Molecular Mechanism of Hepadnavirus Persistence

摘要 乙肝病毒(乙肝病毒)仍然是包括肝硬变在内的慢性肝病的全球病因。 和癌症。目前对慢性乙肝的抗病毒治疗只有部分有效。尤其是，上体 病毒DNA，即所谓的共价闭合环(CCC)DNA，即使在感染后仍留在受感染的细胞核中 多年的抗病毒治疗。CcDNA是所有病毒转录的模板，是 乙肝病毒持续存在的分子基础。因此，消除ccc DNA是任何治愈的先决条件。 感染了乙肝病毒。Ccdna是由病毒基因组dna产生的，基因组dna有一个松弛的环状结构。 (RC)，部分双股结构。要完成RC到CCC DNA的转换过程，需要多个 生化反应是必须发生的，目前对此一无所知。该计划的总体目标 目前的应用是开始分析ccDNA形成的分子机制，使用两种病毒 以鸭乙肝病毒(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，包括可能的中间产物。使用体外细胞 产生HBVccDNA和DHBVccDNA的培养系统和潜在的中间体 积累，我们计划对这些中间体进行详细的鉴定和表征。这一点，加上定向的 在具体目标2和3中提出的扰乱其生产的方法将为以下方面提供重要线索 CcDNA形成的可能途径。特定目标2将确定特定病毒因子的作用， 即病毒被膜蛋白和逆转录酶蛋白，在CCC DNA的形成和调节中， 采用遗传和生化方法相结合的方法。具体目标3将决定 选择寄主因子，特别是细胞DNA修复因子，形成CCC DNA，使用现有的两个细胞 将开发的培养系统和无细胞检测。这些研究应该会带来亟需的见解 探讨ccDNA的形成机制，可能有助于开发新型靶向抗病毒药物 直接在病毒复制的这一关键步骤。此外，它们可能会为我们提供新的线索 细胞DNA损伤修复，它的故障是各种严重的人类疾病的基础 发育缺陷导致癌症。项目叙事 乙肝病毒(HBV)是慢性肝病的全球病因，包括肝硬变和 癌症。我们建议阐明产生该病毒的机制，以及病毒和宿主因素。 核上体病毒DNA，这是乙肝病毒持续存在的分子基础。这些研究应该有助于 针对病毒复制这一关键步骤的新型抗病毒药物的开发 治愈持续性感染。

Abstract 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. Project Narrative 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.