猪流行性腹泻病毒非结构蛋白nsp9互作宿主蛋白对病毒复制的影响

Effects of host proteins interacting with non-structural protein nsp9 of porcine epidemic diarrhea virus on viral replication

猪流行性腹泻病毒(porcineepidemicdiarrheavirus,PEDV)导致仔猪和育肥猪发生急性肠道传染病,是危害养猪业最重要的病原体之一。目前发现PEDV能够编码至少16个非结构蛋白,其中nsp9能够结合至单链RNA中,但是其功能机制还不清楚。本研究通过免疫沉淀联合蛋白质谱分析,筛选出潜在的与PEDV nsp9宿主互作蛋白。通过进一步免疫共沉淀(co-immunoprecipitation,Co-IP)和激光共聚焦技术确认了nsp9与热休克蛋白HSPA8、Toll相互作用蛋白Tollip、热休克蛋白HSPA9、线粒体外膜蛋白TOMM70互作。其中,过表达HSPA8使nsp9的表达量先上调而后下调,并促进PEDV的增殖;过表达Tollip使nsp9的表达量显著上调,并抑制PEDV的增殖;过表达TOMM70使nsp9的表达量显著下调,但对PEDV的增殖无明显影响;过表达HSPA9对nsp9的表达以及PEDV的增殖均无明显影响。该研究为探索nsp9互作蛋白在PEDV感染过程中的功能提供了重要信息。

Porcine epidemic diarrhea virus(PEDV)is a highly pathogenic virus that can cause acute intestinal infectious diseases in both piglets and fattening pigs.The virus encodes at least 16 non-structural proteins,including nsp9,which has been shown to bind to single-stranded RNA.However,its function and mechanism remain unclear.In this study,we aimed to identify potential host proteins that interact with PEDV nsp9 using immunoprecipitation combined with mass spectrometry.The interactions were then confirmed by co-immunoprecipitation(Co-IP)and confocal laser scanning fluorescence techniques.The results showed that nsp9 interacts with HSPA8,Tollip,HSPA9 and TOMM70.Among them,overexpression of HSPA8 resulted in caused first upregulated and then down-regulated expression of nsp9,and promoted the proliferation of PEDV.Overexpression of Tollip significantly upregulated the expression of nsp9 and inhibited the proliferation of PEDV.Overexpression of TOMM70 significantly reduced the expression of nsp9,but did not show significant effect on the proliferation of PEDV.Overexpression of HSPA9 did not show significant effect on the expression of nsp9 and the proliferation of PEDV.These findings may facilitate further investigating the role of nsp9-interacting proteins in PEDV infection.