Advances in
VIRUS RESEARCH VOLUME
74
ADVISORY BOARD DAVID BALTIMORE ROBERT M. CHANOCK PETER C. DOHERTY H. J. GROSS B. D. HARRISON BERNARD MOSS ERLING NORRBY J. J. SKEHEL M. H. V. VAN REGENMORTEL
Advances in
VIRUS RESEARCH VOLUME
74 Edited by
KARL MARAMOROSCH Rutgers University, New Jersey, USA
AARON J. SHATKIN Center for Advanced Biotechnology and Medicine, New Jersey, USA
FREDERICK A. MURPHY University of Texas Medical Branch, Texas, USA
AMSTERDAM • BOSTON • HEIDELBERG • LONDON NEW YORK • OXFORD • PARIS • SAN DIEGO SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO Academic Press is an imprint of Elsevier
Academic Press is an imprint of Elsevier 32 Jamestown Road, London, NW1 7BY, UK Radarweg 29, PO Box 211, 1000 AE Amsterdam, The Netherlands 30 Corporate Drive, Suite 400, Burlington, MA 01803, USA 525 B Street, Suite 1900, San Diego, CA 92101-4495, USA First edition 2009 Copyright # 2009 Elsevier Inc. All Rights Reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone: (þ44) (0) 1865 843830, fax: (þ44) (0) 1865 853333; e-mail:
[email protected]. Alternatively you can submit your request online by visiting the Elsevier web site at http://www.elsevier.com/locate/permissions, and selecting Obtaining permission to use Elsevier material Notice No responsibility is assumed by the publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN: 978-0-12-378587-9 ISSN: 0065-3527 For information on all Academic Press publications visit our website at elsevierdirect.com Printed and bound in USA 09 10 11 12 10 9 8 7 6 5 4 3 2 1
CONTENTS
1. Regulation of HIV-1 Alternative RNA Splicing and Its Role in Virus Replication
1
C. Martin Stoltzfus I. Introduction II. HIV-1 Splicing Regulatory Elements III. Evidence for the Functional Importance of HIV-1
2 3
Splicing Regulatory Elements in Virus Replication
21 30 32 33 33
IV. Strategies to Target HIV-1 Splicing with Antiviral Drugs V. Conclusions and Perspectives
Acknowledgments References
2. New Insights into Flavivirus Nonstructural Protein 5
41
Andrew D. Davidson Introduction The Methyltransferase Domain The RNA-Dependent RNA Polymerase Domain NS5 Interactions NS5 Phosphorylation NS5 Localization Emerging Roles for NS5 in Viral Pathogenesis Conclusions and Future Perspectives Acknowledgments References I. II. III. IV. V. VI. VII. VIII.
3. Replication of the Hepatitis Delta Virus RNA Genome
42 44 61 75 79 81 85 89 92 92
103
John M. Taylor I. II. III. IV. V. VI. VII.
Background Polymerase(s) Promoters and Priming Pausing and Switching Replication in the Nucleus Role(s) of the Delta Antigen Host Factors
104 106 108 110 112 113 115
v
vi
Contents
VIII. Viroid Analogy IX. Conclusions and Outlook
Acknowledgments References
4. Recent Epidemiology of Tick-Borne Encephalitis: An Effect of Climate Change?
116 116 117 117
123
E. I. Korenberg Introduction Major Debatable Issues The Ranges of Main Tick Vectors: Are They Really Expanding? Tick Abundance and TBE Virus Prevalence: Have They Changed? Tick Expansion to the Cities: Is It Related to Climate Change? What Is Known About Newly Formed TBE foci? Anthropurgic TBE foci: What Are the Principles of Their Formation? Since When Has TBE Morbidity Increased in the Cities? What Are the Main Causes of Changes in Parameters of TBE Morbidity? Conclusions Acknowledgment References
I. II. III. IV. V. VI. VII. VIII. IX. X.
Index Color plate section at the end of the book
124 127 128 129 131 133 134 135 136 137 138 138 145
CHAPTER
1 Regulation of HIV-1 Alternative RNA Splicing and Its Role in Virus Replication C. Martin Stoltzfus
Contents
I. Introduction II. HIV-1 Splicing Regulatory Elements A. Cis-regulatory elements that activate and repress metazoan mRNA splicing B. General strategy of HIV-1 RNA splicing C. Intrinsic efficiency of HIV-1 splice sites D. HIV-1 splice sites and their regulatory elements E. Alternative roles of the major splice donor sites 50 ss D1 and D4 in HIV-1 replication F. Effects of inclusion of small exons 2 and 3 on HIV-1 mRNA expression and stability G. Effects of RNA secondary structure on HIV-1 splicing H. Possible roles of HIV-1 Rev, Tat, and Vpr proteins in regulation of viral RNA splicing III. Evidence for the Functional Importance of HIV-1 Splicing Regulatory Elements in Virus Replication A. Sequence comparison of HIV-1 splice sites and regulatory elements B. Mutations of HIV-1 regulatory elements inhibit virus replication C. Overexpression and siRNA inhibition of cellular splicing factors affect HIV-1 splicing and inhibit virus replication
2 3 3 5 6 8 15 17 17 18
21 21 23
28
Department of Microbiology, University of Iowa, Iowa City, Iowa 52242, USA Advances in Virus Research, Volume 74 ISSN 0065-3527, DOI: 10.1016/S0065-3527(09)74001-1
#
2009 Elsevier Inc. All rights reserved.
1
2
C. Martin Stoltzfus
D. Changes in expression of cellular splicing factors during HIV-1 infection IV. Strategies to Target HIV-1 Splicing with Antiviral Drugs V. Conclusions and Perspectives Acknowledgments References
Abstract
29 30 32 33 33
Over 40 different human immunodeficiency virus type 1 (HIV-1) mRNA species, both completely and incompletely spliced, are produced by alternative splicing of the primary viral RNA transcript. In addition, about half of the viral RNA remains unspliced and is transported to the cytoplasm where it is used both as mRNA and as genomic RNA. In general, the identities of the completely and incompletely spliced HIV-1 mRNA species are determined by the proximity of the open reading frames to the 50 -end of the mRNAs. The relative abundance of the mRNAs encoding the HIV-1 gene products is determined by the frequency of splicing at the different alternative 30 -splice sites. This chapter will highlight studies showing how HIV-1 uses exon definition to control the level of splicing at each of its 30 -splice sites through a combination of positively acting exonic splicing enhancer (ESE) elements, negatively acting exonic and intronic splicing silencer elements (ESS and ISS elements, respectively), and the 50 -splice sites of the regulated exons. Each of these splicing elements represent binding sites for cellular factors whose levels in the infected cell can determine the dominance of the positive or negative elements on HIV-1 alternative splicing. Both mutations of HIV-1 splicing elements and overexpression or inhibition of cellular splicing factors that bind to these elements have been used to show that disruption of regulated splicing inhibits HIV-1 replication. These studies have provided strong rationale for the investigation and development of antiviral drugs that specifically inhibit HIV-1 RNA splicing.
I. INTRODUCTION HIV-1 is the etiologic agent of acquired immunodeficiency disease syndrome (AIDS) and currently over 30 million people worldwide are living with HIV-1 infection. Because of its importance in human disease HIV-1 has been the subject of intense study since its discovery in the early 1980s. The knowledge of the basic biology of HIV-1 has led to the development of a number of antiviral drugs that have been targeted to different steps of the virus life cycle. Through the use of a cocktail of several drugs, referred to as highly active antiretroviral therapy (HAART), the treatment
HIV-1 Splicing Regulation and Virus Replication
3
of AIDS has been revolutionized and this therapeutic approach has transformed the disease into a manageable chronic illness. However, one major problem with the antiviral drugs is the high frequency with which HIV-1 mutates to drug resistance. Thus, there is a pressing need to further investigate all steps of the virus life cycle in order to develop new antiviral drugs. One of the steps of the HIV-1 life cycle that has received relatively little attention as a target for antiviral drugs is the process of alternative RNA splicing. This is a complex process by which HIV-1 generates over 40 different spliced mRNAs from the single full-length unspliced RNA which is transcribed from the integrated viral provirus by RNA polymerase II. It is crucial for HIV-1 to maintain appropriate cytoplasmic levels of both spliced mRNAs for viral protein synthesis and unspliced viral RNA for use both as genome RNA and as an mRNA. In addition, the intracellular levels of the different viral mRNA species vary widely reflecting the efficiencies by which splicing occurs at the multiple alternative splice sites in the viral genome. Over the past 15 years, considerable progress has been made in understanding the mechanisms by which HIV-1 regulates its RNA splicing. As described below, this regulation is complex and involves the cooperative action of multiple positive and negative elements acting on the relatively weak core splice sites that characterize the HIV-1 genome. It also involves interaction of these cis-elements with a number of different cellular splicing factors. The purpose of this chapter is first, to briefly review the current knowledge of how HIV-1 cis-splicing elements and the trans-acting cellular and viral factors interacting with these elements regulate HIV-1 splicing. We will then discuss the extent of sequence homology of HIV-1 splice sites and splicing regulatory elements among different HIV-1 strains. This will be followed by a review of genetic evidence supporting the hypothesis that regulation of HIV-1 splicing is essential for efficient virus replication. We will also discuss how overproduction and inhibition of host splicing factors in infected cells affect HIV-1 replication. Finally, we will discuss several antiviral strategies that are being used to target HIV-1 splicing.
II. HIV-1 SPLICING REGULATORY ELEMENTS A. Cis-regulatory elements that activate and repress metazoan mRNA splicing 1. Core splicing signals and exon definition For several recent reviews of RNA splicing and exon definition, the reader is referred to references (Black, 2003; Wang and Burge, 2008; Zheng, 2004). Core splicing signals include three sites which are present in most premRNA introns: the 50 -splice site (50 ss), the 30 -splice site (30 ss), and the
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branch point sequence (BPS). The 50 ss is a binding site for U1 snRNP. The 30 ss, which includes a polypyrimidine (Py) tract and a conserved AG sequence 30 -proximal of the Py tract, is a binding site for the heterodimeric cellular splicing factor U2AF. It is comprised of two subunits: U2AF65 binds specifically to the Py tract and U2AF35 binds to the AG sequence. The BPS has a loose consensus sequence YNCURAY (R is either A or G; Y is C or U; N is any nucleotide; underlined A indicates location of branch point). Early in spliceosome assembly BPS is bound by the branch pointbinding protein (SF1/mBBP) which is subsequently displaced by U2 snRNP as spliceosome formation proceeds. 50 -Splice sites are referred to as ‘‘strong’’ or ‘‘weak’’ depending on the extent of base pairing between U1 snRNA and the 50 ss, that is, the extent of homology to the consensus 50 ss. 30 -Splice sites are referred to ‘‘strong’’ or ‘‘weak’’ depending on the affinity of the splice site for U2AF and SF1/mBBP, that is, the extent of homology to the consensus 30 ss. In mammalian cells, 50 ss and 30 ss are initially recognized in pairs across exons. This interaction between factors at the 50 ss and 30 ss is referred to as ‘‘exon definition’’ or ‘‘exon bridging’’ (Hoffman and Grabowski, 1992; Robberson et al., 1990). Following this initial recognition step, spliceosomes can assemble to early splicing complex (E complex) that results in an irreversible commitment to the splicing reaction. Thus, exon definition is a key step in alternative splicing regulation.
2. Exonic and intronic splicing enhancers and silencers In addition to the core splicing signals, the RNA transcripts of many genes contain additional cis-elements that are necessary to facilitate or repress exon definition (for review, see Matlin et al., 2005; Wang and Burge, 2008). Such regulatory elements are particularly important in alternative splicing pathways. Exonic splicing enhancers (ESEs) are sequence elements within exons that preferentially bind to members of the serine–argininerich protein (SR protein) family. SR proteins have one or more N-terminal RRM domains that bind to ESE sequences and a C-terminal RS domain that acts to facilitate exon definition by interacting with the RS domains of other splicing factors. The serine residues in the RS domains are extensively phosphorylated by several types of protein kinases including SRPK1 and SRPK2, the Clk/Sty family, and DNA topoisomerase. A number of studies have shown that phosphorylation of SR proteins affect their functions in splicing (Graveley, 2000). Exonic splicing silencers (ESSs) are bound by splicing inhibitory proteins and they repress exon definition. ESS sequences are very diverse and most of the sequences are preferential binding sites for members of the cellular heterogeneous ribonuclear protein (hnRNP) families. Intronic splicing silencers and enhancers (ISS and ISE, respectively) have also been identified.
HIV-1 Splicing Regulation and Virus Replication
5
These elements facilitate or repress definition of exons that are surrounded by the intronic splicing elements.
3. Role of secondary structure The secondary structure of pre-mRNAs may also affect alternative splicing by exposing or sequestering core splicing signals and splicing regulatory elements (for review of the role of secondary structure in splicing, the reader is referred to Buratti and Baralle, 2004). A wellcharacterized example of how RNA secondary structure affects splicing is the inclusion of the alternative fibronectin EDA exon. In this case, a downstream sequence stabilizes an upstream ESE sequence within a loop of a stem-loop structure where it is accessible to SR proteins. This results in recognition of the EDA exon. Mutations in the downstream sequence cause a conformational shift such that the ESE is now present in the stem and is relatively inaccessible to SR proteins. This results in failure to recognize the EDA exon (Buratti et al., 2004). Another example is the alternatively spliced tau exon 10 in which the 50 ss is sequestered in a stem-loop element. Disruption of this stem-loop by mutations results in increased binding of U1 snRNP to the 50 ss and increased inclusion of exon 10 (Varani et al., 1999).
B. General strategy of HIV-1 RNA splicing The biogenesis of HIV-1 mRNAs requires the host cell RNA-splicing machinery to produce completely spliced mRNAs, which are transported from the nucleus to the cytoplasm by the endogenous cellular pathway. Splicing of viral RNA is inefficient and results in the accumulation of partially spliced and unspliced RNA whose transport from the nucleus to the cytoplasm is facilitated by the 18-kDa viral regulatory protein Rev. Rev serves as an adapter that targets the viral RNA to the Crm1dependent pathway for nuclear export. The role of Rev in transport of HIV-1 RNA has previously been the subject of a number of reviews and therefore this topic will not be addressed in detail in this chapter (Cullen, 2003; Pollard and Malim, 1998). Rev interacts with a highly structured region in the env gene, the Rev-responsive element (RRE). Early in infection only completely spliced 1.8-kb mRNAs, which encode the viral regulatory proteins Tat, Rev, and Nef, are transported and translated in the cytoplasm. As the infection progresses, sufficient Rev is produced to allow transport of the incompletely spliced 4-kb mRNAs and 9-kb unspliced viral mRNA (Kim et al., 1989; Klotman et al., 1991; Michael et al., 1991). The unspliced mRNA encodes the structural protein precursors Gag and Gag–Pol and also serves as genomic RNA. The incompletely spliced 4-kb mRNAs encode the Env protein as well as accessory proteins Vif, Vpr, and Vpu. Accumulation of incompletely spliced and unspliced
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mRNAs requires retention of the 30 -terminal intron between 50 ss D4 and 30 ss A7, which contains the RRE (Fig. 1B). In general, the identity of the individual HIV-1 mRNAs is determined by the proximity of the open reading frames to the 50 -end of the mRNAs (Fig. 1A). Translation of HIV-1 mRNAs follows the rules of ribosome scanning whereby protein synthesis is most often initiated at the first AUG. (This rule is broken in the case of the Env/Vpu reading frames in which both the 50 -proximal Vpu ORF and the downstream Env ORF are translated from the same set of mRNAs.) Each set of the HIV-1 mRNAs encoding a particular HIV-1 protein is spliced at the 30 ss immediately upstream of the protein open reading frame (Fig. 1B). The extent of splicing at each of the 30 ss is determined by the intrinsic strength of the splice site and by the positive and negative exonic and intronic ciselements that regulate this splice site. Some of the HIV-1 mRNAs are present in relatively high abundance (Env, Nef, Rev) and some are present in low abundance (Vif, Vpr, and Tat; Purcell and Martin, 1993). It has been generally assumed that these differences in mRNA abundance are primarily determined by the different efficiencies by which the splice sites are used.
C. Intrinsic efficiency of HIV-1 splice sites Several approaches have been used to compare the intrinsic strengths of the HIV-1 50 ss and 30 ss compared to the strength of efficient splice sites. One approach was to substitute individual HIV-1 splice sites into a two exon–one intron human b-globin construct. These results indicated that 50 ss D1 and D4 were used as efficiently as the b-globin 50 ss. 50 ss D2 and D3, on the other hand, were used two to three times less efficiently. The efficiency of splicing at the different HIV-1 50 ss was directly related to the relative strengths of the 50 ss predicted by the homologies to the consensus metazoan 50 ss sequence. The HIV-1 30 ss were all shown to be used significantly less efficiently than the b-globin 30 ss (O’Reilly et al., 1995). Since the completion of this study, the roles of additional exonic regulatory elements in the HIV-1 genome have been defined. To determine the effects of these elements, the strengths of the HIV 30 ss were tested with or without their downstream exonic sequences in a one-intron HIV-based env reporter construct. This study indicated that, when the downstream 30 -exonic sequences were absent, 30 ss A1, A4c, A4a, A4b, A5, and A7 were all very inefficient in comparison to an optimized 30 ss. In contrast, 30 ss A2 and A3 were approximately 40% as efficient as the optimized 30 ss. When the exonic sequences were placed downstream of the 30 ss, the efficiencies of 30 ss A1, A4c,a,b, A5, and A7 were greatly increased. On the other hand, the efficiencies of 30 ss A2 and A3 were decreased approximately four- and twofold, respectively. These results emphasized
7
HIV-1 Splicing Regulation and Virus Replication
A
Vpr Nef
Vpu
5⬘ LTR
Vif
Gag Pro
Env
Pol
3⬘ LTR
Tat Rev
B 9-kb
5⬘ss D1
Genomic/ unspliced mRNA
D1a
D2 D3 D4
(D5)
RRE
Gag, pol A1 A2 A3 A5 (A6)
A1a
3⬘ss
A7
A4c,a,b Vif Vpr Tat exon 1 Env/vpu Env/vpu Env/vpu Env/vpu
4-kb mRNA
1
Noncoding exons
Tat exon 1,2 Rev Rev Rev Nef
1.8-kb mRNA
ESEM ESSV ESS2p/ESE2/ ESS2 3 4
ESE-Vif 1
1a 2
4cab G4
2
3
2
3
1.[2].[3].4.7 1.[2].[3].4c.7 1.[2].[3].4a.7 1.[2].[3].4b.7 1.[2].[3].5.7
1
Noncoding exons
C
1.2-I 1.[2].3-I 1.[2].[3].4-I 1.[2].[3].4c-I 1.[2].[3].4a-I 1.[2].[3].4b-I 1.[2].[3].5-I
ISS 6D
(ESE/ESS)
7
ESE2/ESS3
5 GAR
FIGURE 1 Diagrams showing the locations of splice sites, exons, and splicing elements in the HIV-1 genome. (A) Schematic diagram of HIV-1 genome. The open rectangles indicate open reading frames. The long terminal repeats (LTRs) are shown with the three regions comprising the LTRs shown as rectangles: U3-shaded; R-black; U5-open. Full-length RNA transcripts begin at the 50 -end of the R region of the 50 -LTR and poly(A) addition begins at the 30 -end of the R region in the 30 -LTR. Splice sites A6 and D5, which are present only in HXB2 and a few other B clade HIV-1 strains, are shown in parentheses. (B) Locations of 50 and 30 ss in the HIV-1 genome. The location of the RRE is also shown. The exons present in the incompletely spliced 4- and 1.8-kb mRNA species corresponding to the HIV-1 genes are shown as open rectangles. Noncoding exon 1 is present in all spliced HIV-1 mRNA species. Either both or one of the small noncoding exons 2 and 3 shown in black rectangles are included in a fraction of the mRNA species corresponding to the HIV-1 genes. The exon compositions of the RNA species are also shown. Species designated by an ‘‘I’’ are incompletely spliced mRNA species. Brackets indicate that mRNA isoforms containing neither exon 2 or 3, only exon 2 or 3, or both exons 2 and 3 are produced. (C) Locations of known splicing regulatory elements in the HIV-1. The exons are numbered according to the nomenclature shown in (B). Splicing enhancers are designated by white dotted rectangles and splicing silencers are designated by black rectangles.
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the importance of positive exonic sequences for splicing at 30 ss A1, A4c,a,b, A5, and A7 and negative downstream exonic sequences for splicing at 30 ss A2 and A3 (Kammler et al., 2006). One possible reason for the relatively low intrinsic splicing efficiencies of HIV-1 3’ss is the use of nonconsensus branch points at nucleotides other than A residues (Damier et al., 1997; Dyhr-Mikkelsen and Kjems, 1995). Splice sites 30 ss A4c,a,b and A5 have relatively short Py tracts with interspersed purines which could account for their relative low intrinsic efficiency.
D. HIV-1 splice sites and their regulatory elements The mRNAs encoding Vif, Vpr, and Tat are present at low abundance in infected cells or cells transfected with infectious plasmid DNA indicating that 30 ss A1, A2, and A3 are used relatively infrequently. In contrast, the mRNAs for Rev, Env/Vpu, and Nef are present at high abundance indicating that 30 ss A4c,b,a and A5 are used with relatively high frequency. Since approximately half the total spliced mRNA is completely spliced, 30 ss A7 is also used with relatively high efficiency. It has been found that each of the HIV-1 30 ss is regulated by a characteristic set of positive and negative cis-elements that act combinatorially to determine the efficiency by which this splice site is used. Most of these regulatory elements are present in the exonic and intronic sequences immediately downstream of the regulated 30 ss. In most cases, the splicing elements have been shown to be binding sites for cellular RNA-binding proteins. In this section, we summarize our current understanding of how the efficiency of splicing is regulated at each of the HIV-1 30 ss and highlight results indicating that exon definition is the key regulatory step. For a more detailed discussion of HIV-1 ESS, ISS, and ESE, the reader is referred to a previous review (Stoltzfus and Madsen, 2006).
1. Vif mRNA splice site: 30 ss A1 Vif mRNA is an incompletely spliced low abundance mRNA (approximately 1% of the incompletely spliced mRNA in infected cells) which is formed by splicing 50 ss D1 to 30 ss A1 (Fig. 1B). In a fraction of the completely spliced and incompletely spliced vpr, tat, and env/vpu mRNAs, the 50-nt exon defined by 30 ss A1 and 50 ss D2 (exon 2) is included (Purcell and Martin, 1993). Exon 2 does not contain an AUG and becomes part of the 50 -leader region of the mRNAs into which it is included. To test the role of 50 ss D2 on the splicing efficiency at 30 ss A1, mutants in the context of the infectious proviral plasmid pNL4-3 were created in which the predicted affinity of the relatively weak 50 ss D2 for U1 snRNP was increased or decreased relative to the wild-type sequence. These studies indicated that D2-up mutations, with increased affinity to U1 snRNP,
HIV-1 Splicing Regulation and Virus Replication
9
caused greatly increased inclusion of exon 2 as well as increased levels of spliced vif mRNA. This increase in level of vif mRNA was correlated with a corresponding increase in Vif protein levels. D2-down mutants, on the other hand, exhibited decreased levels of vif mRNA and no detectable inclusion of exon 2 into viral mRNAs. In general, the effects on levels of vif mRNA and Vif protein were correlated with the predicted affinity of U1 binding to 50 ss D2. Thus, 50 ss D2 acts as one of the enhancers of splicing at 30 ss A1 and this positive effect on exon definition does not require splicing at 50 ss D2 (Exline et al., 2008). Consistent with this hypothesis, the levels of vif mRNA and Vif protein were restored to wild-type levels by supplying a mutant U1 snRNA in trans whose 50 -end base pairs with the mutated 50 ss D2. The mutant U1 snRNA facilitates wild-type splicing at 30 ss A1 but is incapable of supporting splicing at 50 ss D2 (Mandal et al., 2009). In addition to the downstream 50 ss D2, the definition of exon 2 is facilitated by several other positive elements (Fig. 1B). The first of these elements is localized within the proximal 18 nt of exon 2 and has the properties of an ESE. Mutations within this sequence resulted in a decrease in exon 2 inclusion and greatly reduced levels of vif mRNA when tested in the context of infectious HIV-1 proviral plasmid pNL4-3. In HeLa cell nuclear extracts, this ESE (ESE-Vif) was bound selectively by the SR protein SRp75 (Exline et al., 2008). Two additional elements with the sequence UGGAAAG (M1 and M2) were detected in exon 2 downstream of ESE-Vif. Mutations within either the M1 or M2 sequence reduced exon 2 inclusion when tested using a three exon–two intron construct. Exon 2 sequences bind to the SR protein SF2/ASF; this binding was abrogated by mutations in either M1 or M2. These results were consistent with the hypothesis that M1 and M2 are SF2/ASF-dependent ESE and that both motifs are required for ESE activity. Surprisingly, in the context of pNL4-3, a mutation of M1 alone inhibited exon 2 inclusion into viral mRNAs but did not appear to significantly affect the level of incompletely spliced vif mRNA. This result suggested that the production of incompletely spliced vif mRNA is less dependent on the bipartite M1/M2 ESE than is exon 2 inclusion (Kammler et al., 2006). However, in these experiments, the effect of mutations of M2 alone on vif mRNA splicing were not tested. Mutations within a GGGG motif, which is immediately 30 -proximal of 0 5 ss D2, resulted in an increase in splicing at 30 ss A1, an increase in exon 2 inclusion, and increased vif mRNA and Vif protein when tested in the context of pNL4-3 (Exline et al., 2008). This suggests that the GGGG sequence acts negatively on exon 2 definition. Such 50 ss-proximal GGGG splicing silencers have been recognized in cellular genes and have been hypothesized to operate in concert with hnRNP A1-dependent ESS elements to provide a combinatorial code for splicing silencing in cells. The binding protein responsible for the negative effect on splicing of
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the GGGG motif has not yet been identified. The responsible protein appears not to be hnRNP F or H which do bind to the 50 ss-proximal GGGG motifs but which have been shown to have a positive rather than negative effect on splicing when bound at these sites (Han et al., 2005).
2. Vpr mRNA 30 -splice site: 30 ss A2 Vpr mRNAs are incompletely spliced low abundance mRNAs (approximately 2% of the incompletely spliced mRNA) which are formed primarily by splicing 50 ss D1 to 30 ss A2. There are also minor levels of vpr mRNA in which the 74-nt exon 3, which is defined by 30 ss A2 and 50 ss D3, is included (Fig. 1B). Exon 3 is also included in a fraction of tat, rev, env/vpu, and nef mRNAs (Purcell and Martin, 1993). Exon 3, like exon 2, does not contain an AUG and becomes part of the 50 -leader of these mRNAs. The dominant ciselement regulating splicing at 30 ss A2 is an ESS within exon 3 termed ESSV (Fig. 1C). ESSV is a member of the family of splicing elements with UAGcontaining motifs that preferentially bind to members of the cellular hnRNP A1 protein family (hnRNP A/B proteins; Bilodeau et al., 2001; Caputi et al., 1999; DelGatto-Konczak et al., 1999). Most of the ESSV activity is localized to a 16-nt element containing three (Py/A)UAG motifs (Madsen and Stoltzfus, 2005). Studies in HeLa cell nuclear extracts have indicated that binding of hnRNP A/B proteins to ESSV results in inhibition of binding of U2AF65 to the Py tract of the upstream 30 ss (Domsic et al., 2003). In the context of the infectious proviral plasmid pNL4-3, mutants with lesions in ESSV exhibit greatly increased inclusion of exon 3, an increase in incompletely spliced vpr mRNA, and an increase in Vpr protein (Madsen and Stoltzfus, 2005). In the presence of ESSV, up and down mutations within 30 ss D3 have relatively small effects on splicing at 30 ss A2, which indicates the dominance of the ESS. However when ESSV was inactivated, the 30 ss D3-down mutants dramatically decreased splicing at 30 ss A2 and the level of incompletely spliced vpr mRNA. The extent of this decrease correlated with the reduced predicted binding affinity of 30 ss D3 to U1 snRNA. As has been shown for 50 ss D2, 50 ss D3 acts as an enhancer of splicing and facilitates the production of vpr mRNA. This positive effect on exon 3 definition does not require splicing at 50 ss D3 but is dependent on the strength of U1 snRNP binding to 50 ss D3 ( J. Madsen and C. M. Stoltzfus, unpublished data). Further mutagenesis studies have revealed the presence of an additional positive element or elements within exon 3 downstream of ESSV. Mutations within this element(s) in the context of wild-type or ESSV mutants in subgenomic viral constructs (H. Schaal et al., unpublished data) and in the context of the viral genome (C. M. Stoltzfus et al., unpublished data) cause a decrease in exon 3 inclusion into completely and incompletely spliced mRNAs. The cellular binding protein or proteins interacting with this putative downstream ESE have not yet been identified.
HIV-1 Splicing Regulation and Virus Replication
3. Tat mRNA 30 -splice site: 30 ss A3
11
The tat mRNAs, formed by splicing 50 ss D1, 50 ss D2, or 50 ss D3 to 30 ss A3, are either completely spliced and encode two-exon Tat or incompletely spliced and encode one-exon Tat. The tat mRNAs are relatively low abundance, representing approximately 9% of completely spliced and 5% of incompletely spliced mRNAs (Purcell and Martin, 1993). Tat mRNAs spliced from 50 ss D2 to 30 ss A3 include exon 2 and those spliced from 50 ss D3 to 30 ss A3 include exon 3 or both exons 2 and 3 (Fig. 1B). As shown in Fig. 1C, splicing at 30 ss A3 is repressed by several ESS elements within the first tat coding exon (exon 4). The dominant ESS, an hnRNP A/B-dependent ESS, termed ESS2, is present approximately 70-nt downstream from 30 ss A3 (Amendt et al., 1994). ESS2 was mapped to a 10-nt core sequence containing two PyUAG motifs (Si et al., 1997). A second ESS, termed ESS2p, is present within the 50 -proximal 8-nt region of exon 4 (Jacquenet et al., 2001). ESS2p binds selectively to hnRNP H suggesting that it acts similarly to an hnRNP H-dependent ESS in one of the alternatively spliced exons of the rat b-tropomyosin gene (Chen et al., 1999). Mutagenic inactivation of either ESS2 or ESS2p in the context of the viral genome has indicated that ESS2p is a substantially weaker negative element than ESS2 (P. S. Bilodeau and C. M. Stoltzfus, unpublished data). Further mutagenesis of the 10-nt sequence immediately upstream of ESS2 revealed the presence of an additional cis-element regulating tat mRNA splicing. This region was shown to contain a binding site for the cellular SR protein SC35 which functioned as an ESE in in vitro splicing assays. The element was termed ESE2 (Zahler et al., 2004). Two different groups have shown that the binding sites for SC35 in ESE2 overlap with the hnRNP A1-binding sites in ESS2 and that SC35 and hnRNP A1 compete for these overlapping binding sites (Hallay et al., 2006; Zahler et al., 2004). Based on the data, two alternative models have been proposed to explain how the juxtaposed ESS and ESE elements act to regulate splicing at 30 ss A3. The first model proposes that inhibition of ESS2 activity by depletion of hnRNP A1 or mutations of ESS2 allow binding of SC35 to ESE2. This results in activation of splicing at 30 ss A3 by bridging through the SC35 RS domain to essential splicing factors U2AF and U1 snRNP (Zahler et al., 2004). The second model proposes that SC35 through its RNA-binding domain competes for the overlapping hnRNP A1 sites and blocks cooperative binding of additional hnRNP A1 molecules to exon 4, thus relieving splicing inhibition at 30 ss A3 (Hallay et al., 2006). This model is analogous to a proposed model, summarized below in Section II.D.5, to explain how SF2/ASF relieves hnRNP A/B repression of 30 ss A7.
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4. Rev and env/nef 30 -splice sites: 30 ss A4c, A4a, A4b, and A5
These four 30 ss are contained within a 40-nt region near the middle of the HIV-1 genome (30 ss central cluster) and are used to produce completely spliced rev mRNAs (A4c,a,b), completely spliced nef mRNAs (A5), and incompletely spliced env/vpu mRNAs (Fig. 1B). The mRNAs created by splicing in this region are relatively abundant and represent approximately 90% of the completely spliced mRNAs and 92% of the incompletely spliced mRNAs. Within the incompletely and completely spliced mRNA species arising from splicing in this region are isoforms that include exon 2, exon 3, or both exons 2 and 3. The frequency of splicing at 30 ss A5 is greatest, followed by 30 ss A4a and A4b. The frequency of splicing at 30 ss A4c is very low (Purcell and Martin, 1993). Because the intrinsic strengths of 30 ss A4c, A4a, A4b, and A5 are all very weak, it suggested that activation of these splice sites requires splicing enhancers, the downstream strong 50 ss D4, or both of these positive splicing elements. Indeed, a guanosine–adenosine-rich ESE (GAR ESE) was discovered within exon 5 and downstream of 30 ss A5. The GAR ESE also was shown to activate splicing at the downstream 50 ss D4 and thus is a bidirectional splicing enhancer. The GAR ESE contains two predicted SF2/ASF-binding sites [SF2(1) and SF2(2)] as well as a predicted SRp40binding site. Selective binding of SF2/ASF and SRp40 to GAR ESE was confirmed by experiments to test binding of purified SR proteins (Caputi et al., 2004). In the context of a three-exon, two-intron subgenomic env expression construct, mutations of the SRp40 binding had only a slight effect on splicing within the 30 ss central cluster whereas mutations of both SF2/ASF-binding sites greatly reduced activation of all 30 ss within the 30 ss central cluster. Mutation of only the proximal SF2/ASF-binding site SF2 (1) specifically decreased splicing at 30 ss A5 compared to splicing at 30 ss A4c,a,b; mutation of only the distal site SF2(2) had a smaller effect than mutations of SF2(1) and inhibited splicing at all the 30 ss in the central cluster. A third SF2/ASF-binding site SF2(3), which also contributes to the selective usage of 30 ss A5, was also detected in the region overlapping 30 ss A5. In addition, the region of exon 5 downstream of the GAR, referred to as E42, was shown to be necessary for inclusion of exon 5 and GAR activation of the downstream 50 ss D4. The E42 region by itself does not facilitate U1 snRNP binding in the absence of the GAR enhancer and suggests that E42 may be necessary to recruit additional unidentified factors that mediate interactions between SR proteins bound to GAR and U1 snRNP bound to 50 ss D4 (Asang et al., 2008). In addition to the GAR ESE, splicing at all the 30 ss within the 30 ss cluster was enhanced by the strength of the downstream 50 ss D4, which is used to define exons 4c, 4a, 4b, and exon 5. Mutations with 50 ss D4 that were predicted to decrease affinity for U1 snRNP inhibited splicing at 50 ss D4 and reduced the usage of all 30 ss within the 30 ss cluster. Splicing could be
HIV-1 Splicing Regulation and Virus Replication
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restored upon addition of a compensatory U1 snRNA that re-established complementarity to the mutated 50 ss (Asang et al., 2008). These experiments also showed that, analogous to the results discussed above for 50 ss D2 and D3, exon definition but not splicing at 50 ss D4 is required in order for U1 snRNP to activate the upstream 30 ss splice sites within the 30 ss cluster and enhance the production of incompletely spliced env/vpu mRNAs. Interestingly, in contrast to the effect on exon 5 inclusion, either SF2(1) or SF2(2) alone were not sufficient to activate production of vpu/env mRNA. In addition to the role of cis-acting splicing elements, splicing in the central cluster is affected by overlap of the core splicing signals of the 30 ss. Splicing in the 40-nt region containing 30 ss A4c,a,b and 30 ss A5 involves the usage of different sets of branch points for splicing at each of the four alternative AGs. Two of the branch points used for splicing at 30 ss A5 overlap the AGs used for splicing at 30 ss A4a and A4b (22- and 16-nt upstream of 30 ss A5, respectively; Swanson and Stoltzfus, 1998). Mutations of the 30 ss A4b AG have previously been shown in vitro and in vivo in the context of the viral genome to dramatically increase splicing at 30 ss A5 (Purcell and Martin, 1993; Riggs et al., 1994; Swanson and Stoltzfus, 1998). A possible model for this phenomenon is that factors bound at or near the AG of the A4b splice site may interfere with the formation of spliceosomes at 30 ss A5 by blocking access to the BPS.
5. Nef and tat, rev exon 2 splice site: 30 ss A7
This 30 ss in combination with 50 ss D4 is used to remove the 30 -terminal RRE-containing intron of HIV-1 RNA and generate completely spliced 1.8kb mRNAs for two-exon Tat and Rev as well as Nef (Fig. 1B). Regulation of this splice site is complex and includes several hnRNP A/B-dependent ESS elements, ESE elements, and an intronic splicing silencer (ISS) which was shown to bind hnRNA A1. The ESS was first named ESS3 was mapped to the region 75- to 90-nt downstream from 30 ss A7 (Amendt et al., 1995; Staffa and Cochrane, 1995). Subsequent experiments showed that ESS3 is bipartite and that each of the subelements AGAUC (ESS3a) and UUAG (ESS3b) can inhibit splicing independently (Si et al., 1998). A region upstream of ESS3a with the sequence GAAGAAGAA (GAA3) corresponds to a known ESE element responsive to SF2/ASF and deletion of this element greatly reduced splicing at A7 in vitro and in transfection experiments using a subgenomic construct (Amendt et al., 1995; Staffa and Cochrane, 1995). This suggested its role as an enhancer which was termed ESE3. Subsequently, it was shown that there are additional ESE elements that bind SF2/ASF and SC35 upstream of the (GAA)3 in exon 7 (Mayeda et al., 1999; Tange and Kjems, 2001). Some mutations of the (GAA)3 element increased splicing at A7 rather than decreased splicing as expected for inactivation of an splicing enhancer. These results suggested that the
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(GAA)3 element can act as either an ESS or ESE in the context of exon 7 and for this reason was termed a Janus element (Marchand et al., 2002). Based on in vitro splicing assays and RNA footprinting, it has been proposed that hnRNP A/B proteins exert their negative effect by initiation of binding of hnRNP A/B proteins to ESS3 or (GAA)3 followed by cooperative binding of additional hnRNP A/B proteins to other lower affinity sites within exon 7 and to the ISS. Addition of SF2/ASF either with or lacking the RS domain prevents cooperative hnRNP A/B protein binding initiated at ESS3 or (GAA)3. This failure to initiate cooperative binding of hnRNP A/B proteins is thought to result in increased splicing at 30 ss A7 and binding of U2AF65 to the PPT (Damgaard et al., 2002; Marchand et al., 2002; Zhu et al., 2001). The in vitro splicing data have suggested that in HeLa cell nuclear extracts repression by ESS3 and the intronic ISS dominate activation by exonic ESE elements. In contrast, data obtained by transfection of HeLa cells with a subgenomic construct containing splice sites D4 and A7 and the env gene intron have shown that deletion of ESS3a or deletion of both the (GAA)3 element and ESS3a had little effect on splicing at 30 ss A7. On the other hand, deletion of the (GAA)3 element in the presence of wild-type ESS3a resulted in a dramatic decrease in splicing. These results suggested that one of the functions of the (GAA)3 element is to counteract the effect of ESS3a (Pongoski et al., 2002). It has also been shown that placement of the exon 7 region, which contains ESE3, ESE3a, and ESS3b, downstream of 30 ss A7 resulted in a dramatic increase in splicing at A7 suggesting the dominance of the positively acting ESE3 and other ESE in exon 7 over the negatively acting ESS3 elements (Kammler et al., 2006). These results suggest that there may be differences in the ratio of hnRNP A/B proteins to SF2/ASF in HeLa cell extracts where ESS3 appears to be dominant compared to the same ratio in the nucleus of living cells where ESE3 appears to be dominant.
