禽传染性支气管炎病毒Nsp2蛋白与宿主蛋白eIF2α的互作鉴定  

Identification of eIF2α interacting with Nsp2 protein of avian infectious bronchitis virus

作　　者：李念灵 李永新 樊顺怡 申玉玺 李淑芸[1] 夏静[1] 王安东 崔敏 韩新锋[1] 黄勇[1] 余松城 LI Nianling;LI Yongxin;FAN Shunyi;SHEN Yuxi;LI Shuyun;XIA Jing;WANG Andong;CUI Min;HAN Xinfeng;HUANG Yong;YU Songcheng(College of Veterinary Medicine,Sichuan Agricultural University,Chengdu 61l130,Sichuan,China;Dongpo Bureau of Agriculture and Rural Affairs,Meishan 620010,Sichuan,China)

机构地区：[1]四川农业大学动物医学院,四川成都611130 [2]眉山市东坡区农业农村局,四川眉山620010

出　　处：《微生物学报》2022年第10期4047-4056,共10页Acta Microbiologica Sinica

基　　金：“长江学者和创新团队发展计划”创新团队项目(IRTO848)。

摘　　要：Nsp2蛋白是冠状病毒的非结构蛋白,在病毒早期感染中具有重要作用。【目的】为初步筛选可能与禽传染性支气管炎病毒(avian infectious bronchitis virus,IBV)Nsp2蛋白互作的宿主蛋白,鉴定Nsp2蛋白与真核翻译起始因子2α亚基(eIF2α)的相互作用。【方法】以pCAGGs-Flag-Nsp2和pCAGGs-Flag载体转染后的鸡胚肾(CEK)细胞为研究对象,利用免疫共沉淀(Co-IP)和液相色谱-串联质谱(LC⁃MS/MS)技术筛选出可能与IBV Nsp2蛋白发生互作的宿主蛋白eIF2α,通过免疫共沉淀和间接免疫荧光试验进一步验证二者相互作用。【结果】经免疫共沉淀与质谱分析后筛选到97个可能与Nsp2蛋白互作的宿主蛋白,其中宿主抗病毒反应的关键蛋白eIF2α与Nsp2蛋白的相互作用通过免疫共沉淀和间接免疫荧光试验,表明二者存在直接互作关系,并共定位于细胞质中;此外,Nsp2蛋白表达和IBV感染都能显著提高宿主内源性eIF2α的转录水平。【结论】利用免疫共沉淀联合质谱技术筛选到CEK细胞中存在的97种可能与IBV Nsp2互作的候选蛋白,利用免疫共沉淀与间接免疫荧光证明候选蛋白eIF2α与Nsp2蛋白在细胞质中存在直接互作,同时发现Nsp2蛋白过表达和IBV感染会导致CEK细胞内eIF2α的转录水平显著增强。本研究为进一步探索冠状病毒非结构蛋白Nsp2的生物学功能奠定了基础,并提供了IBV入侵宿主时Nsp2蛋白的作用机制研究的新线索。[Background]Nonstructural protein Nsp2 of coronavirus plays a critical role in the early viral life cycle.[Objective]To explore the host proteins interacting with avian gamma-coronavirus infectious bronchitis virus(IBV)Nsp2 and the interaction of Nsp2 with translation initiation factor 2α(eIF2α).[Methods]We performed the experiment by using the chicken embryo kidney(CEK)cells transfected with recombinant expression vector pCAGGs-Flag-Nsp2 and control vector pCAGGs-Flag.The host cell elF2αinteracting with IBV Nsp2 was obtained by co-immunoprecipitation(Co-IP)and liquid chromatography-tandem mass spectrometry(LC-MS/MS),and then Co-IP and indirect immunofluorescence assay(IFA)were further used to determine the interaction.[Results]A total of 97 candidate proteins were found to interact with Nsp2 through Co-IP and LC-MS/MS,with a high credibility.The key candidate protein elF2αwas further chosen to explore its interaction with Nsp2.The Co-IP and IFA demonstrated that eIF2αand Nsp2 had a direct interrelationship and co-localized in the cytoplasm.Furthermore,the level of elF2αwas upregulated in the overexpression of Nsp2 and IBV-infected CEK cells at the early stage.[Conclusion]In our study,the identification of these candidate proteins interacting with Nsp2 showed that Nsp2 played an important role in IBV infection by manipulating a variety of cellular biological process,and these results provided the basis for further research on the role of Nsp2 in the life cycle of IBV.

关 键 词：禽传染性支气管炎病毒 Nsp2蛋白 质谱 免疫共沉淀 互作蛋白 EIF2Α 

分 类 号：S852.65[农业科学—基础兽医学]