Molecular Mechanisms of HBV cccDNA Formation

 描述(申请人提供)：乙肝病毒(乙肝病毒)共价闭合环(CCC)DNA在确定病毒感染和持久性方面发挥核心作用，是停止治疗后病毒反弹的基础，以及即使在使用当前批准的药物延长治疗后仍难以治愈的基础。HBVcccDNA是在初次感染时通过部分双链松弛环状(RC)DNA Virl基因组的转换而建立的，推测是通过利用宿主细胞在细胞核中的DNA修复机制来建立的。CccDNA Episome水平是通过一条复制途径来维持的，该复制途径涉及将一种被称为前基因组RNA的cccDNA转录物逆转录到后代rcDNA基因组中，其中一些基因组被返回到细胞核中转化为cccDNA。要将rcDNA转化为cccDNA，需要从DNA链的5‘端去除一个共价连接的聚合酶拷贝，这个“去蛋白”步骤会产生一种DNA中间体，即脱蛋白rcDNA(DP-rcDNA)，作为cccDNA形成的前体。此外，我们以前的工作表明，rcDNA去蛋白是一个触发信号，使含有成熟病毒DNA的乙肝病毒核衣壳进入细胞核，在那里发生rcDNA到cccDNA的转换。然而，对于cccDNA的形成和代谢的了解仍有许多细节需要阐明。在这一研究应用中，我们建议利用一系列专门用于cccDNA研究的分子生物学、生物化学和蛋白质组学技术，进一步表征细胞质和核DP-rcDNA的末端结构和存在状态，阐明rcDNA去蛋白的分子机制，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HBVcccDNA形成的分子机制/途径的连贯图景。该项目的完成将填补乙肝病毒分子生物学方面的重大知识空白，并可能为开发新的乙肝治疗药物提供新的抗病毒靶点。

 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.