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

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.