Molecular Mechanism of Hepadnavirus Persistence

描述（由申请人提供）：乙型肝炎病毒（HBV）仍然是慢性肝病的全球原因，包括肝硬化和癌症。目前对慢性乙型肝炎的抗病毒治疗仅部分有效。特别是，附体病毒DNA，即所谓的共价闭合环状（CCC） DNA，即使经过多年的抗病毒治疗，仍然存在于被感染的细胞核中。CCC DNA作为所有病毒转录的模板，是HBV持续存在的分子基础。因此，清除CCC DNA是治愈HBV感染的先决条件。CCC DNA是由病毒基因组DNA产生的，它具有松弛的环状（RC），部分双链结构。为了完成RC到CCC DNA的转化过程，必须发生多种生化反应，目前还不清楚。当前应用的总体目标是开始分析CCC DNA形成的分子机制，使用HBV和鸭HBV （DHBV）作为模型系统。提出了三个具体目标。具体目标1将是确定CCC DNA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，HBV和DHBV CCC DNA形成发生，潜在的中间产物积累，我们计划详细鉴定和表征这些中间产物。这一点，再加上具体目标2和3中提出的干扰其产生的定向方法，将为CCC DNA形成的潜在途径提供重要线索。特异性目标2将确定特定的病毒因子，即病毒包膜和逆转录酶蛋白，在CCC DNA的形成和调控中的作用，采用遗传和生化方法的组合。特异性目标3将确定选定的宿主因子，特别是细胞DNA修复因子，在CCC DNA形成中的作用，使用现有的细胞培养系统和将开发的无细胞测定法。这些研究将为了解CCC DNA形成的机制提供急需的见解，这可能有助于开发直接针对病毒复制这一关键步骤的新型抗病毒药物。此外，它们可能会揭示细胞DNA损伤修复的机制，这种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。公共卫生相关性：乙型肝炎病毒（HBV）是慢性肝病的全球病因，包括肝硬化和癌症。我们建议阐明产生核外泌体病毒DNA的机制，以及病毒和宿主因子参与的机制，这是HBV持续存在的分子基础。这些研究将有助于开发新的抗病毒药物，直接针对病毒复制的这一关键步骤，并能够治疗持续感染。

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.