REPLICATION OF HEPADNAVIRUSES

嗜肝DNA病毒是组织和物种特异性病毒， 引起肝炎并诱导形成原发性肝细胞 carcinoma. 它们通过反向复制小的DNA基因组 转录通过RNA中间体，前基因组。 第一 链DNA由蛋白质引发，第二链DNA由蛋白质引发。 RNA寡聚体，其来源于末端冗余的 部分前基因组RNA。 在第二条链开始之前 DNA合成时，这条RNA引物被易位到第二条引物上。 链DNA引发位点。 为了详细了解 复制方案的嗜肝DNA病毒，我们将分析具体的 复制中间体，进行病毒的遗传分析， 复制并探索基因结构和生物化学 病毒聚合酶基因及其产物的特性。 使用 地松鼠和土拨鼠肝炎病毒，我们将 研究第二链DNA合成的机制 的起源问题，特别是 前基因组RNA上的RNA引物，如何将RNA的3'端 引物是如何产生的，以及RNA引物是如何易位到 第二链DNA引发位点。 为了研究 由病毒聚合酶基因编码的活性，我们将表达 一种细菌中的黄鼠肝炎病毒聚合酶基因 表达系统 具体来说，我们将搜索反向 转录酶和RNaseH活性，并确定 它们的酶结构域在聚合酶开放阅读框上。 使用体外合成的病毒RNA和DNA模板，我们将 探索这些活动的生化特性， 用体外研究补充我们的体内分析， 化学定义的测定条件。 慢性B型肝炎病毒 感染是目前无法治愈主要健康问题 存在. 更好地理解 嗜肝DNA病毒对开发特异性抗病毒药物至关重要 疗法 嗜肝DNA病毒体外检测系统的建立 复制可能对开发具有普遍用途， 逆转录抑制剂的实验测试。

Hepadnaviruses are tissue and species specific viruses that can cause hepatitis and induce formation of primary hepatocellular carcinoma. They replicate their small DNA genomes by reverse transcription via a RNA intermediate, the pregenome. First strand DNA is primed by a protein and second strand DNA by a RNA oligomer which is derived from the terminally redundant portion of pregenome RNA. Prior to initiation of second strand DNA synthesis, this RNA primer is translocated to the second strand DNA priming site. To gain a detailed understanding of the replication scheme of hepadnaviruses, we will analyse specific replicative intermediates, perform a genetic analysis of viral replication and explore the genetic structure and the biochemical properties of the viral polymerase gene and its product(s). Using ground squirrel and woodchuck hepatitis viruses we will investigate the mechanism by which second strand DNA synthesis is initiated, specifically addressing questions of the origin of the RNA primer on pregenome RNA, how the 3' end of the RNA primer is created, and how the RNA primer is translocated to the second strand DNA priming site. To investigate the enzymatic activities encoded by the viral polymerase gene, we will express the ground squirrel hepatitis virus polymerase gene in a bacterial expression system. Specifically we will search for reverse transcriptase and RNaseH activities and identify the location of their enzymatic domains on the polymerase open reading frame. Using in vitro synthesized viral RNA and DNA templates, we will explore the biochemical properties of these activities and complement our in vivo analysis with in vitro studies under chemically defined assay conditions. Chronic hepatitis B virus infection is a major health problem for which presently no cure exists. An improved understanding of the replication scheme of hepadnaviruses is essential for development of specific antiviral therapy. The development of an in vitro system for hepadnavirus replication might be of general use for the development and experimental testing of inhibitors for reverse transcription.