MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS, Mar. 2000, p. 51–68 Vol. 64, No.

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1092-2172/00/$04.00⫹0
Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Hepatitis B Virus Biology

CHRISTOPH SEEGER* AND WILLIAM S. MASON
Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111

INTRODUCTION .........................................................................................................................................................51
THE LIVER AS A TARGET FOR HEPADNAVIRUS INFECTION .....................................................................52
GENOME REPLICATION..........................................................................................................................................53
Introduction ...............................................................................................................................................................53
Infection .....................................................................................................................................................................53
Formation of cccDNA...............................................................................................................................................55
Viral Gene Expression..............................................................................................................................................55
Viral Proteins ............................................................................................................................................................56
Assembly and Reverse Transcription.....................................................................................................................57
Amplification of cccDNA and Production of Infectious Virus............................................................................58
TRANSIENT INFECTIONS........................................................................................................................................58
CHRONIC INFECTIONS............................................................................................................................................60
LIVER CANCER...........................................................................................................................................................61
MAJOR UNRESOLVED ISSUES ..............................................................................................................................62
ACKNOWLEDGMENTS .............................................................................................................................................63
REFERENCES ..............................................................................................................................................................63

INTRODUCTION the principal cell type of the liver. Hepatocytes continuously

shed virus into the bloodstream, ensuring that 100% of the
Hepatitis B virus (HBV) causes transient and chronic infec- hepatocyte population is infected. Also, hepatocytes are nor-
tions of the liver. Transient infections may produce serious mally long-lived, with half-lives estimated at 6 to 12 months or
illness, and approximately 0.5% terminate with fatal, fulminant longer. The combination of a long-lived, usually nondividing
hepatitis. Chronic infections may also have serious conse- host cell and a stable virus-host cell interaction virtually en-
quences: nearly 25% terminate in untreatable liver cancer (10). sures the persistence of an infection in the absence of a robust
Worldwide deaths from liver cancer caused by HBV infection host immune response. Indeed, liver disease in transient and
probably exceed one million per year (52, 162). chronic carriers is thought to be largely due to the host im-
An effective vaccine has been available for nearly 20 years, mune response to the infection. This response can induce a
and attempts at universal vaccination are now under way in high level of hepatocyte destruction (154), leading to scarring,
developed countries, including areas of endemic infection such disruption of blood flow, and obstruction of bile drainage with-
as China, which has about 120 million carriers (163). However, out necessarily eliminating the infection. It remains unknown
vaccination is not a treatment for established infections, and its why this response is unable to clear the virus in many who
effectiveness in preventing blood-borne transmission from an become chronic carriers or, for that matter, why elimination
infected mother to her newborn is about 90% (196). Moreover, sometimes occurs spontaneously after many years of virus pro-
the high cost of the vaccination programs has impeded intro- duction. Therefore, a major focus of HBV research is to un-
duction in many poorer countries where HBV infections are derstand the virus-host interactions which determine whether
prevalent, afflicting 5 to 20% of the population. an infection will persist or terminate. The hope is that this
Attempts at treatment of chronic infections have had only knowledge will lead to better treatments and a reduced inci-
limited success. Alpha interferon therapy is effective in induc- dence of hepatocellular cancer (HCC).
ing virus elimination in a subset of HBV carriers, especially An important development in hepatitis B research was the
those with the most active disease, but is ineffective in more discovery that human HBV virus is the prototype for a family
than 50% of carriers (87). The long-term potential of more
of viruses, referred to as Hepadnaviridae. Those most closely
recent approaches to eliminating the virus, involving therapy
related to HBV have been found in woodchucks (202) and
with nucleoside analogs and other inhibitors of virus replica-
ground squirrels (135, 208, 210). These viruses have about 70%
tion, has yet to be determined. More sophisticated approaches
sequence homology to HBV but are not known to infect hu-
to treatment, involving manipulation of the host immune sys-
mans or other primates; in contrast, HBV is infectious for the
tem to induce the set of responses needed for virus clearance,
are still in development. Thus, in the absence of “spontaneous” great apes. Because of similarities in DNA sequence and ge-
virus clearance, chronic and productive infections in many nome organization, the viruses infecting mammals are grouped
patients will persist for life. The number of carriers worldwide in the genus Orthohepadnavirus. More distantly related viruses,
is estimated at 350 million. with somewhat similar genome organization but almost no
One of the reasons for chronic HBV infections is that the sequence homology, are found in ducks and geese (138, 193).
virus causes chronic, noncytocidal infections of hepatocytes, These are grouped in the genus Avihepadnavirus. Duck hepa-
titis B virus (DHBV) has been used primarily as a model
system to characterize how hepadnaviruses replicate, rather
* Corresponding author. Mailing address: Fox Chase Cancer Cen- than as a disease model, since chronic DHBV infection has
ter, 7701 Burholme Ave., Philadelphia, PA 19111. Phone: (215) 728- generally been studied after in ovo transmission. In ovo trans-
4312. Fax: (215) 728-4329. E-mail: c_seeger@fccc.edu. mission, with exposure to DHBV from the beginning of em-

51
52 SEEGER AND MASON MICROBIOL. MOL. BIOL. REV.

FIG. 1. Anatomy of the liver lobule. The major cell types within the liver are illustrated. Blood enters at the portal tracts (encompassing the portal vein, hepatic
artery, and bile ductules), distributes laterally through smaller veins to the sinusoidal spaces, and flows toward the central vein. The sinusoidal spaces are lined with
endothelial cells and fixed macrophages (Kupffer cells). The endothelial cells are fenestrated, allowing free diffusion between the blood and hepatocytes.

bryonic development and infection of the developing liver may originate during embryonic life from a common progeni-
(221), apparently prevents the development of an effective tor (188, 189, 211) and also may be replaced by proliferation
immune response to infected hepatocytes (78). Neither chronic and differentiation of a common progenitor cell in response to
liver disease nor liver cancer is normally detected, although very acute forms of liver injury (211).
antibodies to the viral nucleocapsid may be produced (A. Jil- Because hepatocytes are the major cell type in the liver, it
bert, personal communication). Woodchuck hepatitis virus might be expected that they would also be the major target of
(WHV) infections have been studied intensely as a model for infection by a liver-tropic virus such as HBV. Indeed, this
chronic liver disease and the attendant liver cancer. Both appears to be the case. Hepatocytes are the only confirmed site
WHV and DHBV have been used to develop antiviral agents of replication for all members of this virus family. Bile ductule
and therapies. The HBV transgenic mouse has also been in- epithelial cells may also be a target of infection, as may a subset
troduced for this purpose, as well as for characterizing the of cells in the pancreas, kidneys, and lymphoid system (16, 74,
susceptibility of HBV, not just to alpha interferon but also to 77, 98, 111, 122, 151, 160, 170, 187, 243). However, the evi-
various other cytokines produced during an immune response dence for replication of the orthohepadnaviruses in bile
to an infection. ductules and at extrahepatic sites is in some cases controversial
The purpose of this review is to introduce the reader to or incomplete, and these sites are not usually considered in
various aspects of hepadnavirus replication, pathogenesis, liver discussions of viral reproduction and pathogenesis. This ap-
cancer, and therapy that have been elucidated over the last proach to infections is at least compatible with the notion that
several years. In addition, we attempt to emphasize the major many of the extrahepatic symptoms of infection that are not
problems that need to be addressed in the future to meet the attributed to liver dysfunction are the result of deposition of
ultimate challenge in this field, which is to provide the basic antibody-antigen complexes. Thus, for the purpose of simplic-
knowledge required to eventually cure chronic infections. As a ity, we discuss infection only in the context of hepatic mani-
prelude, we include a brief description of basic liver structure festations. The implications of extrahepatic infections have yet
and function, which we deem essential for an understanding of to be determined.
hepadnavirus infections. The liver itself is usually considered, for convenience, to be
divided into small compartments called lobules. This subdivi-
THE LIVER AS A TARGET FOR sion emphasizes the role of the liver in relation to blood flow.
HEPADNAVIRUS INFECTION While this view of liver anatomy is somewhat arbitrary, it
provides a convenient way of considering liver function, devel-
The liver plays an essential role in energy storage and con- opment, regeneration, and pathogenesis. An essentially one-
version, blood homeostasis, chemical detoxification, and im- dimensional view of a section of a “classical” lobule is illus-
munity to microbial infections. Although the liver is composed trated in Fig. 1. In this view, blood enters the lobule through
of many different types of cells, much of the functional activity portal veins and hepatic arteries and is distributed by smaller
resides in hepatocytes (which constitute 70% of the liver), bile vessels to enter the sinusoidal spaces, created by plates of
ductule epithelium, and Kupffer cells (macrophages) (62). hepatocytes. The plates are generally one hepatocyte thick in
Among these, hepatocytes and bile ductule epithelial cells are mammals and two hepatocytes thick in birds. Blood passes
unique to the liver and are also closely related. In fact, they through these spaces, which are lined by fenestrated endothe-
VOL. 64, 2000 HEPATITIS B VIRUS BIOLOGY 53

