STRUCTURE AND REPLICATION OF HEPATITIS DELTA VIRUS

HDV是一种人类病原体，通常与更具破坏性的乙肝病毒有关 肝脏感染。长远的目标是理解这部小说 HDV复制机制，重点介绍迄今未知的特征 发生在其他动物病毒的复制过程中。四个目标是 提议：I.体外RNA合成：令人惊讶的是 正常人肝细胞同时转录基因组和反基因组HDV RNA。为了表征这种由宿主酶引导的不同寻常的RNA合成， 将准备核提取液，添加HDV RNA，并对 转录复合体的组装，转录的启动，以及 合成全长或更长的转录本。RNA序列或 启动RNA定向合成所需的结构将是 评估，以及纯化的小三角洲蛋白的效果(如果有的话)。 体内RNA合成：基因组复制可通过以下途径启动 用体外组装的HDV RNA RNP复合体转染细胞 和重组Delta蛋白。这一策略模拟了自然感染 但不需要原代肝细胞(已知的唯一表达 病毒受体)。更改RNP的组件将确定 对RNA和蛋白质的要求，包括对 德尔塔蛋白在启动感染中的作用。5‘-比赛程序 将绘制推测的转录起始点图，有助于确定 改变核糖核酸序列和结构的后果 这样的网站。三.5‘-限制和核输出基因转录本 VIVO：研究将集中在天然HDV mRNA是否具有帽子结构 和顺式作用的核出口信号，将有助于理解核 保留大的HDV RNA。四、德尔塔的翻译后修饰 蛋白质：大的三角洲蛋白可以被磷酸化，是唯一的 蛋白质，来自任何已知法尼化的病毒。实验将会 说明这两个修改之间的明显联系以及它们的 复制中的角色。总而言之，在四个具体目标中提出的研究 将提供有关HDV生命周期的独特方面的信息， 因为HDV可能比其他任何病毒都更依赖于主机功能 动物病毒，关于宿主机器的信息，如宿主如何轮询 II被重定向到复制RNA模板以及如何翻译后 修饰可以被病毒蛋白颠覆。

HDV is a human pathogen typically associated with more damaging HBV infections of the liver. The long-range goal is to understand the novel mechanism of HDV replication, focusing on features not so far known to occur during the replication of other animal viruses. Four aims are proposed: I. RNA synthesis in vitro: Surprisingly nuclear extracts from normal human liver cells transcribe both genomic and anti-genomic HDV RNAs. To characterize this unusual RNA-directed synthesis by host enzymes, nuclear extracts will be prepared, HDV RNAs added, and studies made of the assembly of transcription complexes, initiation of transcription, and synthesis of full-length or longer transcripts. RNA sequences or structures needed for initiation of RNA-directed synthesis will be assessed, and also the effects, if any, of purified small delta protein. II. RNA synthesis in vivo: Genome replication can be initiated by transfecting cells with in vitro assembled RNP complexes, made of HDV RNAs and recombinant delta proteins. This strategy mimics natural infections but does not require primary hepatocytes (the only cells known to express the virus receptor). Altering components of the RNP will determine the requirements of both RNA and protein, including an examination of the roles of delta proteins in the initiation of infection. 5'-RACE procedures will map putative transcription initiation sites help determine the consequences of altering RNA sequence and structure in the vicinity of such sites. III. 5'-Capping and nuclear export of mRNA transcripts in vivo: Studies will focus on whether natural HDV mRNA has a cap structure and a cis-acting nuclear export signal, and will help understand nuclear retention of large HDV RNAs. IV. Post-translational modifications of delta proteins: Large delta protein can be phosphorylated and is the only protein, from any virus, known to be farnesylated. Experiments will address an apparent linkages of these two modifications as well as their roles in replication. In summary, studies proposed in four specific aims will provide information on unique aspects of the HDV life cycle and, because HDV is possibly more dependent on host functions than any other animal virus, information on the host machinery, such as how the host pol II is redirected to copy RNA templates and how post-translational modifications can be subverted by viral proteins.