This document provides a history of the discovery of hepatitis B virus (HBV). HBV was originally identified in the 1960s and associated with serum hepatitis. It was found to be a unique DNA virus that replicates via reverse transcription of an RNA intermediate. Several related hepatitis viruses were later discovered in other species. These viruses were classified in the Hepadnaviridae family based on their small genomes and novel replication strategy. HBV itself exists as different genotypes that tend to have distinct geographic distributions. The structure of the HBV virion is described, consisting of an inner core containing the viral DNA, surrounded by envelope proteins that are acquired from host cells.

1. CHAPTER
[AU1]
68
Christoph Seeger • Fabien Zoulim • William S. Mason
Hepadnaviruses
History
Classification of Viruses within the
Hepadnavirus Family
Virion Structure
Genome Structure and Organization
Stages of Replication
Infection of Hepatocytes
Regulation of Transcription and Translation
Pathogenesis, Pathology, and Epidemiology
Entry into the Host
The Liver and Its Response to HBV Infection
Other Sites of Hepadnavirus Infection
Immune Response to Hepadnavirus Infections
Immune Response in Transient Infections
Clinical Manifestations of Chronic HBV Infections
and Decisions to Treat
Emergence of HBV Variants During a Chronic
Infection
Clinical Assessment of HBV Infections
Laboratory Diagnosis of Infection
Serology of HBV Infections
HBV Genotype and Infection Outcomes
Occult HBV Infections
Diagnosis of Acute Hepatitis B
Diagnosis of Chronic Hepatitis B
Prevention and Control
Antiviral Therapy
Vaccines
Current Vaccines
Animal Models of HBV Infections
Chimpanzee
Woodchuck and Ground Squirrel
Ducks
Transgenic Mice
Chimeric Mice
Hydrodynamic Infection
Tupaia
Hepatocellular Carcinoma
Epidemiology
Viral Factors in HCC
Cellular Factors in HCC
Perspective
Acknowledgments
History
Highly transmissible liver disease has been known for several
thousand years. A major cause is hepatitis A virus (HAV), a
picornavirus that infects the liver and is shed in feces. Evidence for a distinct form of hepatitis, transmitted from blood
and body fluids, began appearing in the nineteenth and early
twentieth centuries. This second form was finally accepted
following outbreaks of hepatitis after vaccination for measles,
mumps, and yellow fever in the 1930s and 1940s. These vaccines all contained convalescent serum or plasma, or human
serum added as a “stabilizer,” which inadvertently contained an
infectious agent. Plasma, blood transfusions, and repeated use
of nonsterile needles were also identified as causes of hepatitis
outbreaks, and the disease was shown to have a viral etiology
(reviewed in 28,268,815). Originally identified as hepatitis B
or serum hepatitis, distinct from the disease caused by hepatitis
A virus, this newly recognized entity was later discovered to
be two separate diseases. Once tests were available for hepatitis B virus (HBV), a unique DNA virus discovered during the
1960s, it became clear that there was a second form of serum
hepatitis, thereafter called nonA nonB hepatitis. A virus with
structural similarities to flaviviruses was identified in the late
1980s as the major cause of nonA nonB hepatitis, and named
hepatitis C virus (HCV).11,60,122,123,128
The discovery of HBV came by an indirect route. To
identify and track genetic differences in human populations, Blumberg and colleagues were using sera from multiply transfused individuals as sources of antibody to human
serum proteins. The idea was that these sera would contain
antibodies that bound to proteins differing in sequence from
those of the transfusion recipients. During the course of
these studies a new antigen, named “Australia antigen,” was
identified in serum from an Australian Aborigine.47 Because
this antigen was found to be common in leukemia patients
and in Down syndrome patients, who have a high risk of
leukemia, it was hypothesized that the antigen predicted
leukemia risk. However, a Down syndrome patient initially
negative for Australia antigen was observed to seroconvert,
and seroconversion was correlated with a mild case of hepatitis. At about the same time, a member of Blumberg’s laboratory experienced a mild case of hepatitis following contact
with contaminated material, again with the appearance of
Australia antigen in the blood.48,50
The Australia antigen was quickly associated with serum
hepatitis in a wider group, including a significant fraction of
post-transfusion hepatitis cases.510 At the time, post-transfusion
hepatitis occurred in at least 10% to 30% of multiply transfused individuals.9,10,339,340,346,558,631 Screening blood banks for
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contaminated blood (Australia antigen-positive) resulted in
an approximately twofold decline in the incidence of posttransfusion hepatitis. The remaining cases were mostly due to
HCV. For this discovery Blumberg received the Nobel Prize in
Medicine in 1976.46
The ability to carry out retrospective studies with assays
for Australia antigen confirmed a long-held suspicion that
HBV was responsible for a chronic hepatitis leading to cirrhosis and liver cancer in many parts of the world.49,215,428,479,
484,560,638,644,673,696,710,738,741,742
The Australia antigen, purified
from the serum of infected individuals, also proved to be an
effective vaccine, with greater than 90% efficacy in inducing an antibody response in adults. However, universal vaccination still remains a goal rather than accomplished fact.
The World Health Organization (WHO) estimates there are
now 400,000,000 individuals worldwide who are chronically
infected with HBV, 25% of whom will die of chronic liver
disease or hepatocellular carcinoma.
Electron microscopic (EM) studies revealed that Australia
antigen is carried by spherical particles, with a diameter of
∼22 nm, and to a lesser extent, by ∼22-nm, rod-like particles
(Fig. 68.1). Sera contain a much smaller amount of spherical
virus particles with a diameter of approximately 42 nm, termed
the Dane particle.149 Australia antigen is a component not just
of the 22-nm particles but also of the virus envelope.258 Treatment with nonionic detergent releases a spherical particle from
virus, the viral capsid, with a diameter of approximately 27
nm. Robinson and colleagues showed that the capsids contain
a circular viral DNA of about 3000 base pairs (bp), as well
as an endogenous DNA polymerase activity that synthesizes
virus DNA when virions are treated with nonionic detergent
and incubated in the presence of dNTPs.314,585–587 Summers
showed that the circular conformation is maintained by a short
cohesive overlap between the 5′ ends of the two DNA strands
and that the circle is only partially double stranded, one strand
being incomplete. This incomplete strand is extended and the
single-strand gap is at least partially filled in by the endogenous
DNA polymerase.674
The endogenous DNA polymerase activity facilitated
the discovery of several HBV-like viruses (Fig. 68.2) including woodchuck hepatitis virus (WHV) in eastern woodchucks (Marmota monax),646,677,678,695,727 duck hepatitis B
virus (DHBV) in domestic ducks in China (Summers, personal communication; 749, 807) and the United States,445
and ground squirrel hepatitis virus (GSHV) in Beechey
A
B
C
D
Figure 68.1. Cryo-electron microscopy of viral particles from a
chronically infected patient. A: 42-nm Dane particles, and 22-nm filamentous and spherical subviral particles are seen. B and C: Particles with
compact and gapped morphology, respectively. D: Particles with mixed
morphology. Gapped areas are delineated in white. (Adapted from Chang
MH, You SL, Chen CJ, et al. Decreased incidence of hepatocellular carcinoma in hepatitis B vaccinees: a 20-year follow-up study. J Natl Cancer
Inst 2009;101:1348–1355, with permission.)
LWBK1180-Ch68_p2185-2221.indd 2186
Figure 68.2. Detection of hepatitis-B like viruses using an endogenous DNA polymerase assay. Serum samples from a woodchuck
and duck were centrifuged to pellet virus. The pellet was suspended in
a DNA polymerase reaction cocktail containing radiolabeled nucleotides
and incubated at 37°C. SDS-pronase was then added, and after digestion at 37°C to free DNA from protein, the products were subjected to
gel electrophoresis in 1.5% agarose. Radiolabeled DNA was detected by
autoradiography. The marker is bacteriophage lambda DNA digested with
the restriction endonuclease Hind III. Woodchuck hepatitis virus (WHV)
DNA migrates faster than duck hepatitis B virus (DHBV) DNA because the
incomplete strand of WHV was only partially filled in by the endogenous
DNA polymerase reaction.
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3. CHAPTER 68
ground squirrels (Spermophilus beecheyi).435 Hepatitis B-like
viruses closely related to the original isolates were later identified in Richardson’s (Spermophilus richardsonii)467,694 and
arctic ground squirrels (Spermophilus parryi kennicotti),699
ducks,236,430,728 wild mallards,135 geese,98,236 cranes,557 storks,565
and herons,651 but the three original nonprimate animal
models—particularly the woodchuck and the domestic
duck—have been the mainstay of hepatitis B research for the
past 30 years.
HBV itself is found in all apes, including chimpanzees,
gorillas, orangutans, and gibbons.280,358,422,500,584,601,658,685,707,
740,753,816
These isolates are closely related in sequence to
human HBV and human isolates were shown to infect chimpanzees and gibbon apes.20–22,39,40,157,270,615 At present, these
primate isolates are considered subtypes of HBV rather than
distinct species. Only the chimpanzee has seen significant
use as an experimental model, though for ethical reasons as
well as cost its use has been limited. A primate virus closely
related to HBV has also been isolated in the New World from
the woolly monkey.357,356 This virus, woolly monkey hepatitis
B virus (WMHV), differs in host range from HBV and has
been designated the prototype for a new species of hepatitis
B-like virus.182
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2187
Classification of Viruses within
the Hepadnavirus Family
All of these hepatitis viruses share remarkable similarities in
genome organization and replication strategy and, with the
Spumaviridae (foamy viruses) (see Chapter 53), are the only [AU2]
DNA viruses of animals known to replicate their DNA by
reverse transcription of a viral RNA. Collectively, the hepatitis B-like viruses are assigned to the family Hepadnaviridae
(hepatitis DNA virus), for which (human) HBV is the prototype. This family contains two genera, the orthohepadnaviruses, infecting mammals, and the avihepadnaviruses,
infecting birds.182 A maximum sequence divergence of about
40% is found among the orthohepadnaviruses,207,618 compared to about 20% among avihepadnaviruses.236 Designation
of the Hepadnaviridae as a new family of viruses is based on
the extremely small size of the viral genomes (3–3.3 kbp), the
novel arrangement of open reading frames, and the unique replication strategy, differing almost completely from other viruses
replicating by reverse transcription. Assignment to two genera
is based on the strong DNA sequence similarities among all
orthohepadnaviruses, and all avihepadnaviruses, but an almost
complete lack of homology between the two groups (Fig. 68.3).
Figure 68.3. Phylogenic tree of avi- (top) and
ortho- (bottom) hepadnaviruses. A dendrogram file
constructed using ClustalX was displayed by Treeview.
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Assignment to separate species within the two genera has been
based primarily on differences in viral host range, which has
also been associated with differences in sequence. Two species
have been assigned in the avihepadnavirus group, DHBV and
heron hepatitis B virus (HHBV). Most newer isolates are as yet
unassigned.182,236 Where sequence and host range data are available, orthohepadnavirus species have also been assigned. These
species include HBV, WHV, GSHV, and WMHV.
