Mechanism of CCC DNA Synthesis in Hepatitis B Virus

乙型肝炎病毒CCC DNA合成机制

• 批准号: 8495929
• 负责人: Christoph Seeger
• 金额: $ 25.17万
• 依托单位: RESEARCH INST OF FOX CHASE CAN CTR
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8495929
• 关键词: Animal Viruses; Antiviral Therapy; Aphidicolin; Biological Assay; Biology; Cells; Circular DNA; DNA; DNA Ligases; DNA Repair; DNA Repair Enzymes; DNA biosynthesis; DNA ligase I; DNA-Directed DNA Polymerase; Development; Duck Hepatitis B Virus; Future; Genes; Genetic Transcription; Genome; Goals; Hepadnaviridae; Hepatitis B Virus; Human; Investigation; Laboratories; Life Cycle Stages; Ligase; Malignant neoplasm of liver; Maps; Modeling; Monitor; Nucleotide Excision Repair; Oncogenic; Pharmaceutical Preparations; Play; Polymerase; Primer Extension; Recruitment Activity; Research; Resistance; Risk; Role; Species Specificity; Staging; System; Viral; Virus Diseases; base; design; dideoxynucleotide; endonuclease; novel; novel strategies; pathogen; repaired; research study; small hairpin RNA; viral DNA; viral RNA

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.

描述（由申请人提供）：对乙型肝炎病毒（HBV）和相关动物病毒，特别是鸭乙型肝炎病毒（DHBV）的研究，提供了肝炎病毒复制周期的详细模型。然而，将松弛环（RC）病毒DNA基因组转化为共价闭合环（CCC） DNA（病毒RNA转录的模板）的机制尚不清楚。同样，哺乳动物肝病毒中调节CCC DNA扩增的机制仍然是未知的。我们实验室的研究表明，DHBV RC转化为CCC DNA的过程独立于病毒的酶功能，而是需要细胞DNA修复酶来抵抗阿帕迪克林和二脱氧核苷酸。与这些结果一致，我们已经获得了令人信服的证据，证明核苷酸切除修复（NER）的核心Y DNA聚合酶kappa是DHBV CCC DNA合成所必需的。我们提出了一个新的假设，即HBV和DHBV使用细胞核苷酸切除修复机制将RC DNA转化为CCC DNA。该假设将通过以下具体目标进行调查：目的1。HBV和DHBV RC转化为CCC DNA所需细胞因子的鉴定。本目的目的是确定HBV CCC DNA合成是否也需要聚合酶¿，并确定NER中涉及的内切酶和DNA连接酶是否在DHBV和HBV RC向CCC DNA的转化中发挥作用。目标2。负链和正链DNA中间产物的鉴定。我们建议通过实验来鉴定预测在缺乏pol的细胞中积累的DNA中间体，并研究负链和正链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肝病毒生物学、CCC DNA合成机制的理解的主要空白，并揭示一种动物病毒复制DNA的全新策略。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治疗超过4亿HBV感染者所需的抗病毒治疗提供靶点。此外，该申请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合成所观察到的物种特异性的机制奠定基础。