6. HXB2 tev splice sites: 30 ss A6 and 50 ss D6
The HXB2 HIV-1 strain contains a novel 30 ss within the env gene (30 ss A6) that is not conserved in other HIV-1 strains and a 50 ss (50 ss D5) 170-nt downstream that is conserved in HXB2 and only a few other B clade HIV-1 strains (Fig. 1B). The usage of these two splice sites in HXB2 results in inclusion of exon 6D and the production of a low abundance spliced mRNA encoding a novel 28-kDa protein Tev whose amino acid sequence corresponds to the first tat coding exon, a portion of the env gene encoded by exon 6D, and the second rev exon. The hybrid Tev protein has functional Tat but not Rev activity (Benko et al., 1990; Salfeld et al., 1990). A naturally arising HXB2 point mutant within exon 6D exhibited a dramatic increase in inclusion of this exon. The mutation was localized to a U-to-C change within exon 6D (Wentz et al., 1997). Caputi and Zahler
HIV-1 Splicing Regulation and Virus Replication
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showed that U-to-C, U-to-A, and U-to-G mutations all increase binding of SR proteins to exon 6D and that the mutant sequences functioned as an SC35-dependent ESE when tested in heterologous dsx in vitro splicing substrates. In addition, they showed that, in the context of the U-to-C mutation, hnRNP H family members bind with increased affinity to a GGGA sequence 3 nucleotides downstream from the mutation and this binding is essential for inclusion of exon 6D into HIV-1 mRNAs. A third element that affects exon 6D inclusion in the context of the U-to-C mutation is a polypurine element further downstream in exon 6D. Paradoxically, this element promotes SR protein binding in HeLa cell nuclear extracts but also serves as an ESS when present in a dsx in vitro splicing substrate (Caputi and Zahler, 2002).
7. Gag–pol splice sites: 30 ss A1A and 50 ss D1A
A novel 50 ss and a 30 ss (50 ss D1A and 30 ss A1A) within a highly conserved region of the pol reading frame were found to define a 190-nt exon (exon 1A) that is included into several HIV-1 mRNA species at a very low level (Fig. 1B; Lutzelberger et al., 2006). The sequence of 50 ss D1A is AG/GUAAGA and differs from consensus only at position þ 6 relative to the splice site. The sequence of 30 ss A1A, on the other hand, has a short Py tract and would be predicted on this basis to be relatively weak. The function of the 50 ss D1A in the context of the HIV-1 genome was tested by a mutation to decrease its affinity for U1 snRNA. This resulted in an approximately threefold decrease in the level of unspliced viral RNA. The wild-type level of unspliced RNA was restored in the 50 ss D1A mutant by expression of U1snRNP with a compensatory change in the U1 RNA sequence. These results suggested that 50 ss D1A may be necessary in the HIV-1 genome to prevent degradation of unspliced viral RNA. The authors speculate that one possible mechanism for this effect is that U1 snRNP bound to 50 ss D1A may recruit SR proteins to the viral RNA. These SR proteins may in turn stabilize binding of Rev and result in more efficient export of unspliced RNA.
E. Alternative roles of the major splice donor sites 50 ss D1 and D4 in HIV-1 replication In addition to the role of the major 50 ss D1 and D4 in defining exon 1 and exons 4, 4a, 4b, and 5, respectively, during HIV-1 RNA splicing there is evidence that these two strong splice sites may play additional roles in the expression of HIV-1 RNA. One function for which the strong HIV-1 50 ss have been implicated is in RNA stability. In the context of a single intron env expression vector, Lu et al. showed that mutations within 50 ss D4 resulted in a drastic inhibition of env mRNA accumulation and loss of Env expression either in the presence or absence of Rev. Env expression
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could be recovered by coexpression of a mutated U1 snRNA that restored wild-type U1 base pairing (Lu et al., 1990). These studies were confirmed and extended by Kammler et al. who showed that the affinity of U1 snRNP for 50 ss D4 was directly related to the level of Env expression. Kammler et al. also showed that the conserved dinucleotide GU in 50 ss D4 was required for splicing at 50 ss D4 but not for stabilizing env mRNA and Env expression (Kammler et al., 2001). A subsequent study by the same group showed that in a three-exon, two-intron env expression context, mutations of 50 ss D4 reduced splicing at the central 30 ss cluster but these mutations did not affect transcript stability. The authors speculated that the difference in the results obtained with the three and two exon constructs is the presence of 50 ss D1 in the two-intron construct which may provide the necessary stabilization function that D4 provides in the two exon construct (Asang et al., 2008). Experiments to test the effect of mutating 50 ss D1 in the context of pNL4-3 have shown that such mutations activate a cryptic 50 ss 4-nt downstream from 50 ss D1. All species of spliced mRNA and proteins accumulated at a reduced rate and the 50 ss D1 mutant displayed a delayed production of virus. Since the cryptic 50 ss is a less strong splice site than 50 ss D1, spliced mRNAs may accumulate at a lower rate than wild-type HIV-1 and this may explain the delayed phenotype (Purcell and Martin, 1993). A later study showed that if both 50 ss D1 and the cryptic 50 ss site were mutated in pNL4-3, only unspliced HIV-1 RNA accumulated in transfected cells. This result supported the authors’ hypothesis that downstream splicing of HIV-1 RNA from 50 ss D4 to 30 ss A7 is dependent on prior splicing of the upstream intron from 50 ss D1 to a downstream 30 ss (Bohne et al., 2005). Although not directly addressed by the authors, these results also suggest that 50 ss D1 is not absolutely required for stabilization of unspliced HIV-1 RNA. It is possible that in this mutant, the unspliced RNA stabilization function may supplied by 50 ss D1A (Lutzelberger et al., 2006). A second function of 50 ss D1 is to suppress one of the two 30 -cleavage and polyadenylation sites in the HIV-1 genome. In HIV-1 as in a number of other retroviruses, the poly(A) signals are duplicated within the R regions of the 50 - and 30 -LTRs of the provirus. This necessitates a mechanism whereby the upstream poly(A) site is suppressed and only the downstream poly(A) is used during the processing of the viral RNA. Ashe et al. found that substitution of the heterologous CMV promoter for the HIV-1 LTR promoter or the closeness of the initiation site for transcription to the poly(A) site did not affect the suppression of the upstream poly(A) site. However, mutations that decreased the affinity of U1 snRNP for 50 ss D1, which is 200-nt downstream of the 50 -LTR poly(A) site, activated the usage of this poly(A) site. Suppression was restored by targeting binding of U1 snRNP to a location near the mutated 50 ss A1 (Ashe et al., 1995, 1997). The suppression activity of U1 snRNP requires stem-loop 1 of U1 snRNA
HIV-1 Splicing Regulation and Virus Replication
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and, since U1-70K binds to this region, it suggests that this protein may be responsible for the effect (Ashe et al., 2000).
F. Effects of inclusion of small exons 2 and 3 on HIV-1 mRNA expression and stability Since the discovery of HIV-1 mRNA isoforms containing one or both of the small noncoding exons 2 and 3, there have been a number of studies intended to define possible functions for these exons in mRNA metabolism or translation. This question has been addressed in several ways. One study created nef cDNA expression clones with exon 2, exon 3, or neither exon 2 or 3 upstream of the nef open reading frame and found that each of these expressed Nef equally well (Schwartz et al., 1990). Muesing et al. assayed several different tat expression constructs by their ability to transactivate the LTR promoter. These results showed that the constructs with either exon 2 or 3 in their 50 -leaders demonstrated small increases in transactivation but was not clear from the data whether these differences were statistically significant (Muesing et al., 1987). Krummheuer et al. investigated the effect in HeLa-T4þ cells of exon 2 or 3 both in the context of a single intron env expression vector and in an LTR CAT expression vector. These studies indicated that constructs with either exon 2 or exon 3 in the 50 -leader of the mRNAs resulted in significantly increased or decreased gene expression, respectively. The effects of exons 2 and 3 appeared to be posttranscriptional, affected RNA stability, and occurred in the nucleus (Krummheuer et al., 2001). Madsen and Stoltzfus (2006) found that the stability of total HIV-1 mRNAs in 293T cells transfected with pNL4-3 mutants overexpressing HIV-1 mRNAs containing either exon 2 or exon 3 did not differ significantly from each other or from wildtype viral mRNAs. Further data have indicated that 50 ss D2-down mutations, which completely prevent inclusion of exon 2, do not significantly affect virus replication in permissive T-cell lines under conditions where Vif is not required (Mandal et al., 2009). To explain these discordant results, it is possible that the differences in the effects of the small noncoding exons on RNA stability or function may be dependent either on the types of cells used in the assays or on the HIV-1 constructs that were used to determine the effects.
G. Effects of RNA secondary structure on HIV-1 splicing The secondary structures of the region surrounding three of the HIV-1 30 ss have been determined by chemical and enzymatic probes and the binding sites for cellular splicing factors have been mapped by RNA footprinting analysis. Based on these secondary structure data, models for protein binding to ESS and ESE elements have been proposed.
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This subject has recently been reviewed and thus will not be discussed in detail in this chapter (Saliou et al., 2009). The exon 3 region defined by 30 ss A2 and 50 ss D3 appears to be folded into a single stem-loop structure. In the structural model, the ESSV core sequence is exposed in the loop region and is accessible to binding of hnRNP A1 proteins (Saliou et al., 2009). The region containing the 50 -part of exon 4 and 30 ss A3 is folded into an extended stemloop structure. HnRNP A1 was shown to bind simultaneously to both ESS2 and ESE2, which is proposed to be the initiation site for cooperative binding of additional hnRNP A1 molecules (Hallay et al., 2006). In the model for the region surrounding 30 ss A7, the RNA appears to be folded into a structure with three stem-loops. Stem-loop 1 contains the ISS element, stem-loop 2 contains the 30 ss and ESE3, and stem-loop 3 contains ESS3. The initiation site for hnRNP A1 binding is proposed to be the ESE3 Janus element discussed above in Section II.D.5. Additional hnRNP A/B proteins are then proposed to bind to the other two stem-loop structures. The RNA–protein binding is thought to be stabilized by protein–protein interactions through the Cterminal glycine-rich domains of the hnRNP A/B proteins (Damgaard et al., 2002; Marchand et al., 2002). For each of the three regulated 30 ss, SR protein-binding sites were shown to overlap the sequence where hnRNP A/B protein binding is initiated. SF2/ASF was shown to bind selectively to the region downstream of 30 ss A2 and A7 whereas SC35 binds selectively to the region downstream of 30 ss A3 (Marchand et al., 2002; Saliou et al., 2009; Tange and Kjems, 2001). When overexpressed, the SR proteins are thought to displace the bound hnRNP A/B proteins and thus abrogate the negative effect of hnRNP A1 on splicing. The major HIV-1 50 ss D1 is predicted to be embedded in a relatively stable stem-loop RNA structure (SD stem-loop). When the SD stem was further stabilized by mutagenesis, virus replication was inhibited due to the failure of the viral RNA to be spliced efficiently. Several revertant viruses were isolated upon long-term passage of the mutated virus in which the SD stem-loop was destabilized. In addition, another type of second site mutation occurred upstream of 50 ss D1 within the RNA dimerlinkage structure (DLS). Further analysis indicated that this mutation created an alternative 50 ss which restored splicing and efficient virus replication (Abbink and Berkhout, 2008). Whether the SD stem-loop normally plays an important role in the regulation of HIV-1 splicing has not yet been established.
H. Possible roles of HIV-1 Rev, Tat, and Vpr proteins in regulation of viral RNA splicing Rev-mediated export of unspliced and incompletely spliced viral mRNA has been shown to compete with HIV-1 splicing and increasing the rate of transcript splicing by strengthening the 30 ss results in a decrease of
HIV-1 Splicing Regulation and Virus Replication
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Rev function (Kammler et al., 2006). Rev interaction with the RRE is able to override the cellular functions that normally act to retain the unspliced and incompletely spliced mRNA in the nucleus. An important unanswered question is whether Rev interaction with the RRE, in addition to its role in HIV-1 RNA transport, plays a direct role in inhibiting HIV-1 splicing by interacting in some way with the cellular splicing machinery. A possible link between Rev and splicing factors was suggested by studies of HIV-1 replication in mouse cells. In infected mouse cells, HIV-1 replication is characterized by a decrease in unspliced and incompletely spliced mRNA, a decrease in Gag protein level, and a decrease in virus production. Expression of human p32 protein in several mouse cell lines was shown to reverse this excessive splicing phenotype. A single Gly to Asp mutation at position 35 of the human p32 sequence to change the protein to the corresponding mouse p32 sequence at this position resulted in the loss of rescue (Zheng et al., 2003). Several laboratories have reported that both murine and human p32 proteins bind to the basic domain of HIV-1 Rev (Luo et al., 1994; Tange et al., 1996). The human p32 protein also binds to SF2/ASF and has been shown to inhibit its phosphorylation and RNA-binding activity (Petersen-Mahrt et al., 1999). It has been proposed that p32 could serve as a bridge between Rev, which is bound to the RRE, and SF2/ASF which is bound to HIV-1 ESE elements in the HIV-1 genome. Such an interaction may inhibit the activity of SF2/ASF and cause an inhibition of HIV-1 splicing (Tange et al., 1996). The HIV-1 14-kDa Tat transactivator Tat binds to the TAR sequence at the 50 -end of the HIV-1 RNA and is necessary for facilitating elongation of viral RNA transcription by recruiting elongation factor P-TEFb to the viral promoter (Price, 2000). It has been proposed that, in addition to its wellcharacterized role in transcription, Tat may also have a role in regulation of HIV-1 splicing (Berro et al., 2006). As shown for HIV-1 Rev, Tat also binds to the p32 protein in vitro; this binding was shown to occur with increased affinity when Tat was acetylated at lysines at amino acids 50 and 51 of Tat. Consistent with a role for acetylated Tat in HIV-1 splicing, transfection of a HeLa cell line containing an integrated full-length Tat-minus HIV-1 provirus with a K50A, K51A-mutated Tat expression plasmid resulted in an approximately twofold reduction in the ratio of unspliced to spliced HIV-1 RNA compared to wild-type Tat. When both Tat and p32 were expressed they were shown to colocalize in the nucleus whereas p32 alone was localized in the cytoplasm (Berro et al., 2006). More recent data from this group have implicated a third component of the complex, the cellular kinase CDK13, which was shown to interact with both acetylated Tat and p32 (Berro et al., 2008). Using several assays for HIV-1 splicing, Berro et al. showed that overexpression of CDK13 resulted in an increase in the ratio of spliced to unspliced viral RNA and a decrease in virus replication. Knockdown of CDK13 using siRNA, on the other hand, increased virus
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production in cells transfected with pNL4-3. CDK13 was further shown to bind to SF2/ASF and, using several in vitro assays, shown to phosphorylate SF2/ASF; this is a possible mechanism by which CDK13 can increase splicing of viral RNA. These results led to a model suggesting that early in HIV-1 infection CDK13 may promote complete splicing of HIV-1 RNA through activation of SF2/ASF-dependent ESE. As acetylated Tat accumulates during infection it recruits binding partner p32 which in turn acts to inhibit phosphorylation of SF2/ASF by CDK13. The effect would be to decrease the activity of SF2/ASF and allow increased accumulation of unspliced and incompletely spliced HIV-1 mRNAs later in HIV-1 infection. It was recently shown that, in the context of a single intron HIV-1 env expression construct, splicing of identical RNA transcripts was increased approximately twofold when the RNA was transcribed under control of the CMV promoter compared to the HIV-1 LTR promoter transactivated by Tat (Bohne and Krausslich, 2004). The authors indicate that this effect was independent of acetylation of Tat since K50A Tat mutants did not behave significantly different from wild-type Tat in this assay but they did not test the double K50A, K51A mutant used by Berro et al. (2006). It was suggested by Bohne and Krausslich that the difference in splicing when RNA is produced from the two promoters may indicate that HIV-1 LTR promoters, as well as other retrovirus LTRs, have been selected to produce excessive amounts of unspliced RNA. It should be noted however that, in the context of full-length infectious plasmid pNL4-3 with mutations that prevented the expression of Tat, Chang and Zhang (1995) found no significant differences in the percentages of unspliced, incompletely spliced, and completely spliced HIV-1 mRNAs when the transcription was driven by either by the Tat-transactivated HIV-1 LTR promoter or the CMV promoter in the absence of Tat. It is not clear if these conflicting results are due to the use of different HIV-1 constructs or to differences in the construction of the hybrid promoters. The HIV-1 vpr gene encodes a 14-kDa protein which is incorporated into virions and which plays a role in infection of macrophages and other nondividing cells by facilitating the nuclear import of preintegration complexes. Vpr also acts to inhibit cells in the G2/M phase of the cell cycle. Vpr was also shown to be a general inhibitor of cellular mRNA splicing when tested by in vitro splicing assays but had significantly reduced effect on splicing in cells transiently transfected with Vpr (Kuramitsu et al., 2005). Subsequent experiments indicated that Vpr binds to a cellular splicing-associated protein SAP145 and prevents association of SAP145 and another splicing-associated protein SAP49. It was proposed that the Vpr cell cycle arrest may be caused, not by the inhibition of splicing, but by inhibiting the formation of the SAP145–SAP49 complex, which in turn induced G2 checkpoint activation (Terada and Yasuda, 2006). However, more recent experiments from a number of
HIV-1 Splicing Regulation and Virus Replication
21
laboratories have indicated that G2 checkpoint activation is caused by interaction of Vpr with a Cul4A-containing E3 ligase complex (for a recent review, see Dehart and Planelles, 2008). Thus, the possible significance for virus replication of the Vpr effect on splicing is not yet clear.
III. EVIDENCE FOR THE FUNCTIONAL IMPORTANCE OF HIV-1 SPLICING REGULATORY ELEMENTS IN VIRUS REPLICATION A. Sequence comparison of HIV-1 splice sites and regulatory elements Since its introduction into the human population, the major HIV-1 group, referred to as group M, which represents over 90% of all HIV-1 strains, has spread into many different areas of the world and has diverged into multiple subtypes or clades. Currently HIV-1 has been divided into nine different clades: A–D, F–H, J, and K. In addition, a number of intraclade recombinants have been isolated. Most HIV-1 strains in the United States and Europe belong to the B clade whereas in Africa, most strains belong to the A and C clades. In addition, there are two other more divergent HIV-1 groups, the outlier (group O) and new (group N). In contrast to the group M viruses, groups O and N viruses, which represent only a small number of HIV-1 isolates, have remained confined to a small region of West Africa. Groups M, N, and O are each thought to have originated from independent transmissions of an HIV-1 progenitor from primates to humans (Sharp et al., 2001). One criterion for the functional importance of HIV-1 splice sites and splicing elements is the extent of sequence homology among the different HIV-1 strains. Most of the results we have discussed so far in this chapter have been obtained using the HIV-1 B clade infectious proviral clone pNL4-3, which has served as a model system for numerous studies on HIV-1 replication (Adachi et al., 1986). Sequence comparisons of the pNL4-3 50 - and 30 -splice sites to those in the other group M clades and to groups N and O are shown in Fig. 2. These data show that most of the 50 -and 30 -splice sites are strongly conserved in all the group M clades as well as in groups N and O. An exception is the region containing pNL4-3 rev 30 -splice sites 4c, 4a, and 4b where there is extensive sequence variation among the different virus clades. This high sequence diversity is accompanied by alternative locations for the rev mRNA splice sites even among virus isolates from the same clade (Bilodeau et al., 1999). The sequences of the known HIV-1 splicing elements are compared in Fig. 3. In general, most of the ESE elements (ESE-Vif, ESE M1 and M2, ESE2, ESE3), ESS2p, and ISS are highly conserved among all of the viruses. The GAR ESE is also reasonably conserved among the group M viruses but
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n* a
c
uu
ag
g *g
u
c
ag
g
c
rg
gg
u
a
ac
u
u
a
u
a
unugu*n
a
u u *
au
u c
a
3⬘ss A7 UUAUCGUUUCAG/A
A1
g
a
A2
g
a
B
r
a
r
ag
au
u* r
5⬘ss D1 NL4-3
G/GUGAGU
5⬘ss D1A G/GUAAGA
c
n
g
ugc
g
g *g
u c
gg
g *g
g *gu
5⬘ss D2/G4 G/GUGAAGGGG
u
a g *
a
5⬘ss D3
5⬘ss D4
G/GUAGGA
A/GUAAGU
A1 A2
n
u
u
u
c
g
u
a
g
uc g
c
a
g
g
c*
u
c
g
ac
g
u
g
N
NL4-3
y
u
u
C
O
3⬘ss A4a
UUUUAAAAUUAG/C ACTGTTTTTCAG/A AUCCAUUUUCAG/A UUCAUUGCCAAG/UUUGUUUCAUAACAAAAG/CCTTAG/GCATCTCCTATGGCAG/G
g
B
C
g
D
c g
r
F1
g
a
r
F1
F2
g
a
r
F2
G
g
a
c
G
H
g
u
C D
a c
g
H
J
g
a
J
K
g
a
K
N
c c
g
N
O
c c
n
O
r
g a
c
c
a g
g
g
a
g
FIGURE 2 Sequence alignment of HIV-1 splice sites in different group M clades (A1-K) and groups N and O. Sequences are compared to the pNL4-3 sequence. The locations of the splice sites are indicated by slash marks. The sequences for each clade or group were obtained from
HIV-1 Splicing Regulation and Virus Replication
23
more divergent in the group O strains. ESSV, an hnRNP A/B-dependent ESS, contains three (Py/A)UAG motifs and all of these motifs have been shown to contribute to its silencer activity. All three of the (Py/A)UAG motifs are conserved among A1, A2, B, C, G, H, and J clade viruses and two of three motifs are conserved in D, F1, F2, and K clades. Only one (Py/A) UAG motif is conserved in the group N strains and none of the motifs are conserved in the group O strains. The ESS activity of the group O sequence was tested by substituting the pNL4-3 ESSV with the corresponding sequence from the group O virus MVP5180. When assayed either by in vitro splicing experiments ( J. M. Madsen and C. M. Stoltzfus, unpublished data) or by transfection in the context of an infectious HIV-1 plasmid, the group O sequence lacked ESS activity (Madsen and Stoltzfus, 2005). ESS2 has two (Py/A)UAG motifs in clade B viruses but only one of these motifs is conserved in the other group M clades. One (Py/A)UAG motif in ESS2 was sufficient for ESS activity when tested by in vitro splicing assays and by transfection of minigene constructs derived from the clade B SF2 HIV-1 strain. The corresponding sequence to ESS in the group O virus has no (Py/A)UAG motifs and the sequence has no ESS activity when assayed by in vitro splicing assays or by transfection of minigene constructs (Bilodeau et al., 1999). The single (Py/A)UAG motif in ESS3b and the AGAUCC sequence in ESS3a, which are both conserved in the group M viruses, are not conserved in the group O strains. Since none of the known ESS elements are conserved in the group O viruses, it was of interest to compare the splicing patterns of RNA isolated from cells infected with the group O strain MVP5180 with RNA from cells infected with the group M virus NL4-3. In spite of the absence of ESSV repression at 30 ss A2 and ESS2 repression at 30 ss A3, the levels of vpr and tat mRNA species were not significantly increased and the overall balance of spliced to unspliced mRNA was maintained MVP5180-infected cells (Madsen and Stoltzfus, 2005). These data suggest that group O viruses may contain alternative ESS elements that compensate for the lack of ESSV, ESS2, and ESS3.
B. Mutations of HIV-1 regulatory elements inhibit virus replication Identification and characterization of the cis-elements regulating HIV-1 splicing allowed targeting these sequences for mutations in the infectious HIV-1 genome to determine the effects of the cis-elements on the HIV-1 Sequence Database (Los Alamos National Laboratory) and the predominant sequence is shown. An ‘‘r’’ indicates that either A or G is present at the position; a ‘‘y’’ indicates that C or U is present at the position; and an ‘‘n’’ indicates that any of the four nucleotides are found at this position.
NL4-3
ESE-VIF
ESEM1
ESEM2
ESSV
ESS2p
GCAGAGAUCC
UGGAAAG
UGGAAAG
AUAGUUAGCCCUAGG
UGGGU
A1
g
A2
u
B
y
C
c
D
c
g a
F2
c
g
G
c
H
c
J
c
K
a
ESS3a
UAGAAGAAGAA
ac ac
A1
c
g
A2
c
g
B
c
C
c
c c
G
c
aug
n
g
u
a c
a ay
ag
ac
c
c
u
cn
auun
c
rcuga c r
a
aca
uc a
c u
u gaga gcc
g g
acag
g
an
a ***
c gc
ISS UAGUGAAUAGAGUUAGGCAGGGA
a r
rrr
r g
F2
c g
a a
D F1
u
ESS3b
AGAUCCAUUCGAUUAG
g
acu
g
ESE3 NL4-3
g
cc
r
c
c
O
nn
a
u
g
N
GAR ESE GAAGAAGCGGAGACAGCGACGAAGA
ac
uu r
F1
ESS2
ESE2
CCAGUAGAUAUCCUAGACUAGA
g
a
r gr
a
r ca
r a
r
a
r
H
r a
J
c
g
K
c
r
N
c
O
c
g
a
g g g
g
r
g gg
c
a
a cc
a agca gc
r aca
FIGURE 3 Sequence alignment of known HIV-1 splicing elements in different group M clades and groups N and O. Sequences are compared to the pNL4-3 sequence. The sequences for each clade or group were obtained from the HIV-1 Sequence Database (Los Alamos National Laboratory) and the predominant sequence is shown. An ‘‘r’’ indicates that either A or G is present at the position; a ‘‘y’’ indicates that C or U is present at the position; and an ‘‘n’’ indicates that any of the four nucleotides are found at this position. The (G/Py)UAG motifs in the ESS elements are underlined.
HIV-1 Splicing Regulation and Virus Replication
25
virus replication. The feasibility of this approach was first shown by Wentz et al. (1997) who studied the replication of the naturally arising point mutant discussed above that activated inclusion of exon 6D. The replication of this mutant in T-cell lines was greatly reduced compared to wild type. This reduction in replication was concomitant with greatly increased levels of tev mRNA and Tev protein as well as a reduction in the levels of Gag and Env. These results suggested that inactivation of the splicing regulatory element may cause a decrease in unspliced and incompletely spliced mRNAs. However, this mutant also expressed fourfold reduced levels of full-length Rev protein. Thus, it was not clear from the results whether the mutant phenotype was primarily due to the to the defect in Rev activity or to excessive splicing of the viral RNA. The importance of an HIV-1 hnRNP A/B-dependent ESS element for virus replication was tested by mutating ESSV, which specifically represses splicing at the vpr 30 ss A2 (Madsen and Stoltzfus, 2005). In 293T cells transfected with infectious pNL4-3 plasmid DNAs, ESSV mutants that did not affect the reading frame of the overlapping vif gene produced an increased level of incompletely spliced vpr mRNA and an almost complete inclusion of noncoding exon 3 which is flanked by 30 ss A2 and 50 ss D3. These mutants also demonstrated a large reduction in unspliced viral RNA, reduced Gag protein levels, and a 10- to 20-fold decrease in production of virus particles. The levels of Env, Rev, and Nef, on the other hand, were comparable or somewhat greater than wild type. Tat mRNA levels were somewhat lower than wild type but cotransfection of the ESSV mutant plasmid with excess amounts of a Tat expression plasmid did not alter the observed virus phenotype (Z. Feng and C. M. Stoltzfus, unpublished data). The results of these experiments indicated that ESSV is required to maintain the appropriate balance of unspliced and spliced mRNA necessary for efficient HIV-1 replication. Consistent with this hypothesis, two types of second site revertants were selected after long-term passage of ESSV mutant virus in Jurkat T cells that restored efficient virus production and balanced splicing (Madsen and Stoltzfus, 2005). The first type of mutation changed the conserved AG at 30 ss A2 such that it was no longer recognized as a splice site. The second type of mutation was a U-to-C change within the conserved GU of the downstream 50 ss D3. The data suggested that, by inhibiting exon 3 definition, this 50 ss D3 mutation acts to inhibit excessive splicing at 30 ss A2 caused by disruption of ESSV. HIV-1 with mutations in ESS2, an hnRNP A/B-dependent ESS which represses splicing at the tat 30 ss A3, exhibits an approximate two- to threefold inhibition of virus production in transfected 293T cells, significantly less inhibition than seen with the ESSV mutants (Z. Feng and C. M. Stoltzfus, in preparation). Interestingly, the ESE2 mutation described by Zahler et al. (2004), which by itself results in an approximately twofold
26
C. Martin Stoltzfus
effect on virus replication, exhibited an approximate 10-fold inhibition of virus production when combined with the ESS2 mutation. These results correlated with increased splicing at 30 ss A3 and a significant reduction in the unspliced mRNA level compared to either the ESE2 or ESS2 single mutants. It appeared from these results that, in the context of the HIV-1 genome expressed in cells, both ESS2 and ESE2 are required to repress splicing at 30 ss A3. The effects on HIV-1 replication of mutations within ESE-Vif and 50 ss D2, both of which regulate splicing at the vif 30 ss A1, have also been tested (Exline et al., 2008). Up mutations of 50 ss D2 and mutations within the GGGG motif silencer caused an excessive splicing phenotype similar to the ESSV mutants. This phenotype was characterized by decreased unspliced RNA, decreased Gag protein levels, and a 10- to 20-fold decrease in virus production. On the other hand, 50 ss D2-down mutations did not significantly affect HIV-1 production in transfected 293T cells or in infected T-cell lines such as CEM-SS cells. CEM-SS cells are referred to as permissive cells for Vif-negative viruses because they do not express ApoBec3G (A3G), which has been shown to inhibit HIV-1 replication at an early step. A3G is a cytidine deaminase which causes C-to-U changes in the HIV-1-negative cDNA strand during reverse transcription, leading to extensive G-to-A mutations in the viral genome. The mechanism by which A3G restricts virus replication is still controversial and may be caused by hypermutation, degradation of newly synthesized HIV-1 DNA, interference with reverse transcription, or a combination of these effects. The restrictive effect of A3G on HIV-1 replication requires packaging of A3G into virions and this is prevented by the expression of Vif, which binds to A3G and targets it for ubiquitination/proteasome-mediated degradation. For a recent review on Vif and APOBEC, the reader is referred to Goila-Gaur and Strebel (2008). Interestingly, in T-cell lines which have been shown to be completely nonpermissive for the replication of vif-minus HIV-1 mutants (e.g., H9 cells), the replication of even the weakest D2-down mutant and the ESE-Vif mutant, both of which produce Vif at levels only approximately 5% that of wild type, were only marginally inhibited. To test the phenotype of the D2-down viruses at elevated A3G levels, a Jurkat T-cell line was created that expressed A3G with a doxycycline-repressible Tet-off promoter. Under no doxycycline conditions, that is, at the highest level of A3G, the A3G-Jurkat T-cell lines produced A3G at levels approximately 15-fold higher than control Tet-off cells. Under these conditions, all of the D2-down and ESE-Vif HIV-1 mutants were shown to be less fit than wildtype virus. The extent of the inhibition of mutant virus replication and their sensitivity to A3G were directly related to their expression levels of Vif. Because 50 ss D2 is highly conserved in all strains of HIV-1 and virus replication does not require 50 ss D2 under permissive conditions,
HIV-1 Splicing Regulation and Virus Replication
27
the major function of this splice site may be to modulate the level of vif mRNA splicing and consequently the level of Vif protein (Mandal et al., 2009). If this is the case, it would further suggest that there may be HIV-1 target cells in which the A3G levels are significantly higher than in the normally nonpermissive T-cell lines. Indeed, it has been shown that A3G is upregulated 10-fold in monocyte-derived macrophages treated with IFN-a (Peng et al., 2006; Stopak et al., 2007). A3G was also induced in resting peripheral blood lymphocytes treated with IL-2, IL7, or IL-15 (Stopak et al., 2007). Treatment of the H9 T-cell line with phorbol myristate acetate was shown to induce A3G by approximately 20-fold (Rose et al., 2004). Thus, higher levels of Vif expression may be necessary for efficient HIV-1 replication in those cells in which A3G is upregulated. HIV-1 mutants with the excessive splicing phenotype also exhibited an additional replication defect: a striking increase in the level of Gag precursor relative to products within infected cells (Madsen and Stoltzfus, 2006; Mandal et al., 2008). This was a surprising finding since the locations of the mutations in the viral genome are far from the gag gene. In the case of some of the D2-up mutants, the mutations in D2 also caused changes in the overlapping integrase reading frame. Thus, it was possible that Gag processing could somehow be altered by the mutated integrase. Indeed, it was previously reported that a glutamic acid-to-lysine change at amino acid residue 247 of the integrase reading frame caused a defect in Gag processing and virus particle production that was unique among all C-terminal integrase mutants (Lu et al., 2005). However, this mutation also changed the overlapping 50 ss D2 sequence from G/GUGAAG to G/GUAAG and this change was shown to activate 50 ss D2 (Mandal et al., 2008). To dissect the effect on splicing from the effect on integrase function, second site U-to-A and U-to-G mutations at the U of the conserved GU sequence were constructed to inhibit splicing at 50 ss D2. Because of the redundancy of the genetic code, these mutations did not change the amino acid sequence of the overlapping integrase reading frame. These second site mutations restored the levels of unspliced viral RNA, virus production, and normal Gag processing. Thus, the effect on Gag processing could clearly be shown to be caused by the activation of 50 ss D2 rather than the mutation of integrase in the overlapping reading frame (Mandal et al., 2008). The mechanism by which the excessive splicing mutations cause defects in Gag processing is not yet understood. It is likely that the mutants are not deficient for viral protease since virions with fully processed Gag are produced in the mutant-transfected cells (Mandal et al., 2008). One possible explanation is that particle assembly is affected by reduced amounts of unspliced RNA available for packaging into virions. This also appears to be unlikely since normal levels of virus particles are produced by HIV-1 mutants that do not specifically package viral RNA
28
C. Martin Stoltzfus
(Aldovini and Young, 1990; Lever et al., 1989). It is possible that the low amounts of intracellular Gag produced by the excessive splicing mutants are below the threshold needed to efficiently drive virus assembly. It is also conceivable that unspliced viral RNA produced in the mutantinfected cells is targeted to locations in the cell that are unfavorable for Gag transport and assembly. This scenario, however, would require some quantitative or qualitative difference in the proteins bound to the wild type and mutant unspliced RNA but the nature of such differences and how they would direct the localization of the viral RNA are unclear.
C. Overexpression and siRNA inhibition of cellular splicing factors affect HIV-1 splicing and inhibit virus replication The profound effects on virus replication seen upon mutating HIV-1 splicing elements suggested that similar effects on replication may occur either upon exogenous overexpression of cellular splicing factors binding to these HIV-1 elements or inhibition of cellular splicing factors by treatment with interfering siRNA. This possibility was first addressed in the context of a gag–pol-deleted viral genome in which overexpression of SR proteins SC35, 9G8, and SRp40 were shown to selectively increase splicing at the tat 30 ss A3 at the expense of other viral mRNAs. It was also shown in this study that overexpression of SF2/ASF under the same conditions resulted in increased splicing at 30 ss A2 (Ropers et al., 2004). These data showing the specific effects on splicing at 30 ss A3 were consistent with protein-binding studies indicating that SC35 and SRp40 selectively bind to the region of ESS2 and ESE2 downstream of 30 ss A3 in exon 4 (Hallay et al., 2006). Furthermore, SC35 was shown to selectively activate in vitro splicing of HIV-1 tat gene minigene substrates (Ropers et al., 2004; Zahler et al., 2004). In the context of the infectious virus plasmid pNL4-3, increased splicing at 30 ss A3 in response to SC35 and 9G8 was concomitant with excessive splicing of viral RNA and a greater than 10-fold decrease in virus production. In addition, there was a decrease in intracellular levels of gp160, probably reflecting reduced production of env mRNAs. Similar to the effect of SC35, increased splicing at 30 ss A2 in cells overexpressing SF2/ASF was accompanied by excessive splicing of viral RNA and a drastic reduction of virus production ( Jacquenet et al., 2005). A more complete study has recently been reported describing the effects on HIV-1 RNA metabolism and virus production of overexpressing SR proteins SC35, SF2, and SRp40 and a collection of hnRNP A/B and hnRNP H proteins (Jablonski and Caputi, 2009). As found previously in the context of the subgenomic HIV-1 plasmids, a selective increase in splicing at 30 ss A3 at the expense of other 30 ss was seen when either SC35 or SRp40 were overexpressed. Overexpression of SF2/ASF, on the other
HIV-1 Splicing Regulation and Virus Replication
29
hand, resulted in a selective increase in splicing at both 30 ss A1 and A2. Also, as previously shown (Jacquenet et al., 2005), virus production was shown to be greatly reduced by overexpression of either SC35 or SF2/ASF. Overexpression of either hnRNP A1 or to a lesser extent hnRNP A2 resulted in drastic decreases in the level of all spliced viral mRNAs and virion production. The unspliced viral mRNA level, on the other hand, was elevated fourfold by hnRNP A1 and twofold by hnRNP A2 protein overexpression. In addition to effects on splicing, hnRNP A1 and A2 overexpression also inhibited nuclear to cytoplasmic transport of unspliced HIV-1 RNA. Interestingly, overexpression of either hnRNP A1 or A2 proteins had unique effects that was demonstrated by accumulation of different incompletely spliced 4-kb mRNA species. These results suggest that in HIV-1-infected cells alternative splicing of viral RNA is affected differently by hnRNP A1 compared to A2. This contrasts with studies based on in vitro splicing assays using HIV-1 substrates which showed that the splicing inhibition functions of different members of the hnRNP A/B protein family were redundant (Bilodeau et al., 2001; Caputi et al., 1999; Zhu et al., 2001). Use of siRNAs to reduce SR protein levels resulted in a decrease in the level of tat mRNAs in cells treated with SC35 siRNA but not with SRp40 siRNA (Jablonski and Caputi, 2009). Knockdown of hnRNP A1 and A2 proteins had less of an effect on HIV-1 splicing than when these proteins were overexpressed. Jablonski and Caputi suggest this difference may be due to incomplete inhibition achieved by the hnRNP A1 and A2 siRNAs. The splicing factor overexpression and siRNA experiments taken together further support the hypothesis that tight regulation of HIV-1 splicing is required for efficient virus replication and that this regulation can be abrogated by changes in the levels of cellular splicing factors.
