The hepatitis B virus (HBV) remains a global cause of chronic liver diseases, including liver cirrhosis and cancer. Current antiviral therapy for chronic hepatitis B is only partially effective. In particular, the episomal viral DNA, the so-called covalently closed circular (CCC) DNA, persists in the infected cell nucleus even after years of antiviral treatment. The CCC DNA serves as the template for all viral transcriptions and is the molecular basis of HBV persistence. Therefore, the elimination of the CCC DNA is a prerequisite for any curing of an HBV infection. The CCC DNA is generated from the viral genomic DNA, which has a relaxed circular (RC), partially double-stranded structure. To complete the RC to CCC DNA conversion process, multiple biochemical reactions have to occur, about which nothing is currently understood. The overall goal of the current application is to begin to analyze the molecular mechanisms of CCC DNA formation, using both HBV and the duck HBV (DHBV) as model systems.
Three Specific Aims are proposed.
Specific Aim 1 will be to determine the potential pathways, including putative intermediates, of CCC DNA formation. Using in vitro cell culture systems where HBV and DHBV CCC DNA formation takes place and potential intermediates accumulate, we plan to identify and characterize these intermediates in detail. This, coupled with directed approaches to perturb their production as proposed in Specific Aims 2 &3, will provide important clues about the potential pathways of CCC DNA formation.
Specific Aim 2 will determine the role of specific viral factors, i.e., the viral envelope and reverse transcriptase proteins, in the formation and regulation of CCC DNA, employing a combination of genetic and biochemical approaches.
Specific Aim 3 will determine the role of selected host factors, particularly cellular DNA repair factors, in CCC DNA formation, using both existing cell culture systems and cell-free assays that will be developed. These studies should bring much needed insights into the mechanism of CCC DNA formation, which may facilitate the development of novel antivirals targeted directly at this critical step of viral replication. In addition, they may shed new light on the mechanisms of cellular DNA damage repair, the malfunction of which underlies a variety of serious human diseases from developmental defects to cancer. Project Narrative The hepatitis B virus (HBV) is a global cause of chronic liver diseases, including liver cirrhosis and cancer. We propose to elucidate the mechanisms of, and viral and host factors involved in, producing the nuclear episomal viral DNA, which is the molecular basis of HBV persistence. These studies should facilitate the development of novel antiviral agents targeted directly at this critical step of viral replication and capable of curing persistent infections.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Virology - B Study Section (VIRB)
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Koshy, Rajen
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Pennsylvania State University
Schools of Medicine
United States
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Hu, Jianming; Liu, Kuancheng (2017) Complete and Incomplete Hepatitis B Virus Particles: Formation, Function, and Application. Viruses 9:
Clark, Daniel N; Hu, Jianming (2015) Unveiling the roles of HBV polymerase for new antiviral strategies. Future Virol 10:283-295
Liu, Kuancheng; Ludgate, Laurie; Yuan, Zhenghong et al. (2015) Regulation of multiple stages of hepadnavirus replication by the carboxyl-terminal domain of viral core protein in trans. J Virol 89:2918-30
Luckenbaugh, L; Kitrinos, K M; Delaney 4th, W E et al. (2015) Genome-free hepatitis B virion levels in patient sera as a potential marker to monitor response to antiviral therapy. J Viral Hepat 22:561-70
Cui, Xiuji; McAllister, Rebecca; Boregowda, Rajeev et al. (2015) Does Tyrosyl DNA Phosphodiesterase-2 Play a Role in Hepatitis B Virus Genome Repair? PLoS One 10:e0128401
Cui, Xiuji; Guo, Ju-Tao; Hu, Jianming (2015) Hepatitis B Virus Covalently Closed Circular DNA Formation in Immortalized Mouse Hepatocytes Associated with Nucleocapsid Destabilization. J Virol 89:9021-8
Jones, Scott A; Clark, Daniel N; Cao, Feng et al. (2014) Comparative analysis of hepatitis B virus polymerase sequences required for viral RNA binding, RNA packaging, and protein priming. J Virol 88:1564-72
Cao, F; Jones, S; Li, W et al. (2014) Sequences in the terminal protein and reverse transcriptase domains of the hepatitis B virus polymerase contribute to RNA binding and encapsidation. J Viral Hepat 21:882-93
Jones, Scott A; Murakami, Eisuke; Delaney, William et al. (2013) Noncompetitive inhibition of hepatitis B virus reverse transcriptase protein priming and DNA synthesis by the nucleoside analog clevudine. Antimicrob Agents Chemother 57:4181-9
Jones, Scott A; Hu, Jianming (2013) Protein-primed terminal transferase activity of hepatitis B virus polymerase. J Virol 87:2563-76

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