of four animals immunized with HDAg/CFA. Before challenge, no signiﬁcant response to any of the peptides was detected. After challenge, all animals of the HDAg/MF59 group showed a speciﬁc response to at least two peptides, one animal of the HDAg/CFA group, and one the control group (three animals) showed also evident responses. As described in detail later (see Sect. 4.1), the detected immune response in either study, however, was not able to protect from HDV superinfection. The differences in the detection of a Th-cell immune response after immunization could be caused by different immunization protocols and/or the outbred status of the woodchucks.

4 Vaccination Studies HDV superinfection of HBV carrier patients is a severe disease; therefore, a vaccine would be important to prevent patients from HDV superinfection. The woodchuck is a good model to test vaccine candidates. The immune response after vaccination can be assessed as far as possible in these outbred animals and the induction of protection can be investigated. Different strategies have been followed to induce a protective immune response against HDV superinfection. Synthetic peptides, incomplete or complete HDAg expressed in E. coli, yeast, or baculovirus, vaccinia virus expressing the small or the large HDAg, and DNA immunization by gene gun have been studied. Table 1 gives an overview of the vaccination studies. Considering the fact that HDAg is a nucleoprotein, one might hypothesize that HDV protein may induce a strong cellular rather than a humoral immune response and, therefore, could be used as a vaccine. It has been shown for several viral infections, including inﬂuenza A virus, rabies virus, HBV, and WHV (Dietzschold et al. 1987; Fu et al. 1999; Murray et al. 1984; Schödel et al. 1993), that immunization with viral nucleoproteins can induce a T-cell response, which is able to suppress viral infections. Our group has shown previously that the mounting of a Th-cell immune response to the nucleoprotein of WHV (WHcAg) after immunization is sufﬁcient for protection from chal-

196

Table 1 Overview of vaccination studies Immunization approach

Number Immune response Challenge of wood- after immunization dose chucks Anti-HDV Th-cell response

peptides

3

3/3

nd

4 HDAg (AS 13–76, E. coli)/CFA 1×0,6–1 ml+2×5-10µg HDAg (baculovirus) 2 3×10 µg 6 HDAg (yeast) 8×40 or 8×100 µg

4/4

Monitoring of viremia

Author

1×105 WID50 RT–PCR, NB

Viremia shorter and lowera

nd

109 genome equivalents

Typical chronic course

Bergmann et al. Conference proceedings 1993: Hepatitis Delta Virus Karayiannis et al. Hepatology 1990

0/2

nd

nd

nd

2×108 genome Nested RT–PCR, Viremia delayed, Karayiannis et al. equivalents J Med Virol 1993 chronic course dot blot RT–PCR 2/6 no viremiab Ponzetto et al. Conference 106 WID50 proceedings 1993: Hepatitis Delta Virus Fiedler et al. Vaccine 2001 nd nd nd

HDAg (yeast)/CFA 4×100 µg HDAg (yeast)/CFA 3×100 µg

4

4/4

2/4

4

4/4

3/4

106 genome equivalents

HDAg (yeast) /CpG 1×100+3×50 µg

4

4/4

3/4

106 genome equivalents

HDAg

Viremia delayed and lower, chronic course Nested RT–PCR, Typical chronic spot blot course RT–PCR, spot blot

D’Ugo et al. Vaccine 2004

Fiedler et al. Vaccine 2001

M. Fiedler · M. Roggendorf

Course after challenge

Immunization approach

Number Immune response Challenge of wood- after immunization dose chucks Anti-HDV Th-cell response

HDAg (yeast)/MF59 3×100 µg

4

4/4

2/4

Vaccinia virus (p24) 4×106 PFU Vaccinia virus (p24) 4×107 PFU

2

0/2

nd

2

0/2

nd

Vaccinia virus (p27) 4×107 PFU

2

0/2

nd

106 WID50

pcDNA3 (p24) 3×20 µg

4

0/4

0/4

106 genome equivalents

106 genome equivalents

Monitoring of viremia

RT–PCR, spot blot

Course after challenge

1/4 viremia lower 2/4 negative in autopsied liver (26 and 113 weeks after challenge) 2×108 genome Nested RT–PCR, Viremia delayed, equivalents chronic course dot blot Typical chronic Blot? 106 WID50 course

Author

D’Ugo et al. Vaccine 2004

Immunology of HDV Infection

Table 1 (continued)

Karayiannis et al. J Med Virol 1993 Eckhart et al. Conference proceedings 1993: Hepatitis Delta Virus Typical chronic Eckhart et al. Conference Blot? proceedings 1993: course Hepatitis Delta Virus Nested RT–PCR, 2/4 viremia later, Fiedler et al. Vaccine 2001 shorter, no Ab spot blot

a

Atypical course in general (no HDAg was seen in the livers of chronically infected woodchucks; no depression of WHV DNA levels in correlation to the increase of HDV RNA) b Monitoring protocol missing and sensitivity of tests not mentioned 197

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lenge (Menne et al. 1997). A Th-cell immune response to the HDV proteins alone seems not to be able to be sufﬁcient to prevent HDV infection. 4.1 Immunization with HDV Protein Prokaryotic and eukaryotic expression systems were used to produce HDV protein for vaccination. Different expression systems result in different protein folding and, therefore, different presentation to the immune system. Karayiannis et al. studied the immunogenicity of HDAg expressed in E. coli in 1990 (Karayiannis et al. 1990). They immunized four WHV carrier woodchucks with a fusion protein consisting of the 64 N-terminal amino acids of HDAg and a part of the MS2 polymerase of the bacterial expression vector (pPL31), which proved to be immunogenic in rabbits. This protocol induced a strong humoral immune response in all woodchucks. After challenge the antibody titers raised signiﬁcantly. However, no protection from infection was observed. The course of infection did not differ from that of the control animals. The HDAg used in this study represented only a part of the total protein. The lack of protection could have been related to the fact that important epitopes for the humoral, or especially the Th and the CTL response, map to the remaining carboxyl end of the protein. Therefore, the same group tested the effect of immunization with the complete S-HDAg expressed in baculovirus using CFA and incomplete Freund’s adjuvant (IFA) in one animal (Karayiannis et al. 1993a, 1993b). This immunization protocol did not induce an antibody response and was not protective, but HDV RNA appeared 4–5 weeks later than in the two control animals and was only detectable by nested PCR, not by dot blot hybridization. Postmortem liver biopsies 6 months after challenge revealed the presence of HDAg in the hepatocytes of this animal and, therefore, a chronic HDV infection. Ponzetto et al. seemed to be more successful using S-HDAg expressed in yeast. They immunized six WHV carrier woodchucks eight times with HDAg without any adjuvant. Two animals seemed to be protected from HDV superinfection. HDV RNA was not detectable by PCR in these animals. Two additional woodchucks presented with viremia of short duration. Unfortunately, the authors did not mention the sensitivity of their tests and the length of the observation period after challenge is missing. This paper is a short communication and has not been published in detail (Ponzetto et al. 1993). The S-HDAg expressed in yeast was also used for immunization of woodchucks by our group (Fiedler et al. 2001). Four animals were immunized with HDAg with immunostimulatory CpG oligonucleotides in IFA and challenged with HDV later on. HDAg/CpG immunization induced a humoral and

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a Th-cell response in WHV carrier woodchucks, which was already described in more detail (see Sect. 3.3.3). The HDAg/CpG immunized woodchucks were challenged with 106 genome equivalents of HDV. Neither the humoral nor the Th-cell immune responses were sufﬁcient to protect the woodchucks from HDV superinfection. The course of HDV superinfection was similar to that in woodchucks that were not immunized; namely, the same level of HDV viremia and the typical ﬂuctuation of viremia with characteristic peaks were observed in both groups. Recently, the same HDAg expressed in yeast was used in another study for vaccination and challenge of eight woodchucks (D’Ugo et al. 2004). Four animals were immunized with HDAg using CFA/IFA as adjuvant, the other four with HDAg using MF59 as adjuvant. MF59 is an oil mineral-water emulsion which has been shown to augment the antigen-speciﬁc humoral immune response and to induce a Th-cell response that is more type 2-like in nature (Verschoor et al. 1999). The humoral immune response was detected earlier and at higher titers in the woodchucks immunized with HDAg/CFA vs. those immunized with HDAg/MF59. The Th-cell immune response was already described (see Sect. 3.3.3). After challenge HDV RNA was detected at 2–4 weeks in all animals, indicating that neither of the vaccines was able to protect from superinfection despite the presence of anti-HDV and a Th-cell immune response to HDAg. However, differences were observed in peak serum HDV RNA levels and persistence; namely, the HDAg/CFA immunized animals presented with a delayed appearance and very low HDV RNA titers. Woodchucks in the HDAg/MF59 group showed a trend to survive longer than those in the HDAg/CFA group and the controls. Histological analysis of liver tissues performed before challenge and after death clearly showed that the control animals developed hepatitis-like massive panacinar hepatic necrosis, which was not present in vaccinated woodchucks. In addition, two woodchucks immunized with HDAg/MF59 had cleared HDV RNA in the livers at autopsy, whereas all other animals still presented with HDV viremia in the liver. The authors speculate that the lack of viremic peak in the HDAg/CFA immunized animals clearly signiﬁed an early anti-viral effect, which could have been accompanied by a cell inﬂammatory response in the liver. In the HDAg/MF59 group the less evident and delayed immune response may have led to an effective anti-viral activity in the absence of an inﬂammatory response. The type of adjuvant could have inﬂuenced the priming of speciﬁc T cells. The combination of HDAg with different adjuvants was able to induce humoral and Th-cell immune responses of different intensities. Protection from superinfection, however, was not achieved. A modulation of the course of infection might have been induced in some, but not all animals.

