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

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。

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

Abstract:

[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 eIF2α interacting 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 eIF2α was further chosen to explore its interaction with Nsp2.The Co-IP and IFA demonstrated that eIF2α and Nsp2 had a direct interrelationship and co-localized in the cytoplasm.Furthermore,the level of eIF2α was 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.

Key words:avian infectious bronchitis virus;nonstructural protein Nsp2;mass spectrometry;co-immunoprecipitation;protein-protein interaction;eIF2α