D. Changes in expression of cellular splicing factors during HIV-1 infection Because of the dramatic effects on HIV-1 replication seen as a result of mutations of the HIV-1 splicing elements or changes in the expression of cellular splicing factors that bind to these elements, it suggests that changes in levels of splicing factors during virus infection may be a mechanism to regulate HIV-1 gene expression. There is surprisingly little information yet available on levels of SR proteins and hnRNP proteins during infection of T cells with HIV-1. A two- to threefold increase in level of SC35 was observed 2 days after infection of H9 T-cell line but this study was not followed up to determine if this change in SC35 level was accompanied by concomitant changes in the HIV-1 splicing profile (Maldarelli et al., 1998). It has also been found that 9G8 mRNA was downregulated 60 h after HIV-1 infection of the MT4 T-cell line but
30
C. Martin Stoltzfus
again it was not clear if this decrease in 9G8 was accompanied by changes in HIV-1 splicing (Ryo et al., 2000). Macrophages are also a major target for HIV-1 and are an important virus reservoir in infected humans. HIV-1 infections of cultured monocytederived macrophages have been used to study viral gene expression and replication of the virus. Macrophage infections are characterized by several weeks of productive infection followed by a progressive decline over several weeks in virus production. This decline in virus production was correlated with a specific decrease in mRNA species encoding Tat which could be restored by expression of exogenous Tat (Sonza et al., 2002). The decrease in Tat was shown not to be caused by changes in tat mRNA stability but is likely due to changes in the levels of splicing factors. Levels of proteins hnRNP A1, A2, and H were found to decrease relative to uninfected cells for 1–2 weeks after infection followed by a return to uninfected levels during the subsequent 2–3 weeks. There also was a large increase in relative expression of SC35 in the first week of infection followed by a progressive relative decrease in SC35 over the next 4 weeks of infection. The levels of SF2/ASF, on the other hand, remained more constant during infection (Dowling et al., 2008). These results are consistent with previous results discussed in Section III.C, showing a selective effect of SC35 overexpression and knockdown on tat mRNA splicing. Another possible effect of HIV-1 infection are changes in the phosphorylation of SR proteins. These modifications could affect the activity of the proteins as splicing regulators. This possibility was investigated by analysis of SR proteins in pNL4-3-transfected 293T cells (Fukuhara et al., 2006). This study indicated that there was indeed a decrease in phosphorylated SR proteins but further analysis showed this was due to a decrease in the levels of the SR proteins.
IV. STRATEGIES TO TARGET HIV-1 SPLICING WITH ANTIVIRAL DRUGS Because the data highlighted above indicate that regulation of splicing is essential for efficient HIV-1 replication, it suggests that disruption of this exquisitely balanced system is a reasonable approach for development of novel antiviral drugs. Inhibition of virus production could be accomplished either by selectively inhibiting HIV-1 splicing, as seen with D2-down and ESE-Vif mutants, or by selectively enhancing HIV-1 splicing, as seen with the D2-up and ESSV mutants. To date, two general antiviral approaches have been used; both are directed toward specific inhibition of HIV-1 splicing. First, investigators have searched for specific inhibitors of SR proteins and SR protein kinases. Second, antisense nucleic acid approaches have been used to inhibit HIV-1 splicing,
HIV-1 Splicing Regulation and Virus Replication
31
either with antisense oligonucleotides or with modified snRNPs to target HIV-1 sequences. Fukuhara et al. (2006) investigated the antiviral activity of a specific isonicotinamide compound termed SRPIN340 which inhibits the SR protein kinases SRPK1 and SRPK2. SRPIN340 was shown to inhibit SRp75 phosphorylation and cause a decrease in the stability of the protein. Virus production in pNL4-3-transfected 293T cells appeared to be limited for phosphorylated SRp75 since overexpression of SRp75 resulted in a 10-fold increase in virus production and this effect was enhanced by coexpression of SRPK2. However, subsequent studies showed that SRPIN340 was not an effective inhibitor of HIV-1 replication when assayed by standard T-cell line infections. It is possible that further testing of compounds related to SRPIN340 may lead to more effective inhibitors of virus replication. Soret et al. (2005) showed that an indole derivative IDC16, which interferes with the enhancer activity of SF2/ASF by binding to the RS domain, specifically suppressed the accumulation of completely spliced HIV-1 mRNA without significant effects on accumulation of unspliced viral RNA. These authors found that HIV-1 virus replication in PBLs and macrophages was inhibited approximately fivefold at IDC16 concentrations that did not affect cell viability. Early events of virus replication (early and late reverse transcription and integration) were not affected in the presence of the drug. Although additional studies must be done to assess the effects of IDC16 on expression of cellular genes, these preliminary studies are interesting and suggest that compounds of this class may have promise as therapeutically important anti-HIV drugs. In early studies, antisense oligonucleotides directed against HIV-1 splice sites were shown to have only limited ability to inhibit virus replication (Goodchild et al., 1988). A more recent study has reported a novel antisense strategy based on derivatives of cellular U7 snRNP targeted to splice sites 30 ss A4, A4c, A4ab, A5, and D4 (Asparuhova et al., 2007). U7 constructs targeted against A5/D4 or GAR ESE/D4 were the most effective of any of the targets in inducing exon skipping and thus, inhibiting the splicing of Tat and Rev mRNAs. The modified U7 constructs were inserted into lentiviral vectors which were then used to create CEM-SS T-cell lines in order to assay the effects on HIV-1 replication. Although most of the constructs tested showed little if any effect on virus replication, the strongest effect on wild-type HIV-1 replication (approximately twofold inhibition) was seen in cells expressing a U7 snRNA targeted to GAR ESE and 50 ss D4. The inhibition was significantly enhanced when a Vif-deficient HIV-1 instead of wild-type virus was targeted in the assay. Based on this result, the authors suggest that the U7 strategy may only be effective in combination with other antivirals targeted to other steps of the virus life cycle.
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V. CONCLUSIONS AND PERSPECTIVES We now have a reasonable picture of the cis-elements and trans-acting cellular factors that regulate splicing at each of the HIV-1 alternative splice sites. This picture has been further enhanced by models of RNA secondary structure and the mapping of protein-binding sites in the regions of the HIV-1 30 ss. These models will continue to be further refined by determinations of three-dimensional RNA–protein structures using NMR. The importance for HIV-1 replication of some of the ESS elements and ESE elements have been established by genetic studies. In addition, the highly conserved splice sites downstream of the regulated exons (50 ss D2, D3, and D4) have been shown to be important elements determining the production levels of the incompletely spliced vif, vpr, and env mRNAs, respectively. These studies taken together have established that the exquisite balance of unspliced and spliced mRNA is necessary for efficient virus replication and has provided a strong rationale for developing antivirals specifically targeting HIV-1 splicing. Most of the studies of HIV-1 splicing regulation to date have been performed using HIV-1 strains that belong to the major virus group, group M HIV-1, which represent most of the HIV-1 strains in the world. Sequence comparisons and splicing assays indicated that, although the splice sites of group M and the outlier groups N and O are highly homologous, most of the ESS elements that have been discovered in group M viruses are not conserved at the corresponding positions in the genomes of the group O viruses. These data suggest that the outlier virus strains may contain novel splicing elements at alternative locations in the genome to compensate for the lack of silencers and enhancers that are present in the group M strains. It is now well established that pre-mRNA processing in eukaryotic cells (capping, splicing, and 30 -cleavage/polyadenylation) normally occurs cotranscriptionally (for review, see Kornblihtt et al., 2004; Pandit et al., 2008). This has important implications for retroviruses that depend on the production of large amounts of unspliced and incompletely spliced mRNAs for their survival. It is a particularly relevant issue with a complex retrovirus such as HIV-1 that undergoes extensive splicing with numerous alternative splicing pathways. Little is yet known about the extent to which retroviruses are cotranscriptionally spliced. Using a splice junction in situ hybridization probe, Zhang et al. (1996) could not detect spliced tat mRNA in the nucleus of HeLa cells transfected for 12 h with a subgenomic HIV-1 env expression plasmid. Using another specific probe, on the other hand, these investigators were able to easily detect unspliced RNA in the nucleus in the same cells. Based on this result, it was suggested that efficient splice sites and long transcription units may favor
HIV-1 Splicing Regulation and Virus Replication
33
cotranscriptional splicing whereas inefficient splice sites and relatively short transcription units which characterize retroviruses may favor posttranscriptional splicing. These studies need to be confirmed and extended using more sensitive techniques to investigate the possibility of cotranscriptional splicing in the context of the HIV-1 genome. It will be of interest, for instance, to compare the extent of cotranscriptional splicing occurring with wild-type HIV-1 to the excessive splicing mutants described above in which the frequency of splicing is significantly increased. The potential roles of the small exons 2 and 3 in HIV-1 replication also remain an unresolved issue. As discussed above, conclusions based on results from expression of subgenomic constructs have been contradictory. The results in the context of infectious virus indicate that HIV-1 can replicate in T-cell lines under conditions where either exon 2 or 3 is not included into any viral mRNAs. Also in the context of the viral genome, the stability and function of mRNAs in some cell types were not significantly affected by the presence or absence of these small exons. Thus, mRNA species containing the small exons may be byproducts resulting from the necessity for 50 ss D2 and D3 to serve as regulators of vif and vpr mRNA splicing as well as for the maintenance of an adequate supply of unspliced RNA. This is a delicate balancing act where small changes in the U1 snRNP-binding affinities to the downstream 50 ss can dramatically shift the HIV-1 splicing patterns. Because of the dramatic effects of splicing element mutants and effects of cellular splicing factors on virus replication, the search for additional antiviral drugs that specifically inhibit HIV-1 splicing should be pursued. It is possible that additional drugs that target specific SR proteins required for the activity of HIV-1 ESEs will be discovered. Use of antisense nucleic acids to target HIV-1 ESE and ESS as well as specific HIV-1 splice sites should also be tested using new generation antisense oligonucleotides such as locked nucleic acids (LNAs), peptide nucleic acids (PNAs), and morpholinos. These types of antisense oligonucleotides have been found more stable and effective than standard antisense oligonucleotides (for a recent review, see Karkare and Bhatnagar, 2006).
ACKNOWLEDGMENTS This work was supported by Public Health Service grant AI36073 from the National Institute of Allergy and Infectious Disease. I thank the past and present members of my laboratory and other colleagues for their contributions to the work described in this chapter.
REFERENCES Abbink, T. E., and Berkhout, B. (2008). RNA structure modulates splicing efficiency at the human immunodeficiency virus type 1 major splice donor. J. Virol. 82:3090–3098.
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2 New Insights into Flavivirus Nonstructural Protein 5 Andrew D. Davidson
Contents
I. Introduction II. The Methyltransferase Domain A. 50 -RNA cap formation B. MTase enzymatic activities C. MTase structure D. MTase structure–function studies E. A model for flavivirus cap methylation III. The RNA-Dependent RNA Polymerase Domain A. Flavivirus RNA synthesis B. RdRp activity of NS5 C. RdRp structure D. Structure–function analysis IV. NS5 Interactions A. NS5 intramolecular interactions B. The interaction of NS5 with viral RNA C. The interaction of NS3 and NS5 D. The interaction of NS5 with host proteins V. NS5 Phosphorylation VI. NS5 Localization A. Cellular localization of NS5 B. NS5 nuclear localization VII. Emerging Roles for NS5 in Viral Pathogenesis VIII. Conclusions and Future Perspectives Acknowledgments References
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Department of Cellular and Molecular Medicine, School of Medical and Veterinary Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom Advances in Virus Research, Volume 74 ISSN 0065-3527, DOI: 10.1016/S0065-3527(09)74002-3
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2009 Elsevier Inc. All rights reserved.
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Abstract
Andrew D. Davidson
Disease caused by flavivirus infections is an increasing world health problem. Flavivirus nonstructural protein 5 (NS5) possesses enzymatic activities required for capping and synthesis of the viral RNA genome and is essential for virus replication. NS5 is comprised of two domains. The N-terminal domain binds GTP and can perform two biochemically distinct methylation reactions required for RNA cap formation. The C-terminal domain contains RNA-dependent RNA polymerase activity. As such, NS5 is an interesting target against which antiviral drugs could be developed and research toward this goal has accelerated our understanding of NS5 structure and function in recent years. The production and purification of recombinant versions of either the full-length NS5 or the two individual NS5 domains has led to detailed enzymatic studies on NS5 and the determination of structures of the two NS5 domains. In turn, studies using a combination of structural, biochemical, and reverse genetic approaches are revealing how NS5 performs its multifunctional roles in genome replication. Aside from its localization in the membranebound replication complex, NS5 can be found free in the cytoplasm and for some flaviviruses in the nucleus of virus-infected cells. NS5 is phosphorylated which may potentially regulate NS5 function and trafficking. Recently, NS5 of a number of flaviviruses has been shown to interact with cellular pathways involved in the host immune response, suggesting that NS5 may play a role in viral pathogenesis. This chapter reviews recent advances in our understanding of the multifunctional roles played by NS5 in the virus lifecycle.
I. INTRODUCTION Flaviviruses are small enveloped RNA viruses that comprise one of the three genera in the Flaviviridae family together with the Hepaciviruses and the Pestiviruses. The Flavivirus genus contains at least 53 recognized viral species, which are predominantly transmitted by arthropod vectors. There are 40 flaviviruses capable of causing disease in humans (Gubler et al., 2007). A number of these are medically important pathogens causing significant mortality and morbidity including; the four serotypes of dengue virus (DENV types 1–4), Japanese encephalitis virus ( JEV), tick-borne encephalitis virus (TBEV), West Nile virus (WNV), and yellow fever virus (YFV). Control measures against flaviviruses are limited to vaccines against JEV, TBEV, and YFV and vector control. Currently, there are no antiviral compounds in clinical use against flaviviruses. Difficulties in controlling flaviviral vectors, particularly mosquitoes, societal changes and lapses in vaccine coverage have made DENV, JEV, and WNV among the most important examples of emerging and reemerging viruses (Mackenzie et al., 2004).
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The flavivirus particle consists of two outer membrane proteins, envelope (E) and membrane (M, processed from the precursor prM), surrounding a nucleocapsid containing the capsid (C) protein and a positive sense single-stranded RNA genome. The genome is approximately 11 kb in size with a type I cap structure at the 50 -end but lacks a 30 -polyadenylate tail. The viral RNA contains a single long open reading frame flanked by 50 - and 30 -untranslated regions (UTRs). The UTRs contain a number of cis-acting signals required for viral replication including conserved stem-loop structures. Complementary sequences in the 50 - and 30 -terminal regions (TRs) have been identified that are able to interact and cyclize the genome, a prerequisite for genome replication (reviewed in Markoff, 2003; Villordo and Gamarnik, 2009). The open reading frame is translated as a single polyprotein which is cleaved by a combination of cellular signal peptidase and the virally encoded two component serine protease NS2B/NS3. Proteolysis yields the three structural proteins (C, prM, and E) and seven nonstructural (NS) proteins; NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5. The nonstructural proteins are all involved in viral RNA replication with NS3 and NS5 possessing the enzymatic activities required for RNA capping and genome replication. In addition, specific nonstructural proteins have been shown to play roles in viral assembly and perturbation of host defense mechanisms (Lindenbach and Rice, 2003; Lindenbach et al., 2007). Flavivirus infection results in extensive reorganization and proliferation of cytoplasmic endoplasmic reticular (ER) membranes, leading to the formation of characteristic structures, late in infection, described as vesicle packets (VPs), convoluted membranes (CM), and paracrystalline arrays (PC). Ultrastructural, immunological, and biochemical studies have shown that newly synthesized viral RNA is found in VPs in association with NS1, NS2A, NS3, NS4A, NS4B, and NS5 while NS2B, NS3, and NS4A are found in CM structures (Mackenzie et al., 1998; Miller et al., 2006; Westaway et al., 1997). It has been proposed that the VPs are double-membrane structures that house the replication complex and are the sites of RNA synthesis, the newly synthesized RNA is then exported to the CM/PC for translation and proteolytic processing (Mackenzie, 2005; Westaway et al., 2002, 2003). NS5 is the largest (900 amino acids) and most conserved of the flaviviral proteins. NS5 plays a key role in viral replication, containing enzymatic activities required for capping and synthesis of the RNA genome. Sequence analysis first suggested that NS5 is comprised of an methyltransferase N-terminal S-adenosyl-L-methionine-dependent domain (Koonin, 1993) and a C-terminal RNA-dependent RNA polymerase (RdRp) domain (Koonin, 1991; Poch et al., 1989; Rice et al., 1985; Sumiyoshi et al., 1987). The N-terminal domain has since been shown to have the ability to bind GTP (Egloff et al., 2002) and perform two biochemically distinct methylation reactions required for RNA cap formation
44
Andrew D. Davidson
(Egloff et al., 2002; Ray et al., 2006). Recombinant versions of NS5, including the C-terminal domain alone, have been demonstrated to have RdRp activity in in vitro assays (Ackermann and Padmanabhan, 2001; Guyatt et al., 2001; Selisko et al., 2006; Steffens et al., 1999; Tan et al., 1996). The X-ray crystal structures of the two individual domains have been determined for representative flaviviruses (Assenberg et al., 2007; Egloff et al., 2002; Malet et al., 2007; Mastrangelo et al., 2007; Yap et al., 2007a; Zhou et al., 2007). Recent advances in our understanding of the enzymatic activities of NS5, coupled with structure–function and interaction studies are beginning to reveal how NS5 performs its multifunctional roles in viral replication, making NS5 an interesting target for antiviral drug development (reviewed in (Dong et al., 2008c; Malet et al., 2008; Rawlinson et al., 2006)). In addition to its role in the viral replication complex, recent studies have shown that NS5 may also play a role in pathogenesis. NS5 is not only found associated with the membrane-bound replication complex but free in the cytoplasm and for some flaviviruses in the nucleus where it potentially interacts with host factors. The NS5 of specific flaviviruses can perturb interferon signaling and cytokine production (Best et al., 2005; Lin et al., 2006; Medin et al., 2005; Werme et al., 2008). NS5 is known to be phosphorylated, providing a mechanism by which NS5 enzymatic activity, molecular interactions, and trafficking could be regulated (Lindenbach et al., 2007). This chapter will focus on reviewing recent advances in our understanding of NS5 replicative function and trafficking and the possible role of NS5 in pathogenesis. As the functional and structural properties of the N- and C-terminal domains have largely been investigated in isolation, studies on each domain will be reviewed separately.
II. THE METHYLTRANSFERASE DOMAIN A. 50 -RNA cap formation Cellular and many viral mRNAs contain a modified 50 -terminal guanosine ‘‘cap’’ structure covalently linked to the 50 -end of the mRNA. The formation of the 50 -RNA cap structure requires three sequential enzymatic reactions: (1) the 50 -terminal triphosphate of the nascent RNA is hydrolyzed to a diphosphate by the enzyme RNA triphosphatase, (2) the RNA is capped with GMP in a 50 –50 -triphosphate linkage by mRNA guanyltransferase, and (3) the guanosine is methylated at the N7 position by a (guanine-N7)-methyltransferase (N7 MTase) using S-adenosyl7 L-methionine (AdoMet) as a methyl donor to form a type 0 (m GpppN) cap structure. Nucleotides adjacent to the cap structure may be further methylated by nucleoside-20 -O-methyltransferases (20 -O-MTase), to give type I (m7GpppNm) or type II cap structures (m7GpppNmNm) (Furuichi
Flavivirus NS5
45
and Shatkin, 2000; Shuman, 2001). Vector-borne flaviviruses have identical type I cap structures (m7GpppAmG) (Cleaves and Dubin, 1979; Wengler et al., 1978) as the first two nucleotides (AG) of the genome are strictly conserved (Markoff, 2003). Formation of the flavivirus cap structure is believed to involve NS3 which has RNA triphosphatase activity (Bartelma and Padmanabhan, 2002; Benarroch et al., 2004b; Wengler, 1993) and NS5 that has both N7 and 20 -O-MTase activities (Egloff et al., 2002; Ray et al., 2006). As yet, the source of the mRNA guanyltransferase activity has not been identified. Although the mechanism by which the cap structure is formed is functionally conserved in eukaryotes and many viruses, the architecture of the capping enzymes and sequence of capping reactions varies (Furuichi and Shatkin, 2000). In the simplest case, such as for yeast, each of the enzymatic activities required for RNA capping resides in an individual protein. In contrast, for vaccinia virus, the best characterized viral system, capping is performed by a heterodimeric enzyme. The larger D1 subunit has RNA triphosphatase and mRNA guanyltransferase activities, while full N7 MTase activity requires the formation of a complex with the smaller D12 subunit (Shuman, 1995). A third protein, VP39, possesses 20 -O-MTase activity. For dsRNA viruses of the family Reoviridae and negative strand RNA viruses of the order Mononegavirales, all of the enzymatic activities have been detected in single large multidomain proteins (Furuichi and Shatkin, 2000; Hercyk et al., 1988; Ogino et al., 2005; Ramadevi et al., 1998). Structural studies on Reoviridae proteins have assigned distinct enzymatic activities to individual protein domains (Reinisch et al., 2000; Sutton et al., 2007). Interestingly, the L protein of vesicular stomatitis virus (VSV), similar to the flavivirus NS5, exhibits both N7 and 20 -O-MTase activities but contains a single AdoMet-binding site (Li et al., 2006).
B. MTase enzymatic activities The identification of a sequence motif that is conserved in AdoMetdependent MTases first suggested that the N-terminal region of NS5 may have MTase activity (Koonin, 1993). Further bioinformatic analysis delineated a potential MTase domain in the first 296 amino acids of the DENV-2 NS5 (DENV-2 MTase) (Fig. 1). Examination of the MTase activity of the bacterially expressed DENV-2 MTase, using short capped (GpppAC5 and m7GpppAC5) and noncapped (pppAC5) RNA substrates, showed that the MTase had cap-dependent 20 -O-MTase activity (Egloff et al., 2002). Under the experimental conditions used, N7 MTase activity was not detectable. More recently, the MTase activities of a bacterially expressed, full-length WNV NS5 and a truncated NS5, containing the MTase domain (amino acids 1–300), were examined using a capped RNA transcript corresponding to the first 190 nucleotides of the WNV
MA1 DV-1 DV-3 DV-2 DV-4 JEV MVEV WNV KUNV YFV TBEV LGTV MEAV MoDV CFA
MA2
MB1
MA3
MaX
X
Mb2
DV-1 DV-3 DV-2 DV-4 JEV MVEV WNV KUNV YFV TBEV LGTV MEAV MoDV CFA
MaA
Mb1
+ 20 + 40 + 60# + 80 + ---GTGAQGETLGEKWKRQLNQLSKSEFNTYKRSGIIEVDRSEAKEGLKRGE-PTKHA--VSRGTAKLRWFVERNLVKPEGKVIDLGCGRGGWSYYCAGLKKV ---GTGSQGETLGEKWKKKLNQLSRKEFDLYKKSGITEVDRTEAKEGLKRGE-TTHHA--VSRGSAKLQWFVERNMVVPEGRVIDLGCGRGGWSYYCAGLKKV ---GTGNIGETLGEKWKSRLNALGKSEFQIYKKSGIQEVDRTLAKEGIKRGE-TDHHA--VSRGSAKLRWFVERNMVTPEGKVVDLGCGRGGWSYYCGGLKNV ---GTGTTGETLGEKWKRQLNSLDRKEFEEYKRSGILEVDRTEAKSALKDGS-KIKHA--VSRGSSKIRWIVERGMVKPKGKVVDLGCGRGGWSYYMATLKNV ----GRPGGRTLGEQWKEKLNAMSREEFFKYRREAIIEVDRTEARRARRENNIVGGHP--VSRGSAKLRWLVEKGFVSPIGKVIDLGCGRGGWSYYAATLKKV ----GRAGGRTLGEQWKEKLNAMGKEEFFSYRKEAILEVDRTEARRARREGNKVGGHP--VSRGTAKLRWLVERRFVQPIGKVVDLGCGRGGWSYYAATMKNV ----GGAKGRTLGEVWKERLNQMTKEEFTRYRKEAIIEVDRSAAKHARKEGNVTGGHP--VSRGTAKLRWLVERRFLEPVGKVIDLGCGRGGWCYYMATQKRV ----GGAKGRTLGEVWKERLNQMTKEEFIRYRKEAITEVDRSAAKHARKERNITGGHP--VSRGTAKLRWLVERRFLEPVGKVIDLGCGRGGWCYYMATQKRV ----GSANGKTLGEVWKRELNLLDKRQFELYKRTDIVEVDRDTARRHLAEGKVDTGVA--VSRGTAKLRWFHERGYVKLEGRVIDLGCGRGGWCYYAAAQKEV ----GGSEGDTLGDLWKRRLNNCTREEFFVYRRTGILETERDKARELLRRGETNVGLA--VSRGTAKLAWLEERGYATLKGEVVDLGCGRGGWSYYAASRPAV ----GGSEGDTLGDMWKARLNSCTKEEFFAYRRAGVMETDREKARELLKRGETNMGLA--VSRGTSKLAWMEERGYVTLKGEVVDLGCGRGGWSYYAASRPAV ---GPGSTGASLGMMWKDKLNAMTKEEFTRYKRAGVMETDRKEARDYLKRGDGKTGLS--VSRGTAKLAWMEERGYVELTGRVVDLGCGRGGWSYYAASRPHV ---GICSSAPTLGEIWKRKLNQLDAKEFMAYRRRFVVEVDRNEAREALAKGKTNTGHA--VSRGTAKLAWIDERGGVELKGSVVDLGCGRGGWSYYAASQPNV MHIAARALGAVAPFNQFRALEKSTTIGLGMKWKMTLNALDGDAFTRYKSRGVNETERGDYVSRGGLKLNEIISKYEWRPSGRVVDLGCGRGGWSQRAVMEETV * **** * * * ********* *
Mb3
I
MaD
Mb4
Mb5 #
MaE
100 + 120 + 140 # + 160 + 180 + TEVKGYTKGGPGHEEPIPMATYGWNLVKLYSGKDVFFTPPEKCDTLLCDIGESSPNPTIEEGRTLRVLKMVEPWLRGN---QFCIKILNPYMPSVVETLEQMQ TEVRGYTKGGPGHEEPVPMSTYGWNIVKLMSGKDVFYLPPEKCDTLLCDIGESSPSPTVEESRTIRVLKMVEPWLKNN---QFCIKVLNPYMPTVIEHLERLQ REVKGLTKGGPGHEEPIPMSTYGWNLVRLQSGVDVFFTPPEKCDTLLCDIGESSPNPTVEAGRTLRVLNLVENWLNNNT--QFCIKVLNPYMPSVIEKMEALQ TEVKGYTKGGPGHEEPIPMATYGWNLVKLHSGVDVFYKPTEQVDTLLCDIGESSSNPTIEEGRTLRVLKMVEPWLSSKP--EFCIKVLNPYMPTVIEELEKLQ QEVRGYTKGGAGHEEPMLMQSYGRNLVSLKSGVDVFYKPSEPSDTLFCDIGESSPSPEVEEQRTLRVLEMTSDWLHRGP-REFCIKVLCPYMPKVIEKMEVLQ QEVRGYTKGGPGHEEPMLMQSYGWNIVTMKSGVDVFYKPSEISDTLLCDIGESSPSAEIEEQRTLRILEMVSDWLSRGP-KEFCIKILCPYMPKVIEKLESLQ QEVRGYTKGGPGHEEPQLVQSYGWNIVTMKSGVDVFYRPSECCDTLLCDIGESSSSAEVEEHRTIRVLEMVEDWLHRGP-REFCVKVLCPYMPKVIEKMELLQ QEVRGYTKGGPGHEEPQLVQSYGWNIVTMKSGVDVFYRPSECCDTLLCDIGESSSSAEVEEHRTLRVLEMVEDWLHRGP-KEFCVKVLCPYMPKVIEKMELLQ SGVKGFTLGRDGHEKPMNVQSLGWNIITFKDKTDIHRLEPVKCDTLLCDIGESSSSSVTEGERTVRVLDTVEKWLACGV-DNFCVKVLAPYMPDVLEKLELLQ MSVRAYTIGGKGHEAPKMVTSLGWNLIKFRSGMDVFSMQPHRADTVMCDIGESSPDAAVEGERTRKVILLMEQWKNRNPTAACVFKVLAPYRPEVIEALHRFQ MSVRAYTIGGKGHESPRMVTSLGWNLIKFRAGMDVFSMEPHRADAILCDIGESNPDAVVEGERSRRVILLMEQWKNRNPTATCVFKVLAPYRPEVIEALHRFQ MDVRAYTLGVGGHEVPRITESYGWNIVKFKSRVDIHTLPVERTDVIMCDVGESSPKWSVESERTIKILELLEKWKVKNPSADFVVKVLCPYSVEVMERLSVMQ REVKAYTLGTSGHEKPRLVETFGWNLITFKSKVDVRKMEPFQADTVLCDIGESNPTAAVEASRTLTVLNVISRWLEYNQGCGFCVKVLNPYSCDVLEALMKMQ SSALGFTIGGAEKENPQRFVTKGYNLATLKTGVDVHRLTPFRCDTIMCDIGESDPSPIKEKTRTLKVLQLLENWLLVNPGAHFVCKILSPYSLEVLRKIESLQ * * * * * * * * ** *** * * * * * ** * *
II
III
IV
FIGURE 1
V
(Continued)
97 97 97 97 97 97 97 97 97 97 97 98 98 103
VI
VII
197 197 198 198 199 199 199 199 199 200 200 201 201 206
DV-1 DV-3 DV-2 DV-4 JEV MVEV WNV KUNV YFV TBEV LGTV MEAV MoDV CFA
MaE Mb6 MB2 Pa1 MA4 Mb6 200 + # 220 + 240 + 260 + 280 + RKHGGMLVRNPLSRNSTHEMYWVSCGTGNIVSAVNMTSRMLLNRFTMAHRK-PTYERDVDLGAGTRHVAVEPEV-ANLDIIGQRIENIKNGHKSTWHYDEDNP RKHGGMLVRNPLSRNSTHEMYWISNGTGNIVSSVNMVSRLLLNRFTMTHRR-PTIEKDVDLGAGTRHVNAEPET-PNMDVIGERIKRIKEEHNSTWHYDDENP RKHGGALVRNPLSRNSTHEMYWVSNASGNIVSSVNMISRMLINRFTMRHKK-ATYEPDVDLGSGTRNIGIESEI-PNLDIIGKRIEKIKQEHETSWHYDQDHP RKHGGNLVRCPLSRNSTHEMYWVSGASGNIVSSVNTTSKMLLNRFTTRHRK-PTYEKDVDLGAGTRSVSTETEK-PDMTIIGRRLQRLQEEHKETWHYDQENP RRFGGGLVRLPLSRNSNHEMYWVSGAAGNVVHAVNMTSQVLLGRMDRTVWRGPKYEEDVNLGSGTRAVGKGEVH-SNQEKIKKRIQKLKEEFATTWHKDPEHP RRFGGGLVRVPLSRNSNHEMYWVSGASGNIVHAVNMTSQVLIGRMDKKIWKGPKYEEDVNLGSGTRAVGKGVQH-TDYKRIKSRIEKLKEEYAATWHTDDNHP RRYGGGLVRNPLSRNSTHEMYWVSRASGNVVHSVNMTSQVLLGRMEKRTWKGPQYEEDVNLGSGTRAVGKPLLN-SDTSKIKNRIERLRREYSSTWHHDENHP RRYGGGLVRNPLSRNSTHEMYWVSRASGNVVHSVNMTSQVLLGRMEKKTWKGPQYEEDVNLGSGTRAVGKPLLN-SDTSKIKNRIERLRREYSSTWHHDENHP RRFGGTVIRNPLSRNSTHEMYYVSGARSNVTFTVNQTSRLLMRRMRRPTGK-VTLEADVILPIGTRSVETDKGP-LDKEAIEERVERIKSEYMTSWFYDNDNP LQWGGGLVRTPFSRNSTHEMYYSTAVTGNIVNSVNVQSRKLLARFGD--QRGPTKVPELDLGVGTRCVVLAEDK-VKEQDVQERIRALREQYSETWHMDEEHP LQWGGGLVRTPFSRNSTHEMYFSTAITGNIVNSVNIQSRKLLARFGD--QRGPTRVPEIDLGVGTRCVVLAEDK-VKEKDVMERIQALKDQYCDTWHEDHEHP RKWGGGLVRNPYSRNSTHEMYFTSRAGGNIIGAVTACTERLLGRMAR--RDGPVVVPELNLGTGTRCVTLAEDK-VSRDLIDERLAKIKSQYAASWLEDENHP RRFGGGLIRVPLSRNSTHEMYFVSGIKNNIMGNVTAVSRQLLKRMEE--QGGERVVPDYKFSTGTRSNLTQKIE-VPEEEVQMRVDKIKAEKSGTWCFDSNHP HLYNGRLVRLSHSRNSSVEMYYISGARSNVVRTTYMTLAALMARFSR--HLDSVVLPSPVLPKGTRADPAASVASMNTSDMMDRVERLMNENRGTWFEDQQHP * * **** *** * * * *** * * * *
VIII
DV-1 DV-3 DV-2 DV-4 JEV MVEV WNV KUNV YFV TBEV LGTV MEAV MoDV CFA
298 298 299 299 301 301 301 301 300 300 300 301 301 307
Interdomain Linker
Pa3 Pa5 Pa4 Pa6 Pa7 Pa2 L2 L1 Pb1 301 + 320 + 340 + 360 + 380 + YKTWAYHGSYEVKPSGSASSMVNGVVRLLTKPWDVIPMVTQIAMTDTTPFGQQRVFKEKVDTRTPKAKRGTAQIMEVTARWLWGFLSRNK-KPRICTREEFTR YKTWAYHGSYEVKATGSASSMINGVVKLLTKPWDVVPMVTQMAMTDTTPFGQQRVFKEKVDTRTPRPLPGTRKVMGITAEWLWRTLGRNK-RPRLCTREEFTK YKTWAYHGSYETKQTGSASSMVNGVVRLLTKPWDVVPMVTQMAMTDTTPFGQQRVFKEKVDTRTQEPKEGTKKLMKITAEWLWKELGKKK-TPRMCTREEFTR YRTWAYHGSYEAPSTGSASSMVNGVVKLLTKPWDVIPMVTQLAMTDTTPFGQQRVFKEKVDTRTPQPKPGTRMVMTTTANWLWALLGKKK-NPRLCTREEFIS YRTWTYHGSYEVKATGSASSLVNGVVKLMSKPWDAIANVTTMAMTDTTPFGQQRVFKEKVDTKAPEPPAGAKEVLNETTNWLWAHLSREK-RPRLCTKEEFIK YRTWTYHGSYEVKPSGSASTLVNGVVRLLSKPWDAITGVTTMAMTDTTPFGQQRVFKEKVDTKAPEPPQGVKTVMDETTNWLWAYLARNK-KARLCTREEFVK YRTWNYHGSYDVKPTGSASSLVNGVVRLLSKPWDTITNVTTMAMTDTTPFGQQRVFKEKVDTKAPEPPEGVKYVLNETTNWLWAFLAREK-RPRMCSREEFIR YRTWNYHGSYEVKPTGSASSLVNGVVRLLSKPWDTITNVTTMAMTDTTPFGQQRVFKEKVDTKAPEPPEGVKYVLNETTNWLWAFLAREK-RPRMCSREEFIR YRTWHYCGSYVTKTSGSAASMVNGVIKILTYPWDRIEEVTRMAMTDTTPFGQQRVFKEKVDTRAKDPPAGTRKIMKVVNRWLFRHLAREK-NPRLCTKEEFIA YRTWQYWGSYRTAPTGSAASLINGVVKLLSWPWNAREDVVRMAMTDTTAFGQQRVFKDKVDTKAQEPQPGTRVIMRAVNDWILERLAQKS-KPRMCSREEFIA YRTWQYWGSYKTAATGSSASLLNGVVKLLSWPWNAREDVVRMAMTDTTAFGQQRVFKDKVDTKAQEPQPGTKIIMRAVNDWLLERLVKKS-RPRMCSREEFIA YRTWQYWGSYRCADSGSAASLINGIVKMMSWPWNNREDVCLMAMTDTTAFGQQRVFKDKVDTKAQEPRVGTRVVMRTVNNWLLERLSRKS-KPRLCTREEFIQ YRTWNYHGSYRVRDVGTRASAVNHVVKLLSWPWGKMEKVLAMSMTDTTAFGQQRVFKQKVDTKAPEPNIQVKKVMRKVFKWLIERIKTKGGKVRTCTKEEFIQ YKSFKYFGSFVTDDVKVGGQAVNPLVRKIMWPWETLTSVVGFSMTDVSTYSQQKVLREKVDTVIPPHPQHIRRVNRTITKHFIRLFKNRNLRPRILSKEEFVA * * ** * ** * *** ** * **** * ***
“bNLS”
FIGURE 1
“a/bNLS”
(Continued)
400 400 401 401 403 403 403 403 402 402 402 403 404 410
DV-1 DV-3 DV-2 DV-4 JEV MVEV WNV KUNV YFV TBEV LGTV MEAV MoDV CFA
Pa9 Pa7 Pa8 Pa10 Pa11 L3 403 + 420 + 440 + 460 + 480 + 500 KVRSNAAIGAVFVDENQWNSAKEAVEDERFWDLVHRERELHKQGKCATCVYNMMGKREKKLGEFGKAKGSRAIWYMWLGARFLEFEALGFMNEDHWFSRENSL KVRTNAAMGAVFTEENQWDSAKAAVEDEEFWKLVDRERELHKLGKCGSCVYNMMGKREKKLGEFGKAKGSRAIWYMWLGVRYLEFEALGFLNEDHWFSRENSY KVRSNAALGAIFTDENKWKSAREAVEDSRFWELVDKERNLHLEGKCETCVYNMMGKREKKLGEFGKAKGSRAIWYMWLGARFLEFEALGFLNEDHWFSRENSL KVRSNAAIGAVFQEEQGWTSASEAVNDSRFWELVDKERALHQEGKCESCVYNMMGKREKKLGEFGRAKGSRAIWYMWLGARFLEFEALGFLNEDHWFGRENSW KVNSNAALGAVFAEQNQWSTAREAVDDPRFWEMVDEERENHLRGECHTCIYNMMGKREKKPGEFGKAKGSRAIWFMWLGARYLEFEALGFLNEDHWLSRENSG KVNSHAALGAMFEEQNQWKNAREAVEDPKFWEMVDEERECHLRGECRTCIYNMMGKREKKPGEFGKAKGSRAIWFMWLGARFLEFEALGFLNEDHWMSRENSG KVNSNAALGAMFEEQNQWRSAREAVEDPKFWEMVDEEREAHLRGECHTCIYNMMGKREKKPGEFGKAKGSRAIWFMWLGARFLEFEALGFLNEDHWLGRKNSG KVNSNAALGAMFEEQNQWRSAREAVEDPKFWEMVDEEREAHLRGECHTCIYNMMGKREKKPGEFGKAKGSRAIWFMWLGARFLEFEALGFLNEDHWLGRKNSG KVRSHAAIGAYLEEQEQWKTANEAVQDPKFWELVDEERKLHQQGRCRTCVYNMMGKREKKLSEFGKAKGSRAIWYMWLGARYLEFEALGFLNEDHWASRENSG KVKSNAALGAWSDEQNRWASAREAVEDPAFWRLVDEERERHLMGRCAHCVYNMMGKREKKLGEFGVAKGSRAIWYMWLGSRFLEFEALGFLNEDHWASRESSG KVRSNAALGAWSDEQNKWKSAREAVEDPEFWSLVEAERERHLQGRCAHCVYNMMGKREKKLGEFGVAKGSRAIWYMWLGSRFLEFEALGFLNEDHWASRASSG KVRSNAAIGAWLDEQNQWKNAREAVEDPRFWRMVDEERELHLQGRCATCVYNMMGKREKKAGEFGKAKGSRAIWYMWLGSRFLEFEALGFLNEDHWASREKSG KVRSHAAIGAWSSDMEGWSSAVEAVDDPRFWNMVQKERDLHLQGKCEMCVYNLMGKSEKKPGDFGVAKGSRTIWYMWLGSRFLEFESFGFLNEEHWASRELSG NVRNDAAVGSWSRDVP-WRDVQEAIQDQCFWDLVGKERALHLQGKCEMCIYNTMGKKEKKPSLAGEAKGSRTIWYMWLGSRFLEFEALGFLNADHWVSREHFP * ** * * * * ** * ** * * * * ** *** *** * ***** ** **** * **** ** * ** *