With the availability of polymerase chain reaction (PCR)based assays, numerous studies have been performed to gain
information on the number and geographic distribution of
HBV genotypes and naturally occurring HBV mutants infecting humans. Eight HBV genotypes, A to H, have been identified,15,206,499 with isolates belonging to different genotypes
showing pairwise differences greater than 8% and less than
17%. A ninth genotype, I, has been proposed but remains
controversial as the divergence is about or slightly less than
8%, with a close relationship of half the I genome to genotype
C HBV.515,793 An isolate possibly defining a tenth genotype, J,
has also been described.689 Distinct genotypes have also been
found in great apes. Different genotypes tend to have distinct
geographic distributions and possibly distinct clinical manifestations (Fig. 68.3).
Virion Structure
HBV is a spherical virus with an outer diameter of approximately
42 nm (Fig. 68.1). The inner shell of the virus has a diameter of
∼22 nm and is made up of 120 dimers of the core protein. The
dimers form the icosahedral capsid with a triangulation number
T = 4. A small fraction of capsids consists of only 90 dimers
with a triangulation number T = 3.55,132,140,167,772 It is not known
whether virions with the smaller capsids are infectious or represent
Figure 68.4. Model of HBV virions. A: HBV
virion with a T = 4 icosahedral capsid (blue)
with 120 spikes and an outer envelope with
protein projections. B: X-ray crystal structure
of a capsid docked into the cryo-electron
microscopy density map of the virion capsid
(left). S, M, and L refer to the three envelope
proteins described in the text. Amino acids
around the base of the spikes in core proteins, which are important for envelopment of
core particles, are shown in green.370,522 (From
Dryden KA, Wieland SF, Whitten-Bauer C,
et al. Native hepatitis B virions and capsids
visualized by electron cryomicroscopy. Mol
Cell 2006;22:843–850, with permission.)
LWBK1180-Ch68_p2185-2221.indd 2188
dead-end products caused by an aberrant assembly process. The
capsid is covered with a lipoprotein membrane made up of three
forms of the viral envelope protein, large (L), middle (M) and
small (S) (Fig. 68.4), acquired together with host lipids during
budding into multivesicular bodies (Fig. 68.5). The L, M, and
S proteins are present in the virus envelope at a ratio of about
1:1:4.258 A model based on the analyses of virions and capsids
by electron cryomicroscopy (cryoEM) predicts that T = 4 capsids carry 180 dimers formed by envelope proteins.167,621 The
proportions of homo- and heterodimers is unknown. Notably,
virions from patient sera exhibit morphologic variation: they
appear either as compact or as gapped particles, which differ in
the distance between the capsid and membrane621 (Fig. 68.1).
Capsids contain a single copy of the partially double-stranded
DNA (dsDNA) genome, which is covalently linked to the viral
reverse transcriptase (RT) at the 5′ end of the complete strand
(Fig. 68.6). RT provides the endogenous DNA polymerase
activity, discussed earlier314,674 (Figs. 68.2 and 68.6). There is
also evidence for the presence of cellular proteins including one
or more serine kinases within the virus.7 The virus has a buoyant
density of 1.24 to 1.26 g cm−3 in CsCl and an s20,w of 280S. The
titers of HBV can vary significantly among patients, ranging up
to 1010 per ml in blood.
As noted earlier, HBV infections also lead to the production of noninfectious subviral particles. The 22-nm spheres
can reach titers as high as 1012 per ml and represent the most
abundant particle released into the blood from infected liver
cells. CryoEM studies of isometric particles isolated from sera of
transgenic mice revealed that they have an octahedral symmetry, different from the icosahedral structure of Dane particles.325
These spheres are composed of 48 dimer subunits that assemble into two classes of particles that differ in size, presumably
caused by the heterogeneity of the subunits. Spheres contain M
and S proteins at a ratio of about 1:2 and only trace amounts
A
B
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5. CHAPTER 68
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SVP
Exosome
2-adaptin
DSL
RC
Integration
CCC
Assembly
L,M,S
C/pg
preS
S
Vps4
ESCRT
An
MVB
Nedd4
2-adaptin
C,POL
HSP90/70
Virus assembly
Packaging
DNA synthesis
CCC DNA amplification
[AU7]
Figure 68.5. Model for the life cycle of hepadnaviruses, as described in the text.522 Envelope proteins are shown in green,
DNA-containing capsids in blue, and RNA-containing capsids in red. Early in infection, when envelope protein concentrations are low,
capsids enter the CCC DNA amplification pathway. Envelope proteins enter the endoplasmic reticulum and assemble into subviral
particles (SVP) or transfer to MVBs where virion assembly is believed to occur. Mature virions might exit cells through exosomes (for
details and references see the text).
of L.259 The rod-like particles (tubes, filamentous particles) contain approximately equal amounts of M and L. Their surface
exhibits spike-like features composed of homo- and heterodimers of L, M, and S proteins which, like virus, are in the ratio
1:1:4, with a diameter of about 22 nm.635 However, in contrast
to octahedral isometric particles, tubes isolated from patient
sera do not have an ordered structure.635 Subviral particles contain 40% lipid and sugar by mass and have a buoyant density
of 1.18 g cm−3 in CsCl. Their exact role in the HBV life cycle is
not known. One possibility is that, by adsorbing virus-neutralizing antibodies, they facilitate virus spread and maintenance
in the host.
Genome Structure and Organization
The structure of the HBV genome and organization of open
reading frames on viral DNA is shown in Figure 68.6. All of
the ORFs are in the same direction (clockwise in this illustration), defining minus and plus strands of viral DNA.
Within virions, minus-strand DNA is complete and spans the
entire genome, in contrast to plus strands, which extend to
LWBK1180-Ch68_p2185-2221.indd 2189
about two-thirds of the genome length and have variable 3′
ends.420,674 In this regard, avihepadnaviruses differ from orthohepadnaviruses because they normally extend plus strands
almost all the way to the location of the modified 5′ end.400
The primers of both plus- and minus-strand DNA synthesis
remain attached throughout virus maturation. Minus strands
are covalently linked to the viral reverse transcriptase through
a phosphotyrosine bond. Plus strands contain a short RNA
oligomer derived from the 5′ end of pregenome (pg) RNA,
the template for minus-strand DNA synthesis. Minus strands
exhibit a small 8- to 9-nucleotide-long terminal redundancy,
termed r, which is required for the formation of relaxed circular
(RC) DNA during plus-strand DNA synthesis.400,617,764 A small
fraction (5%–20%) of virus contains double-stranded linear
(DSL) DNA in lieu of RC DNA, a consequence of in situ
priming of plus-strand DNA synthesis.657 Virions with DSL
DNA are infectious, but can lose important sequences from
their ends during initiation of infection and appear, therefore,
to play only a minor role in hepadnavirus replication.782,784
The genetic organization of HBV is complex. The genome
contains four promoters, two enhancer elements, and a single
polyadenylation signal to regulate transcription of viral RNAs.
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A
B
Figure 68.6. Genome structure and organization. The relaxed-circular DNA genome of HBV with a complete minus strand and
incomplete plus strand is shown in A (inner circle), along with the major mRNAs, all of which end at a common polyadenylation signal
located in the core open reading frame. All open reading frames have a clockwise direction. The single-stranded gap in the plus strand
is filled in by the viral RT, which is covalently attached to the 5′ end of minus-strand DNA. The proteins produced from each open
reading frame are illustrated in B, using pgRNA, a terminally redundant mRNA that is reverse transcribed to produce viral DNA, as a
map reference. Map coordinates are from the sequence reported by Valenzuela et al.735 (accession number X02763), with numbering
from a unique EcoR1 restriction endonuclease site. R, large terminal redundancy; pgRNA, pregenomic RNA; DR, direct repeat; EN,
enhancer, PRE, post-transcriptional regulatory element.
In addition, there are four open reading frames and several
cis-acting signals required for viral DNA replication (Fig. 68.6).
All viral transcripts are encoded by the minus strand, and are
capped and polyadenylated. Transcription regulatory regions
are present within open reading frames and are active following
the transport of the genome into the cell nucleus, where it is
converted into a covalently closed circular DNA form, called
CCC DNA.
The major transcripts that are detected by Northern blot
analyses of HBV-infected livers are 3.5 kb, 2.4 kb and 2.1 kb
[AU3] in length and termed pre-C/C, pre-S and S messenger RNAs
(mRNAs), respectively.90,175 In addition, a minor transcript,
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X mRNA, about 0.7 kb, has occasionally been detected in
infected tissues and more consistently in cells transfected with
subgenomic HBV DNA.221,599,717 The existence of an X mRNA
would provide the most plausible mechanism for the translation
of this gene, although internal initiation of translation from
pre-C/C RNAs, pre-S or S mRNAs cannot be excluded as
alternative mechanisms. Avihepadnaviruses express three major
transcripts analogous in length to the three major mRNAs of
mammalian hepadnaviruses.78 All hepadnavirus transcripts share
a common 3′ end created by a polyadenylation signal located
in the core gene. Fine mapping of the 3.5-kb preC/C mRNAs
revealed three different 5′ ends bracketing the initiation codon
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7. CHAPTER 68
of the pre-C gene, indicating that translation of the overlapping pre-C and core genes occurs from separate transcripts
(Fig. 68.6).90,175 The two longer RNAs, beginning upstream of
the pre-C initiation codon, are referred to as pre-core (pre-C)
mRNAs and the shorter, beginning downstream, is pregenome
RNA. PgRNA is the template for the translation of the core
and RT proteins and, as the name indicates, is also the template for viral DNA synthesis via reverse transcription. In contrast, the function of the pre-C mRNAs appears to be limited
to the translation of the pre-C gene. As with the large, terminally redundant mRNAs, S mRNAs also have heterogeneous 5′
ends flanking the initiation codon of pre-S2 and, hence permitting the translation of either the M or S protein.89,656 The third
major transcript, pre-S, has a unique 5′ end and supports the
translation of L. The 5′ end of X mRNA is heterogeneous.775 In
addition to the four promoters, two enhancers, EN1 and EN2,
regulate transcription of the viral RNAs (Fig. 68.6).
The core protein is a cytoplasmic, basic phosphoprotein
with a molecular weight (MW) of 21 kd that assembles into
subviral capsids. Early on, its antigenicity was recognized and
diagnostic assays to monitor ongoing or resolved infections were
developed. The pre-C protein is best known by its serologic
name: e-antigen, or HBeAg. Although the pre-C gene includes
the entire core protein open reading frame and upstream coding
sequences, the polypeptide is shorter than the core protein due
to posttranslational processing, and has a MW of only 15 kd.
The mature pre-C protein exhibits distinct antigenic properties
from the core protein. Pre-C does not play a role in viral replication, but might exert a role in the regulation of the immune
response against HBV, particularly against the core protein.109
HBeAg is an important diagnostic tool that can be used to
determine the status of ongoing HBV infections.
The pol gene encodes the viral DNA polymerase, which is
the sole enzyme encoded by hepadnaviruses. It consists of three
functional domains and a hinge region, known as the “spacer,”
and has an MW of about 90 kd. The N-terminus encodes the
terminal protein (TP) domain, which acts as the primer for
minus-strand DNA synthesis. The C-terminal region encodes
the reverse transcriptase and RNAseH (RT) domains.
