Mechanism of CCC DNA Synthesis in Hepatitis B Virus

描述（由申请方提供）：对B型肝炎病毒（HBV）和相关动物病毒，特别是鸭B型肝炎病毒（DHBV）的研究提供了嗜肝DNA病毒复制周期的详细模型。然而，负责将松弛环状（RC）病毒DNA基因组转化为共价闭合环状（CCC）DNA（病毒RNA转录的模板）的机制尚不清楚。同样，哺乳动物嗜肝DNA病毒中CCC DNA扩增的调控机制仍然是难以捉摸的。我们实验室的研究表明，DHBV RC转化为CCC DNA的发生独立于病毒酶功能，而是需要对aphidicolin和双脱氧核苷酸具有抗性的细胞DNA修复酶。与这些结果一致，我们已经获得了令人信服的证据表明，Y DNA聚合酶κ，核心核苷酸切除修复（NER），是所需的DHBV CCC DNA合成。我们提出了一个新的假设，规定HBV和DHBV，使用细胞的核苷酸切除修复机制的转换RC DNA到CCC DNA。该假设将通过以下具体目标进行研究：目标1。HBV和DHBV RC转化为CCC DNA所需细胞因子的鉴定本研究的目的是确定聚合酶是否也是HBV CCC DNA合成所必需的，并确定NER中涉及的核酸内切酶和DNA连接酶是否在DHBV和HBV RC转化为CCC DNA中发挥作用。目标二。正、负链DNA中间体的鉴定。我们提出实验来鉴定DNA中间体，预测在缺乏pol的细胞中积累，并研究负链和正链DNA的修复是否通过相同或不同的机制发生。这项提议将填补我们对嗜肝DNA病毒生物学、CCC DNA合成机制的理解中的一个重大空白，并揭示动物病毒复制DNA所使用的一种全新策略。从我们对CCC DNA合成机制的研究中获得的信息可以用于鉴定新的细胞基因，这些基因可能为未来的抗病毒治疗提供靶点，治愈超过4亿HBV感染者。此外，该应用程序的目的代表了一种探索性的努力，将为未来研究HBV招募NER系统的机制以及控制HBV CCC DNA合成的观察到的种属特异性的机制奠定基础。

DESCRIPTION (provided by applicant): Investigations on hepatitis B virus (HBV) and related animal viruses, particularly duck hepatitis B virus (DHBV), have provided a detailed model of the hepadnavirus replication cycle. However, the mechanism responsible for the conversion of the relaxed circular (RC) viral DNA genome into covalently closed circular (CCC) DNA, the template for viral RNA transcription, is not yet known. Likewise, the mechanism regulating amplification of CCC DNA in mammalian hepadnaviruses is still elusive. Research from our laboratory showed that the conversion of DHBV RC into CCC DNA occurs independently of viral enzymatic functions, and requires instead cellular DNA repair enzymes that are resistant to aphidicolin and dideoxynucleotides. Consistent with these results, we have obtained compelling evidence that the Y DNA polymerase kappa, central to nucleotide excision repair (NER), is required for DHBV CCC DNA synthesis. We propose a novel hypothesis stipulating that HBV, and DHBV, use the cellular nucleotide excision repair machinery for the conversion of RC DNA into CCC DNA. The hypothesis will be investigated through the following specific aims: Aim 1. Identification of cellular factors required for the conversion of HBV and DHBV RC into CCC DNA. The purpose of this aim is to determine if polymerase ¿ is also required for HBV CCC DNA synthesis and to determine if the endonucleases and DNA ligases involved in NER play a role in the conversion of DHBV, as well as HBV RC to CCC DNA. Aim 2. Identification of intermediates of minus and plus strand DNA. We propose experiments to identify DNA intermediates predicted to accumulate in cells lacking pol ¿ and investigate whether the repair of minus and plus strand DNA occurs by the same or different mechanisms. This proposal will fill a major gap in our understanding of hepadnavirus biology, the mechanism of CCC DNA synthesis, and reveal a completely novel strategy used by an animal virus to replicate DNA. The information gained from our research into the mechanism of CCC DNA synthesis could be exploited for the identification of novel cellular genes that might provide targets for future antiviral therapies, required for the cure of over 400 million HBV infected people. Moreover, the aims of the application represent an exploratory effort that will set the stage for future investigations into he mechanism used by HBV to recruit the NER system and on the mechanism controlling the observed species specificity of HBV CCC DNA synthesis.