GENETICS AND BIOCHEMISTRY OF POTATO VIRUS X REPLICATION

马铃薯 X 病毒复制的遗传学和生物化学

• 批准号: 2187379
• 负责人: CYNTHIA L HEMENWAY
• 金额: $ 7.36万
• 依托单位: NORTH CAROLINA STATE UNIVERSITY RALEIGH
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-09-01 至 1999-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2187379
• 关键词: Escherichia coli; RNA binding protein; RNA biosynthesis; RNA virus; gel mobility shift assay; gene deletion mutation; genetic regulatory element; helicase; host organism interaction; molecular genetics; nucleic acid sequence; nucleic acid structure; plant virus; protein structure function; protoplast /spheroplast; replicase; site directed mutagenesis; tissue /cell culture; virus genetics; virus protein; virus replication

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.

RNA指导的RNA合成对于许多原核生物的生存是必不可少的。 和真核RNA病毒。 大部分真核病毒 在宿主细胞的细胞质中复制的正链RNA病毒， 其中许多会导致医学和农业疾病， 意义 尽管它们很重要，但只有少数动物和植物 病毒已经在复制方面进行了研究， 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 RNA复制和疾病发展所必需的。 这种病毒 含有一个功能上为单顺反子的基因组RNA。 病毒 复制酶P1是RNA合成所需的唯一病毒蛋白质， 潜在地编码复制所需的若干活动。 因此，在本发明中， PVX P1是复制模块的优秀模型， 协调实体。 PVX可用于生物化学和遗传学研究 因为病毒在烟草植物和原生质体中复制良好， 感染性转录物可以从PVX cDNA克隆产生， 突变分析 该系统将用于确定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 P1的多功能性通过解决三个问题。 有哪些 RNA所必需的顺式作用调节序列和结构 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 cDNA克隆末端的缺失。 成绩单 将来自这些克隆的细胞接种到烟草原生质体上， 评估RNA的合成。 预测次级的重要性 通过分析RNA， 在接种有含有位点- 定向突变，以及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 RNA 结合结构域和PVX RNA中与P1或P2结合的精确区域 将确定宿主蛋白质。 P1的功能意义 将通过突变分析来分析复制的RNA结合结构域 在原生质体中。 哪些生化反应是复制所必需的 是由P1或含有P1的提取物催化的？ P1可以作为 RNA结合蛋白、RNA依赖性RNA聚合酶、解旋酶和加帽 酵素 感染的烟草提取物和分离自烟草的P1或P1的部分 E.大肠杆菌或昆虫细胞将测定与每种相关的活性。 这些功能。 这些研究将为今后的研究奠定基础 通过分析P1功能结构域，宿主蛋白功能， 复制复合体形成所需的相互作用，潜在的 P1/宿主膜相互作用，以及体外 复制系统来定义RNA复制机制。