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4.2 Immunization with Synthetic Peptides Bergmann et al. identiﬁed epitopes on HDAg by peptide mapping, which react with HDV antibody positive sera of humans, chimpanzees, and woodchucks (see Sect. 5 and the chapter by J.L. Casey and J.L. Gerin, this volume). These experiments resulted in the deﬁnition of a presumably immunodominant epitope of the N-terminal region of HDAg (aa 52–93) (Bergmann et al. 1989). Four WHV carrier woodchucks were immunized with three synthetic peptides corresponding to this region. The peptides were conjugated to keyhole limpet hemocyanin to improve the immune response (Bergmann et al. 1993). The immunization induced antibodies to the synthetic peptides and also to HDAg, but only in low titers. After challenge HDV RNA became detectable by PCR in all vaccinated and all control animals, but two of three vaccinated woodchucks (one died before challenge) stayed HDV RNA negative by northern blot analysis, a method approximately 100-fold less sensitive than the PCR. The authors speculate that HDV replication was limited in these two animals due to the immune response induced by immunization. However, the HDV infection in all these woodchucks, controls and vaccines, presented an atypical course. The authors observed no depression of the WHV DNA levels in correlation to the increase of HDV RNA, which is in contrast to other studies (Fiedler et al. 2001, Karayiannis et al. 1990, Karayiannis et al. 1993b, Ponzetto et al. 1984, Schlipköter et al. 1990). In addition, the authors cannot explain the discrepancy that some of their animals presented with HDV RNA positivity in sera, but were HDAg negative in the liver. 4.3 DNA Immunization Immunization with a DNA vaccine expressing viral antigens can induce a broad range of immune responses that provide protection from several viral infections, e.g. SIV or lymphocytic choriomeningitis virus (Yasutomi et al. 1994, Yokoyama et al. 1995). As already described above (see Sect. 3.3.3), in mice a CTL response against HDV could be induced, which was able to protect against tumor challenge (Mauch et al. 2002). Our group immunized four woodchucks with a DNA vector (pcDNA3 ) encoding S-HDAg (p24) (Fiedler et al. 2001). The DNA vaccine was administered by a gene gun in order to facilitate the induction of a strong cellular immune response (Raz et al. 1994; Shimizu et al. 1998; Siegel et al. 2001). This immunization protocol did not result in a measurable humoral or Th-cell immune response. In a previous series of vaccination studies in woodchucks, which were immunized with a DNA vaccine encoding WHV core antigen (WHcAg),

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the animals were protected from WHV challenge despite the absence of a detectable immune response to the core antigen (Lu et al. 1999). These results encouraged us to challenge the DNA-vaccinated woodchucks despite the absence of a measurable Th-cell response. After three immunizations with HDV DNA we observed a modulation of the course of HDV superinfection in two of three DNA vaccinated animals. However, immunization failed to protect WHV carrier woodchucks from infection. The modulation of the course of infection observed in two woodchucks was characterized by the following parameters: (1) the time of appearance of HDV viremia was delayed for 3 and 7 weeks according to PCR and for 3 and 10 weeks according to spot blot hybridization, respectively; (2) the typical ﬂuctuation of the HDV RNA level with several peaks was absent in these animals; (3) both woodchucks remained anti-HDV negative up to week 52 after challenge. This lack of seroconversion is unusual. The other two animals produced low levels of anti-HDV at weeks 10 and 11 after challenge. In previous studies conducted in our lab more than 20 woodchucks superinfected with HDV always showed seroconversion to anti-HDV (Rasshofer et al. 1990; Schlipköter et al. 1990). 4.4 Immunization with Vaccinia Virus Expressing HDAg Vaccination with recombinant vaccinia virus expressing viral antigens, e.g., antigens of the human or simian immunodeﬁciency viruses, is able to induce a speciﬁc CTL response (Allen et al. 2000; Dorrell et al. 2000). Two groups tested the efﬁcacy of immunization with vaccinia virus expressing either S- or L-HDAg (Eckart et al. 1993; Karayiannis et al. 1993a, 1993b). Altogether, six WHV carrier woodchucks were immunized. None of these animals produced a measurable humoral immune response after immunization; the cellular immune response was not measurable at this time in the woodchuck model. After challenge all animals developed chronic HDV infection. Karayiannis et al., however, described a modulation of the course of infection. Viremia was detected some weeks later and presented at lower levels in the immunized woodchucks in comparison to the control animals. Eckart et al. observed no differences in the course of infection between vaccinated and control woodchucks. It is difﬁcult to discern the reasons for the different course of infection in these studies. The amount of the vaccinia virus given is comparable, but Karayiannis et al. immunized twice intradermally, whereas Eckart et al. immunized once intravenously. Probably, the intradermal protocol was more effective and was able to modulate the course of infection, however, all animals became chronically infected.

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4.5 HDV Is a Poor Immunogenic Protein In several studies immunization of mice or woodchucks with DNA vaccines failed to induce a detectable humoral immune response. In the study of Mauch et al. none of more than 120 mice of different haplotypes that were immunized with plasmids expressing either S- or L-HDAg developed a detectable anti-HDAg response. Failure to detect anti-HDAg was not due to lack of HDV speciﬁc T-cell help, as a CD4+ Th-cell immune response was induced by all plasmids. Seizer et al. demonstrated that immunization with low amounts of HDV protein in mice induced no antibody response, which could be signiﬁcantly enhanced by combination with heterologous antigens such as HBsAg or HBcAg (Seizer et al. 2005). Only DNA constructs expressing another antigen (e.g., GFP) beside HDAg were able to elicit a strong antibody response. The authors demonstrated that the help of speciﬁc CD4+ T-cells induced by heterologous proteins was necessary for the induction of an anti-HDAg response. In the woodchuck model vaccination with DNA or vaccinia virus-expressing HDAg was also not able to mount a measurable anti-HDV response in six and four woodchucks, respectively (see Sects. 4.3 and 4.4) (Eckart et al. 1993; Fiedler et al. 2001; Karayiannis et al. 1993a). These data are in contrast with results obtained after intramuscular immunization of mice with a plasmid containing replication competent head-to-tail cDNA dimers of HDV that induced a humoral immune response (Polo et al. 1995). Also Huang et al. were able to detect speciﬁc antibodies in mice after DNA immunization (Huang et al. 2000, 2003). Plasmids encoding the small HDAg induced high titer antiHDAg, whereas plasmids expressing the L-HDAg induced only low antibody titers in 70% of the mice. The differences in antibody detection in these studies may be partially due to different expression plasmids, protein detection systems and other unknown factors. In conclusion, it could be stated that HDAg itself is a poor B-cell immunogen. HBV/HDV-infected patients develop serum antibody responses to HDAg, presumably because the extracellular forms of HDAg are always associated with immunogenic HBsAg. 4.6 Conclusions on Vaccination Studies Different immunization approaches have been tested to induce a protective immune response against HDV superinfection in chronically WHV-infected woodchucks: immunization with peptides, with prokaryortic and eukaryortic expressed protein, or with vaccinia virus or DNA expressing HDAg. None of these protocols was able to prevent HDV superinfection in all animals.

