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 Advances in
VIRUS RESEARCH

VOLUME 63
 ADVISORY BOARD

DAVID BALTIMORE BERNARD MOSS

ROBERT M. CHANOCK ERLING NORRBY
PETER C. DOHERTY J. J. SKEHEL
H. J. GROSS R. H. SYMONS
B. D. HARRISON M. H. V. VAN REGENMORTEL
PAUL KAESBERG
 Advances in
VIRUS RESEARCH

Edited by

KARL MARAMOROSCH AARON J. SHATKIN

Department of Entomology Center for Advanced Biotechnology
Rutgers University and Medicine
New Brunswick, New Jersey Piscataway, New Jersey

VOLUME 63
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 CONTENTS

Duck Hepatitis B Virus: An Invaluable Model System For

HBV Infection
URSULA SCHULTZ, ELIZABETH GRGACIC, AND MICHAEL NASSAL
I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... 1
II. Animal Models of HBV. . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... 3
III. The Hepadnaviral Infectious Cycle: An Overview . . . . . . . . ...... 7
IV. DHBV Proteins and Their Basic Functions in Replication
and Morphogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... 12
V. Experimental DHBV Infection . . . . . . . . . . . . . . . . . . . . . . ...... 37
VI. DHBV as a Model to Study Host Responses to and Control
of HBV Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... 46
VII. DHBV and the Development of Hepadnaviral
Transduction Vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... 53
VIII. Conclusions and Perspectives . . . . . . . . . . . . . . . . . . . . . . . ...... 54
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... 55

Novel Insights into Hepatitis C Virus Replication

and Persistence
RALF BARTENSCHLAGER, MICHAEL FRESE, AND THOMAS PIETSCHMANN
I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
II. Genomic Organization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
III. Virus Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
IV. Experimental Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
V. Replication Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
VI. Virus–Host Interaction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
VII. Antiviral Therapies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146
VIII. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149

v
vi CONTENTS

The Regulation and Maturation of Antiviral

Immune Responses
J. LINDSAY WHITTON, MARK K. SLIFKA, FEI LIU, ALEXANDER K. NUSSBAUM,
AND JASON K. WHITMIRE

I. Overview of the Immune Response to Viral Infection ........... 182

II. B Lymphocytes and Their Role in Antiviral
Immune Responses . . . . . . . . . . . . . . . . . . . . . . . . . ........... 184
III. Antiviral T Cells: A Primer . . . . . . . . . . . . . . . . . . . ........... 192
IV. CD8þ T Lymphocytes and Their Role in Antiviral
Immune Responses . . . . . . . . . . . . . . . . . . . . . . . . . ........... 194
V. CD4þ T Lymphocytes and Their Role in Antiviral
Immune Responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
VI. Immunopathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
VII. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223

Prospects for the Therapy and Prevention of

Dengue Virus Infections
ELSA B. DAMONTE, CARLOS A. PUJOL, AND CELIA E. COTO
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239
II. The Agent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240
III. The Human Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242
IV. DENV as a Global Reemerging Agent . . . . . . . . . . . . . . . . . . . . . . 245
V. Virus Structure and the Replicative Cycle: Possible Targets
for Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246
VI. Viral Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254
VII. Strategies for Vaccine Development . . . . . . . . . . . . . . . . . . . . . . . . 268
VIII. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273

Bacteriophage T4: Structure, Assembly, and Initiation

Infection Studied in Three Dimensions
VADIM V. MESYANZHINOV
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287
II. Bacteriophage T4 Head Structure and Assembly . . . . . . . . . . . . . . 291
III. The Tail . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303
IV. The Fibers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305
V. The Baseplate Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311
VI. Structure of the Star-Shaped Baseplate . . . . . . . . . . . . . . . . . . . . . 337
VII. The Mechanism of the Baseplate Conformational Transition
and Initiation of the Infection . . . . . . . . . . . . . . . . . . . . . . . ..... 339
 CONTENTS vii

VIII. Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341

Genomic Organization, Biology, and Diagnosis of Taura

Syndrome Virus and Yellowhead Virus
of Penaeid Shrimp
ARUN K. DHAR, JEFF A. COWLEY, KENNETH W. HASSON,
AND PETER J. WALKER