lial cells and fixed macrophages (Kupffer cells), where the

various functional interactions take place. The blood is then
collected in central veins and exits the liver. (It should be noted
that because of the rather homogeneous nature of liver anat-
omy, blood from each portal vein and artery will flow to several
surrounding central veins; a more thorough discussion of liver
structure and function is presented in reference 55. Generally,
the distance from a portal vein to a central vein is 20 to 30
hepatocytes. During catabolism of heme produced by red
blood cell breakdown in the liver, bile is formed and secreted
into bile canaliculi, small channels formed at the junctions of
hepatocytes. Bile flows in the opposite direction from blood,
passing through a region known as the canal of Hering to enter
bile ducts. From there, it flows into larger ducts and is even-
tually transported to the gallbladder and intestine.
Although hepatocytes are “terminally” differentiated, they
retain the capacity for extensive proliferation in response to
liver injury. Under normal conditions, hepatocytes may have
lifetimes exceeding 6 to 12 months. However, when required,
virtually the entire population may enter the cell cycle and
divide. Following partial hepatectomy of 70% of the liver,
virtually every hepatocyte passes through the cell cycle at least
once, and the liver cell mass is restored within a few days (67,
132). In an even more extreme example of liver cell prolifer-
ation, Rhim et al. (175) observed, under conditions of very
acute liver injury, the replacement of virtually the entire he-
patocyte population of the mouse by clonogenic outgrowth of
mature hepatocytes. This required an average of at least 12
cycles of cell division.
Hepatocyte replacement may also occur via proliferation of FIG. 2. Hepadnavirus life cycle. The details of the replication cycle are dis-
progenitor cells, a situation that appears in the context of cussed in the text. Briefly, during initiation of infection the viral rcDNA (or linear
DNA) genome, with a protein (the viral reverse transcriptase) attached to the 5⬘
long-term liver injury and/or acute injury (e.g., caused by some end of the minus strand and a short RNA attached to the 5⬘ end of the plus
hepatotoxic drugs) in which hepatocyte proliferation is re- strand, is converted into cccDNA. During this process, both the protein and the
tarded. These progenitors are thought to be a population of RNA are removed. The cccDNA serves as the template for transcription of viral
mRNAs (see Fig. 4). One of these, the pregenome, serves as the mRNA for the
facultative stem cells present in the region of the portal tracts, synthesis of core protein (nucleocapsid subunit) and the viral reverse transcrip-
possibly closely associated with or identical to cells in bile ducts tase. The reverse transcriptase binds to the 5⬘ end of its own mRNA template,
or the canals of Hering (36, 42, 54, 56, 212). When induced to and the complex is then packaged into nucleocapsids, where viral DNA synthesis
occurs (see Fig. 3). Once partially double-stranded DNA has been produced,
proliferate, they may first appear as the so-called oval cells nucleocapsids can undergo a maturation event that facilitates their acquisition of
(176) and may then differentiate to form hepatocytes. an outer envelope via budding into the ER. These nucleocapsids can also migrate
Thus, while hepatocytes may represent a fairly homoge- to the nucleus to increase the copy number of cccDNA. Since cccDNA does not
neous population of quiescent cells at the time of infection, the undergo semiconservative replication, all cccDNA is derived from viral DNA
made in the cytoplasm via the reverse transcription pathway (217). Accumulation
situation can rapidly change as the immune system attacks of viral envelope proteins prevents excessive cccDNA formation, which can be
infected cells. This means that an understanding of transient toxic to hepatocytes (200, 201).
infections and chronic infections requires an understanding of
how the liver regenerates, as well as how components of the
virus replication machinery, including transcriptionally active GENOME REPLICATION
viral DNAs, behave as the infected cells begin to divide. For
example, any explanation of transient infections requires some Introduction
explanation of the fate of infected hepatocytes as the virus is The life cycle of hepadnaviruses is characterized by the syn-
cleared. If the entire hepatocyte population is infected, virus thesis of a ⬃3-kb partially double-stranded, relaxed-circular
clearance requires either a mechanism for eliminating virus DNA (rcDNA) genome by reverse transcription of an RNA
from hepatocytes or complete replacement of infected hepa- intermediate, the pregenome (Fig. 2 and 3). The mechanism of
tocytes by proliferation of hypothetically uninfected popula- RNA-directed DNA synthesis has been well characterized
tion of progenitor cells. While these two alternatives appear through genetic as well as biochemical studies, which have
clear-cut, they are not in practice that easy to distinguish. been described in several reviews (61, 182). In contrast, early
Likewise, understanding of chronic infections and how they events of the viral life cycle, including entry, uncoating, and
may be treated requires some knowledge of liver cell prolifer- delivery of the viral genome into the cell nucleus, are not well
ation during an infection and how cell proliferation affects the understood. This is, in part, due to the absence of cell lines that
virus life cycle. Again, the answers are either unavailable or not are susceptible to hepadnavirus infection.
clear-cut. Nonetheless, as we attempt to stress later in this
review, without answers to these questions, our knowledge of Infection
hepatitis B is at best superficial and the possibility of effectively Since hepatocytes are the primary site of viral DNA repli-
dealing with disease progression in patients is probably limited. cation, it is tempting to conclude that tissue specificity is con-
54 SEEGER AND MASON MICROBIOL. MOL. BIOL. REV.

FIG. 3. Hepadnavirus DNA replication. (A) Virus particles contain predominantly rcDNA with a complete minus strand and a partially synthesized plus strand.
A small amount of linear DNA, formed as a result of aberrant plus-strand priming (in situ priming) is also found in the virus. (B) During initiation of infection, both
forms of virion DNA are converted to a cccDNA; however, conversion of the linear form involves illegitimate recombination, which can lead to subsequent defects in
the ability of the virus to replicate. cccDNA serves as template for the transcription of the pregenome. The reverse transcriptase binds to the epsilon stem-loop structure
near the 5⬘ end of its own mRNA to facilitate packaging into nucleocapsids and initiation of reverse transcription by a protein-priming mechanism, utilizing a tyrosine
located near the amino terminus of the reverse transcriptase itself. (C) Following the synthesis of 4 bases, the polymerase translocates to the 3⬘ end of the RNA
template, where the 4 bases can anneal with complementary sequences. During the elongation of the minus strand to the 5⬘ end of the pregenome, all but the 5⬘ 17
to 18 bases of the pregenome, including the CAP and DR1, are degraded by the viral RNase H. (D) The remaining fragment then serves as the primer for plus-strand
synthesis, usually following its translocation to DR2, with which it can hybridize because of the sequence identity between DR1 and DR2. A third translocation occurs
when the plus strand reaches the 5⬘ end of the minus strand, to circularize the molecule and allow continued plus-strand elongation. This translocation may be facilitated
by the short (⬃9-base) terminal redundancy on the minus strand. The plus strand is not completed prior to virion release; a repair reaction to produce a fully double
stranded DNA occurs during initiation of a subsequent round of infection. (E) A fraction of the virions have linear genomes because priming of plus-strand DNA
synthesis occurs in the absence of primer translocation.

trolled primarily by a hepatocyte-specific receptor. However, whether the detection of viral nucleic acids in lymphocytes
hepatotropism is also manifested on the level of viral gene reflects active viral replication or is the result of phagocytic
expression. Indeed, a variety of studies indicated a selectivity of activity of immune system cells is still a matter of debate. Some
the virus for productive infection of only very specific cells. In recent studies have suggested, for example, that HBV nucleic
vivo, viral DNA replication outside the liver has been most acids found in lymphocyte preparations are due to adsorbed
thoroughly documented in DHBV-infected ducks. It was found virus (108), while others have asserted that the HBV nucleic
in a subset of exocrine cells and in the endocrine islets of the acids detected in these cell preparations include viral RNA
pancreas and in the proximal tubular epithelium of the kidney transcribed in the cells from a covalently closed circular DNA
(75–77, 98, 204). While it has been straightforward to identify (cccDNA) template (197). Indeed, the difficulty in resolving
certain extrahepatic tissues as possible sites of virus replica- this question may arise in large part from the need to use very
tion, it has been much harder to rule out other sites. sensitive techniques such as PCR and reverse transcriptase
For instance, as noted above, periodic reports suggested that PCR to detect any virus signal. A more biological approach
lymphocytes are targets of infection by the hepadnaviruses and suggested that WHV can infect woodchuck lymphocytes in
could provide a second reservoir for virus to persist. However, vivo. This can be deduced from the observation that virus
VOL. 64, 2000 HEPATITIS B VIRUS BIOLOGY 55

replication is induced upon mitogen stimulation of lympho-

cytes cultured from WHV-infected woodchucks (111, 143).

