William S. Mason

Hepatitis B Virus Biology

SUMMARY Hepadnaviruses (hepatitis B viruses) cause transient and chronic infections of the liver. Transient infections run a course of several months, and chronic infections are often lifelong. Chronic infections can lead to liver failure with cirrhosis and hepatocellular carcinoma. The replication strategy of these viruses has been described in great detail, but virus-host interactions leading to acute and chronic disease are still poorly understood. Studies on how the virus evades the immune response to cause prolonged transient infections with high-titer viremia and lifelong infections with an ongoing inflammation of the liver are still at an early stage, and the role of the virus in liver cancer is still elusive. The state of knowledge in this very active field is therefore reviewed with an emphasis on past accomplishments as well as goals for the future.

Replication of the genome of a hepatitis B-like virus by reverse transcription of an RNA intermediate

Duck hepatitis B virus, a DNA virus closely related to human hepatitis B virus, was studied in infected duck liver. Subviral particles resembling the viral nucleocapsid cores were isolated from persistently infected liver and shown to have a DNA polymerase activity that utilizes an endogenous template and synthesizes both plus- and minus-strand viral DNA. Synthesis of the viral minus-strand DNA utilized an RNA template that was degraded as it was copied. Viral plus-strand synthesis occurred on the completed minus-strand DNA. A pathway for the replication of the DNA genome of hepatitis B-like viruses by reverse transcription of an RNA intermediate is proposed.

miR-122, a mammalian liver-specific microRNA, is processed from hcr mRNA and may downregulate the high affinity cationic amino acid transporter CAT-1.

These studies show that miR-122, a 22-nucleotide microRNA, is derived from a liver-specificnon-coding polyadenylated RNA transcribed from the gene hcr. The exact sequence of miR-122as well as the adjacent secondary structure within the hcr mRNA are conserved from mammalianspecies back to fish. Levels of miR-122 in the mouse liver increase to half maximal valuesaround day 17 of embryogenesis, and reach near maximal levels of 50,000 copies per averagecell before birth. Lewis et al (2003) predicted the cationic amino acid transporter (CAT-1 orSLC7A1) as a miR-122 target. CAT-1 protein and its mRNA are expressed in all mammaliantissues but with lower levels in adult liver. Furthermore, during mouse liver development CAT-1mRNA decreases in an almost inverse correlation with miR-122. Eight potential miR-122 targetsites were predicted within the human CAT-1 mRNA, with six in the 3’-untranslated region.Using a reporter construct it was found that just three of the predicted sites, linked in a 400-nucleotide sequence from human CAT-1, acted with synergy and were sufficient to stronglyinhibit protein synthesis and reduce mRNA levels. In summary, these studies followed theaccumulation during development of miR-122 from its mRNA precursor, hcr, through toidentification of what may be a specific mRNA target, CAT-1. Link to supplemental material: http://www.landesbioscience.com/supplement/changRNA1-2-sup.pdf

Molecular biology of hepatitis B virus infection.

Human hepatitis B virus (HBV) is the prototype of a family of small DNA viruses that productively infect hepatocytes, the major cell of the liver, and replicate by reverse transcription of a terminally redundant viral RNA, the pregenome. Upon infection, the circular, partially double-stranded virion DNA is converted in the nucleus to a covalently closed circular DNA (cccDNA) that assembles into a minichromosome, the template for viral mRNA synthesis. Infection of hepatocytes is non-cytopathic. Infection of the liver may be either transient (<6 months) or chronic and lifelong, depending on the ability of the host immune response to clear the infection. Chronic infections can cause immune-mediated liver damage progressing to cirrhosis and hepatocellular carcinoma (HCC). The mechanisms of carcinogenesis are unclear. Antiviral therapies with nucleoside analog inhibitors of viral DNA synthesis delay sequelae, but cannot cure HBV infections due to the persistence of cccDNA in hepatocytes.

Genetic recombinants and heterozygotes derived from endogenous and exogenous avian RNA tumor viruses.

Abstract A selective technique based on infective centers is described for isolating host range recombinants of avian RNA tumor viruses. This method was exploited to obtain recombinants between the genome of chick helper factor ( chf ) and nondefective strains of Rous sarcoma virus (RSV), including temperature-sensitive mutants. Recombinants of chf with the defective Bryan strain RSV could not be isolated. A majority of nondefective RSV particles selected as carrying the chf host range also carried the parental RSV host range; i.e., were of dual host range, but the progeny segregated into parental or recombinant genotypes. These observations suggested that the particles were at least partially diploid or polyploid and represented unstable “heterozygotes.” Genotypic mixing was not evident in chf -negative cells which contain viral DNA but not viral RNA, suggesting that genetic reassortment occurs among RNA molecules. A model is proposed in which reassortment of independent genome segments may be converted into stable recombinants following provirus formation in the next replicative cycle.