I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353
II. Taura Syndrome Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354
III. Yellowhead Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383
IV. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 410
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 411

Viruses of the Chestnut Blight Fungus, Cryphonectria parasitica

BRADLEY I. HILLMAN AND NOBUHIRO SUZUKI
I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... 424
II. Fungi as Hosts for Virus Infection . . . . . . . . . . . . . . . . . . . ...... 425
III. History of C. parasitica as a Virus Host . . . . . . . . . . . . . . . ...... 427
IV. Viruses as the Cause of Hypovirulence and a
Description of CHV1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... 427
V. General Properties and Procedures for Studying
C. parasitica Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... 433
VI. Structure and Characteristics of the CHV1 Genome . . . . . . ...... 435
VII. Host Genes and Signaling Pathways Affected by
CHV1 Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 444
VIII. Other Cryphonectria Hypoviruses . . . . . . . . . . . . . . . . . . . . . . . . . . 447
IX. Defective and Satellite RNAs of Hypoviruses . . . . . . . . . . . . . . . . . 451
X. Other Viruses of C. parasitica . . . . . . . . . . . . . . . . . . . . . . . . . . . . 453
XI. Population Biology and Evolution of Cryphonectria Viruses. . . . . . . 458
XII. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 462
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 462

Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 473
 ADVANCES IN VIRUS RESEARCH, VOL 63

DUCK HEPATITIS B VIRUS: AN INVALUABLE MODEL

SYSTEM FOR HBV INFECTION

Ursula Schultz,* Elizabeth Grgacic, and Michael Nassal*

*University Hospital Freiburg, Department of Internal Medicine II/Molecular Biology

D-79106 Freiburg, Germany
Macfarlane Burnet Institute for Medical Research and Public Health
Hepatitis Laboratory, Melbourne, Victoria 3004, Australia

I. Introduction
II. Animal Models of HBV
A. Primate Orthohepadnaviruses
B. Non-Primate Orthohepadnaviruses
C. Bird HBVs (Avihepadnaviruses)
III. The Hepadnaviral Infectious Cycle: An Overview
IV. DHBV Proteins and Their Basic Functions in Replication and
Morphogenesis
A. The Core Protein
B. The Reverse Transcriptase (P Protein)
C. Envelope Proteins
V. Experimental DHBV Infection
A. In Vitro Infection of Primary Duck Hepatocytes
B. Experimental In Vivo Infection
VI. DHBV as a Model to Study Host Responses to and Control of HBV Infection
A. Cytokines and Their Role in Controlling Hepadnaviral Infection
B. Chemotherapy and Vaccination
VII. DHBV and the Development of Hepadnaviral Transduction Vectors
VIII. Conclusions and Perspectives
References

I. INTRODUCTION

Hepatitis B virus (HBV) is the causative agent of acute and chronic

hepatitis B in humans. More than 350 million people worldwide are
chronic virus carriers and face a significantly increased risk of develop-
ing liver cirrhosis and primary hepatocellular carcinoma (Blumberg,
1997). Effective prophylactic vaccines based on noninfectious empty
envelopes (termed S particles or subviral particles), originally purified
from the plasma of carriers and later produced in recombinant form
in yeast or mammalian cell lines, have been available since the 1980s;
for a comprehensive review on clinical aspects, including various vac-
cines, see Hollinger and Liang (2001). Nonetheless, for many develop-
ing countries, large-scale vaccination programs were hardly affordable.

1 Copyright 2004, Elsevier Inc.

All rights reserved.
0065-3527/04 $35.00
2 URSULA SCHULTZ ET AL.

