REPLICATION OF HEPADNAVIRUSES

借助地鼠肝炎病毒和土拨鼠肝炎 作为乙肝病毒的模型，我们已经确定了主要的 复制的DNA中间体和识别的主要信号 控制正链和负链DNA合成。此外，我们还拥有 开发了一种新的系统，允许同步复制 培养细胞中的鸭乙型肝炎病毒，使用磷酸甲酸盐，a 庚型肝炎病毒逆转录酶的可逆抑制物。这 系统允许积累和随后分离复制的DNA 中间体在不同的成熟阶段，因此，允许 对庚型核糖核酸病毒复制周期几个方面的检查 以前不能进行生化分析的物质。为 这项建议我们主要关注四个目标，其中一些目标直接 从我们之前对病毒DNA的遗传和生化研究中发现 一等奖支持的综合。我们将调查身体状况 核心颗粒中前基因组RNA的结构和排列。这些 研究将首次揭示最早的 前基因组RNA、聚合酶和核心蛋白的病毒复制周期 组装成起始复合体，开始病毒DNA合成。在……里面 此外，我们将研究RNA在哪些生化条件下 含有核心颗粒的体外合成DNA并使用一种新策略 用于表达具有酶活性的逆转录酶。我们 还将继续不断努力，以期了解 启动正链DNA合成并执行一种 核心颗粒的生化和结构研究，其中DNA 当正链启动发生时，合成就停止了。 我们将确定正链DNA合成的启动是否取决于 核心粒子中负标准DNA的精确空间排列。 最后，我们将提出一种试验性策略来确定 在DNA合成阶段，胞质核心颗粒与 病毒包膜成分以及控制这一过程的信号是什么 反应。 从这些调查中获得的信息不仅有助于 全面了解庚型肝炎病毒复制的机制 同时也为设计有效的抗病毒药物提供了新的靶点 超过2亿的处理所需的化合物 慢性感染乙肝病毒的个人。

With the help of ground squirrel hepatitis virus and woodchuck hepatitis virus as models for hepatitis B virus, we have identified the major replicative DNA intermediates and identified the major signals controlling minus and plus strand DNA synthesis. Furthermore, we have developed a novel system allowing for the synchronized replication of duck hepatitis B virus in cultured cells, employing phosphonoformate, a reversible inhibitor of the hepadnavirus reverse transcriptase. This system permits accumulation and subsequent isolation of DNA replicative intermediates at different stages of maturation and therefore, allows for an examination of several aspects of the hepadnavirus replication cycle that have previously not been accessible for a biochemical analysis. For this proposal we have focused on four aims, some of which have directly emerged from our previous genetic and biochemical studies on viral DNA synthesis supported by the FIRST award. We will investigate the physical structure and arrangement of pregenomic RNA in core particles. These studies will for the first time shed light on the earliest events of the viral replication cycle where pregenomic RNA, polymerase and core protein assemble into an initiation complex to begin viral DNA synthesis. In addition, we will examine the biochemical conditions under which RNA containing core particles synthesize DNA in vitro and use a new strategy for the expression of enzymatically active reverse transcriptase. We will also continue ongoing efforts aimed at an understanding of the mechanism by which plus strand DNA synthesis is primed and perform a biochemical and structural investigation of core particles, in which DNA synthesis has been arrested at a step when plus strand priming occurs. We will determine whether priming of plus strand DNA synthesis depends on a precise spatial arrangement of minus stand DNA in core particles. Finally, we will present an experimental strategy to determine at what stage during DNA synthesis cytoplasmic core particles assemble with the viral envelope components and what the signals are that control this reaction. The information gained from these investigations will not only help to complete our understanding of the mechanism of hepadnavirus replication but also reveal new target sites for the design of effective antiviral compounds required for the treatment over more than 200 million individuals chronically infected with hepatitis B virus.