Identification Of Host Factors Interacted With Non-Structural Protein 3 Of Chikungunya Virus

Background Chikungunya Virus(CHIKV)is a member of Alphavirus transmitted by Aedes mosquitoes.It is highly infectious and belongs to Risk Group II pathogens.CHIKV infection causes Chikungunya fever(CHIKF),which is characterized by high fever,rash,and joint pain,and can lead to death in severe cases.Currently,CHIKV is present in more than 100 countries and regions around the world,and about 1 million people are infected every year,posing a huge threat to global public health.In 2017,World Health Organization(WHO)called for CHIKF to be a priority pathogen in research and development.However,CHIKV is often overlooked due to its misdiagnosis as dengue virus(DENV),and there are no commercially approved vaccines or drugs against CHIKV.Furthermore,the study of CHKV is greatly hampered by requirement of the high biosafety level laboratory as a Risk Group Ⅱ pathogen.The non-structural Protein 3(CHKnsP3)of CHIKV,a major non-structural protein,is a key determinant of host factor recruitment to viral replication complex assembly sites and other cytoplasmic complexes on the plasma membrane.Therefore,in this study,CHKnsP3 was overexpressed in mammal cells,and host factors interacted with CHKnsP3 and related signaling pathways were identified by transcriptomics,proteomics,coimmunoprecipitation(Co-IP),GST pull-down and mass spectrometry,with a view to finding potential host factors associated with CHIKV infection and provide ideas for research and development of vaccines or drugs against CHIKV.Methods 1.The expression of CHKnsP3 in eukaryotic system.Firstly,we compared the expression of CHKnsP3 in mammalian cells via two different expression vectors;And then we compared expression levels of CHKnsP3 protein at different time points by Western Blot(WB)and selecting the optimal expression time.2.Transcriptomics and proteomics analysis of HEK293T overexpressing CHKnsP3.Firstly,we prepared CHKnsP3-overexpressing samples and sent them for analysis of transcriptomics and proteomics;Then we used qPCR and WB to verify the differentially expressed gene of transcriptomics(|Foldchange|>2)and protein of proteomics(|Foldchange|>1.5);And the analysis of GO and KEGG enrichment is for signal pathways;3.The coimmunoprecipitation analysis of candidate host factors interacted with CHKnsP3.At first,we overexpressed flag-tagged protein CHKnsP3-Flag in HEK293T cells;Then,we used Co-IP to analysis of host factors interacted with CHKnsP3 and determined candidate host factor proteins in Co-IP by WB and mass spectrometry analysis;4.Identifying candidate host factors directly interacted with CHKnsP3 by GST pull-down.Firstly we compared the expression of CHKnsP3 gene and GST fusion protein in the prokaryotic system before and after codon optimization;Then,we optimized expression temperature for GST-optCHKnsP3 protein expression to obtain high levels of expression and solubility;We expressed and purified GST-optCHKnsP3 protein and GST protein with glutathione preloaded column;And WB and mass spectrometry combined with omics analysis of host factors directly interacting with CHKnsP3 captured by GST pull-down.5.Analysis of CHKnsP3-related signaling pathways.First we used WB to validate related proteins expression in mTOR signaling pathway and regulation of related downstream proteins of S6K signaling;Then the expression of autophagy associated proteins was analyzed by WB and autophagy analysis of subcellular structures was observed by transmission electron microscopy(TEM).Results At first,the gene CHKnsP3 constructed into the plasmid vector pMD2.G was successfully expressed in the cell line HEK293T.The optimal time for sample collection was 48h according to the peak of CHKnsP3 protein expression.Secondly,transcriptomic analysis of HEK293T cells with overexpression of CHKnsP3 showed that CHKnsP3 expression induced upregulation of 460 genes and downregulation of 153 genes,and the foldchange was>2.Quantitative PCR validation of expression of 13 genes showed that differential expressions of genes were consistent with those of transcriptomic analysis.In proteomic analysis,38 proteins with up-regulated expression and 3 proteins with downregulated expression were found,with a difference ratio of>1.5,among which TUFT1 protein showed the most significant upregulation(2.38 times),and its mRNA was also up-regulated by 1.4 times.The transcriptional levels of HSPA1B,ATF3 and SESN2 were up-regulated more than two-fold,and their protein expressions were also up-regulated.G3BP2,reported to interact with CHKnsP3,and ATF6 involved in CHKnsP3-related signaling pathway were also up-regulated in this study.WB verified these 6 differentially expressed proteins,which was consistent with those in proteomic analysis.soon afterwards,combined analysis of Co-IP,WB and mass spectrometry showed that only G3BP2 and HSPA1B interacted with CHKnsP3 among these 6 differentially expressed proteins.Finally,Using GST-CHKnsP3 fusion protein expressed in prokaryotic cells,combined analysis of GST pull-down,WB and mass spectrometry showed that HSPA1B had direct interaction with CHKnsP3 and was a potential host factor for CHKnsP3 interaction.5.Analysis of signal pathways affected by CHKnsP3 overexpression showed that mTOR signal pathway was significantly inhibited,and its downstream S6K signaling was significantly down-regulated.Furthermore,autophagy pathway was obviously activated.The overexpression of CHKnsP3 induced the formation of autophagosomes and autophagolysosomes by transmission electron microscopy.Conclusion In this study,host factor proteins interacting with non-structural protein 3 of CHIKV and related signaling pathways were identified in CHKnsP3-overexpressed HEK293T cells by transcriptome,proteome,immunoprecipitation and GST pull-down techniques.These findings lay a foundation for further study on the mechanism of CHKnsP3 involved in CHIKV infection of cells,and provides important information for screening antiviral drugs targeting CHKnsP3.