Structure and Function of a Viral RNA-Dependent RNA Polymerase

花王病毒RNA复制是病毒发病的一个基本过程。本实验室研究了一种模型（+）链RNA病毒——溴代花叶病毒（BMV），以表征RNA依赖的RNA聚合酶（RdRp）亚基之间的相互作用和RNA合成的机制。本研究将同时开展三个项目。首先，通过化学诱变、选择在双杂交试验中失去蛋白质-蛋白质相互作用的稳定突变体，以及对突变进行测序，剖析BMV复制酶中发现的蛋白质相互作用的要求。第二个项目旨在阐明有助于启动（-）链病毒RNA合成的序列，并研究这些序列如何影响（-）链RNA合成的步骤。第三个项目通过鉴定最小功能启动子和构建核苷酸替换启动子来检测基因组（+）链RNA合成，以鉴定影响病毒RNA合成各个步骤的RNA序列。从这个项目的执行中获得的知识将有助于对病毒如何复制的基本理解。使用雀科花叶病毒作为实验模型，将直接增加对研究较少的病毒的了解，其中一些病毒会引起疾病。对病毒RNA复制的不同步骤的详细了解最终可能会发现设计高度特异性抗病毒化合物的靶标。

Kao Viral RNA replication is a fundamental process in viral pathogenesis. This laboratory studies brome mosaic virus (BMV), a model (+)-strand RNA virus, in order to characterize the interactions between RNA-dependent RNA polymerase (RdRp) subunits and the mechanism of RNA synthesis. Three projects will be carried out simultaneously in this research. The first is designed to dissect the requirements for the interaction of the proteins found in the BMV replicase through chemical mutagenesis, selection for stable mutants that have lost protein-protein interaction in the two-hybrid assay, and sequencing the mutations. The second project is designed to elucidate the sequences that contribute to the initiation of (-)-strand viral RNA synthesis and to examine how these sequences affect steps in (-)-strand RNA synthesis. The third project examines genomic (+)-strand RNA synthesis by identifying the minimal functional promoter and constructing promoters with nucleotide substitutions, to identify RNA sequences that affect the various steps in viral RNA synthesis.Knowledge gained from the execution of this project will contribute to the basic understanding of how viruses replicate. The use of a plant virus, brome mosaic virus, as an experimental model, will directly increase the understanding of less well-studied viruses, some of which cause disease. The detailed understanding of the different steps in viral RNA replication could ultimately uncover targets for the design of highly specific antiviral compounds.