Hepatitis B virus (HBV) covalently closed circular (ccc) DNA plays a central role in the establishment of viral infection and persistence, and is the basis for viral rebound after the cessation of therapy, as well as the elusiveness of a cure even after extended treatment with current approved medications. HBV cccDNA is established upon initial infection through conversion of the partially double stranded relaxed circular (rc) DNA virl genome, presumably through employment of the host cell's DNA repair mechanisms in the nucleus. The cccDNA episome levels are maintained through a replication pathway that involves retrotranscription of a cccDNA transcript, termed pregenomic RNA, into progeny rcDNA genomes, some of which are returned to the nucleus for conversion into cccDNA. The conversion of rcDNA into cccDNA requires the removal of a covalently-linked copy of the polymerase from the 5' end of one of the DNA strands, and this deproteinization step generates a DNA intermediate, the deproteinized rcDNA (DP-rcDNA), as precursor for cccDNA formation. In addition, our previous work suggested that the rcDNA deproteinization is a trigger signal for transportation of HBV nucleocapsid containing mature viral DNA into nucleus, where the rcDNA to cccDNA conversion takes place. However, there are many details yet to be elucidated in the understanding of cccDNA formation and metabolism. In this research application, by making use of a battery of molecular biology, biochemistry, and proteomics technologies specially designed for cccDNA study, we propose to further characterize the terminal structure and the state of existence of both cytoplasmic and nuclear DP-rcDNA, elucidate the molecular mechanisms of rcDNA deproteinization, and systematically identify host DNA repair genes involved in rcDNA to cccDNA conversion. Our ultimate goal is to illustrate a coherent picture of the molecular mechanisms/pathway for HBV cccDNA formation. The accomplishment of this project will fill a significant knowledge gap in HBV molecular biology, and potentially provide new antiviral targets for development of novel therapeutics for hepatitis B.
Chronic hepatitis B remains a significant public health burden affecting approximately 300 million individuals worldwide, and those patients have a high risk of occurrence of cirrhosis and liver cancer. Chronic HBV infection is maintained by a persistent viral genome called cccDNA, which could not be eradicated by currently approved drugs. We propose in this application to elucidate the molecular mechanisms and pathway of cccDNA formation, which will discover new antiviral targets for future development of novel HBV therapeutics.
|Long, Quanxin; Yan, Ran; Hu, Jieli et al. (2017) The role of host DNA ligases in hepadnavirus covalently closed circular DNA formation. PLoS Pathog 13:e1006784|
|Hong, Xupeng; Kim, Elena S; Guo, Haitao (2017) Epigenetic regulation of hepatitis B virus covalently closed circular DNA: Implications for epigenetic therapy against chronic hepatitis B. Hepatology 66:2066-2077|
|Zhang, Yongmei; Mao, Richeng; Guo, Haitao et al. (2017) Detection of HBV cccDNA Methylation from Clinical Samples by Bisulfite Sequencing and Methylation-Specific PCR. Methods Mol Biol 1540:73-84|
|Pang, Jinke; Zhang, Geng; Lin, Yong et al. (2017) Transforming growth factor ?-activated kinase 1 transcriptionally suppresses hepatitis B virus replication. Sci Rep 7:39901|
|Liu, Yuanjie; Nie, Hui; Mao, Richeng et al. (2017) Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA. PLoS Pathog 13:e1006296|
|Iwamoto, Masashi; Cai, Dawei; Sugiyama, Masaya et al. (2017) Functional association of cellular microtubules with viral capsid assembly supports efficient hepatitis B virus replication. Sci Rep 7:10620|
|Yan, Ran; Cai, Dawei; Liu, Yuanjie et al. (2017) Detection of Hepatitis B Virus Particles Released from Cultured Cells by Particle Gel Assay. Methods Mol Biol 1540:193-202|
|Liu, Chunlan; Cai, Dawei; Zhang, Lin et al. (2016) Identification of hydrolyzable tannins (punicalagin, punicalin and geraniin) as novel inhibitors of hepatitis B virus covalently closed circular DNA. Antiviral Res 134:97-107|
|Mitra, Bidisha; Guo, Haitao (2016) Hepatitis B virus X protein crosses out Smc5/6 complex to maintain covalently closed circular DNA transcription. Hepatology 64:2246-2249|
|Cai, Dawei; Wang, Xiaohe; Yan, Ran et al. (2016) Establishment of an inducible HBV stable cell line that expresses cccDNA-dependent epitope-tagged HBeAg for screening of cccDNA modulators. Antiviral Res 132:26-37|