Identification and Analysis of a Potyviral RNA-dependent RNA-polymerase

马铃薯病毒RNA依赖性RNA聚合酶的鉴定和分析

• 批准号: 8902610
• 负责人: William Dougherty
• 金额: $ 24.5万
• 依托单位: Oregon State University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-09-01 至 1993-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=8902610&HistoricalAwards=false
• 关键词: Identification; Analysis; Potyviral; RNA; dependent

Tobacco etch virus (TEV) is a member of the potato virus Y (potyvirus) group. Potyviruses are frequently encountered pests and result in significant crop loss. New sources of virus resistance are needed. The work detailed in this proposal will identify the virus gene product(s) involved in viral RNA replication. The plan makes use of a defined system to ask questions about the role played by the TEV polymerase during the template elongation reaction. The polymerase gene will be expressed in heterologous cell systems and polymerase activity will be purified and assayed in a cell-free system using defined TEV templates. Protein domains responsible for polymerase activity will be identified using immounological and molecular genetic approaches. Monoclonal antibodies will be used to identify domains when bound by antibody, eliminate polymerase activity. These regions become candidates for a molecular genetic analysis using site directed mutagenesis. Likewise, an attempt will be made to construct hybrid polymerase molecules using gene segments from two closely related potyviruses. Polymerase domains responsible for the template elongation reaction are proposed to be common between these two viruses should be interchangable. Successful completion of the plan should identify a virus specific product which can be targeted for disruption in the design of new resistance strategies. These studies are important, both in terms of their potential for eventual control of this enocomically important pest and as an resource extremely useful for comparitive studies of RNA replication factors.

烟草蚀刻病毒（TEV）是马铃薯病毒Y（Potyvirus）组的成员。 托管病毒经常遇到害虫，并导致大量作物损失。 需要新的病毒抗性来源。 该提案中详细介绍的工作将确定与病毒RNA复制有关的病毒基因产物。 该计划利用定义的系统来询问有关TEV聚合酶在模板伸长反应中起作的作用的问题。 聚合酶基因将在异源细胞系统中表达，聚合酶活性将在使用定义的TEV模板中纯化并在无细胞系统中进行测定。 将使用免疫和分子遗传学方法鉴定负责聚合酶活性的蛋白质结构域。 单克隆抗体将用于鉴定抗体结合时，消除了聚合酶活性。 这些区域成为使用位点的诱变进行分子遗传分析的候选者。 同样，将尝试使用来自两个密切相关的托管病毒的基因段构建杂交聚合酶分子。 提出负责模板伸长反应的聚合酶结构域在这两种病毒之间是常见的。 该计划的成功完成应确定特定于病毒的产品，该产品可以针对新的阻力策略设计。 这些研究都很重要，无论是在最终控制这种重要的害虫方面的潜力而言，作为对RNA复制因子的比较研究非常有用的资源。