大菱鲆呼肠孤病毒非结构蛋白NS22诱导细胞融合关键功能域鉴定及其对病毒感染影响的研究

项目名称： 大菱鲆呼肠孤病毒非结构蛋白NS22诱导细胞融合关键功能域鉴定及其对病毒感染影响的研究

项目编号： No.31302227

项目类型： 青年科学基金项目

立项/批准年度： 2014

项目学科： 农业科学

项目作者： 柯飞

作者单位： 河南城建学院

项目金额： 25万元

中文摘要： 水生呼肠孤病毒是一类危害鱼类养殖的重要病毒性病原，其感染和致病机制备受关注，而细胞融合则是水生呼肠孤病毒感染细胞所引起的主要特征之一。本项目以新分离的一株水生呼肠孤病毒-大菱鲆呼肠孤病毒为研究对象，在鉴定了一个诱导细胞融合蛋白（FAST蛋白）NS22的基础上，首先利用片段缺失突变和定点突变技术对NS22进行改造，以融合细胞形成能力为依据，查明NS22引起细胞融合的功能结构域和必需氨基酸位点，并通过基因过表达技术验证上述功能结构域诱导细胞融合的情况；进一步，采用RNA干扰技术抑制NS22的表达，分析NS22下调表达对病毒侵染的影响，另外，在已表达NS22的细胞中接种病毒，研究NS22过表达对病毒侵染的影响。通过本项目研究，不仅能深入了解NS22各功能区对其诱导细胞融合能力的影响，而且能查明细胞融合与水生呼肠孤病毒感染的关系，对揭示水生呼肠孤病毒的复制与致病机制具有重要的意义。

中文关键词： 大菱鲆呼肠孤病毒；细胞融合；功能区域；病毒复制；共定位

英文摘要： Aquareoviruses have been isolated from a wide variety of aquatic animals. These viruses represent a great threat to the aquaculture industry in China and East Asia. One of the typical cytopathic effects in aquareovirus infection is the formation of syncytium, which is caused by cell-cell fusion. Recently, a new Scophthalmus maximus reovirus (SMReV) was isolated and identified from a diseased turbot. The complete genome of SMReV has been sequenced, which was the first isolated and completely sequenced aquareovirus from marine fish in China. Sequence and functional analysis of genome sequences of SMReV identified a fusion associated small transmembrane (FAST) protein NS22, whose expression caused cell-cell fusion and syncytium formation in fish cells. However, how this protein caused cell-cell fusion and its function in virus replication remained unknown. In the present study, the functional domains and amino acids of NS22 would be analyzed to investigate the mechanism and significance of cell-cell fusion. Functional domains and amino acids of NS22 would be truncated or mutated. The ability of syncytium formation would be analyzed among NS22 truncations and mutants to identify the key domains and amino acids. NS22 truncations that did not cause cell-cell fusion would be coexpressed with full-length NS22 to further

英文关键词： Scophthalmus maximus reovirus；cell-cell fusion；functional region；virus replication；colocalization