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（由申请人提供）：B肝炎病毒（HBV）共价闭合环状（ccc）DNA在病毒感染和持续性的建立中起着核心作用，是治疗停止后病毒反弹的基础，以及即使在使用当前批准的药物进行延长治疗后也难以治愈。HBV cccDNA在初始感染时通过部分双链松弛环状（rc）DNA病毒1基因组的转化建立，推测是通过在细胞核中利用宿主细胞的DNA修复机制。cccDNA附加体水平通过复制途径维持，该复制途径涉及将cccDNA转录物（称为前基因组RNA）逆转录成子代rcDNA基因组，其中一些返回细胞核转化为cccDNA。将rcDNA转化为cccDNA需要从DNA链之一的5'端去除聚合酶的共价连接拷贝，并且该“脱蛋白”步骤产生DNA中间体，脱蛋白rcDNA（DP-rcDNA），作为cccDNA形成的前体。此外，我们以前的工作表明，rcDNA脱蛋白是将含有成熟病毒DNA的HBV核衣壳转运到细胞核中的触发信号，在细胞核中发生rcDNA到cccDNA的转化。然而，在理解cccDNA形成和代谢方面还有许多细节有待阐明。在本研究应用中，通过利用一系列专门为cccDNA研究设计的分子生物学、生物化学和蛋白质组学技术，我们建议进一步表征胞质和核DP-rcDNA的末端结构和存在状态，阐明rcDNA脱蛋白的分子机制，并系统地鉴定参与rcDNA向cccDNA转化的宿主DNA修复基因。我们的最终目标是阐明HBV cccDNA形成的分子机制/途径的一致性。该项目的完成将填补HBV分子生物学的重大知识空白，并可能为开发新型B型肝炎治疗药物提供新的抗病毒靶点。