GENETICS AND BIOCHEMISTRY OF POTATO VIRUS X REPLICATION

RNA指导的RNA合成是许多原核生物生存所必需的 和真核生物RNA病毒。很大比例的真核病毒是 在宿主细胞的细胞质中复制的正链RNA病毒， 其中许多会导致医疗和农业方面的疾病 意义。尽管它们很重要，但只有少数动物和植物 病毒已经从复制的角度进行了研究，但很少有人研究 了解复制机制。马铃薯X病毒(PVX)是一种 了解病毒/宿主相互作用的优秀模型系统 是RNA复制和疾病发展所必需的。这种病毒 含有一种具有单顺反子功能的基因组RNA。病毒式传播 复制酶P1是RNA合成所需的唯一病毒蛋白，并且 潜在地编码了复制所需的几个活动。因此， PVX P1是复制模块的优秀模型，该模块演变为 协调实体。PVX在生化和遗传学研究中很有用 因为病毒在烟草植株和原生质体中复制良好，而且 PVX cDNA克隆可产生具有感染性的转录本 突变分析。该系统将被用来确定RNA/RNA 以及与PVX RNA合成相关的RNA/蛋白质相互作用， 宿主蛋白参与复制的程度，以及 通过回答三个问题来解决P1的多功能性质。什么是 RNA所需的顺式作用调控序列和结构 复制？重要的序列和结构 正链和负链RNA的合成将通过引入 感染性PVX基因克隆末端的缺失。文字记录 从这些克隆中衍生出来的将被接种到烟草原生质体上 对RNA的合成进行了评估。预测的次要事件的意义 调控区域的结构将通过分析RNA来确定 转录本接种原生质体中的合成 定向突变，并通过分析溶液中的结构。做P1 和/或宿主蛋白与PVX RNA特异性相互作用？P1核糖核酸 结合结构域和PVX RNA的精确区域，该区域与P1或 宿主蛋白将被确定。P1的功能意义 将通过突变分析来分析与复制相结合的RNA结构域 在原生质体中。复制需要哪些生化反应 是由P1或含P1的提取物催化的？P1可以作为一个 RNA结合蛋白、依赖于RNA的RNA聚合酶、解旋酶和封顶 酵素。受感染的烟草提取物和从中国分离的P1或部分P1 大肠杆菌或昆虫细胞将被检测与每种细胞相关的活动 这些功能中的一个。这些研究将构成未来研究的基础。 对P1功能区、宿主蛋白功能、 复制复合体形成所需的相互作用，潜力 P_1/宿主膜相互作用与AFP的体外发育 复制系统，以定义RNA复制的机制。

RNA directed RNA synthesis is essential for survival of many procaryotic and eucaryotic RNA viruses. a large percentage of eucaryotic viruses are plus-strand RNA viruses that replicate in the cytoplasm of host cells, and many of these cause diseases that are of medical and agricultural significance. Despite their importance, only a few animal and plant viruses have been studied in terms of replication and very little is known about the replication mechanisms. Potato virus X (PVX) is an excellent model system for understanding the virus/host interactions necessary for RNA replication and disease development. This virus contains one genomic RNA that is functionally monocistronic. The viral replicase, P1, is the only viral protein required for RNA synthesis, and potentially encodes several activities required for replication. Thus, PVX P1 is an excellent model for a replication module that evolved as a coordinated entity. PVX is useful for biochemical and genetic studies because the virus replicates well in tobacco plants and protoplasts, and infectious transcripts can be generated form a PVX cDNA clone for mutational analyses. This system will be used to determine the RNA/RNA and RNA/protein interactions associated with PVX RNA synthesis, the extent of host protein involvement in replication, and the multifunctional nature of P1 by addressing three questions. What are the cis-acting regulatory sequences and structures necessary for RNA replication? The sequences and structures that are important for synthesis of plus and minus strand RNAs will be determined by introducing deletions at the termini of an infectious, PVX cDNA clone. Transcripts derived from these clones will be inoculated onto tobacco protoplasts and evaluated for synthesis of RNA. The significance of predicted secondary structures in regulatory regions will be determined by analyzing RNA synthesis in protoplasts inoculated with transcripts containing site- directed mutations, and by analysis of structures in solution. Do P1 and/or host proteins interact specifically with PVX RNA? The P1 RNA binding domain and the precise region of PVX RNA that is bound by P1 or host proteins will be determined. The functional significance of the P1 RNA binding domain to replication will be analyzed by mutational analysis in protoplasts. Which biochemical reactions necessary for replication are catalyzed by P1 or P1-containing extracts? P1 could function as an RNA binding protein, RNA dependent RNA polymerase, helicase and capping enzyme. Infected tobacco extracts and P1 or portions of P1 isolated from E. coli or insect cells will be assayed for activities related to each of these functions. These studies will form a basis for future research on analysis of P1 functional domains, host protein functions, interactions required for replication complex formation, potential P1/host membrane interactions, and the development of an in vitro replication system to define mechanisms of RNA replication.