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

烟草蚀刻病毒(TEV)是马铃薯Y病毒(马铃薯Y病毒)组的成员。马铃薯Y病毒是一种常见的害虫，对农作物造成重大损失。需要新的病毒抗性来源。这项提案中详细介绍的工作将鉴定参与病毒核糖核酸复制的病毒基因产物(S)。该计划利用一个确定的系统来询问关于TEV聚合酶在模板延伸反应中所起的作用的问题。聚合酶基因将在异种细胞系统中表达，聚合酶活性将被提纯，并在无细胞系统中使用定义的TEV模板进行分析。负责聚合酶活性的蛋白质结构域将使用免疫学和分子遗传学方法进行鉴定。单抗将被用来识别与抗体结合的结构域，消除聚合酶活性。这些区域成为使用定点突变进行分子遗传分析的候选区域。同样，将尝试使用两种密切相关的马铃薯Y病毒的基因片段来构建杂交聚合酶分子。负责模板延长反应的聚合酶结构域被认为是这两种病毒之间共同的，应该是可以互换的。成功完成该计划应确定一种特定于病毒的产品，该产品可在设计新的抵抗策略时作为干扰的目标。这些研究是重要的，因为它们可能最终控制这种重要的害虫，并且作为一种非常有用的资源，对RNA复制因子的比较研究非常有用。

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.