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It is difﬁcult to assess and to compare the studies, particularly because the monitoring after immunization and after challenge is described insufﬁciently in some studies and the sensitivity of the tests to measure HDV viremia is not comparable. Together these results allow us, however, to conclude that immunization against HDV with conventional vaccines is not possible. Neither a good humoral nor a weak Th-cell immune response is sufﬁcient to protect from HDV superinfection. Immunizations with vaccinia virus or DNA expressing HDAg were at least able to modulate the course of infection. In general, these approaches are known to induce a CTL response, which may be essential for protection. In further studies HDV vaccination schemes should be optimized to enhance the cellular immune response, e.g., by using different combinations of DNA vaccines with woodchuck cytokine (Lohrengel et al. 1998, 2000). This approach has been shown to enhance the cellular immune response to other hepadnavirus antigens (Mauch et al. 2002; Siegel et al. 2001). In future vaccination studies the monitoring of the immune response with respect to B- and T-cell responses has to be improved to see whether vaccination induced a measurable immune response prior to challenge.

5 Immunogenic Domains of HDAg In the late 1980s and early 1990s some groups investigated the B-cell epitopes of HDV. With the current knowledge about the humoral and cellular immune responses against HDV it has to be stated that the antibody response and, therefore, the corresponding epitopes, do not play an important role in the defense of HDV. Figure 2 gives an overview of the different epitopes characterized by different authors. Bergmann et al. identiﬁed immunogenic epitopes on HDAg by the use of 15 synthetic peptides covering the whole open reading frame (ORF) encoding HDAg (Bergmann et al. 1989). Antisera of humans, chimpanzees, and woodchucks infected with HDV reacted with the peptides. The pattern was distinct for each serum. Many of the sera from all three species recognized sequences from the whole ORF. The unique reactivity proﬁle of each serum could be related to genetic variations among HDV isolates. Residues 52–93 were shown to induce a major immunodominant response in humans by the use of anti-peptide sera in a competition enzyme immunoassay, in immunoprecipitation, and in immunoblotting. Furthermore, in this region a peptide is located which all human sera recognized. Peptides derived from this domain were later used for the vaccination of woodchucks (see Sect. 4.2 and Fig. 2).

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In another study 209 overlapping hexapeptides spanning the entire 214 amino acid residues of HDAg were used for the investigation of immunogenic domains on HDAg (Wang et al. 1990). Screening of the peptides with ﬁve hightiter anti-HDV human sera by ELISA resulted in the identiﬁcation of seven antigenic domains. Although the carboxy-terminal 50 residues of the HDAg reacted with each of the human sera, evident variation was found between individual sera in respect to the degree to which certain hexapeptides were bound by antibody. To conﬁrm the results three oligopeptides were tested for antigenic activity by microdilution ELISA. Maximal antigenic activity was found with the peptide spanning the residues 156–184, less antigenic activity for peptide 197–211, and only limited activity for peptide 2–17. These results were in concordance with the ELISAs with the hexapeptides (Fig. 2). On the basis of these results Poisson et al. investigated the antigenic activity of 80 sera of HDV-infected humans with four oligopeptides (Poisson et al. 1993). In agreement with the results of Wang et al., all peptides were recognized; however, the frequency of reactivity was low with peptide 2–17 (Wang et al. 1990). The peptide reacting with most sera (aa 155–172) corresponds to one of the two regions (amino acids 84–111 and aa 155–172) found to be highly conserved between different isolates (Chao et al. 1991). The binding activity of peptide 168–182 was signiﬁcantly greater than that of the other three peptides. The authors found differences in the immune response to the HDAg-derived peptides between HDV–HBV coinfection or HDV superinfection and also according to the delay between onset of infection and time point of serum sampling (Fig. 2). In a more recent study a dominant HDAg-speciﬁc antibody domain was localized on the N terminus of HDAg (residues 1–83) (Seizer et al. 2005). Balb/c mice were immunized with plasmids encoding three overlapping residues of HDAg fused to the hsp73-binding for efﬁcient expression. Only in mice immunized with the plasmid expressing the N-terminal residues a detectable antibody response (ELISA and western blot) was induced (Fig. 2). Altogether, four predominant B-cell epitopes have been characterized in the above described studies: residues 2–17, 52–93, 156–184, and 189–211. Bichko et al. showed that three of these domains, corresponding to the nuclear localization signal, the putative assembly domain, and the 19 aa C-terminal extension unique to the L-HDAg, are exposed on HDV. Only the domain between aa 2–17 could not be characterized ﬁnally in this study (Bichko et al. 1996). Patients infected with HDV have been shown to develop antibodies to different antigenic sites of HDAg. However, neutralizing anti-HDAg antibodies which were able to provide protection, have not been found.

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6 Closing Notes HDV superinfection of chronically HBV-infected patients results in acute, sometimes severe, hepatitis and in a high frequency of persistence of both viruses. The disease could be triggered by a HDV speciﬁc CTL response, although no data on the cellular immune response in acute HDV infection has been reported so far. Cellular immune responses (Th cells and CTLs) were found in patients with resolved HDV superinfection (without detection of HDV RNA), but not in chronically HDV-infected patients. Although these results were seen only in a small number of patients, one might conclude that a proper T-cell response seems to be the prerequisite for the elimination of HDV. This mechanism was shown for chronic HBV infection: a higher frequency of HBV-speciﬁc CD8+ T-cells was detected in patients with a low level of HBV replication than in those with a high level of HBV replication (Webster et al. 2004). The HBV-speciﬁc CD8+ T-cell response was overall weak in the blood of patients with chronic HBV infections. Beside the cellular immune response a humoral one is mounted in all patients. However, antibodies are apparently not able to modulate the course of infection. Like HBcAg, HDV is encapsidated by HBsAg; therefore, antibodies to HDV are not able to neutralize the virus, comparable to the inability of anti-HBc antibodies to neutralize HBcAg. Many questions about the immunopathogenesis of HDV still remain open. It is still unanswered whether the balance between viral load (high versus low) and the strength of the HDV-speciﬁc T-cell response play a role for the preferential establishment of either immunity or immunopathology in HDV superinfection. Cytopathic effects of HDV replication in human livers have not been ﬁnally excluded and, therefore, HDV itself may contribute to liver disease. Genetic variations during the chronic course of infection and its role for an immunevasion have also not been extensively studied so far (see the chapter by J.L. Casey and J.L. Gerin, this volume). Probably studies in the woodchuck model investigating the kinetics of the immune response and looking for immunological escape during the course of infection could give us some more insight in the immune response to HDV.

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Lu M, Hilken G, Kruppenbacher J, Kemper T, Schirmbeck R, Reimann J, Roggendorf M (1999) Immunization of woodchucks with plasmids expressing woodchuck hepatitis virus (WHV) core antigen and surface antigen supresses WHV infection. J Virol 73:281–289 Mauch C, Grimm C, Meckel S, Wands J, Blum H, Roggendorf M, Geissler M (2002) Induction of cytotoxic T lymphocyte responses against hepatitis delta virus antigens which protect against tumor formation in mice. Vaccine 20:170–180 Menne S, Maschke J, Tolle TK, Lu M, Roggendorf M (1997) Characterization of the T-cell response to woodchuck hepatitis virus core protein and protection of woodchucks from infection by immunization with peptides containing a T-cell epitope. J Virol 71:65–74 Murray K, Bruce SA, Hinnen A, Wingﬁeld P, van Eerd PM, de Reus A, Schellekes H (1984) Hepatitis B virus antigens made in microbial cells immunise against viral infection. EMBO J 3:645–650 Negro F, Shapiro M, Satterﬁeld WC, Gerin JL, Purcell RH (1989) Reappearance of hepatitis D virus (HDV) replication in chronic hepatitis B virus carrier chimpanzees rechallenged with HDV. J Infect Dis 160:567–571 Nisimi R, Paroli M, Accapezzato D, Borino F, Rosina F, Santantonio T, Sallusto F, Amoroso A, Houghton M, Barnebo V (1997) Human CD4+ T-cell response to hepatitis delta virus: identiﬁcation of multiple epitopes and characterization of T-helper cytokine proﬁles. J Virol 71:2241–2251 Poisson F, Baillou A, Dubois F, Janvier B, Roingeard P, Goudeau A (1993) Immune response to synthetic peptides of hepatitis delta antigen. J Clin Microbiol 31:2343– 2349 Polo J, Lim B, Govindarajan S, Lai M (1995) Replication of hepatitis delta virus RNA in mice after intramuscular injection of plasmid DNA. J Virol 69:5203–5207 Ponzetto A, Eckart M, D’Urso N, Negro F, Silvestro M, Bonino F, Wang K-S, Chien D, Choo Q-L, Houghton M (1993) Towards a vaccine for the prevention of hepatitis delta virus superinfection in HBV carriers. Prog Clin Biol Res 382:207–210 Ponzetto P, Cote PJ, Popper H, Hoyer BH, London WT, Ford EC, Bonino F, Purcell RH, Gerin JL (1984) Transmission of the hepatitis B virus associated delta agent to the eastern woodchuck. Proc Natl Acad Sci USA 81:2208–2212 Rasshofer R, Choi SS, Wölﬂ P, Manneck K, Weisensee U, Roggendorf M (1990) Inhibition of HDV RNA replication in vitro by Ribavirin and Suramin. In: Hollinger FB, Lemon SM, Margolis H (eds.) Viral hepatitis and liver disease. Boltimore, Williams & Wilkins, pp 659–662 Raz E, Carson DA, Parker SE, Parr TB, Abai AM, Aichinger G, Gromkowski SH, Singh M, Lew D, Yankauckas MA and others (1994) Intradermal gene immunization: the possible role of DNA uptake in the induction of cellular immunity to viruses. Proc Natl Acad Sci USA 91:9519–9523 Rizetto M (1981) Biology and characterization of the delta agent. In: Maynard JE, editor. Viral hepatitis. New York: The Franklin Institute Press pp 355–360 Rizetto M, Hoyer BH, Purcell RH, Gerin JL (1984) Hepatitis delta virus infection. In: Hoofnagle JH, editor. Viral Hepatitis and Liver Disease. Orlando: Grune & Stratton Inc., pp 371–379

