猪流行性腹泻病毒非结构蛋白Nsp8与宿主细胞互作蛋白的筛选与鉴定

Screening and identification of host proteins interacting with Nsp8 of porcine epidemic diarrhea virus

摘要:

猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)是一种能引起严重腹泻、脱水的肠道病毒，PEDV的广泛流行对生猪养殖产业造成巨大经济损失，目前仍无有效的预防和治疗手段。Nsp8是一种参与PEDV复制的重要非结构蛋白，其存在相互作用的宿主蛋白尚不清楚。【目的】筛选与PEDV Nsp8互作宿主蛋白，初步探究互作宿主蛋白对PEDV复制的影响，为寻找PEDV新的关键性治疗靶点提供理论基础。【方法】利用真核表达载体pcDNA3.1(+)成功构建PEDV Nsp8真核表达质粒，通过免疫共沉淀、质谱分析及激光共聚焦等技术筛选能够与其相互作用的宿主蛋白，进一步在LLC-PK细胞中通过过表达、敲低等方法探究互作宿主蛋白对PEDV复制的影响。【结果】质谱检测筛选到Nsp8潜在互作宿主蛋白36个，验证了宿主蛋白热休克蛋白成员8(heat shock protein member 8,HSPA8)与Nsp8相互作用，在LLC-PK细胞中过表达HSPA8能够剂量依赖性抑制Nsp8蛋白过表达，而且在蛋白质和转录水平显著剂量依赖性抑制PEDV复制；干扰内源性HSPA8表达能够显著促进PEDV复制，TCID₅₀和间接免疫荧光试验检测(indirect immunofluorescent assay,IFA)结果进一步证明了HSPA8抑制PEDV复制。【结论】本研究筛选出PEDV Nsp8的互作宿主蛋白HSPA8，并且证明HSPA8能显著抑制PEDV复制，为设计以HSPA8为靶点的预防或治疗药物提供新思路。

Abstract:

Porcine epidemic diarrhea virus (PEDV) is an enterovirus that can cause severe diarrhea and dehydration.The widespread epidemic of PEDV has caused huge economic losses to the pig breeding industry,which,however,lacks effective means for prevention and treatment.Nsp8 is an important non-structural protein involved in the replication of PEDV,while the host proteins interacting with Nsp8 remains unclear.[Objective] To screen the host proteins interacting with PEDV Nsp8 and explore the effects of the host proteins on the replication of PEDV,so as to provide a theoretical basis for discovering new key functional receptors or therapeutic targets of PEDV.[Methods] The eukaryotic expression plasmid of PEDV Nsp8 was successfully constructed with the eukaryotic expression vector pcDNA3.1(+).The host proteins interacting with PEDV Nsp8 were screened by co-immunoprecipitation,mass spectrometry,and laser confocal microscopy.The effects of the host proteins on PEDV replication were explored by overexpression and knockdown in LLC-PK cells.[Results] Thirty-six potential host proteins interacting with Nsp8 were screened by mass spectrometry,and the interaction between heat shock protein member 8(HSPA8) and Nsp8 was verified.The overexpression of HSPA8 in LLC-PK cells inhibited the overexpression of Nsp8 in a dose-dependent manner.Meanwhile,it significantly inhibited the replication of PEDV in a dose-dependent manner at the protein and transcriptional levels.Interfering with endogenous HSPA8 expression significantly promoted the replication of PEDV.The 50% tissue culture infectious dose (TCID₅₀) and indirect immunofluorescence further proved that HSPA8 inhibited PEDV replication.[Conclusion] This study screened out the host protein HSPA8 interacting with PEDV Nsp8 and proved that HSPA8 could significantly inhibit PEDV replication,which provided a new idea for the design of HSPA8-targeted drugs for the prevention or treatment of PEDV.