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

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 investigate the function of these domains in syncytium formation. On the other hand, expression of NS22 in virus infection would be suppressed by RNA interference (RNAi), and then the virus multiplication including cytopathic effect, virus ultrastructure and virus virulence would be analyzed to investigate the effects caused by inhibition of NS22 expression. Similar observations would also be done in virus infected full-length NS22 expressed cells to investigate the effects caused by NS22 overexpression. The significance of cell-cell fusion caused by NS22 in virus infection would be analyzed based on the results from inhibition of NS22 expression and NS22 overexpression. Taken together, results of the above would help us to understand the mechanism and significance of cell-cell fusion caused by NS22. It would give us new insights into the mechanisms of pathogenesis of aquareoviruses and new approaches in the prevention of aquareovirus infection.

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。

大菱鲆呼肠孤病毒（Scophthalmus maximus reovirus, SMReV）感染时引起鱼类培养细胞的融合。前期研究表明细胞融合是由SMReV编码的非结构蛋白NS22介导的。氨基酸序列分析表明，NS22分为N端（1-34氨基酸）、跨膜区（35-57）和C端（58-198）。其中，N端包括1个十四烷基化位点，C端包括2个多碱性氨基酸（polybasic，PB；61-68，82-95）区域、2个多精氨酸、脯氨酸和组氨酸（RPH；114-136，162-172）区域、1个疏水（HP，140-150）区域。为确定NS22诱导细胞融合的关键功能区域，本项目首先构建了N端、跨膜区、C端及C端各区域分别缺失的重组质粒。转染实验表明，N端、跨膜区、C端、PB区，RPH1区、HP区对于NS22诱导细胞融合的能力不可或缺。十四烷基化位点的突变实验表明其对于细胞融合也是必需的。进一步对N端、跨膜区和C端PB区的氨基酸残基进行逐一点突变，分析了各氨基酸残基突变后NS22诱导细胞融合能力的变化，明确了影响细胞融合的关键位点。同时，对细胞融合的药物抑制实验表明病毒复制不依赖于细胞融合。另外，共转染及免疫荧光分析表明NS22在细胞内与内质网和高尔基体共定位，其可能通过内质网-高尔基体途径转运到细胞膜上。NS22诱导细胞融合的能力与培养基中钙离子浓度有关，受到钙离子的调节，提示NS22诱导的细胞融合可能需要钙依赖蛋白的参与。相关研究为深入理解NS22诱导细胞融合的分子机制和意义提供了新的线索。