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Schirmbeck R, Melber K, Reimann J (1999) Adjuvants that enhance priming of cytotoxic T cells to a Kb-restricted epitope processed from exogenous but not endogenous hepatitis B surface antigen. Int Immunol 11:1093–1102 Schlipköter U, Ponzetto A, Fuchs K, Rasshofer R, Choi SS, Roos S, Rapicetta M, Roggendorf M (1990) Different outcome of chronic hepatitis delta virus infection in woodchucks. Liver 20:291–301 Schödel F, Neckermann G, Peterson D, Fuchs K, Fuller S, Will H, Roggendorf M (1993) Immunization with recombinant woodchuck hepatitis virus nucleocapsid antigen or hepatitis B virus nucleocapsid antigen protects hepatitis virus infection. Vaccine 11:624–628 Seizer P, Riedl P, Reimann J, Schirmbeck R (2005) Different sources of “help” facilitate the antibody response to hepatitis D virus d antigen. J Mol Med 83:225–234 Shimizu Y, Guidotti LG, Fowler P, Chisari FV (1998) Dendritic cell immunization breaks cytotoxic T lymphocyte tolerance in hepatitis B virus transgenic mice. J Immunol 161:4520–4529 Siegel F, Lu M, Roggendorf M (2001) A single immunization of woodchucks with plasmids expressing woodchuck hepatitis virus (WHV) core antigen and IFN-γ suppresses WHV infection. J Virol 75:5036–5042 Verschoor E, Mooij P, Oostermeijer H, Kolk Mvd, Haaft Pt, Verstrepen B, Sun Y, Morein B, Akerblom L, Fuller D and others (1999) Comparison of immunity generated by nucleic acid-, MF59-, and ISCOM-formulated human immunodeﬁciency virus type 1 vaccines in Rhesus macaques: evidence for viral clearance. J Virol 73:3292– 3300 Wang D, Pearlberg J, Liu Y-T, Ganem D (2001) Deleterious effects of hepatitis delta virus replication on host cell proliferation. J Virol 75:3600–3604 Wang JG, Jansen RW, Brown EA, Lemon SM (1990) Immunogenic domains of hepatitis delta virus antigen: peptide mapping of epitopes recognised by human and woodchuck antibodies. J Virol 64:1108–1116 Webster G, Reignat S, Brown D, Ogg G, Jones L, Seneviratne S, Williams R, Dusheiko G, Bertoletti A (2004) Longitudinal analysis of CD8+ T cells speciﬁc for structural and nonstructural hepatitis B virus proteins in patients with chronic hepatitis B: implications for immunotherapy. J Virol 78:5707–5719 Wu J, Chen T, Huang Y, Yen F, Ting L, Sheng W, Tsay S, Lee S (1995) Natural history of hepatitis D viral superinfection: signiﬁcance of viremia detected by polymerase chain reaction. Gastroenterology 108:796–802 Yasutomi Y, Robinson HL, Lu S, Mustafa F, Lekutis C, Arthos J, Mullins JI, Voss G, Manson K, Wyand M and others (1994) Simian immunodeﬁciency virus-speciﬁc cytotoxic T-lymphocyte induction through DNA vaccination of rhesus monkeys. J Virol 70:678–681 Yokoyama M, Zhang J, Whitton JL (1995) DNA immunization confers protection against lethal lymphocytic choriomeningitis virus infection. J Virol 69:2684–2688

CTMI (2006) 307:211–225 c Springer-Verlag Berlin Heidelberg 2006

The Woodchuck Model of HDV Infection J. L. Casey1 (u) · J. L. Gerin2 1 Department of Microbiology and Immunology, Georgetown University Medical

Center, Washington, DC 20007, USA [email protected] 2 Department of Microbiology and Immunology and Division of Molecular Virology and Immunology, Georgetown University Medical Center, Washington, DC 20007, USA

1

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212

2

A Woodchuck-HDV Inoculum Derived from a Molecular Clone: Analysis of Genetic Changes Occurring During Acute and Chronic Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213

3

Vaccine Strategies for HDV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218

4

Therapy for HDV Based on Inhibition of the Helper Hepadnavirus . . . . . 220

5

Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222 Abstract The Eastern woodchuck, Marmota monax, has been a useful model system for the study of the natural history of hepadnavirus infection and for the development and preclinical testing of antiviral therapies. The model has also been used for hepatitis delta virus (HDV). In this chapter several new applications of the woodchuck model of HDV infection are presented and discussed. The development of a woodchuck HDV inoculum derived from a molecular clone has facilitated the analysis of viral genetic changes occurring during acute and chronic infection. This analysis has provided insights into one of the more important aspects of the natural history of HDV infection—whether a superinfection becomes chronic. These results could renew interest in further vaccine development. An effective therapy for chronic HDV infection remains an important clinical goal for this agent, particularly because of the severity of the disease and the inability of current hepadnaviral therapies to ameliorate it. The recent application of the woodchuck model of chronic HDV infection to therapeutic development has yielded promising results which indicate that targeting the hepadnavirus surface protein may be a successful therapeutic strategy for HDV.

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1 Introduction The dependence of hepatitis delta virus (HDV) infection on the presence of hepatitis B virus (HBV) imposes restrictions on the types of animal models available to study the natural history of HDV infection and to develop vaccine and therapeutic strategies. Primate models of hepadnavirus infection have included the chimpanzee, which can be infected with HBV, and either the woolly monkey or spider monkey, which can be infected with the closely related woolly monkey hepatitis B virus (WMHBV) (Lanford et al. 1998). The utility of the chimpanzee is severely limited by the scarcity of animals and ethical concerns; similar issues pertain to the woolly monkey, an endangered species. The natural history of WMHBV in spider monkeys is not fully understood, and the ability to establish chronic WMHBV infection followed by HDV superinfection has not been demonstrated. While the duck has been a useful model for HBV, duck hepatitis B virus supports neither HDV packaging nor infection (see the chapter by C. Sureau, this volume). Our efforts have focused on the eastern woodchuck, which has been a valuable naturally occurring animal model of hepadnavirus infection and disease (Tennant and Gerin 1994). There is no evidence that HDV infection occurs naturally in woodchucks, but several laboratories have shown that woodchucks chronically infected with woodchuck hepatitis virus (WHV) can be infected with HDV. Initially, infection was produced by inoculation of woodchucks with HDV derived from chimpanzees (Ponzetto et al. 1984). Consistent with the requirement of hepatitis B surface antigen (HBsAg) for the HDV replication cycle (Rizzetto et al. 1980), the HBsAg envelope protein was replaced by the WHV surface antigen following the initiation of infection (Ponzetto et al. 1984). It was later demonstrated that WHsAg can efﬁciently package the HDV genome in cell culture (Ryu et al. 1992). Woodchuck-derived HDV has been passaged serially in woodchucks, and was shown to be infectious in cultured woodchuck hepatocytes (Choi et al. 1988, Taylor et al. 1987). The natural history of HDV infection in woodchucks is similar to that in humans: infection results in acute and chronic infection in a high percentage of animals (Casey et al. 2005, Ponzetto et al. 1984, 1987, Schlipkoter et al. 1990). Finally, HDV replication is restricted to the liver in infected woodchucks (Negro et al. 1989), as it is in chimpanzees and humans. Thus, the woodchuck has been a valuable animal model for HDV. There are two modes of HDV infection: superinfection of an individual with chronic HBV infection; and coinfection with both viruses of an individual who has not been exposed previously to HBV. While the latter type of exposure

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typically leads to recovery from both viral infections, the former results in chronic HDV infection with a frequency of 70%–80% (Gerin et al. 2001). Chronic HDV infection is similar to chronic HBV infection and is typically characterized by necroinﬂammatory lesions, but the disease is more severe, and frequently progresses rapidly to cirrhosis and liver failure. Chronic HDV infection is thus disproportionately associated with terminal liver disease and an indication for liver transplantation (Rizzetto et al. 1983, Smedile et al. 1994). In this chapter we discuss the use of the woodchuck model of HDV infection for the analysis of HDV genetic changes occurring during acute and chronic infection, vaccine studies (also see the chapter by M. Fiedler and M. Roggendorf, this volume), and for the development of therapeutic strategies to combat chronic HDV infection (also see the chapter by J.S. Glenn, this volume).

