与猪流行性腹泻病毒M蛋白互作的宿主蛋白的鉴定

【背景】猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)膜蛋白(M)在病毒粒子的组装、膜融合和病毒复制等方面具有重要的作用,但M蛋白与宿主细胞的互作机制尚不清楚。【目的】利用免疫沉淀技术和液质联用技术筛选细胞内与PEDVM蛋白相互作用的蛋白,为揭示M蛋白在病毒增殖过程中发挥的功能提供研究基础。【方法】将MOI=0.1的PEDV DR13疫苗株接种于长成单层的Vero细胞,感染36 h后,收集细胞并进行裂解。利用抗M的单克隆抗体沉淀与M相互作用蛋白复合物,通过液相色谱串联质谱(LC-MS/MS)进行鉴定并利用细胞功能富集分析(Gene ontology,GO)对感染组鉴定到的细胞蛋白进行分析,确定两个细胞内源性蛋白为候选蛋白,进行免疫共沉淀(Co-IP)验证和共定位分析。【结果】基于鉴定蛋白的肽段数的方法分析显示,感染组与对照组相比,鉴定了218个与M蛋白相互作用的细胞内源性蛋白,分别与蛋白质合成、代谢、细胞信号通路转导等密切相关,选择细胞分裂周期蛋白42 (Cell division cycle 42,CDC42)、真核翻译起始因子3亚基L蛋白(eIF3L)为候选蛋白进行Co-IP(Co-immunoprecipitation)验证和共定位分析,结果证实CDC42、eIF3L蛋白分别与M蛋白在细胞内存在相互作用。【结论】鉴定出PEDV M蛋白能够与宿主细胞CDC42和eIF3L蛋白相互作用,并鉴定出其他可能与M蛋白发生相互作用的宿主蛋白60个,为开展PEDV与宿主细胞蛋白相互作用研究提供了重要理论依据。

Identification of host cellular proteins interacting with porcine epidemic diarrhea virus M protein

Background] The membrane protein (M) of porcine epidemic diarrhea virus (PEDV) plays an important role in the viral assembly process, membrane fusion and viral replication, but the mechanism of interaction between M protein and host cells is still unclear. Objective] Co-immunoprecipitation technique coupled with LC-MS/MS were used to screen cellular proteins interacting with PEDV M protein, which can provide a foundation for revealing the function of M in viral multiplication. Methods] PEDV DR13 vaccine strain was inoculated into monolayer of Vero cells at a MOI of 0.1. After 36 hours of infection, the cells were collected and lysed. Host cellular proteins that interact with the M protein of PEDV were immunoprecipitated using the M monoclonal antibody, then identified by LC-MS/MS, and analyzed by gene ontology (GO) annotation. Among them, two interested cellular proteins were further confirmed with Co-IP and cellular colocalization. Results] Based on the analysis of the number of peptide segments, 218 cellular proteins interacting with M protein were identified. These host cellular proteins are closely related to protein synthesis, metabolism and cell signaling pathway transduction. Cell division cycle 42 (CDC42) and eukaryotic translation initiation factor 3 subunit L (eIF3L) were chosen for reverse Co-IP reconfirmation and colocalization analysis. The results showed that both CDC42 and eIF3L protein interact with M protein. Conclusion] This study identified that PEDV M protein could interact with CDC42 and eIF3L proteins in host cells, and identified 60 other host proteins that might interact with M protein. The study is to provide an important theoretical basis for the study of the interaction between PEDV and host cell proteins.