Hepadnavirus envelope proteins regulate covalently closed circular DNA amplification.

Primary duck hepatocytes were infected with a mutant duck hepatitis B virus defective in envelope protein but competent for viral DNA synthesis. Cells infected by this mutant accumulated higher levels of viral covalently closed, circular DNA (cccDNA) than those infected by wild-type virus. The accumulation of high levels of cccDNA was due to a failure of the mutant-infected cells to suppress de novo cccDNA synthesis compared with suppression by cells infected by the wild type. The envelope-defective virus failed to establish a persistent infection in vitro, possibly because of a virus-mediated cell death. Therefore, one or both viral envelope proteins are required for regulation of cccDNA synthesis and for maintenance of persistent infection in vitro.     

Roles of the envelope proteins in the amplification of covalently closed circular DNA and completion of synthesis of the plus-strand DNA in hepatitis B virus

Covalently closed circular DNA (cccDNA), the nuclear form of hepatitis B virus (HBV), is synthesized by repair of the relaxed circular (RC) DNA genome. Initially, cccDNA is derived from RC DNA from the infecting virion, but additional copies of cccDNA are derived from newly synthesized RC DNA molecules in a process termed intracellular amplification. It has been shown that the large viral envelope protein limits the intracellular amplification of cccDNA for duck hepatitis B virus. The role of the envelope proteins in regulating the amplification of cccDNA in HBV is not well characterized. The present report demonstrates regulation of synthesis of cccDNA by the envelope proteins of HBV. Ablation of expression of the envelope proteins led to an increase (>6-fold) in the level of cccDNA. Subsequent restoration of envelope protein expression led to a decrease (>50%) in the level of cccDNA, which inversely correlated with the level of the envelope proteins. We found that the expression of L protein alone or in combination with M and/or S proteins led to a decrease in cccDNA levels, indicating that L contributes to the regulation of cccDNA. Coexpression of L and M led to greater regulation than either L alone or L and S. Coexpression of all three envelope proteins was also found to limit completion of plus-strand DNA synthesis, and the degree of this effect correlated with the level of the proteins and virion secretion.     

Generation of Covalently Closed Circular DNA of Hepatitis B Viruses via Intracellular Recycling Is Regulated in a Virus Specific Manner

Persistence of hepatitis B virus (HBV) infection requires covalently closed circular (ccc)DNA formation and amplification, which can occur via intracellular recycling of the viral polymerase-linked relaxed circular (rc) DNA genomes present in virions. Here we reveal a fundamental difference between HBV and the related duck hepatitis B virus (DHBV) in the recycling mechanism. Direct comparison of HBV and DHBV cccDNA amplification in cross-species transfection experiments showed that, in the same human cell background, DHBV but not HBV rcDNA converts efficiently into cccDNA. By characterizing the distinct forms of HBV and DHBV rcDNA accumulating in the cells we find that nuclear import, complete versus partial release from the capsid and complete versus partial removal of the covalently bound polymerase contribute to limiting HBV cccDNA formation; particularly, we identify genome region-selectively opened nuclear capsids as a putative novel HBV uncoating intermediate. However, the presence in the nucleus of around 40% of completely uncoated rcDNA that lacks most if not all of the covalently bound protein strongly suggests a major block further downstream that operates in the HBV but not DHBV recycling pathway. In summary, our results uncover an unexpected contribution of the virus to cccDNA formation that might help to better understand the persistence of HBV infection. Moreover, efficient DHBV cccDNA formation in human hepatoma cells should greatly facilitate experimental identification, and possibly inhibition, of the human cell factors involved in the process.     

Inhibition of Hepatitis B Virus cccDNA by siRNA in Transgenic Mice

The elimination of viral covalently closed circular DNA (cccDNA) from the nucleus of infected hepatocytes is an obstacle to achieving sustained viral clearance during antiviral therapy of chronic hepatitis B virus (HBV) infection. The aim of our study was to determine whether treatment with siRNA is able to suppress viral cccDNA amplification using a HBV-transgenic mice model. The experimental results revealed that siRNAs can serve as efficient alternative anti-HBV agents, because they showed better inhibitory effect on viral replication and antigen expression in transgenic mice. More importantly, the siRNA markedly inhibited HBV cccDNA amplification.     

