FUNCTIONAL ANALYSIS OF THE HEPADNA VIRUS GENOME

在之前的资助期间，我的实验室开发了一种体外培养技术 一种酶活性逆转录酶的合成体系 鸭乙型肝炎病毒--一种已建立的人类乙肝模型 病毒(乙肝病毒)。在这个系统的帮助下，我们已经获得了 对这种聚合酶的机制有了实质性的新见解 以蛋白质为引子，启动逆转录。这些结果 导致提出了一个修订的肝炎病毒复制模型，该模型 涉及病毒RNA上的一个序列，称为epsilon，作为 逆转录。 我们现在的研究范围是开发我们的体外系统 黄曲霉毒素的生化特性和功能研究 庚型肝炎病毒DNA聚合酶。具体来说，我们将调查这一角色 宿主因子，包括初步出现的伴侣HSP90 生产具有酶活性的POL基因所必需的数据 产品。我们的观察表明，宿主因素对 逆转录酶的酶活性是史无前例的 为反转录反应的第一步提供了新的见解 在庚型核糖核酸病毒中。此外，我们将确定决定因素 聚合酶和控制它们组装成 核糖核蛋白复合体，被发现对蛋白质启动至关重要 反应和RNA包装。因为聚合酶不携带已知的RNA 认可的主题，这些调查的结果不仅将 有助于更好地理解病毒复制，但也会产生 关于控制RNP形成的决定因素的新信息。其他内容 建议进行实验，以研究该反应 控制聚合酶和病毒RNA组装到核衣壳中。 为此，我们试图将我们的体外聚合酶检测发展成 该系统将允许组装完整的病毒核衣壳。 最后，我们将探讨松弛的形成机制。 环状病毒DNA成为共价闭合的环状DNA，这是第一次 病毒DNA复制周期的一步。利用遗传和生化 我们将确定病毒聚合酶或细胞 酶是这一反应所必需的，并确定细胞内的位置 这个反应发生在什么地方。除了我们的整体做法外，我们还将 建立病毒聚合酶的详细遗传和功能图谱 多肽，它的可获得性将为 成功地进行了本申请中提出的实验。 有了这项研究计划取得的预期成果，我们将 不仅获得了关于病毒DNA机制的重要信息 复制，但将进一步有助于识别小说 抗病毒治疗的靶点。乙肝病毒是一种具有全球意义的病原体 会导致急性和慢性肝炎，并导致肝细胞 癌症。而主要的乙肝病毒感染可以通过接种疫苗来预防， 到目前为止，超过2亿慢性感染者还没有治疗方法。 个人。

During the previous funding period my laboratory developed an in vitro system for the synthesis of enzymatically active reverse transcriptase of duck hepatitis B virus (DHBV), an established model for human hepatitis B virus (HBV). With the help of this system we have already gained substantial new insight into the mechanism by which this polymerase initiates reverse transcription with protein as a primer. These results led tot he proposal of a revised model for hepadnavirus replication, which implicates a sequence on viral RNA, termed epsilon, as the origin of reverse transcription. The scope of our research program now is to exploit our in vitro system for investigations on the biochemical properties and functions of the hepadnavirus DNA polymerase. Specifically, we will investigate the role of host factors, including chaperone hsp90, which appears from preliminary data to be essential for the production of enzymatically active pol gene product. Our observations that host-factors play a critical role for the enzymatic activity of a reverse transcriptase are unprecedented and will provide new insight into first steps of the reverse transcription reaction in hepadnaviruses. Furthermore, we will identify the determinants on the polymerase and on epsilon RNA that control their assembly into a ribonucleoprotein complex, found to be critical for the protein priming reaction and RNA packaging. Since the polymerase does not bear a known RNA recognition motif, results from these investigations will not only contribute to a better understanding of viral replication but also yield new information about determinants that control RNP formation. Additional experiments are proposed for the investigation of the reaction that controls assembly of the polymerase and viral RNA into the nucleocapsid. For this purpose we seek to develop our in vitro polymerase assay into a system that will allow for assembly of intact viral nucleocapsids. Finally, we will investigate the mechanism for the formation of relaxed circular virion DNA into covalently closed circular DNA, which is the first step of the viral DNA replication cycle. Using genetic and biochemical approaches we will determine whether athe viral polymerase or cellular enzymes are required for this reaction and identify the cellular locale in which this reaction occurs. Apart of our overall approach, we will also establish a detailed genetic and functional map of the viral polymerase polypeptide, the availability of which will provide a valuable tool for the successful conduct of the experiments proposed in this application. With the anticipated results obtained through this research program we will not only gain significant information about the mechanism of viral DNA replication, but will further contribute to the identification of novel targets for antiviral therapy. HBV is a pathogen of global significance that can cause acute and chronic hepatitis and induce hepatocellular carcinoma. Whereas primary HBV infection can be prevented by vaccination, so far no treatment is available to over 200 million chronically infected individuals.