503 503 504 504 506 506 506 506 505 505 505 506 507 512
F
DV-1 DV-3 DV-2 DV-4 JEV MVEV WNV KUNV YFV TBEV LGTV MEAV MoDV CFA
Pa12 a13 Pa15 Pa14 Pb3 Pb2 506 + 520 + 540 + 560 + 580 + 600 SGVEGEGLHKLGYILRDISKIPGGNMYADDTAGWDTRITEDDLQNEAKITDIMEPE--HALLATSIFKLTYQNKVVRVQRPA--KNG-TVMDVISRRDQRGSG SGVEGEGLHKLGYILRDISKIPGGAMYADDTAGWDTRITEDDLHNEEKIIQQMDPE--HRQLANAIFKLTYQNKVVKVQRPT--PTG-TVMDIISRKDQRGSG SGVEGEGLHKLGYILRDVSKKEGGAMYADDTAGWDTRITLEDLKNEEMVTNHMEGE--HKKLAEAIFKLTYQNKVVRVQRPT--PRG-TVMDIISRRDQRGSG SGVEGEGLHRLGYILEEIDKKDGDLMYADDTAGWDTRITEDDLQNEELITEQMAPH--HKILAKAIFKLTYQNKVVKVLRPT--PRG-AVMDIISRKDQRGSG GGVEGSGVQKLGYILRDIAGKQGGKMYADDTAGWDTRITRTDLENEAKVLELLDGE--HRMLARAIIELTYRHKVVKVMRPA--AEGKTVMDVISREDQRGSG GGVEGAGIQKLGYILRDVAQKPGGKIYADDTAGWDTRITQADLENEAKVLELMEGE--QRTLARAIIELTYRHKVVKVMRPA--AGGKTVMDVISREDQRGSG GGVEGLGLQKLGYILREVGTRPGGKIYADDTAGWDTRITRADLENEAKVLELLDGE--HRRLARAIIELTYRHKVVKVMRPA--ADGRTVMDVISREDQRGSG GGVEGLGLQKLGYILREVGTRPGGRIYADDTAGWDTRITRADLENEAKVLELLDGE--HRRLARAIIELTYRHKVVKVMRPA--ADGRTVMDVISREDQRGSG GGVEGIGLQYLGYVIRDLAAMDGGGFYADDTAGWDTRITEADLDDEQEILNYMSPH--HKKLAQAVMEMTYKNKVVKVLRPA--PGGKAYMDVISRRDQRGSG AGVEGISLNYLGWHLKKLSTLNGGLFYADDTAGWDTKVTNADLEDEEQILRYMEGE--HKQLATTIMQKAYHAKVVKVARPS--RDGGCIMDVITRRDQRGSG AGVEGISLNYLGWHLKKLASLSGGLFYADDTAGWDTKITNADLDDEEQILRYMDGD--HKKLAATVLRKAYHAKVVRVARPS--REGGCVMDIITRRDQRGSG GGVEGMGLHYLGWLVKDLAELEGGKLYADDTAGWDTRVTNSDLEDEEEILNHLEGE--HKKLAEAIMKLAYHAKVVKVARPA--SDGGTVMDIISRRDQRGSG GGVEGIPLNYLGYHLREMAQKPG-VLYADDTAGWDTRITMADLEDEGMLLDMMSGE--HKKLASALFSKAYKVKVALCPRPG--PKGGTLMDVISRTDQRGSG GGVGGVGVNYFGYYLKDIA-SRGKYLIADDIAGWDTKISEEDLEDEEALLTALTEDPYHRALMAATMRLAYQNIVAMFPRTHSKYGSGTVMDVVGRRDQRGSG ** * * * *** ***** ** * * * * * ** * ******
A
B
FIGURE 1
(Continued)
601 601 602 602 605 605 605 605 604 604 604 605 605 614
DV-1 DV-3 DV-2 DV-4 JEV MVEV WNV KUNV YFV TBEV LGTV MEAV MoDV CFA
a17 Pa18 Pa16 a19 Pa20 Pb4 Pb5 604 + 620 + 640 + 660 + 680 + 700 QVGTYGLNTFTNMEAQLIRQMESEGIFSPSELETP-NLAER-VLDWLKKHGTERLKRMAISGDDCVVKPIDDRFATALTALNDMGKVRKDIPQWEPSKGWNDW QVGTYGLNTFTNMEAQLVRQMEGEGVLTKADLENP-HLLEKKITQWLETKGVERLKRMAISGDDCVVKPIDDRFANALLALNDMGKVRKDIPQWQPSKGWHDW QVGTYGLNTFTNMEAQLIRQMEGEGVFKSIQHLT--VTEEIAVQNWLARVGRERLSRMAISGDDCVVKPLDDRFASALTALNDMGKVRKDIQQWEPSRGWNDW QVGTYGLNTFTNMEVQLIRQMEAEGVITQDDMQNP-KGLKERVEKWLKECGVDRLKRMAISGDDCVVKPLDERFGTSLLFLNDMGKVRKDIPQWEPSKGWKNW QVVTYALNTFTNIAVQLVRLMEAEGVIGPQHLEQLPRKTKIAVRTWLFENGEERVTRMAISGDDCVVKPLDDRFATALHFLNAMSKVRKDIQEWKPSHGWHDW QVVTYALNTFTNIAVQLVRLMEAEAVIGPDDIESIERKKKFAVRTWLFENAEERVQRMAVSGDDCVVKPLDDRFSTALHFLNAMSKVRKDIQEWKPSQGWYDW QVVTYALNTFTNLAVQLVRMMEGEGVIGPDDVEKLTKGKGPKVRTWLFENGEERLSRMAVSGDDCVVKPLDDRFATSLHFLNAMSKVRKDIQEWKPSTGWYDW QVVTYALNTFTNLAVQLVRMMEGEGVIGPDDVEKLTKGKGPKVRTWLSENGEERLSRMAVSGDDCVVKPLDDRFATSLHFLNAMSKVRKDIQEWKPSTGWYDW QVVTYALNTITNLKVQLIRMAEAEMVIHHQHVQDCDESVLTRLEAWLTEHGCDRLKRMAVSGDDCVVRPIDDRFGLALSHLNAMSKVRKDISEWQPSKGWNDW QVVTYALNTLTNIKVQLIRMMEGEGVIEAADAHNP---RLLRVERWLKEHGEERLGRMLVSGDDCVVRPLDDRFGKALYFLNDMAKTRKDIGEWEHSAGFSSW QVVTYALNTITNIKVQLVRMMEGEGVIEVADSHNP---RLLRVEKWLEEHGEERLSRMLVSGDDCVVRPVDDRFSKALYFLNDMAKTRKDTGEWEPSTGFASW QVVTYALNTITNIKVQLIRMMEGEGVIGPADMTEP---RIIRVERWLERHGEERLGRLLVSGDDCVVKPIDDRFAEAVHFLNDMSKTRKDIGEWSPSVGYTNW QVVTYALNTLTNIKVQLIRMAEAEGVLGATFEDFG-------IDRWLQEHGEDRVERMLVSGDDCVVNAIDERFGSSLNWLNAMEKVRKDIDLWKPSPSFRNW QVVTYALNTITNGKVQVARVLESEGLLQAD---------ESVLDAWLEKHLEEALGNMVIAGDDVVVSTDNRDFSSALEYLELTGKTRKNVPQGAPSRMESNW ** ** *** ** * * * * ** *** ** * * * ** * *
B
DV-1 DV-3 DV-2 DV-4 JEV MVEV WNV KUNV YFV TBEV LGTV MEAV MoDV CFA
C
D
Pa21 Pa22 Pa23 Pb6 Pb 7 705 + 720 + 740 + 760 + 780 + 800 QQVPFCSHHFHQLIMKDGREIVVPCRNQDELVGRARVSQGAGWSLRETACLGKSYAQMWQLMYFHRRDLRLAANAICSAVPVDWVPTSRTTWSIHAHHQWMTT QQVPFCSHHFHELIMKDGRKLVVPCRPQDELIGRARISQGAGWSLRETACLGKAYAQMWSLMYFHRRDLRLASNAICSAVPVHWVPTSRTTWSIHAHHQWMTT TQVPFCSHHFHELIMKDGRVLVVPCRNQDELIGRARISQGAGWSLRETACLGKSYAQMWSLMYFHRRDLRLAANAICSAVPSHWVPTSRTTWSIHAKHEWMTT QEVPFCSHHFHKIFMKDGRSLVVPCRNQDELIGRARISQGAGWSLRETACLGKAYAQMWSLMYFHRRDLRLASMAICSAVPTEWFPTSRTTWSIHAHHQWMTT QQVPFCSNHFQEIVMKDGRSIVVPCRGQDELIGRARISPGAGWNVKDTACLAKAYAQMWLLLYFHRRDLRLMANAICSAVPVDWVPTGRTSWSIHSKGEWMTT QQVPFCSNHFQEVIMKDGRTLVVPCRGQDELIGRARISPGSGWNVRDTACLAKAYAQMWLVLYFHRRDLRLMANAICSSVPVDWVPTGRTTWSIHGKGEWMTT QQVPFCSNHFTELIMKDGRTLVVPCRGQDELVGRARISPGAGWNVRDTACLAKSYAQMWLLLYFHRRDLRLMANAICSAVPVNWVPTGRTTWSIHAGGEWMTT QQVPFCSNHFTELIMKDGRTLVTPCRGQDELVGRARISPGAGWNVRDTACLAKSYAQMWLLLYFHRRDLRLMANAICSAVPVNWVPTGRTTWSIHAGGEWMTT ENVPFCSHHFHELQLKDGRRIVVPCREQDELIGRGRVSPGNGWMIKETACLSKAYANMWSLMYFHKRDMRLLSLAVSSAVPTSWVPQGRTTWSIHGKGEWMTT EEVPFCSHHFHELVMKDGRTLVVPCRDQDELVGRARISPGCGWSVRETACLSKAYGQMWLLSYFHRRDLRTLGLAINSAVPADWVPTGRTTWSIHASGAWMTT EEVPFCSHHFHELVMKDGRALVVPCRDQDELVGRARVSPGCGWSVRETACLSKAYGQMWLLSYFHRRDLRTLGFAICSAVPVDWVPTGRTTWSIHASGAWMTT EEVPFCSHHFHRLVMKDGRELIVPCRDQDELIGRARVSPGCGWTVRETAGLSKAYAQMWLLSYFHRRDLRLMGFGICSAVPVDWVPTGRTTWSIHGKGEWMTT ERVEFCSNHFHEMTMKDGRVIVAPCRGQTELIARGTVNQGGCVGVESTGCLAKAYAQMWLLLYFHRRDLRTLALAVMSAVPSNWIPTGRTTWSLMVKGEWMTD EKVEFCSHHYHEMSLKDGRIIIAPCRHENEVLGRSRLQKGGVVSISESACMAKAYAQMWALYYFHRRDLRLGFIAISSAVPTNWFPLGRTSWSVHQYHEWMTT * *** * **** *** * * * * * ** *** ** * * ** * * ** ** ***
E
Priming loop
FIGURE 1 (Contined)
702 703 703 704 708 708 708 708 707 704 704 705 701 708
805 806 806 807 811 811 811 811 810 807 807 808 804 811
50
899 900 900 900 905 905 905 905 905 903 903 906 898 906
Priming loop
FIGURE 1 Comparative alignment of the flavivirus NS5 amino acid sequences. Secondary structural elements are indicated by boxes (a-helices), arrows (b-sheets), or dots (important loop regions) above the sequences. Elements in the MTase and POL domains follow those described for the DENV-2 MTase (Egloff et al., 2002) and DENV-3 POL (Yap et al., 2007a,b) structures, respectively. The elements are labeled as M for the MTase or P for the POL domains. Conserved MTase sequence motifs I–X (Malone et al., 1995) based on the DENV-2 MTase structure (Egloff et al., 2002) are shown below the sequence alignment as are the positions of conserved RdRp motifs A–F defined by Poch et al. (1989). The positions of four catalytic amino acids conserved among 20 -O-MTases are indicated by # above the sequence. Invariant amino acids are marked below the sequences as ‘‘*’’. The virus sequences and their GenBank accession numbers are as follows: DENV-1 strain Western Pacific 74 (DV-1) (DVU88535), DENV-3 strain Singapore (DV-3) (AY662691), DENV-2 strain NGC (DV-2) (AF038403), DENV-4 strain 814669 (DV-4) (M14931), Japanese encephalitis virus (JEV) strain JaOArS982 (M18370), Murray Valley encephalitis virus (MVEV) strain 1–51 (AF161266), WNV strain NY 2000-crow3356 (AF404756), WNV strain Kunjin (KUNV) (AY274504), yellow fever virus (YFV), strain 17D (X03700), tick-borne encephalitis virus (TBEV) strain Neudorfl (U27495), Langat virus (LGTV) strain E5 (AF253420), Meaban virus (MEAV) (DQ235144), Modoc virus (MoDV) (AJ242984), and Cell fusing agent (CFA) (M91671). The alignments were performed using ClustalW (Thompson et al., 1994).
Andrew D. Davidson
DV-1 DV-3 DV-2 DV-4 JEV MVEV WNV KUNV YFV TBEV LGTV MEAV MoDV CFA
Pa 24 a 25 Pa 26 Pa 27 808 + 820 + 840 + 860 + 880 + EDMLSVWNRVWIEENPWM--EDKTHVSSWEDVPYLGKREDRWCGSLIGLTARATWATNIQVAINQVRRLIGNEN-----YLDFMTSMKRFKNESDPEGALW-EDMLTVWNRVWIEENPWM--EDKTPVTTWENVPYLGKREDQWCGSLIGLTSRATWAQNIPTAIQQVRSLIGNEE-----FLDYMPSMKRFRKEEESEGAIW-EDMLTVWNRVWIQENPWM--EDKTPVESWEEIPYLGKREDQWCGSLIGLTSRATWAKNIQTAINQVRSLIGNEE-----YTDYMPSMKRFRREEEEAGVLW-EDMLKVWNRVWIEDNPNM--TDKTPVHSWEDIPYLGKREDLWCGSLIGLSSRATWAKNIHTAITQVRNLIGKEE-----YVDYMPVMKRYSAPSESEGVL--EDMLQVWNRVWIEENEWM--MDKTPITSWTDVPYVGKREDIWCGSLIGTRSRATWAENIYAAINQVRAVIGKEN-----YVDYMTSLRRYEDVLIQEDRVI-EDMLSVWNRVWILENEWM--EDKTTVSDWTEVPYVGKREDIWCGSLIGTRTRATWAENIYAAINQVRSVIGKEK-----YVDYVQSLRRYEETHVSEDRVL-EDMLEVWNRVWIEENEWM--EDKTPVEKWSDVPYSGKREDIWCGSLIGTRARATWAENIQVAINQVRAIIGDEK-----YVDYMSSLKRYEDTTLVEDTVL-EDMLEVWNRVWIEENEWM--EDKTPVEKWSDVPYSGKREDIWCGSLIGTRARATWAENIQVAINQVRSIIGDEK-----YVDYMSSLKRYEDTTLVEDTVL-EDMLEVWNRVWITNNPHM--QDKTMVKKWRDVPYLTKRQDKLCGSLIGMTNRATWASHIHLVIHRIRTLIGQEK-----YTDYLTVMDRYSVDADLQLGELIEDMLDVWNRVWILDNPFM--QNKERVMEWRDVPYLPKAQDMLCSSLVGRRERAEWAKNIWGAVEKVRKMIGPEK-----FKDYLSCMDRHDLHWELRLESSII EDMLEVWNRVWIYDNPFM--EDKTRVDEWRDTPYLPKSQDILCSSLVGRGERAEWAKNIWGAVEKVRRMIGPEH-----YRDYLSSMDRHDLHWELKLESSIF EDMLEVWNRVWIEDNPFMPCEKKRWITDWRDVPYLPKAQDQICGSLIGTSSRASWAENIWSTVEKVRGMVGAEN-----YRDYLSVMDRYGGGTPVPMTSDIL EDMLAVWNRVWIEDNPFM--EDKREVERWSEVPYLPRNQDKSCGSLIGTTARAEWAKLLPGAVEKVRNIFGKQR-----FRNYLRNMGRYESQEEAPFSMY-DDMLRVWNDVWVHNNPWM--LNKESIESWDDIPYLHKKQDITCGSLIGVKERATWAREIENSVISVRRIIDAETGVLNTYKDELSVMSRYRRGNDVI-----*** *** ** * * * * ** * * ** * ** ** * *
Flavivirus NS5
51
genome as a substrate. Both proteins exhibited 20 -O-MTase activity, and in addition, N7 MTase activity (Ray et al., 2006). MTase activity could not be detected using a capped nonspecific RNA substrate, demonstrating that both MTase activities relied on the presence of specific flaviviral sequences. Examination of the MTase activities of the corresponding MTase domains of DENV-1, YFV, and Powassan virus (PWV) using the WNV RNA substrate, revealed that all of the proteins possessed both N7 and 20 -O-MTase activities (Dong et al., 2007; Zhou et al., 2007). Furthermore, kinetic analysis of the methylation of the substrate RNA (GpppA-RNA) by the WNV, DENV-1, and YFV MTases resulted in the initial detection of the guanine N7-methylated product (m7GpppA-RNA). The 20 -O-methylated product (m7GpppAm-RNA) was detected only after sufficient N7-methylated substrate had accumulated, suggesting that flavivirus cap methylation occurs in the order GpppA ! m7GpppA ! m7GpppAm (Ray et al., 2006; Zhou et al., 2007). Analysis of the substrate preferences of the WNV MTase showed that the N7 MTase was equally active on nonmethylated (GpppA-RNA) or 20 -O-methylated (GpppAm-RNA) substrates, whereas the 20 -O-MTase was more active on a N7-methylated substrate (m7GpppARNA) than a nonmethylated substrate (GpppA-RNA). The preference of the 20 -O-MTase for a N7-methylated substrate may therefore determine the sequential order of cap methylation (Dong et al., 2008a). The DENV-2 MTase has recently been shown to exhibit N7 MTase activity using a capped RNA transcript corresponding to the first 211 nucleotides of the DENV-2 genome as a substrate (Kroschewski et al., 2008). As the assay conditions were similar to those used for the WNV N7 MTase activity assay, the results confirm that flavivirus N7 MTase activity is reliant on the use of a viral-specific RNA substrate. Detailed analysis of the RNA substrate requirements for WNV MTase activity, by mutation of the 190-nucleotide RNA substrate, has revealed that distinct viral sequences are required for N7 and 20 -O-MTase activities (Dong et al., 2007). The first 190 nucleotides of the flavivirus genome contains three stem-loop structures termed ‘‘stem-loop A’’ (SLA), ‘‘stem-loop B’’ (SLB), and ‘‘capsid hairpin’’ (cHP) that are highly conserved (Markoff, 2003; Villordo and Gamarnik, 2009). N7 MTase activity was reliant on a substrate containing at least the first 74 nucleotides of the WNV genome, which includes three 50 -terminal nucleotides, the second (G), and third (U) of which had to be of wild-type sequence and SLA. Systematic mutagenesis of the stem-loop structure revealed that N7 activity primarily relied on the presence of two specific helical stem regions at the base of the stem-loop structure but not sequences within the stemloop structure. In contrast, 20 -O-MTase activity required a substrate of at least 20 nucleotides in length, containing the wild-type viral sequence at the first (A) and second (G) nucleotides (Dong et al., 2007). The importance of the 50 -terminal nucleotides for both N7 and 20 -O-MTase activities
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Andrew D. Davidson
was supported by the finding that the binding of small RNAs to the DENV-2 MTase was increased when the RNA substrates contained the first two authentic viral nucleotides (Egloff et al., 2007). It should, however, be noted that studies using different experimental systems to assay flavivirus MTase activities have shown that whereas N7 MTase activity appears to be strictly dependent on a viral RNA substrate containing at least the 50 -conserved structure SLA and specific buffer conditions, the template and buffer conditions for 20 -O-methylation show more variability. WNV 20 -O-MTase activity was optimal at pH 10 using a viral substrate consisting of the 50 -terminal 20 nucleotides and 5–10 mM MgCl2. By contrast, for other flaviviral MTases, 20 -O-MTase activity has been more often demonstrated using short (<10 nucleotides) nonviral RNA substrates with nucleotides other than G at the second position and is optimal at neutral pH (Egloff et al., 2002; Kroschewski et al., 2008; Mastrangelo et al., 2007; Peyrane et al., 2007). More recently, detailed biochemical characterization of the DENV-2 MTase 20 -O-methylation activity has shown that although pH 10 is optimal for activity in the absence of metal ions, maximal activity was achieved at pH 7–8 in the presence of 10 mM KCl, 2 mM MgCl2, and 2 mM MnCl2 (Lim et al., 2008).
C. MTase structure The X-ray crystal structure of the DENV-2 MTase (amino acids 1–296) at ˚ resolution was the first flavivirus MTase structure to be determined 2.4-A (Egloff et al., 2002). The structures of the bacterially expressed MTase domains of WNV (Zhou et al., 2007), Murray Valley encephalitis virus (MVEV) (Assenberg et al., 2007), and Meaban virus (MEAV) (Mastrangelo ˚, et al., 2006, 2007) have since been determined to 2.8, 2.0, and 2.9 A respectively, making the flavivirus MTase one of the best structurally characterized viral MTases. Comparison of the flavivirus MTase structures has shown that they are well conserved, even though the primary amino acid sequence of the MTase of MEAV, a tick-borne virus, is evolutionary divergent from those of the other mosquito-borne flaviviruses (Mastrangelo et al., 2007; Fig. 1). The flavivirus MTase structure contains three subdomains, a N-terminal subdomain, a core subdomain, and a C-terminal subdomain (Fig. 2A). The N-terminal subdomain contains a GTP-binding pocket that accommodates the guanosine of the cap structure. The core MTase subdomain is responsible for AdoMet binding and catalytic activity and is linked to the GTP-binding pocket by a stretch of positively charged amino acids that are predicted to bind viral RNA (Fig. 2B). The function of the C-terminal subdomain has not been elucidated and is only visible to residue(s) 267–269 in the MTase structures, defining the boundary of the MTase domain. Structural determination of the MTase in complex with small molecules, coupled with
53
Flavivirus NS5
A
C
αD αE
A4
AdoHcy β5 β4 β6 β7
A1
AdoHcy
Val-132
β1 β2 β3
Lys-105 Ile-147
Thr-104 His-110
αX
αA Glu-111
B1
Trp-87 Gly-86
B2 GTP
Ser-56
A3
B
AdoHcy
D
Lys-14
Pro-152
Ser-151 Ser-150
Asn-18
GTP Leu-20 GTP RNA binding groove
Phe-25
FIGURE 2 Structure of the NS5 MTase domain. (A) A schematic representation of the X-ray structure of the DENV-2 MTase in complex with GTP and AdoMet (PDB code: 2P1D; Egloff et al., 2007) is shown with the N-terminal, core MTase, and C-terminal subdomains colored in red, cyan, and yellow, respectively. The bound AdoHcy and GTP molecules are shown in stick representation and colored purple. Secondary structural elements are labeled according to Egloff et al. (2002). (B) A surface representation of the DENV-2 MTase shown in (A). Positively, neutral, and negatively charged amino acids are shown in blue, white, and red, respectively. The AdoHcy and GTP are molecules are shown in stick representation and arrowed. The positively charged surface predicted to bind RNA is arrowed. (C) Amino acids involved in AdoMet binding are numbered and colored in gray, blue, and red for C, N, and O, respectively. Hydrogen bonds are shown by dashed lines. (D) Amino acids involved in GTP binding are numbered and colored in gray, blue, and red for C, N, and O, respectively. The schematics shown in (A), (C), and (D) were produced using PYMOL (DeLano, 2002) while the surface representation was produced using the CCP4 molecular graphics software (Potterton et al., 2002).
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structure–function analysis, has delineated three functional regions in the MTase structure that correlate to the architecture described earlier: (1) the AdoMet-binding pocket, (2) the GTP-binding pocket, and (3) a positively charged surface groove predicted to bind RNA (Fig. 2A and B).
1. The core MTase subdomain The core MTase subdomain (amino acids 55–222; unless otherwise indicated, the numbering used refers to DENV-2) is comprised of a sevenstranded b-sheet surrounded by four a-helices (Figs. 1 and 2A). The fold of the flavivirus core subdomain closely resembles those of other structurally characterized AdoMet-dependent MTases (Fauman et al., 1999; Martin and McMillan, 2002) except that an alpha helix typically located between strands b3 and b4 is absent and another between b2 and b3 is shortened to a single turn (Egloff et al., 2002). Structural comparison of DNA MTases led to the identification of nine conserved motifs involved in AdoMet binding and catalysis (Malone et al., 1995). Structure-based alignment of the DENV-2 MTase with other MTases, including the vaccinia virus VP39 20 -O-MTase, positioned eight of the nine motifs in the MTase sequence (Egloff et al., 2002). Residues in motif I, involved in AdoMet binding, showed the best sequence conservation. Indeed, the presence of motif I in NS5 led to the prediction that NS5 possessed MTase activity (Koonin, 1993). Four putative active site residues (Lys-61, Asp-146, Lys-181, and Glu-217) required for 20 -O-MTase catalytic activity, were identified in regions corresponding structurally to motifs X, IV, VI, and VIII (Fig. 1). Superimposition of the DENV-2 and VP39 20 -OMTases (Hodel et al., 1996) revealed a close spatial conservation of the predicted active site residues, confirming biochemical studies showing that the flavivirus MTase was a 20 -O-MTase (Egloff et al., 2002). However, there appeared to be no resemblance to the N7 MTase structure of the reovirus l2 protein (Reinisch et al., 2000), the only N7 MTase structure available at the time. Determination of the Encephalitozoan cuniculi (Ecm1) (Fabrega et al., 2004) and vaccinia virus (De la Pena et al., 2007) N7 MTase structures has since facilitated a more wide ranging structural comparison of the flavivirus MTase with N7 MTase structures. Although it is possible to align the flavivirus MTase with other N7 MTase structures (Assenberg et al., 2007), overall, the MTase most closely resembles other 20 -O-MTases. The structure of the AdoMet-binding pocket is the most conserved feature of MTases, although the residues involved, apart from those in motifs I and II which are highly conserved, may vary (Fig. 1; Martin and McMillan, 2002). In the DENV-2 and WNV structures, the AdoMet-binding pocket was defined by the cocrystallization of S-adenosyl-L-homocysteine (AdoHcy), a byproduct of the methylation reaction, that had copurified with the MTase. By contrast, the MEAV and MVEV MTase
Flavivirus NS5
55
proteins were crystallized in the presence of either AdoMet or both AdoHcy and AdoMet, respectively, which appear to bind identically (Assenberg et al., 2007). AdoMet is stabilized in the binding pocket by a network of hydrogen bonds (H-bond) and van der Waals contacts to specific residues (Fig. 2C). Thus the adenosine ring is accommodated in a hydrophobic pocket defined by the side chains of residues Thr-104, Lys-105, Val-132, and Ile-147 and further stabilized by H-bonds involving residues Lys-105, Asp-131, and Val-132. Overall, these residues are highly conserved among flaviviruses apart from Lys-105 which varies between mosquito and tick-borne viruses. Compared to the adenosine ring, the ribose moiety of AdoMet was bound differently in the different flavivirus structures. The ribose 20 - and 30 -hydroxyl groups formed H-bonds with His-110 and Glu-111, respectively, in the WNV and MVEV structures. In the MEAV structure only a single H-bond between the 20 -hydroxyl and His-110 was detected, whereas in the DENV-2 structure a sulfate ion, arising from the crystallization conditions, was found to form H-bonds with the 20 - and 30 -hydroxyl groups in place of His-110 and Glu-111. The rest of the AdoHcy molecule was stabilized by H-bonds involving Ser-56, Gly-86, and Trp-87. These residues are invariant among flaviviruses (Figs. 1 and 2C).
2. N-terminal GTP-binding subdomain The search for a cap recognition domain in the DENV-2 MTase, using GTP-binding studies in combination with structural characterization of crystals soaked with a GTP analog, identified a novel GTP-binding subdomain in an N-terminal extension of the core MTase subdomain (Egloff et al., 2002). Since this initial finding, the structures of the DENV-2 MTase domain complexed with the GTP analog ribavirin triphosphate (Benarroch et al., 2004a) and the cap analogs GpppA, m7GpppA, GpppG, m7GpppG, m7GpppGm (Egloff et al., 2007), and the MVEV MTase domain in complex with the cap analogs m7GTP, GpppA, and GpppG (Assenberg et al., 2007) have been determined. These studies have clearly defined the GTP-binding pocket and the contacts made to the guanine ring, ribose, and a-phosphate (Fig. 2D). Compared to cellular GTP-binding proteins, which form at least two contacts to the guanine ring, the flavivirus MTase binds GTP in a unique manner. The guanine ring is stabilized by base stacking with the aromatic ring of Phe-25 and electrostatic interactions between the 2-amino group of guanine and residues Leu-17, Asn-18, and Leu-20. This mechanism of binding is selective for guanine which has been confirmed in nucleotide-binding assays (see Section II.D.1). The limited contacts with guanine allow both methylated and unmethylated GTP to bind the MTase with equal specificity. Interestingly, ribavirin triphosphate was found to compete with GTP for binding of the DENV-2 MTase by mimicking the interactions made
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Andrew D. Davidson
between the 2-amino group of guanine and the MTase (Benarroch et al., 2004a). The specificity for ribonucleotides is determined by the formation of H-bonds between the ribose 20 -hydroxyl group with Lys-14 and Asn-18 and the 30 -hydroxyl group with Lys-14 and Ser-151. Ser-151 is contained in a loop protruding from the core MTase domain that packs against the ribose. Both Lys-14 and Asn-18 are invariant among flaviviruses. Although the interaction of the MTase with guanosine is clearly defined, structural analysis of complexes of the flavivirus MTase with cap analogues containing dinucleotides (i.e., GpppN) gave a variety of results. In all cases, the cap guanosine bound identically to the GTP-binding site; however, the following bases were found to adopt different conformations. In a complex between MVEV and GpppG, the second guanine was disordered. The MVEV MTase complexed with GpppA and AdoMet took the form of a dimer. Two GpppA molecules bound to each monomer, one in the GTPbinding site and the other adjacent to it in the putative positively charged RNA-binding site (Assenberg et al., 2007). Although this may have interesting implications for MTase function (see Section II.E), it is unlikely this structure is physiologically relevant. The structures of the DENV-2 MTase in complex with a range of cap analogs revealed at least three possible conformations for cap binding. Two of the conformations involved further base stacking in which the second nucleotide stacked over the cap guanosine in a hairpin-like conformation. Although some of the conformations may not occur under physiological conditions, it was postulated that a base-stacked conformation adopted by GpppA may mimic the end product of guanyltransferase activity that would act on pppG and pppAGN to form the capped sequence GpppA, which is conserved in all flaviviruses. These studies have led to the suggestion that NS5 may also possess guanyltransferase activity (Egloff et al., 2007).
3. RNA-binding groove
˚ away from the The GTP-binding pocket is located approximately 12–13 A AdoMet-binding pocket, separated by a positively charged groove on the MTase surface (Fig. 2B). Comparison of the structure of the DENV-2 MTase with that of vaccinia virus VP39 in complex with a capped RNA substrate and AdoHcy, suggested that the capped flavivirus RNA could adopt a similar positioning with the positively charged surface groove interacting with the negatively charged RNA (Egloff et al., 2002). Attempts to determine the structure of the MTase in complex with small capped RNA molecules (m7GpppAC3 and m7GpppAC5) have so far proven unsuccessful (Egloff et al., 2007). However, structural models of the MTase in complex with small capped RNAs (i.e., m7GpppA-RNA) in a conformation suitable for 20 -O-methylation, suggest that a maximum of five nucleotides after the adenosine could be accommodated in the
Flavivirus NS5
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positively charged amino acid groove (Egloff et al., 2007; Mastrangelo et al., 2007).
D. MTase structure–function studies The structural and biochemical features of the flavivirus MTase show it plays a multifunctional role in cap formation. However, the presence of a single AdoMet-binding site suggests that the capped RNA must be repositioned between each methylation step. The elucidation of the mechanisms involved in flavivirus cap formation and the distinct regions of NS5 involved, have been the subject of a number of recent studies.
1. GTP-binding studies The GTP-binding ability of the flavivirus MTase was first confirmed in a biochemical UV crosslinking assay using radiolabeled nucleotides and the recombinant DENV-2 MTase. As predicted from the structural studies, the MTase selectively binds GTP but none of the other nucleotides. The guanosine analog m7GTP and cap analog GpppA bound to the MTase with a similar affinity to GTP, whereas m7GpppA had an increased dissociation constant, suggesting that the unmethylated cap structure may bind preferentially to the MTase (Egloff et al., 2002). Binding studies conducted with an immobilized DENV-2 MTase and small capped and uncapped RNAs revealed that (a) the presence of a cap, (b) the use of transcripts containing the flavivirus consensus sequence GpppAG, and (c) the RNA chain length were all important factors in binding (Egloff et al., 2007). The presence of a cap and the wild-type flavivirus sequence at the first and second nucleotides served to increase binding. For each cap structure and RNA sequence there was an optimal chain length; decreasing the chain length from the optimum decreased binding whereas an increase had no effect. The effects of mutating DENV-2 residues predicted to be involved in GTP binding were assessed using the GTP crosslinking assay. Mutation of Phe-25 to Ala eliminated GTP binding whereas mutation of Asn-18, Lys-29, and Ser-150 to Ala severely decreased GTP binding, confirming the importance of these residues in GTP binding (Egloff et al., 2002). Interestingly, mutation of these residues and additionally Lys-13 (equivalent to Lys-14 in DENV-2) in the WNV MTase was found to differentially affect the MTase activities. Mutation of Phe-24 to Ala significantly affected both N7 and 20 -O-MTase activities. The remaining mutations had little effect on N7 activity while all caused varying reductions in 20 -O-MTase activity (Dong et al., 2008b). Analysis of the WNV MTase activities in the presence of GTP and GpppA revealed that both molecules inhibited 20 -O-MTase activity in a dose-dependent manner whereas N7 activity was not affected in the dose range tested. Similarly, the 20 -O-MTase activity of DENV-2
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was found to be inhibited by ribavirin triphosphate, which was shown to occupy the GTP-binding site (Benarroch et al., 2004a). These results suggest that 20 -O-methylation of the substrate RNA requires correct placement of the cap in the GTP-binding pocket.
2. MTase activities
Previous studies have shown that N7 and 20 -O-methylation reactions require distinct chemistries. 20 -O-Methylation requires the presence of four conserved catalytic residues that are involved in the transfer of the AdoMet methyl group to the substrate using a direct in line SN2 nucleophilic transfer mechanism (Hager et al., 2002; Hodel et al., 1998). By contrast, N7 activity appears to depend on optimal alignment of the methyl donor and substrate in a manner that promotes methyl transfer (Fabrega et al., 2004; Hausmann et al., 2005; Zheng et al., 2006). Structural alignment of the DENV-2 MTase with proteins involved in 20 -O-methylation identified four residues (Lys-61, Asp-146, Lys-181, and Glu-217, or Lys-182 and Glu-218 in WNV) corresponding to the catalytic active site residues (Egloff et al., 2002; Fig. 1). These residues were mutated to Ala or residues having a similar or dissimilar charge to that of the wild-type residue, in the WNV MTase, to test their effects on N7 and 20 -O-MTase activities (Ray et al., 2006; Zhou et al., 2007). All of the mutations to the putative active site residues were found to abolish 20 -O-methylation, confirming their role in flavivirus 20 -O-MTase activity. However, the mutations were found to have variable effects on N7 MTase activity. Only Asp-146 was found to be essential for N7 MTase activity. Lys-61, Lys-182, and Glu-218 were found to contribute to, but were not essential for WNV N7 MTase activity (Zhou et al., 2007). These results suggested that distinct regions of the MTase are involved in N7 and 20 -O-MTase activities, although only one AdoMet-binding pocket could be detected in the MTase structure. Accordingly, further mutagenesis studies have been conducted using the WNV and DENV2 MTase domains to identify residues commonly and distinctly required for methylation of RNA substrates (Dong et al., 2008b; Kroschewski et al., 2008). Site-specific mutation of DENV-2 and WNV residues, predicted from the structural studies to be involved in AdoMet binding, including; Ser-56, Trp-87, Lys-105, His-110, Glu-111, Asp-131, and Ile-147 (Fig. 2C) were all found to reduce and in some cases abolish either N7 or 20 -Omethylation. However, analysis of the effects of the WNV mutations on AdoMet binding, using a UV crosslinking assay, showed that with the exception of the mutation Asp-131 to Ala, single point mutations that severely decreased one or both of the MTase activities had little effect on AdoMet binding. Subtle differences in the binding conformation of AdoMet may therefore have large effects on MTase activity. In the case of DENV-2, multiple clustered mutations to residues in motif I totally
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abolished both MTase activities. Collectively the results confirmed that both N7 and 20 -O-MTase activities are reliant on the use of a single AdoMet-binding site. Twenty amino acid residues in the WNV MTase, predicted to interact with RNA through electrostatic or base-stacking interactions, were mutated to investigate the importance of RNA binding to MTase activity (Dong et al., 2008b). Examination of the effects of the mutations on the WNV MTase activities revealed that only three residues (Arg-37, Arg-57, and Trp-87) affected both activities, the remainder of the mutations had no effect or differentially affected either N7 or 20 -O-MTase activity. The mutations-affecting 20 -O-MTase activity were more numerous and dispersed on the MTase structure than those effecting N7 MTase activity, suggesting that the RNA substrate binds to different regions of the MTase during the two methylation reactions. Examination of the effects of the mutations on RNA binding revealed that single mutations were not sufficient to inhibit RNA binding which must rely on multiple contacts. A number of the MTase mutations described earlier were introduced into DENV-2 and WNV infectious clones to examine the requirement for N7 and 20 -O-MTase activities in the context of the viral lifecycle (Dong et al., 2008b; Kroschewski et al., 2008; Zhou et al., 2007). Mutations targeting residues in the AdoMet-binding pocket and the catalytic residue Asp-146, which were found to abolish or severely decrease both N7 and 20 -O-MTase activities in vitro, proved lethal when introduced into the DENV-2 and WNV genomes. This demonstrated a strict requirement for one or both MTase activities for virus replication. However, the introduction of mutations that abolished or severely decreased 20 -O-MTase activity while retaining at least moderate levels of N7 MTase activity resulted in the production of viable viruses. In the case of DENV-2, recombinant viruses deficient in 20 -O-MTase activity replicated to wild-type levels in Vero cells (Kroschewski et al., 2008) whereas for WNV, viruses deficient in 20 -O-MTase activity showed delayed replication kinetics in Vero cells and attenuation in C6/36 mosquito cells (Zhou et al., 2007). Furthermore, experiments testing the effects of different cap structures on the translation and replication of a WNV replicon showed that N7 methylation but not 20 -O-methylation of the cap was required for efficient translation and that defective translation led to a defect in RNA replication (Ray et al., 2006). These results suggested that N7 but not 20 -O-MTase activity was strictly required for virus replication. Interestingly, investigation of the virulence properties of WNV mutants that retained N7 MTase activity but were deficient in 20 -O-MTase activity, in mice, revealed that the mutant viruses were attenuated and did not cause mortality at any inoculation dose. The mice were protected from subsequent challenge with the wildtype WNV. The elucidation of the role of 20 -O-MTase activity in the virus lifecycle is therefore an intriguing question.
