Screening of the Interacting Proteins with BTV NS4 by Co-immunoprecipitation Combining with Mass Spectrometry
[Objective]The purpose of this study was to screen the endogenous interaction protein of NS4 protein of Bluetongue virus(BTV)against interferon(IFN)signaling pathway,in order to further investigate the mechanism of NS4 inhibition of IFN signaling.[Method]On the basis of previous studies on the gene expression of IFN signaling pathway induced by Sendai virus(SeV),NS4 interacting proteins were extracted from HEK-293T cells transfected with NS4 fusion eGFP label expression vector pcDNA3.1-NS4-eGFP and empty vector pcDNA3.1-eGFP by co-immunoprecipitation method.The immunoprecipitates were analyzed by SDS-PAGE,and the immunocoprecipitated eluents were identified by mass spectrometry and bioinformatics.Real-time quantitative PCR was used to detect the expression characteristics of candidate protein-coding genes.[Result]A total of 189 differentially expressed proteins were obtained by co-immunoprecipitation,mass spectrometry analysis and database comparison.The results of bioinformatics analysis showed that the candidate proteins were mainly involved in protein transcription,translation,viral infection and immune regulation.The results of subcellular localization analysis showed that the differentially expressed proteins were mainly located in the nucleus,cytoplasm and cytoplasm-nucleus,and four candidate proteins interacting with BTV NS4 protein were screened out,which were polypyrimidine bunch-binding protein 1(PTBP1),cell cycle dependent protein kinase 9(CDK9),N-terminal myristylase 1(NMT1)and Y-box binding protein 1(YBX1).Real-time quantitative PCR results showed that compared with control group,the mRNA expressions of PTBP1,NMT1,YBX1 and CDK9 genes in experimental groups were significantly or extremely significantly up-regulated after 72 h of SeV stimulation(P＜0.05 or P＜0.01).[Conclusion]The antagonistic IFN signaling pathway of BTV NS4 protein was related to the up-regulation of PTBP1,NMT1,YBX1 and CDK9 proteins,which provided a target reference for further research on the molecular mechanism of BTV NS4 protein antagonizing host innate immune response.
Bluetongue virus(BTV)co-immunoprecipitationmass spectrometryNS4 protein
万方数据
维普