Therapeutic Antiviral Effect of the Nucleic Acid Polymer REP 2055 against Persistent Duck Hepatitis B Virus Infection

Previous studies have demonstrated that nucleic acid polymers (NAPs) have both entry and post-entry inhibitory activity against duck hepatitis B virus (DHBV) infection. The inhibitory activity exhibited by NAPs prevented DHBV infection of primary duck hepatocytes in vitro and protected ducks from DHBV infection in vivo and did not result from direct activation of the immune response. In the current study treatment of primary human hepatocytes with NAP REP 2055 did not induce expression of the TNF, IL6, IL10, IFNA4 or IFNB1 genes, confirming the lack of direct immunostimulation by REP 2055. Ducks with persistent DHBV infection were treated with NAP 2055 to determine if the post-entry inhibitory activity exhibited by NAPs could provide a therapeutic effect against established DHBV infection in vivo. In all REP 2055-treated ducks, 28 days of treatment lead to initial rapid reductions in serum DHBsAg and DHBV DNA and increases in anti-DHBs antibodies. After treatment, 6/11 ducks experienced a sustained virologic response: DHBsAg and DHBV DNA remained at low or undetectable levels in the serum and no DHBsAg or DHBV core antigen positive hepatocytes and only trace amounts of DHBV total and covalently closed circular DNA (cccDNA) were detected in the liver at 9 or 16 weeks of follow-up. In the remaining 5/11 REP 2055-treated ducks, all markers of DHBV infection rapidly rebounded after treatment withdrawal: At 9 and 16 weeks of follow-up, levels of DHBsAg and DHBcAg and DHBV total and cccDNA in the liver had rebounded and matched levels observed in the control ducks treated with normal saline which remained persistently infected with DHBV. These data demonstrate that treatment with the NAP REP 2055 can lead to sustained control of persistent DHBV infection. These effects may be related to the unique ability of REP 2055 to block release of DHBsAg from infected hepatocytes.     

Coordinate regulation of replication and virus assembly by the large envelope protein of an avian hepadnavirus.

We have used linker scanning and site-directed mutagenesis in an attempt to distinguish among the known functions of the duck hepatitis B virus large envelope protein, p36. We found that linker-encoded amino acid substitutions in at least one region of the pre-S envelope protein p36 produced defects in both the production of enveloped virus and the regulation of covalently closed circular DNA (cccDNA) synthesis. Most linker substitutions, typically in the 5' two-thirds of the pre-S region of the p36 gene did not affect either cccDNA regulation or enveloped virus production but did destroy the infection competence of the enveloped particles produced. Single amino acid substitutions of residues 128 and 131 demonstrated a similar correlation between defects in the ability of p36 to support enveloped virus production and to control cccDNA levels. We concluded from these studies that virus production and cccDNA regulation probably require a common activity of p36.     

Characterization of the Host Factors Required for Hepadnavirus Covalently Closed Circular (ccc) DNA Formation

Synthesis of the covalently closed circular (ccc) DNA is a critical, but not well-understood step in the life cycle of hepadnaviruses. Our previous studies favor a model that removal of genome-linked viral DNA polymerase occurs in the cytoplasm and the resulting deproteinized relaxed circular DNA (DP-rcDNA) is subsequently transported into the nucleus and converted into cccDNA. In support of this model, our current study showed that deproteinization of viral double-stranded linear (dsl) DNA also took place in the cytoplasm. Furthermore, we demonstrated that Ku80, a component of non-homologous end joining DNA repair pathway, was essential for synthesis of cccDNA from dslDNA, but not rcDNA. In an attempt to identify additional host factors regulating cccDNA biosynthesis, we found that the DP-rcDNA was produced in all tested cell lines that supported DHBV DNA replication, but cccDNA was only synthesized in the cell lines that accumulated high levels of DP-rcDNA, except for NCI-H322M and MDBK cells, which failed to synthesize cccDNA despite of the existence of nuclear DP-rcDNA. The results thus imply that while removal of the genome-linked viral DNA polymerase is most likely catalyzed by viral or ubiquitous host function(s), nuclear factors required for the conversion of DP-rcDNA into cccDNA and/or its maintenance are deficient in the above two cell lines, which could be useful tools for identification of the elusive host factors essential for cccDNA biosynthesis or maintenance.     

Protease-induced infectivity of hepatitis B virus for a human hepatoblastoma cell line.

The human hepatoblastoma cell line HepG2 produces and secretes hepatitis B virus (HBV) after transfection of cloned HBV DNA. Intact virions do not infect these cells, although they attach to the surface of the HepG2 cell through binding sites in the pre-S1 domain. Entry of enveloped virions into the cell often requires proteolytic cleavage of a viral surface protein that is involved in fusion between the cell membrane and the viral envelope. Recently, we observed pre-S-independent, nonspecific binding between hepatitis B surface (HBs) particles and HepG2 cells after treatment of HBs antigen particles with V8 protease, which cleaves next to a putative fusion sequence. Chymotrypsin removed this fusion sequence and did not induce binding. In this study, we postulate that lack of a suitable fusion-activating protease was the reason why the HepG2 cells were not susceptible to HBV. To test this hypothesis, virions were partially purified from the plasma of HBV carriers and treated with either staphylococcal V8 or porcine chymotrypsin protease. Protease-digested virus lost reactivity with pre-S2-specific antibody but remained morphologically intact as determined by electron microscopy. After separation from the proteases, virions were incubated with HepG2 cells at pH 5.5. Cultures inoculated with either intact or chymotrypsin-digested virus did not contain detectable levels of intracellular HBV DNA at any time following infection. However, in cultures inoculated with V8-digested virions, HBV-specific products, including covalently closed circular DNA, viral RNA, and viral pre-S2 antigen, could be detected in a time-dependent manner following infection. Immunofluorescence analysis revealed that 10 to 30% of the infected HepG2 cells produced HBV antigen. Persistent secretion of virus by the infected HepG2 cells lasted at least 14 days and was maintained during several reseeding steps. The results show that V8-digested HBV can productively infect tissue cultures of HepG2 cells. It is suggested that proteolysis-dependent exposure of a fusion domain within the envelope protein of HBV is necessary during natural infection.     