This situation is improving, but an enormous number of chronic HBV

carriers will be in need of better medication for decades to come. Cur-
rent therapies are based on the systemic administration of high doses of
interferon- (IFN-) or, more recently, on nucleoside analogs, such as 3-
thiacytidine (lamivudine) and adefovir. However, both therapies have a
sustained response rate of only about 30%, combinations exert no clear
synergism, and lamivudine therapy leads to the rapid emergence of
resistant virus variants (Pumpens et al., 2002; Zoulim, 2001).
A full understanding of the molecular biology of HBV and its infec-
tious cycle is hampered by experimental limitations: as of yet there is
no feasible small animal infection model, and only a few aspects of its
replication cycle are amenable to biochemical methods. The focus of
this review is on one of two established animal virus models, namely
duck hepatitis B virus (DHBV). Although humans and ducks are only
distantly related hosts, HBV and DHBV, which are the type members
of the orthohepadnaviruses and avihepadnaviruses (hepatotropic DNA
viruses), share fundamental common features. In fact, many of the
principles of hepadnavirus replication have been established using
DHBV. Its major advantages are the ready availability of ducks, allow-
ing experimental infections with wild-type and mutant viruses in vivo
as well as in cultured primary hepatocytes, and the recent develop-
ment of in vitro systems to study biochemically the intricate mecha-
nism of hepadnaviral replication. Thus, unlike other hepadnaviruses
DHBV offers the full range of experimental approaches, from the test
tube to animal studies, to investigate fundamental as well as selected
medical aspects of hepadnavirus biology.
The emphasis here is on the value of DHBV as a model for HBV, but
the differences between the human and the duck viruses should not be
neglected. DHBV can cause acute and chronic infections but is at
variance with HBV and mammalian viruses in several aspects:
(i) DHBV does not appear to be pathogenic for its host; (ii) DHBV
probably lacks a regulatory protein comparable to the mammalian
virus HBx gene product, a promiscuous transactivator that, by acting
on the host cell, appears to be essential for establishment of infection
and has been implicated in carcinogenesis; and (iii) a host-cell encoded
glycoprotein, carboxypeptidase D (CPD), appears to be critical for
DHBV infection, yet no evidence supports a similar role for its human
homologue. This chapter will address these differences as well as
peculiarities concerning the structure and function of individual viral
proteins. Additional information on DHBV can be found in several
recent reviews describing the general features of hepadnavirus biology
(Ganem and Schneider, 2001; Nassal, 1999, 2000; Seeger and Mason,
 DUCK HEPATITIS B VIRUS 3

2000) and describing DHBV-specific immunological aspects (Jilbert

and Kotlarski, 2000).

II. ANIMAL MODELS OF HBV

Two salient features of hepadnaviruses are their pronounced liver

tropism and their narrow host range. In general, therefore, the closer
the hosts are related, the closer the respective viruses are related.
Overall, however, the mammalian orthohepadnaviruses and the avian
avihepadnaviruses share a very similar genome organization and rep-
lication characteristics (Section III); hence, numerous aspects of one
type of hepadnavirus are also applicable to the other types.

A. Primate Orthohepadnaviruses
After the discovery of human HBV in 1970 (Dane et al., 1970) and
the discovery that chimpanzees are susceptible to infection with hu-
man HBV and cloned HBV DNA (Will et al., 1982), HBV-like viruses
were detected in hominoid primates, such as in all great apes. Their
genome sequences are extremely close to the variants circulating in
the human population. Therefore, it has long been disputed whether
they are true animal viruses or have been contracted by contact with
humans; recent data provide accumulating evidence that hominoid
primates do represent a natural reservoir for HBV (Robertson and
Margolis, 2002). Obviously, none of these primates, with the limited
exception of chimpanzees, is a feasible experimental animal. A new
HBV discovered in woolly monkeys (Lagothrix lagotricha), a New
World primate, is clearly distinct from human HBV (Lanford et al.,
1998). Woolly monkeys are an endangered species and therefore are
not suited as experimental animals. Successful transmission of woolly
monkey HBV (WMHBV) to the related, nonendangered spider monkey
(Ateles geoffrey) raised hopes for the establishment of a new, lower
primate infection model. However, viral titers appear to be low (less
than 105 virions per milliliter of serum), which is unpractical. At-
tempts to infect primary hepatocytes from marmosets (Callithrix
jacchus), a close relative of woolly monkeys, remained negative (Köck,
McNelly, and Nassal, unpublished data). In contrast, primary hepato-
cytes from tree shrews (Tupaia belangeri) can clearly be infected by
HBV and WMHBV (Köck et al., 2001) although the animals are not
true primates, and their assumed phylogenetic closeness to pri-
mates remains controversial (Murphy et al., 2001; Schmitz et al.,
4 URSULA SCHULTZ ET AL.