The PreS/S genes overlap the hinge region and RT domains
of the pol gene, albeit in a different reading frame. They encode
three integral transmembrane envelope glycoproteins with distinct N-terminal domains. The shortest, the S protein, is the
most abundant envelope protein in virions and in the subviral
spheres and rods. It contains the major antigenic determinants
of Australia antigen, which led to the discovery of HBV and
provided the reagent for the development of diagnostic tools for
the detection of HBV infections and of vaccines against HBV
infections.46 A fraction of S protein is modified by asparagine
(N)-linked oligosaccharides, increasing the MW of the protein
from 24 to 27 kd.437,543 The 31-kd M protein represents a larger
form of HBsAg with a 55 amino acid N-terminal, glycosylated
extension, referred to as PreS2, translated from an in-frame initiation codon. It represents about 10–15% of total envelope
proteins in infected cells. A specific function for this protein
is not yet known, as it is not essential for virion assembly.73
Recent studies suggest that the 55 amino acids that distinguish
M from S may serve primarily a spacer function between S and
the PreS1 domain of L.493
The 42 kd L protein is a myristoylated polypeptide translated from the first initiation codon of the PresS/S open reading
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2191
frame.539 The extra amino acids at the N-terminus, relative to
M, define the PreS1 domain. Although it represents only 1%
to 2% of total surface proteins in infected cells, L is enriched in
virions, where it represents approximately 17% of the envelope
proteins.258 In contrast, subviral particles are primarily composed of M and S proteins. Consistent with its distribution,
L protein provides the primary ligand for the viral receptor.
In contrast to the mammalian hepadnaviruses, avihepadnaviruses encode only two nonglycosylated envelope proteins, corresponding to L and S of HBV. DHBV subviral particles are
pleomorphic, roughly spherical, with diameters ranging from
35 to 60 nm,445 as compared to the ∼40-nm DHBV virion.
The smallest viral gene, found exclusively in mammalian hepadnaviruses, encodes HBx or X with a MW of 17
kd. DHBV has been reported to express an X-like protein,99
but a functional significance for this ORF was not supported
by in vivo studies.455 The X gene overlaps, in a different reading frame, the C-terminal portion of the polymerase and two
transcriptional control elements, EN2 and core promoter. X is
predominantly a soluble cytoplasmic protein with a short halflife in the range of 15 to 20 minutes.147,148,166,260,605 However, it
has also been found associated with the cytoskeleton363 and in
the nucleus.54,166,260 Modifications of the protein (phosphorylation, acetylation) have been observed under selected cell-culture
conditions,378,732 but not yet in infected liver tissue. Except for
the spacer region in the polymerase, X is the least conserved
hepadnavirus protein. While X is required for efficient infection
in vivo,106,804,814 its exact role in the viral life cycle is not known.
Since the report that HBx has transcription factor–like activity,649,726 experiments in tissue culture cells revealed many other
functions of HBx that will be summarized later in this chapter.
Stages of Replication
Infection of Hepatocytes
The mechanisms by which HBV and other hepadnaviruses
infect hepatocytes are still not well understood. Efforts to
investigate this problem have been hampered by the lack of
widely available cell lines that are permissive for infection. As
a consequence, studies have been limited to the use of primary
hepatocyte cultures (PHC) that typically remain susceptible to
infection for only a few days following their preparation from
liver tissue,8,225,503,725 or to a cell line that, under extreme culture conditions, becomes susceptible to HBV.226
Investigations into the identification of envelope components that play a role in infection revealed that the PreS1
domain of L has a critical role. The most compelling results
stem from genetic experiments with chimeric envelope proteins between closely related hepadnaviruses that exhibit different host-range specificity, such as DHBV and its close relative,
heron hepatitis B virus (HHBV), or HBV and WMHBV.124,292
These studies revealed that the specificity of these viruses for
their cognate hepatocytes segregates with the N-terminal half
of the PreS1 domain. Consistent with these observations, infectivity of DHBV and HBV can be neutralized by anti-PreS1
antibodies,114,354 and infection of hepatocytes can be blocked
by peptides homologous to portions of the PreS1 region of the
L protein.224,542,731 A study with hepatitis delta virus (HDV,
Chapter 70), a viroid-like satellite of HBV that requires HBV [AU4]
envelope proteins to infect hepatocytes, provided evidence that
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a second determinant, mapping to the external hydrophilic
loop of the S protein is required for infectivity.298 More recent
work with HBV confirmed this finding. It remains unclear how
this determinant functions during infection of hepatocytes.44,600
Infection of duck hepatocytes by DHBV has been intensely
studied. It is a slow process that takes place over a period of
at least 16 hours.564 Internalization rather than binding to the
receptor appears to be the rate-limiting step because the latter
can rapidly occur at 4°C. An estimated 104 receptors with highaffinity binding sites for DHBV are present on primary cultures
of duck hepatocytes.326,562
Infection by DHBV and HBV is most likely pH independent because it can occur in the presence of lysosomotropic agents or after pretreatment of virus at low pH.246,328,580
Moreover, for DHBV, infection may depend on a conserved
peptide translocation motif (TLM), which is located near the
N-terminus of preS1.662 TLM might become functional following cleavage of envelope proteins on virus particles by one
or several proteases present in endosomes. Curiously, the corresponding TLM motif in HBV is not required for infection,44
suggesting that avian and mammalian hepadnaviruses might
differ in their mechanisms of infection.
Several cellular and serum proteins that bind to HBV,
DHBV, subviral HBsAg particles or recombinant envelope
components have been identified since the mid-1980s as possible virus receptors.77,152,199,261,454,486,487,551,598,691,715,716,769 Many
of these studies were of a descriptive nature and yielded only
preliminary evidence of a role for the respective protein in viral
entry. Exceptions are studies on a receptor candidate for DHBV,
carboxypeptidase D (CPD). CPD was originally identified as
a cell glycoprotein that binds DHBV particles and recombinant L protein.347,348 The DHBV-CPD interaction occurs in a
species-specific manner, requires the PreS1-specific domain of
the envelope protein, and is inhibited by PreS1 specific neutralizing antibodies.347,712 Many of the characteristics of CPD are
consistent with its proposed role as a viral receptor. It is a type I
transmembrane protein that cycles between the trans-Golgi network and the plasma membrane, and provides a high-affinity
binding site for L within one of its three extracellular domains
(domain C).63,650,734 This domain does not exhibit enzymatic
activity. Both soluble CPD and antibodies against domain C
can block infection of primary duck hepatocytes.733,734 Interestingly, CPD is downregulated in DHBV infected hepatocytes, which could contribute to the resistance of hepatocytes
to superinfection.62 Such resistance could also be explained by
other factors, including blocking of receptors by viral envelope
proteins produced in the infected cell.745 However, while the
evidence for a role of CPD in DHBV infections is compelling, proof for its role as a receptor is still lacking. The fact that
CPD is expressed in cells that are nonpermissive for DHBV
infections indicates that additional cell components must participate in the formation of a functional receptor complex and
explains why transfection of cells with recombinant CPD does
not confer susceptibility to DHBV infections, although particle
internalization may occur.711 Glycine decarboxylase has been
shown to bind truncated L protein and might represent a tissuespecific co-factor that plays a role for establishment of DHBV
infections following the binding of virus particles to their cellsurface receptor(s).387,388 More recently, heparan sulfate proteoglycans have been invoked as low-affinity receptors for HBV
that “capture” virus particles to facilitate binding to the high-
LWBK1180-Ch68_p2185-2221.indd 2192
affinity, tissue-specific receptor(s).379 Moreover, evidence has
been obtained for a role of caveolin-1 in entry of HBV in a
tissue culture system.424
After entry and uncoating of virus, capsids must be transported to the cell nucleus.310 Experiments with capsids produced
in hepatoma cells and in Escherichia coli provided evidence for
a model in which core particles migrate along microtubules to
the nuclear periphery. From there, capsids enter the nuclear basket in an importin a/b-dependent process and bind to nucleoporin 153.568,612 The model predicts that capsids disintegrate
within the nuclear pore complex and release RC DNA into the
nucleus,569 where it is converted into CCC DNA, the template
for transcription of the viral RNAs.
Regulation of Transcription and Translation
Under physiologic conditions, CCC DNA is associated with
histones and other proteins to form a mini-chromosome.51,52,488
Orthohepadnaviruses contain four promoters that control the
transcription of the 3.5-kb preC and pgRNAs and the subgenomic 2.4-kb, 2.1-kb, and 0.8-kb RNAs, PreS1, S and X,
respectively (Fig. 68.6). All promoters, except for preS1, lack a
TATA box and hence produce transcripts with heterogeneous 5′
ends, which in the case of the S and preC/C promoters encode
distinct proteins: M and S, and pre-C and core/pol, respectively.
The possibility that the PreC/C promoter actually consists of
two distinct promoter elements has been suggested by genetic
experiments and by naturally occurring mutants that fail to
express HBeAg.76,205,508,603,798
A single polyadenylation signal located in the core gene
regulates the formation of the 3′ ends of all four transcripts.639 In
the case of Pre-C/C RNAs, RNA polymerase II bypasses the poly
A signal once, leading to the formation of terminally redundant
transcripts (Fig. 68.6). Sequences located close to the 5′ end of
the transcript play a role in the suppression of premature polyadenylation during the first passage by RNA polymerase II.597
The two enhancers, EN1, located upstream of the X
region and EN2, overlapping the pre-C/C promoter, regulate
the transcription of the four promoters.625,785 Consistent with
the hepatotropic nature of hepadnaviruses, all transcriptional
regulatory elements of HBV, except for the S promoter, contain
binding sites for liver-enriched transcription factors (Fig. 68.7;
for a more comprehensive description, see 341,604,624). For
instance, the PreS1 promoter contains binding sites for the
liver-enriched factors HNF1 and HNF3.134,240,413,573
EN1, a highly complex enhancer less than 300 nucleotides
in length, harbors binding sites for liver-enriched factors HNF1,
HNF3, and C/EBP.108,514,719 In addition, the pre-C/C promoter
and both enhancers contain binding sites for nuclear receptors
(NRs) including HNF4a, retinoid X receptor alpha (RXRa),
peroxisome proliferator–activated receptor alpha (PPARa), the
chicken ovalbumin upstream promoter transcription factors
(COUP-TF) 1 and 2, and others (reviewed in 341). Note that a
clear separation between the binding sites on the pre-C/C promoter and En2 is not possible because of the overlap between
the two elements. Ectopic expression of RXRa and PPARAa
in NIH3T3 cells can induce the expression of pgRNA and the
accumulation of HBV replication intermediates that otherwise
are not produced in these cells, underscoring the significance
of NRs in the control of viral gene expression.281,688,797 Curiously, substantial differences in the organization of the transcriptional control elements seem to exist among mammalian
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9. CHAPTER 68
A
B
C
D
Figure 68.7. Transcription factor binding sites. The figure shows
the binding sites for transcription factors on the preS1 and preS2 promoters (A, B), enhancer 1 and the X promoter (C) and enhancer 2 and the
pre-core/core promoter (D) (adapted from (341). Transcription start sites
are indicated with arrows. For details see text. (D adapted from Kosovsky
MJ, Qadry I, Siddiqui A. The regulation of hepatitis B virus gene expression: an overview of the cis- and trans-acting components. In: R. Koshy R,
Caselman WH, eds. Hepatitis B virus: molecular mechanism in disease
and novel strategies for antiviral therapy. London: Imperial College Press,
[AU8] 1998.)
hepadnaviruses. For example, WHV does not bear an element
corresponding to HBV enhancer EN1.159
The exact mechanism by which these factors regulate
transcription from CCC DNA is not well understood and
major unresolved questions remain about the regulation of
transcription from CCC DNA under physiologic conditions.
