Molecular Mechanisms of HBV cccDNA Formation

HBV cccDNA形成的分子机制

• 批准号: 10049281
• 负责人: Haitao Guo
• 金额: $ 36.25万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-11-01 至 2021-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10049281
• 关键词: Molecular; Mechanisms; HBV; cccDNA; Formation

 DESCRIPTION (provided by applicant): 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.

 描述（申请人提供）：乙型肝炎病毒（HBV）共价闭合环状（ccc）DNA在病毒感染和持续存在的过程中发挥着核心作用，是治疗停止后病毒反弹的基础，也是治疗后病毒反弹的基础。即使在使用目前批准的药物进行长期治疗后，在初次感染时，通过部分双链松弛环状（rc）DNA病毒基因组的转化（可能是通过宿主的使用），也难以治愈。细胞核中的 cccDNA 附加体水平通过复制途径维持，该途径涉及将 cccDNA 转录物（称为前基因组 RNA）逆转录为子代 rcDNA 基因组，其中一些返回细胞核以转化为 rcDNA 的 cccDNA。进入 cccDNA 需要从 DNA 链之一的 5' 端去除聚合酶的共价连接副本，并且此“去蛋白化”步骤生成DNA中间体，即脱蛋白的rcDNA（DP-rcDNA），作为cccDNA形成的前体。此外，我们之前的工作表明，rcDNA脱蛋白是含有成熟病毒DNA的HBV核衣壳转运至细胞核的触发信号，其中rcDNA进入细胞核。然而，在理解 cccDNA 形成和代谢方面，还有许多细节需要阐明。在本研究应用中，通过利用一系列分子生物学，结合专为cccDNA研究而设计的生物化学和蛋白质组学技术，我们建议进一步表征DP-rcDNA的末端结构和细胞质和核的存在状态，阐明rcDNA脱蛋白的分子机制，并以某种方式鉴定参与的宿主DNA修复基因rcDNA 到 cccDNA 的转换。我们的最终目标是阐明 HBV cccDNA 形成的分子机制/途径的连贯图景，该项目的完成将填补 HBV 分子的重大知识空白。生物学，并可能为乙型肝炎新疗法的开发提供新的抗病毒靶点。