2000). Moreover, transduction of HBV genomes into tree shrews using

replication-defective adenovirus vectors as vehicles (Ren and Nassal,
2001) led to a long-lasting HBV viremia in vivo. It has not yet been
proven, however, whether the circulating HBV was derived from a true
HBV infection or solely from the AdHBV vector. Given that tree shrews
are relatively easily bred in captivity, establishing a self-
sustained HBV infection in these squirrel-sized animals would indeed
represent a major advance; however, the full potential of the system
remains to be explored.
In the absence of a practical HBV infection model, several surrogate
systems have been established to study the human virus. The ability of
certain human hepatoma cell lines, such as HepG2 and Huh7, to
support HBV replication upon transfection of cloned wild-type, and
mutant, HBV DNA is a highly valuable tool to investigate replication
per se; most of our current knowledge is derived from this system.
Because these cell lines cannot be infected, however, the early steps
of the replication cycle, such as entry and genome uncoating, are dif-
ficult to study. This restriction may be overcome by the recent discov-
ery of a new human hepatoma line that, under specialized conditions of
culture and inoculation, appears to be susceptible to HBV infection
although infectibility seems to be lost after a number of passages
(Gripon et al., 2002).
Another model that has been highly useful in particular for immu-
nological studies uses HBV transgenic mice (reviewed in Chisari,
1996). The animals contain a chromosomally integrated and linear
1.3 overlength HBV genome (the terminal duplication mimics the
circular form of the natural virus genome; hence, transcription is
controlled by the authentic regulatory elements). The mice produce
substantial amounts of viral antigens and complete virions, but these
cannot infect mouse hepatocytes; in addition, there is no clear-cut
evidence that the central intracellular intermediate of the authentic
infection, a covalently closed circular (ccc) DNA molecule that is main-
tained in an episomal state and serves as the natural template for
transcription of the viral RNAs, is formed in the mice except, perhaps,
in a very special genetic background (Raney et al., 2001). Nonetheless,
based on the advanced state of mouse genetics and on the availability
of various knockout mice as well as cloned genes of and antibodies
against all immunologically relevant gene products, fundamentally
new insights into the immune response against HBV have been gained
(Section VI). Most notably, T cells can suppress HBV replication in a
noncytolytic fashion, mainly mediated by type I and type II interferons
and TNF- (Guidotti and Chisari, 1999). Alternatively, mice have been
 DUCK HEPATITIS B VIRUS 5

xenotransplanted with hepatocytes from other species (Dandri et al.,

2001a), including humans (Dandri et al., 2001b), and studies have
shown that these mice can be infected with HBV. Though powerful
for some applications, the chimeric animals are technically difficult
to generate and, by necessity, the recipients have to be severely im-
munocompromised to avoid transplant rejection. This also limits the
immunological conclusions that can be drawn.
Therefore, a combined approach integrating biochemical in vitro
analysis, genetics and infection in cell culture, and in vivo biology is
currently not available for HBV.

B. Non-Primate Orthohepadnaviruses
Shortly after the discovery of human HBV, several related viruses
were found in a few nonprimate mammals: the North American wood-
chuck (Summers et al., 1978), the Beechey ground squirrel: (Marion
et al., 1980), and the arctic ground squirrel (Testut et al., 1996). No HBV-
like viruses were found in species that are commonly used as laboratory
animals, such as mice and rats. Woodchuck hepatitis B virus (WHV) has
therefore become the model of choice for mammalian HBVs, as reviewed
in Roggendorf and Tolle (1995) and Tennant and Gerin (2001). Its
sequence is about 60% similar to that of the human virus and, impor-
tantly, WHV causes chronic hepatitis and hepatocellular carcinoma,
just as does HBV. WHV is therefore used for tracking fundamental
pathogenetic and therapeutic aspects of hepadnaviral infection (Mason
et al., 1998; Yamamoto et al., 2002; Zhou et al., 2000); with cloning of
several key factors of the innate and acquired immune response (Guo
et al., 2000; Lohrengel et al., 1998; Salucci et al., 2002; Yang et al., 2000),
immunological studies can now be conducted. Experiments in chroni-
cally WHV-infected woodchucks have revealed some unexpected differ-
ences to data obtained in HBV transgenic mice and in acutely infected
humans and chimpanzees; for instance, conventional treatment or gene
therapy with IFN-, a major player in suppressing HBV replication in
mice and in clearing acute infection, had no significant impact on chron-
ic WHV infection (Jacquard et al., 2004; Lu et al., 2002). The reasons are
not clear but certainly worth being followed. The advantage of WHV’s
remarkable similarity to human HBV related disease is, however, par-
tially offset by several practical limitations. Adult woodchucks weigh
about 4 to 5 kg, and they are difficult to handle. They do not breed easily
in captivity and, therefore, many experiments are performed with wild
animals trapped in their natural habitat in the northeastern part of
the United States. These animals are outbred, and many of them are
6 URSULA SCHULTZ ET AL.