Formation of cccDNA
The first event in virus DNA replication accessible to exper-
imental investigation is the conversion of the viral rcDNA
genome into cccDNA (Fig. 2 and 3). Since cccDNA is the
template for the transcription of viral mRNAs, its formation
indicates a successful initiation of infection. Conversion of
rcDNA to cccDNA in the liver is detected within the first 24 h
following virus inoculation (137, 203). The mechanism of DNA
repair involved in this conversion is not known. The ability of
inhibitors of the viral DNA polymerase to block initiation of
infection by an orthohepadnavirus (2, 146) implies that this
enzyme plays a role. However, we can also anticipate that
general concepts of cellular DNA repair may apply to the
hepadnavirus system in some aspect of the completion of plus-
strand DNA and the ligation of the ends of the two DNA
strands. The removal of the covalently attached reverse tran-
scriptase (Fig. 3) from the 5⬘ end of minus-strand DNA is, in
contrast, a unique reaction and, depending on the mechanism,
has consequences for our understanding of cccDNA forma-
tion. For instance, ligation of the two ends of minus-strand
DNA first requires the removal of the reverse transcriptase FIG. 4. Transcriptional and translational map of HBV. The figure shows the
from the 5⬘ end. Is this step catalyzed by the polymerase itself, physical map of the HBV genome. The inner circle depicts the rcDNA with the
reverse transcriptase attached to the 5⬘ end of the complete minus-strand DNA
and does it require the hydrolysis of the DNA-protein bond, or (solid sphere) and a capped RNA oligomer attached to the 5⬘ end of the
does a cellular endonuclease cleave viral DNA close to the 5⬘ incomplete plus-strand DNA (solid half sphere). The positions of the direct
end? Also, does this step occur before or after the transloca- repeats, DR1 and DR2, as well as the positions of the two enhancers, EN1 and
tion of the DNA across the nuclear membrane? EN2, are indicated. The outer circle depicts the three major viral RNAs, the core
(C) or pgRNA, the pre-S (L) mRNA, and the S mRNA. The common 3⬘ ends of
Recent reports describing the ability of peptides to direct the three mRNAs are indicated by the letters A. Not shown in the figure is the
plasmid DNA into the nucleus via the nuclear import pathway putative X mRNA that spans the X coding region and terminates at the site
raise questions about a similar role for the reverse transcrip- indicated for the other three mRNAs. The four protein-coding regions are shown
tase in nuclear transport of hepadnavirus DNA (249). Some between the inner and outer circles. They include the precore (PC) and core
genes, the polymerase gene, and the X gene. The envelope genes pre-S1 (L),
experimental data reported by Kann et al. seem to support pre-S2 (M), and surface (S) overlap with the polymerase open reading frame.
such a possibility (103), but the evidence has been derived from
an in vitro transport system with a very low efficiency and thus
awaits further confirmation. Interestingly, Kann et al. recently
single template for the synthesis of all these mRNAs is relieved
found that phosphorylated core protein can bind to the nuclear
by template specialization.
core complex in a karyopherin-dependent reaction, suggesting
Control of gene expression has been studied most thor-
that the core could also act as a vector for the nuclear transport
oughly for HBV. Basically, this research has identified ele-
of viral DNA (104). Whatever the mechanism for the nuclear
ments in the viral genome that display promoter and/or en-
transport of virion DNA may be, formation of cccDNA, which
hancer activity in cell culture systems and/or liver extracts.
accumulates only in the nucleus, completes the initiation of
Thus, it is now known that each gene of HBV has one or more
infection. cccDNA then acts as a template for the transcription
promoters regulating its activity and that these promoters are
of all the viral mRNAs.
in turn regulated by one or both of the viral enhancer elements,
En1 and En2, that are located upstream of the core promoter
Viral Gene Expression (Fig. 4). Details about transcription factors capable of operat-
The viral RNAs include pregenomic RNA (pgRNA), which ing through the two enhancers and the four promoters of HBV
serves as the template for reverse transcription, as well as three have been obtained primarily by studying expression in liver-
subgenomic mRNAs (two for the avihepadnaviruses) neces- derived cell lines, which mimic some but certainly not all of the
sary for the translation of the envelope proteins and the transcriptional regulation of mature hepatocytes. In brief, the
mRNA for the X protein. X is encoded by all known mamma- results confirm the idea that HBV transcription is dependent
lian hepadnaviruses (Fig. 4) but not by the avihepadnaviruses. to a large extent upon liver-enriched transcription factors.
Because the cccDNA template is circular and because all these These results have been reviewed in detail elsewhere (178,
RNAs have a common polyadenylation signal, use of a single 184) and will not be further considered here. Preliminary stud-
template with multiple promoters raises obvious but unre- ies with DHBV (40, 126) and WHV (48, 232) have also sug-
solved questions about how potential problems, such as pro- gested the importance of liver-specific transcription factors,
moter occlusion, are avoided. For instance, transcription of although very little else is known about the specific factor
pregenomic RNA can suppress transcription of the down- interactions that take place in the hosts of these two viruses.
stream envelope mRNAs of DHBV (95). However, as dis- A problem of particular importance with a circular DNA
cussed below, by the time virus production begins, infected template is termination of transcription, especially for the pre-
cells contain multiple copies of cccDNA in the nucleus, and genome and precore mRNAs. For these, transcription must
these, at least for DHBV, exist as a heterogeneous population pass through the polyadenylation signal on the first pass in
of small chromosomes (149). It is thus formally possible that order to make a full-size, terminally redundant RNA. Studies
part of the potential structural impediments posed by using a with DHBV indicated that this is facilitated by a sequence on
56 SEEGER AND MASON MICROBIOL. MOL. BIOL. REV.