Formation of covalently closed circular DNA in Hep38.7-Tet cells,a tetracycline inducible hepatitis B virus expression cell line

Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) plays a central role in chronic HBV infection. However, analysis of the molecular mechanism of cccDNA formation is difficult because of the low efficiency in tissue cultured cells. In this study, we developed a more efficient cccDNA expression cell, Hep38.7-Tet, by subcloning from a tetracycline inducible HBV expression cell, HepAD38. Higher levels of cccDNA were produced in Hep38.7-Tet cells compared to HepAD38 cells. In Hep38.7-Tet cells, the cccDNA was detectable at six days after HBV induction. HBV e antigen (HBeAg) secretion was dependent upon cccDNA production. We screened chemical compounds using Hep38.7-Tet cells and HBeAg secretion as a marker. Most of the hit compounds have already been reported as anti-HBV compounds. These data suggested that Hep38.7-Tet cells will be powerful tools for analysis of the molecular mechanism of cccDNA formation/maintenance and development of novel therapeutic agents to control HBV infection.     

Distinct Distribution Pattern of Hepatitis B Virus Genotype C and D in Liver Tissue and Serum of Dual Genotype Infected Liver Cirrhosis and Hepatocellular Carcinoma Patients

Aims

The impact of co-infection of several hepatitis B virus (HBV) genotypes on the clinical outcome remains controversial. This study has for the first time investigated the distribution of HBV genotypes in the serum and in the intrahepatic tissue of liver cirrhotic (LC) and hepatocellular carcinoma (HCC) patients from India. In addition, the genotype-genotype interplay and plausible mechanism of development of HCC has also been explored.

Methods

The assessment of HBV genotypes was performed by nested PCR using either surface or HBx specific primers from both the circulating virus in the serum and replicative virus that includes covalently closed circular DNA (cccDNA) and relaxed circular DNA (rcDNA) of HBV from the intrahepatic tissue. The integrated virus within the host chromosome was genotyped by Alu-PCR method. Each PCR products were cloned and sequences of five randomly selected clones were subsequently analysed.

Results

HBV/genotype D was detected in the serum of all LC and HCC patients whereas the sequences of the replicative HBV DNA (cccDNA and rcDNA) from the intrahepatic tissue of the same patients revealed the presence of both HBV/genotype C and D. The sequences of the integrated viruses exhibited the solo presence of HBV/genotype C in the majority of LC and HCC tissues while both HBV/genotype C and D clones were found in few patients in which HBV/genotype C was predominated. Moreover, compared to HBV/genotype D, genotype C had higher propensity to generate double strand breaks, ER stress and reactive oxygen species and it had also showed higher cellular homologous-recombination efficiency that engendered more chromosomal rearrangements, which ultimately led to development of HCC.

Conclusions

Our study highlights the necessity of routine analysis of HBV genotype from the liver tissue of each chronic HBV infected patient in clinical practice to understand the disease prognosis and also to select therapeutic strategy.     

Analysis of host range phenotypes of primate hepadnaviruses by in vitro infections of hepatitis D virus pseudotypes

Hepatitis B virus (HBV) and woolly monkey hepatitis B virus (WMHBV) have natural host ranges that are limited to closely related species. The barrier for infection of primates seems to be at the adsorption and/or entry steps of the viral replication cycle, since a human hepatoma cell line is permissive for HBV and WMHBV replication following transfection of cloned DNA. We hypothesized that the HBV and WMHBV envelope proteins contain the principal viral determinants of host range. As previously shown by using the hepatitis D virus (HDV) system, recombinant HBV-HDV particles were infectious in chimpanzee as well as human hepatocytes. We extended the HDV system to include HDV particles pseudotyped with the WMHBV envelope. In agreement with the natural host ranges of HBV and WMHBV, in vitro infections demonstrated that HBV-HDV and WM-HDV particles preferentially infected human and spider monkey cells, respectively. Previous studies have implicated the pre-S1 region of the large (L) envelope protein in receptor binding and host range; therefore, recombinant HDV particles were pseudotyped with the hepadnaviral envelopes containing chimeric L proteins with the first 40 amino acids from the pre-S1 domain exchanged between HBV and WMHBV. Surprisingly, addition of the human amino terminus to the WMHBV L protein increased infectivity on spider monkey hepatocytes but did not increase infectivity for human hepatocytes. Based upon these data, we discuss the possibility that the L protein may be comprised of two domains that affect infectivity and that sequences downstream of residue 40 may influence host range and receptor binding or entry.