For example, does transcription from all three major promoters occur simultaneously from the same copy of CCC DNA or
does CCC DNA differentiate and support transcription from a
subset of the four promoters? Because hepatocytes contain several copies of CCC DNA, it is possible that each CCC DNA
undergoes a developmental process that leads to the inactivation of all but one promoter. Such a model would predict that
the first CCC DNA molecule, derived from virion DNA, produces pgRNA, sufficient for initiation of viral DNA synthesis, and that CCC DNA derived from the intracellular CCC
DNA amplification process (see Amplification and Stability
of CCC DNA section) supports the transcription of either
LWBK1180-Ch68_p2185-2221.indd 2193
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Hepadnaviruses
2193
pre-C, pg, X, or envelope mRNAs. For instance, COUP-TF1
was shown to suppress transcription of PreC/C transcripts in
cell culture and might play such a role in vivo.797 Also, the viral
core and X proteins could be involved in differentiation of
CCC DNA. DHBV core proteins co-localize with pgRNA in
infected hepatocytes and might play a role in the regulation of
pgRNA synthesis.421,613 Moreover, HBx was shown to bind the
HBV CCC DNA minichromosome and to act as a transactivator of the viral promoters.36
Major mammalian and avian hepadnavirus transcripts
are unspliced. In HBV and WHV, transport of unspliced
viral RNA from the nucleus to the cytoplasm is regulated by
a posttranscriptional cis regulatory element (PRE) on viral
RNAs.164,165,283,288 PRE co-localizes with EN1 and a portion of
the X gene. Avian hepadnaviruses appear to lack PRE. Instead,
they contain positive and negative effectors of transcription
(pet, net) that regulate the synthesis of pgRNA.284 Pet spans
a 60-nucleotide-long sequence near the 5′ end of pgRNA,
whereas net is located downstream of the polyadenylation
signal. Pet prevents net-induced termination of transcription
of nascent pregenomes during the first passage of the RNA
polymerase through the polyadenlylation site. Like PRE, pet
acts in an orientation-specific fashion, but its mode of action
is unknown.
Evidence for the presence of a spliced transcript has been
obtained with DHBV.502 It contains a short sequence from the
5′ end of pgRNA fused to PreS mRNA. The exact role of this
transcript, if any, for viral replication is unclear. The observation of a spliced HBV transcript, this time encoding a fusion
of S and pol, was made in chronically infected patients429,648,698
but, again, its functional significance is unknown.
Translation of HBV proteins is controlled by initiation
codons located closest to the 5′ end of the relevant mRNA.
An exception is the polymerase protein. It is translated from
an internal AUG codon on pgRNA located at the beginning
of the pol gene (Fig. 68.6). Although many other viruses (i.e.,
picornaviruses, hepaciviruses) control internal initiation of
translation with internal ribosome entry sites (IRES), hepadnaviruses do not have an IRES. Moreover, pol is not translated
by a mechanism depending on a plus one frameshift from the
core to the polymerase gene, as it has been described for certain retroviruses,295 since stop codons or frame shift mutations
placed upstream of the initiation site have no effect on translation.95,608 One model, consistent with some experimental data,
predicts that a small fraction of ribosomes recruited at the 5′
end of pgRNA bypass the core AUG codon and scan the transcript until they reach the initiation AUG of the pol ORF.193,401
Another, more recent, model suggests that ribosomes bind at
the 5′ end but are then shunted to the initiation codon for pol
without scanning the intervening codons.623 Whatever the correct mechanism, core and polymerase polypeptides accumulate
at a constant ratio, which is believed to be in the order of 200
to 300 to 1.
Viral Proteins
Core
The core protein of HBV is a 183- to 185-aa-long polypeptide of MW 21 kd with an arginine-rich “protamine” domain
located at its C-terminus (residues 150–183) (Fig. 68.8). Avihepadnaviruses encode core proteins that are ∼80 aa longer than
HBV core, with similar properties except for an approximately
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Figure 68.8. Structures of the HBV capsid. The figure shows the dimeric structure
of core proteins with Cys-61 (green) forming
the disulfide bridge between the monomers
and amino acid sequences of arginine-rich
C-terminal domains of HBV and DHBV. The figure also shows the arrangement of the dimers
in icosahedral capsids. (From Wynne SA,
Crowther RA, Leslie AG. The crystal structure
of the human hepatitis B virus capsid. Mol Cell
1999;3:771–780, with permission.)
SPRRRTPSPRRRRSQSPRRRRSQSREPQC
DHBV
SKSRERRAPTPQRAGSPLPRSSSSHHRSPSPRK
45-aa-long insertion in the central domain of the polypeptide
that forms the “spike” characteristic of viral capsids, and additional amino acids in the arginine-rich carboxy terminus.65 CryoEM and X-ray crystallography helped reveal the structure of
capsids produced in Escherichia coli.55,132,772 The folding of the
core polypeptide chain is primarily a−helical and, unlike other
viral capsid proteins, lacks b-sheets (Fig. 68.8). Two juxtaposed
alpha helices (a3, a4) connected by a loop represent the central domain of the monomeric structure. Dimerization leads to
the formation of a 4-helix bundle that assumes the shape of an
inverted T, where the stem constitutes the dimer interface linked
by a disulfide bridge and forms the spikes on the surface of capsids. The tips of the arms form the contact points, primarily
located in a5, for the polymerization of the dimers into capsids.
During an infection, the majority of capsids are assembled from
120 dimer subunits into a T = 4 structure. Capsids used for
the structural studies were comprised of core proteins lacking
about 30 aa from the C-terminus including serine phosphorylation sites and, as a consequence, exhibited an increased fraction
of particles consisting of 90 dimers with T = 3. A structural
analysis of the peptide that links the shell-forming core domain
with the C-terminal region was consistent with a model predicting that the C-terminal “protamine” domain provides a mobile
platform for viral DNA synthesis inside capsids.757 Nevertheless, a conformational change at the exterior surface of capsids
could still occur as a result of DNA replication in the interior,
and provide a signal for the assembly of cores with envelope
components, a step known to depend on DNA synthesis.534,671
The C-terminal domain (CTD) contains three serine phosphorylation sites that are located at the beginning
of arginine-rich motifs harboring nuclear localization signals.172,311,355,386,392,786 One or several cellular enzymes must
mediate the phosphorylation because none of the viral proteins
exhibit kinase activity. In cellular extracts the SR protein-specific kinases 1 and 2 (SRPK1, SRPK2) associate with cores and
phosphorylate the three serine residues in the SPRRR motif.151
LWBK1180-Ch68_p2185-2221.indd 2194
HBV
In addition, several kinases, including cyclin-dependent kinase
2 (Cdc2), protein kinase C, and a 46-kd serine kinase can
phosphorylate cores in vitro.213,312,319 However, which kinases
play a role in the HBV life cycle is still unknown. Moreover, it
is not certain which kinase represents the protein kinase activity associated with Dane particles, which was first described
more than 30 years ago.7 Experiments with DHBV revealed
that DNA replication is accompanied by the gradual dephosphorylation in the core protein, which might contribute to a
reorganization of the C-terminus.29,535,561,794,795 As with DHBV,
all steps of HBV DNA synthesis may be regulated by serine
phosphorylation in the core protein, indicating that this mechanism is shared by all members of the Hepadnaviridae.355,385,456
Finally, genetic studies demonstrated that the arginine clusters
in the SPRRR motifs play a role in packaging of pregenomic
RNA, and in DNA replication and perhaps in the recruitment of SRPK1 and SRPK2.385 Although the exact role of the
charged arginine residues in DNA replication is not known,
they might play a role in regulating the spatial organization
of pregenomic RNA and minus-strand DNA to facilitate viral
DNA synthesis.370
As mentioned previously, a second product derived from
the core region is pre-core or HBeAg. It is translated from pre-C
mRNA with 5′ ends located a few nucleotides upstream of the first
AUG in the pre-C/core open reading frame (Fig. 68.6). A signal
sequence directs the translation of HBeAg to ER membranes. As
a consequence, the protein enters the secretory pathway, where
it undergoes a second cleavage event that removes about 34 aa
from its C-terminus, before it is secreted from infected cells as
a 15-kd protein.74,210,316,518,520,655,806 Expression of pre-core is not
required for the establishment of productive infections in experimentally infected woodchucks and ducks.92,107 Consistent with
these observations, HBV mutants that are defective for HBeAg
production have been detected in patients with chronic infections.68 One possible role for this protein could be to transiently
suppress the immune response to the virus, thereby increasing
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11. CHAPTER 68
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Hepadnaviruses
2195
Figure 68.9. Proposed membrane topology of HBV envelope proteins. The orientation of the transmembrane domains (TM) is
identical in S and M and the refolded L proteins. The external hydrophilic domain of S between amino acids 99 and 168 is illustrated
at the left, along with the “a” determinant and the glycosylation site (branched structure) at asparagine 146. All proteins share a
hydrophobic C-terminal domain that is embedded in membranes. M bears a second glycosylation site at its N-terminal extension
(thin line). In the primary product of L, TM I is located in the cytosol where the N terminus is attached to membranes by the modification with myristic acid (for details see text). (Adapted from Bruss V. Envelopment of the hepatitis B virus nucleocapsid. Virus Res
2004;106:199–209; and Tagawa M, Omata M, Okuda K. Appearance of viral RNA transcripts in the early stage of duck hepatitis B
virus infection. Virology 1986;152:477–482.)
the frequency of chronic infections, which might explain the
conservation of this gene. It is assumed that this function is no
longer required once a chronic infection is established.108
Envelope
The three envelope proteins, L, M and S, encoded by the mammalian hepadnaviruses have two principal functions: (a) they
provide the protein components of the virus envelope and (b)
they assemble into aggregates that are secreted as subviral particles. L and M differ from S in their N-terminal regions (Figs.