cccDNA, named PET (positive effector of transcription), lo- response to the infected hepatocytes (144, 145). This idea has
cated between the transcription initiation site and the poly(A) not yet been tested in a natural host for a hepadnavirus.
signal (Fig. 4) (95). PET somehow facilitates passage of tran- The function of the structural genes is more apparent. The
scription complexes through the termination site on the first core gene encodes the viral capsid protein, known as hepatitis
but not the second passage. If PET is deleted, transcription of B virus core antigen (HBcAg). Assembly of cores into the
the smaller, envelope mRNAs is increased. A second signal icosahedral subviral core particles requires the formation of
sequence, NET (negative effector of transcription), which is dimers of the core subunit (29), stabilized through two disul-
needed for termination, is suppressed by PET. If NET is de- fide bonds, followed by the assembly of 120 dimers into a shell
leted, longer transcripts accumulate due to additional transits with a diameter of 36 nm and a triangulation number (T) of 4
around the cccDNA template (13). (41). Expression of cores with small truncations at their C
Spliced transcripts have sometimes been detected, but there termini can induce the formation of smaller shells consisting of
is so far no evidence that RNA splicing plays an important role 90 dimer subunits and a triangulation number of 3 (252). The
in HBV gene expression. On the other hand, a spliced form of structure of the cores was resolved first to 7.4 Å resolution by
the pregenome has been detected in DHBV-infected cells and cryoelectron microscopy (19, 39) and then to 3.3 Å resolution
could serve as a second mRNA for the synthesis of the large of X-ray crystallography (235). The folding of the protein is
envelope protein of DHBV. It was reported that mutation of characterized by four ␣-helices and the absence of ␤-sheets. In
the splice donor site to block production of the spliced RNA agreement with previous biochemical analyses, the structural
did not inhibit virus release from a transfected cell line whereas data revealed two regions required for the dimerization of core
virus release from primary hepatocyte cultures infected with monomers and for the subsequent assembly of the dimers into
the mutant virus was blocked (156). There is no a priori ex- core particles (110, 253). Two separate regions of the core
planation for the mutant phenotype. The authors did not in- polypeptide are exposed on the surface of core particles and
dicate if the mutation affected early viral DNA synthesis and/or thus provide targets for interaction with envelope components
cccDNA amplification in the primary hepatocyte cultures. An (see “Assembly and reverse transcription” below). One of the
effect on DNA synthesis might have occurred because of the two sites forms spikes that extend from the interface of the
location of the splice donor site in a region critical for initiation dimerization sites; the other is located just downstream of the
of DNA synthesis, and an effect on early cccDNA amplification ␣-helix believed to mediate the multimerization of dimers (38).
might have been anticipated if the new mRNA had primarily a The polymerase polypeptide, translated from an internal
regulatory function. initiator codon on pgRNA (Fig. 4), consists of two major do-
Regulation of viral gene expression also occurs at the level mains that are tethered by an intervening spacer region. The
of translation. The pregenome (Fig. 4) serves as the mRNA amino-terminal domain (also called the terminal protein [TP]
not only for the viral core protein but also for the viral poly- domain) plays a critical role in the packaging of pgRNA and in
merase, which initiates from an AUG located in the distal the priming of minus strand DNA, as discussed below. The
portion of the core gene, although not in the same reading carboxy-terminal domain is a reverse transcriptase that, like
frame as core (30, 161). Reading of the polymerase open the retroviral polymerases, is endowed with an RNase H ac-
reading frame appears to be inefficient compared to that of the tivity. Structural and biochemical investigations of the hepad-
core open reading frame. However, since core particles are navirus polymerases have been complicated by the apparent
assembled from 240 core protein subunits (41) and only one or requirement of cellular factors for enzymatic activity. For ex-
perhaps two polymerase proteins (7), pgRNA may serve as an ample, the protein-priming reaction of DHBV depends on the
mRNA for the translation, on average, of approximately 200 to molecular chaperone hsp90 and some of its cofactors including
300 core polypeptides before allowing the translation of a p23 (93). For these and perhaps other reasons, purification of
polymerase polypeptide. Since the polymerase preferentially the polymerase from cellular extracts has so far almost always
binds to the 5⬘ end of its own mRNA to initiate reverse tran- resulted in the loss of the protein-priming and DNA polymer-
scription and packaging (6, 224), synthesis of the polymerase is ase activities (however, see reference 155).
apparently sufficient to stop further translation of the prege- Orthohepadnaviruses express three envelope components
nome. called S, M, and L. S (226 amino acids long), the smallest,
defines the S domain. The two larger proteins contain S plus
amino-terminal extensions of S created by initiation at up-
Viral Proteins stream start codons. For HBV, these codons are located ap-
proximately 165 (M) and 489 (L) nucleotides upstream of the
With a genome that is only 3 kbp in length, hepadnaviruses initiation codon for S. The extra domain of M is known as
express a very limited repertoire of proteins. The core and pre-S2, while the domain unique to L is called pre-S1. All three
polymerase genes are essential for viral DNA replication, and envelope components are glycosylated, type II transmembrane
the envelope proteins, which consists of two or three subspe- proteins that can form multimers stabilized by disulfide bridges
cies, depending on the hepadnavirus, are essential for envel- formed by cysteine residues present in the S domain. S, L, and
opment of nucleocapsids. Two additional gene products ex- M are all found as components of the 42-nm-diameter infec-
pressed during natural infections are of unknown function, tious viral particles, also known as Dane particles after the
hepatitis X protein (HBx) and hepatitis e-antigen (HBeAg). X author of a paper describing their appearance in the electron
is required for the establishment of an infection in vivo (32, microscope (45). L and M are present in roughly equal
254) but is dispensable for virus replication in transfected cells amounts in Dane particles and together constitute approxi-
(17, 237). HBeAg is dispensable for in vivo infections (28, 33). mately 30% of the envelope protein content (82). S by itself,
Curiously, avian hepadnaviruses do not possess an X gene, and together with the larger envelope proteins, also forms
although DNA sequence comparisons have suggested that this filamentous and spherical “surface antigen” particles that are
gene may have been present at some time in the evolution of secreted from infected cells in at least 100-fold excess over
these viruses (148). Experiments in mice suggest that HBeAg virions. These spheres and filaments can accumulate to con-
may cause depletion of Th1 helpers cells, thereby suppressing centrations of several hundred micrograms per milliliter in the
the ability to mount a strong cytotoxic T-lymphocyte (CTL) blood of HBV-infected patients. Complexes of these particles
VOL. 64, 2000 HEPATITIS B VIRUS BIOLOGY 57

with their cognate antibodies are probably responsible for the physiological role of X during the course of an infection re-
immune complex syndromes that sometimes occur during tran- mains a major unresolved issue in hepadnavirus biology.
sient infections.
Antibodies to surface antigen particles composed of S pro- Assembly and Reverse Transcription
tein alone are sufficient to provide protection against HBV
infection. However, there is good reason to believe that the Inferring from genetic experiments, it has been suggested
pre-S1 domain is, at least in part, the substrate for the still that translation of the polymerase and pgRNA packaging into
elusive viral receptor. Epitopes in pre-S1 displayed on the viral nucleocapsids are tightly coupled events (6, 84, 101).
outside of surface antigen can also elicit virus-neutralizing an- These experiments revealed not only that the reverse transcrip-
tibodies (115, 116, 247) and alter the host range of the virus tase is required for RNA packaging but also that packaging
upon genetic recombination (97). occurs in cis. It is now well established that the polymerase
Besides its role in infection, the pre-S1 domain provides the binds to an RNA stem-loop structure at the 5⬘ end of pgRNA,
ligand for core particles during the assembly of the viral enve- termed epsilon, and that this event triggers the sequestration of
lope (see below), which requires display of pre-S1 epitopes on viral RNA and polymerase into core particles (167, 226) (Fig.
the cytosolic side of the viral membrane. As a consequence of 2 and 3).
this dual function, a fraction of L polypeptides must maintain RNA packaging also depends on host factors, in particular
the pre-S1 domain in the virus interior while the remaining L polypeptides belonging to the molecular chaperone complex of
polypeptides undergo a conformational shift to transfer the hsp90 (92). hsp90 appears to stabilize a transient conformation
receptor binding pre-S1 region across the lipid bilayer to the of the polymerase that facilitates the binding to epsilon on
outside of the virus particle (24, 72). This transfer appears to pgRNA. Thus, drugs that interfere with hsp90 function, such as
occur as viruses mature during their passage along the secre- the ansamycin geldanamycin, inhibit RNA packaging and con-
tory pathway. The function of the M polypeptide is not well sequently the replication of both DHBV and HBV (93).
understood, and the pre-S2 domain is apparently not essential In addition to its role in RNA packaging, epsilon plays a
for virus infection of hepatocyte cultures (58). major role in the initiation of viral DNA synthesis. This reac-
In addition to glycosylation, L protein of HBV is modified by tion is unique to hepadnaviruses, since it uses the reverse
N-terminal myristylation (114). Myristylation is not required transcriptase itself as a protein primer for DNA synthesis (118,
for efficient virion assembly but is required for infectivity (23, 207, 225). A hydroxyl residue of a tyrosine located near the
66). amino terminus of the polymerase polypeptide serves as a
The avihepadnaviruses encode only two envelope proteins, substrate for the formation of a phosphodiester linkage with
dGMP (230, 255). The template for this reaction is located in
analogous to S and L. Glycosylation of these proteins has not
the proposed bulge region of epsilon RNA that is believed to
been detected (174). The pre-S domain is phosphorylated and
separate the lower and upper stem regions (168, 224). The
is also myristylated at its N terminus. Phosphorylation has not
priming reaction terminates following the synthesis of only 4
been shown to play a role in envelope assembly and infectivity
nucleotides, but it somehow triggers the transfer of the nascent
(18). As with HBV, myristylation is not required for DHBV
DNA strand to the 3⬘ end of the pgRNA, where the 4 nucle-
assembly but is required for infectivity (133).
otides base pair with complementary sequences (Fig. 3). Ex-
Unique to orthohepadnaviruses is the presence of a fourth actly how this transfer occurs is not known, since information
gene, termed X. The function of X during a natural viral about the spatial organization of nucleic acids and polymerase
infection is not known. As mentioned above, X is essential for inside the core particles is not available.
virus replication in animals but dispensable for viral DNA Plus-strand priming, like minus-strand priming, also involves
synthesis in transfected tissue culture cells (32, 254). Evidence a transfer reaction. In this case, an RNA oligomer derived
that the gene is expressed in vivo stems from the detection of from the 5⬘ end of pgRNA recombines with an internal accep-
antibodies present in sera of HBV-infected humans and natu- tor site on minus-strand DNA (123, 179). Annealing of the
rally infected animals (166, 223) and from protein analysis of primer with complementary sequences at the acceptor is pos-
liver samples obtained from WHV-infected woodchucks (44). sible due to an 11- to 12-nucleotide sequence homology re-
In vitro, X exhibits a plethora of activities. From cell culture ferred to as direct repeats 1 and 2 (DR1 and DR2) (Fig. 3).
studies, it is believed that X can activate the transcription of The 3⬘ end of the RNA primer most probably represents the
host genes, including the major histocompatibility complex (94, final RNase H cleavage site on pgRNA (194). Plus-strand
219, 251) and c-myc (4), as well as viral genes (37, 192), and DNA synthesis involves a template switch from the 5⬘ end to
one investigator has even reported that X stabilizes viral RNAs the 3⬘ end of minus-strand DNA, which is facilitated by short
(186). Most investigators agree that X is not a DNA binding sequence repetitions at the ends of the DNA template (79).
protein and that it is therefore not a typical transactivator. The final result of the reverse transcription reaction is an
Thus, its effects on transcription are thought to be indirect. rcDNA genome with two modified 5⬘ ends and, in mammalian
Suggestions have included interacting with and altering the viruses, with a less-than-genome-length plus-strand DNA,
DNA binding of cyclic AMP-responsive element binding pro- leaving up to 50% of the rcDNA single stranded (89, 117, 131,
tein and ATF-2 (134), activating NF-␬ B (218), and contacting 199). With DHBV, the plus strand is more nearly complete in
basal transcription factors (80). viral rcDNA, with completion apparently blocked at DR2 be-
Aside from the transactivation of many promoters, activities cause of the RNA primer of plus-strand synthesis that is an-
linked to X include stimulation of signal transduction (14) and nealed to the minus strand (124).
binding, to various degrees, to well-known protein targets such In approximately 10% of cases, the transfer of RNA primer
as p53 (57, 206, 229), proteasome subunits (190, 205), and from DR1 to DR2 does not occur, leading to an in situ priming
UV-damaged DNA binding protein (119). Many different ex- reaction (194). The result is a double-stranded linear DNA
planations for these and other effects of the X protein have (dslDNA) genome rather than rcDNA (Fig. 3). Although the
been proposed. Whether all or any point to the role of X in the significance of dslDNA in a natural infection is not known,
virus life cycle is unknown. It is clear that X-defective virus is genetic experiments showed that variants of DHBV producing
unable to initiate infection in vivo (32, 254). However, the only dslDNA can infect primary hepatocyte cultures and
58 SEEGER AND MASON MICROBIOL. MOL. BIOL. REV.