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E. A model for flavivirus cap methylation Based on the structural, biochemical, and structure–function studies described earlier, a number of models have been proposed to explain how the flavivirus MTase may function in cap formation (Assenberg et al., 2007; Dong et al., 2008b; Egloff et al., 2007; Zhou et al., 2007). The following evidence has been taken into account developing these models: (a) the MTase can bind both GTP and m7GTP, (b) m7GpppA binds to the MTase in a conformation suggesting that it could be the product of as yet unidentified guanyltransferase activity, (c) once formed, the cap structure is sequentially methylated by the demonstrated N7 and 20 -O-MTase activities in the order GpppA ! m7GpppA ! m7GpppAm, (d) the two MTase activities are dependent on the use of a single AdoMet-binding site but differ in the sets of amino acids required for MTase activity. 20 -O-MTase activity appears to depend on larger number of specific residues that are more widely dispersed over the surface of the MTase than for N7 MTase activity, and (e) 20 -O-MTase activity is cap dependent and inhibited by compounds that can occupy the cap-binding pocket, whereas N7 activity is not affected. All the models agree that cap methylation involves repositioning of the substrate between methylation reactions as originally proposed by Shi and colleagues (Dong et al., 2008b; Zhou et al., 2007). Canard and colleagues (Egloff et al., 2007) have suggested that GTP first binds in the cap-binding pocket and is subsequently transferred to the end of the nascent RNA by an as yet unidentified guanyltransferase activity, possibly resident in NS5 or a complex containing NS5. The capped RNA is then repositioned such that the N7 position of guanine is positioned correctly for methylation using the bound AdoMet. Once methylated, the capped RNA is again repositioned so that the methylated guanine is accommodated in the cap-binding pocket and the 20 -O-position of the adenosine ribose is now positioned next to AdoMet for the second methylation. Two mechanisms have been proposed to explain how the N7-methylated capped RNA could be repositioned for 20 -O-methylation. In the first, 20 -O-methylation could occur using the same AdoMet-binding pocket. This would require AdoHcy, the byproduct of methylation to be replaced with AdoMet which may coincide with repositioning of the capped RNA. Alternatively, the N7methylated capped RNA could be transferred to the cap-binding site on a second MTase, already charged with AdoMet, which then performs the 20 -O-methylation reaction. The structure of the MVEV MTase in complex with GpppA and AdoMet was observed as a dimer. It was postulated that this may represent the structure of methylation complex (Assenberg et al., 2007). In addition, Dong et al. (2008a) have shown that full methylation of a capped RNA substrate can occur using two mutant MTase proteins, each defective in either N7 or 20 -O-MTase activity. These results support
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the hypothesis that the functional MTase may be comprised of two molecules of NS5 that can function in trans. Further experimentation is required to fully define the mechanism of flavivirus cap formation.
III. THE RNA-DEPENDENT RNA POLYMERASE DOMAIN A. Flavivirus RNA synthesis In flavivirus-infected cells, viral RNA synthesis occurs in the perinuclear region of the cytoplasm in association with ER membranes (Westaway et al., 2002). Three viral RNA species have been detected in infected cells, a single-stranded genomic RNA of 40–44 S, a double-stranded replicative form (RF) of 20–22 S, and a partially single-stranded replicative intermediate (RI) of 20–28 S (Chu and Westaway, 1985; Cleaves et al., 1981). Examination of the kinetics of synthesis of the viral RNA species led to the proposal that late in infection, the (þ) strand RNA genome is replicated asymmetrically in a semiconservative manner using a () strand RNA intermediate as a recycling template (Chu and Westaway, 1985, 1987). Negative strand RNA synthesis was found to occur continuously throughout infection. Thus, the ratio of (þ) to () RNA strands rose to a level of approximately 10:1 late in infection (Chu and Westaway, 1985; Cleaves et al., 1981). Early studies showed that cytoplasmic membrane containing fractions from flavivirus-infected cells, containing the three viral RNA species, could be used to establish in vitro RdRp activity assays (Bartholomeusz and Wright, 1993; Chu and Westaway, 1985, 1987; Grun and Brinton, 1986). RdRp activity was demonstrated by either the incorporation of radioactive nucleotides into the endogenous viral RNA species or the conversion of an exogenous 32P-labeled RF template to the RI and 44 S genomic RNA species, which suggested that initiation of RNA synthesis occurred in vitro (Bartholomeusz and Wright, 1993; Chu and Westaway, 1987). It was first demonstrated that an exogenous subgenomic viral RNA transcript, comprised of the DENV-2 50 - and 30 -terminal regions (TRs), could be used as template for RNA synthesis using cytoplasmic extracts from DENV-2-infected cells (You and Padmanabhan, 1999). Two predominant products were synthesized in the assay. Analysis of the size of the products by denaturing agarose gel electrophoresis showed that one of the products was the same size as the input template RNA (1 product) while the other was twice the size of the input template (termed the 2 product). Characterization of the products by RNase A digestion revealed that before denaturation, the 1 product was present in a dsRNA form, suggesting that it represented a newly synthesized () strand RNA copy bound to the input template. The 2 product was also
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found to be dsRNA; however, after RNase A digestion and denaturation it migrated to the same size as the input template. This suggested that it was comprised of a dsRNA hairpin containing a single-stranded RNA loop susceptible to RNase A. This type of ‘‘copy-back’’ product arises when the 30 -end of the template folds back on itself to act as an intramolecular primer for () strand RNA synthesis as has been observed for other viral systems (Kao et al., 2001; van Dijk et al., 2004). Although it was likely that the 1 product was synthesized by primer independent de novo initiation of RNA synthesis, this could not be conclusively demonstrated due to the possible presence of nucleases in the extracts.
B. RdRp activity of NS5 1. NS5 has RdRp activity Although the development of a cell-based RdRp assay that could use exogenous templates was very useful for examining the template requirements for viral RNA synthesis (see Section III.B.4), it had limited use for examining the role of specific viral proteins in RNA synthesis. Comparative analysis of flavivirus sequences showed that the C-terminal region of NS5 contained sequence motifs conserved in other viral RdRp proteins, suggesting that it was the viral RdRp (Koonin, 1991; Poch et al., 1989; Rice et al., 1985; Sumiyoshi et al., 1987). This was supported by the findings that antisera specific to DENV-2 and JEV NS5 and NS3 inhibited RNA synthesis in in vitro RdRp assays (Bartholomeusz and Wright, 1993; Edward and Takegami, 1993). The RdRp activity of flavivirus NS5 in the absence of other viral and host cell components was first shown using a bacterially expressed full-length DENV-1 NS5 (Tan et al., 1996). The DENV-1 NS5 was able to use templates corresponding to either the 30 -end (629 or 3200 nucleotides in size) of the DENV-1 genome or a nonviral RNA in combination with a specific primer, to produce template size products of negative strand polarity. The products were demonstrated not to be covalently linked to the input template (i.e., not ‘‘copy-back’’ products). By contrast, baculovirus-expressed WNV NS5, contained in insect cell extracts, exhibited RdRp activity on nonspecific templates employing either an exogenously added primer or using a ‘‘copy-back’’ mechanism leading to the suggestion that the flavivirus RdRp was primer dependent (Steffens et al., 1999). Baculovirus-expressed WNV strain Kunjin (WNVKUN) NS5, purified from insect cells, was found to have nonspecific RdRp activity using replication competent WNVKUN or Semliki Forest virus subgenomic replicons as templates. The products of the reaction consisted of dsRNA twice the size of the input templates (Guyatt et al., 2001). Collectively, these studies showed that recombinant full-length NS5 had low nonspecific RdRp activity but did not conclusively determine whether the flavivirus RdRp was primer dependent or could initiate RNA synthesis de novo.
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2. The flavivirus RdRp can initiate RNA synthesis de novo Analysis of the RNA synthesizing ability of bacterially expressed DENV-2 NS5 confirmed for the first time that the flavivirus RdRp was template specific and could initiate () strand RNA synthesis de novo (Ackermann and Padmanabhan, 2001). Similar to the study of You and Padmanabhan (1999) described earlier, the addition of a DENV-2 subgenomic template containing the 50 - and 30 -TRs to an RdRp assay containing recombinant DENV-2 NS5 led to the synthesis of two products equivalent in size to the previously characterized 1 and 2 products. Analysis of the products demonstrated that the 1 product was a () strand RNA complementary to the input template whereas the 2 product was a dsRNA hairpin. Blockage of the 30 -hydroxyl on the 30 -TR of the subgenomic template by periodate oxidation led to the production of only the 1 product, confirming that DENV-2 NS5 was capable of de novo synthesis of () strand RNA (Ackermann and Padmanabhan, 2001). Application of the same experimental approach to WNV confirmed that purified bacterially expressed WNV NS5 also could initiate de novo () strand RNA synthesis from a subgenomic WNV template containing the 50 - and 30 -TRs (Nomaguchi et al., 2004). Purified bacterially expressed JEV NS5 has also been shown to have RdRp activity (Kim et al., 2007; Yu et al., 2007). Using RNA templates corresponding to the terminal 1000 nucleotides of the (þ) and () strand 30 -ends, it was confirmed that JEV NS5 can also initiate RNA synthesis de novo (Yu et al., 2007). Characterization of the enzymatic properties of JEV NS5 revealed a preference for Mn2þ over Mg2þ for optimal activity and that Ca2þ inhibited RdRp activity (Kim et al., 2007; Yu et al., 2007), features reported for other viral RdRps (van Dijk et al., 2004). The determination of the X-ray structures of the hepacivirus hepatitis C virus (HCV) and pestivirus bovine viral diarrhea virus (BVDV) RdRps facilitated a structure-based sequence analysis of the flavivirus NS5 that delineated the extent of the RdRp domain (Selisko et al., 2006). Contrary to earlier predictions, the RdRp domain encompassed a region of NS5 that had been described as an interdomain linker and shown for DENV-2 to contain functional nuclear localization signals (Brooks et al., 2002; Forwood et al., 1999; see Section VI.B). Bacterial expression of truncated DENV-2 (amino acids 272–900) and WNV (amino acids 274–905; strains Kunjin and IS-98-STI) NS5 containing the delineated domains (termed POL domains) with an N-terminal hexahistidine tag, resulted in the production of soluble proteins that had RdRp activity. Similar to the recombinant full-length NS5 (Ackermann and Padmanabhan, 2001), the DENV-2 POL was able to synthesize de novo initiated (1) and copyback (2) RNA products from a subgenomic DENV-2 template containing the 50 - and 30 -TRs. In addition, the DENV-2 and WNV POLs were able to initiate RNA synthesis de novo using the homopolymeric RNA template
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oligo(C), but only in the presence of Mn2þ ions. Mn2þ was also preferred over Mg2þ for DENV POL activity using the subgenomic RNA template. Kinetic analysis of the RdRp activities of the POL domains and the corresponding full-length NS5 revealed that they behaved identically (Selisko et al., 2006). Bacterial expression of a DENV-3 POL domain (amino acids 273–900) also resulted in the production of a soluble protein that had RdRp activity using a homopolymeric oligo(C) primer. In this case, the POL domain was less active in RNA synthesis than the fulllength NS5 (Yap et al., 2007b). Optimization of the enzymatic properties of the POL domains of a number of flaviviruses using homopolymeric templates has now led to the development of high throughput RdRp assays that are very useful for the identification of flavivirus polymerase inhibitors (reviewed by (Malet et al., 2008)).
3. Requirements for de novo initiation During the analysis of the in vitro RdRp activity of a recombinant DENV-2 NS5, it was found that the mechanism used to prime RNA synthesis was temperature dependent (Ackermann and Padmanabhan, 2001). At lower temperatures (18 C) there was a preference for de novo initiation of () strand RNA synthesis. As the temperature was raised, there was a shift to self-primed initiation such that the ratio of the 1 and 2 reaction products changed from 2:1 to 1:4 over the temperature range 20–40 C. Using a modified RdRp assay in which the initiation and elongation phases of RNA synthesis could be uncoupled, it was found that de novo initiation required a low temperature, but once a stable de novo preinitiation complex had formed, the elongation step of RNA synthesis was insensitive to temperature (Ackermann and Padmanabhan, 2001). Further investigation of the requirements for de novo initiation of RNA synthesis established that the minimum components required for the formation of the preinitiation complex were; template RNA, ATP, a high concentration of GTP (>500 uM), and NS5 (Nomaguchi et al., 2003). A similar high concentration of GTP, compared to the other nucleotides, has also been found to be required for de novo initiation of RNA synthesis by the RdRp proteins of BVDV (Ranjith-Kumar et al., 2002), HCV (Luo et al., 2000), and other viruses (Kao et al., 2001; van Dijk et al., 2004). Structural studies have now shown that all of the Flaviviridae RdRps have a binding site for GTP (Bressanelli et al., 2002; Choi et al., 2004; Yap et al., 2007b) that is postulated to stabilize the priming nucleotide. The initiation of DENV-2 RNA synthesis by self-priming at higher temperatures suggested that an exogenous primer may also function to prime RNA synthesis at higher temperatures. Accordingly, addition of the primer AGAA to the RdRp assays performed at 35 C caused a significant shift toward the synthesis of the 1 product rather than the hairpin product. A four nucleotide primer was found to be optimal for RNA synthesis, suggesting that this
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may be the size of the RNA product formed before the transition to RNA elongation (Nomaguchi et al., 2003). Based on these studies, it was proposed that the RdRp could exist in two states (Ackermann and Padmanabhan, 2001; Nomaguchi et al., 2003). At lower temperature, the RdRp was in a closed form which could not accommodate a template with a folded back 30 -end and synthesized RNA de novo on a single-stranded RNA template. Initial de novo synthesis or an increase in temperature caused a change in the conformation of the RdRp to a more mobile open form that either elongated the de novo initiation product or could accommodate a folded back 30 -structure to initiate synthesis of the hairpin-like RNA. However, as previous analysis of flaviviral RNA species had not detected any hairpin structure (Chu and Westaway, 1985) it was concluded that this form of initiation was an artifact of in vitro RdRp assays (Kao et al., 2001). Therefore, the change in RdRp conformation from a closed to an open form, observed with temperature change in vitro, most likely reflects the conformational changes that occur in the transition from de novo initiation of RNA synthesis to elongation of RNA. To identify putative rate-limiting steps that may occur during NS5 RNA synthesis, RNA products synthesized by the DENV-2 and WNVKUN POLs and the HCV and BVDV RdRps were compared over time (Selisko et al., 2006). RNA synthesis by the HCV RdRp is known to undergo several distinct rate-limiting steps during the transition from de novo initiation to elongation. Analysis of the accumulation of reaction products of different sizes produced by the different POLs using an oligo(C) template showed that the HCV RdRp and WNVKUN POL produced a higher percentage of short abortive products compared to the BVDV RdRp and DENV-2 POL. This suggested that there were fewer ratelimiting steps in the transition from de novo RNA synthesis to elongation by the DENV-2 POL compared to that of WNVKUN and the HCV RdRp. This could be explained by the DENV-2 POL possessing a higher conformational flexibility than the WNV POLKUN and HCV RdRp.
4. Template requirements for RNA synthesis The demonstration that extracts from DENV-2-infected cells had RdRp activity using an exogenous subgenomic RNA template containing the viral 50 - and 30 -TRs (You and Padmanabhan, 1999) has led to studies investigating the template requirements of recombinant flavivirus NS5. Analysis of the template requirements for () strand RNA synthesis using DENV-2 RdRp assays based on either extracts from DENV-2-infected cells or a recombinant DENV-2 NS5 (Ackermann and Padmanabhan, 2001) revealed that the 30 -TR could only act as a template when the 50 -TR was present, either in cis or in trans. By contrast, the 50 -TR alone could be used as a template to synthesize both 1 de novo initiated and
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2 hairpin products. A functional interaction between the 50 - and 30 -TRs, mediated by complementary regions in the two regions that are known as cyclization (CYC) sequences was shown to be required for the initiation of () strand synthesis from the 30 -TR. The CYC sequences are critical for virus viability in vivo (Corver et al., 2003; Khromykh et al., 2001; Kofler et al., 2006). In addition to the CYC sequences, highly conserved flavivirus stem-loop structures in the 50 - and 30 -TRs were found to be important for RNA synthesis using the 30 -TR template. (You and Padmanabhan, 1999; You et al., 2001). Analysis of the template requirements of a bacterially expressed WNV NS5 showed that a subgenomic WNV RNA transcript containing the WNV 50 - and 30 -TRs was active as a template for () strand RNA synthesis, confirming the results of the DENV-2 NS5 study. The WNV NS5 also had RNA synthesizing activity using a template of () strand polarity complementary to the subgenomic template (Nomaguchi et al., 2004). In contrast to the products obtained using the (þ) strand template, nearly all of the product synthesized from the () strand RNA template was the result of de novo initiation. In addition, the use of capped (þ) strand transcripts was found to inhibit the synthesis of the 2 hairpin product but had little effect on RNA synthesis initiated de novo or on RNA synthesis using a capped () strand template. The use of (þ) and () strand templates with mutated CYC sequences showed that whereas the presence of the 50 -CYC and to a lesser extent the 30 -CYC was required for RNA synthesis using the (þ) transcript, mutation of the CYC sequences had no effect on RNA synthesis using the () strand template. Furthermore, analysis of the efficiency of RNA synthesis from the individual (þ) 30 -TR, () 30 -TR, and (þ) 50 -TRs revealed that as for DENV-2, the (þ) 30 -TR alone was inactive in RNA synthesis. RNA synthesis from the () strand 30 -TR template resulted primarily in de novo initiated 1 products whereas the use of the (þ) strand 50 -TR resulted in the production of a mixture of 2 and 1 products. These results suggested that cyclization of the genome was an important prerequisite for () but not for (þ) strand RNA synthesis by flavivirus NS5. Investigation of the specific viral RNA sequences recognized by the DENV-2 POL (NS5 amino acids 270–900) revealed that NS5 bound specifically to the conserved 50 -stem-loop structure SLA (Filomatori et al., 2006). As in previous studies, the DENV-2 POL was able to use a template consisting of either the 50 -terminal 160 nucleotides or the 50 -terminal 160 nucleotides and the (þ) 30 -TR, but not the (þ) 30 -TR alone, to synthesize RNA. Mutation and deletion analysis of the 50 -terminal 160 nucleotides, in combination with electrophoretic mobility shift and filter-binding assays, using the DENV-2 POL, localized the element responsible for the 50 template activity to the highly conserved SLA structure. Mutagenesis of SLA in the context of the DENV-2 genome and replicons revealed that
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mutations that disrupted the base of the stem or a top loop abolished or severely decreased viral replication but not initial translation (Filomatori et al., 2006). A physical interaction between the DENV-2 POL and the 50 -TR, mediated by SLA, was confirmed by atomic force microscopy and RNA-binding assays. The DENV-2 POL did not interact with the 30 -TR confirming that it is not an active template for RNA synthesis. However, the 50 -TR with an intact SLA could promote RNA synthesis in trans on a (þ) 30 -TR template. Based on these results, it was proposed that SLA acts a promoter to recruit NS5 for RNA synthesis. Following genome cyclization via long-range interactions between sequences in the 50 - and 30 -TRs, the NS5-SLA complex is positioned adjacent to the 30 -end of the genome facilitating de novo synthesis of the () strand genomic RNA. In contrast to DENV-2 and WNV NS5, JEV NS5 was shown to initiate RNA synthesis de novo using templates corresponding to both the (þ) and () 30 -regions. The yield of product from the 30 1-kb (þ) template was detectable but much less than that using the () template (Kim et al., 2007; Yu et al., 2007). NS5 was shown to bind to and use the terminal 83 nucleotides of the genome to produce a template size product. Sizing of the product showed it was 81 nucleotides in length suggesting that internal initiation had occurred (Kim et al., 2007). It may be the case for JEV NS5 that the 30 -terminal 83 nucleotides are sufficient for the initiation of () RNA synthesis but that RNA synthesis is enhanced by genome cyclization. The difference in template specificity could also be explained by the reaction conditions used in the various experimental systems but requires further investigation.
C. RdRp structure 1. Overall NS5 RdRp structure The crystal structures of the WNV and DENV-3 RdRp domains (described as POL domains below) have recently been determined. Truncated WNV NS5 fusion proteins consisting of a N-terminal hexahistidine tag fused to either amino acids 273–905 (POL1) or 316–905 (POL2) of NS5, expressed in E. coli were soluble and could be crystallized. The ˚ resolution and used as X-ray structure of POL2 was determined to 2.35 A ˚ a basis to determine the POL1 structure to 3 A resolution by molecular replacement (Malet et al., 2007). Plasmid constructs encoding N-terminal truncations of NS5 for all four DENV serotypes were expressed in E. coli and screened for proteins suitable for crystallization. A truncated DENV-3 protein containing amino acids 273–900 of NS5 yielded crystals that ˚ resolution (Yap et al., 2007a). Using the structure of diffracted at 1.85 A the WNV POL as a guide, the DENV-3 POL structure was determined by molecular replacement (Yap et al., 2007b). Not surprisingly, the overall fold of the WNV and DENV-3 POL structures is well conserved and most
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closely related to RdRp structures determined for the Flaviviridae members, HCV (Ago et al., 1999; Bressanelli et al., 1999, 2002; Lesburg et al., 1999) and BVDV (Choi et al., 2004, 2006). However, superimposed on the overall core structure are a number of important differences which distinguish the flavivirus RdRp from those of other viruses. The structures of the WNV and DENV-3 POLs revealed that they have a roughly spherical shape and adopt an architecture typical of previously characterized viral RdRp structures that resemble a ‘‘cupped right hand’’ with subdomains that have been termed the ‘‘fingers,’’ ‘‘palm,’’ and ‘‘thumb’’ ( Joyce and Steitz, 1995; Ng et al., 2008; Fig. 3A). Similar to other viral RdRps, the fingers and thumb subdomains of the flavivirus POL interconnect via the N-terminal region of the RdRp and through loops protruding from the fingers domain to encircle the active site on the palm domain, forming a closed structure (Ferrer-Orta et al., 2006). Two tunnels that run perpendicular to each other can be observed on the structure. One tunnel is located at the interface between the fingers and thumb domain and is predicted to allow access of the single-stranded RNA template to the active site on the palm domain. A second tunnel runs perpendicular to the first, intersecting at the active site and opens to the back of the structure, allowing diffusion of dNTPs to the active site (Fig. 3B).
2. The palm subdomain The palm subdomain is the most highly conserved feature of RdRp structures and contains the active site. Four of the six conserved sequence motifs (A to F (Fig. 1)) that define RdRps are located in the palm domain and residues from these motifs are involved in the binding of metal ions, nucleotides and RNA, and phosphoryl transfer. The palm contains three strictly conserved aspartic acid residues (located in motifs A and C containing the sequences Asp–X4–Asp and Gly–Asp–Asp, respectively) that coordinate two Mg2þ ions and catalyze phosphoryl transfer ( Joyce and Steitz, 1995; Ng et al., 2008). The palm domain of the flavivirus POL structures closely resembles those of other structurally characterized RdRps; however, some important differences were observed (Malet et al., 2007; Yap et al., 2007b). Typically, the palm domain is comprised of a central b-sheet formed from three antiparallel b-strands surrounded by a-helices. By contrast, the central b-sheet of the flavivirus POL consists of only two b-strands (Fig. 1; b4 and b5; unless stated the nomenclature used and amino acid numbering follows that described for the DENV-3 structure (Yap et al., 2007b)) surrounded by eight a-helices. The two b-strands were much shorter than those found in other RdRp structures ˚ compared with 20 A ˚ in the HCV and BVDV RdRps). The active (i.e., 10 A site Gly–Asp–Asp residues (Motif C) are located in a turn between the b4 and b5 strands. Conversely, there was an unusually long insertion between motifs B and C which is absent in the HCV and BVDV RdRp
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RNA template tunnel
A
Priming loop Thumb
Fingers
Palm
B a/bNLS
bNLS
Fingers
Fingers Thumb
180° Palm
“Front”
dNTP access tunnel
Palm
“Rear”
RNA entry C
Core MTase subdomain
Thumb Fingers
N-term subdomain
RNA exit
Palm
FIGURE 3 Structure of the NS5 POL domain. (A) A schematic representation of the X-ray structure of the DENV-3 POL (PDB code: 2J7U; Yap et al., 2007a,b) is shown with the fingers, palm, and thumb subdomains colored in blue, green, and salmon, respectively. The a/bNLS and bNLS are colored in yellow and purple, respectively. The priming loop is colored in black and arrowed. (B) Front and rear surface views of the DENV-3 POL structure. The coloring scheme is the same as in (A). (C) A hypothetical model of the overall WNV NS5 structure generated as described in Malet et al. (2007).
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structures. Structural comparison of the flavivirus POLs with those of other viruses identified the three conserved Asp residues as Asp-533 (motif A), Asp-663, and Asp-664 (motif C). However, neither of the flaviviral POL structures contained metal ions coordinated to the Asp residues in a catalytic conformation. Soaking of the DENV-3 and WNV POL crystals in MgCl2 resulted in the binding of a Mg2þ ion to Asp-533 and Asp-664 in a noncatalytic conformation. The function of the noncatalytic metal ion is unknown. By analogy to other RdRp structures in which noncatalytic metal ions have been observed, it was suggested that it may play a role in the initiation of RNA synthesis (Malet et al., 2007).
3. The fingers subdomain The fingers subdomain serves to shape a tunnel that guides the template RNA to the active site cleft. Similar to the HCV and BVDV RdRps, the fingers subdomain of flavivirus POLs contained a core domain and extended loops termed the ‘‘fingertips’’ that interconnect with the thumb subdomain to enclose the active site (Bressanelli et al., 1999; Choi et al., 2004). However, major differences in the fingers subdomain of flaviviruses and those of HCV and BVDV were found in (a) the N-terminal region of the fingers subdomain, (b) a loop region termed the ‘‘G loop,’’ and (c) the orientation of secondary structures encompassing conserved motif F (Malet et al., 2007, 2008; Yap et al., 2007b). The fingers subdomain also contains a region that has been shown for DENV-2 to contain functional nuclear localization sequences, the so-called ‘‘bNLS’’ and ‘‘a/bNLS’’ (Brooks et al., 2002; Forwood et al., 1999; see Section VI.B). Elements in this region play an important role in the RdRp structure. In the flavivirus, POL structures there is an extra N-terminal stretch of 35-amino acids containing an a-helix (a1) and a b-strand (b1) (Fig. 1), which is absent in the HCV and BVDV structures. Interestingly, the WNV POL2 protein, which had a 44-amino acid N-terminal truncation compared to POL1, lacked RdRp activity, despite the proteins having very similar structures. The b1 strand in the additional N-terminal region contributes to the formation of a three-stranded b-sheet that is absent in the HCV and BVDV RdRps. It was predicted that the b-sheet may stabilize the fingers domain and contribute to the formation of the RNA template tunnel therefore playing an important role in the structure of the POL domain (Malet et al., 2007). The coordinates for the model were kindly provided by Dr. Bruno Canard and colleagues. The WNV POL subdomains are colored as for the DENV-3 POL in (A). The WNV MTase is shown with the N-terminal, core MTase, and C-terminal subdomains colored in red, cyan, and yellow, respectively. The directions for the entry of the template and exit of the nascent RNA are shown. All schematics were produced using PYMOL (DeLano, 2002).
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A flexible loop (termed L1) extended from the N-terminal structure in the fingers subdomain to connect with three a-helices (a2–a4) that encompass a helix-turn-helix motif, and lay on top of the thumb domain. Another loop (L2) extended back to helix a5 in the fingers subdomain, providing an interdomain link (Figs. 1 and 3A and B). Interestingly, a-helices 2–5 are all located in a region shown for DENV-2 to bind both the intracellular tranport protein importin-b and NS3 (termed the ‘‘bNLS’’; Johansson et al., 2001; see Section VI.B). Loop L2 and helix a5, in particular, are found in a 20-amino acid stretch that is highly conserved between flaviviruses (Fig. 1) and predicted to be mobile in the structure. It was suggested that the two flexible loops are likely to be important in transmitting conformational changes between the fingers and thumb domains and maintaining the POL structure (Yap et al., 2007b). Furthermore, the loops may serve to modulate a conformational change in the POL, from a closed to an open structure. For DENV-2, a region adjacent to the bNLS, termed the ‘‘a/bNLS’’ has been shown to contain a functional nuclear localization signal and bind to importin-a/b (Brooks et al., 2002; Forwood et al., 1999). The DENV-3 POL structure revealed that the a/ bNLS is comprised of helix a6, located between the fingers and palm subdomains and helix a7 which is buried in the finger subdomain. Extending from helix a7 is a highly mobile loop (L3) termed the ‘‘G loop’’ as it corresponds to a loop found in primer-dependent RdRps containing a conserved motif (G motif) (Malet et al., 2008). However in the flavivirus POL, loop L3 is found in a unique conformation compared to other characterized RdRps. The loop protrudes toward the active site and is well placed to regulate access of the ssRNA substrate at the entrance to the template channel and could contribute to closure of the active site. The position of the loop coincides with the positioning of C-terminal regions of the HCV and BVDV RdRps. For HCV, the C-terminal region is known to be able to regulate RdRp activity (Leveque et al., 2003; Vo et al., 2004) suggesting loop L3 may play a similar role. Motif F is a conserved feature unique to RdRps that contains positively charged residues that mediate interactions with incoming NTPs (Bruenn, 2003; Ferrer-Orta et al., 2006; Lesburg et al., 1999). In the flavivirus POL, motif F is found in a second fingertip loop. This region was partially disordered and amino acids 454–466 preceding motif F were not visible in the POL structures. However, it was observed that the ordered part of the fingertip contained an a-helix rather than a b-strand present in other RdRps. In addition, this structure was orientated perpendicular to the corresponding structures of other RdRps, shifting the localization of motif F such that it cannot bind incoming NTPs. It was suggested that a conformational shift in the POL would have to occur for motif F to play a role in catalysis (Malet et al., 2007).
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The largest variation between the WNV and DENV-3 structures occurred in the fingers subdomain at residues 296–301, 419–423, 543–545, and 556–559. In addition to local changes, the overall orientation of the fingers subdomain of the WNV POL was rotated closer to the thumb subdomain in comparison to the DENV-3 POL, giving rise to a more closed conformation for the WNV POL. Overall, the fingers subdomain of the flavivirus POL is highly mobile and by comparison to other RdRps, has some important structural differences that necessitate conformational changes occur, before RNA synthesis can be initiated (Malet et al., 2008).
4. The thumb subdomain The C-terminal 187 amino acids of the flavivirus RdRp constitute the thumb subdomain, the most diverse feature among viral RdRp structures. The size and complexity of the thumb subdomain distinguishes RdRps that initiate RNA synthesis de novo from those that require a primer, which have a much smaller thumb subdomain (Ferrer-Orta et al., 2006). The thumb subdomain of RdRps that initiate RNA synthesis de novo possess two features not found in primer-dependent RdRps: (1) a characteristic loop (often termed the initiation or priming loop) that is predicted to form a platform stabilizing the RNA initiation complex and (2) unique C-terminal regions that can fold back into the active site cleft to regulate RNA synthesis (Ng et al., 2008; van Dijk et al., 2004). While the flavivirus thumb subdomain resembles those of other RdRps that initiate synthesis de novo, its overall topology is distinct from that of the HCV thumb subdomain, and more closely resembles the BVDV thumb subdomain. Aside from two antiparallel b-strands that form the interface between the thumb and palm subdomains and constitute conserved motif E, the thumb subdomain consists of a-helices connected by large loops. Two of these loops are of particular importance for flaviviral RNA synthesis. The loop connecting a21 and a22 projects toward the fingers domain and in association with the fingertips contributes to the shape of the RNA template tunnel. A second loop, connecting a23 and a24, was identified as the flavivirus priming loop (amino acids 792–804; Fig. 1; Malet et al., 2007; Yap et al., 2007b). Although clearly recognizable on the structure, it was previously not possible to identify the priming loop by sequence and secondary structure analysis (Kao et al., 2001). The flavivirus priming loop originates from the same part of the thumb subdomain as in the HCV and BVDV RdRps, but is larger in size. Unlike the priming loop of HCV, which takes the form of a b-hairpin, the flavivirus priming loop contains no structural elements. The priming loop is stabilized by internal electrostatic (Thr-794, Ser-796, Glu-807, Arg-815) and base-stacking (Arg-749, Trp-787) interactions with amino acids that are well conserved in the flavivirus POL sequence. The priming loop is likely
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to shape the upper part of the template tunnel and, in association with loop L3 from the fingers subdomain, regulate the entry and exit of the template from the active site (Yap et al., 2007b). The C-terminal regions of HCV, BVDV, Norwalk virus, and bacteriophage F6 can all fold back into the active site cleft. In the case of HCV, this has been shown to regulate RNA synthesis (Leveque et al., 2003; Vo et al., 2004). The C-terminal regions of the flavivirus POLs were not visible in the structures. However, the distance between the last visible residues (Met-883 and Leu-899 for DENV-3 and WNV, respectively) and the active site appeared to be too great for the C-terminus to fold back into it. In addition, a C-terminally truncated WNV POL, lacking the last 23 amino acids, had normal RdRp activity, indicating that the C-terminus of flavivirus NS5 does not regulate RNA synthesis, unlike the HCV and BVDV RdRps. It was suggested that the L3 loop, extending from the fingers subdomain may instead perform this function (Malet et al., 2007).
5. Initiation of RNA synthesis The determination of the structures of a number of viral RdRps in complex with metal ions, ssRNA and/or nucleoside triphosphates has identified specific regions of RdRps important for the initiation of RNA synthesis (van Dijk et al., 2004). Although it has not yet been possible to determine the structure of a flavivirus POL in complex with ssRNA, the structure of the DENV-3 POL in complex with the nucleoside analog 30 dGTP has been determined (Yap et al., 2007b). Biochemical studies have previously shown that flavivirus de novo RNA synthesis requires high concentrations of rGTP. By analogy with other RdRps, it was suggested that rGTP is required for formation of the preinitiation complex (Nomaguchi et al., 2003). The 30 dGTP molecule was found to bind in the ˚ from the active site. Three amino acid vicinity of the priming loop, 7 A residues, strictly conserved in flaviviruses (Ser-710, Arg-729, and Arg-737) were found to bind to the triphosphate component of 30 dGTP. Superimposition of the DENV-3 complex with other RdRps bound to rGTP suggested that Trp-795 stabilized 30 dGTP by base stacking. Based on the structures of the HCV and F6 RdRps bound to ssRNA and rNTPs, the flavivirus RdRp initiation complex has been modeled (Malet et al., 2007; Yap et al., 2007b). A ssRNA template of 5–7 nucleotides could be modeled into a template tunnel formed between the fingers and thumb subdomains such that the 30 -end of the RNA was placed in the catalytic site in a position suitable for interaction with rNTPs involved in the initiation of RNA synthesis. The tunnel is shaped by loops L1, L2, and L3 projecting from the fingers subdomain and the priming loop and the loop connecting a21 and a22, projecting from the thumb subdomain. Binding of the ssRNA was stabilized by electrostatic interactions made with residues in the fingers subdomain. Residue Trp-795, in the priming
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loop, was well placed to stabilize an interaction between the priming nucleotide and the RNA template, therefore providing a platform for the initiation of RNA synthesis. Although a strand of ssRNA could fit in the template tunnel, the tunnel was not wide enough to accommodate a RNA duplex. This supports biochemical studies, suggesting that conformational changes in the RdRp, from a closed to an open form are required during RNA synthesis.
6. Structure of the full-length NS5 Despite intensive effort, it has not yet been possible to determine the structure of the full-length NS5 for any flavivirus. However, the elucidation of multiple flavivirus structures for the MTase and POL domains, in combination with genetic data has facilitated the production of a model for the full-length WNV NS5 using an in silico docking approach (Malet et al., 2007; Fig. 3C). Reverse genetic analysis of the DENV-2 MTase domain led to the identification of a genetic interaction between the MTase and POL domains (Kroschewski et al., 2008; Malet et al., 2007). Mutation of the DENV-2 MTase residues Lys-46, Arg-47, and Glu-49 to Ala, in the context of the viral genome abolished virus replication. Repeated attempts to rescue virus containing this mutation, resulted in the identification of a compensatory mutation in the POL domain (Leu-512 to Val) that restored virus replication. As the MTase and POL mutations lay outside of active site regions, it was proposed that they defined regions in the two domains that interact. Using this data, a homology model of the WNV MTase (based on the DENV-2 MTase structure) and the WNV POL were docked in silico. Spatial constraints imposed by the C-terminus of the MTase and N-terminus of the POL domains (amino acids 264 and 278, respectively) were applied during the modeling process. The model of the full-length WNV NS5 places the RNA-binding region of the MTase in close proximity to the RNA exit tunnel of the POL, suggesting that capping of the newly synthesized positive strand RNA could occur as it leaves the POL domain.
D. Structure–function analysis By contrast to the MTase, there have been fewer studies specifically investigating the structure–function relationship of the flavivirus POL domain, although studies on other viral RdRps can be more easily extrapolated to the flavivirus POL. The essential requirement of conserved RdRp motifs A–D for NS5 function has been confirmed by mutagenesis, either by assaying the RdRp activity of wild-type and mutant recombinant NS5 (Yu et al., 2007) or by the introduction of mutations into the viral genome (Khromykh et al., 1998; Westaway et al., 2002). Eighty clustered charged to Ala mutations have been introduced into the NS5 gene of
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DENV-4 and their effects on viral replication examined to identify attenuated viruses (Hanley et al., 2002). A number of mutations conferring lethal or temperature sensitive viral phenotypes map to regions of NS5 predicted to be important for function by structural analysis. The recent determination of the WNV and DENV-3 POL structures has raised many questions regarding the importance of specific POL elements for RNA synthesis that can now be tested using biochemical and genetic approaches.