68.6 and 68.9). As a consequence of the common S region,
the three proteins share several features: they contain two topogenic signal—I and I—that determine their orientation in lipid
bilayers, a hydrophobic C-terminal region that is most likely
embedded in ER membranes and a common N-glycosylation
site (Asn-146) in S. Like a conventional signal sequence, signal I
is located at the N-terminus of S, but is not proteolytically processed. Signal II is a hydrophobic domain acting as a stop-transfer
sequence and a signal sequence. As a result, the two hydrophobic domains form a hairpin structure with a cytosolic loop.170,171
The presence of a third hydrophobic domain invokes the theoretical possibility of two additional transmembrane passages. In
addition to the common glycosylation site located downstream
from signal II, the M protein harbors a second N-glycosylation
site (Asn-4) near its N-terminus.258 This site is also present in L,
but not used, because of the cytoplasmic location of the preS2
domain of L. Some HBV genotypes contain O-glycans (Thr37) in the preS2 domain of both L and M proteins.611 L and
M proteins contain modified N-termini. L carries a myristate
group at Gly-2,539 whereas M is N-terminally acetylated.610
The role of M in HBV replication is not yet understood,
because this protein is not required for the production of Dane
particles.185 In avihepadnaviruses, L-proteins are also myristoylated, but not glycosylated.563 Instead they become phosphorylated in the preS1 domain.223
A major feature of L is that it exists in two conformations that differ in the localization of the N-terminal domain
LWBK1180-Ch68_p2185-2221.indd 2195
(Fig. 68.9). In the first, the N-terminus, including signal I, is
located in the cytosol,75,516,556 where it is required for binding
of capsids and for the assembly of virions. In the second, the
N-terminus is present in the ER lumen and, as a consequence,
exposed on the surface of viral particles where it plays a role
in the infection of hepatocytes.556 The conformational change
is facilitated by interactions of L with the molecular chaperones Hsc70/Hsp40 and BiP.353 However, the details of the
mechanism regulating this step are not yet understood. The
major determinants for infectivity of HBV are located in the
N-terminal 75 amino acids of L (44, 371, reviewed in 218).
Evidence for this conclusion is derived from genetic experiments with mutant envelope proteins. In addition, peptides
spanning different regions of the envelope proteins have been
useful in mapping domains critical for infection. Experiments
with peptides demonstrated the importance of N-terminal
myristoylation. Myristoylated peptides are much more potent
inhibitors of HBV infection than peptides with normal Ntermini.23,219,224,542 A second region overlapping with the socalled “a” determinant or antigenic loop located between transmembrane domain II and the hydrophobic C-terminus of S
contains a second infectivity determinant.367,600 However, the
function of this determinant in infection is not yet known.
Following integration into membranes, envelope proteins form intermediates that include homo- and heterodimers stabilized by covalent disulfide bridges between different
cysteine residues in the S domain, and subsequently assemble
into either subviral or Dane particles290,637 (Fig. 68.5). S and
M proteins contain the necessary signals for the export process
because they can be secreted independently. In contrast, when
synthesized in the absence of the other two envelope proteins,
L is retained in membranes, suggesting that it carries a retention signal that prevents, in the absence of S and M, the export
process controlled by its S domain.113,538
In addition to their roles as envelope proteins, L and M
can activate, in trans, the transcription from selected promoters
in transfected cells.88,322 This function was initially described
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12. 2196
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for naturally occurring mutants of these proteins with C terminal truncations. Because truncated forms of L and M were
initially identified in tissues from chronically infected patients,
it has been speculated that they contribute to the development
of hepatocellular carcinoma (HCC). Later work showed that
the complete L protein could also transactivate selected promoters.190,262,263 This may be indirectly related to the fact that
accumulation of L protein in the ER can lead to ER stress and
consequently increase expression of M and S.287,773 This presumably facilitates L secretion and relieves ER stress. Thus, the
transactivating potential of the HBV envelope proteins may
ultimately reflect adaptations to facilitate survival of infected
hepatocytes and might, but only in the long term, lead to
transformation of rare infected hepatocytes.352
Reverse Transcriptase
Hepadnaviral polymerases have an approximate MW of 90 kd
and consist of three functional domains: the terminal protein
(TP) required for the priming of minus-strand DNA synthesis,
and the reverse transcriptase (RT) and RNAseH for DNA synthesis and degradation of pgRNA. A spacer (hinge) separates
the TP from the other two domains (Fig. 68.10). The spacer
region appears to have no other function than to provide a flexible connection between the TP and RT domains.27,94 As will be
discussed later in this chapter, RT is the target for all currently
approved antiviral therapies with the exception of interferon.
A1
Y65
Unlike retroviral RT, hepadnaviral polymerases are strictly
template specific, which is a direct consequence of the mechanism for the activation of the enzyme. It requires binding of the
polypeptide to the packaging signal, termed epsilon, located at
the 5′ end of pgRNA (a second copy of epsilon is located near
the 3′ end, but does not serve as a binding site for Pol; Fig.
68.11B). As is discussed later in this chapter, binding of the
polymerase to epsilon leads to the priming of reverse transcription from a tyrosine residue (Y65) in the TP domain.362,747,758
This results in the formation of a covalent link between the
polymerase and the nascent viral minus strand. However, this
priming activity of Pol also requires cellular factors, explaining
why early attempts to demonstrate polymerase activity with Pol
expressed in bacteria were not successful, although similar strategies yield functional retroviral polymerases.686 Enzymatically
active polymerase was first produced with the DHBV enzyme
translated in rabbit reticulocyte lysates, and led to the discovery that the interaction between the polymerase and epsilon
RNA is dependent on chaperones including heat shock proteins 90 (Hsp90), Hsp70, and p23.276,278,654 Consistent with
these observations, both DHBV and HBV replication are sensitive to geldanamycin and its derivatives, which bind to the Nterminal ATP binding domain of Hsp90.275,278 Similarly, novabiocin, which binds to the C-terminus of Hsp90, inhibits HBV
replication.275 Moreover, Hsp90, p23, and three additional
chaperones—Hsp70, Hsp40, and Hop—can activate DHBV
348:349 F A
179:180
spacer
TP
B C DE
692:693
845
RNaseH
RT
1
345
SNLSWLS…IPMGVGLSPFLLAQFTSAICS…… AFSYMDDVVLG…SLGIHLNPNKTK…LNFMG
75
80
B
173
Drug
180
A
200
184
B
204
230
C
D
236
247
250
E
LAM/Ldt L80I……… V173L……… M204V/I
L180M
A181T/V
ADV
A181T/V…………………….. N236T
ETV
L180M……….M204V/I
T184*……… .S202C/G/I…………….. M250I/V
I169T
*S/A/I/L/G/C/M
Figure 68.10. Physical organization of the HBV polymerase and resistance to nucleoside analogs. A: Domains of the pol
protein. The polymerase contains three main functional domains, the terminal protein domain (TP), the reverse transcriptase (RT)
domain, and the RNaseH domain (A). Functional domains within the RT have been assigned based on structural modeling using the
crystal structure of the HIV RT as a guide.150a By this analogy, domains A, C and D would appear to be involved in deoxynucleotide
binding and polymerization. The B domain is thought to participate in template binding and the E domain in binding of the primer
strand. B: Resistance mutations. The figure shows the location of amino acid mutations that confer resistance to lamivudine (LAM)
and telbivudine (LdT), adefovir (ADV) and entecavir (ETC). (Adapted from Sall AA, Starkman S, Reynes JM, et al. Frequent infection of
Hylobates pileatus (pileated gibbon) with species-associated variants of hepatitis B virus in Cambodia. J Gen Virol 2005;86:333–337.)
LWBK1180-Ch68_p2185-2221.indd 2196
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13. CHAPTER 68
B
1
2
Φ
cap DR1
DR2 DR1
3
cap DR1
cap DR1
DR2
r3
RT
Φ
G
T
A
dNTP A
4
5
cap
DR1
r3
DR2 r5
cap
DR1
DR2
and HBV polymerases to bind to epsilon RNA in vitro and, in
the case of the DHBV polymerase, restore the protein-priming
activity.275,277 With HBV, the protein-priming activity has been
demonstrated in insect cells,361 but so far not in bacteria or the
reticulocyte lysate system. With DHBV enzymatically active
polymerase can also be expressed in yeast.690
The function of the chaperones is not completely understood, because structural information about the polymerases
of hepadnaviruses is lacking. It is possible that they stabilize
an energetically unfavorable conformation of the polymerase
and, in this way, facilitate the binding of polymerase with epsilon RNA84,653 (Fig. 68.11A). Consistent with such a model is
the observation that DHBV polymerases with deletions of the
RNAseH domain can exhibit protein-priming activities in the
absence of cellular factors.750 Thus, these domains might hold
the polymerase in a “closed” conformation that, in the absence
of chaperones, prevents interaction with the packaging signal.
It is likely that a single polymerase polypeptide catalyzes
one complete round of DNA replication because assembly of the
polymerase into capsids depends on its interaction with epsilon
sequences on pgRNA, which would indicate that polymerase
and RNA templates are present at equimolar amounts in subviral particles.25,803 Consistent with such a model, experiments
meant to quantify polymerase levels in HBV capsids revealed a
molar ratio of ∼0.7 polymerase molecules per virion DNA.25,26
HBx
X is an enigmatic protein of the orthohepadnaviruses that is
required for efficient infection and replication in vivo.106,415,804,814
LWBK1180-Ch68_p2185-2221.indd 2197
2197
pA
pA
cap DR1
primer translocation
hsp70/90
Hepadnaviruses
primer translocation
A
TP
|
r5
Figure 68.11. Genome replication. A: Binding
of epsilon RNA to the TP and RT domains of the
polymerase facilitated by chaperones (hsp70/90)
and initiation of reverse transcription at the bulge
of epsilon RNA as described in the text. Phi (F)
depicts the RNA sequence on epsilon proposed to
be required for circularization of pgRNA (see B).
B: The figure depicts five important steps in viral
DNA synthesis: 1) Transfer of the DNA primer
from epsilon to DR1 near the 3′ end of pgRNA;
2) Elongation of minus-strand DNA and digestion of pgRNA by RNaseH of the polymerase; 3)
Transfer of the capped RNA primer from DR1 to
DR2 and synthesis of plus-strand DNA to the 5′
end of minus-strand DNA; 4) Template switch of
the nascent plus strand with the help of the small
terminal redundancies r5 and r3, resulting in circularization of the genome; 5) Completion of plusstrand DNA as described in the text. The figure
was not drawn to scale. For steps 1 through 3,
pgRNA and minus-strand DNA are depicted in a
linear confirmation.
Expression of this polypeptide has been assessed in the livers and
primary hepatocyte cultures from WHV-infected woodchucks
where it can accumulate to 40,000 to 80,000 copies per cell in
the cytoplasm.148 In contrast, in hepatoma cell lines HBx can
be detected in both the cytoplasm and the nucleus.166 However,
the exact role of X activity has been difficult to elucidate because
the protein interacts with many cellular factors, including the
proteasome, and its activity varies depending on the cell lines
used for a study (for more detailed reviews see 57, 475).