Kinetics of Hepadnavirus Loss from the Liver during Inhibition of Viral DNA Synthesis

ABSTRACT Hepadnaviruses replicate by reverse transcription, which takes place in the cytoplasm of the infected hepatocyte. Viral RNAs, including the pregenome, are transcribed from a covalently closed circular (ccc) viral DNA that is found in the nucleus. Inhibitors of the viral reverse transcriptase can block new DNA synthesis but have no direct effect on the up to 50 or more copies of cccDNA that maintain the infected state. Thus, during antiviral therapy, the rates of loss of cccDNA, infected hepatocytes (1 or more molecules of cccDNA), and replicating DNAs may be quite different. In the present study, we asked how these losses compared when woodchucks chronically infected with woodchuck hepatitis virus were treated with L-FMAU [1-(2-fluoro-5-methyl-β-l-arabinofuranosyl) uracil], an inhibitor of viral DNA synthesis. Viremia was suppressed for at least 8 months, after which drug-resistant virus began replicating to high titers. In addition, replicating viral DNAs were virtually absent from the liver after 6 weeks of treatment. In contrast, cccDNA declined more slowly, consistent with a half-life of ∼33 to 50 days. The loss of cccDNA was comparable to that expected from the estimated death rate of hepatocytes in these woodchucks, suggesting that death of infected cells was one of the major routes for elimination of cccDNA. However, the decline in the actual number of infected hepatocytes lagged behind the decline in cccDNA, so that the average cccDNA copy number in infected cells dropped during the early phase of therapy. This observation was consistent with the possibility that some fraction of cccDNA was distributed to daughter cells in those infected hepatocytes that passed through mitosis.

In hepatocytes infected with duck hepatitis B virus, the template for viral RNA synthesis is amplified by an intracellular pathway

During the productive phase of chronic hepadnaviral infections, virion DNA synthesis occurs in the cytoplasm of the infected hepatocyte, but viral RNA is synthesized in the nucleus, apparently from a covalently closed, circular (CCC) viral DNA. J. Tuttleman, C. Pourcel, and J. Summers (1986a, Cell 47, 451-460) have shown that the intracellular levels of CCC DNA can increase during initiation of infection of duck hepatocytes in vitro with duck hepatitis B virus and during long term culture of infected duck hepatocytes in vitro. This amplification of CCC DNA occurs through the reverse transcription pathway. To distinguish between an entirely intracellular process of amplification and amplification due to multiple infections by extracellular virus in the virus producing cultures, suramin was added to the infected cultures to block superinfection. We found that CCC DNA amplification occurred at least as efficiently in the presence of suramin as in its absence. First, there was a net increase in the total amount of CCC DNA in the cultures both in the presence and in the absence of suramin. Second, synthesis of CCC DNA in the presence and absence of suramin was observed by density labeling of this viral DNA by growth of the cultures in medium containing BUdR. Amplification was also demonstrable in the presence of neutralizing duck antibodies. These results support the hypothesis of Tuttleman et al. (1986a) that CCC DNA amplification in chronically infected cultures and, by inference, the mechanism of persistent infection involves primarily intracellular regulatory mechanisms.

Apoptosis and Regeneration of Hepatocytes during Recovery from Transient Hepadnavirus Infections

ABSTRACT It is well known that hepatitis B virus infections can be transient or chronic, but the basis for this dichotomy is not known. To gain insight into the mechanism responsible for the clearance of hepadnavirus infections, we have performed a molecular and histologic analysis of liver tissues obtained from transiently infected woodchucks during the critical phase of the recovery period. We found as expected that clearance from transient infections occurred subsequent to the appearance of CD4+ and CD8+ T cells and the production of interferon gamma and tumor necrosis factor alpha in the infected liver. These events were accompanied by a significant increase in apoptosis and regeneration of hepatocytes. Surprisingly, however, accumulation of virus-free hepatocytes was delayed for several weeks following this initial influx of lymphocytes. In addition, we observed that chronically infected animals can exhibit levels of T-cell accumulation, cytokine expression, and apoptosis that are comparable with those observed during the initial phase of transient infections. Our results are most consistent with a model for recovery predicting replacement of infected hepatocytes with regenerated cells, which by unknown mechanisms remain protected from reinfection in animals that can be cured.

Towards an HBV cure: state-of-the-art and unresolved questions—report of the ANRS workshop on HBV cure

HBV infection is a major cause of liver cirrhosis and hepatocellular carcinoma. Although HBV infection can be efficiently prevented by vaccination, and treatments are available, to date there is no reliable cure for the >240 million individuals that are chronically infected worldwide. Current treatments can only achieve viral suppression, and lifelong therapy is needed in the majority of infected persons. In the framework of the French National Agency for Research on AIDS and Viral Hepatitis ‘HBV Cure’ programme, a scientific workshop was held in Paris in June 2014 to define the state-of-the-art and unanswered questions regarding HBV pathobiology, and to develop a concerted strategy towards an HBV cure. This review summarises our current understanding of HBV host-interactions leading to viral persistence, as well as the roadblocks to be overcome to ultimately address unmet medical needs in the treatment of chronic HBV infection.

Hepatocyte turnover during resolution of a transient hepadnaviral infection

We estimated the amount of hepatocyte turnover in the livers of three woodchucks undergoing clearance of a transient woodchuck hepatitis infection by determining the fate of integrated viral DNA as a genetic marker of the infected cell population. Integrated viral DNA was found to persist in liver tissue from recovered animals at essentially undiminished levels of 1 viral genome per 1,000–3,000 liver cells, suggesting that the hepatocytes in the recovered liver were derived primarily from the infected cell population. We determined the single and multicopy distribution of distinct viral cell junctions isolated from small pieces of liver after clearance of the infection to determine the cumulative amount of hepatocyte proliferation that had occurred during recovery. We estimated that proliferation was equivalent to a minimum of 0.7–1 complete random turnovers of the hepatocyte population of the liver. Our results indicated that during resolution of the transient infections a large fraction of the infected hepatocyte population was killed and replaced by hepatocyte cell division.