Background & Aims: Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) is a

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Tenofovir is an acyclic nucleotide analog with activity against human immunodeficiency virus (HIV) and hepatitis B virus (HBV). Tenofovir disoproxil fumarate (tenofovir DF), a bis-alkoxyester prodrug of tenofovir, is approved for the treatment of HIV and is currently being developed to treat chronic hepatitis B. In this report, we further characterize the in vitro activity of tenofovir against HBV as well as its metabolism in hepatic cells. We show that tenofovir is efficiently phosphorylated to tenofovir diphosphate (TFV-DP) in both HepG2 cells and primary human hepatocytes. TFV-DP has a long intracellular half-life (95 h) and is a potent and competitive inhibitor of HBV polymerase (K i ‫؍‬ 0.18 M). Tenofovir has a 50% effective concentration of 1.1 M against HBV in cell-based assays, and potency is improved >50-fold by the addition of bis-isoproxil progroups. Tenofovir has previously demonstrated full activity against lamivudine-resistant HBV in vitro and clinically. Here we show that the major adefovir resistance mutation, rtN236T, confers three-to fourfoldreduced susceptibility to tenofovir in cell culture; the clinical significance of this susceptibility shift has not yet been determined. The rtA194T HBV polymerase mutation recently identified in tenofovir DF-treated HIV/ HBV-coinfected patients did not confer in vitro resistance to tenofovir as a single mutation or in a lamivudineresistant viral background. Overall, the antiviral and metabolic profile of tenofovir supports its development for the treatment of chronic hepatitis B.

The structural maintenance of chromosome 5/6 complex (Smc5/6) is a restriction factor that represses hepatitis B virus (HBV) transcription. HBV counters this restriction by expressing HBV X protein (HBx), which targets Smc5/6 for degradation. However, the mechanism by which Smc5/6 suppresses HBV transcription and how HBx is initially expressed is not known. In this study we characterized viral kinetics and the host response during HBV infection of primary human hepatocytes (PHH) to address these unresolved questions. We determined that Smc5/6 localizes with Nuclear Domain 10 (ND10) in PHH. Co-localization has functional implications since depletion of ND10 structural components alters the nuclear distribution of Smc6 and induces HBV gene expression in the absence of HBx. We also found that HBV infection and replication does not induce a prominent global host transcriptional response in PHH, either shortly after infection when Smc5/6 is present, or at later times post-infection when Smc5/6 has been degraded. Notably, HBV and an HBx-negative virus establish high level infection in PHH without inducing expression of interferon-stimulated genes or production of interferons or other cytokines. Our study also revealed that Smc5/6 is degraded in the majority of infected PHH by the time cccDNA transcription could be detected and that HBx RNA is present in cell culture-derived virus preparations as well as HBV patient plasma. Collectively, these data indicate that Smc5/6 is an intrinsic antiviral restriction factor that suppresses HBV transcription when localized to ND10 without inducing a detectable innate immune response. Our data also suggest that HBx protein may be initially expressed by delivery of extracellular HBx RNA into HBV-infected cells.

Therapy of chronic hepatitis B virus (HBV) infection with the polymerase inhibitor lamivudine frequently is associated with the emergence of viral resistance. Genotypic changes in the YMDD motif (reverse transcriptase[rt] mutations rtM204V/I) conferred resistance to lamivudine as well as reducing the in vitro replication efficiency of HBV. A second mutation, rtL180M, was previously reported to partially restore replication fitness as well as to augment drug resistance in vitro. Here we report the functional characterization of a third polymerase mutation (rtV173L) associated with resistance to lamivudine and famciclovir. rtV173L was observed at baseline in 9 to 22% of patients who entered clinical trials of adefovir dipivoxil for the treatment of lamivudine-resistant HBV. In these patients, rtV173L was invariably found as a third mutation in conjunction with rtL180M and rtM204V. In vitro analyses indicated that rtV173L did not alter the sensitivity of wild-type or lamivudine-resistant HBV to lamivudine, penciclovir, or adefovir but instead enhanced viral replication efficiency. A molecular model of HBV polymerase indicated that residue rtV173 is located beneath the template strand of HBV nucleic acid near the active site of the reverse transcriptase. Substitution of leucine for valine at this residue may enhance polymerization either by repositioning the template strand of nucleic acid or by affecting other residues involved in the polymerization reaction. Together, these results suggest that rtV173L is a compensatory mutation that is selected in lamivudine-resistant patients due to an enhanced replication phenotype.Until the recent approval of adefovir dipivoxil, lamivudine (a dideoxycytidine analog in the unnatural L configuration) was the only approved oral therapy for the treatment of chronic hepatitis B. Antiviral therapy for chronic hepatitis B with famciclovir and lamivudine has been limited by the emergence of viral resistance in significant proportions of patients. Although lamivudine therapy results in potent reductions in viremia, relapse is common, as resistant viruses emerge in approximately 24% of patients after 1 year of therapy and 70% after 4 years of therapy (20). Sequencing of hepatitis B virus (HBV) isolates from patients for whom lamivudine treatment failed revealed a mutation of methionine to valine or isoleucine at position rt204 (rtM204V/I) in the YMDD motif of the C subdomain of HBV polymerase (3, 21); amino acid residues in HBV polymerase are numbered according to the consensus nomenclature developed by Stuyver et al. (34). A second mutation, of leucine 180 to methionine (rtL180M), in the upstream B subdomain of HBV polymerase frequently accompanies rtM204 mutations. The rtM204V mutation almost invariably occurs in tandem with rtL180M, while rtM204I can occur as a single mutation or in conjunction with rtL180M.In vitro analyses have confirmed and characterized the role of the major HBV polymerase mutations in lamivudine resistance. Cell culture and enzyme assays have revealed that rtM20...