IV. NS5 INTERACTIONS The key processes in viral replication performed by NS5 undoubtedly require intramolecular changes to the conformation of NS5 itself, in addition to interactions with viral and presumably host proteins and viral RNA in the replication complex. As described earlier, both the MTase and RdRp activities of NS5 require specific interactions with the viral genome which have been mapped. Interactions between the MTase and POL have also been identified. The multifunctional NS3 protein contains RNA helicase, nucleoside triphosphatase (NTPase), and RNA triphosphatase activities, which are assumed to act in combination with the MTase and RdRp activities of NS5 to replicate and cap the viral genome. A number of studies have detected an association between NS3 and NS5 and regions of the proteins which interact have been identified. In addition, interactions with an increasing number of cellular proteins not currently known to be directly required for replication, are being defined, suggesting that NS5 plays a number of roles in the infected host cell.
A. NS5 intramolecular interactions The enzymatic activities of the MTase and POL domains are active in isolation; however, recent evidence suggests that the conformation or enzymatic activity of one domain may influence the function of the other. As described earlier (see Section III.C.6) reverse genetic analysis of the DENV-2 MTase domain identified a genetic interaction between residues Lys-46, Arg-47, and Glu-49 in the MTase domain and Leu-512 in the POL domain (Malet et al., 2007). In silico docking of the two domains fitted a MTase loop containing residues Lys-46, Arg-47, and Glu-49 into a groove formed by the fingers and thumb subdomains. A loop protruding from the palm subdomain containing residue Leu-512 was located at the base of the groove suggesting a direct interaction between the residues could occur, although structural analysis could not provide an explanation for the effects mutations at these residues had on NS5 function. Reverse genetic analysis has also identified a genetic interaction between
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the WNV MTase and POL domains (Zhang et al., 2008). Mutation of the catalytic site residue Asp-146 to Ala in the WNV MTase abolished both N7 and 20 -O-MTase activities and viral replication. Examination of the effects of additional mutations at Asp-146 revealed that the introduction of a Ser substitution restored minimal levels of N7 MTase activity and allowed low-level replication. Continued passaging of the mutant virus resulted in the isolation of large plaque variants which were found to have second site mutations in the MTase (Lys-61 to Gln/Thr) and POL domains (Trp-751 to Arg) and the 50 -stem-loop structure (G35U or a U insertion at nucleotide 38). Analysis of the mutations revealed that the substitutions at Lys-61 increased N7 MTase activity while the change Trp751 to Arg enhanced RdRp activity. Trp-751 was shown to be surface exposed, at the opening of the template tunnel, but unlikely to directly interact with residue Asp-146 in the MTase domain, suggesting that the increase in RdRp activity may compensate for decreased cap methylation.
B. The interaction of NS5 with viral RNA A number of studies have investigated the interaction of NS5 with the 50 - and 30 -TRs. As described earlier (see Section III.B.4), a conserved stemloop structure (SLA) at the 50 -end of the flavivirus genome has been shown to be essential for both MTase and RdRp activities and to bind recombinant full-length WNV NS5 (Dong et al., 2007) and DENV-2 POL (Filomatori et al., 2006) in vitro. Mapping of the site of interaction of WNV NS5 with the 50 -terminal 190 nucleotides of the genome by RNA foot print analysis showed that NS5 protected the lower half of the SLA stem-loop structure and a number of nucleotides in SLB. Analysis of NS5 binding when the 50 -TR was complexed with the 30 -TR revealed an identical pattern, except that nucleotides in SLB were no longer protected. The results suggested that NS5 binds to the 50 -TR primarily through SLA. The importance of the nucleotides implicated in NS5 binding to viral replication was confirmed by mutagenesis studies using a WNV replicon system (Dong et al., 2008d). The full-length WNV NS5 and the DENV-2 POL were not found to bind specifically to the 30 -TR, by contrast, an interaction between JEV NS5 and the 30 -TR has been detected. A UV crosslinking assay was used to identify proteins in JEV-infected BHK-21 cell lysates that bound to the 30 -terminal 585 nucleotides of the JEV RNA genome (Chen et al., 1997). Two proteins identified as the NS3 and NS5 proteins by immunoprecipitation were found to bind the RNA. The NS5-binding site was mapped to the terminal 83 nucleotides of the RNA, containing a conserved stem-loop. This result was supported by the finding that the 30 -terminal 83 nucleotides could be used as a template for RNA synthesis by a recombinant JEV NS5 (Kim et al., 2007). Interestingly, it was reported that although WNV NS5 bound
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to the 50 -TR, the WNV POL showed minimal binding (Dong et al., 2008d). Overall, these results suggest that there may be flavivirus-specific differences in the interaction of NS5 with the 50 - and 30 -TRs.
C. The interaction of NS3 and NS5 Interaction of NS3 and NS5 during viral infection was first demonstrated for DENV-2 by radioimmunoprecipitation using antibodies against the NS3 and NS5 proteins (Kapoor et al., 1995). NS3 and NS5 also interacted in HeLa cells coinfected with recombinant vaccinia viruses expressing the two proteins, showing that additional viral proteins were not required for the interaction. A recombinant C-terminally hexahistidine-tagged NS5, immobilized to Ni-agarose beads, bound recombinant NS3 present in cell lysates, demonstrating that NS3 and NS5 interacted in vitro. In the study of Kapoor et al. (1995) two forms of NS5 were detected, a hyperphosphorylated form that was predominantly localized to the nucleus of infected cells and a hypophosphorylated form present in the cytoplasm. NS3 only interacted with the cytoplasmic hypophosphorylated form, suggesting that differential phosphorylation may regulate the interaction between NS3 and NS5 (see Section V). The association between NS3 and NS5 in vivo has since been confirmed by coimmunoprecipitation experiments using lysates from JEV- (Chen et al., 1997) and DENV-1 (Cui et al., 1998)-infected cells and NS3- and NS5-specific antisera. The physical association of NS3 and NS5 potentially leads to an alteration in the enzymatic activities of one or both proteins. The NTPase activity of a recombinant full-length DENV-1 NS3 was stimulated by the addition of purified recombinant DENV-1 NS5 to the assays, an effect that was specific to NS5 among the viral proteins (Cui et al., 1998). The stimulatory effect of NS5 on NS3 NTPase activity was confirmed using recombinant full-length DENV-2 NS5 and NS3 (Yon et al., 2005). The DENV-2 NS5 stimulated NS3 NTPase activity in a dose-dependent manner until a 1:1 stoichiometry was reached, after which there was no effect, suggesting that a NS3/NS5 complex was the active unit for NTPase activity. The presence of the DENV-2 NS5 was also found to stimulate the 50 -RNA triphosphatase activity of NS3 by fivefold, providing evidence that flavivirus cap formation involves a complex between NS3 and NS5 (Yon et al., 2005). Using the yeast two-hybrid system, it was shown that amino acids 303–618 of the DENV-2 NS3, located in the C-terminal helicase domain, interacted with amino acids 320–368 of DENV-2 NS5 located in the RdRp domain (Johansson et al., 2001). Interestingly, the nuclear import factor importin-b was also found to interact with the same region of NS5 using the yeast two-hybrid system. Competitive binding analysis using the fulllength NS3 and importin-b showed that the NS5-binding sites for NS3 and importin-b either overlap or are closely related. NS5 residues 320–368
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encompass a 20-amino acid stretch that is highly conserved among all flaviviruses. In contrast to the interacting NS3 and NS5 regions defined by biochemical studies, a genetic interaction between the DENV-2 NS3 protease domain and the NS5 MTase domain has also been demonstrated. Mutation of NS5 amino acids Glu-192, Lys-193, and Glu-195 to Ala in the context of a DENV-2 infectious clone led to undetectable levels of virus replication but had little effect on N7 and 20 -O-MTase activities (Kroschewski et al., 2008). The mutated residues are surface exposed in helix aE of the MTase, well removed from the AdoMet- and GTP-binding sites (Fig. 2A). Repeated attempts at virus recovery resulted in the isolation of a virus containing the clustered Glu-192, Lys-193, and Glu-195 to Ala mutation and additionally an Ala to Gly substitution at amino acid residue 70 in the protease domain of NS3, suggesting that helix aE of the MTase, encompassing residues 187–202, interacts with the NS3 protease domain. This finding is supported by a previous study showing that NS5 amino acids 1–316 and NS3 amino acids 1–178 could not be complemented in trans using a WNVKUN replicon system (Khromykh et al., 1999, 2000); therefore, the genetic analysis may have identified regions of NS3 and NS5 that interact in cis during the formation of the replication complex in vivo. Substitution of Glu-192 and Glu-193 with Ala in a DENV-4 infectious cDNA clone abolished viral replication confirming the importance of helix aE for virus replication (Hanley et al., 2002).
D. The interaction of NS5 with host proteins There are a number of points during the virus lifecycle where NS5 is believed to interact with host proteins. It has been proposed that NS5 interacts with viral RNA sequences and proteins during the formation and functioning of the replication complex (Villordo and Gamarnik, 2009; Westaway et al., 2003). Host proteins also presumably play a role in the viral replication complex. Experiments analyzing the interaction of the DENV-4 50 - and 30 -TRs with cellular and viral proteins led to the identification of an interaction between NS5 and the La protein (Garcia-Montalvo et al., 2004). UV crosslinking experiments using RNA transcripts representing the DENV-4 50 - and 30 -TRs and lysates from infected U937 monocytic cells identified seven proteins that interacted with both the 50 - and 30 -TRs, one of the proteins was the La protein, a protein previously shown to interact with the 30 -TR of the DENV-4 genome in human monocytes (YocupicioMonroy et al., 2003). The La protein could be immunoprecipitated from lysates from infected cells both in complex with the DENV-4 50 - and 30 -TRs and NS5 (Garcia-Montalvo et al., 2004). A recombinant La protein was found to inhibit DENV RdRp activity in a dose-dependent manner, both in assays using recombinant DENV-2 NS5 or extracts from DENV-4-infected C6/36 cells, suggesting that the La protein may play a role in regulating positive and negative strand synthesis (Yocupicio-Monroy et al., 2007).
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NS5 is found not only in the replication complex but also free in the cytoplasm and for some flaviviruses in the nucleus of infected cells (see Section VI). In addition, NS5 is known to be phosphorylated (see Section V). This suggests that NS5 interacts at least transiently with host proteins involved in intracellular trafficking and phosphorylation/ dephosphorylation. Evidence for such interactions is described in Sections V and VI. Recent investigations have also shown that NS5 can influence cellular host immune responses leading to the identification of flavivirus-specific interactions with host proteins. These interactions are described in Section VII.
V. NS5 PHOSPHORYLATION For a number of flaviviruses, NS5 has been shown to be phosphorylated. As the phosphorylation of a protein can change its enzymatic activity, subcellular localization or ability to interact with other macromolecules (Cohen, 2000), phosphorylation provides a means to regulate the multifunctional roles of NS5 in the virus lifecycle. It is well established that the function of replicative proteins of negative strand viruses can be regulated by phosphorylation and there is accumulating evidence that this may also be the case for positive strand RNA viruses ( Jakubiec and Jupin, 2007). Immunoprecipitation of NS5 from flavivirus-infected cells metabolically labeled with 32P-orthophosphate established that NS5 of DENV-2 (Kapoor et al., 1995), YFV (Reed et al., 1998), and WNVKUN (Mackenzie et al., 2007) can be phosphorylated during viral infection. In addition, the TBEV NS5 present in lysates from infected cells or immunoprecipitates could be phosphorylated using an in vitro kinase assay (Morozova et al., 1997). Ectopic expression of the DENV-2 and YFV NS5 genes in mammalian cells resulted in the production of phosphorylated NS5, showing that NS5 phosphorylation can occur in the absence of other virus proteins. Phosphoamino acid analyses of 32P-labeled NS5 from DENV-2-, TBEV-, and YFV (Kapoor et al., 1995; Morozova et al., 1997; Reed et al., 1998)infected cells and Western blot analysis of the WNVKUN NS5, using phospho-specific antibodies, revealed that phosphorylation is primarily restricted to Ser, although a low level of Thr phosphorylation was also observed for YFV. Phosphorylation of DENV-2 NS5 was proposed to occur on at least four distinct Ser residues. More recently, mass spectrometry has been used in combination with site-specific mutagenesis to identify a specific amino acid residue (Ser-56) that is phosphorylated in YFV NS5 (Bhattacharya et al., 2008). Mass spectrometry analysis of a recombinant hexahistidine-tagged YFV NS5, expressed in and purified from HEK-293 cells, resulted in the identification of six phosphopeptides containing Ser and/or Thr residues. Detailed analysis of the phosphorylation
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of one of the peptides (amino acids 49–61), by mutagenesis and biochemical analysis, identified Ser-56 as a phosphoamino acid. Interestingly, Ser56 has been shown to be essential for MTase activity (Dong et al., 2008b; Kroschewski et al., 2008; see Section II.D.2). Substitution of Ser-56 with Ala (ablating phosphorylation) or Asp (mimicking phosphorylation) both in a recombinant bacterially expressed YFV MTase protein and in the context of a YFV replicon, abolished 20 -O-MTase activity and replication of the YFV replicon, respectively, confirming the importance of Ser-56 in the viral lifecycle (Bhattacharya et al., 2008). It is tempting to speculate that phosphorylation of Ser-56, a residue strictly conserved among flaviviruses and essential for MTase activity, may regulate the function of NS5. However, it remains to be demonstrated whether Ser-56 is phosphorylated during the virus lifecycle and manifests a change in NS5 function in vivo. Little is known concerning the kinases or phosphatases that may regulate the phosphorylation of NS5. A study comparing the phosphorylation of the NS5A proteins of HCV and BVDV with YFV NS5, using an in vitro kinase assay, suggested that the same or closely related kinases phosphorylated all three proteins and that the kinase responsible may be a member of the CMGC family, which includes casein kinase II (CKII) and proline-directed kinases such as the mitogen-activated protein kinases (MAPKs), glycogen synthase kinase 3 (GSK3), and cyclin-dependent kinases (CDKs). In addition, in vitro phosphorylation of YFV NS5 was found to be much more sensitive to the broad spectrum kinase inhibitor staurosporine, indicating a number of kinases may be involved in its phosphorylation (Reed et al., 1998). However, subsequent studies on the HCV 5A protein have shown that casein kinase I-a (CKI-a) rather than a CMGC kinase is most likely to phosphorylate the 5A protein in vivo (Huang et al., 2007; Quintavalle et al., 2007). Differentially phosphorylated forms of DENV-2 NS5 could be detected in both infected cells and cells expressing NS5 alone. Cell fractionation experiments revealed that hypophosphorylated NS5 was confined to the cytoplasm whereas a hyperphosphorylated form was found predominantly in the nucleus. As mentioned earlier, NS3 associated with the hypo but not the hyperphosphorylated form of NS5 (Kapoor et al., 1995). These results led to a model predicting that the association of NS3 and NS5 in the viral replication complex is regulated by phosphorylation. Hyperphosphorylation of NS5 results in dissociation of NS5 from NS3 and in the case of DENV-2, nuclear import of NS5 (Kapoor et al., 1995). However, phosphorylation of NS5 may also inhibit NS5 nuclear import. Examination of the DENV-2 NS5 sequence led to the identification of a consensus CKII site (Thr-395/Arg/Glu/Glu) within a 37-amino acid stretch containing a functional nuclear localization signal. A fusion protein consisting of the 37-amino acid stretch fused to b-galactosidase could
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be phosphorylated in vitro by CKII; however, phosphorylation inhibited rather than enhanced nuclear import (Forwood et al., 1999).
VI. NS5 LOCALIZATION A. Cellular localization of NS5 Fractionation of WNV-infected cells to enrich for RdRp activity led to progressive depletion of NS5 from membrane fractions but had little effect on the RdRp activity exhibited by these fractions (Grun and Brinton, 1986, 1987, 1988). Further studies demonstrated that WNVKUN RdRp activity was predominantly associated with cytoplasmic ‘‘heavy’’ membrane fractions which could be sedimented at >16,000g (termed the ‘‘16K fraction’’) (Chu and Westaway, 1987, 1992; Chu et al., 1992). The heavy membrane fraction was found to be enriched for NS3, NS2A, NS2B, and NS4A. By contrast, NS5 was either depleted or could not be detected in the heavy membrane fractions. The majority of NS5 was found in soluble fractions which retained little RdRp activity. More recently, 16K membrane fractions produced from 35S-labeled JEV-, DENV-2-, and WNV-infected mammalian cells were found to possess RdRp activity which was associated with detectable amounts of labeled NS3 and NS5 (Uchil and Satchidanandam, 2003a,b). Extensive treatment of the 16K membrane fraction with trypsin decreased NS3 and NS5 to undetectable amounts whereas there was no reduction in RdRp activity. When the 16K membrane fractions were first solubilized with the ionic detergent sodium deoxycholate before trypsin treatment, RdRp activity was destroyed. Collectively these results suggested that only very small amounts of catalytically active NS3 and NS5 are found in the membrane-bound replication complex and are required for RdRp activity. Whereas NS3 is primarily membrane associated, NS5 may be localized in soluble cytoplasmic or nuclear fractions. Immunolocalization studies have shown that, during infection, the NS5 of DENV-2 (Kapoor et al., 1995; Mackenzie et al., 2007; Malet et al., 2007; Miller et al., 2006; Pryor et al., 2007), YFV (Buckley et al., 1992), and JEV (Uchil et al., 2006) can be detected in the nucleus of a range of mammalian cell types. Nuclear localization of DENV-2 NS5, in infected Vero and BHK-21 cells, could be detected as early as 14–16 h postinfection and increased as the infections progressed (Miller et al., 2006; Pryor et al., 2007). By contrast, NS5 of WNV strains Kunjin and Sarafend could not be detected in the nucleus of infected cells either by immunofluorescence assay or by immunogold labeling (Mackenzie et al., 2007; Malet et al., 2007). Detailed studies of DENV-2 NS5 have shown that the nuclear localization of NS5 is an active process dependent on the cellular nuclear
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import and export pathways (Alvisi et al., 2008; Brooks et al., 2002; Forwood et al., 1999; Pryor et al., 2006; see Section VI.B). Recently, it has been suggested that nuclear-localized NS5 may be involved in the synthesis of viral RNA. In early studies investigating flaviviral RdRp activity, a proportion of the RdRp activity was often found in crude nuclear pellets. However, it was not determined whether the RdRp activity was contained within the nucleus or was associated with contaminating outer nuclear membranes that are a rich source of RdRp activity. The possible involvement of the nucleus in flavivirus RdRp activity was re-examined by Uchil et al. (2006). Examination of the RdRp activity of cytoplasmic, heavy membrane, and nuclear fractions from mammalian cells infected with JEV, WNV, and DENV revealed that while the heavy membrane fractions contained the majority of the RdRp activity, as previously reported, 30–40% of the RdRp activity was associated with the nuclear pellet. Treatment of virus-infected cells with the microtubule depolymerizing drug nocodazole, to separate cytoplasmic membranes from the nucleus, did not affect the proportion of the RdRp activity found in the nuclear fraction. Biochemical analysis using techniques that separate nuclei from nuclear membranes revealed that the JEV NS3 and NS5 proteins and newly synthesized RNA were present in the nuclear preparations (Uchil et al., 2006). NS5 was found diffusely throughout the nucleus whereas NS3 and viral RNA were tightly localized to the inner nuclear membrane. By contrast, colocalization studies using WNVKUN-infected cells have detected NS3, NS5, and newly synthesized RNA only in the cytoplasm in association with perinuclear membranes (Mackenzie et al., 2007). Further investigations are required to determine whether nuclear-localized NS5 is involved in flavivirus RNA synthesis.
B. NS5 nuclear localization The nuclear localization of proteins greater than 45 kDa is an active process requiring recognition of a nuclear localization signal (NLS) or nuclear export signal (NES) on the cargo protein by members of the importin superfamily. In the ‘‘classical’’ NLS import pathway, the positively charged NLS is first recognized by importin-a which serves as an adaptor to indirectly link the cargo protein to importin-b. The complex is then translocated through the nuclear pore complex. Binding of Ran-GTP to importin-b on the nucleoplasmic side of the pore results in dissociation of the complex and release of the cargo protein in the nucleus. Typically, NLSs recognized by importin-a consist of either a single stretch of basic amino acids (monopartite NLS) such as that for the SV40 large T antigen (PKKKRKV) or two clusters of basic amino acids separated by 10–12 amino acids (bipartite NLS). However, most importin family members,
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including importin-b, can interact directly with a NLS on the cargo protein and translocate it through the nuclear pore complex. Conversely, proteins can also be actively exported from the nucleus by specific importin superfamily members termed exportins. Typically, a leucine-rich NES on the protein is recognized by an exportin complexed with Ran-GTP. Disassociation of Ran-GTP occurs upon hydrolysis of GTP to GDP, leading to nuclear export. The best characterized exportin is CRM-1, responsible for export of the HIV-1 Rev protein (Alvisi et al., 2008; Conti and Izaurralde, 2001; Lange et al., 2007). Following the finding that DENV-2 NS5 could be detected in the nucleus of infected cells (Kapoor et al., 1995), analysis of the DENV-2 NS5 sequence identified three clusters of basic amino acids (Lys-371/ 372, Lys-388/389/390, and Arg-401/Lys-402) encompassed within amino acids 369–405 that resembled one or more bipartite NLSs (Fig. 1). Fusion of the 37-amino acid region N-terminally to b-galactosidase (NS5NLS-b-gal) resulted in the nuclear localization of b-galactosidase, both in vivo in microinjected cells and in vitro in mechanically perforated cells, confirming that DENV-2 NS5 contained a functional NLS (Forwood et al., 1999). The NS5-NLS-b-gal protein bound a mouse importina/b-heterodimer with high affinity in an ELISA-based binding assay, suggesting that nuclear transport of NS5 depended on the conventional importin-a/b import pathway. Site-directed mutagenesis and deletion analysis of the 37-amino acid region containing the NLS identified a minimal NLS (amino acids 369–389, termed the a/bNLS), which retained the b-gal nuclear-targeting ability and importin-a/b-binding activity of amino acids 369–405. Within this region, Lys-371/372 and to a lesser extent Lys-388/389 were found to be most important for the function of the NLS (Brooks et al., 2002). A second functional NLS was identified adjacent to the a/bNLS. Yeast two-hybrid analysis identified an interaction between amino acids 320–368 of the DENV-2 NS5 (termed the bNLS) and importin-b1 (Johansson et al., 2001). A fusion protein consisting of the bNLS fused N-terminally to b-galactosidase (NS5-bNLS-b-gal) accumulated in the nucleus and bound importin-b1 with high affinity in an ELISA assay (Brooks et al., 2002; Johansson et al., 2001). As mentioned previously, the C-terminal region of NS3 was also found to interact with NS5 amino acids 320–368 and compete with importin-b for binding of the bNLS using pulldown assays. Interestingly, the bNLS contains a stretch of 20 amino acids (amino acids 342–361; Fig. 1) that are highly conserved among flaviviruses. Although the bNLS and a/bNLS were found individually to be functional, a b-gal fusion protein containing both sequences showed markedly reduced nuclear accumulation and binding to importin-b1 and importina/b. This suggested that in the context of the full-length NS5, the function of the NLSs may be regulated by their conformation or interaction with
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other molecules (e.g. NS3). To investigate whether both NLSs were required for nuclear localization of the full-length NS5 protein and assess the role of the NLSs in the viral lifecycle, the NLSs were mutated, in the context of both a plasmid encoding the green fluorescent protein N-terminally fused to the full-length NS5 (GFP-NS5) and a DENV-2 infectious cDNA clone (Pryor et al., 2007). Two clusters of charged residues in the bNLS (Arg-353/Lys-358 and Glu-357/Lys-358/Asp-360), predicted to play a role in the binding of importin-b (Brooks et al., 2002) were mutated to Ala. The mutations had little effect on the nuclear localization of the transiently expressed GFP-NS5; however, when introduced into the viral genome, the mutations abolished viral replication. By contrast, mutation of each of the two basic clusters of amino acids, previously shown to be important for the function of the a/bNLS (Lys-371/372 and Lys-387/388/ 389) confirmed that both clusters were required for nuclear localization of GFP-NS5 with the second cluster being most important. Introduction of the individual clustered mutations into the viral genome resulted in the production of viable viruses whereas the introduction of both clusters abolished viral replication. Analysis of the localization of NS5 in cells infected with virus containing the Lys-387/388/389 to Ala mutations revealed that there was a delay in NS5 localization compared to the wild-type virus that was most pronounced early in the infection. The delay in NS5 localization was found to correlate with a delay in viral growth and a 100-fold decrease in peak viral titer, suggesting that NS5 nuclear localization plays a role in DENV-2 replication. Although originally proposed to lie in an interdomain linker region of NS5 (Forwood et al., 1999), structural studies have now shown that the bNLS and a/bNLS are actually important structural components of the RdRp domain (Fig. 3B; Malet et al., 2007; Yap et al., 2007b; see Section III. C.3). Therefore, it is not surprising that when mutations in the bNLS were introduced into the viral genome they abolished virus replication without affecting nuclear localization. In addition, it is possible that mutations in the a/bNLS could also affect viral replication through mechanisms distinct to its role in nuclear localization. The accumulation of DENV-2 NS5 in the nucleus is not only dependent on nuclear import but also on nuclear export. The drug leptomycin B inhibits CRM-1-dependent nuclear export. Treatment of DENV-2-infected Vero cells with leptomycin B resulted in an increase in the accumulation of nuclear-localized NS5, particularly early in the infection which correlated with an increase in virus production (Pryor et al., 2006). This effect was also shown when a GFP-NS5 was expressed alone in Vero cells. An NES has been identified in the bNLS and it has been reported that site-specific mutagenesis of the NES results in increased NS5 accumulation and nonresponsiveness to leptomycin B (Alvisi et al., 2008; Pryor et al., 2006; Rawlinson et al., 2009).
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The role of NS5 in the nucleus has not yet been elucidated. As NS5 nuclear localization is not a property shared by all flaviviruses, it cannot be strictly required for replication. It has been shown that DENV-2 NS5 can induce the production of the cytokine IL-8 (see Section VII). A mutation in the a/bNLS which decreased nuclear localization of NS5 and virus production also altered IL-8 secretion, providing evidence that the nuclear trafficking of NS5 may influence host cell processes (Pryor et al., 2007). Further studies on NS5 localization using a range of flaviviruses and cell types relevant to disease are required to define the role of NS5 localization in the virus lifecycle.
VII. EMERGING ROLES FOR NS5 IN VIRAL PATHOGENESIS A number of recent studies suggest that NS5 has the ability to interfere with key processes involved in the host immune response, but that interestingly, the pathways involved may be flavivirus specific. The interferon (IFN) response is a key host defense to viral infection including flaviviruses. Like many viruses, flaviviruses have evolved strategies to evade the IFN response. A common theme is the ability of flaviviruses to block cellular signaling by the Janus-activated kinase– signal transducer and activator of transcription (JAK–STAT) pathway in response to IFN stimulation. A number of different flavivirus proteins have been shown to be capable of inhibiting IFN signaling including, recently, the NS5 of JEV, LGTV, and TBEV. IFN-a and -b (Type I IFNs) and IFN-g (Type II IFN) bind to heterodimeric receptors on the cell surface known as the type I IFN receptor (comprised of the IFNAR1 and IFNAR2 subunits) and type II IFN receptor (comprised of the IFNGR1 and IFNGR2 subunits), respectively. The subunits of the receptors are constitutively associated with distinct cellular tyrosine kinases belonging to the JAK family; IFNAR1 and IFNAR2 are associated with tyrosine kinase 2 (Tyk2) and JAK1, respectively, while IFNGR1 and IFNGR2 are associated with JAK1 and JAK2, respectively. Binding of the IFNs to their specific receptors causes rearrangement and oligomerization of receptor subunits leading to autophosphorylation and activation of the associated tyrosine kinase, which in turn regulates the phosphorylation and activation of STAT proteins. The phosphorylated STAT proteins form homodimers or heterodimers with other STAT proteins (STAT1–STAT2 for IFN-a/b and STAT1–STAT1 for IFN-g) and then translocate to the nucleus to activate the transcription of IFN-stimulated genes (ISGs). The production of the ISG gene products leads to an antiviral state in the cell (Platanias, 2005; Randall and Goodbourn, 2008).
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IFN-a/b signaling was found to be inhibited in JEV-infected cells due to a block in Tyk2 phosphorylation (Lin et al., 2004). By contrast, in LGTVinfected cells, the phosphorylation of Tyk2 and JAK1 was blocked, leading to inhibition of both IFN-a/b and IFN-g signaling (Best et al., 2005). Analysis of STAT1 phosphorylation and nuclear translocation in response to IFN-a/b treatment of cells expressing the individual JEV and LGTV gene products revealed that NS5 alone inhibited JAK–STAT signaling (Best et al., 2005; Lin et al., 2006). Further analysis of the effects of NS5 on JAK–STAT signaling showed that JEV NS5 inhibited Tyk2 phosphorylation and the downstream induction of several IFN-a-inducible gene products while LGTV NS5 could inhibit transcription from IFN-a/b and IFN-g-responsive reporter gene constructs. To determine whether the effects of NS5 on JAK–STAT signaling were the result of a direct physical interaction with components of the IFN signaling pathways, protein interaction studies were done. Immunoprecipitation of the IFN receptor subunits from Vero cells transiently expressing the LGTV NS5 demonstrated that NS5 bound to the IFN-a/b receptor subunit IFNAR2 and possibly the IFN-g receptor subunit IFNGR2 (Best et al., 2005). The interaction of LGTV NS5 with IFNAR2 and IFNGR2 was confirmed in a more relevant context using lysates from LGTV-infected human and murine monocyte-derived dendritic cells. Once again, the LGTV NS5 coprecipitated with IFNAR2 but not JAK1, Tyk2 or STAT1. The interaction of LGTV NS5 with IFNAR2 occurred both with and without IFN treatment. The JEV NS5 was found not to interact with Tyk2 or JAK1 using a mammalian two-hybrid system (Lin et al., 2006). However, pretreatment of JEV NS5-expressing cells with sodium orthovanadate, a broad spectrum inhibitor of protein tyrosine phosphatases that are known to negatively regulate JAK–STAT signaling, resulted in suppression of the effects of NS5 on IFN signaling. Based on these results, it was suggested that JEV NS5 might activate inhibitors of JAK–STAT signaling rather than directly perturbing the components themselves. The minimal regions of the JEV and LGTV NS5 proteins required for inhibition of JAK–STAT signaling were defined by examining STAT1 phosphorylation and/or nuclear translocation in response to IFN treatment of cells expressing truncated versions of NS5. A truncated JEV NS5 protein containing the N-terminal 1–762 amino acids was capable of inhibiting STAT1 nuclear translocation, similar to the full-length protein. However, deletion of the C-terminal region to residue 667 reduced the block to STAT1 nuclear translocation while further deletion of either the C-terminus to residue 584 or the N-terminal 83 or 166 amino acids abolished the ability of the truncated NS5 proteins to inhibit STAT1 nuclear translocation (Lin et al., 2006). These results suggested that JEV NS5 did not require functional MTase or RdRp activities to inhibit IFN signaling. The examination of 11 N- and C-terminally truncated LGTV NS5 proteins
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defined a minimal region of NS5 (amino acids 355–735) that was required for wild-type inhibition of JAK–STAT signaling (Park et al., 2007). A truncated LGTV NS5 comprising amino acids 342–735 was then subjected to extensive random and site-directed mutagenesis including potential phosphorylation and active site residues and the resultant proteins screened for their ability to inhibit JAK–STAT signaling. Two regions were found to contribute to the inhibition of JAK–STAT signaling when evaluated in the context of the full-length NS5; residues 374–380 with Arg-376 and Asp-380 being most critical and residues 624–647 with residues Glu-326, Glu-328, and Trp-647 being the most critical. When residues 374–380 and 624–647 were modeled on the WNV POL crystal structure, it was found that the two regions lay in close proximity and were surface exposed at the junction of the finger and palm subdomains, suggesting that this region of the protein may be directly involved in binding the IFNAR2 receptor complex (Park et al., 2007). Interestingly, residues 374–389 in LGTV NS5 overlaps the a/bNLS identified in DENV-2 NS5 (see Section VI.B), suggesting that this region of NS5 may be specifically suited to interact with host proteins. Neither NS5 phosphorylation nor RdRp activity appeared to be required for NS5-mediated inhibition of JAK–STAT signaling. It will be of interest to determine whether mutation of these regions abrogate IFNAR2 binding and effect the sensitivity of LGTV to IFNs when introduced into the viral genome. Using a different approach, it has recently been shown that the TBEV NS5 is also able to interfere with JAK–STAT signal transduction. The TBEV NS5 was found to interact with the human scribble (hScrib) protein in a yeast two-hybrid screen (Werme et al., 2008). hScrib is highly concentrated at epithelial cell junctions where it is involved in establishing and maintaining cell polarity (Dow et al., 2003) and belongs to the LAP family of adaptor proteins which are characterized by a combination of 16 leucine repeats at the N-terminal region of the protein and either 1 or 4 PDZ domains at the C-terminal region (Santoni et al., 2002). Each PDZbinding domain mediates binding of the protein to C-terminally located sequences in proteins conforming to the PDZ-binding motif (S/T–X–L/ V/I). The TBEV NS5 protein contains a PDZ-binding motif in its C-terminal region; however, deletion analysis of TBEV NS5 followed by site-specific mutagenesis showed that Tyr-222 and Ser-223 and not the residues in the PDZ-binding motif mediated hScrib binding. The association between NS5 and hScrib was confirmed by pull-down assays using bacterially expressed proteins and lysates from HeLa cells expressing NS5. Investigation of the colocalization of endogenous hScrib and transiently expressed wild-type and mutant (Tyr-222/Ser-223 to Ala) NS5 in MDCK cells showed that there was substantial enrichment of wild-type but not mutant NS5 at the cell–cell contacts where hScrib was found. Knockdown of hScrib led to relocalization of NS5 to the site of the mutant
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NS5, demonstrating that hScrib was able to target NS5 to the cell periphery. To determine if the membrane-localized NS5 may play a role in perturbing IFN signaling, HeLa cells expressing the wild-type and mutant TBEV NS5 were examined for their IFN responsiveness by the analysis of STAT-1 phosphorylation and reporter gene assays. The results showed that both IFN-a/b and IFN-g signaling pathways were inhibited by NS5 whereas the mutant NS5 had a reduced ability to block JAK–STAT signaling. Overall, the results of this work suggested that the association between hScrib and NS5 was important for inhibition of JAK–STAT signaling (Werme et al., 2008). In comparison to the TBEV, JEV, and LGTV NS5, the NS5 proteins of DENV-2 and 4, which were included as controls in the studies described previously, did not inhibit JAK–STAT signaling (see Footnote for recent studies showing that DENV-2 NS5 can perturb type I IFN signalling)1. However, DENV-2 NS5 has been shown to be capable of inducing the production of the cytokine interleukin-8 (IL-8). Elevated levels of IL-8 have been detected in the serum of DENV-infected patients (Juffrie et al., 2000; Medin et al., 2005; Raghupathy et al., 1998) and in the culture supernatants of a variety of DENV-infected cultured cells including dendritic cells and monocytes (Bosch et al., 2002; Moreno-Altamirano et al., 2004), which are of relevance to DENV infection in vivo. Expression of individual DENV genes in HEK-293 cells identified NS5 alone as being capable of increasing IL-8 gene expression and secretion (Medin et al., 2005). A number of transcription factors are required to activate IL-8 transcription including, activating protein 1 (AP-1), NF-kB and CAAT/ enhancer-binding protein (c/EBP). Using promoter reporter constructs activated by the three transcription factors, it was shown that NS5induced IL-8 expression was predominantly reliant on c/EBP and to a lesser extent NF-kB while DENV-2 infection activated all three factors suggesting NS5 alone was not responsible for full IL-8 induction (Medin et al., 2005). To determine whether the localization of NS5 played a role in IL-8 induction, IL-8 secretion from HEK-293 cells infected with DENV2 or a recombinant virus containing a mutation in the NS5 a/bNLS, which delayed NS5 localization was compared. It was found that delayed nuclear accumulation of NS5 led to an increase in IL-8 secretion and decrease in virus production. The effects on IL-8 secretion were confirmed by transient expression of the wild-type and mutant NS5 (Pryor et al., 2007). The results suggested that an increase in cytoplasmic NS5 may contribute to IL-8 induction. However, it remains to be determined how the change in NS5 nuclear localization alters the IL-8 response. 1
During the publication of this article, two manuscripts (listed below) describing the interaction of DENV-2 NS5 with STAT-2 have been published. Both articles provide evidence that like the NS5 of JEV, LGTV and TBEV, the DENV-2 NS5 can also perturb type I IFN signalling. (Ashour et al., 2009; Mazzon et al., 2009)
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Nitric oxide radicals released during inflammatory processes have been shown to inhibit DENV replication in a strain-specific fashion (Charnsilpa et al., 2005; Ubol et al., 2008). Using in vitro RdRp assays based on both crude extracts from DENV-2-infected cells and bacterially expressed DENV-2 NS5, it was shown that de novo synthesis of RNA was inhibited by nitric oxide radicals, suggesting that NS5 may play a role in nitric oxide sensitivity (Takhampunya et al., 2006). Based on this premise the NS5 genes from DENV strains showing differences in susceptibility or resistance to nitric oxide were sequenced. Although sequence changes were detected, a firm correlation with nitric oxide sensitivity was not established (Ubol et al., 2008). Collectively, these studies demonstrate that NS5 has a role outside of the replication complex in viral-infected cells. The effects of NS5 on the host cell described to date involve perturbation of the host immune response and therefore NS5 has the potential to play a role in viral pathogenesis. Future studies using relevant cell types and disease models will be required to establish the contribution of NS5 to viral pathogenesis.
VIII. CONCLUSIONS AND FUTURE PERSPECTIVES Flavivirus NS5 is essential for virus replication, possessing a number of viral-specific enzymatic properties. NS5 is therefore a very interesting target against which antiviral drugs can be developed and research in this field has accelerated our understanding of NS5 structure and function in recent years. The production and purification of recombinant versions of either the full-length NS5 or the two individual NS5 domains has led to detailed enzymatic studies on NS5 and the determination of the structures of the two individual NS5 domains. The original prediction, over 20 years ago, that NS5 was the viral RdRp has been well substantiated. In addition, NS5 is now known to play a major role in RNA cap formation as it can bind the cap structure and perform two sequential methylation reactions required for the formation of a type I cap structure. The structural characterization of the MTase and POL domains of NS5 has had a major impact on our understanding of NS5. The structures provide a basis for functional studies and understanding the enzymatic properties of NS5 at the atomic level. Enzymatic assays have now been developed using recombinant MTase and POLs that can be adapted for high throughput screening of antiviral compounds. Coupled with in silico structure-based compound screening and structure-guided drug design, compounds have already been identified which hold promise as antiflaviviral agents. It is anticipated that in the future, our increased understanding of NS5 will translate into antiviral therapies against flaviviruses that are urgently needed.