ducks. cccDNA formation seems to occur through illegitimate proteins that are displayed by major histocompatibility com-
recombination of the ends of dslDNA (242). plex type I on the surface of infected cells. Many cells are then
killed before virus production can initiate, thereby preventing
Amplification of cccDNA and Production of Infectious Virus the spread of viruses to other susceptible cells. Further control
of virus spread is provided by a rapid production of virus-
The most intriguing aspect of the hepadnavirus replication neutralizing antibodies, which serve to protect susceptible
cycle is the regulation of cccDNA synthesis. It is the basis for cells. Local protection of susceptible cells is also provided by
the establishment and persistence of hepadnaviruses in in- interferons and other cytokines, which may induce resistance
fected hepatocytes and consequently can be expected to play a to infection in nearby cells. In brief, the infection is prevented
major role in recovery from an infection. The concept that from spreading to all potential targets.
cccDNA synthesis is regulated is consistent with the observa- This model is not easily adapted to transient hepadnavirus
tion that the nuclei of DHBV-infected hepatocytes contain on infections, which generally run a course of 1 to 6 months,
average 10 to 50 copies of cccDNA while 10 times as much of including an asymptomatic incubation period, often accompa-
the rcDNA precursor to cccDNA may reside in the cytoplasm. nied by a high-titer viremia (up to 1010 per ml), which may
In addition, amplification of cccDNA levels takes place in the itself last several weeks. During this period, the immune system
first few days following infection of primary duck hepatocyte is either unable or not activated to gain control of the infection,
cultures with DHBV (217). By using DHBV variants that fail and the entire hepatocyte population of the liver may be in-
to express the large envelope polypeptide, Summers et al. fected. The lack of symptoms in HBV-infected patients for the
showed that cccDNA accumulation was indeed a regulated first few weeks suggests that activation of the immune response
process (200, 201). In the absence of envelope proteins, accu- is delayed, although there are very few objective data on which
mulation continued to levels toxic to primary hepatocyte cul- to base this conclusion. Thus, the virus may spread from only
tures as well as hepatocytes in vivo (121, 201). These experi- a few hepatocytes to involve, in at least some cases, the entire
ments pointed to a role for the envelope in the regulation of hepatocyte population of the liver (5, 15, 71, 88, 100, 102, 171).
cccDNA. A direct implication of such a model is that cccDNA The virus is then rapidly cleared from the serum, often in a few
amplification preferentially occurs during the early phase of an weeks, with either coincident or delayed clearance of infected
infection, when the concentration of the envelope is still low. hepatocytes from the liver.
Once higher levels are reached, subviral core particles are Hepadnavirus infections, and transient infections in partic-
segregated into the secretory pathway through their interaction ular, therefore raise difficult issues. First, why is there not a
with envelope polypeptides residing in membranes of the en- more effective control of virus spread during the early period?
doplasmic reticulum (ER). Of note is the general observation Second, how is the immune system later able to clear an in-
that virion formation depends on viral DNA synthesis, i.e., that fection, after it has spread to essentially the entire hepatocyte
cores have to “mature” in order to interact with viral envelope population? A possible immunosuppressive role of the e-anti-
to form virions. Thus, virus is enriched for the partially double- gen has been suggested (144, 145) but has not yet been proven
stranded rcDNA while infected cells contain approximately in an experimental model of infection. The concept that e-an-
equal amounts of partially double-stranded and single- tigen is immunosuppressive in a natural infection is consistent
stranded DNAs (198, 231). The molecular signals that emerge with the observation that ducks infected with DHBV that is
on the surface of cores and presumably regulate the interaction unable to produce e-antigen develop and sustain higher anti-
with envelope components leading to virion formation are body titers to viral core antigen than do ducks infected with
unknown. Changes in the phosphorylation of the DHBV core wild-type DHBV (250; J. Summers, personal communication).
protein are believed to play a role, since core protein in viral This may happen because of suppression of the antibody re-
particles has a different phosphorylation pattern than core sponse by e-antigen, which is cross-reactive with the core, or
protein within cells (173). However, mutation from serine to because infections with e-antigen-deficient strains produce
alanine at one of the phosphorylation sites (Ser-259) that is more liver disease than do infections with wild-type strains,
apparently essential for the intracellular pattern (173) did not thereby increasing the amount of antigen stimulation. Both
block virus assembly, although the resultant virus was unable to mechanisms would be consistent with the proposal that e-an-
infect primary duck hepatocyte cultures (245, 246). In brief, tigen functions to suppress the antiviral immune response.
there is still no evidence that the interaction between nucleo- The second issue is equally difficult. How is the immune
capsids and envelope proteins is actually regulated by changes system finally activated, and virus eliminated, after infection of
in core protein phosphorylation. the entire hepatocyte population? In approaching this issue, it
Binding of cores to the N-terminal domain of the large is necessary to at least take account of various beliefs and
envelope polypeptides results in the translocation (budding) of observations about the virus and the host. (i) Hepatocytes
the core particles across the ER membrane. Enveloped parti- have, as mentioned above, a very long half-life (6 to 12 months
cles containing all three envelope proteins are thought to be or more). Indirect evidence suggests that the hepatocyte half-
transported through the ER into the Golgi complex (96). Gly- life may be as little as 10 days in some chronically infected
cosylation at an asparagine residue located in the S domain of patients with active liver disease (154). However, even this high
the envelope proteins occurs during this phase of the assembly a turnover would not by itself be sufficient to explain the
process, which is completed with the secretion of mature viri- clearance of infected cells during resolution of transient infec-
ons into the bloodstream. tions (see below). (ii) Viral cccDNA is thought to be stable in
infected and nonproliferating hepatocytes (130, 146; however,
TRANSIENT INFECTIONS see reference 35), although virus expression can be suppressed
and degradation of viral proteins and nucleic acids in the
The majority of hepadnavirus infections (90 to 95%) in cytoplasm can be induced by certain cytokines (68). (iii) Clear-
adults are transient, while ⬃90% of perinatal infections are ance of virus from the liver during recovery from a transient
chronic (195). One view of the immunoregulation of viral in- infection does not appear to involve the replacement of the
fections, generally applied to infections by cytocidal viruses, is infected cell population by proliferation and differentiation
that CTL responses quickly recognize epitopes of early viral into hepatocytes of a hypothetical population of uninfected
VOL. 64, 2000 HEPATITIS B VIRUS BIOLOGY 59

