IBV Nsp2-interacting Host Factor EIF2α To Affect Viral Replication

Infectious bronchitis virus（IBV）,which causes Infectious bronchitis（IB）,is an important pathogen threatening poultry farms worldwide.IBV contains 15 non-structural proteins（Nsp2-16）,they are not only involved in viral replication,assembly and maturation but also play an essential role in antagonizing the host antiviral response.Nsp2is one of the first translated and processed replicative proteins.However,its function in viral infection is currently unknown.To reveal the biological function of non-structural protein Nsp2 in the process of IBV infection,in the study a comprehensive bioinformatics analysis of Nsp2 was conducted first to predict the biological function of Nsp2.Then,we used Co-immunoprecipitation（Co-IP）to screen the candidate host proteins interacting with Nsp2 by protein tagging,combined with high-performance liquid chromatography-tandem mass spectrometry（LC-MS/MS）for protein identification and obtained eIF2α,a key protein in the host protein-protein interaction network.Subsequently,the interaction between Nsp2 and host cell protein eIF2αwas further verified,and the molecular mechanism of its influence on IBV replication was analyzed at m RNA levels,The specific contents of this study are as follows:1.Bioinformatics characteristics of IBV Nsp2 proteinIn the study,the gene sequence of IBV Nsp2 was obtained by sequencing from previous studies in our laboratory.The basic physical and chemical properties of IBV Nsp2protein were analyzed by using bioinformatics online analysis tools according to the amino acid composition,fat index,instability index,hydrophilicity and other indexes.Analysis and prediction using bioinformatics online tools showed that the Nsp2 protein is not a transmembrane protein.By the way,the precursor protein Nsp2-3 has no signal peptide,and there is no transport of the mature Nsp2 after it has been cleaved by the viral protein Nsp3.So Nsp2 has function in the cytoplasm.Based on the secondary structure,the tertiary structure with high confidence was predicted,indicating that IBV Nsp2 has a Zn²⁺binding site.Therefore,it further binds DNA/RNA,and it is predicted that Nsp2 is involved in the viral m RNA transcription or translation.2.Screening and analysis of proteins interacting with IBV Nsp2 and host cellsTo screen for host proteins that interact with IBV Nsp2 and have regulatory transcriptional or translational functions,chicken embryonic kidney cells expressing Flag-Nsp2 protein were used for the study.Using Co-IP and LC-MS/MS,97 host proteins that might interact with IBV Nsp2 protein were screened out.Bioinformatics software was used to analyze candidate host proteins for GO function and KEGG enrichment.We found that the Nsp2 protein was involved in Protein synthesis、Nucleotide binding and other biological processes.Especially,we found that the candidate host protein eIF2α,is a key protein in the host protein-protein interaction network and for translation initiation.We will focus on exploring the interaction between them in depth.It lays a foundation for further study on the biological function of Nsp2 in the course of IBV infection.3.Identification of interaction between IBV Nsp2 and host cell protein eIF2αand the molecular mechanism affecting IBV replicationIn order to reveal the interaction between Nsp2 and eIF2αand investigate the effect of the interaction on viral replication,Co-ip and indirect immunofluorescence assay（IFA）were performed to verify the interaction between Nsp2 and eIF2α;and the effects of IBV and Nsp2 on eIF2αm RNA expression in the host to investigate whether IBV regulation of eIF2αcould be related to Nsp2.The effect of Nsp2 on IBV replication was investigated by Real-time fluorescent quantitative PCR（q PCR）of IBV m RNA levels,followed by a sea kidney luciferase activity assay to test the effect of Nsp2 on host protein synthesis to investigate the effect of Nsp2 on the host protein synthesis system;finally,the effect of Nsp2 on m RNA levels of key factors of the eIF2α-related signaling pathway in the host was investigated by q PCR to further investigate the mechanism of Nsp2 action.The results showed that Nsp2 directly interacts with eIF2α,demonstrating the reliability of mass spectrometry identification and that IBV and Nsp2 protein significantly increased the level of endogenous eIF2αtranscription in cells,suggesting that Nsp2 protein plays an important role in increasing eIF2αtranscription at the early stage of IBV infection;co-expression of Nsp2 and eIF2αfacilitated the increase in IBV m RNA.The relatively low luciferase activity in sea cucumber host cells overexpressing Nsp2 suggested that Nsp2 affected the host’s exogenous protein synthesis,and both results indicated that Nsp2 could hold host translation machinery hostage by interacting with eIF2α,which could enhance viral replication to some extent.In addition,the ability of Nsp2 to activate the key eIF2αsignaling pathway was investigated,and the results showed that Nsp2 significantly increased the expression levels of endogenous eIF2αand ATF4 in cells without activating the ER stress PERK pathway,so it was hypothesized that viral Nsp2 promotes IBV replication through the eIF2α-ATF4 pathway.In conclusion,IBV Nsp2 plays an important role in viral replication,interacting with eIF2αand participating in the regulation of translation.and it was speculated that Nsp2could improve the viral m RNA transcription level by activating the eIF2α-ATF4 pathway,which further indicated that eIF2αwas the target factor of IBV infection.The results show that IBV has developed a novel combination of mechanisms,that Nsp2 activates the eIF2α-ATF4 pathway to maintain viral protein synthesis for better virus growth.The findings provide an important reference for basic research on the interaction between coronaviruses and host cells,and for elucidating the biological functions of the non-structural protein Nsp2 during IBV infection.