Originally, HBx was identified as a relatively weak transactivator for promoters with NF-kB, AP-1, AP-2, c/EBP, ATF/
CREB, or NFAT binding sites in tissue culture cells. Because
HBx does not bind directly to DNA, it regulates transcription
either directly by binding to transcription factors and chromatin
(e.g., on CCC DNA), or indirectly through activation of signal
transduction pathways in the cytoplasm (Fig. 68.12).36,57,594,649,726
For example, it has been reported that HBx enhances the binding affinity of CREB, a member of the basic leucine zipper
family, for the CREB/ATF2 binding site in HBV enhancer I
(Fig. 68.7).427,765 Other reports provided evidence for binding of HBx to p53,751 the RNA polymerase subunit RPB5681
and the DNA helicase components, ERCC2 and ERCC3 of
TFIIH.751 Whether in vivo HBx is essential for transcription
of viral RNAs or regulation of cellular genes required for the
viral life cycle is difficult to assess. Experiments with hydrodynamically injected mice demonstrated that nuclear (but not
cytoplasmic) HBx acts as a transactivator of the viral promoters
and, by inference, might exert this activity in the natural life
cycle of the virus.320,321
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HBx
Nucleus
Gene expression
DNA repair
ATF-2/CREB
p53
RPB5
TFIIH
TC-NER
GG-NER
Cytoplasm
Signal transduction
Ca2+release
???
Pyk2/FAK
NFκB
Cullin-DDB1
Src-K
Figure 68.12. Model for HBx functions. The figure
depicts three major proposed activities of HBx: regulation
of gene expression, inhibition of DNA repair and activation of several signal transduction pathways described in
the text.
Cell proliferation/apoptosis
Replication
Investigations on the cytoplasmic activity of HBx led to
a model predicting that expression of HBx induces the release
of calcium ions (Ca2+) into the cytosol, either from the ER or
from mitochondria.58 The transient influx of Ca2+ then activates the nonreceptor tyrosine kinases proline-rich tyrosine
kinase 2 (Pyk2) and focal adhesion kinase (FAK), which in
turn activate the Src family kinases (Src-K) (Fig. 68.12). The
latter are known to activate the downstream Ras-Raf-MAP
kinase signal transduction pathways. In HepG2 cells, where
X expression can significantly stimulate viral DNA synthesis,
drugs that chelate intracellular Ca2+ or prevent activation of
Src-K can inhibit HBV replication.56 Conversely, compounds
known to induce the efflux of Ca2+ into the cytosol can increase
the replication levels of HBx negative mutants. Nevertheless,
it needs to be determined whether expression of HBx in natural infections triggers the transient Ca2+ fluxes that have been
observed in transfected hepatoma cell lines, and how this activity is involved in chronic infection of liver cells in vivo. Notably, the effects on viral replication appear to be independent of
the Ras pathway, suggesting that Ras-dependent activation of
transcription is not required. One possibility is that X-activated
pathways play a direct role in viral replication by regulating
the phosphorylation of the viral core protein.456 While a model
invoking a role for HBx in the release of Ca2+ is consistent
with many activities attributed to this polypeptide, including
activation of transcription, cell-cycle control, and apoptosis, it
cannot explain all the features of the protein, most notably, the
reported activation of NF-kB.116,383,416,666
Several groups reported that expression of X proteins
from both HBV and WHV can inhibit nucleotide excision
repair (NER) through binding to DNA damage-binding protein 1 (DDB1),35,301,375,642 though it remains unclear if inhibition is specific to global or transcription-coupled NER, or
to both. More recent studies provided evidence for an HBxDDB1 complex binding to cullin4A-RING (CUL4A) ubiquitin ligase. This association is mediated by DDB1 acting as
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Liver cancer
an adaptor protein.544 Structural data has revealed an a-helical
motif at position 88 to 100 in HBx that binds to the BPABPC domains of DDB1,390 and validated the HBx-CUL4A
interaction.391 Thus, HBx might act as a bridge linking the
CUL4A-DDB1 complex to a substrate that is then ubiquitinated by the CUL4A-RING ubiquitin ligase. The substrate
for the CUL4A-DDB1-HBx complex is not known. Notably,
the V proteins of simian virus 5 and human parainfluenzavirus
type 2 can redirect the CUL4A-DDB1 ligase to STAT proteins, promoting polyubiquitination and degradation of these
transcription factors.160,729 Hence, it is possible that HBx plays
a role in inhibiting innate immune pathways. Results from
recent work indicated that HBx can inhibit dsDNA-induced
activation of the RIG-I pathway.786 Alternatively, the protein
might play a role in regulating a cellular pathway required for
a specific step in viral life cycle, such as the formation of CCC
DNA. In addition to its role in HBV replication, HBx might
also play a role in the development of liver cancer, as discussed
later in this chapter.
Identification of the physiologic role of HBx in viral replication and pathogenesis remains one of the most challenging problems in HBV biology. The lack of mouse models or
hepatocyte cell lines permissive for HBV infections has stymied
efforts to investigate relevant HBx-dependent host–virus interactions that play a role in viral replication during natural infections. Nevertheless, many important properties of HBx have
been uncovered with available experimental systems. It is certain that HBx is required for viral replication in vivo and that
HBx expression may effect signal transduction and other gene
expression pathways that alter the physiology of hepatocytes in
a fashion that promotes viral replication and persistence. The
use of primary hepatocyte cultures in lieu of immortalized or
transformed cell lines might be required to unravel the true
function of HBx. For example, recent studies with primary
cultures provided evidence for a role of HBx in promoting progression of the cell cycle from G0 to G1.386
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15. CHAPTER 68
Replication of Genomic Nucleic Acid
Formation of CCC DNA
The first step in the hepadnavirus replication cycle is the conversion of genomic RC DNA into CCC DNA (Fig. 68.5).
Although the details of the mechanisms responsible for this
step are not understood, a comparison of the two DNA forms
can be used to establish a model for CCC DNA formation. It
predicts that the polymerase and one of the terminally redundant segments on the minus strand, termed r, are removed
prior to the ligation of the two ends. Similarly, a capped RNA
oligomer present at the 5′ end of plus-strand DNA must be
removed and the incomplete plus strands extended before the
ends can be joined. Whether the viral DNA polymerase or a
cellular polymerase elongates the 3′ end of plus strands is not
known. Reverse genetic experiments with DHBV suggested
that an endonuclease removes the 5′ end of minus strand DNA
prior to CCC DNA formation, leading to the formation of a
protein-free RC DNA, which can be detected in tissue culture
cells.209,235,330,647 Most likely, cellular DNA repair enzymes are
responsible for the conversion of RC to CCC DNA. Consistent with this view, CCC DNA formation following infection
of primary hepatocyte cultures with DHBV or WHV is not
blocked by inhibitors of the viral polymerase.198,470 Whatever
the exact mechanism of CCC DNA synthesis, it must be
extremely efficient, because in natural infections virions have a
specific infectivity close to one.16,305
Packaging of pgRNA
pgRNA (C mRNA) has dual functions in viral replication. It acts
both as the mRNA for the translation of the core and polymerase polypeptides and as the template for genome replication. The
transition from the first to the second function is triggered by
the binding of the polymerase to the packaging signal, epsilon,
at the 5′ end of the mRNA (Fig. 68.11A). In turn, this reaction
creates a signal for the assembly of this ribonucleoprotein complex (RNP) into capsids. Polymerases preferentially bind to their
own mRNA, possibly while translation is still ongoing, which
increases the chance for packaging and replication of biologically
intact pgRNA.26,265,285 The interaction between the polymerase
and epsilon RNA requires cellular chaperones, as described earlier. While structurally intact polymerase is required for RNA
packaging, DNA polymerization activity per se is not required
for this process, indicating that the RNP can induce assembly
without a requirement for DNA synthesis.24,94,111,265,596 Nevertheless, as will be discussed, packaging and initiation of reverse
transcription are intimately linked events.
Based on secondary structure predictions, epsilon contains
two inverted repeats that can fold into an RNA hairpin with a
basal and apical stem that are separated by a bulge (Fig. 68.11).
The upper stem is capped by a loop.33,189,279,307 RNA footprint
analysis of free and bound epsilon RNA suggested that binding
of the polymerase could induce a single-stranded conformation
in the upper stem.34 Again, it should be noted that due to a terminal redundancy (R), epsilon RNA is present at both ends of
pgRNA. However, genetic experiments showed that only the 5′
copy provides a binding site for the polymerase and is required
for packaging.307,327,546 Consistent with these results, evidence
has been obtained that the nearby cap structure on pgRNA may
play a role in RNA packaging. These results also evoked the
possibility that the polymerase might interact with translation
factors to induce a transition from translation to replication.300
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Interestingly, epsilon maps upstream of the AUG for core, and
evidence has been obtained that translation of pre-C RNA,
with passage of 80S ribosomes through epsilon, prevents its
packaging.483
In contrast to the mammalian hepadnaviruses, packaging in the avian viruses requires, in addition to epsilon, a second pregenome sequence, termed region II, located about 900
nucleotides downstream of epsilon and spanning approximately
300 nucleotides.83,266,517 The role of this downstream sequence
in RNA packaging is not yet understood.
In addition to the chaperones required for RNA packaging described previously, the human cytidine deaminase
APOBEC3G is incorporated into viral particles through binding to the viral reverse transcriptase.490,491 Ectopic expression of
several members of the APOBEC family of proteins inhibits
HBV replication.592 Notably, unlike the inhibition by APOBEC
of retrovirus and retroposon replication,593 inhibition of HBV
was not dependent on the catalytic activity of the deaminase.
Instead, it appears that the protein inhibits virus replication
during an early step of minus-strand DNA synthesis, perhaps
by binding to viral RNA or the polymerase.490
Minus-strand DNA Synthesis
The first step in minus-strand DNA synthesis is the priming
reaction that leads to the formation of a covalent link between
a tyrosine residue in the TP domain of the polymerase and
dGMP (Fig. 68.11A).359,747,758,813 The template for this proteinpriming reaction is a C residue located in the bulge of epsilon.
Although in vitro priming can occur immediately following the
binding of the RT to epsilon in the absence of core protein, the
exact sequence of events in infected cells is not known. Thus,
priming could occur prior to, during, or after the assembly of
capsids. To complete the priming reaction the polymerase copies the next two or three nucleotides from the bulge of epsilon.
As a consequence of this mechanism, the polymerase remains
covalently linked to the 5′ end of minus-strand DNA during
all subsequent steps of viral DNA synthesis, virus assembly,
and release.214,468,469
To continue DNA synthesis, the 3- to 4-nt DNA oligomer is transferred to the 3′ end of pgRNA, where it anneals
with a complementary sequence motif located in a 10- to
12-nucleotide-long region known as DR1 (Fig. 68.11B, step
1).746 However, the 3- to 4-nucleotide acceptor site by itself is
too short to specify the transfer to DR1. Additional sequences
on pgRNA are necessary to control the translocation of the
DNA primer to DR1. The selection of the natural site is most
likely facilitated by the structural arrangement of pgRNA in
the capsid. The acceptor site and epsilon RNA must be held
in close physical proximity to facilitate the transfer of the 3- to
4-nt oligomer across the pregenome. Indeed, a short cis-acting
element, termed phi, located upstream of the acceptor site at
DR1 is required for accurate minus-strand DNA synthesis
from DR1 in HBV, but not DHBV.2,431,633,687 Phi can basepair with the 5′ region of epsilon RNA, thereby stabilizing the
predicted structural conformation of pgRNA required for the
transfer of the short minus strand. Following the translocation
reaction, minus-strand DNA synthesis continues all the way to
the 5′ end of the RNA template (step 2). During this reaction,
pgRNA is degraded by the RNaseH activity present near the
C-terminus of the polymerase.110,570,671 Due to the location of
DR1 within the terminal redundancy on the pregenome, the
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completed minus-strand DNA bears the short terminal redundancy, r. As described below, r plays a role in the circularization
of the viral genome.400,617,764
Plus-strand DNA Synthesis
Plus-strand DNA is primed by an 18-nucleotide-long, capped
RNA oligomer derived from the 5′ end of pgRNA. The oligomer contains a complete copy of DR1 and represents a product of the RNAseH activity of the polymerase (Fig. 68.11B,
step 3).399,410 As a consequence of this priming mechanism, plusstrand DNA synthesis can only begin after minus-strand DNA
synthesis is complete. To prime plus-strand DNA synthesis, the
RNA oligomer must first translocate to and anneal with DR2,
located near the 5′ end of the minus-strand DNA and identical in sequence to DR1.399,617,657,764 As expected, mutations that
disrupt the homology between DR1 and DR2 block the formation of RC DNA and instead favor an in situ DNA priming
reaction from the nontranslocated primer, leading to the formation of double-stranded linear (dsl) DNA.130,657,783 DSL DNA
is produced even under natural conditions, albeit with a low
frequency of about 5% to 20% of RC DNA (Fig. 68.5).