2 A Woodchuck-HDV Inoculum Derived from a Molecular Clone: Analysis of Genetic Changes Occurring During Acute and Chronic Infection The determinants of the outcome of HDV superinfection are not known. Analyses of the viral genetics of hepatitis C virus have indicated that progression to chronic infection is correlated with sequence changes and increased sequence diversity in the hypervariable region of the envelope protein (Farci et al. 2000). Previous studies of genetic changes that occur during the course of HDV infection have either analyzed sequence modiﬁcations that occur over time in HDV RNA isolated from the sera of chronically infected patients (Chao et al. 1994, Imazeki et al. 1990, Lee et al. 1992), or from the liver of an infected woodchuck at the end of several serial passages (Netter et al. 1995). However, none of these studies addressed the role of viral genetic changes in the establishment of chronic infection. In order to study the natural history of HDV infection in the woodchuck model, including analysis of genetic changes that occur during acute and chronic infection, we have developed a woodchuck HDV inoculum derived from an HDV cDNA molecular clone (Casey et al., unpublished results). This inoculum was created by pooling sera collected weekly following the injection of plasmid DNA containing a 1.2 overlength HDV cDNA insert into the liver of a woodchuck chronically infected with WHV. Infection of chronic WHVcarrier woodchucks with this molecularly derived inoculum led to patterns of HDV viremia and a chronicity rate typical of human infection (Fig. 1).

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Fig. 1 Time course of HDV viremia in WHV carrier woodchucks infected with a woodchuck HDV inoculum derived from a molecular clone. HDV RNA levels were determined by a semiquantitative RT–PCR assay; the scale is approximately logarithmic. Dashed vertical lines indicate 27 and 73 weeks, at which times HDV RNA sequences were analyzed. Consensus sequence changes in the HDAg coding sequence are indicated. Partial changes are indicated in parenthesis

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Analysis of HDV sequences obtained postinfection with the molecularly derived inoculum indicated that sequence diversity increased over the course of 73 weeks in chronically infected animals. Eight weeks postinfection, clones of 695-bp RT–PCR products encompassing the HDAg coding region averaged just 1 nucleotide change, a rate that is nearly indistinguishable from the error rate of the RT–PCR ampliﬁcation. However, by 73 weeks, the average number of sequence modiﬁcations had increased to 4.8 per clone. U to C and A to G transitions dominated the changes observed, accounting for 67% of all changes. In an analysis of sequence diversity occurring during the course of serial acute phase passage of HDV in woodchucks, Netter et al. (1995) also observed higher rates of U to C and A to G sequence changes. These modiﬁcations are consistent with RNA editing by adenosine deaminase-1 (ADAR1), which speciﬁcally edits the antigenomic RNA at the amber/W site in an integral part of the HDV replication cycle (see the chapter by J.L Casey, this volume). Deamination by ADAR1 converts adenosines in RNA to inosine; because inosine base pairs preferentially with C rather than U, subsequent viral RNA replication changes the corresponding position on the complementary RNA strand from U to C (and subsequently to G on the RNA strand that was originally edited). The occurrence of A to G transitions on the genome suggests that the genomic RNA may also be susceptible to editing by ADAR1. Editing on the HDV RNA by ADAR1 has been shown to be highly speciﬁc in vitro and in cultured cells; that is, modiﬁcations at nonamber/W sites occur at very low levels during the course of a typical 1- or 2-week cell culture experiment. Thus, the functional signiﬁcance of the possible editing by ADAR1 at particular nonamber/W sites is not clear. Some sites consistent with ADAR1 editing were found modiﬁed in many of the 40 clones obtained from week 73 PCR products. In such cases, the high degree of modiﬁcation could be due to either selective pressure or the accumulation of editing events at these positions, which likely have somewhat greater than average activity as substrates for ADAR1. Most modiﬁcations, however, were found in just a single clone. Perhaps, in addition to editing at moderate to low efﬁciency sites at which editing accumulates slowly over time, ADAR1 editing potentiates genetic changes at other positions by providing an additional mechanism besides polymerase error by which genome diversity increases. Genomes containing modiﬁcations at these sites then either increase or decrease in the viral RNA population based on whether these changes confer a growth advantage or disadvantage. One of the more interesting observations of the genetic changes occurring during the course of infection was that in nearly all animals, a limited number of modiﬁcations had occurred in the consensus sequence that altered the sequence of the viral protein, hepatitis delta antigen (HDAg) (Fig. 1). Of

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particular note, the timing and degree of these changes was correlated with the outcome of infection. Five of ﬁve chronically infected woodchucks exhibited consensus sequence changes at 73 weeks postinfection, but just one of these animals exhibited sequence substitutions at earlier times (Fig. 1). In contrast, consensus sequence changes were apparent by 27 weeks in two of three animals that eventually recovered from HDV infection (Fig. 1). Moreover, the number of positions changed was generally greater in the animals that recovered compared with those that developed chronic infection (Fig. 1). While consensus changes at some positions in some animals were due to modiﬁcations in all clones, some sites were characterized by clusters of modiﬁcations, such that all clones were modiﬁed within a 15-nucleotide region, but at different positions (Fig. 2). It is difﬁcult to distinguish between the inﬂuences of negative selective pressure of immune responses and positive selective pressure of increased replication efﬁciency on the observed changes in the consensus sequence. Most likely, both processes play a role. We suggest that selective pressure imposed by immune responses is important because of the following observations (Figs. 1 and 2): (a) the varied patterns of sequence changes in different animals (the woodchucks used in this study were not inbred); (b) the clustering of sequence changes around certain locations in different clones obtained from the same animal; and (c) the different time course of consensus sequence changes in the animals that became chronically infected compared with those that recovered from HDV infection. It is not clear to what extent a previous study of the genetic stability of HDV in woodchucks contributes to the above discussion (Netter et al. 1995). Although the reported absence of consensus sequence changes occurring during serial passages over a similar total length of time (253 days) could argue against a dominant role for positive selective pressure due to replication advantages conferred by sequence changes, only a portion of the HDAg coding region was analyzed in that study—some changes could have been missed. Furthermore, that study used an HDV clone that was derived from an HDV passage series in woodchucks, and thus might have been derived from an isolate that had already undergone changes adaptive to this animal. We suggest that our data are consistent with a model in which HDV sequence changes are selected by host immune responses. One explanation of the differences observed in animals that recovered vs. those that became chronically infected is that the virus sequence changed at the earlier time in the animals that eventually recovered because of a more vigorous host immune response in these animals compared with those that became chronically infected. In the animals that recovered from infection, either the change in the HDAg sequence was insufﬁcient to avoid immune pressure, or the host

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Fig. 2 Changes occurring in the predicted HDAg sequence at 73 weeks postinfection. Eight clones were obtained from RT–PCR products from each of ﬁve chronically infected animals. Each horizontal bar corresponds to the sequence of one clone. Vertical lines indicate positions at which the predicted HDAg sequence deviated from that of the inoculum. Boxes highlight the clustering of sequence changes

was able to recognize additional epitopes elsewhere to aid in viral clearance. In the animals that became chronically infected, the immune response was apparently weak or delayed, and limited to just one or two epitopes. Thus, chronic HDV infection in woodchucks may result from a delayed and weak immune response that is limited to a small number of epitopes on HDAg.

