REPLICATION OF HEPADNAVIRUSES

在黄鼠肝炎病毒和土拨鼠肝炎的帮助下 作为B型肝炎病毒的模型，我们已经确定了主要的 复制的DNA中间体，并确定了主要的信号 控制负链和正链DNA合成。 此外，我们有 开发了一种新的系统，允许同步复制 鸭肝炎B病毒在培养的细胞，采用膦甲酸， 嗜肝DNA病毒逆转录酶的可逆抑制剂。 这 系统允许DNA复制的积累和随后的分离 不同成熟阶段的中间体，因此， 嗜肝DNA病毒复制周期的几个方面的检查 以前无法进行生化分析。 为 我们的建议集中在四个目标上，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得出 第一个奖项支持的合成。 我们会调查 核心颗粒中前基因组RNA的结构和排列。 这些 这些研究将首次揭示人类最早期的事件， 病毒复制周期中前基因组RNA、聚合酶和核心蛋白 组装成起始复合物以开始病毒DNA合成。 在 此外，我们还将研究RNA产生的生化条件， 含有核心颗粒的体外合成DNA并使用新策略 用于表达酶活性逆转录酶。 我们 还将继续努力， 正链DNA合成的启动机制， 核心颗粒的生物化学和结构研究，其中DNA 合成在正链引发发生的步骤中被阻止。 我们将确定正链DNA合成的启动是否取决于 核心粒子中负链DNA的精确空间排列。 最后，我们将提出一个实验策略，以确定在什么情况下， 在DNA合成的阶段，细胞质核心颗粒与 病毒包膜成分以及控制它的信号是什么 反应 从这些调查中获得的信息不仅有助于 完成我们对嗜肝DNA病毒复制机制的理解 还揭示了设计有效抗病毒药物的新靶点， 治疗所需的化合物超过2亿 慢性感染B型肝炎病毒的个体。

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.