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: 6636091
• 负责人: CYNTHIA L HEMENWAY
• 金额: $ 21.98万
• 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 2005-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6636091
• 关键词: RNA binding protein; RNA biosynthesis; RNA virus; gel mobility shift assay; genetic regulatory element; genetic transcription; helicase; host organism interaction; immunoprecipitation; plant virus; protein protein interaction; protein sequence; protein structure function; protoplast /spheroplast; virus genetics; virus protein; virus replication; yeast two hybrid system

DESCRIPTION: (Adapted from the Investigator's abstract): The potato virus X (PVX) system is an excellent model for understanding general mechanisms of RNA replication and potentially for designing novel antiviral strategies. PVX has a relatively small genome from which only a few subgenomic RNAs are produced. Both a tobacco protoplast replication system and a soluble template-dependent extract will be used to answer four questions. Which cis-acting sequences and structures affect PVX RNA accumulation in vitro? Sequences and structures that are important for synthesis of plus-and minus-strand RNAs will be determined by analysis of RNA accumulation in tobacco protoplasts inoculated with wild-type and mutant PVX transcripts. What are the template requirements for PVX RNA synthesis in vitro? The PVX template-dependent in vitro transcription system will be optimized and utilized to define template requirements for minus-and plus-strand RNA synthesis. What are the protein constituents and protein-protein interactions required for PVX RNA synthesis in vivo? The protein constituents required for PVX RNA synthesis in vitro will be purified and sequenced. Corresponding cDNA clones obtained by sequence analysis or from a yeast two-hybrid library screen will be used to assess protein-protein interactions required for RNA synthesis. Do host and/or viral proteins interact with regulatory elements in PVX RNA? The extent of binding of proteins from uninfected and infected plant or protoplast extracts to wild-type and mutant PVX transcripts will be determined by gel shift and immunoprecipitation assays. Purified binding proteins will be isolated by affinity chromatography for sequencing. Further analyses of RNA-protein interactions will utilize the cDNAs and proteins corresponding to sequenced proteins. Data obtained from these specific aims will define the components and mechanisms involved in RNA synthesis.

描述：（改编自研究者摘要）：马铃薯X病毒 (PVX)系统是理解RNA一般机制的极好模型 复制和潜在的设计新的抗病毒策略。PVX具有 相对较小的基因组，仅产生少量亚基因组RNA。 烟草原生质体复制系统和可溶性模板依赖的 我们会用摘录来回答四个问题。哪些顺式作用序列和 结构影响PVX RNA在体外的积累？序列和结构， 对于正链和负链RNA的合成是重要的， 用野生型烟草原生质体接种的烟草原生质体中RNA积累的分析 和变异的PVX转录物。PVX RNA的模板要求是什么 体外合成？PVX模板依赖的体外转录系统 将被优化和利用，以定义模板要求， 正链RNA合成。什么是蛋白质成分和 体内PVX RNA合成所需的蛋白质-蛋白质相互作用？的 将纯化PVX RNA体外合成所需的蛋白质组分 和序列测定相应的cDNA克隆通过序列分析或从 酵母双杂交文库筛选将用于评估蛋白质-蛋白质 RNA合成所需的相互作用。宿主和/或病毒蛋白质是否相互作用 PVX RNA中的调控元件蛋白质的结合程度 未感染和感染的植物或原生质体提取物转化为野生型和突变体 将通过凝胶迁移和免疫沉淀试验测定PVX转录物。 纯化的结合蛋白将通过亲和色谱法分离， 测序RNA-蛋白质相互作用的进一步分析将利用cDNA 和对应于测序蛋白质的蛋白质。数据来源于这些 具体的目标将确定RNA的组成部分和机制， 合成.