FIG. 5. CTL killing and hepatocyte replacement. The kinetics of clearance of infected hepatocytes have been calculated by assuming that CTL killing is a first-order
reaction. Each point indicates the fraction of the initial number of infected hepatocytes that were killed in the preceding 24-h period. The different curves were
determined using t1/2 values for killing that would produce 99% loss of infected hepatocytes in the indicated times.

progenitor cells. In particular, periportal hepatocytes, located iments with woodchucks and ducks, we have obtained liver
near the putative origin of progenitor cells at or adjacent to biopsy specimens during the partial clearance phase of an
bile ductules, can be the last in the lobule to clear an infection infection only three times (73, 100, 102). In one example, a
(99, 102). (iv) Massive cell destruction has not so far been liver biopsy specimen taken during the recovery phase of the
linked to virus clearance even after the entire hepatocyte pop- infection revealed ⬃20% infected cells and a rate of hepato-
ulation appears to have been infected, although it has also not cyte replacement (and, by inference, cell death), estimated
been definitively ruled out. (v) Hepatocytes can actively pro- from BUdR and proliferating-cell nuclear antigen labeling in-
liferate to maintain liver cell mass. dices, of ⬃3 to 6% per day. Such a rate would be compatible
If we assume that clearance involves replacement of infected with half-lives of ⬃2.25 to 4.5 days for infected hepatocytes.
hepatocytes via proliferation of initially rare uninfected hepa- These rates would be sufficient to produce a 99% decline in the
tocytes and that infected cells do not divide, it is possible to number of infected hepatocytes in 2 to 4 weeks.
generate a reasonably simple model for clearance. In particu- Unfortunately, the actual rate of virus clearance is unknown
lar, if elimination of infected cells occurs by CTL killing via a and methods for accurately determining rates of cell replace-
first-order reaction, a series of curves can be drawn relating the ment are not available. In addition, this simple model does not
kinetics of clearance to the rate of hepatocyte replacement take account of recent observations made with an HBV trans-
(Fig. 5). Thus, a single time point determination of the fraction genic mouse. These studies revealed that cytokines, particu-
of infected cells and the rate of liver cell regeneration during larly alpha and gamma interferons and tumor necrosis factor
the clearance phase will yield an estimate of the half-life for alpha (TNF-␣) play key roles in pathways that induce the
infected hepatocytes. The first measurement is fairly straight- apparently noncytocidal loss of virus replication intermediates,
forward. While there is no simple way to precisely measure including proteins, replicating DNA, and RNAs, from nonpro-
rates of cell turnover, it is possible to estimate this by counting liferating hepatocytes (26, 68, 216). Analogous effects on
mitotic figures, apoptotic cells, or proliferating-cell nuclear cccDNA, if any, were not identified, since cccDNA is not
antigen-positive hepatocytes or by in vivo pulse-labeling with synthesized in this transgenic mouse. Thus, although hepato-
bromodeoxyuridine (BUdR). cyte death appears to be a characteristic feature of transient
If this model were correct and if complete clearance (e.g., to infections, there is at least a possibility that virus is completely
less than 1% infected hepatocytes) occurred in less than 15 to eliminated without this killing. Recent data on HBV infections
30 days (Fig. 5), as suggested by studies with the woodchuck of chimpanzees appear consistent with this possibility (71).
and duck models (100, 102, 171), the half-life of infected hepa- The idea that virus can be lost from hepatocytes is also
tocytes could be only 2.25 to 4.5 days or less during the clear- consistent with studies of WHV infections suggesting that in-
ance phase. Thus, extremely high levels of hepatocyte loss and fected hepatocytes, which were BUdR pulse-labeled at the
replacement would take place only for a short period, perhaps peak of a transient infection, were still present after the virus
a week or less, at the times when significant numbers (e.g., had been eliminated (102). However, another explanation of
⬎10%) of the hepatocytes were still infected. To our knowl- this result is that the BUdR-labeled hepatocytes lost the virus
edge, such measurements have not been made in patients due to a combination of cytokine-induced destruction of rep-
recovering from transient HBV infections. In our own exper- lication intermediates, which form the precursors to cccDNA,
60 SEEGER AND MASON MICROBIOL. MOL. BIOL. REV.

and the loss of cccDNA itself during cell division. One of the assays (e.g., ⬎105 to 106 per ml of serum) also no longer have
unknown factors is whether cccDNA is passed to daughter cells active liver disease, defined by ongoing infiltration of leuko-
when hepatocytes divide. cytes and active destruction of hepatocytes. Viremic individu-
If cccDNA is lost during mitosis or passes inefficiently to als with the highest titers (109 to 1010 per ml) usually are
daughter cells, it is possible to generate models for virus clear- thought to have a low immunologic response to the viral anti-
ance in which infected hepatocytes are lost with less cumulative gens and a low disease activity, while those with somewhat
cell death than in the model described above, in which all lower titers (107 to 108 per ml) appear to have the highest
infected hepatocytes (up to 100% of hepatocytes) are de- disease activity.
stroyed. Thus, if both uninfected and infected hepatocytes di- Chronic infection begins when the immune response that
vide to replace the infected hepatocytes that are killed by CTLs normally clears the infection fails to take place or is too weak
and if infected hepatocytes are no longer infected as a result of to be effective. Thus, infections are almost always chronic fol-
cell division, a 99% loss of infected hepatocytes will occur after lowing exposure of children younger than 1 year or of immu-
only 70% are killed by CTLs (139). However, 70% cell replace- nocompromised individuals. Chronic infections also occur in
ment in only a few weeks is still a major change compared to about 5% of otherwise healthy adults. Patients infected as
the quiescent nature of the healthy liver. Thus, unless there is adults often have the most severe and progressive liver disease
a novel mechanism for virus elimination from quiescent cells, (142). These patients may also experience recurrent bouts of
cell death plays an obligatory role. This conclusion, if correct, acute liver disease, possibly because their immune system at-
has major implications not just for transient infections but also tempts to produce the response that normally leads to virus
for the design of antiviral treatments to terminate chronic clearance (86, 127).
infections. The prognosis for many chronic carriers of HBV who were
Irrespective of the mechanism of virus elimination, an obvi- infected with HBV as adults is poor. About 10 to 25% will die
ous question remains. What triggers an effective antiviral re- of either liver cancer or cirrhosis (tissue scarring). Smaller
sponse after a delay of many weeks or even months (158)? numbers, particularly those infected as adults, will die or re-
Most probably the delay is due, in part, to interactions between quire liver transplantation due to rapidly progressing liver dis-
the immune system and the liver. In particular, similarly pro- ease leading to hepatic failure. Attempts at antiviral therapy to
longed time courses and extensive infection of the hepatocyte eliminate the virus and reduce disease activity have had the
population are observed with two other liver-specific viruses, highest success rate in individuals with active, immune system-
hepatitis A virus (a picornavirus) and hepatitis E virus (a cali- mediated liver disease (22, 87). This is probably because their
civirus), neither of which produces a chronic infection. Hepa- immune system is already close to producing a response lead-
titis delta virus follows a similar time course as well, although ing to viral clearance. Antiviral therapy to eliminate the virus
its life cycle is more complicated, since it depends upon HBV has not been as successful for the vast majority of viremic
for its envelope proteins. It has been proposed that the liver carriers, probably because their immune system mounts a
may suppress CTL responses in favor of antibody production, weaker response to the infection. Immune system-mediated
since the liver plays a major role in antibody-mediated protec- liver disease may be less active in these carriers because infec-
tion against prokaryotic and eukaryotic pathogens. However, tion occurred during birth or in the first year or two of life,
whether this explains the delay in mounting a successful anti- producing a degree of tolerance to the viral antigens. Unfor-
viral response is not yet known. tunately, most carriers fall into this group, which therefore
represents the largest target for antiviral intervention. In these
CHRONIC INFECTIONS carriers, the interval between initiation of the HBV infection
and the peak incidence of HCC is ⬃30 to 50 years (11, 12).
Chronic infection is defined as the persistent presence of Interestingly, the majority of untreated carriers, who were
HBsAg in the serum of an individual for 6 months or longer infected early in life, do not die of liver disease or liver cancer,
(52). Commercial assays can detect as little as a few nanograms and, indeed, cancer incidence may decline as carriers age (11,
of HBsAg per milliliter of serum, and most chronically infected 12). One explanation is that some carriers differ in their risk
individuals have titers exceeding 1 ␮g per ml (53). Thus, the due to their genetic makeup. Indeed, a case-control study
assay has the advantage of great sensitivity. One disadvantage carried out in China indicated that a family history of HCC was
of defining chronic infection on the basis of this assay alone is a risk factor for HCC among HBV carriers (129). One study
that HBsAg may be produced at high levels even if virus rep- even suggested that carriers lacking the gene for glutathione
lication in the liver has virtually ceased. The probable reason S-transferase M1 might be at increased risk for HCC, possibly
for this continued HBsAg production in the absence of detect- because of increased sensitivity to DNA adduct formation by
able levels of virus replication is that viral DNA may integrate aflatoxin B1 (141).
into host chromosomes during the course of infection and Another factor that has been considered to increase the risk
provide a template for transcription of HBsAg mRNAs. The of liver disease and HCC is persistent high-titer virus replica-
predominant precursor to integrated DNA is likely to be the tion. The incidence of high-titer virus production (where the
linear double-stranded DNA produced as a result of in situ virus is readily detectable by nucleic acid hybridization tech-
priming (63, 64, 241, 242). The result of integration is that viral niques [e.g., ⬎106 per ml of serum]) declines with increasing
genes, particularly for the core and polymerase, may be dis- age, at least among individuals infected in childhood (53). For
rupted (Fig. 4) while the coding regions and promoters for the instance, about 25% of male carriers in China maintain high-
envelope proteins remain intact. (Integrated DNA may also be titer virus production past the age of 30 years whereas virtually
highly rearranged. It is unclear if this rearrangement occurs none do in Senegal. Moreover, the HCC risk in the Chinese
before or after integration or if both are possible [159, 177].) population was about 10-fold higher than in the Senegalese
Thus, chronic infection as defined by HBsAg production may cohort. Thus, a correlation between HCC and prolonged high-
encompass a broad spectrum of virologic and pathogenic titer virus replication might be deduced. Unfortunately, al-
states. though the correlation may seem correct when populations are
It is generally the case that HBsAg carriers who no longer examined, it was not observed among individuals who were
make virus that is detectable by conventional hybridization first investigated near the time of the HCC diagnosis, even
VOL. 64, 2000 HEPATITIS B VIRUS BIOLOGY 61