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3 Vaccine Strategies for HDV If the immune response explanation of the genetic changes discussed above is correct, we could conclude that the stimulation of a potent immune response, which is inferred by the 27 week sequence changes in the animals that recovered from HDV infection, could be achieved by vaccination and that this response could clear the virus. Analysis of antibody responses to HDAg in patients and experimentally infected woodchucks indicated an immunodominant domain between amino acids 52 and 93 (Bergmann et al. 1989; Wang et al. 1990). A preliminary report (Bergmann et al. 1993) described a vaccine strategy in which woodchucks were vaccinated with three HDAg peptides conjugated individually to keyhole limpet hemocyanin. Vaccinated woodchucks were challenged with a woodchuck-adapted HDV pool. All animals became infected with HDV, based on the ability to detect HDV RNA in serum (Bergmann et al. 1993). However, preliminary analysis of viral RNA levels suggested that viremia was lower and of shorter duration in the vaccinated animals compared to controls. Further, it was found that antiHD antibody responses were not typical, particularly in the vaccinated animals. Following challenge, none of the three animals that had received the vaccine exhibited increases in antiHD, while four of ﬁve unvaccinated control animals produced increasing levels of antiHD following challenge. It was speculated that the lack of a signiﬁcant increase in antiHD in the vaccinated animals was due to a limited infection that did not elicit a strong antiHD response (Bergmann et al. 1993). Follow-up analysis of HDV RNA by a sensitive RT–PCR assay has conﬁrmed and extended the original observations (Fig. 3). The three vaccinated animals exhibited low levels of viremia of limited duration. In contrast, all three of the surviving animals in the control group were still viremic 32 weeks post-challenge. Thus, while the peptide vaccine did not prevent HDV infection, it did modulate the course of the viremia. Other studies using different vaccine strategies directed at speciﬁcally stimulating cell mediated responses, including infection with recombinant HDAg-vaccinia or injection of DNA constructs designed to express HDAg, have reported similar ﬁndings of modulation of the course of HDV superinfection (D’Ugo et al. 2004; Fiedler et al. 2001; Karayiannis et al. 1993; see also the chapter by M. Fiedler and M. Roggendorf, this volume). That these vaccines do not prevent brief viremia should not necessarily be viewed as a failure, because most of the damage done by HDV occurs during chronic infection, which some vaccines appear to have limited. Frequently, reduced, delayed and shortened periods of viremia correlated with weak or absent humoral antibody responses. That humoral antibody

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Fig. 3 Time course of HDV viremia in vaccinated and control animals following challenge with a woodchuck HDV inoculum. HDV RNA levels were determined by a semiquantitative RT–PCR assay; the scale is approximately logarithmic

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responses are not protective against HDV infection (Karayiannis et al. 1990) is not particularly surprising given that HDAg is not exposed on the surface of the virion. However, it appears that some form of immunity follows acute, self-limiting infection because hepatitis B carrier chimpanzees superinfected with HDV were resistant to rechallenge with HDV 6 months later (Purcell et al. 1987). Most likely, the alteration of the course of infection by some vaccines is due to the ability to elicit appropriate cell mediated immune responses. However, there appears to be no correlation between vaccine efﬁcacy and antiHD T-cell proliferative responses (Fiedler et al. 2001). Perhaps, cytotoxic T lymphocyte activity is the more important contributor to clearance of virally infected cells. Future vaccine studies may beneﬁt from measurement of cytotoxic T lymphocyte activity induced by vaccines and/or HDV infection. Most vaccines tested thus far have been closely related, if not identical, to the sequence of HDAg in the inoculum. If minor sequence changes provide an escape mechanism for the virus during the development of chronic infection (see Sect. 2) , the occurrence of such sequence differences between vaccines and inocula could be an important factor to consider in the likely success of vaccines. Perhaps the ability to stimulate a potent response to multiple epitopes will be critical for the success of an HDV vaccine outside of the laboratory setting. In future vaccine studies, the relationship between the HDAg sequence used and that of the inoculum may be an additional important consideration. Such studies may beneﬁt from a combined analysis of immune responses to infection/vaccination with inspection of genetic changes occurring on the viral genome.

4 Therapy for HDV Based on Inhibition of the Helper Hepadnavirus There is currently no generally accepted effective therapy for type D hepatitis (see Niro et al. 2005 for a review), and liver transplantation is the only option for the associated end-stage liver disease (Wright and Pereira 1995). The dependence of HDV on HBV could suggest that successful treatment of HDV infection would follow successful treatment of the supporting HBV infection. Unfortunately, this does not always appear to be the case. Although treatment of chronic HBV carriers with lamivudine (b-l-2 ,3 -dideoxy-3 -thiacytidine, 3TC) leads to decreased levels of HBV in serum and improved liver histology (Dienstag et al. 1995; Lai et al. 1998; Nevens et al. 1997), in patients with chronic delta hepatitis prolonged lamivudine therapy neither lowers HDV RNA levels nor ameliorates disease activity, even though HBV viremia is reduced (Lau et al. 1999; Wolters et al. 2000). Similarly, treatment with famciclovir was not effective against HDV infection (Yurdaydin et al. 2002). The most likely

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explanation for the failure of these treatments to affect HDV is that HDV requires the HBsAg function of HBV, and lamivudine treatment does not typically reduce HBsAg levels. The nucleoside analog L-FMAU [2 -ﬂuoro-5-methyl-β-l-arabinofuranosyluridine, clevudine] has substantial antiviral activity against WHV replication in chronically infected woodchucks (Menne et al. 2002; Peek et al. 2001; Zhu et al. 2001), and has recently exhibited potent activity in HBV-infected patients in a phase II clinical trial (Marcellin et al. 2004). Of particular interest, about 75% of animals treated with 10 mg/kg of clevudine exhibited 100-fold or greater decreases in serum levels of WHsAg (Korba et al. 2004; Peek et al. 2001). The reason for the different patterns of WHsAg response is not clear, but may be related to the effects of clevudine treatment on hepatic levels of WHV covalently closed circular DNA (Peek et al. 2001). Because of the high frequency of substantial reductions in surface antigen levels in woodchucks treated with clevudine, we sought to determine whether clevudine therapy could be effective in reducing levels of HDV viremia in chronically infected woodchucks. Four woodchucks chronically infected with HDV for at least 11 months were given 10 mg/kg clevudine (L-FMAU) orally, once daily for 20 weeks. As expected, all four animals exhibited marked decreases in serum WHV DNA after 4 weeks of treatment (>107 -fold reduction). Consistent with the effects of clevudine on WHsAg levels observed in other studies (Korba et al. 2004; Peek et al. 2001), there was an approximate 1,000-fold decrease in WHsAg levels in three of the four treated animals by 12 weeks of treatment. In all three of these animals HDV RNA became undetectable by 16 weeks of treatment. Of particular note, in the one animal that did not exhibit decreased levels of WHsAg, HDV RNA remained at high levels. Thus, there was a strong temporal correlation between the decrease in the levels of WHsAg and the drop in HDV viremia. The correlation of the suppression of HDV viremia with the reduction of surface antigen in individual animals is consistent with the concept that targeting surface antigen expression is a useful antiviral strategy for HDV. Thus, we suggest that any therapy that lowers WHV or HBV surface antigen levels sufﬁciently may be useful as a therapeutic option to control chronic HDV infection. Although HBsAg levels were not measured in previous reports of lamivudine therapy of chronic HDV carriers (Lau et al. 1999; Wolters et al. 2000), treatment was unsuccessful most likely because HBsAg was not reduced. Indeed, neither lamivudine nor adefovir dipovoxil, the two nucleoside analogues currently licensed for the treatment of HBV infection, routinely have a signiﬁcant effect on circulating levels of HBsAg, even though both reduce HBV replication sufﬁciently to ameliorate HBV-induced disease (Dienstag et al. 1995; Feld and Locarnini 2002; Lai et al. 1998; Nevens et al. 1997).

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There is no long-standing cellular repository for HDV as there is for HBV (Smedile et al. 1998), and the half-life of HDV-infected cells may be brief; in mice infected cells survive for as little as 2 weeks (Netter et al. 1993). While in this study it was not possible to determine the long term outcome of clevudine therapy on the course of chronic HDV infection or disease, the sustained reduction of HDV to undetectable levels by therapy with this potent nucleoside analog suggests that HDV disease in clinical patients would be reduced and that treatment has the potential to eliminate HDV infection in chronically infected individuals. In this regard, it is important to note that a recent phase II clinical trial indicated potent activity of clevudine in HBV-infected patients, although effects on HBsAg levels were not reported (Marcellin et al. 2004).