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As for other viral proteins involved in RNA capping, the study of NS5 has illustrated the many variations on a theme that exist for formation of the RNA cap structure. Typically, different proteins are required for N7 and 20 -O-methylation of the cap structure. N7 and 20 -O-MTases are structurally distinct and methylate RNA using different reaction mechanisms. However, for flaviviruses, both activities reside in NS5 although there is only one binding site for AdoMet, the methyl donor. Structure–function studies have been instrumental in identifying regions of NS5 involved in each of the MTase activities and suggesting mechanisms to explain how NS5 may perform both methylation reactions. It has recently been proposed that separate molecules of NS5 may perform the individual methylation reactions. In addition, cap formation also requires RNA triphosphatase, and guanyltransferase activities. NS3 has been shown to possess RNA triphosphatase activity. The guanyltransferase activity required for flavivirus cap formation has not yet been identified, although it has been suggested that NS5 may also carry out this function. By analogy with other capping systems, it may be that the full complement of enzyme activities required for flavivirus cap formation will only be identified through examination of a multiprotein complex rather than the individual protein constituents. The next challenge in understanding the role of NS5 in capping will therefore be to determine how NS3 and NS5 interact to facilitate cap formation and determine the stoichiometry of NS5 in the capping complex. Intriguingly, studies have shown that N7 but not 20 -O-MTase activity is strictly required for virus viability. Little is known concerning the role of 20 -O-methylation in the RNA cap structure either for flaviviruses or in general. Animal studies suggest that the 20 -O-MTase activity of NS5 may play a role in viral pathogenesis which is an interesting future area of research. Despite intensive research, it has not yet been possible to produce a structure for the entire NS5. The determination of the structures for the two individual NS5 domains has had a major impact on our understanding of NS5 as the enzymatic functions of the two domains are independently active. However, evidence suggests that NS5 undergoes a number of conformational changes when carrying out its enzymatic activities and that the two domains operate in concert during flaviviral replication. In the absence of a full-length NS5 structure, a speculative structural model has been produced using an in silico docking approach. The model suggests that once the newly synthesized RNA exits the POL domain, it can engage with the MTase domain. Viral sequences and structures in the 50 -TR are required for N7 and to a lesser extent 20 -O-MTase activities, providing a mechanism to discriminate between (þ) strand RNA viral transcripts and () strand viral RNA transcripts that
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are not believed to be capped. However, the relationship between RNA capping and RNA synthesis is currently unknown and it is not clear how many viral nucleotides are synthesized before the nascent transcript is capped. Current and future research will be directed at understanding how the full-length NS5 functions to coordinately synthesize and cap the RNA genome in combination with other viral and presumably host proteins. Many studies have demonstrated that flaviviral RNA synthesis occurs in association with host perinuclear ER membranes. However, it appears that only catalytic amounts of NS5 are required for genome replication. Viral RNA sequences and regions of NS3 that are believed to interact with NS5 during viral replication have been defined using in vitro studies, but the exact composition of the replication complex and the interactions that take place within it, in virally infected cells remains unknown, as does the contribution of host proteins. Much of the NS5 in flavivirus-infected cells is found free in the cytoplasm or for a number of flaviviruses in the nucleus. It is not known whether only a specific subset of NS5 molecules are recruited to the replication complex or alternatively if modification of NS5 in the replication complex leads to its redistribution in the host cell. Recent studies have shown that aside from its role in replication, NS5 can interact with host macromolecules involved in the host immune response, raising the possibility that NS5 may be involved in viral pathogenesis. Little is still known about either the trafficking of NS5 in infected cells and its interaction with host proteins or the how these processes are regulated. Phosphorylation is one means by which NS5 function could be regulated and a potential NS5 phosphorylation site has recently been identified that has the potential to regulate MTase function. The role of NS5 in the nucleus is presently unclear, although recent studies on DENV-2 suggest NS5 localization is important for virus replication. Nuclear-localized NS5 has the potential to alter host processes or play a role in viral RNA synthesis. The trafficking of NS5 to the nucleus has been studied in detail for DENV-2, but it remains to be determined whether the trafficking mechanisms established for DENV-2 are common to other flaviviruses. Compared to the enzymatic and structural properties of NS5, there are likely to be many more flaviviral-specific differences in the way in which NS5 interacts with the host cell. The elucidation of the role NS5 in pathogenesis will ultimately require studies in relevant systems in the context of virus infection. Due to the pleiotropic functions of NS5 in viral replication this will not be trivial, as disrupting one function of NS5 may have unforeseen consequences on other functions. Nevertheless, the investigation of NS5–host cell interactions is an exciting area of research which promises to expand our understanding of the role played by NS5 in the flavivirus lifecycle.
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ACKNOWLEDGMENTS The author wishes to acknowledge research support from the Medical Research Council, UK; the Novartis Institute for Tropical Diseases, Singapore; and the National Health and Medical Research Council of Australia. The author is grateful to Stuart Siddell for critical reading of the manuscript; former laboratory members Rebecca Butcher and Helga Kroschewski for their input into flavivirus NS5 research; and Siew Pheng Lim, Subhash Vasudevan, Peter Wright, David Jans, Melinda Pryor, Marie-Pierre Egloff, Barbara Selisko, Bruno Canard and Mike Jacobs for engaging in stimulating collaborative investigations.
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Westaway, E. G., Mackenzie, J. M., and Khromykh, A. A. (2002). Replication and gene function in Kunjin virus. Curr. Top. Microbiol. Immunol. 267:323–351. Westaway, E. G., Mackenzie, J. M., and Khromykh, A. A. (2003). Kunjin RNA replication and applications of Kunjin replicons. Adv. Virus Res. 59:99–140. Yap, T. L., Chen, Y. L., Xu, T., Wen, D., Vasudevan, S. G., and Lescar, J. (2007). A multi-step strategy to obtain crystals of the dengue virus RNA-dependent RNA polymerase that diffract to high resolution. Acta Crystallograph. Sect F Struct. Biol. Cryst. Commun. 63:78–83. Yap, T. L., Xu, T., Chen, Y. L., Malet, H., Egloff, M. P., Canard, B., Vasudevan, S. G., and Lescar, J. (2007). Crystal structure of the dengue virus RNA-dependent RNA polymerase catalytic domain at 1.85-angstrom resolution. J. Virol. 81:4753–4765. Yocupicio-Monroy, R. M., Medina, F., Reyes-del Valle, J., and del Angel, R. M. (2003). Cellular proteins from human monocytes bind to dengue 4 virus minusstrand 30 untranslated region RNA. J. Virol. 77(5):3067–3076. Yocupicio-Monroy, M., Padmanabhan, R., Medina, F., and del Angel, R. M. (2007). Mosquito La protein binds to the 30 untranslated region of the positive and negative polarity dengue virus RNAs and relocates to the cytoplasm of infected cells. Virology 357:29–40. Yon, C., Teramoto, T., Mueller, N., Phelan, J., Ganesh, V. K., Murthy, K. H., and Padmanabhan, R. (2005). Modulation of the nucleoside triphosphatase/RNA helicase and 50 -RNA triphosphatase activities of Dengue virus type 2 nonstructural protein 3 (NS3) by interaction with NS5, the RNA-dependent RNA polymerase. J. Biol. Chem. 280:27412–27419. You, S., and Padmanabhan, R. (1999). A novel in vitro replication system for Dengue virus. Initiation of RNA synthesis at the 30 -end of exogenous viral RNA templates requires 50 - and 30 -terminal complementary sequence motifs of the viral RNA. J. Biol. Chem. 274:33714–33722. You, S., Falgout, B., Markoff, L., and Padmanabhan, R. (2001). In vitro RNA synthesis from exogenous dengue viral RNA templates requires long range interactions between 50 - and 30 -terminal regions that influence RNA structure. J. Biol. Chem. 276:15581–15591. Yu, F., Hasebe, F., Inoue, S., Mathenge, E. G., and Morita, K. (2007). Identification and characterization of RNA-dependent RNA polymerase activity in recombinant Japanese encephalitis virus NS5 protein. Arch. Virol. 152:1859–1869. Zhang, B., Dong, H., Zhou, Y., and Shi, P. Y. (2008). Genetic interactions among the West Nile virus methyltransferase, the RNA-dependent RNA polymerase, and the 50 stem-loop of genomic RNA. J. Virol. 82:7047–7058. Zheng, S., Hausmann, S., Liu, Q., Ghosh, A., Schwer, B., Lima, C. D., and Shuman, S. (2006). Mutational analysis of Encephalitozoon cuniculi mRNA cap (guanine-N7) methyltransferase, structure of the enzyme bound to sinefungin, and evidence that cap methyltransferase is the target of sinefungin’s antifungal activity. J. Biol. Chem. 281:35904–35913. Zhou, Y., Ray, D., Zhao, Y., Dong, H., Ren, S., Li, Z., Guo, Y., Bernard, K. A., Shi, P. Y., and Li, H. (2007). Structure and function of flavivirus NS5 methyltransferase. J. Virol. 81:3891–3903.
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3 Replication of the Hepatitis Delta Virus RNA Genome John M. Taylor
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Contents
I. Background II. Polymerase(s) III. Promoters and Priming IV. Pausing and Switching V. Replication in the Nucleus VI. Role(s) of the Delta Antigen VII. Host Factors VIII. Viroid Analogy IX. Conclusions and Outlook Acknowledgments References
Abstract
Hepatitis delta virus (HDV) is a subviral agent dependent upon hepatitis B virus (HBV). HDV uses the envelope proteins of HBV to achieve assembly and infection of target cells. Otherwise, the replication of the RNA genome of HDV is totally different from that of its helper virus, and involves redirection of host polymerase activity. This chapter is concerned with recent developments in our understanding of the genome replication process.
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I. BACKGROUND Hepatitis delta virus (HDV) was discovered in 1977 in studies of patients with a more severe form of hepatitis B virus (HBV) infection (Rizzetto et al., 1977). Much has since been learnt about its molecular biology and for detailed information the reader is directed to recent reviews (Lai, 2005; Taylor et al., 2007) and monographs (Casey, 2006a; Handa and Yamaguchi, 2006). The focus of this chapter is on recent developments in our understanding of the genome replication process. But before considering the recent studies, a brief introduction is needed. HDV is a subviral agent that depends upon HBV as a helper virus. In cells infected with both HDV and HBV, HBV shares its envelope proteins so as to allow the assembly and release of particles containing the HDV genome. These HDV particles can then infect new susceptible hepatocytes, presumably in very much the same manner as HBV infects such cells (Sureau, 2006). Subsequent rounds of HDV replication continue to require HBV to provide the necessary envelope proteins. The HDV genome is a small 1700-nucleotide (nt) long single-stranded RNA. As represented in Fig. 1, this RNA is circular in conformation and is predicted to make about 74% intramolecular base pairing so as to form an unbranched rod-like structure. During replication a second circular RNA, exactly complementary to the genome is produced. This antigenome contains the coding region for the one protein of HDV, the small delta Genome
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FIGURE 1 Representation of three processed RNAs detected during HDV genome replication. The model shown is reproduced with permission (Taylor, 2006). The antigenome is an exact complement of the genome. The circular genomic and antigenomic RNAs have significant intramolecular pairing and form unbranched rod-like folding. They each contain a sequence that acts in vitro, as a ribozyme that produces a site-specific cleavage. The upper and lower ends of these RNA foldings are referred to here, as the top and bottom, respectively. The mRNA, which is of antigenomic polarity, contains the open reading frame (ORF) for the delta antigen (dAg). Processing of this RNA depends upon a poly(A) signal.
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antigen, dAg-S. This antigen is translated from a third and less abundant RNA, a mRNA of about 900 nt in length and like cellular mRNAs, in that it possesses a 50 -cap structure and a 30 -poly(A) tail (Gudima et al., 2000). The only cells that are susceptible to HDV infection are hepatocytes. However, many different cell types can support HDV genome replication; initiation in other cell types requires either delivery into the cells of HDV RNA or expression from DNA of the HDV RNA sequences. As discussed further in Section VI, HDV genome replication is somehow dependent upon a source of dAg-S (Chao et al., 1990). The genome, antigenome, and mRNA are all processed RNA transcripts. The processing to make the genomic and antigenomic RNA circles is mediated by two site-specific ribozymes (Kuo et al., 1988b; Sharmeen et al., 1988) that are thought to release unit-length linear RNAs from multimeric transcripts produced in what is referred to as rolling-circle replication, as has been previously used to describe the replication of plant viroid RNAs (Branch and Robertson, 1984). Such an adapted double rolling-circle model is presented in Fig. 2. In this particular model, all the transcription is considered to take place in the nucleus and all transcription is mediated by the redirection of host RNA polymerase II, pol II (Taylor, 2006). Genomic RNA in the nucleus is transcribed to make RNAs that are either processed to become mRNA, steps 1–2, or undergo cleavage and ligation to become new antigenomic RNAs, steps 3–6. The new antigenomic RNA is transcribed to make RNAs that are processed to become new genomic RNAs, steps 6–8. Sections II–VII consider in more detail this and alternative replication schemes. dAg-S binds to both the genomic and antigenomic rod-like RNAs, but only the genome is assembled into new virus particles (Sureau, 2006). As represented in Fig. 3, dAg-S is 195 amino acids in length. During HDV genome replication some of the nascent antigenomic RNA transcripts undergo a site-specific editing by an adenosine deaminase (Casey, 2006a). This leads to a change in the amber termination codon of the mRNA, allowing the translation of a somewhat longer protein, the large delta antigen (dA-L). dAg-L does not support genome replication, and can be a dominant negative inhibitor (Chao et al., 1990). However, its unique C-terminus undergoes modification by farnesylation (Glenn et al., 1992), producing a protein that has an essential role in the assembly of new particles as mediated by the HBV envelope proteins (Chang et al., 1991). The two forms of the dAg have shared sequences and not surprisingly shared features, as represented in Fig. 3 and discussed further in Section VI. Questions still arise as to in what ways, if any, does the dAg-S contribute to the HDV RNA-directed RNA transcription process. Unlike for HBV, HDV has no DNA intermediates. All HDV replication is via RNA-directed RNA transcription. This chapter will focus on progress relating to how we consider the HDV RNA genome is replicated,
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FIGURE 2 A double rolling-circle model of HDV genome transcription and processing. This is an extension of models proposed earlier for plant viroids RNAs (Branch et al., 1990). The model shown is reproduced with permission (Taylor, 2006). Implied is that all transcription takes place using one enzyme, pol II, and that there are alternative processing pathways based on the two ribozymes and the polyadenylation signal. A more complicated model has been proposed by Li et al. that incorporates the concept that transcription of genomic RNA to make new antigenomes can occur with pol I and in the nucleolus (Li et al., 2006).
along with some discussion of associated questions not yet resolved. The reader is directed elsewhere for reviews of other important topics, such as RNA editing (Casey, 2006b; Linnstaedt et al., 2009), the two ribozymes (Doudna and Lorsch, 2005; Perrotta and Been, 2007), the process of infection of susceptible cells (Urban, 2008), the assembly of new virus particles using HBV envelope proteins (Sureau, 2006), and the multiple genotypes of HDV (Deny, 2006; Radjef et al., 2004).
II. POLYMERASE(S) For some time, it was asked which host polymerase is used for the RNA-directed transcription of HDV RNAs. Several different approaches were used that indicated host RNA polymerase II (pol II) was necessary. (i) The mRNA possessed a 50 -cap structure consistent with pol II
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FIGURE 3 Features on the two forms of the delta antigen. CCD at positions 12–60 is the coiled-coil dimerization domain based upon the structural study of Zuccola et al. (1998). However, more recent studies using phylogenetics and structural predictions suggest a shorter region (Deny, 2006; Enomoto et al., 2006). See also Fig. 5. NLS at positions 66–75 is the nuclear localization signal based upon Alves et al. (2008) whereas earlier studies advocate an additional facilitating domain (Lai, 2006). RBD at positions 97–146 is drawn as a single RNA-binding domain, although studies have shown that some of the central sequences are not needed (Lai, 2006); that is, the RNA-binding domain can be considered as bipartite. Near the C-terminus of the large delta, antigen is a cysteine that becomes farnesylated.
transcription (Gudima et al., 1999; Nie et al., 2004). (ii) The mRNA also contained a 30 -poly(A) as directed by a AAUAAA poly(A)-signal, features typical of processed host mRNAs transcribed by pol II. (iii) In cells undergoing HDV replication inhibition could be achieved with concentrations of amanatin consistent with inhibition of host pol II (Chang et al., 2006; Moraleda and Taylor, 2001). (iv) In nuclear run-on reactions, the synthesis of genomic RNA was again sensitive to pol II inhibition (Chang et al., 2006; Moraleda and Taylor, 2001). (v) Studies with nuclear extracts under conditions optimized for DNA-directed transcription by pol II indicated that exogenous HDV RNAs could be transcribed into full-length transcripts (Fu and Taylor, 1993); however, it has subsequently not been possible to repeat such results (unpublished). Nevertheless, other studies have detected short transcripts (less than 100 nt) and as discussed in Section III, such were primer-dependent transcripts. (vi) Studies with purified pol II or with nuclear extracts have indicated that pol II will bind HDV RNA sequences (Greco-Stewart et al., 2007). However, a recent extension of such in vitro studies has shown that pol I and pol III will also bind such RNAs (Greco-Stewart et al., 2009).
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Along the way, the story has become more complicated with reports from the laboratory of Michael Lai (Li et al., 2006; Macnaughton et al., 2002; Modahl et al., 2000; Tseng et al., 2008). It was reported that the transcription of new antigenomic RNA in nuclear run-on was resistant to amanitin. This suggested a role for an enzyme like pol I, the enzyme which acts in the nucleolus in the transcription of ribosomal RNA precursors. Actually, there is no direct evidence that pol I will transcribe HDV RNAs whereas there is evidence to the contrary, using nuclear runon with an endogenous template, that the synthesis of new antigenomic RNAs, like that of genomic RNAs, is sensitive to low doses of amanitin, consistent with pol II transcription (Chang et al., 2008). Nevertheless, as discussed further in Section III, such contrary findings provoke more objectivity, in that the observed mechanism of HDV RNA-directed transcription might actually vary according to the experimental situation that is used to study such transcription.
III. PROMOTERS AND PRIMING In terms of DNA-directed RNA transcription, a promoter is typically defined as the specific sequences on the DNA template that are recognized by transcription factors which recruit RNA polymerase at the site. Thus, it seems reasonable to ask whether some form of promoters can be defined for HDV RNA-directed transcription? In an early study, cells were transfected with a reporter construct containing as promoter, double-stranded cDNA of sequences corresponding to the top of the HDV genomic RNA (Macnaughton et al., 1993). In this way, bidirectional initiation of transcription of genomic and antigenomic RNAs was detected from a region corresponding to the top of the rod-like fold. This transcription was from DNA; thus, this study did not directly address the question of whether some sequence or structure on the RNAs functions as a promoter. Already there was the suspicion of at least one such promoter because the HDV mRNA species has a unique initiation site (Chen et al., 1986; Gudima et al., 1999, 2000). As indicated in Fig. 1, this site corresponds to initiation at sequences near to the top of the rod-like folding of the genomic RNA. Furthermore, mutagenesis that alters the sequence and/or structure of the stem-loop region at and around this site was found to interfere with HDV replication ability (Gudima et al., 1999; Wu et al., 1997). Greco-Stewart et al. (2007) have since shown that both the top and bottom stem-loop structures of the genomic and antigenomic RNAs are capable of pol II binding. Only in one case, at the top of the genomic RNA, did they report initiation in vitro (Abrahem and Pelchat, 2008), and this
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was at and around the observed site for the 50 -end of in vivo mRNA (Gudima et al., 2000). Maybe the other three sites are capable of polymerase binding but do not lead to initiation. And, as mentioned earlier, Greco-Stewart et al. (2009) have since reported that pol I and pol III will also bind HDV stem-loop structures, but no data were presented for initiation. Implicit in the above discussion of promoters for HDV RNA-directed transcription is the assumption that some or all of the sites of initiation will be at specific locations and transcription will be initiated de novo, that is, without a primer. In this respect, the assumption is that RNA-directed transcription will resemble host DNA-directed RNA transcription, which is always unprimed. Contrary to this assumption, there is already evidence that some HDV RNA-directed transcription, as studied in vitro or even in vivo, can involve priming. Already two forms of RNA priming have been observed for in vitro transcription of HDV RNA sequences. The first is the process of addition to the 30 -end of the RNA templates. The additions have been achieved with purified pol II and/or extracts containing pol II (Beard et al., 1996; Filipovska and Konarska, 2000; Gudima et al., 2000). A second form of priming is one that is preceded by an endonucleolytic cleavage to produce a novel 30 -end. Filipovska et al. reported that for some in vitro transcription of HDV RNA there was initiation at a site somehow created by a prior cleavage. The RNA transcript was characterized and found to be quite short. In a subsequent study, it was shown that the length increased if dAg-S was present, but still the transcript was <100 nt (Yamaguchi et al., 2001). More recently, Lehmann et al. (2007) reported a similar cleavage followed by initiation. However, in this case it was shown that the transcription factor TFIIS was somehow able to enhance the prior cleavage reaction. In all these RNA-primed transcriptions the templates were not full length and the length of the transcripts was <100 nt. In a test of whether such priming can occur in vivo as well as in vitro, Haussecker et al. applied an RT-PCR assay to RNAs from transfected cells. They detected a band interpreted as evidence for a RNA-primed transcription as described in the earlier studies (Haussecker et al., 2008). Further characterization and confirmation of such a primed species is needed. In both of the above methods of RNA-primed HDV RNA transcription, even if the problem of limited strand elongation were solved, there remains the need for a model that would resolve the covalent linkage between template and transcript; that is, between genomic and antigenomic sequences. There are two more forms of RNA priming that one could speculate might occur for HDV. The first would be to use some preexisting RNA of the host as a primer. Such a primer might be as small as a microRNA, which are known to be 20–22 nt in length. However, other host RNA
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species might provide 30 -ends to function as primers. The second form of priming would be via a nascent host RNA; that is, one that is still being transcribed. Specifically, a host RNA being transcribed by a host polymerase from a DNA template might undergo template switching and continue transcription of an HDV RNA. Such a possibility is consistent with the observed ability for template switching during HDV RNA-directed transcription, as discussed in the following section.
IV. PAUSING AND SWITCHING During DNA-directed RNA transcription host pol II is also known to initiate and then come to discrete pause sites. Such pausing can be caused by waiting for specific positive transcription factors such as pTEFb or for relief from negative regulation such as by NELF (Yamaguchi et al., 2002). Yamaguchi et al. have asserted that dAg-S binds to one of the four subunits of NELF, and prevents what might otherwise be negative regulation of HDV RNA-directed transcription (Yamaguchi and Handa, 2006; Yamaguchi et al., 2002). With such pausing in mind, it should be noted that short RNAs corresponding to the 50 -end of the HDV mRNA have been reported by Haussecker et al. (2008). Furthermore, these short 50 -capped RNAs were in 10-fold greater abundance than the polyadenylated mRNA (Chen et al., 1986). Thus, one interpretation is that more RNA-directed HDV RNA transcripts are paused relative to those that proceed further and get processed to accumulate as mRNA. If this is correct, it will be important to determine the cause of the pausing, and the mechanism by which it is relieved. Certainly, this is consistent with the reports of Yamaguchi et al. (2001) that dAg-S acts a mediator of such relief. In addition, Haussecker et al. also reported that the small capped antigenomic RNAs are also present in HDV particles assembled from transfected cultured cells. They speculate that such RNAs, although presumably generated without a primer, might facilitate initiation of new rounds of infection by acting as primers for transcription on genomic RNA templates (Haussecker et al., 2008). In the absence of a direct test of this hypothesis, a more likely possibility is that they are short aborted transcripts, and that they do not function as primers for further transcription. They might in fact be analogous to the 18-nt tiny RNAs (tiRNA) recently shown to be associated with highly expressed host RNA transcripts and sites of pol II binding (Taft et al., 2009). One can ask whether the predicted rod-like folding or other structural features on HDV RNA templates can cause transcriptional pausing. As an approach for this, we attempted transcription of HDV RNA in vitro, using the RNA-directed RNA polymerase of phage phi6 (Butcher et al., 2001;
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Laurila et al., 2005; Salgado et al., 2004). Unlabeled transcripts were detected by strand-specific northern analysis and for a separate reaction, products were detected following incorporation of 32P-UTP. We were readily able to produce new unit-length HDV RNAs (Fig. 4 and unpublished observations) that we interpret as arising via primer-independent initiation adjacent to the 30 -end of the unit-length template. And, we were able to detect molecules of twice unit length, consistent with primerdependent elongation from the 30 -end of the template; a process sometimes called back-priming (Laurila et al., 2005). Thus, we interpret that under these experimental conditions, the HDV RNA template allowed both primer-independent and primer-dependent transcription. At the same time, there was no evidence of significant pausing that might be mediated by intramolecular structures in the RNA template. Consider now the special situation of template switching on HDV RNAs. The first evidence that template switching could occur during HDV replication was a report that a linear HDV RNA transcribed in vitro, when transfected into cells containing dAg-S, could initiate genome replication (Glenn et al., 1990). Subsequent studies with other linear RNAs, even ones slightly shorter than unit length, were also found to be capable of initiating replication. Sequence comparisons of the input linear RNAs relative to the rescued replicating RNAs revealed specific deletions and/or insertions that could be interpreted as errors during template switching (Chang and Taylor, 2002). Further studies showed Northern G rNTPs: − +
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FIGURE 4 HDV genomic RNA can be readily copied in vitro using the RNA polymerase of phage phi6. This was readily performed in vitro in the absence () or presence (þ) of rNTPs, and in the absence of dAg. For the lanes indicated as northern, the rNTPs were unlabelled and detection was via use of strand-specific 32P-labeled probes (G and aG). For the lane labeled 32P, the reaction mixture also contained 32P-UTP. The transcripts that doubled the length of the template (2) are considered to be primed on the 30 -end of the template. This is supported by the observation that the species hybridizes equally to G and aG probes. The transcripts that are of same length as the template (1) could have arisen in a primer-independent manner and/or as a consequence of cleavage at or near the middle of a double-length transcript.
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that even pairs of less-than unit-length HDV RNAs when cotransfected into cells could lead to the reconstitution and accumulation of full-length HDV RNA circles (Gudima et al., 2005). Consistent with such template switching between separate RNA molecules, Chao and coworkers have reported that recombination between HDV genomes can occur in vivo, in patients, and also in cells replicating HDV RNA genomes (Chao, 2007; Chao et al., 2006; Wang and Chao, 2005).
V. REPLICATION IN THE NUCLEUS It is important to understand the intracellular localizations of HDV RNAs and dAg during replication. It is possible to localize dAg and HDV RNA during the process of replication. To avoid artifact associations or rearrangements, the cells can be fixed prior to examination, whether by in situ hybridization, immunostaining, or immunoprecipitation. However, interpretation of such data in terms of the HDV replication process has numerous caveats. For example, the localizations might reflect sites of accumulation of already processed RNAs or sites where dAg can accumulate independent of the replication process. During replication most of the genomic and antigenomic RNAs are detected in the nucleus (Tavanez et al., 2002; Taylor et al., 1987). Even after cell fractionation of unfixed cells, most of the antigenomic RNA is nuclear although genomic RNA can be found in the cytoplasm (Macnaughton and Lai, 2002). Observation and interpretation of the intracellular localization of dAg can be complicated not only by the two forms, small and large, but also because during replication altered forms of antigen arise that can have altered patterns of localization within the cell (Bichko and Taylor, 1996). To simplify the situation one can express just the forms of dAg in the absence of replication. And, to avoid mutations during replication, one can express the essential dAg-S from a separate cDNA and use an HDV genome that has been mutated so that it cannot express any dAg. As now considered, even after such simplifications, the story is still complex. Expression of small dAg-S along with replication of an HDV RNA genome that cannot make this protein leads to a stable nucleoplasmic distribution. This distribution is very similar to that of pol II (Bichko and Taylor, 1996; Chang et al., 2008; Han et al., 2009). Expression of dAg-S, in the absence of HDV RNAs, typically leads to accumulation predominantly in the nucleolus, the site of transcription by pol I of rRNA precursors and of their maturation. However, for cultured cells this pattern can change to become predominantly nucleoplasmic. This happens slowly if the cells progress to stationary phase or promptly, and reversibly, if the cells are treated with inhibitors of ribosomal
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RNA processing (Chang et al., 2008; Han et al., 2009). It can also happen if nonreplicating forms of HDV RNA are expressed in the cell (Han et al., 2009). The significance of dAg-S in the nucleolus remains unclear. At one extreme, it might be considered as an irrelevant association of an RNAbinding protein with a large amount of newly synthesized rRNA. Maybe transit to the nucleolus does not occur when replication is possible. The other extreme is that dAg-S in the nucleolus is actually needed for transcription of genomic RNA templates by pol I (Li et al., 2006). As a part of this nucleolar story, a recent study has suggested that dAg-S in the nucleolus lacks posttranslational modifications, including phosphorylation, acetylation, and methylation (Tseng et al., 2008).
VI. ROLE(S) OF THE DELTA ANTIGEN The small form is essential for RNA accumulation. The large form acts as a potent dominant negative inhibitor of the small, supporting the interpretation that the small form needs to function as a multimer (Chao et al., 1990). In proceeding further with the question of the role(s), if any, of small dAg in the HDV RNA-directed RNA transcription process, some additional introduction is required. As summarized in Fig. 3, the small and large forms of dAg share some common features. The isolated region from positions 12–60 was studied as a synthetic peptide. It was demonstrated to have significant a-helical structure. It readily formed crystals and the structure determined was that of dimers in an antiparallel coiled-coil arrangement (Zuccola et al., 1998). This region has thus become referred to as the coiledcoil domain, CCD. It has been shown to act in vivo as a facilitator of dimer and higher multimer formation. However, as now explained, the structure derived from crystals of the isolated peptide might be an overestimate relative to what is present in the context of the whole protein. Figure 5 is an analysis for all eight clades of HDV of the total protein intrinsic disorder using the program PONDR (Goh et al., 2008a,b). Note that most of the dAg is predicted to be disordered and only a small part of the CCD is actually predicted to be ordered. Adjacent to the CCD is a nuclear localization signal, NLS. This was originally considered to be larger and composed of two separated parts (Lai, 2006). However, the one part indicated in Fig. 3 is sufficient for nuclear localization (Alves et al., 2008). Adjacent to the NLS is an RNA-binding domain. This can be divided into two separated parts, each arginine-rich, with a central spacer region that is also essential (Chang et al., 1988). Overall, the delta antigens demonstrate affinity for structured RNAs relative to unstructured (Chao
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Location on delta antigen (amino acid)
FIGURE 5 The delta antigen is largely in a disordered structure. Currently, based on nucleotide sequence differences, there are considered to be eight clades of HDV (Deny, 2006). The predicted large delta protein sequences for these (in colors), along with the sequence used in this lab (in black) were analyzed by the program PONDR, which predicts regions of protein disorder (Goh et al., 2008b). Note that the proteins show significant similarity, are mainly disordered, and that there is only a relatively small patch within the CCD that in most cases is predicted to have less disorder.
et al., 1991). In vitro, they will bind to RNAs with HDV rod-like folding, but also to non-HDV forms of double-stranded RNA. A recent study has suggested that at least 311 nt of HDV RNA in rod-like folding, is needed for dAg binding to occur (Defenbaugh et al., 2009). Also, in vitro, dAg or even fragments thereof, can facilitate ribozyme cleavage (Huang and Wu, 1998). When multimers of HDV RNA are expressed in cells from transfected DNA constructs, the presence of dAg can facilitate the accumulation of processed HDV RNA species (Lazinski and Taylor, 1995). dAg can also mediate the transfer of HDV RNAs to the nucleus (Chou et al., 1998). Such positive effects can be observed independent of HDV RNA replication. In summary, the properties of the small dAg do support the observation that this protein is essential for the accumulation of processed HDV RNA species, but as now discussed, what remains controversial is whether dAg directly participates in and contributes to the RNA-directed transcription process, and if so, by what mechanism.
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Others and we have been unable to demonstrate in vivo, the role of small dAg in the transcription process (Chang et al., 2008; Haussecker et al., 2008). As mentioned in Sections II and III, some of the studies of in vitro transcription by pol II have been achieved in the absence of dAg. When dAg was added, the transcripts were increased in length, but only modestly (Yamaguchi et al., 1998, 2001, 2002). It may be relevant that the small dAg with a predicted charge of þ 12 might exert some nonspecific effects for reactions involving negative-charged nucleic acids (Kuo et al., 1988a). Also, it has been asserted that dAg has a role in transcriptional fidelity (Yamaguchi et al., 2007). And, Nedialkov et al. (2003) have reported that dAg can enhance pol II transcription that is DNA-directed. In summary, while the small dAg is essential in vivo for the accumulation of processed HDV RNAs, one interpretation is that it has not yet been proven to have a direct role in the transcription process itself.
VII. HOST FACTORS While HDV replication will depend upon dAg and one or more host polymerases, it is likely to need many other host proteins. As discussed here, several proteins have been suggested based either on observed associations or on the impact of depleting a particular protein, such as with siRNA. Maybe the first host protein partner described for dAg was DIP-A (Brazas and Ganem, 1996). Other partners include nucleolin (Lee et al., 1998) and B23 (Huang et al., 2001). Haussecker et al. (2008) refer to a large screen in which they identified many host proteins that bind to dAg. One of their candidates, MOV10, is a homolog of a plant helicase, and they proceeded to show that reduction of MOV10 also reduced HDV RNA accumulation threefold. They also show that suppression of AGO4, a protein that is both a translation factor and one involved in RNA silencing, reduces HDV RNA accumulation. The mechanism behind such reductions in HDV RNA accumulation is not yet clear. Yamaguchi et al. (2001) report in vitro studies which indicate not only a direct interaction of dAg with pol II but also the ability of delta antigen to bind to subunit A of NELF, thereby inactivating the ability of NELF to act as a negative regulator of pol II transcription. That is, dAg is interpreted as an elongation factor. In summary, until we are clear as to which host polymerases are involved in the HDV RNA transcription as it occurs in vivo, and until we can accurately reconstitute major aspects of this in vitro, we will have to be cautious about interpreting which host factors, either positively or negatively, regulate the RNA-directed transcription process.
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VIII. VIROID ANALOGY The viroids are subviral pathogens of plants. They have RNA genomes of 246–401 nt that are even smaller than HDV. They have no known encoded proteins and no helper viruses. However, like HDV, they depend upon redirection of host RNA polymerases to achieve RNA-directed RNA replication. Therefore, there is significant potential value to considering what is known about viroid RNA transcription and replication (Flores et al., 2008). The known viroids are divided into two families: Pospoviroidae and Avsunviroidae, with five genera and >24 species, or two genera with two species each, respectively (Tabler and Tsagris, 2004). The Pospoviroid RNAs are often circular, have no known ribozymes, and are reported to replicate in the nucleus by redirection of pol II. The Avsunviroid RNAs are typically circular, but contain two ribozymes referred to as hammerheads, replicate in the plant chloroplast, and redirect not pol II but another nucleus-encoded polymerase. Several studies have characterized the requirements for the initiation of viroid RNA transcription. Initiation sites have been mapped by different strategies (Daros et al., 2006; Flores et al., 2008). In some cases, a single initiation site has been mapped per template, while in other cases as many as three sites. No examples of RNA-primed initiation have been reported; it seems all transcription is without a primer. Some of the viroids have predicted intramolecular folding that is referred to as rod-like or pseudorod-like. In some cases, it is considered that terminal stem-loop structures are recognized by the host polymerase to achieve initiation of transcription. Such recognition can be achieved in vitro, and in one study even the RNA polymerase of Escherichia coli was shown to bind to the same stem-loop and initiate with the same specificity as observed in infected plants (Pelchat et al., 2002). Thus, sequence and structural features of the RNA templates are major players in determining the specificity of initiation. In summary, there are diverse answers regarding the transcription of the plant viroid RNAs and yet they do give us what may be valuable precedents for what to expect for RNA-directed transcription of HDV RNAs.
IX. CONCLUSIONS AND OUTLOOK This chapter has considered the mechanism by which host polymerase activity is redirected from DNA templates to the transcription of HDV RNAs. It has been pointed out that there are many associated questions and controversies yet to be resolved. Once the HDV-specific transcription
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answers are obtained we would hope to know to what extent they apply, if at all, to the host RNAs acting as templates for transcription. Also, we would like to see to what extent there is similarity with the host polymerase redirection that is achieved in plants for viroid RNA replication. In all such RNA-directed transcription, we have so far only been considering the survivor RNAs; that is, those RNAs that for reasons such as circular conformation, intramolecular base pairing, and protein protection, are able to resist degradation by host nucleases, such as the 30 –50 activity of the exosome. Recent studies showed that when the exosome is suppressed, unexpected, novel host RNA transcripts can be detected (Preker et al., 2008). We should therefore expect some surprises when such suppression is tested during replication of HDV and plant viroids.
ACKNOWLEDGMENTS The author has been supported by NIH grants AI-26522 and CA-06927 and by an appropriation from the Commonwealth of Pennsylvania. This chapter is based on a talk given in March 2009 for the ISVHLD Meeting held in Washington, DC. Certain unpublished studies cited were performed in collaboration with Severin Gudima, Roland Dunbrack, Ziying Han, and Carolina Alves. Severin Gudima and William Mason gave constructive comments on the chapter.
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4 Recent Epidemiology of Tick-Borne Encephalitis: An Effect of Climate Change? E. I. Korenberg
Contents
Abstract
I. Introduction II. Major Debatable Issues III. The Ranges of Main Tick Vectors: Are They Really Expanding? IV. Tick Abundance and TBE Virus Prevalence: Have They Changed? V. Tick Expansion to the Cities: Is It Related to Climate Change? VI. What Is Known About Newly Formed TBE foci? VII. Anthropurgic TBE foci: What Are the Principles of Their Formation? VIII. Since When Has TBE Morbidity Increased in the Cities? IX. What Are the Main Causes of Changes in Parameters of TBE Morbidity? X. Conclusions Acknowledgment References
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Consideration is given to the opinion of some specialists that the rise in tick-borne encephalitis (TBE) morbidity at the turn of the century has been accounted for by new features of TBE epidemiology as well as by global climate change. It is shown that neither the
Gamaleya Research Institute for Epidemiology and Microbiology, Gamaleya St. 18, Moscow 123098, Russia Advances in Virus Research, Volume 74 ISSN 0065-3527, DOI: 10.1016/S0065-3527(09)74004-7
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reputed current expansion of the ranges of main TBE vectors, the taiga (Ixodes persulcatus) and sheep (Ixodes ricinus) ticks, nor the significant rise of their abundance and TBE virus prevalence in them are confirmed by any objective data. The concept of recent tick expansion to large cities and human TBE infection in newly formed urban foci disagrees with the facts repeatedly described during the past four decades. There is no reliable information on the expansion of TBE nosological range. The influence of newly formed anthropurgic foci and of changes in the contribution of city dwellers to the general morbidity structure on the current epidemiological situation is estimated. As in the case of any other zoonosis with natural focality, the level of epidemiological manifestation of TBE foci is determined by two main parameters: the intensity of virus circulation in the foci (i.e., their loimopotential) and the frequency of human contact with them. Attention is paid to the character of interaction between these two factors, which accounted for a major outbreak of TBE morbidity at the end of the twentieth century, followed by a long-term decrease in its level.