infested with other pathogens. In addition, woodchucks hibernate, dur-

ing which time no experiments can be performed. Another fundamental
limitation of the WHV system is the current lack of cell lines that
efficiently support replication of cloned WHV DNA; hence, the power
of reverse genetics cannot be applied to WHV.

C. Bird HBVs (Avihepadnaviruses)

In 1980, Mason et al., discovered an HBV-related virus, duck HBV
(DHBV), in Pekin ducks (Anas platyrhynchos forma domestica; derived
from mallard ducks); since then, the virus has been detected in a
substantial fraction of commercially bred flocks, with no signs of overt
pathogenicity or progression of chronic infection into liver cancer,
except possibly in the presence of carcinogens such as aflatoxin (Cova
et al., 1994). The presence of HBV in ducks is generally viewed as the
result of a long-lasting coevolution between virus and host and is
supported by the rapid replacement by noncytopathic viruses of an
artificial, cytopathic DHBV variant (Lenhoff et al., 1998).
Further avihepadnaviruses have been isolated from grey herons
(Ardea cinerea) (Sprengel et al., 1988), snow geese (Anser caerulescens)
(Chang et al., 1999), white storks (Pult et al., 2001), and, most recently,
cranes (several species of the genus Grus) (Prassolov et al., 2003); the
latter reference also contains comprehensive sequence comparisons as
well as a phylogenetic tree of the host birds. In general, avihepadna-
viruses have a narrow host range (Section V.A.4), similar to the mam-
malian viruses, such that DHBV does not infect chickens (a desirable
host in view of the advanced genetic and immunological knowledge on
this species) (Marion et al., 1987) or even Muscovy ducks (Cairina
moschata) (Pugh and Simmons, 1994), which belong to the same order
as Pekin ducks (Anseriformes). In turn, the viruses from storks and
herons (order Ciconiiformes) are not detectably infectious for ducks
in vivo; however, primary duck hepatocytes can be infected with heron
HBV (HHBV) (Ishikawa and Ganem, 1995) though with a low efficien-
cy, as seen for DHBV infection of primary Muscovy duck hepatocytes.
Unexpectedly, crane HBV (CHBV) appears to efficiently infect primary
duck hepatocytes although its natural host is more closely related to
storks and herons (Prassolov et al., 2003); the genome sequence of
CHBV, by contrast, is closer to that of DHBV and snow goose HBV
(SGHBV). The evolutionary background of this disparity is unclear;
hence, further investigations are certainly warranted.
In general practical terms, however, DHBV will remain the most
important of the avihepadnaviruses. Protocols for the reproducible
 DUCK HEPATITIS B VIRUS 7

