Mechanism of CCC DNA Synthesis in Hepatitis B Virus

乙型肝炎病毒CCC DNA合成机制

• 批准号: 8355689
• 负责人: Christoph Seeger
• 金额: $ 22.31万
• 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 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8355689
• 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. PUBLIC HEALTH RELEVANCE: Over 400 million people are currently infected with hepatitis B virus and are at significantly elevated risk for the development of liver cancer. Our research into the mechanism of CCC DNA synthesis is directed at identification of cellular genes that represent novel targets for future antiviral therapies that, in combination with existing drugs, might cure HBV infections.

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）的研究提供了嗜肝DNA病毒复制周期的详细模型，然而，负责转化的机制。同样，哺乳动物嗜肝DNA病毒中调节CCC DNA扩增的机制尚不清楚。我们实验室的研究表明，DHBV RC 转化为 CCC DNA 的过程与病毒酶功能无关，并且需要对阿非迪霉素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。 Y DNA 聚合酶 kappa 是核苷酸切除修复 (NER) 的核心，是 DHBV CCC DNA 合成所必需的。我们提出了一个新的假设，规定 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研究： 目标 1. 鉴定 HBV 和 DHBV RC 转化为 CCC DNA 所需的细胞因子。这个目的是确定聚合酶是否HBV CCC DNA 合成以及确定 NER 中涉及的核酸内切酶和 DNA 连接酶是否在 DHBV 以及 HBV RC 向 CCC DNA 的转化中发挥作用也需要 目标 2. 负链和正链 DNA 中间体的鉴定。我们提出实验来鉴定预计会在缺乏 pol 的细胞中积累的 DNA 中间体 ¿并研究负链和正链 DNA 的修复是否通过相同或不同的机制发生。该提议将填补我们对嗜肝DNA病毒生物学、CCC DNA 合成机制理解的重大空白，并揭示动物使用的全新策略。我们从 CCC DNA 合成机制的研究中获得的信息可用于识别新的细胞基因，这些基因可能为未来的抗病毒治疗提供靶点，从而治愈 4 亿多 HBV 感染者。的目标该申请代表了一项探索性工作，将为未来研究 HBV 用于招募 NER 系统的机制以及控制 HBV CCC DNA 合成的观察到的物种特异性的机制奠定基础。 公共卫生相关性：目前有超过 4 亿人感染乙型肝炎病毒，并且患肝癌的风险显着升高。 CCC DNA 合成机制旨在识别细胞基因，这些基因代表未来抗病毒疗法的新靶标，与现有药物相结合，可能治愈 HBV 感染。