Zoulim chapitre fields virology 2013 lwbk1180-ch68 p2185-2221


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Zoulim chapitre fields virology 2013 lwbk1180-ch68 p2185-2221

  1. 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 2185 LWBK1180-Ch68_p2185-2221.indd 2185 25/02/13 7:05 PM
  2. 2. 2186 SECTION II | SPECIFIC VIRUS FAMILIES 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. 25/02/13 7:05 PM
  3. 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 | Hepadnaviruses 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. LWBK1180-Ch68_p2185-2221.indd 2187 25/02/13 7:05 PM
  4. 4. 2188 SECTION II | SPECIFIC VIRUS FAMILIES 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 25/02/13 7:05 PM
  5. 5. CHAPTER 68 | Hepadnaviruses 2189 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. 25/02/13 7:05 PM
  6. 6. 2190 SECTION II | SPECIFIC VIRUS FAMILIES 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, LWBK1180-Ch68_p2185-2221.indd 2190 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 25/02/13 7:05 PM
  7. 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 LWBK1180-Ch68_p2185-2221.indd 2191 | Hepadnaviruses 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 25/02/13 7:05 PM
  8. 8. 2192 SECTION II | SPECIFIC VIRUS FAMILIES 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 25/02/13 7:05 PM
  9. 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 | 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 25/02/13 7:05 PM
  10. 10. 2194 SECTION II | SPECIFIC VIRUS FAMILIES 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 25/02/13 7:05 PM
  11. 11. CHAPTER 68 | 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 25/02/13 7:05 PM
  12. 12. 2196 SECTION II | SPECIFIC VIRUS FAMILIES 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 25/02/13 7:05 PM
  13. 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 25/02/13 7:05 PM
  14. 14. 2198 SECTION II | SPECIFIC VIRUS FAMILIES 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 LWBK1180-Ch68_p2185-2221.indd 2198 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 25/02/13 7:05 PM
  15. 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 LWBK1180-Ch68_p2185-2221.indd 2199 | Hepadnaviruses 2199 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 25/02/13 7:05 PM
  16. 16. 2200 SECTION II | SPECIFIC VIRUS FAMILIES 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 LWBK1180-Ch68_p2185-2221.indd 2200 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 25/02/13 7:05 PM
  17. 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 LWBK1180-Ch68_p2185-2221.indd 2201 | Hepadnaviruses 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 25/02/13 7:05 PM
  18. 18. 2202 SECTION II | SPECIFIC VIRUS FAMILIES 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. LWBK1180-Ch68_p2185-2221.indd 2202 25/02/13 7:05 PM
  19. 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 LWBK1180-Ch68_p2185-2221.indd 2203 | Hepadnaviruses 2203 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 25/02/13 7:05 PM