Mechanism of CCC DNA Synthesis in Hepatitis B Virus

描述(由申请人提供)：对乙肝病毒和相关动物病毒的研究，特别是对鸭乙型肝炎病毒的研究，提供了肝炎病毒复制周期的详细模型。然而，松弛环状(RC)病毒DNA基因组转化为共价闭合环状(CCC)DNA的机制尚不清楚，后者是病毒RNA转录的模板。同样，调控哺乳动物肝病毒中CCC DNA扩增的机制仍然难以捉摸。我们实验室的研究表明，DHBVRC转化为CCC DNA的过程不依赖于病毒的酶功能，而需要耐药的细胞DNA修复酶。与这些结果一致，我们获得了令人信服的证据，即Y DNA聚合酶kappa是核苷酸切除修复(NER)的核心，是DHBVccDNA合成所必需的。我们提出了一个新的假设，即乙肝病毒和乙肝病毒利用细胞核苷酸切除修复机制将RC DNA转化为CCC DNA。这一假说将通过以下具体目标进行研究：目的1.鉴定将HBVRc和DHBVRC转化为CCC DNA所需的细胞因子。其目的是确定聚合酶是否也是合成HBVccDNA所必需的，并确定NER所涉及的内切酶和DNA连接酶是否在DHBVc和HBVRC向ccDNA的转化中发挥作用。目的2.正链和负链DNA中间体的鉴定。我们建议进行实验，以确定DNA中间体预计会在缺乏POL的细胞中积累，并调查负链和正链DNA的修复是通过相同还是不同的机制发生的。这一建议将填补我们对庚型核糖核酸病毒生物学、CCC DNA合成机制的了解的一个重大空白，并揭示一种全新的动物病毒复制DNA的策略。从我们对ccDNA合成机制的研究中获得的信息可以被用来鉴定新的细胞基因，这些基因可能为未来的抗病毒治疗提供靶点，所需的抗病毒治疗需要治愈超过4亿乙肝病毒感染者。此外，该应用的目的是一种探索性的努力，将为未来研究乙肝病毒招募NER系统的机制以及控制观察到的HBVccDNA合成的物种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.