Molecular Mechanisms Of Hepatitis B Viral infection, Pathogenesis And Persistence

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

Hepatitis B virus (HBV) consists of 9 major genotypes (A to I), one minor strain (designated J) and multiple subtypes, which may have different natural history, disease progression and treatment response. As only cell lines expressing genotype D3 have been established, here we aim to establish stable cell lines producing high-titer cell culture-generated HBV (HBVcc) of different genotypes and to explore their infectivity, virological features and treatment response. Stable cell lines producing high titer of HBV with genotypes A2, B2, C1, E, F1b and H were generated by transfecting replication-competent 1.3Xlength HBV genome in a plasmid containing an antibiotic marker in HepG2 cells that can support HBV replication. Clones with highest levels of HBV DNA and/or HBeAg were selected and expanded for large-scale purification of HBVcc. HBVcc of different genotypes were tested in cells and humanized chimeric mouse model. All HBVcc infected mouse-passaged primary human hepatocytes (PXB cells) and genotypes exhibit different responses to human IFN-a with variable kinetics of reduction in HBV DNA, HBeAg and HBsAg. HBVcc of all genotypes were infectious in humanized chimeric mice but with variable kinetics of viremia and viral antigen production. Treatment of infected mice with human IFN-a resulted in modest and variable reductions of viremia and viral antigenemia. HBVcc passaged in humanized chimeric mice (HBVmp) infected PXB cells much more efficiently than that of the original HBVcc viral stock. Here we generate stable cell lines producing HBV of various genotypes that are infectious in vitro and in vivo. We observe genotype-associated variations in viral antigen production, infection kinetics and responses to human IFN- treatment in these models. HBV infects hepatocytes and establishes itself within the nucleus as a mini-chromosome referred to as covalently closed circular DNA (cccDNA), which serves as the transcriptional template for all viral products and is capable of replenishing itself. Its stability accounts for the long-term persistence of HBV in the liver of infected patients treated with nucleos(t)ide inhibitors, which are effective in blocking viral replication but have little or no impact on eliminating HBV cccDNA. Therefore, identifying cccDNA-associated host factors and their functions is not only important in understanding the basic biology of HBV but also relevant to designing novel strategies to target cccDNA for potential curative therapy. Recently we isolated and purified cccDNA-associated host proteins from HBV-infected cells and identified by mass spec Nucleolin as a key cccDNA-binding protein that plays an important role in HBV infection. Nucleolin is an integral component of the HBV cccDNA mini-chromosome and exerts epigenetic regulation of HBV transcription. The mechanism of epigenetic regulation whereby Nucleolin interacts with and functionally modulates the epigenetic machinery of cccDNA remains to be further elucidated. Current antiviral therapies do not cure CHB due to their failure in the eradication of HBV covalently closed circular DNA (cccDNA) that is the persistent form of HBV genome in hepatocytes and template of viral transcript synthesis. Smc5/6 has been identified as a restriction factor for HBV that directly binds cccDNA and blocks its transcription. The HBV protein HBx directs the Smc5/6 for ubiquitination and degradation by the proteasome, thus allowing productive HBV gene expression from the cccDNA. p97/VCP (valosin-containing protein), a cytosolic ATPase protein, plays an essential role in targeting ubiquitinated proteins and directs them to the proteasome for degradation. Thus p97 plays an important role in HBV cccDNA transcription and replication. Primary human hepatocytes (PHH) were transfected with nontargeting control (siNT), p97 and Smc5/6 siRNAs. Cells were then infected with WT or HBx-deficient HBV and treated with various inhibitors targeting the ATPase function of p97. All HBV markers as well as host mRNAs and proteins expressions were quantified at day 6 post-infection. A cccDNA HBV reporter system (mcHBV-Gluc) was used to assess the direct effects of various treatments on the transcriptional activity of cccDNA. HBV-infected cells treated with sip97 showed substantially reduced secreted and intracellular HBV markers without showing cell toxicity, when compared to the siNT-treated cells. siSmc5/6 treatment markedly increased HBV replication in cells infected with HBVx- virus, consistent with the role of HBx in Smc5/6-mediated restriction of HBV infection. Sip97 treatment further suppressed HBV replication in HBVx- infected and siSmc5/6 treated cells. Sip97 treatment resulted in similarly suppressed HBV reporter activities in the mcHBV-Gluc transfected cells. The transcriptional effect of sip97 seemed to rely on the native episomal form of cccDNA as other reporter constructs directed by HBV promoters/enhancer in either episomal or chromosomal form. Interestingly, p97 ATPase inhibitors had no effect on HBV transcription and replication. These results demonstrate that p97 is a proviral host factor for HBV and important for cccDNA transcription. This function of p97 in HBV infection appears to be independent of its ATPase activity and HBx, and is only active on the native HBV cccDNA. p97 may serve as a novel target for HBV therapeutic development. HBV depends heavily on host factors for infection and replication. Comprehensive knowledge of critical host dependencies could provide valuable insight into the molecular mechanisms of viral replication and facilitate the development of novel host targeting agents. To systematically identify host factors involved in HBV infection, we used a HepG2 cell line that overexpressed NTCP, a recently identified host receptor for HBV. This cell line was then optimized and miniaturized for a high-throughput AlphaLisa screen to detect viral antigen. An arrayed genome-wide screen targeting approximately 21,500 genes using three unique siRNA sequences per gene was performed using this HepG2-NTCP cell line and AlphaLisa assay to detect viral anti