when the sensitivity of the virus assay was increased through at the beginning of the expansion of the tumor. By analogy to
PCR. The hypothesis remains attractive because antiviral ther- the model of oncogene activation by insertional mutagenesis,
apy would also prevent liver cancer if the risk of HCC were coined by Hayward et al. (81) and Payne et al. (164), it was
related to the duration of high-titer virus replication. hypothesized that HBV could act as an insertional mutagen,
There are currently two approved therapeutic agents for causing the activation of a proto-oncogene. Results obtained
chronic HBV, alpha interferon and lamivudine (a cytosine with the woodchuck model supported this hypothesis. They
analog). Alpha interferon has been used for more than a revealed that WHV sequences are present at or near N-myc2,
decade. It probably acts directly to suppress virus replication a pseudogene of N-myc, in almost every liver tumor arising in
within hepatocytes by inducing the degradation of viral WHV-infected animals (59, 60). In almost all cases examined
mRNAs (69) and indirectly to stimulate the immune system. so far, integration of WHV results in activation of transcription
Indeed, as noted above, patients with an active liver disease of the N-myc2 locus. In the few cases where N-myc2 is not
due to their immune response to HBV are the best candidates transcribed, WHV integration is found at the c-myc locus (91)
for successful therapy with alpha interferon (22, 165, 234). A or the N-myc1 locus (60). In contrast to the observations with
probable reason for the failure of alpha interferon therapy in woodchucks, similar efforts with tumors obtained from HBV-
most other carriers is that their ongoing immune response to infected patients did not reveal a common cellular target for
the infection is quite low (128). HBV integration. Over the past 20 years, only a handful of
Recently, lamivudine has been approved for treatment of “interesting” HBV insertions have been reported. They were
HBV carriers. In theory, suppression of virus replication for a found adjacent to or within the genes encoding cyclin A and
sufficiently long time should lead to clearance as infected hepa- the retinoic acid receptor beta (46, 227, 228). However, these
tocytes and other reservoirs for the virus die off. In practice, findings have not been generalized beyond the original tumor
lamivudine-resistant variants may replace wild-type virus after samples and thus did not represent the hoped-for bonanza
a year or more of therapy (3, 8, 9, 31, 125, 152, 154, 213). concealing a general mechanism for HBV-induced oncogene-
Lamivudine-resistant variants of the HBV polymerase are sen- sis.
sitive to adefovir, another nucleoside analog inhibitor of viral There are other obvious differences between WHV and
DNA synthesis that is now undergoing clinical evaluation, and HBV associated cancers. First, liver cancer in humans is almost
the two may prove useful in combination (236). always associated with cirrhosis whereas in woodchucks fibrosis
Interestingly, lamivudine alone can induce a rapid reduction of the liver is uncommon. Second, the time for the develop-
in symptoms of liver disease even though the virus is still ment of liver neoplasia in humans is on the average 20 to 40
present in the host (154). It is not known whether this reduc- years whereas in woodchucks it is only 1 to 3 years.
tion in disease activity is due to a decline in the number of An important factor to be considered in models for cancer is
productively infected hepatocytes as targets for the immune the frequency of cell regeneration. Regeneration of hepato-
system or whether it occurs because viral DNA replication per cytes is a direct consequence of cell killing caused by the
se increases the susceptibility of hepatocytes to injury. Another immune response of the host to the infection. Cell killing
hypothetical explanation is that lamivudine alters the immune occurs by apoptosis of hepatocytes or by necrosis due to liver
response to HBV, as suggested for the therapeutic effects of inflammation. As mentioned above, HBV-infected patients
the nucleoside ribavirin (153) in the treatment of chronic hep- can exhibit severalfold-increased rates of hepatocyte turnover
atitis C. Reports that several months can elapse between the compared to uninfected healthy people.
appearance of low levels of lamivudine-resistant HBV in the What, then, are other factors that could play a role in hepa-
serum and a major elevation in HBV titers suggest that the tocarcinogenesis? As the search for common cellular targets
spread of the mutants in the liver may be blocked by wild-type for HBV DNA integration remained unproductive, two alter-
HBV through a superinfection resistance mechanism (31). In native models that emerged from research on retroviruses and
other words, the therapeutic effect is not dependent on clear- colon cancer were adapted to explain HBV-induced oncogen-
ance of HBV from hepatocytes. esis. The first model predicted that HBV, like certain tumor
viruses, contains an oncogene. The second model invoked a
LIVER CANCER role for tumor suppressor genes, in particular p53, in HCC
formation.
Epidemiological studies established a link between HBV The major prediction of the oncogene model is that expres-
infection and liver cancer. In humans, the lifetime risk for a sion of one or several HBV gene products induces malignant
chronically infected person is approximately 10 to 25%. In transformation in tissue culture cells or in transgenic mice.
woodchucks chronically infected with WHV, the lifetime risk is Transformation of cells expressing intact hepadnavirus ge-
essentially 100%. An obvious question raised by these obser- nomes has generally not been observed. An exception is a
vations concerns the role of the virus in liver cancer. Although report by Hohne et al. (85), who transfected a nonmalignant
this question has been addressed experimentally in a variety of immortalized mouse hepatocyte line harboring metallothio-
settings over the past two decades, the amount of solid infor- nein promoter-driven simian virus 40 large tumor antigen with
mation is still disappointingly sparse. One exception is the HBV DNA. They observed that all transfected clones which
work by Buendia and colleagues on the genetic analysis of replicated HBV displayed malignant growth characteristics in
tumors from woodchucks, which implicated the myc family of soft agar and were tumorigenic upon inoculation in nude mice.
genes as contributors to liver cancer (for a review, see refer- A notable difference between these cell lines and infected liver
ence 181). tissue was that the cell lines expressed a transcript correspond-
The quest to unravel the mechanism of HBV induced liver ing to the viral X gene at much higher levels than were ob-
cancer was originally spurred by reports from a number of served under physiological conditions in vivo. Indeed, subse-
laboratories (20, 27, 51, 136) that HBV DNA was present in quent studies by the same laboratory with the same cell line
high-molecular-weight DNA extracted from HCC or from cell showed that overexpression of the viral X gene was required
lines derived from HCC. Since the pattern of DNA integration for cell transformation (183).
was random with regard to the cellular DNA flanking the viral Expression of the viral X gene can also disturb normal cell
sequences, it was concluded that integration occurs prior to or growth in transgenic mice. For example, overexpression of
62 SEEGER AND MASON MICROBIOL. MOL. BIOL. REV.

