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

烟草蚀纹病毒是马铃薯Y病毒的成员 （马铃薯Y病毒）组。 Potyviruses经常遇到 害虫，造成重大作物损失。 的新来源 需要抗病毒。 本书中详细介绍的工作 提案将确定涉及的病毒基因产物， 病毒RNA复制 该计划利用一个定义的 询问有关TEV所扮演角色的问题的系统 聚合酶在模板延伸反应中的作用。 的 聚合酶基因将在异源细胞中表达 系统和聚合酶活性将被纯化和测定 在无细胞系统中使用定义的TEV模板。 蛋白 负责聚合酶活性的结构域将是 通过免疫学和分子遗传学鉴定 接近。 单克隆抗体将用于鉴定 当被抗体结合时，消除聚合酶 活动 这些区域成为分子的候选者， 使用定点诱变进行遗传分析。 同样地， 将尝试构建杂交聚合酶 分子使用基因片段从两个密切相关的 马铃薯Y病毒 聚合酶结构域负责 模板延伸反应被认为是常见 这两种病毒之间应该可以互换。 成功完成该计划应确定一个病毒 特定的产品，可以有针对性地破坏 设计新的抵抗策略。 这些研究很重要，无论是从其潜力来看， 为了最终控制这种重要的经济害虫， 作为一个资源非常有用的比较研究， RNA复制因子

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.