preparation and in vitro infection of primary duck hepatocytes have

been established (Section V.A). In addition, the chicken hepatoma cell
line LMH fully supports DHBV replication after transfection with
cloned DHBV DNA, yielding infectious virions, and such recombinant
viruses can be analyzed for infectivity in duck hepatocytes and ducks.
Finally, some biochemical aspects of replication can as of yet only be
investigated with DHBV (Section IV.B). Hence, the DHBV–duck vi-
rus–host pair is at present the only practical system in which all facets
of hepadnaviral replication and infection, from molecular interactions
to virus fitness in vivo, can be addressed. Many fundamental discov-
eries have first been made with DHBV, such as hepadnaviruses repli-
cation by reverse transcription (Summers and Mason, 1982), the
mechanisms of cccDNA formation (Tuttleman et al., 1986a), and initi-
ation of reverse transcription, including its cell-free reconstitution
(Wang and Seeger, 1992). Host-range determinants (Ishikawa and
Ganem, 1995) and putative receptors have also been defined (Breiner
et al., 1998; Ishikawa and Ganem, 1995; Kuroki et al., 1995; Urban
et al., 1998). In addition, the link between viral persistence and
cccDNA following antiviral treatment was detected, and the first dem-
onstration of the in vivo efficacy of antisense approaches against a
viral infection was made with DHBV (Offensperger et al., 1993), re-
cently shedding new light on the antisense mechanism as such (Thoma
et al., 2001). Finally, DHBV was crucial for the development of hepad-
navirus-based liver-specific gene delivery systems (Protzer et al.,
1999).

III. THE HEPADNAVIRAL INFECTIOUS CYCLE: AN OVERVIEW

All hepadnaviruses are DNA viruses (i.e., infectious virions contain

DNA). Summers and Mason were the first to demonstrate, using
DHBV, that this DNA is generated by reverse transcription (Summers
and Mason, 1982). Hence, hepadnaviruses are related to retroviruses
although the latter, based on the genome form present in infectious
virions, are RNA viruses. Retroviral RNA is reverse transcribed upon
infection of a new cell (i.e., as an early event of the cycle), whereas in
hepadnaviruses, reverse transcription occurs late in the originally
infected cell. In addition, integration of proviral DNA into the host
chromosome is an obligatory step in retroviral but not hepadnaviral
replication. To account for these fundamental differences, hepadna-
viruses, together with a few plant viruses that use a similar strategy
such as cauliflower mosaic virus, have been termed pararetroviruses.
8 URSULA SCHULTZ ET AL.

Foamy viruses may form an evolutionary link between the two groups
since their virions probably contain mostly DNA, as in hepadna-
viruses, but replication requires integration (Linial, 1999).
A schematic comparison of the DHBV virion and the DHBV genome
with that of HBV is shown in Fig. 1; with small modifications, all avian
hepadnaviruses are similar to DHBV, and all mammalian hepadna-
viruses are similar to HBV. All hepadnavirions possess an outer enve-
lope, that is, a host-derived lipid bilayer into which the large (L)
proteins and a small (S) surface protein are embedded; the mammalian
viruses have an additional middle (M) surface protein (Section IV.C).
The envelope encloses the icosahedral nucleocapsid formed by the
capsid, or core, protein. The viral genome is a partially duplex and
circular (but not covalently closed) DNA (relaxed circular, or RC DNA)
of about 3.0 kb (avian) or 3.2 kb (mammalian HBVs) in length. The
( )-strand DNA is at full length, and its 50 -terminal nucleotide is
covalently linked to the terminal protein (TP) domain of the reverse
transcriptase, called the P protein; the (þ)-strand is incomplete to
various extents. A typical feature of all hepadnavirus genomes is their
compact organization. All nucleotides (nt) have coding capacity in one
open reading frames (ORFs); many nt have this capacity in two ORFs.
In addition, all regulatory elements such as promoters and enhancers,
as well as various other cis-elements, overlap with coding information.
The three major ORFs, C, P, and S, encode the core protein, the P
protein, and the small surface protein (S). C and S are preceded by the
preC and preS (preS1 and preS2 in orthohepadnaviruses) regions that
give rise to N-terminally extended proteins. The precore protein is a
secreted nonassembling core protein variant that, after N- and C-
terminal processing, is found in serum as HBeAg (or DHBeAg in
DHBV). Its function might be to modulate the immune response to
the core protein, but this is poorly understood (Section IV.A.3). Simi-
larly, cotranslation of the preS region with S yields the L protein
(which in HBV is the product of the complete preS1/preS2/S ORF;
preS2/S gives the M protein). Three major internal promoters drive
transcription of the C, S, and preS/S mRNAs, which all end after a
common polyadenylation signal; the C and, in mammalian hepadna-
viruses, the S transcripts have staggered 50 -ends bracketing the up-
stream preC and preS2 ATGs. The C mRNA encompasses the entire
genome plus a terminal redundancy. The P protein is also translated
from this transcript which, in addition, is packaged into nucleocapsids
for reverse transcription; the transcript is therefore also termed RNA
pregenome, or pregenomic RNA (pgRNA). One major difference be-
tween the avian and mammalian viruses is the presence, in the latter,
 DUCK HEPATITIS B VIRUS 9