5 Perspective The studies summarized in this chapter demonstrate that the WHV/woodchuck model of experimental chronic hepatitis infection can be applied to the analysis of aspects of the natural history of HDV infection, the development of HDV vaccine strategies, and therapeutic studies of chronic HDV superinfection. The relatively rapid progression to hepatocellular carcinoma in WHV-infected woodchucks does pose challenges for the use of this model for evaluating drug efﬁcacy against chronic HDV disease. Further, HDV infection appears to increase the risk of hepatocellular carcinoma in patients with compensated cirrhosis type B (Fattovich et al. 2000) and the inﬂuence of chronic HDV on progression of end-stage liver disease in the woodchuck model has not been established. Indeed, many of the woodchucks in the clevudine study progressed to hepatocellular carcinoma, which precluded post-treatment follow-up studies. Perhaps these limitations can be overcome by the use of younger WHV-carrier animals or use of less virulent WHV strains. Clearly, further studies of the natural history of HDV disease in this model are needed before many important therapeutic issues can be addressed.

References Bergmann KF, Casey JL, Tennant BC, Gerin JL (1993) Modulation of hepatitis delta virus infection by vaccination with synthetic peptides: a preliminary study in the woodchuck model. Prog Clin Biol Res 382:181–187

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Bergmann KF, Cote PJ, Moriarty A, Gerin JL (1989) Hepatitis delta antigen. Antigenic structure and humoral immune response [published erratum appears in J Immunol 1990 Feb 1;144(3):1151]. J Immunol 143:3714–3721 Casey JL, Cote PJ, Toshkov IA, Chu CK, Gerin JL, Hornbuckle WE, Tennant BC, Korba BE (2005) Clevudine inhibits hepatitis delta virus viremia: a pilot study in chronically infected woodchucks. Antimicrob Agents Chemother 49: 4396–4399 Chao YC, Tang HS, Hsu CT (1994) Evolution rate of hepatitis delta virus RNA isolated in Taiwan. J Med Virol 43:397–403 Choi SS, Rasshofer R, Roggendorf M (1988) Propagation of woodchuck hepatitis delta virus in primary woodchuck hepatocytes. Virology 167:451–457 D’Ugo E, Paroli M, Palmieri G, Giuseppetti R, Argentini C, Tritarelli E, Bruni R, Barnaba V, Houghton M, Rapicetta M (2004) Immunization of woodchucks with adjuvanted sHDAg (p24): immune response and outcome following challenge. Vaccine 22:457–466 Dienstag JL, Perrillo RP, Schiff ER, Bartholomew M, Vicary C, Rubin M (1995) A preliminary trial of lamivudine for chronic hepatitis B infection. N Engl J Med 333:1657–1661 Farci P, Shimoda A, Coiana A, Diaz G, Peddis G, Melpolder JC, Strazzera A, Chien DY, Munoz SJ, Balestrieri A and others (2000) The outcome of acute hepatitis C predicted by the evolution of the viral quasispecies. Science 288:339–344 Fattovich G, Giustina G, Christensen E, Pantalena M, Zagni I, Realdi G, Schalm SW (2000) Inﬂuence of hepatitis delta virus infection on morbidity and mortality in compensated cirrhosis type B. The European Concerted Action on Viral Hepatitis (Eurohep). Gut 46:420–426 Feld J, Locarnini S (2002) Antiviral therapy for hepatitis B virus infections: new targets and technical challenges. J. Clin. Virol. 25:267–283 Fiedler M, Lu M, Siegel F, Whipple J, Roggendorf M (2001) Immunization of woodchucks (Marmota monax) with hepatitis delta virus DNA vaccine. Vaccine 19:4618–4626 Gerin JL, Casey JL, Purcell RH (2001) Hepatitis Delta Virus. In: Knipe DM, Howley PM, (eds) Field’s Virology. 4 ed. Lippincott, Williams & Wilkins, Philadelphia, PA, pp 3037–3050 Imazeki F, Omata M, Ohto M (1990) Heterogeneity and evolution rates of delta virus RNA sequences. J Virol 64:5594–5599 Karayiannis P, Saldanha J, Jackson AM, Luther S, Goldin R, Monjardino J, Thomas HC (1993) Partial control of hepatitis delta virus superinfection by immunisation of woodchucks (Marmota monax) with hepatitis delta antigen expressed by a recombinant vaccinia or baculovirus. J Med Virol 41:210–214 Karayiannis P, Saldanha J, Monjardino J, Goldin R, Main J, Luther S, Easton M, Ponzetto A, Thomas HC (1990) Immunization of woodchucks with recombinant hepatitis delta antigen does not protect against hepatitis delta virus infection. Hepatology 12:1125–1128 Korba BE, Cote PJ, Menne S, Toshkov IA, Baldwin BH, Wells FV, Tennant BC, Gerin JL (2004) Clevudine therapy with vaccine inhibits progression of chronic hepatitis and delays onset of hepatocellular carcinoma in chronic woodchuck hepatitis virus infection. Antiviral Therapy 9:937–952

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Lai CL, Chien RN, Leung NW, Chang TT, Guan R, Tai DI, Ng KY, Wu PC, Dent JC, Barber J and others (1998) A one-year trial of lamivudine for chronic hepatitis B. Asia Hepatitis Lamivudine Study Group. N Engl J Med 339:61–68 Lanford RE, Chavez D, Brasky KM, Burns RB, 3rd, Rico-Hesse R (1998) Isolation of a hepadnavirus from the woolly monkey, a New World primate. Proc Natl Acad Sci U S A 95:5757–5761 Lau DT, Doo E, Park Y, Kleiner DE, Schmid P, Kuhns MC, Hoofnagle JH (1999) Lamivudine for chronic delta hepatitis. Hepatology 30:546–549 Lee CM, Bih FY, Chao YC, Govindarajan S, Lai MM (1992) Evolution of hepatitis delta virus RNA during chronic infection. Virology 188:265–273 Marcellin P, Mommeja-Marin H, Sacks SL, Lau GK, Sereni D, Bronowicki JP, Conway B, Trepo C, Blum MR, Yoo BC and others (2004) A phase II dose-escalating trial of clevudine in patients with chronic hepatitis B. Hepatology 40:140–148 Menne S, Roneker CA, Korba BE, Gerin JL, Tennant BC, Cote PJ (2002) Immunization with surface antigen vaccine alone and after treatment with 1-(2-ﬂuoro-5-methylβL-arabinofuranosyl)-uracil (L-FMAU) breaks humoral and cell-mediated immune tolerance in chronic woodchuck hepatitis virus infection. J Virol 76:5305– 5314 Negro F, Korba BE, Forzani B, Baroudy BM, Brown TL, Gerin JL, Ponzetto A (1989) Hepatitis delta virus (HDV) and woodchuck hepatitis virus (WHV) nucleic acids in tissues of HDV-infected chronic WHV carrier woodchucks. J Virol 63:1612– 1618 Netter HJ, Kajino K, Taylor JM (1993) Experimental transmission of human hepatitis delta virus to the laboratory mouse. J Virol 67:3357–3362 Netter HJ, Wu TT, Bockol M, Cywinski A, Ryu WS, Tennant BC, Taylor JM (1995) Nucleotide sequence stability of the genome of hepatitis delta virus. J Virol 69:1687– 1692 Nevens F, Main J, Honkoop P, Tyrrell DL, Barber J, Sullivan MT, Fevery J, De Man RA, Thomas HC (1997) Lamivudine therapy for chronic hepatitis B: a six-month randomized dose- ranging study. Gastroenterology 113:1258–1263 Niro GA, Rosina F, Rizzetto M (2005) Treatment of hepatitis D. J Viral Hepat 12:2–9 Peek SF, Cote PJ, Jacob JR, Toshkov IA, Hornbuckle WE, Baldwin BH, Wells FV, Chu CK, Gerin JL, Tennant BC and others (2001) Antiviral activity of L-FMAU (1(2-ﬂuoro-5-methyl-b_L-arabinofuranosyl) uracil) against WHV replication and gene expression in chronically infected woodchucks. Hepatology 33:254–266 Ponzetto A, Cote PJ, Popper H, Hoyer BH, London WT, Ford EC, Bonino F, Purcell RH, Gerin JL (1984) Transmission of the hepatitis B virus-associated delta agent to the eastern woodchuck. Proc Natl Acad Sci U S A 81:2208–2212 Ponzetto A, Forzani B, Smedile A, Hele C, Avanzini L, Novara R, Canese MG (1987) Acute and chronic delta infection in the woodchuck. Prog Clin Biol Res 234:37–46 Purcell RH, Satterﬁeld WC, Bergmann KF, Smedile A, Ponzetto A, Gerin JL (1987) Experimental hepatitis delta virus infection in the chimpanzee. In: Rizzetto M, Gerin JL, Purcell RH, (eds) The hepatitis delta virus and its infection. Alan R. Liss, New York, pp 27–36 Rizzetto M, Hoyer B, Canese MG, Shih JW, Purcell RH, Gerin JL (1980) delta Agent: association of delta antigen with hepatitis B surface antigen and RNA in serum of delta-infected chimpanzees. Proc Natl Acad Sci U S A 77:6124–6128

