Molecular Mechanism of Hepadnavirus Persistence

描述(申请人提供)：乙肝病毒(乙肝病毒)仍然是慢性肝病的全球病因，包括肝硬变和癌症。目前对慢性乙肝的抗病毒治疗只有部分有效。特别是，异体病毒DNA，即所谓的共价闭合环(CCC)DNA，即使在多年的抗病毒治疗后仍留在受感染的细胞核中。CCC DNA是所有病毒转录的模板，也是乙肝病毒持续存在的分子基础。因此，消除ccc DNA是治愈乙肝病毒感染的先决条件。CCC DNA是由病毒基因组DNA产生的，具有松弛的环状(RC)、部分双链结构。为了完成从RC到CCC的DNA转换过程，必须发生多个生化反应，目前对此一无所知。目前应用的总体目标是开始分析CCC DNA形成的分子机制，以乙肝病毒和鸭乙肝病毒(DHBV)为模型系统。提出了三个具体目标。具体目标1将是确定ccDNA形成的潜在途径，包括假定的中间产物。利用体外细胞培养系统，在其中发生乙肝病毒和DHBVCCC DNA的形成和潜在的中间体积累，我们计划详细地鉴定和表征这些中间体。这一点，再加上特定目标2和3中提出的干扰其生产的定向方法，将提供关于CCC DNA形成的潜在途径的重要线索。具体目标2将采用遗传和生物化学方法相结合的方法，确定特定病毒因素，即病毒包膜和逆转录酶蛋白，在CCC DNA的形成和调节中的作用。具体目标3将利用现有的细胞培养系统和即将开发的无细胞分析方法，确定选定的宿主因素，特别是细胞DNA修复因子，在CCC DNA形成中的作用。这些研究将带来对CCC DNA形成机制的迫切需要的见解，这可能有助于开发直接针对病毒复制的这一关键步骤的新型抗病毒药物。此外，它们可能为细胞DNA损伤修复机制提供新的线索，细胞DNA损伤修复机制的故障是从发育缺陷到癌症等各种严重人类疾病的基础。与公共卫生相关：乙肝病毒(乙肝)是慢性肝病的全球病因，包括肝硬变和癌症。我们建议阐明产生核上体病毒DNA的机制，以及病毒和宿主因素参与其中，这是乙肝病毒持续存在的分子基础。这些研究应该有助于开发直接针对病毒复制的这一关键步骤并能够治愈持续性感染的新型抗病毒药物。

DESCRIPTION (provided by applicant): The hepatitis B virus (HBV) remains a global cause of chronic liver diseases, including liver cirrhosis and cancer. Current antiviral therapy for chronic hepatitis B is only partially effective. In particular, the episomal viral DNA, the so-called covalently closed circular (CCC) DNA, persists in the infected cell nucleus even after years of antiviral treatment. The CCC DNA serves as the template for all viral transcriptions and is the molecular basis of HBV persistence. Therefore, the elimination of the CCC DNA is a prerequisite for any curing of an HBV infection. The CCC DNA is generated from the viral genomic DNA, which has a relaxed circular (RC), partially double-stranded structure. To complete the RC to CCC DNA conversion process, multiple biochemical reactions have to occur, about which nothing is currently understood. The overall goal of the current application is to begin to analyze the molecular mechanisms of CCC DNA formation, using both HBV and the duck HBV (DHBV) as model systems. Three Specific Aims are proposed. Specific Aim 1 will be to determine the potential pathways, including putative intermediates, of CCC DNA formation. Using in vitro cell culture systems where HBV and DHBV CCC DNA formation takes place and potential intermediates accumulate, we plan to identify and characterize these intermediates in detail. This, coupled with directed approaches to perturb their production as proposed in Specific Aims 2 & 3, will provide important clues about the potential pathways of CCC DNA formation. Specific Aim 2 will determine the role of specific viral factors, i.e., the viral envelope and reverse transcriptase proteins, in the formation and regulation of CCC DNA, employing a combination of genetic and biochemical approaches. Specific Aim 3 will determine the role of selected host factors, particularly cellular DNA repair factors, in CCC DNA formation, using both existing cell culture systems and cell-free assays that will be developed. These studies should bring much needed insights into the mechanism of CCC DNA formation, which may facilitate the development of novel antivirals targeted directly at this critical step of viral replication. In addition, they may shed new light on the mechanisms of cellular DNA damage repair, the malfunction of which underlies a variety of serious human diseases from developmental defects to cancer. PUBLIC HEALTH RELEVANCE: The hepatitis B virus (HBV) is a global cause of chronic liver diseases, including liver cirrhosis and cancer. We propose to elucidate the mechanisms of, and viral and host factors involved in, producing the nuclear episomal viral DNA, which is the molecular basis of HBV persistence. These studies should facilitate the development of novel antiviral agents targeted directly at this critical step of viral replication and capable of curing persistent infections.