Hepatitis B virus (HBV) is a small, double-stranded DNA virus and is the prototype of the hepadnavirus family. HBV is a human pathogen capable of causing both acute and chronic hepatitis. The World Health Organization currently estimates that 350 million people are chronically infected with HBV. Persistent HBV infection is also associated with an increased risk of cirrhosis and hepatocellular carcinoma. 1 Although a tremendous amount is known about HBV, our knowledge of the virus is by no means complete. Historically, major obstacles in the study of HBV have been the inability of the virus to infect cells in vitro, and the lack of animal model systems due to a strict virus-host range. Thus, many aspects of HBV biology have been unraveled by studying related hepadnaviruses, such as the duck hepatitis virus which is capable of in vitro infection, 2 and the woodchuck hepatitis virus which allows for the in vivo study in an animal model system. 3 The duck hepatitis virus and woodchuck hepatitis virus systems were instrumental in developing an understanding of the hepadnavirus lifecycle and remain valuable models for HBV infection. However, many significant differences exist between animal hepadnaviruses and HBV. For example, avian hepatitis viruses do not encode the X gene, 4 and major transcriptional differences between woodchuck hepatitis virus and HBV have been reported. 5 Within the last decade, several HBV expressing cell lines have been established by transfecting viral DNA into liverderived human cell lines and by selecting novel cell lines containing stably integrated HBV genomes. [6][7][8] The most widely used are the HepG2 2.2.15 cell line (2.2.15) derived from the HepG2 hepatoblastoma cell line and HB611 derived from the HuH6 hepatoma cell line. These and other cell lines have led to considerable progress in the study of HBV in vitro. However, there are some inherent drawbacks which preclude the use of these cell lines in studying some aspects of HBV biology. (1) Many HBV expressing cell lines were created using constructs containing strong heterologous promoters proximal to the HBV genome. The effect those promoters have on HBV transcription and replication is unclear but could differ substantially from what occurs in a natural infection in vivo in which HBV gene expression is driven solely by endogenous HBV promoters. (2) Cell lines commonly used to study HBV contain multiple copies of integrated HBV DNA. Unlike retroviruses, which integrate viral DNA into the host genome, hepadnavirus genomes are not integrated routinely but, instead, are maintained in the nucleus of infected cells in vivo as a pool of episomal covalently closed circular (CCC) DNA molecules. 9 Although the integration of HBV DNA in human liver has been reported, 10 it is not an obligatory part of the HBV lifecycle. HBV does not encode any machinery for integration into the host genome, and integration is not required for HBV replication. In addition, when integrated HBV DNA is found,