The mechanism responsible for the transfer of the RNA
primer to DR2 is not completely understood. The most likely
scenario is that the regions encompassing DR1 and DR2 on
minus-strand DNA are juxtaposed to facilitate the transfer of
the primer from DR1 to DR2. Studies with DHBV and HBV
revealed the presence of three sequence motifs on the minus
strands, which have the potential to form short duplexes that
might stabilize a secondary structure required for plus-strand
primer translocation.241,256,384,408,473 Mutations that would be
expected to disrupt the formation of these duplexes inhibited
RC DNA, but not dsl DNA synthesis.384,407,408 In addition,
capsid proteins might impose certain structural constraints on
minus strands and thereby play a role in primer transfer.
Following the priming reaction at DR2, plus-strand DNA
synthesis ensues until it reaches the 5′ end of minus-strand
DNA. At this point, a template switch (i.e., circularization) is
required for the continuation of DNA synthesis (Fig. 68.11B,
step 4). The template switch is facilitated by the aforementioned
terminally redundant sequences, r, in minus-strand DNA. The
structural requirements for this reaction must be complicated,
because the polymerase attached to the 5′ end of the minus
strand accommodates both ends of the minus-strand DNA in
close proximity. In spite of the expected steric constraints, the
polymerase can copy the entire r5 region, including the dGMP
residue that is covalently linked to the RT, and then induce the
necessary template switch to r3.409 As with priming at DR2,
this recombination event also depends on the formation of
small duplexes on distant sites in minus-strand DNA, indicating that the two critical steps in plus-strand DNA synthesis
might be controlled by the same mechanism.255,472
In mammalian hepadnaviruses, plus-strand DNA synthesis
is incomplete and reaches approximately half the genome length
prior to virion formation.585,671 The cause and significance of
the premature termination of plus-strand synthesis remains
obscure. Perhaps the arrest in DNA synthesis is caused by steric
factors imposed by the capsid and by the polymerase itself. For
instance, capsids of HBV assembled from core proteins with
truncated C-termini accumulate virion DNA with a greater
deficiency in plus-strand synthesis, and a shift to in situ priming of plus-strand elongation482; with DHBV, capsids assembled
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with core proteins with truncated C-termini are also defective
in plus-strand elongation.794 Thus, capsid structure can influence elongation of plus-strand DNA. In addition, the coating of
capsids with envelope proteins probably leads to the depletion
of the dNTP pool prior to the completion of DNA synthesis.
This latter possibility is supported by the fact that plus-strand
DNA can be extended in an in vitro reaction in the presence of
precursor dNTPs and nonionic detergents that disrupt the viral
envelope314,671 (Fig. 68.2). In contrast to orthohepadnaviruses,
plus-strand DNA synthesis in wild-type avihepadnaviruses is
virtually complete,400 except that the polymerase does not displace the RNA primer from DR2, so DR2 has to be copied
prior to CCC DNA formation.
Amplification and Stability of CCC DNA
The fate of DNA-containing capsids in the cytoplasm of infected
cells is twofold: The particles either enter the cell nucleus and
release RC DNA or assemble with envelope proteins into virions
and enter the secretory pathway (Fig. 68.5). The first pathway
amplifies the copy number of CCC DNA.724,770 Using DHBV,
CCC DNA amplification in cultures of nondividing hepatocytes was shown to occur early in an infection when the cytoplasmic concentration of viral envelope proteins is still low.675,676
The final, average CCC DNA copy number per nucleus in vivo
is usually between 1 and 50.306,308,466,802 The route and mechanism of transport of capsids to the nucleus are unknown. Transport of newly made capsids from the cytoplasm to the nucleus
does not require envelope proteins because CCC DNA amplification occurs when hepatocytes are infected with viral mutants
that are unable to synthesize these proteins.675,676 Instead, signals
generated on capsids during their maturation might play a role
in retrograde transport.
A critical issue with important implications for antiviral
therapies with inhibitors of viral DNA replication is whether
CCC DNA has a half-life and, therefore, whether ongoing CCC
DNA synthesis is required to maintain a steady state within a
nondividing cell. An early study126 addressed CCC DNA stability with a BUDR pulse/chase protocol using primary, nondividing cultures of hepatocytes derived from ducks infected with
DHBV. CCC DNA labeled in a pulse chase appeared completely stable. However, if BUDR was instead added later and
the fate of unlabeled CCC DNA present prior to BUDR addition was followed, a shorter half-life of 3 to 5 days was observed.
This discrepancy is not yet understood. In another study, CCC
DNA stability in the presence of reverse transcription inhibitors
was analyzed following infection of primary woodchuck hepatocyte cultures with WHV. These experiments suggested a halflife greater than 30 days in nondividing hepatocytes.470,809 CCC
DNA also appeared to survive mitosis in primary woodchuck
hepatocyte cultures that were treated with adefovir dipivoxil to
block viral DNA synthesis.145 A fourth study took a different
approach, examining CCC DNA stability in chicken hepatoma
cells expressing DHBV from an inducible promoter.238 This
study, like the earlier study in duck hepatocytes, reported a short
CCC DNA half-life: ∼2 days. Moreover, it suggested that CCC
DNA could survive cell division and partition to daughter cells
and thus, that in the absence of new viral DNA synthesis, CCC
DNA would be gradually lost from cells through dilution, as
also demonstrated in a chimeric mouse model with implanted
human hepatocytes.419 A fifth study demonstrated a CCC DNA
half-life of ∼14 days in HepG2 cells transduced with HBV using
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17. CHAPTER 68
a baculovirus vector.1 Given the possibility that cell loss might
have contributed to CCC DNA loss in the LMH and HepG2
cell-line experiments, published studies seem to suggest, on balance, that CCC DNA is stable in nondividing cells in culture,
and perhaps in dividing cultures as well.145,238 Some,5,418,809 but
not all759,771 in vivo studies of antiviral therapy with nucleoside
analogs that inhibit viral DNA synthesis suggest that CCC
DNA may be stable in the chronically infected liver and survive
through mitosis. The idea that CCC DNA stability is high is
also supported by studies of competition in the fully infected
liver between strains of DHBV with different replication rates.
Competition between the strains essentially stops in the fully
infected adult liver, where cell turnover is low, again suggesting
that CCC DNA has a lower turnover rate in infected cells.801
Virus Assembly and Release
Assembly of hepadnaviruses is still a poorly understood process. Assembly of Dane particles occurs in at least two distinct
steps: the formation of capsids that contain pgRNA and RT and
the formation of enveloped virus particles that contain the viral
DNA genome. During DNA synthesis, capsids gain the ability to
interact with envelope proteins290,531 (Fig. 68.5). The N-terminal
domain of L plays an important role in this interaction because
M and S proteins alone cannot support translocation of capsids across membranes.381 Recent reports provided evidence for
a mechanism in which DNA containing core particles assemble
with envelope proteins at membranes of multivesicular bodies
(MVPs).323,754 This model is supported by data demonstrating
a requirement for vacuolar protein sorting proteins AIP1 and
VPS4B in assembly and release of virus (Dane) particles.754 In
contrast, formation and secretion of SVPs occur through the
ER-Golgi compartment and do not depend on MVPs. Other
factors reported to interact with capsids and envelope protein
that could play a role in assembly include the ubiquitin ligase
Nedd4,595 gamma2-adaptin, a clathrin adaptor protein,254
and thioredoxin-related transmembrane protein 2,708 a protein involved in clathrin-mediated endocytosis and, hence, the
early endocytic pathway. The observed interaction of capsids
with Nedd4 is particularly interesting because members of the
Nedd4 family of ubiquitin ligases play a role in linking capsid
proteins of certain retroviruses and RNA viruses with components of the ESCRT (endocytic sorting complexes required for
transport) machinery that sorts cargo proteins into the luminal
vesicles of MVPs and facilitates budding.200
Pathogenesis, Pathology, and
Epidemiology
Entry into the Host
HBV is found at high titers, sometimes up to 1010 Dane particles per ml, in the blood of infected individuals. Thus, the main
routes of infection involve exposure to blood or blood-derived
products, such as during childbirth from an HBV-positive
mother, blood transfusion, or other potential sources of percutaneous exposure, including sexual intercourse.377
Perinatal Infections
The greatest sources of infection worldwide are from infected
mothers to newborns, or among very young children. The risk
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2201
of vertical transmission varies depending on geographic regions.
In Asia, the rate of perinatal transmission from infected mothers
is as high as 90%, because many of the pregnant women that
are chronically infected have high titers of circulating HBV. In
infants infected by HBV, the rate of chronicity reaches 90%659
without vaccination. In North America, Western Europe, and
Africa, the risk of vertical transmission from chronically infected
mothers is about 10% with any preventative therapy. This lower
risk is consistent with reports that infected mothers in these
locales usually have a low viral load. These different viral loads
are likely a result of different natural histories of chronic infection in the different locales, with infections at the time of birth
that become chronic, leading to a more persistent high-level
viremia than infections later in life. That is, women infected at
birth would have a higher viremia as they aged and would be
more likely to pass the infections to the neonate than women
infected later during the first few years of life. Thus, the high rate
of chronicity in Africa appears mostly due to horizontal spread
to young children from playmates and adults involved in their
care, rather than directly from infected mothers during birth.
Additional Routes of Transmission: Blood Transfusion,
Intravenous Drug Use, Sexual Transmission and
Nosocomial Infections
The risk of HBV transmission by blood transfusion has decreased
dramatically since the early 1970s because of the exclusion of
paid donors and the introduction of serologic screening of volunteer blood donors for serum HBsAg and anti-HBc immunoglobulins. In the United States, the risk of HBV transmission
via blood products is now one out of 63,000 transfusions,
down from 15% in the 1960s.12,220 The current incidence may
be attributed to the failure to identify infected blood donors
because of the serologic window during the incubation period
following infection, the presence of some rare HBsAg variants
that are not detected by the serologic assay for HBsAg, particularly when concurrent testing for anti-HBc is not performed,
and the problem of so-called occult HBV infections, in which
neither HBsAg or anti-HBc are detected.