HBx in transgenic CD-1 mice, which are susceptible to the (21, 90). Most importantly, in 80 to 90% of the cases with p53
development of spontaneous hepatomas, increased the fre- mutations, the G residue of codon 249 was changed to T. Such
quency of liver tumors at least 10-fold compared to that in G-to-T transversions are known to occur by guanosine adduct
normal mice of the same strain (105, 109). In contrast, trans- formation caused by certain chemical carcinogens such as af-
genic mice expressing lower, most probably more physiologi- latoxin B1. Patients from both areas could have been exposed
cal, levels of X did not develop hepatomas at an increased rate to aflatoxins through contaminated foods. Consistent with a
compared to normal CD-1 mice (109). Also, another strain of role of environmental factors in p53 mutagenesis, several other
transgenic mice (ICR ⫻ B6C3F1) expressing X did not de- reports indicated that the incidence of p53 mutations in HCC
velop hepatic tumors, and, most importantly, hepatomas have is generally much lower, ranging from 12 to 30% (25, 150).
not yet been observed in transgenic mice (C57BL/6 and These numbers are below the rate observed in other human
B10.D2) expressing replication-competent HBV genomes cancers, where p53 mutations are estimated to occur in roughly
(120). Several laboratories have reported that transgenic mice 50% of all cases (65). Whether p53 mutations are generally
expressing X exhibit an increased rate of liver cancer formation lower in HCC than in other malignancies or whether the lower
when exposed to known carcinogens such as diethylnitro- incidence is characteristic of HBV infection is an important
samine or p-dimethylaminoazobenzene (50, 191). Similarly, X unanswered question. Observations made with HCC samples
has been shown to reduce the latency period for HCC devel- from HCV-infected individuals indicated that the relatively
opment in transgenic mice expressing a woodchuck c-myc gene low rate of p53 mutations in HCC is not dependent on the
(209). etiology of the disease. For example, Pontisso et al. found p53
Thus, most of the experimental evidence that has accumu- mutations in less than 10% of HCC samples obtained from
lated so far suggests that X is not, by itself, the cause of HCV-infected patients of Caucasian background (169). Simi-
HBV-associated carcinogenesis. Indeed, it should be kept in larly, Sheu et al. found p53 mutations in approximately 25% of
mind that the effects of X may be strongly dependent on the HCC samples obtained from Taiwanese patients that were
experimental system, which in no case was the natural host for negative for HBsAg but positive for HCV markers (185).
HBV infection. For example, Kim et al. showed that in another In agreement with observations made in colon and other
experimental system, X induced apoptosis rather than cell cancers, p53 mutations in HCC are considered late events
growth (106). It therefore remains possible that X, particularly associated with a dedifferentiated phenotype of the tumor
if overexpressed, is a contributing factor in tumor promotion. (107). For example, a report by Oda et al. showed that tumor
It is also quite possible that other viral gene products can nodules growing out of a parent nodule (nodules-in-nodule)
exhibit a tumor-promoting activity in hepatocytes that are ex- exhibited distinct p53 genotypes from each other (157).
posed to an altered environment, as occurs in a chronically Information about early markers for HCC development is
infected liver. still scanty, mainly because liver cancers are generally diag-
Different from the observations reported for HBx, overex- nosed during the late phase of the disease. However, in hu-
pression of the large envelope protein of HBV in transgenic mans as well as in the woodchuck model, insulin-like growth
mice causes hepatocyte injury and death and induces liver factor II (IGFII) is activated in altered hepatic or dysplastic
regeneration. These mice ultimately develop HCC (34). Injury foci as well as in HCCs (47, 240). Consistent with its role as a
is believed to be due to the accumulation of filamentous ag- target for WHV integration, N-myc-2 can also be activated in
gregates formed by L and HBsAg in the ER of hepatocytes, altered hepatic foci in WHV-infected woodchucks (238). Al-
resulting in a storage-like disease, such as observed in patients though it is not known whether IGFII plays a role in HCC
with ␣1-antitrypsin deficiency or in mice expressing mutant development, it is conceivable that it promotes cell prolifera-
␣1-antitrypsin. Since L protein in natural infections does not tion in an autocrine fashion (238). On the other hand, IGFII
accumulate to levels observed in transgenic mice, it is unlikely expression may be required to suppress apoptosis induced by
that this model is relevant for liver cancer in chronic infections. activated myc genes, including N-myc-2 (220, 239).
Surface antigen filaments can overaccumulate in a fraction of
infected hepatocytes, sometimes known as ground-glass hepa- MAJOR UNRESOLVED ISSUES
tocytes, but this contribution to liver injury is probably small
compared with the injury caused by the antiviral immune re- During the past two decades, the major principles of hep-
sponse. adnavirus infections have been resolved. Infectious viral ge-
The second model predicting that HCC development pro- nomes have been cloned and sequenced, the major viral gene
ceeds in multiple steps is based on the identification of specific products have been characterized, and the mechanism of viral
lesions that are visible in livers of patients with HCC as well as DNA replication has been uncovered. In addition, the course
in animal models of liver cancer. Such lesions include altered of viral infections in vivo has been characterized with the help
hepatic foci, dysplastic (neoplastic) nodules, and low- and of animal models and, to some extent, with samples obtained
high-grade HCCs exhibiting different levels of cell differentia- from HBV-infected patients. In spite of these accomplish-
tion. It is generally believed that the development of carcino- ments, many gaps in our knowledge of the viral life cycle still
mas progresses through these individual stages, as described exist.
for the progression of colon cancer. While the progression of On the level of viral replication, two major unresolved issues
neoplasia in colon cancer has been characterized on the mo- concern early events of the replication cycle and the function
lecular level, i.e., through the correlation of phenotypic alter- of the X gene. As alluded to above (see “Infection”), the viral
ations with mutations or deletions in specific genes, a molec- receptor is not known. It is generally believed that the viral
ular characterization of liver cancer is still at an early stage receptor is a major determinant for the observed tissue tropism
(107). of hepadnaviruses. Similarly, it is believed that the resistance
The major focus, so far, has been on the genetic analysis of of tissue culture cell lines to viral infection is due to the ab-
the tumor suppressor p53. Interest in this gene has been sence of the receptor on the surface of these cells. A major
spurred by two reports demonstrating that p53 mutations were impediment to the isolation of the receptor lies in the difficulty
present in approximately half of HCC samples obtained from of constructing a replication-competent hepadnavirus vector
HBV-infected patients in southern Africa and Qidong in China with a gene that can be used for positive selection in mamma-
VOL. 64, 2000 HEPATITIS B VIRUS BIOLOGY 63

lian cells. This is mainly due to the small genome size and the degradation of viral DNA replication intermediates (68, 69).
compactness of cis-acting sequences dispersed over the entire However, the mechanism responsible for the apparent deple-
genome. Such recombinant viral vectors have been essential tion of intracellular virus particles is not yet known. One pos-
for an unambiguous identification of retroviral receptors in- sibility is that certain cytokines activate the cellular pathway(s)
cluding those for Moloney murine leukemia virus, avian leu- used for the disintegration of core particles following virus
kosis virus, and human immunodeficiency virus (1, 43, 140, penetration. Unfortunately, very little is known about the bio-
244). Recently, however, the production of HBV and DHBV chemical steps that are responsible for virus disassembly. Re-
variants expressing green fluorescent protein or gamma inter- search aimed at resolving this interesting problem could pro-
feron has been reported (172). An alternative approach could vide the basis for the future design of effective antiviral
be the production of pseudotypes with viruses such as vesicular therapies required to cure the enormous number of patients
stomatitis virus or certain retroviruses carrying hepadnavirus with chronic HBV infections.
envelope components that could be used for the identification
or selection of cells expressing the viral receptor(s), as shown ACKNOWLEDGMENTS
for the identification of CD4 as the receptor for human immu-
nodeficiency virus. We are grateful to W. T. London for critical reading of the manu-
script and to Jesse Summers (University of New Mexico, Albuquer-
Using a biochemical approach, Kuroki et al. (112, 113) iden- que) for helpful discussions.
tified a novel carboxypeptidase as a potential DHBV receptor. Work in our laboratories is supported by grants from the National
The polypeptide was shown to interact with both virions and Institutes of Health and by an appropriation from the Commonwealth
recombinant envelope polypeptides and could be blocked with of Pennsylvania.
neutralizing antibodies. While these findings have been con-
firmed by other investigators (214, 222), the observation that REFERENCES
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