REPLICATION OF HEPADNAVIRUSES

肝炎病毒是特定于组织和物种的病毒，它可以 引起肝炎和诱导形成原发性肝细胞 癌症。他们通过反向复制他们的小DNA基因组 通过RNA中间体，前基因组进行转录。第一 链DNA由一种蛋白质启动，第二链DNA由一种 从末端冗余衍生的RNA低聚物 前基因组RNA的一部分。在启动第二链之前 DNA合成时，这个RNA引物被转移到第二个 链DNA启动点。要详细了解 庚型核糖核酸病毒复制方案，我们将分析特定的 复制中间体，执行病毒的基因分析 复制和探索遗传结构和生化 病毒聚合酶基因及其产物的性质(S)。vbl.使用 地鼠和土拨鼠肝炎病毒我们将 研究第二链DNA合成的机制 被发起，特别是解决了 关于前基因组RNA的RNA引物，RNA的3‘端如何 引物被创建，以及RNA引物如何转移到 第二链DNA启动点。为了研究酶的作用 由病毒聚合酶基因编码的活动，我们将表达 一株细菌中的地松鼠肝炎病毒聚合酶基因 表达系统。具体地说，我们将搜索反向 转录酶和RNAseH活性并确定其位置 它们在聚合酶开放阅读框架上的酶结构域。 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，并 补充我们的体内分析与体外研究 化学定义的分析条件。慢性乙肝病毒 感染是目前无法治愈的主要健康问题 是存在的。提高了对复制方案的理解 庚型核糖核酸病毒是开发特异性抗病毒药物所必需的 心理治疗。人肝细胞病毒体外检测系统的研制 复制可能对开发和 反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.