VSV核衣壳蛋白上关键氨基酸的功能研究及弱化的重组VSV病毒株的筛选

水泡性口炎病毒（VSV）是研究不分节负链RNA病毒的的模式病毒,该类病毒在细胞质内的复制与转录是依赖于自身所携带的聚合酶来完成的。本研究在之前生物信息学分析的基础上，以核衣壳蛋白（N蛋白）为主要研究对象，利用丙氨酸扫描、微基因组检测、免疫共沉淀等方法来寻找并解析VSV核衣壳蛋白上关键氨基酸位点突变后对病毒复制转录的影响。为此，我们将首先通过丙氨酸扫描以及微基因组复制子报告基因表达检测明确N蛋白上的关键氨基酸位点；然后，鉴定这些关键氨基酸对N蛋白寡聚化和聚合酶形成的调节；最后，我们将明确关键氨基酸突变后N蛋白在细胞内的分布、是否能形成病毒RNA合成所需的功能性模板以及重组病毒的表型。本研究的开展不仅明确了N蛋白在VSV转录复制过程中的具体作用机制，而且将尝试得到一些弱化的或对温度敏感的重组VSV病毒株，用于疫苗和溶瘤病毒的研发。

Vesicular stomatitis virus (VSV) is the model virus for studying nonsegmented negative strand RNA virus. The transcription and replication of VSV depend on the polymerase of itself. Based on previous bioinformatic analysis, our research is focused on the viral Nucleocapsid protein. In this study, we will use alanine-scanning mutation, VSV minigenome system and co-immunoprecipitaion to find out the key amino acids of the nucleocapsid protein. Therefore, we will first check the effect of amino acids in C-terminal of N protein on the expression of CAT in minigenome system; then, we will further identify if the key amino acids can regulate the oligomerization of N protein or the formation of RNA polymerase complex; Finally, we will define the distribution of N mutants in host cell, the effect of N mutants on the formation of functional N-RNA template and the phenotype of recombinant VSV viruses. Therefore, this study will not only make clear that the role about key amino acids of N protein in VSV life cycle, but also will try to get some attenuate or temprature sensitive recombinant VSV for the development of vaccines and oncolytic viruses.