Hepadnaviruses, or hepatitis B viruses, are small, hepatotropic DNA viruses that replicate through an RNA intermediate (the pregenomic RNA or pgRNA) by reverse transcription. A critical early step in viral replication is the assembly of a ribonucleoprotein (RNP) complex comprised of the viral reverse transcriptase (RT) and pgRNA, which triggers both the assembly of nucleocapsids, the site of viral DNA synthesis, and the initiation of reverse transcription, which is primed by RT itself (the protein priming reaction). Through a complex multi- step reverse transcription pathway, the pgRNA is subsequently converted, within the context of the nucleocapsids, into the characteristic, relaxed circular DNA genome which can then exit the cell. The broad, long term objective of this proposal is to elucidate the molecular mechanisms of hepadnavirus replication, focusing on the virus-cell interactions critical for viral assembly and replication. The first specific aim is to identify host factors required for the interaction between RT and pgRNA and for protein priming. A combined approach based on physical association with RT, pgRNA and viral particles and biochemical fractionation and reconstitution will be used for this purpose, taking advantage of an established cell-free system that can recapitulate these early events in viral replication.
The second aim i s to elucidate the determinants on both RT and pgRNA responsible for specific RNA-protein interaction. Optimal conditions for RT- binding and crosslinking will be sought. The RT and contact sites in the RNP complex will then be identified through protein and RNA sequence analyses. This will, in turn, guide further mutagenesis studies to elucidate the specific requirements of RT and pgRNA for RNP formation.
The third aim i s to determine the requirements for individual steps of the viral reverse transcription pathway, using a recently developed synchronized viral replication system. Nucleocapsids from every stage of reverse transcription will be isolated to determine their structural changes, which accompany, and may be required for, the progression of viral DNA synthesis. Furthermore, specific host factors required for the different steps of reverse transcription will be identified by (1) carrying out viral DNA synthesis with the isolated nucleocapsids under cell-free reconstitution conditions and (2) treating virus-producing cells, at different stages of reverse transcription, with agents that target candidate host factors. The accomplishment of these goals should reveal novel cellular, as well as viral, targets for developing effective antivirals which is urgently needed for the treatment of over 300 million patients worldwide infected with the hepatitis B virus, who are at great risk of developing hepatic failure and hepatocellular carcinoma. In addition, studies on how host factors facilitate viral replication will also provide important insights into the normal cellular functions of these factors.
|Zhao, Qiong; Hu, Zhanying; Cheng, Junjun et al. (2018) Hepatitis B Virus Core Protein Dephosphorylation Occurs during Pregenomic RNA Encapsidation. J Virol 92:|
|Alter, Harvey; Block, Timothy; Brown, Nathaniel et al. (2018) A research agenda for curing chronic hepatitis B virus infection. Hepatology 67:1127-1131|
|Hu, Jianming; Lin, You-Yu; Chen, Pei-Jer et al. (2018) Cell and Animal Models for Studying Hepatitis B Virus Infection and Drug Development. Gastroenterology :|
|Liu, Kuancheng; Luckenbaugh, Laurie; Ning, Xiaojun et al. (2018) Multiple roles of core protein linker in hepatitis B virus replication. PLoS Pathog 14:e1007085|
|Ning, Xiaojun; Luckenbaugh, Laurie; Liu, Kuancheng et al. (2018) Common and Distinct Capsid and Surface Protein Requirements for Secretion of Complete and Genome-Free Hepatitis B Virions. J Virol 92:|
|Liu, Kuancheng; Hu, Jianming (2018) Host-regulated Hepatitis B Virus Capsid Assembly in a Mammalian Cell-free System. Bio Protoc 8:|
|Clark, Daniel N; Jones, Scott A; Hu, Jianming (2017) In Vitro Assays for RNA Binding and Protein Priming of Hepatitis B Virus Polymerase. Methods Mol Biol 1540:157-177|
|Hu, Jianming; Liu, Kuancheng (2017) Complete and Incomplete Hepatitis B Virus Particles: Formation, Function, and Application. Viruses 9:|
|Clark, Daniel N; Flanagan, John M; Hu, Jianming (2017) Mapping of Functional Subdomains in the Terminal Protein Domain of Hepatitis B Virus Polymerase. J Virol 91:|
|Luo, Jun; Cui, Xiuji; Gao, Lu et al. (2017) Identification of Intermediate in Hepatitis B Virus CCC DNA Formation and Sensitive and Selective CCC DNA Detection. J Virol :|
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