Molecular Mechanisms of HBV cccDNA Formation

摘要 乙肝病毒共价闭合环(CCC)DNA在病毒的建立中起着核心作用 感染和持久性，是停止治疗后病毒反弹的基础，以及 即使在使用目前批准的药物进行延长治疗后，也难以治愈。HBVcccDNA是 通过转化部分双链松弛环状(RC)DNA而在初始感染时建立 含有末端特性的病毒基因组，通过利用宿主细胞的DNA修复机制 在原子核中。CccDNA Episome水平在复制周期中保持不变，包括 将cccDNA转录物，称为前基因组RNA，逆转录到后代rcDNA基因组中，其中一些 它们被返回到细胞核以转化为cccDNA。Rcdna到cccDNA的转化需要 从其中一条DNA链的5‘端移除共价连接的聚合酶拷贝，并且这 “去蛋白”步骤产生一种dna中间体，去蛋白rcDNA(dp-rcDNA)，作为 CccDNA形成。Rcdna去蛋白是乙肝病毒核衣壳转运的触发信号 将含有成熟病毒DNA的DNA送入细胞核，在那里发生rcDNA到ccDNA的转换。我们有 最近绘制了胞质DP-rcDNA的末端，这证明了病毒聚合酶和RNA 在去蛋白的过程中，引物从rcdna上完全去掉，正链dna进一步延长，但 末端冗余序列被保留在DP-rcDNA上。此外，我们和其他人最近的研究 已经确定了几个参与cccDNA形成的宿主DNA修复因子。然而，有很多 为了更好地理解cccDNA的生物合成，分子细节还有待阐明，以及 免疫活性小动物乙型肝炎病毒感染模型的建立受阻 CCDNA在小鼠肝细胞中的形成。在这项研究应用中，通过利用分子电池 生物学、生物化学、蛋白质组学和基因组学技术，我们建议进一步阐明分子 Dp-rcDNA(Aim 1)和cccDNA(Aim 2)的生物发生机制，并确定宿主 小鼠肝细胞CCDNA形成失败的决定子(S)(目的3)。我们的最终目标是 图解了乙肝病毒ccDNA形成的分子机制/途径的连贯图解。这个 该项目的完成将揭示治疗乙肝的新的潜在抗病毒靶点，并帮助 建立完全易感乙肝病毒感染的小鼠模型。

ABSTRACT 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 viral genome containing terminal peculiarities, through employment of the host cell’s DNA repair mechanisms in the nucleus. The cccDNA episome levels are maintained through a replication cycle 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 “deproteination” step generates a DNA intermediate, the deproteinated rcDNA (DP-rcDNA), as precursor for cccDNA formation. The rcDNA deproteination is a trigger signal for transportation of HBV nucleocapsid containing mature viral DNA into nucleus, where the rcDNA to cccDNA conversion takes place. We have recently mapped the termini of cytoplasmic DP-rcDNA, which demonstrated that the viral polymerase and RNA primer are completely removed from rcDNA during deproteination, the plus strand DNA is further elongated but the terminal redundant sequence is maintained on DP-rcDNA. In addition, recent studies by us and others have identified a handful of host DNA repair factors involved in cccDNA formation. However, there are many molecular details yet to be elucidated for a better understanding of cccDNA biosynthesis, and the establishment of immunocompetent small animal model for HBV infection is hampered by the inability of cccDNA formation in mouse hepatocyte. In this research application, by making use of a battery of molecular biology, biochemistry, proteomics and genomics technologies, we propose to further elucidate the molecular mechanisms underlying the biogenesis of DP-rcDNA (Aim 1) and cccDNA (Aim 2), and to define the host determinant(s) for the failure of cccDNA formation in mouse hepatocyte (Aim 3). Our ultimate goal is to illustrate a coherent picture of the molecular mechanisms/pathway for HBV cccDNA formation. The accomplishment of this project will reveal new potential antiviral targets for treatment of hepatitis B and aid the development of a mouse model fully susceptible to HBV infection.