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Rizzetto M, Verme G, Recchia S, Bonino F, Farci P, Arico S, Calzia R, Picciotto A, Colombo M, Popper H (1983) Chronic hepatitis in carriers of hepatitis B surface antigen, with intrahepatic expression of the delta antigen. An active and progressive disease unresponsive to immunosuppressive treatment. Ann Intern Med 98:437–441 Ryu WS, Bayer M, Taylor J (1992) Assembly of hepatitis delta virus particles. J Virol 66:2310–2315 Schlipkoter U, Ponzetto A, Fuchs K, Rasshofer R, Choi SS, Roos S, Rapicetta M, Roggendorf M (1990) Different outcomes of chronic hepatitis delta virus infection in woodchucks. Liver 10:291–301 Smedile A, Casey JL, Cote PJ, Durazzo M, Lavezzo B, Purcell RH, Rizzetto M, Gerin JL (1998) Hepatitis D viremia following orthotopic liver transplantation involves a typical HDV virion with a hepatitis B surface antigen envelope. Hepatology 27:1723–1729 Smedile A, Rizzetto M, Gerin JL (1994) Advances in hepatitis D virus biology and disease. Prog Liver Dis 12:157–175 Taylor J, Mason W, Summers J, Goldberg J, Aldrich C, Coates L, Gerin J, Gowans E (1987) Replication of human hepatitis delta virus in primary cultures of woodchuck hepatocytes. J Virol 61:2891–2895 Tennant BC, Gerin JL (1994) The woodchuck model of hepatitis B virus infection. In: Shafritz DA, (ed) The Liver: Biology and Pathology. 3rd ed. Raven Press, Ltd., New York, pp 1455–1466 Wang JG, Jansen RW, Brown EA, Lemon SM (1990) Immunogenic domains of hepatitis delta virus antigen: peptide mapping of epitopes recognized by human and woodchuck antibodies. J Virol 64:1108–1116 Wolters LM, van Nunen AB, Honkoop P, Vossen AC, Niesters HG, Zondervan PD, de Man RA (2000) Lamivudine-high dose interferon combination therapy for chronic hepatitis B patients co-infected with the hepatitis D virus. J Viral Hepatitis 7:428– 434 Wright TL, Pereira B (1995) Liver transplantation for chronic viral hepatitis. Liver Transplant Surg 1:30–42 Yurdaydin C, Bozkaya H, Gurel S, Tillmann HL, Aslan N, Okcu-Heper A, Erden E, Yalcin K, Iliman N, Uzunalimoglu O and others (2002) Famciclovir treatment of chronic delta hepatitis. J Hepatol 37:266–271 Zhu Y, Yamamoto T, Cullen J, Saputelli J, Aldrich CE, Miller DS, Litwin S, Furman PA, Jilbert AR, Mason WS (2001) Kinetics of hepadnavirus loss from the liver during inhibition of viral DNA synthesis. J Virol 75:311–322

Subject Index

acetylation 93, 101–103, 106 acid-base catalysis 58, 59, 61 acute infection 212, 213, 220 adenosine deaminase that acts on RNA (ADAR) 7, 17, 215 adenosine deamination 70, 71, 77, 84 Africa 152, 156, 157, 160, 162, 164–166 amber/W site 69–84 antigenomic RNA 70, 71 antigenomic RNA replication 29–32, 34–38 antiviral therapy 134, 138, 141, 142 arginine methylation 30, 35, 37, 104–106

cytotoxic T-cell (CTL) 188, 190, 193, 194, 198, 200, 201, 203, 205

B-cell 193, 202–204 budding 116, 118–121, 123, 124, 126

genetic changes 213, 215, 218, 220 genetic variability 155, 156, 160, 166 genomic RNA replication 29–32, 34–38 genotype 152, 155–157, 160, 164–166 genotype I 73–76, 79, 81–84, 134, 139, 155–157, 162, 166, 175, 178–182 genotype II 155–157, 166, 175–181 genotype IIb 155, 156, 160, 164 genotype III 74, 76, 79, 81–84, 134, 139, 155, 157, 162, 164, 166, 167 geography 164, 166 geranylgeranyltransferase 93

catalytic site 56 cellular intermediate compartment (IC) 116, 118, 119, 125 chronic infection 212, 213, 215–218, 220–222 circular RNA 3–5, 10, 11 clades 156, 159–161, 165, 166 clevudine 221, 222 clinical signiﬁcance 175 crystal structure 51, 57, 60 cytopathic 188, 190 cytopathic effect 16 cytosine 75 (Cyt75) 49, 51–53

delta protein 3, 6, 16 dicer 15 divalent metal 48 divalent metal ion 52, 55, 60 DNA immunization 188, 194, 195, 200–202 editing

2, 6–8, 17

farnesylation 95, 96, 98, 120, 121 farnesyltransferase 94, 95 farnesyltransferase inhibitor (FTI) 139–144

Subject Index

HDAg interaction 173, 178, 179 HDV assembly 122, 123 HDV infectivity 124–126 hepatitis B surface antigen (HBsAg) 114 hepatitis B virus (HBV) 114–126, 152, 156, 164–167 hepatitis delta antigen (HDAg) 27–39, 92, 97–102 hepatitis delta antigen long form (L-HDAg) 68, 69, 71, 74, 75, 77–80, 82–84 histone acetyltransferase (HAT) 101, 103 IgM 188, 191, 192 immune response 216–218, 220 immunization 188, 194, 195, 198–204 immunogenic domain 204 immunopathogenesis 188, 190, 192, 205 in-line orientation 54, 55 inhibition of replication 6, 8 initiation 3, 4, 9–12, 16 inoculum 213–215, 217, 219, 220 interferon 14, 138 isoprenylation 93, 95, 96 kinase

97–100

L-HBsAg 115–119, 124–126 lamivudine 138, 220, 221 large hepatitis delta antigen (L-HDAg) 114, 117, 120–124, 126 linear RNA 3–5, 10–12, 17 M-HBsAg 115, 116, 118, 119, 124, 125 mechanism of transcription 35–38 Mg2+ 53, 55, 56, 61 molecular clone 213, 214 mouse 194 mouse model 138–140 mRNA transcription 33, 34

227

negative feedback regulation 82 nucleobase 53, 54, 57, 58, 61 phosphorylation 97–100 phylogenetic analysis 156, 160 post-translational modiﬁcation (PTM) 92, 93, 106, 107 pre-S1 117–119, 124 prenylation 134, 137, 139–143 prenylation inhibition 138, 139, 141, 142 prenyltransferase 137, 142, 144 protein arginine methyltransferase (PRMT) 104, 105 pseudoknot structure 50 recombination 13, 14 regulation of editing 77, 80 replication 2–4, 6–18, 26, 28–41, 68–72, 75–84 ribavirin 14 ribonucleoprotein (RNP) 114–117, 120–126 ribozyme 48–61 RNA editing 69–71, 79, 84, 174, 180 RNA packaging 177, 179, 180 RNA polymerase I 26, 40 RNA polymerase II 8, 9, 17, 26, 36–38, 40 RNA replication 177–179 RNA secondary structure 160, 162 RNA sequence changes 8, 16, 17 RNA structural dynamics 82, 84 RNA structure 84 S-HBsAg 114–119, 121–126 selective pressure 215, 216 sequence comparison 175 siRNA 15 small hepatitis delta antigen (S-HDAg) 114, 115, 120, 121, 124, 162, 166, 167 subcellular localization 96, 101–103

228

Subject Index

subviral particle (SVP) 115, 123, 126 surface antigen 212, 221 T-cell 188–190, 192, 194, 195, 202, 203, 205 template-switching 12, 13, 17 therapy 220–222 transactivation 174, 177, 179 transmembrane domain (TMD) 117–119, 122, 123 unbranched rod structure

68, 76, 80

vaccination 195, 198–203 vaccine 212, 213, 218, 220, 222 viral genetics 155, 160 virion packaging 71 viroid 11, 15, 17 virus-like particle (VLP) 138, 139 woodchuck 188–190, 192, 194, 195, 198–203, 205, 212–214, 216–219, 221, 222 woodchuck hepatitis virus (WHV) 212–214, 221, 222

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