Multiple subunits of the hepatitis B virus (HBV) core protein (HBc) assemble into an icosahedral capsid that packages the viral pregenomic RNA (pgRNA). The N-terminal domain (NTD) of HBc is sufficient for capsid assembly, in the absence of pgRNA or any other viral or host factors, under conditions of high HBc and/or salt concentrations. The C-terminal domain (CTD) is deemed dispensable for capsid assembly although it is essential for pgRNA packaging. We report here that HBc expressed in a mammalian cell lysate, rabbit reticulocyte lysate (RRL), was able to assemble into capsids when (low-nanomolar) HBc concentrations mimicked those achieved under conditions of viral replication in vivo and were far below those used previously for capsid assembly in vitro. Furthermore, at physiologically low HBc concentrations in RRL, the NTD was insufficient for capsid assembly and the CTD was also required. The CTD likely facilitated assembly under these conditions via RNA binding and protein-protein interactions. Moreover, the CTD underwent phosphorylation and dephosphorylation events in RRL similar to those seen in vivo which regulated capsid assembly. Importantly, the NTD alone also failed to accumulate in mammalian cells, likely resulting from its failure to assemble efficiently. Coexpression of the full-length HBc rescued NTD assembly in RRL as well as NTD expression and assembly in mammalian cells, resulting in the formation of mosaic capsids containing both full-length HBc and the NTD. These results have important implications for HBV assembly during replication and provide a facile cell-free system to study capsid assembly under physiologically relevant conditions, including its modulation by host factors. IMPORTANCE Hepatitis B virus (HBV) is an important global human pathogen and the main cause of liver cancerworldwide. An essential component of HBV is the spherical capsid composed of multiple copies of a single protein, the core protein (HBc). We have developed a mammalian cell-free system in which HBc is expressed at physiological (low) concentrations and assembles into capsids under near-physiological conditions. In this cell-free system, as in mammalian cells, capsid assembly depends on the C-terminal domain (CTD) of HBc, in contrast to other assembly systems in which HBc assembles into capsids independently of the CTD under conditions of nonphysiological protein and salt concentrations. Furthermore, the phosphorylation state of the CTD regulates capsid assembly and RNA encapsidation in the cell-free system in a manner similar to that seen in mammalian cells. This system will facilitate detailed studies on capsid assembly and RNA encapsidation under physiological conditions and identification of antiviral agents that target HBc.

Hepatitis B e antigen (HBeAg) negative chronic hepatitis B (CHB) is frequently caused by a mutation (G1896A) in the hepatitis B virus (HBV) precore (PC) reading frame that creates a stop codon, causing premature termination of the PC protein. During lamivudine treatment, drug resistance develops at a similar rate in HBeAg positive and HBeAg negative CHB. Lamivudine-resistant HBV mutants have been shown to replicate inefficiently in vitro in the absence of PC mutations, but it is unknown whether the presence of PC mutations affects replication efficiency or antiviral sensitivity. This study utilized the recombinant HBV baculovirus system to address these issues. HBV baculoviruses encoding the G1896A PC stop codon mutation were generated in wild-type (WT) and lamivudine-resistant (rtM204I and rtL180M ؉ rtM204V) backgrounds, resulting in a panel of 6 related recombinant baculoviruses. In vitro assays were performed to compare the sensitivities of the PC mutant viruses with lamivudine and adefovir and to compare relative replication yields. The PC mutation did not significantly affect sensitivities to either adefovir or lamivudine. WT HBV and PC mutant HBV showed similar replication yields, whereas the replication yields of the lamivudine-resistant mutants were greatly reduced in HBeAg positive HBVs, confirming previous observations. However, the presence of the PC mutation was found to compensate for the replication deficiency in each of the lamivudine-resistant mutants, increasing the replication yields of each virus. In conclusion, the PC stop codon mutation appears to increase the replication efficacy of lamivudine-resistant virus but does not affect in vitro drug sensitivity. (HEPATOLOGY 2003;37:27-35.) H epatitis B e antigen (HBeAg) negative chronic hepatitis B (CHB), a phase in the natural history of CHB, is marked by the selection of hepatitis B viruses (HBV) unable to secrete HBeAg and has become the major form of disease presentation in many parts of the world. 1 The most common of several mutations that can cause HBeAg negativity is a guanine to adenine transition at nucleotide position 1,896 (G1896A), which creates a TAG stop codon at codon 28 of the precore (PC) protein. [1][2][3][4][5] Interferon alfa and lamivudine are the only therapeutic agents approved for treatment of CHB. 6 Lamivudine is regarded as safe and as efficacious as interferon alfa, but the percentage of patients with HBeAg positive CHB who undergo HBeAg seroconversion increases with the duration of treatment. 7 Seroconversion rates between 11% and 15% per year have been reported over treatment periods up to 3 years. [7][8][9][10] Unfortunately, the frequency of antiviral drug resistance increases with the duration of therapy, rising to as high as 66% after 3 years. 10,11 The most common mutations conferring lamivudine resistance affect the active site YMDD (tyrosine-methionine-aspartate-aspartate) motif in the C domain of the HBV polymerase protein, causing the methionine (M) residue (amino acid 204) to be replaced with either isole...