FIG 1. Comparison between DHBV and HBV. (A) Virion structure. The surface pro-
teins (L and S in avihepadnaviruses; L, M, and S in orthohepadnaviruses) are embedded
into the lipid envelope that enwraps the nucleocapsid formed by the core protein. Virions
contain the cellular heat-shock protein Hsc70 and, possibly, other cellular factors. The
genome inside the capsid is shown in its DNA form with one complete strand, with the
covalently linked TP domain of P protein, and the incomplete second strand. (B) Genome
organization. The different circles represent, from the periphery to the center, (i) the
various transcripts with the arroweads indicating start sites; (ii) the partially double-
stranded DNA genome with the circles numbered 1 and 2 representing the direct repeat
(DR) and Enh as the enhancer elements; and (iii) the open reading frames C, P, S, and X.
The " and D" denote the RNA stem-loops that act as encapsidation signals and replica-
tion origins for reverse transcription. The D" II is a second RNA element essential for
encapsidation in avihepadnaviruses that has no known counterpart in the mammalian
viruses. The SD and SA are the major splice donor and acceptor sites in DHBV. (See Color
Insert.)
10 URSULA SCHULTZ ET AL.

of an additional open reading frame called X. This frame encodes a

regulatory protein whose exact function is still incompletely under-
stood although a stimulatory effect on HBV replication has recently
been reconstituted in transfected hepatoma cell lines (Bouchard et al.,
2001). Stimulation correlates with the ability of HBx to release Ca2þ
from internal stores, which then leads to activation of kinases
(Bouchard et al., 2001; Nassal, 2002) that possibly target the capsid
protein (Section IV.A). Avian hepadnaviruses apparently lack an X
ORF although a ‘‘hidden’’ X-like ORF in DHBV has recently been
shown to be present and expressed in cultured cells (Chang et al.,
2001). It is unclear, however, from what RNA the X-like product would
be translated, and recent in vivo experiments in ducks did not provide
evidence for a functional importance of the gene product in DHBV
infection (Meier et al., 2003). A second obvious difference is the size
of the core protein, which consists of only about 180 amino acids (aa)
in the mammalian hepadnaviruses but about 260 aa in the avian
hepadnaviruses (Section IV.A).
A simplified view of the hepadnaviral infectious cycle is shown in
Fig. 2. The individual steps are briefly outlined in the figure legend.
Only those steps whose understanding has been greatly enhanced by
using the DHBV system and those that are addressed in the following
sections are listed. Enveloped virions bind, via exposed preS domains,
to specific receptor(s) on the hepatocyte surface and are internalized
(Section V.A.3). The nucleocapsid is released into the cytoplasm and
transported to the nucleus by way of nuclear localization signals
(NLSs) in the core protein (Section IV.A.1). The intact nucleocapsid
can apparently traverse the nuclear pore whose functional diameter,
with about 39 nm, appears to be significantly larger (Pante and Kann,
2002) than previously estimated (Dworetzky et al., 1988). The disas-
sembly of the capsid shell and release of the genomic RC DNA into the
nucleoplasm are not well understood but, eventually, the RC DNA is
converted into episomal cccDNA, which then acts as a transcriptional
template for cellular RNA polymerase II. Nuclear export of the ortho-
hepadnaviral transcripts is mediated by a posttranscriptional regu-
latory element (PRE) (Zang and Yen, 1999) whose counterpart, if any,
in avihepadnaviruses has not been defined; notably, a major spliced
transcript is produced from the pgRNA of avihepadnaviruses but not
orthohepadnaviruses (Obert et al., 1996).
Once in the cytoplasm, all viral RNAs are translated. The pgRNA
packaging relies on a specific chaperone-mediated interaction between
the P protein and an RNA stem-loop structure on the pgRNA called ",
which also acts as replication origin for RT (Section IV.B.2.1). The
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