I. INTRODUCTION The tick-borne encephalitis (TBE) virus was discovered in 1937 in the Russian Far East by a combined expedition headed by Professor L. A. Zil’ber. Within the spring–summer months of 1937, these specialists managed to determine the etiology of TBE disease and its vector, to analyze its clinical, pathoanatomical, and histopathological characteristics, and to isolate 25 strains of previously unknown virus (Zil’ber, 1939, 1945, 1957). Studies on this pathogen have more than a 70-year history, and, according to a rough estimation, their results have been described and discussed in no less than 6000 articles and about 50–60 monographs dealing with various aspects of TBE. However, since most of these publications are in Russian, they unfortunately remain poorly unknown to the international scientific community. As a result, it may sometimes appear that the structure of natural TBE foci has been studied mostly in a few countries of Central Europe (Kunz, 1992). It is now well known that natural TBE foci are distributed so that they form a continuous band along the southern forest zone of nontropical Eurasia, from the Mediterranean Sea to the Pacific Ocean. Ixodes persulcatus and Ixodes ricinus ticks are the only vectors of TBE virus, and the geographic range of the disease coincides with the distribution of these ticks. The tick I. persulcatus has a vast Eurasian range, which is mostly confined to the territory of Russia and only slightly extends west of its border. The range of I. ricinus, in contrast, covers all countries of western, central, and eastern Europe and, as a narrow zone, stretches out to northern Africa and the
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Near East. Its eastern part is in Russia, where it occupies a vast territory from the western state border approximately to the middle reaches of the Volga River. In a broad zone of Eastern Europe, the ranges of I. persulcatus and I. ricinus ticks overlap (Korenberg, 1979). According to the zoning of the TBE range, this vast area includes eight groups of focal regions: Central European–Mediterranean, Crimean–Caucasian, Eastern European, Western Siberian, Kazakh–Central Asian, Central Siberian–Transbaikalian, Khingan–Amur, and Pacific. Each focal region is characterized by an individual combination of abiotic and biotic factors, which determine the main epizootiological features of its natural foci, and by a specific spectrum of social conditions, which also have an effect on the parameters of epidemic manifestations in these foci (Korenberg and Kovalevskii, 1981, 1999). On the basis of differences in the structure of outer surface protein E, specialists have distinguished three genotypes (subtypes) of TBE virus, namely, the Far Eastern, Siberian (Ural–Siberian), and European subtypes (Ecker et al., 1999; Votyakov et al., 2002; Zlobin et al., 2001, 2007). Recent data (Demina et al., 2007; Pogodina et al., 2004a; Verkhozina et al., 2008; Zlobin et al., 2007; and others) confirm our concept that all these genotypes or at least two of them occur throughout the TBE virus range, but their frequencies differ depending on geographic region (Korenberg, 2003). In other words, different virus subtypes can circulate in the same natural focus, with the geographic name of a subtype reflecting no more than its prevalence in a given region. Although some data indicate that the surface protein E is a major factor of TBE virus virulence (Holzmann et al., 1997), this character is controlled by several genes and is generally multivariate (Heinz, 2003). Different virus subtypes distinguished by specific features of protein E structure are similar in antigenic properties, display cross-protection between each other (Holzmann et al., 1992), and do not differ in tropism and pathogenicity for humans (this is true at least for the Siberian and Far Eastern subtypes; Pogodina, 2008). Each subtype can cause the entire spectrum of clinical manifestations of TBE, from inapparent to focal forms with lethal outcome (Pogodina et al., 2004a,b, 2007). In our opinion, the TBE pathogen is a widespread polytypic species characterized by considerable geographic and intrapopulation variation in a series of genetic and phenotypic traits (Korenberg, 1976; Korenberg and Kovalevskii, 1981; Kucheruk et al., 1969). Some studies demonstrated the existence of a cline in TBE virus variation across its range (Gould et al., 1997; Zanotto et al., 1995), and this is a strong argument in favor of our concept. An approximately 20-year period of relatively low TBE morbidity was followed by its sharp increase in the early 1990s (Korenberg, 2003). The maximum values were recorded in Russia in 1996 and 1999: the totals of 10,298 and 9955 cases or 7.0 and 6.8 cases per 100,000 populations, respectively (Fig. 1). In about the same period, an increase in TBE
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8 7 6 5 4 3 2 1 0 1950
1955
1960
1965
1970
1975
1980
1985
1990
1995
2000
2005
FIGURE 1 Parameters of tick-borne encephalitis morbidity in Russia (number of cases per 100,000 population) between 1950 and 2008.
morbidity was also recorded in some Baltic and Central European countries (Randolph, 2004, 2008). The annual average values of morbidity in Russia between 1996 and 2002 proved to be 2.5–3 times higher than in all other European countries taken together (Gritsun et al., 2003). The long-term dynamics of TBE morbidity in Russia and the factors that have had the strongest effect on it in different periods have been considered in my previous study (Korenberg, 2003; Korenberg and Kovalevskii, 1999; Korenberg and Likhacheva, 2006). To explain the current epidemiological situation, specialists often revive old hypotheses that have already proved ineffectual, with long-known and relatively well-studied phenomena being regarded as emergent factors responsible for its complication. Such interpretations usually disregard the complexity of ecological processes underlying TBE epizootiology and epidemiology, which have received serious consideration in the context of the study and prevention of this disease. To foretell the possibility of such an ‘‘unexpected’’ and ‘‘unprecedented’’ peak of morbidity, it is important to reveal its actual causes and, in particular, to understand whether they include some new features of TBE natural focality that have emerged during the past 15 years. This chapter is an attempt at critical analysis of current opinions and of the validity of facts on which these opinions are based. Our concepts concerning specific features of TBE distribution, basic aspects of its epizootiology and epidemiology, and crucial stages in the study of this infection in Russia have been reviled in previous publications (Korenberg, 1976, 1979, 2003; Korenberg and Kovalevskii, 1981, 1994, 1999).
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II. MAJOR DEBATABLE ISSUES The most widespread explanations to the unusual rise of TBE morbidity in the late twentieth century are as follows (Alekseev, 2004, 2007; Gray et al., 2009; Korotkov, 2008; Pogodina et al., 2007; Zlobin, 2004, 2006; and others): (a) Global climate warming, due to which Geographic ranges of the main TBE vectors (I. persulcatus and I. ricinus ticks) have expanded. Their abundance and TBE virus prevalence in them have markedly increased. The ticks have entered big cities. Natural TBE foci dangerous for the population have appeared in areas where no such foci ever existed, including big cities. Properties of TBE virus itself have changed. (b) The formation of numerous anthropurgic foci (c) A drastic increase in the contribution of city dwellers to the general morbidity structure Most of these factors, especially those attributed to climate change, operate on a long time scale and by no means can provide for a sharp increase and then for a similarly sharp decrease in morbidity (Korenberg, 2004), but it is exactly what has taken place during the past 8 years both in individual regions and in Russia as a whole: by 2006, the TBE morbidity rate in the country decreased to 2.44 cases per 100,000 population (Onishchenko et al., 2007). This is 2.8–2.9 times lower than in 1996 and 1999 and approximately equal to the rate recorded in 1989, the year preceding a 10-year-long increase in TBE morbidity (Korenberg, 2003). Parameters of TBE morbidity recorded in 2008 proved to be still lower: 2817 cases, or 1.98 cases per 100,000 population (Fig. 1). No conclusive results have been obtained in attempts to reveal correlations between the TBE morbidity rate and climatic conditions (mainly temperature parameters) in certain regions over the period from 1970 to 2006 (Korotkov et al., 2007a). The opinion concerning the stimulating effect of global warming on TBE foci and their epidemic manifestation has become fairly popular. Some specialists predict that changes in summer climatic conditions will promote the spread of TBE to high latitudes and upper altitudinal belts of mountain systems, with its range expanding by the 2080s to southern Scandinavia, including southern Finland (Randolph, 2000; Randolph and Rogers, 2000), where, by the way, TBE has been recorded since the late 1950s (Korenberg and Kovalevskii, 1981; Kucheruk et al., 1969). This idea is by no means new. As early as 1965, after a 10-year period of increased TBE morbidity in the former Soviet Union, a hypothesis was proposed that
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the main factor providing for this increase was ‘‘global climate warming in the previous century and particularly in the previous decade’’ (Birulya and Zalutskaya, 1965). However, a long period of decrease in TBE morbidity began already next year, 1966, that is, even before the first critical comments on this hypothesis were published (Korenberg and Ivanova, 1967).
III. THE RANGES OF MAIN TICK VECTORS: ARE THEY REALLY EXPANDING? To confirm the increasing expansion of main TBE vectors, researchers often refer to single findings of these ticks far north and, for some reasons, even south of the ‘‘former’’ boundaries of their ranges. However, such data on the I. persulcatus tick has long been known. For example, this species was found almost at the 66th parallel, near Turukhansk, and even north of the 72nd parallel, on the Taimyr Peninsula (Korenberg et al., 1969). Findings of I. ricinus ticks beyond the accepted boundaries of the species range have also been repeatedly reported in the literature (Korenberg et al., 1971). It is apparent that similar findings will periodically take place in the future. However, such data themselves cannot be regarded as evidence for the shift of range boundaries under the effect of climate change; they only confirm the possibility of tick transfer to a new area. This transfer is characteristic of many arthropod species parasitizing highly mobile hosts (e.g., birds). Such ticks may even contain TBE virus or its DNA sequences, but this does not prove the possibility of virus circulation, the formation of a natural TBE focus in the corresponding area, and the expansion of its range. Climate warming is not necessarily favorable for the ticks and TBE virus (see below). For example, there are data that I. persulcatus ticks can hardly tolerate winter warmings, which are common in areas with average January temperatures above 5 C (Korotkov, 2008). The combination of temperature and humidity is known to be of great ecological significance for arthropods (Azzi, 1956; Korenberg and Kovalevskii, 1981; Yakhontov, 1964). The boundaries of TBE vector ranges are determined by the hydrothermal conditions necessary and sufficient for the development of ticks (Korenberg, 1979). However, the mechanism of synchronization of their complex life cycle is based on the response to changes in the daylight period. Therefore, the possibility of their existence under illumination conditions characteristic of certain latitude depends not only on the total sum of temperatures. Ticks must complete each stage of the cycle within a certain period of time, and this is possible only upon receiving a sufficient amount of heat during a specified season (Korenberg, 2000; Randolph et al., 2000). This is especially important for the I. persulcatus tick, since the summer season within its range is
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relatively short (Korenberg, 1985; Korotkov, 2005). Near the range boundary, the tick population has a specific type of spatial structure: it is such that tick abundance in the absolute majority of cases is estimated at zero (Korenberg and Kovalevskii, 1986), and only the results of a representative, randomized census can confirm that this population really exists in the study area. Except for the study by Livanova and Livanov (2006), such data are absent from the recent literature on the subject.
IV. TICK ABUNDANCE AND TBE VIRUS PREVALENCE: HAVE THEY CHANGED? Since the mid-twentieth century, many specialists working in different parts of the vast I. persulcatus range have conclusively shown that this vector is characterized by a cyclic pattern of population dynamics, with a peak usually occurring every third year (Korenberg, 1976). The recent increase in TBE morbidity is attributed to the general growth of tick abundance (Onishchenko et al., 2007), which, in turn, is explained by anthropogenic transformation of forests (which has indeed taken place; see below) and global warming (Zlobin, 2006). Such a conclusion should have been based on the results of long-term data on tick abundance obtained by standard methods at the same observation points. In most regions, however, the monitoring of TBE foci at permanent stations came to a standstill 10–15 years ago. The data obtained in areas where such observations are still being performed show that, in general, no significant increase in tick abundance has taken place in areas not exposed to a strong anthropogenic impact. For example, in mountain taiga forests of Perm Province (the Cisural region), the abundance of I. persulcatus ticks at the 3-year population peak of 2005 was even lower than at a similar peak in 1993: 445 versus 835 unfed adult ticks per hectare; in the next years after the peaks (2006 and 1994), these values were quite similar: 610 and 663 ind./ha, respectively (Yu. V. Kovalevskii et al., unpublished data). According to the results of 10-year monitoring in southern taiga forests of Krasnoyarsk Province (Eastern Siberia), the seasonal average abundance of ticks along a 1-km census route in 2006 was almost the same as in 1997: 14.4 and 14.8 ind., respectively (Khazova, 2007). There is information that the total period of seasonal activity of adult I. persulcatus ticks in some regions (e.g., in Eastern Siberia) has become 60–70 days longer in recent years, which is attributed to climate warming (Nikitin and Antonova, 2005). This conclusion is based on the records of the earliest and latest dates of visits to medical facilities for removing an attached tick, rather than on the results of direct censuses of questing ticks. As noted by these authors, the dates of the first attacks by ticks have changed insignificantly, usually remaining within the first 10-day period
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of April. On the other hand, the cessation of attacks has shifted to considerably later dates, but it should be noted that this shift coincides with the period of activity in tick larvae and nymphs of the genus Dermacentor and in adult Haemaphysalis concinna ticks, which consistently begins in August or slightly later. Meanwhile, the species of ticks removed from humans have not been indicated in medical records and, hence, there is no reason to claim that the activity season of I. persulcatus ticks has lengthened. It is known that fluctuations of TBE virus prevalence in ticks barely correlate with their population dynamics (Kovalevskii et al., 1988; and others). In previous years, opinions concerning this parameter in different regions were usually based on the results of virus isolation with the use of laboratory animals. In the recent decade, TBE virus in ticks has been identified by means of ELISA, which is far more sensitive. The results obtained by these two methods are absolutely incomparable, which should be taken into account while concluding (Bespyatova et al., 2008; Korotkov et al., 2007b) that TBE virus prevalence in I. persulcatus and I. ricinus ticks has increased due to climatic changes over the past 30 years. A high TBE virus prevalence observed in some regions of Russia, for example, in 2006 (Onishchenko et al., 2007) does not by itself confirm that such an increase has taken place. The results of 10-year observations at a permanent station in Eastern Siberia show that, against the background of usual annual fluctuations, parameters of TBE virus prevalence in ticks between 2003 and 2006 did not increase but, on the contrary, decreased considerably as compared to those between 1997 and 2002 (Khazova, 2007). Likewise, the data kindly supplied by Dr. V. Romanenko show that no increase in virus prevalence in taiga ticks between 1999 and 2007 took place in the Cisural region. A comparative data analysis by Danielova´ et al. (2002) shows that, taking into account differences in the sensitivity of methods used for isolating the TBE virus, it may be concluded that its prevalence in I. ricinus ticks in the foci of Central Europe in 2000 remained approximately the same as in the late 1970s. It is important to note in this context that the direct effect of temperature conditions (as well as of other abiotic factors) on the TBE virus population is practically unknown, and climate warming should not necessarily be favorable for it. For example, experiments showed that keeping I. ricinus nymphs at different temperatures (15 or 24 C) had no effect on the proportion of TBE-infected individuals among the emerging adult ticks, whereas changes in relative humidity at the same temperatures proved to produce such an effect (Danielova´, 1990). In another series of experiments, TBE virus titers in unfed adult I. ricinus ticks kept at 18–23 C proved to decrease gradually; long-term exposure at high temperature (37 C) resulted in an abrupt drop of virus titers, whereas at lower temperatures of 9–14 C the virus was preserved in ticks for up to
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1 year; at 22 C and gradually decreasing humidity, virus titers markedly dropped after no more than 2 months, decreasing to zero within 7 months (Mishaeva, 1975; Mishaeva and Erofeeva, 1979). Long-term keeping of I. ricinus ticks at 20 C reduced their ability to acquire the TBE virus upon feeding on infected hosts (Mishaeva, 1976). Some specialists consider that low temperatures in winter and a prolonged cold season contribute to the development of TBE virus pathogenicity (Bolotin and Gorkovenko, 1998; Korotkov, 2005). It is known that climate continentality in Eurasia decreases in an east–west direction. In particular, this is manifested in an increasing duration of the warm season and a considerable rise of winter monthly temperatures. In general, TBE virus virulence and the severity of human disease also decrease in the same direction (Bolotin, 1999; Korotkov, 2005). This fact provided a basis for our hypothesis (Litvin and Korenberg, 1999) that the temperature regime of the period when the main TBE vectors are inactive and the parameters of climate continentality are the main factors accounting for the observed clinal variation and evolution of viruses of the TBE group (Gould et al., 1997; Zanotto et al., 1995).
V. TICK EXPANSION TO THE CITIES: IS IT RELATED TO CLIMATE CHANGE? The existence of populations of the main TBE vectors and its foci in urban areas is not a new aspect of TBE epizootiology and epidemiology. Large park complexes within the city of Praha, formed in places of original phytocenoses in the 1840s to 1890s, have long been inhabited by all phases of I. ricinus ticks; therefore, the complete life cycle of these ticks takes place ˇ erny´, 1986). TBE foci have long been known to exist in there (Daniel and C park forests within many large Russian cities (Kucheruk, 1980; Sapegina et al., 1985). For example, I. persulcatus and I. ricinus ticks have been found in 58 sites of the St. Petersburg green belt. Many of these sites have long been isolated and are inhabited by insular tick populations with a high level of abundance (up to 108 ind./flag hour) and virus prevalence (up to 10%). Between 1971 and 1986, the number of citizens complaining of tick attachment after visits to these sites varied from 121 to 910 per year, with 33 persons falling ill with TBE (Vershinskii et al., 1988). A quarter century ago, we generalized data on the effect of urbanization on I. persulcatus and I. ricinus ticks, which had already been available by that time (Korenberg et al., 1984), and found that, under appropriate conditions, populations of these species can live for a long time not only in new districts but also in older, historically established parts of the cities (Table I). Therefore, global climate warming is by no means the factor determining the possibility for the populations of TBE vectors to exist in urbanized areas.
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Possible variants of the influence of urbanization on the populations of I. ricinus and I. persulcatus ticks (Korenberg et al., 1984) State of ixodid tick populations
Urbanized biocenoses
With initial presence of ticks With initial absence of ticks
Stages of urbanization
Under favorable conditions
Under unfavorable conditions
Initial period Final period (situation within a developed city) Initial period
Population persists Population persists
(a) Population vanishes (b) Ticks are absent or single broughtin specimens are encountered
(a) Brought-in ticks appear (b) Autonomous ticks population is formed Autonomous tick population present for a long time
(a) Ticks are absent (b) Single brought-in ticks may appear
Final period (situation within a developed city)
E. I. Korenberg
TABLE I
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VI. WHAT IS KNOWN ABOUT NEWLY FORMED TBE foci? There is also the opinion that the existence of reputedly emergent natural foci of TBE foci and the possibility of their epidemic manifestation in some regions of Germany, France, Greece, Albania, Denmark, Sweden, Finland, and Mongolia confirm the recent expansion of the TBE nosological range, but most of these facts have been known since the 1950s or 1960s (Korenberg and Kovalevskii, 1981; Kucheruk et al., 1969). This opinion is usually underlain by drawbacks in the system of keeping records of diseases in these countries (Bro¨ker and Gniel, 2003) and by poor knowledge of available literature, rather than by the actual expansion of TBE foci. This also concerns the recent fact of TBE virus isolation from I. persulcatus ticks collected in the Kokkola Archipelago (Finland) located only 300 km south of the Arctic Circle (Ja¨a¨skela¨inen et al., 2006): natural TBE foci in this area have been known since the 1960s, as justly noted by the authors themselves. Within the TBE nosological range, especially at its periphery (in approximately one-third of administrative regions of Russia), local TBE cases are usually recorded in only 1 out of 10 randomly chosen years (Korenberg et al., 1986). Taking into account the long-term complete developmental cycle of tick vectors (from 3 to 5 or 6 years in different regions; Shashina, 1985) and periodic fluctuations of their abundance, a retrospective comparative analysis of this range as a whole or of its parts should cover the period of no less than 10–12 years. Since published data of this kind are practically absent, the concepts concerning the expansion of TBE range in recent years (e.g., Zlobin, 2006) have no sound foundation. Some researchers conclude even that ‘‘evolution’’ of the properties of TBE virus populations has taken place over the past 50–60 years under the effect of climatic and other aforementioned factors (Pogodina, 2005, 2008). Moreover, several ‘‘types of evolutionary transformations’’ are distinguished. Thus, it is assumed that, in some areas of the Cisural region and Western Siberia, the Far Eastern subtype (genotype) of the TBE virus has been ‘‘displaced’’ by the Siberian subtype; in certain regions, only the Siberian subtype circulates consistently, with its mutant forms accumulating with time; in other regions, the Far Eastern and Siberian subtypes coexist (it remains unclear in this case, what is regarded as an alleged population transformation). Finally, one more type of ‘‘evolutionary transformation’’ is that specific variants of TBE virus appear in the foci where its different subtypes circulate together: in these variants, the genome contains gene fragments from both subtypes, which control the synthesis of E and MS1 proteins. This concept is based on the results of genotyping a relatively small number of randomly chosen virus isolates and RNA amplicons from ticks collected in different foci of a certain region in different years (Pogodina et al., 2007). However, the virus population of a certain TBE focus, as well as a natural population of any
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zoonosis agent (Korenberg, 1989; Korenberg et al., 2006; and many others), is markedly heterogeneous, with its genotype changing from year to year under the effect of numerous biotic and abiotic factors (Belikov et al., 2001; Casati et al., 2006; Karganova, 2008; Korenberg and Kovalevskii, 1994, 1999; Vereta and Vorobyova, 1990; Vereta et al., 1983; Verkhozina et al., 2008; Votyakov et al., 2002; and others). Therefore, any reliable conclusions about changes in the genotype structure of a TBE virus populations and especially about virus evolution (i.e., its vectorial and irreversible transformation) can be drawn only on the basis of studies on a large number of isolates obtained in a certain focus over many years, with samples for virus isolation being randomized. As noted by Heinz (2003), the greater the number of samples included in analysis, the clearer the resultant picture reflecting the possibility of circulation of different TBE virus subtypes in the same region. Today, any representative data on the long-term dynamics of the genotype structure of TBE virus population (by any marker) in natural foci are simply absent. Similarly, inconclusive are claims that, as compared with the 1940s– 1970s, the proportion of TBE cases with the febrile and meningeal forms of the disease has increased in recent years, with the consequent decrease in the proportion of focal forms and general TBE mortality. Indeed, a formal analysis of data from hospitals allows such a conclusion (Ierusalimskii, 2001; Volkova and Obraztsova, 2002; Zhukova et al., 2002). However, it is a long established fact (Karpov et al., 1960) that the ratio of different clinical forms of the disease among recorded TBE cases largely depends on the current level of laboratory diagnosis. Today, progress in this field allows timely diagnosis of mild TBE forms, which have been largely overlooked in previous years. Thus, as noted previously (Korenberg, 2004), there are no reliable facts confirming the effect of climatic changes on TBE distribution, morbidity, vectors, and basic components of parasitic systems. The authors of the revived hypothesis concerning this effect could not help to arrive at the same conclusion: an analysis of the situation in the northwestern part of TBE range showed that climate change could account neither for the spatiotemporal heterogeneity of TBE epidemiology nor for a peak of TBE morbidity in the Baltic region between 1994 and 2004, which was apparently a consequence of socioeconomic changes (Randolph, 2004, 2008a,b; Sˇumilo et al., 2007, 2008; Vasilenko et al., 2008).
VII. ANTHROPURGIC TBE foci: WHAT ARE THE PRINCIPLES OF THEIR FORMATION? The emergence of numerous anthropurgic foci is regarded as a distinctive feature of recent TBE epidemiology that has a considerable effect on the level of morbidity. Indeed, this process developed at a sharply increased
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rate between the mid-1980s and mid-1990s (see below). It is known, however, that different forms of economic activity at different stages may account for the opposite trends in its development; moreover, similar anthropogenic factors may produce opposite effects at the northern and southern boundaries of the ranges of TBE virus and its vector (Korenberg, 1985). These facts are considered in detail in dozens of special and review articles. Here, it would be expedient to mention only the most general trends in the impact of human activities on natural TBE foci. At the initial stage, anthropogenic impact always leads to increasing landscape patchiness, and this provides for the enhancement of pathogen circulation and activation of natural foci, which may take root near populated areas, especially in pastures (Kucheruk, 1980). At later stages, when the land is generally settled, the activity of these foci begins to cease gradually (Kucheruk, 1980; Pavlovsky, 1947; Petrishcheva, 1964). These processes have been analyzed in detail in the course of studies on the impact of forest cutting and subsequent overgrowing of cutover areas on natural TBE foci (Tupikova and Korenberg, 1965). Today, they can also be observed in certain regions (Bespyatova et al., 2008; Danchinova et al., 2008), but this is hardly related to global climate change. The same pattern of development is also characteristic of the situation in forest biocenoses exposed to anthropogenic factors in Central Europe and Baltic countries (Randolph, 2008) and in the greater part of TBE range around many large Russian cities, where large areas of forests and open woodland have been allotted for country houses and garden plots since the mid-1980s. In the suburbs of Irkutsk (Eastern Siberia), the abundance of adult I. persulcatus ticks increased by a factor of more than 80 between 1986 and 2006 but has already shown a tendency to decrease since then (Korotkov et al., 2007c; Nikitin and Antonova, 2005). On the other hand, their abundance between 1970 and 1985 remained consistently low, irrespective of changes in climatic parameters. Therefore, the climatic factor cannot fully account for the long-term dynamics of vector abundance.
VIII. SINCE WHEN HAS TBE MORBIDITY INCREASED IN THE CITIES? As early as in 1961, it was noted that the contribution of city dwellers to the overall structure of TBE morbidity had increased throughout the Russian Federation (Ivanova, 1961). In 1970, however, city dwellers accounted for no more than 44% of recorded TBE cases. Their proportion began to increase more rapidly in the early 1970s, mainly on account of large cities with populations of more than 400,000, reaching 68% in 1978, long before the record-breaking morbidity peaks of 1996 and 1999. In the 1990s, this parameter in some years was even slightly higher, but its value
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in the peak year 1999 was only 64.2%, with those in the first years of the twenty-first century varying from 60% to 67% (Korenberg, 2003; Zlobin, 2004). Hence, the increasing proportion of city dwellers among TBE patients and their present-day prevalence among them are not specific to the most recent TBE epidemiology. This proportion has remained relatively stable over the past three decades, and, therefore, neither the significant increase in TBE morbidity nor its no less significant subsequent decrease can be explained by variation in this proportion.
IX. WHAT ARE THE MAIN CAUSES OF CHANGES IN PARAMETERS OF TBE MORBIDITY? The level of epidemic manifestations in natural foci of any zoonosis is eventually determined by two major parameters (Fig. 2): the intensity of virus circulation in the foci and the frequency of contact with them (Korenberg and Yurkova, 1983). As follows from the data presented above, the state of natural foci has not undergone any changes significant enough to explain the last outbreak of TBE morbidity followed by its current gradual decrease. On the other hand, the frequency of contact with the foci has drastically increased within a short period of time because of the aforementioned large-scale land allotment for country houses and garden plots in areas with natural TBE foci. According to official statistical data, the area of such plots in many regions of Russia Block model for predicting the epidemic manifestation of natural foci of human diseases
Anthropogenic impact
Ecological state of parasitic system components
Sociodemographic condition of population
Frequency of human contact with natural foci
Epizootic potential of natural foci ? Epidemic manifestation of natural foci
FIGURE 2 Block model for predicting the epidemic manifestation of natural foci of human diseases (Korenberg and Yurkova, 1983).
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increased by a factor of approximately 2.0–2.2 between 1984–1985 and 1991–1992 and by a factor of almost 3 by 1995. This purely social factor, resulting in increasing landscape patchiness, provided at the initial stage for the growing abundance of ticks (and, probably, other components of the parasitic system). A quite expectable epidemiological result of these processes was the increase in morbidity accounted for by TBE (and some other infections with natural focality) to the record-breaking levels in 1996 and 1999. The intensity of land development in the vicinities of large cities ceased to increase by mid-1992, and TBE foci in already settled areas began to degrade. It became clear that morbidity will cease to grow, stabilizing (with usual fluctuations by years) at the level determined by the existing frequency of people’s contact with the natural foci. Observations made in the past 8 years confirm the validity of this epidemiological prognosis. Attributing the increasing incidence of TBE cases to ‘‘the collapse of communism’’ in Eastern Europe (Randolph, 2008; Randolph and Rogers, 2000) is a straightforward simplification with a feeble epidemiological basis, since such episodes repeatedly took place during the deeply communist regime. For example, it is hardly logical to draw a connection between vast outbreaks of tularemia in the Soviet Union in 1938 to 1957 (Olsufiev and Dunaeva, 1970) and its communist rule, which collapsed only 35 years later, or between the increasing Lyme borreliosis morbidity and the political system in the United States. However, socioeconomic factors may exert a powerful effect on the frequency of population contact with agents of all zoonoses with natural focality, which leads to an explosive increase in general morbidity (Fig. 2). This is what accounted for the peak of TBE morbidity recorded in the mid-1960s not only in Russia (Fig. 1) but also in some other countries. A relevant example is also that socioeconomic transformations in Russia resulted in the decline of goat stocks and, as a consequence, previously frequent cases of alimentary TBE infection have become rare within a relatively short time (Korenberg, 2003).
X. CONCLUSIONS Thus, there are no reliable data confirming the effect of climatic changes on the spread and level of TBE morbidity, vectors, and main components of parasitic systems. The drastic increase in TBE morbidity in the 1990s in Russia and some other European countries was accounted for by social rather than biological factors. An outbreak of morbidity caused by TBE and other infections with natural focality may take place upon a new increase in the frequency of population contact with their natural foci. Such an increase may be conditioned by certain socioeconomic factors relevant for all people living in a large part of the forest zone.
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ACKNOWLEDGMENT This study was supported by the Russian Foundation for Basic Research (Project No. 07-04-00286).
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INDEX A Acquired immunodeficiency disease syndrome (AIDS), 2 Antiviral drugs and HIV-1 splicing, 30–31. See also Human immunodeficiency virus type 1 (HIV-1) Avsunviroid RNAs, 116. See also Hepatitis delta virus (HDV) B Bovine viral diarrhea virus (BVDV), 63–64, 70–71 Branch point sequence (BPS), 4 C CAAT/enhancer-binding protein (c/EBP), 88 Capsid hairpin (cHP), 51 Casein kinase II (CKII), 80 Cis-regulatory elements. See also Human immunodeficiency virus type 1 (HIV-1) core splicing signals, 3–4 exonic and intronic splicing enhancers, 4–5 secondary structure role, 5 Climate change and TBE, 131–132. See also Tick-borne encephalitis (TBE) morbidity, 127 Core splicing signals, 3. See also Human immunodeficiency virus type 1 (HIV-1)
Cyclin-dependent kinases (CDKs), 80 Cyclization (CYC) sequences, 66 D Delta antigen, role, 113–115. See also Hepatitis delta virus (HDV) Dengue virus (DENV), 42, 72, 81 Dimer linkage structure (DLS), 18 E Encephalitozoan cuniculi, 54 Env protein, 5 Exonic splicing enhancers (ESEs), 4, 13–14, 26 Exonic splicing silencers (ESSs), 4, 13–14, 25 F Flavivirus cap methylation, model, 60–61. See also Methyl transferase domain Flavivirus MTase GTP-binding ability, 57–58 structure, 52–54 core MTase subdomain, 54–55 N-terminal GTP-binding subdomain, 55–56 RNA-binding groove, 56–57 Flavivirus NS5, 42 amino acid sequence, 46–50 flaviviral protein, 43 future perspectives, 89–91 interaction host proteins, 78–79
145
146
Index
Flavivirus NS5 (cont.) intramolecular interactions, 75–76 and NS3, 77–78 and viral RNA, 76–77 localization cellular localization, 81–82 nuclear localization, 82–85 MTase domain, structure, 53 phosphorylation, 79–81 POL domain, 69 RdRp activity de novo initiation, 64–65 flavivirus RdRp, 63–64 RdRp activity and NS5, 62 RNA synthesis, 65–67 RdRp structure, 67–68 structure, 74 in viral pathogenesis, 85–89 Flavivirus RNA synthesis, 61–62. See also RNA-dependent RNA polymerase (RdRp) domain G Gag-pol splice sites, 15. See also Human immunodeficiency virus type 1 (HIV-1) GAR ESE. See Guanosine–adenosine-rich ESE Glycogen synthase kinase 3 (GSK3), 80 Guanosine–adenosine-rich ESE, 12 H Haemaphysalis concinna, 130 Hepatitis B virus (HBV), 104 Hepatitis C virus (HCV), 63, 65, 70 Hepatitis delta virus (HDV), 104–106 delta antigen, 113–115 host factors, 115
pausing and switching, 110–112 polymerase, 106–108 promoters and priming, 108–110 replication in nucleus, 112–113 viroid analogy, 116 Heterogeneous ribonuclear protein (hnRNP), 4 Highly active antiretroviral therapy (HAART), 2 Human immunodeficiency virus type 1 (HIV-1), 2 antiviral drugs, 30–31 Cis-regulatory elements core splicing signals, 3–4 exonic and intronic splicing enhancers, 4–5 secondary structure role, 5 exons 2 and 3, 17 Rev, Tat and Vpr proteins, 18–21 RNA secondary structure effects, 17–18 RNA splicing, 5–6 splice sites and regulatory elements Gag-pol splice sites, 15 HXB2 tev splice sites, 14–15 Nef and tat, rev exon 2 splice site, 13–14 Rev and env/nef 30 -splice sites, 12–13 tat mRNA 30 -splice site, 11 Vif mRNA splice site, 8–10 Vpr mRNA 30 -splice site, 10 splicing regulatory elements cellular splicing factors expression, 29–30 and regulatory elements, 21–23 siRNA inhibition, 28–29 virus replication inhibition, 23–28 50 ss D1 and D4, 15–17
147
Index
Human scribble (hScrib) protein, 87–88 HXB2 tev splice sites, 14–15. See also Human immunodeficiency virus type 1 (HIV-1) I IFN-stimulated genes (ISGs), 85 Interferon (IFN), 85 Intronic splicing enhancers (ISE), 4 Intronic splicing silencer (ISS), 4, 13 Ixodes persulcatus, 129 abundance, 135 distribution, 124–125 TBE virus prevalence, 130 urbanization effect, 131–132 Ixodes ricinus distribution, 124–125 TBE virus prevalence, 130 urbanization effect, 131–132 J Janus-activated kinase–signal transducer and activator of transcription ( JAK-STAT), 85–86 Japanese encephalitis virus (JEV), 42 L Locked nucleic acids (LNAs), 33 M Meaban virus (MEAV), 52 Metazoan mRNA splicing. See also Human immunodeficiency virus type 1 (HIV-1) core splicing signals, 3–4 exonic and intronic splicing enhancers, 4–5 secondary structure role, 5 Methyl transferase domain
flavivirus cap methylation model, 60–61 flavivirus MTase structure, 52–54 core MTase subdomain, 54–55 N-terminal GTP-binding subdomain, 55–56 RNA-binding groove, 56–57 MTase enzymatic activities, 45–52 MTase structure-function studies GTP-binding studies, 57–58 MTase activities, 58–59 50 -RNA cap formation, 44–45 Mitogen-activated protein kinases (MAPKs), 80 Murray Valley encephalitis virus (MVEV), 52 N Nef and tat, rev exon 2 splice site, 13–14. See also Human immunodeficiency virus type 1 (HIV-1) NES. See Nuclear export signal NLS. See Nuclear localization signal Nonstructural protein 5 (NS5), 42 flaviviral protein, 43 future perspectives, 89–91 interaction host proteins, 78–79 intramolecular interactions, 75–76 and NS3, 77–78 and viral RNA, 76–77 localization cellular localization, 81–82 nuclear localization, 82–85 MTase domain, structure, 53 phosphorylation, 79–81 POL domain, 69 RdRp activity
148
Index
Nonstructural protein 5 (NS5) (cont.) de novo initiation, 64–65 flavivirus RdRp, 63–64 RdRp activity and NS5, 62 RNA synthesis, 65–67 RdRp structure, 67–68 structure, 74 in viral pathogenesis, 85–89 NS3 and NS5 interaction, 77–78. See also Nonstructural protein 5 (NS5) N-terminal GTP-binding subdomain, 55–56. See also Flavivirus MTase Nuclear export signal, 82–84 Nuclear localization signal, 82–84 Nucleoside triphosphatase (NTPase), 75
flavivirus RdRp, 63–64 RdRp activity and NS5, 62 RNA synthesis, 65–67 flavivirus RNA synthesis, 61–62 structure fingers subdomain, 70–72 NS5 RdRp structure, 67–68 NS5 structure, 74 palm subdomain, 68–70 RNA synthesis initiation, 73–74 thumb subdomain, 72–73 structure-function analysis, 74–75 RNA synthesis, template, 65–67. See also Nonstructural protein 5 (NS5) RRE. See Rev-responsive element Russia, TBE morbidity, 126
P
S
Peptide nucleic acids (PNAs), 33 Polypyrimidine (Py), 4 Pospoviroid RNAs, 116. See also Hepatitis delta virus (HDV) Powassan virus (PWV), 51 R Rev and env/nef 30 -splice sites, 12–13. See also Human immunodeficiency virus type 1 (HIV-1) Rev protein, 5 Rev-responsive element, 5–6, 19 RNA-binding groove, 56–57. See also Methyl transferase domain 50 -RNA cap formation, 44–45. See also Methyl transferase domain RNA-dependent RNA polymerase (RdRp) domain, 43–44 activity de novo initiation, 64–65
S-adenosyl-L-homocysteine (AdoHcy), 54 S-adenosyl-L-methionine (AdoMet), 44, 55 Serine–argininerich protein (SR protein), 4 SiRNA inhibition and HIV-1 splicing, 28–29. See also Human immunodeficiency virus type 1 (HIV-1) SRPIN340, role, 31 50 ss D1 and D4, 15–17. See also Human immunodeficiency virus type 1 (HIV-1) Stem loop structure (SLA), 76 T 0
Tat mRNA 3 -splice site, 11. See also Human immunodeficiency virus type 1 (HIV-1) Tat protein, in viral RNA splicing, 18–21. See also Human
149
Index
immunodeficiency virus type 1 (HIV-1) Terminal regions (TRs), 43 Tick-borne encephalitis (TBE), 42, 124–126 abundance and prevalence, 129–131 anthropurgic foci, 134–135 climate change, 131–132 distribution, 125 foci, 133–134 issues, 127–128 morbidity, 135–137 vectors, 128–129 Tiny RNAs (tiRNA), 110 Tyrosine kinase 2 (Tyk2), 85–86 U Untranslated regions (UTRs), 43 V Vesicular stomatitis virus, 45 Vif mRNA splice site, 8–10. See also Human immunodeficiency virus type 1 (HIV-1) Viral pathogenesis, NS5 role, 85–89. See also Nonstructural protein 5 (NS5)
Virus replication. See also Human immunodeficiency virus type 1 (HIV-1) cellular splicing factors expression, 29–30 and regulatory elements, 21–23 siRNA inhibition, 28–29 virus replication inhibition, 23–28 Vpr mRNA 30 -splice site, 10. See also Human immunodeficiency virus type 1 (HIV-1) Vpr protein, in viral RNA splicing, 18–21. See also Human immunodeficiency virus type 1 (HIV-1) VSV. See Vesicular stomatitis virus W West Nile virus (WNV), 42, 72, 76–77 WNV strain Kunjin (WNVKUN), 62 Y Yellow fever virus (YFV), 42, 79