In contrast to blood transfusion, percutaneous infection
of young people and adults via intravenous drug use, tattoos,
acupuncture, ear piercing, sharing razors, and other avenues,
remain as major modes of HBV transmission. Sexual transmission still represents 40% of the new cases of acute hepatitis B
in many developed countries,12,220 while the role of intravenous
drug use seems to be decreasing with time, currently representing 6% to 10% of new cases. HBV can also be transmitted by
accidental needle stick in the healthcare setting.12,220 Nosocomial transmission represents approximately 10% of the new
cases of HBV infection, usually as the consequence of invasive treatment or diagnostic procedures. The risk of accidental
transmission by percutaneous route is estimated to be 30%
from highly viremic patients. Transmission from healthcare
worker to patients may also occur.253 Other cases of nosocomial transmission have been reported in hemodialysis centers,
and in the setting of organ transplantation, even from donors
who only have anti-HBc antibodies. When found alone, antiHBc antibodies are usually a marker of a past infection from
which an individual has recovered. HBV infection of the liver
graft recipient, presumably virus reactivation in the donor liver,
is observed in more than 50% of cases when the donor has
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antibodies to HBcAg but no other serologic markers of HBV
infection.153 As will be discussed later, this is consistent with
other studies indicating that residual amounts of HBV remain
for years or decades after clearance of transient infections.
Horizontal transmission can be observed among institutionalized persons via close bodily contact, leading to HBV infection
through minor skin breaks and mucous membranes.737
In brief, high-risk groups for HBV infection include
healthcare workers, especially surgeons and physicians working
in hemodialysis, oncology, or AIDS units; laboratory workers
in contact with blood or human fluids; institutionalized handicapped persons, their attendants, and family; patients requiring
frequent blood product transfusions in countries where blood
screening procedures are inadequate; patients on hemodialysis;
patients with organ transplantation; intravenous drug users;
men who have sex with men; and promiscuous heterosexuals.
The Liver and Its Response to HBV Infection
The main cellular target of HBV is the hepatocyte, which in
humans is the only cell type convincingly shown to replicate
the virus. However, the belief that other cells replicate the virus
in humans has persisted, despite a lack of conclusive evidence.
The liver has a central role in synthesizing plasma proteins,
storing and metabolizing glycogen as a source of energy, removing dead and dying cells from the blood stream, and detoxifying
harmful chemicals, among other things. Structurally, the liver
is comprised of microscopic lobules into which blood enters
from the hepatic artery and portal vein, which are situated in
a region known as the portal triad, and exits via the hepatic
vein. The lobule itself is not an anatomically defined structure
but a region arbitrarily defined by the positioning of the portal
tracts and central vein. The structure of a small portion of a
lobule is illustrated in a two-dimensional view in Figure 68.13.
The parenchymal cell of the lobule, comprising 60% to 70% of
liver cell mass, is the hepatocyte. Other cells include bile ductule
epithelial cells, sinusoidal endothelial cells, hepatic stellate cells
(Ito cells), and Kupffer cells, the resident liver macrophages.
In addition to the portal vein and the hepatic artery, the triad
also contains lymphatics as well as the bile duct, through which
bile, produced by hepatocytes during breakdown of bilirubin,
is exported to the gall bladder and small intestine. In contrast
to blood, which flows away from the portal tracts to the central
vein, lymph507 and bile flow towards the tracts. Lymph flows
through the space of Disse, between hepatocytes and the overlying endothelial cell layer, and bile is excreted into and flows
via channels (canaliculi) formed at the interface of adjacent
hepatocytes. Bile enters the bile ductules through a specialized
structure known as the Canal of Hering. Destruction of large
numbers of hepatocytes during immune clearance of hepadnavirus infections leads in some patients to jaundice (icterus) due
Figure 68.13. Structure of the liver lobule. Two-dimensional representation of a small portion of a liver lobule with various
cell types present (hepatic stellate cells, localized between sinusoidal endothelial cells and hepatocytes, within the space of Disse,
are not shown). Blood enters the lobule from the hepatic artery and portal vein, and flows through the sinusoids bounded by plates
of hepatocytes, exiting at the central vein. Hepatocytes produce bile, which is released into bile canaliculi, small channels formed
where the apical surfaces of hepatocytes make contact, flows to the canals of Hering and then to bile ductules. From there it flows to
larger ducts and exits the liver. The origin of hepatic progenitor cells, which normally only appear during certain conditions of acute
and chronic liver injury, is also illustrated. The exact location of progenitor cells in the healthy liver is uncertain, with different lines
of evidence pointing to either bile duct epithelium or cells in the canals of Hering. In the actual lobule, many plates of hepatocytes
connect the portal triad to the central vein, though only two are shown here.
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19. CHAPTER 68
to a buildup of bilirubin in the blood, producing a yellowing of
the skin, eyes, and mucous membranes.
Two liver cell types, hepatocytes and bile duct epithelial
cells, differentiate from a common precursor during embryonic development.634 In the duck both cell types are targets of
hepadnavirus infection.192,249,373,494 One early study45 suggested
this was also true in humans, though later reports have not yet
confirmed this observation. Other liver cell types do not appear
to be infected.
Hepatocytes are long lived, with a half-life exceeding 6
months under normal conditions, and a correspondingly low
proliferation rate.227,342,423,622,739,766 Though hepatocytes are a
self-renewing population in the normal liver, under conditions
of severe injury or where hepatocyte proliferation is blocked—
for instance by a toxic chemical—facultative progenitor cells,
considered to be located in the Canal of Hering, may give rise
to oval cells that proliferate and ultimately differentiate into
hepatocytes.143,180,183,640,705,706 Progenitor cells are also found
in the bone marrow,489 though their quantitative contribution to hepatocyte replacement and relationship to progenitor
cells attributed to the Canals of Hering is unclear. Hepatocyte
replacement in response to killing of infected hepatocytes by
antiviral cytotoxic T cells (CTL) during acute, transient infections appears to be primarily through division of other hepatocytes.306,308,670 Replacement from progenitor cells, with the
appearance of oval cells in the lobule, is more evident during
late stages of chronic infections, by which time the liver may
be highly damaged,203,272,589 but hepatocyte proliferation also
occurs, and the relative contribution of the two pathways to
hepatocyte replacement during late phases of chronic infections has not been determined.
When the liver is injured through killing of hepatocytes,
hepatic stellate cells, located in the space of Disse, will respond
by producing collagen fibers.448,545 During chronic infections
the persistent injury due to CTL killing of hepatocytes leads
to persistent deposition of collagen, building up fibrous tissue
that can evolve to cirrhosis, a condition that distorts the lobular
structure, disrupts normal blood flow through the liver, and
can lead to death due to liver failure. The progression to cirrhosis may be interrupted, and even reversed, if the infection is
controlled by antiviral therapies.396,524,581,588,788
A number of studies suggest that the liver can regulate or
at least protect itself against the host immune response. First, in
some species including rats, pigs and mice, liver transplantation
between allogeneic animals induces tolerance to grafts of other
tissues from the same donor that would normally be rejected,
suggesting that the liver has immunoregulatory properties,
possibly attributable to hepatic dendritic cells.702,704 Second, a
number of different cell types in the liver appear to have the
ability to present antigen in a suboptimal context, in some cases
leading to immune tolerance or a weak immune response.139,703
Third, the immune response to a number of human viruses
that appear to productively infect only hepatocytes—including
HAV, HBV, HCV, and hepatitis E virus (HEV) in humans—
only becomes robust enough to induce high levels of cell death
and virus clearance 4 to 8 weeks after infection, during which
time the entire hepatocyte population may become infected.
A similar pattern has been seen following DHBV infection of
ducks and WHV infection of woodchucks. These observations
suggest that scanning of hepatocytes by the immune system
is low, and has been attributed to low expression of major
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histocompatibility class I (MHC I) genes and poor access of
circulating lymphocytes to hepatocytes, despite the occurrence
of fenestrations in the liver endothelial cells. The possibility
that liver cells other than hepatocytes may induce at least partial tolerance to viral antigens could also contribute to the prolonged course of transient and chronic infections.
Other Sites of Hepadnavirus Infection
The best evidence for replication in cells other than hepatocytes comes from studies of ducks infected with DHBV.
Replication in the extrahepatic sites has been observed during chronic DHBV infections established in ovo163,251 or following inoculation of young ducklings.192,201,302,304,305,441 CCC
DNA and typical DHBV DNA replication intermediates,
with abundant single-stranded DNA (ssDNA), are found not
only in liver, but also in pancreas and kidney of chronically
infected ducks,201,249,271,304,683 as well as in the yolk sac during
embryonic development.684 Viral DNA also accumulates in the
spleen, due to passive accumulation of virus by follicular dendritic cells304; evidence for DNA replication intermediates683
and CCC DNA235 in the spleen has also been reported, though
DNA replication intermediates were not observed in the latter
study. The site of DNA replication in the kidney appears to be
the proximal tubular epithelium, though infection of glomeruli
is also suggested (Fig. 68.14).201,249,304 Viral DNA replication in
the pancreas appears restricted to a small subset (∼1%) of exocrine cells but a majority of endocrine cells,142,176,201,249,248,304
which in ducks are localized to alpha and beta islets. Infection
of bile duct epithelial cells of the liver also occurs249,494 and
studies with primary cell cultures suggest that these are sites of
DHBV reproduction in vivo.373
Extrahepatic infection has also been studied in chronically
infected woodchucks. Gel electrophoresis and Southern blot
analysis demonstrated typical viral DNA replication intermediates in the liver and, at an approximately 10-fold lower level,
in the spleen. Though typical replicative intermediates were
not demonstrated at other sites, ∼1,000-fold lower amounts of
RNA and total episomal viral DNA than in the liver have been
reported in kidney, pancreas, thymus, bone marrow, testes, and
ovary,336,337,506 suggesting possible infection; in addition, some
cells in these latter tissues appeared to contain viral nucleic
acids by in situ hybridization. It remains unclear, however, if
these observations of low levels of viral nucleic acids, outside the
woodchuck liver, reflect actual infection or passive accumulation. The most convincing evidence of extrahepatic infection in
the woodchuck was obtained from studies with PBLs of WHVinfected woodchucks. Although PBLs did not replicate WHV
in vivo, they produced typical viral DNA replication intermediates and released virus when stimulated with lipopolysaccharide
in vitro, thus appear to be latently infected in vivo.333,335
Early immunohistochemical studies of human tissues
other than the liver suggested that, as in the duck, exocrine
and endocrine cells of the pancreas may be infected.632,789
Evidence for DNA replication intermediates in the spleen
was also described for humans158 and chimpanzees,397 though
we are unaware of any recent follow-up studies. Evidence
for infection,91,267,364,398,527,528,550,590,627 gene transcription, and
viral replication in peripheral blood mononuclear cells has
been reported.19,59,242,497 Data in support of infection in bone
marrow, as evidenced by the presence of HBsAg and 3 kbp
RC DNA, but not DNA replication intermediates, has also
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