Study On Identification And Function Of Key Phosphoproteins Regulating Senecavirus A Infection

Senecavirus disease caused by Senecavirus A(SVA),which is a viral infectious disease that mainly infects pigs and it poses a threat to the pig industry.As an emerging pathogen of swine infectious diseases,the pathogenesis of SVA is still unclear.Phosphorylation is a kind of widespread post-translational modification that regulates numerous biological processes.SVA infection can alter the physiological activities of host cells to promote virus replication,and regulating phosphorylation is one of the mechanisms.Identifying the key phosphorylated proteins on regulating SVA infection and studying their functions and mechanisms in the process of SVA infection are helpful to understand the interaction mechanism between SVA and the host.Moreover,it contributes to reveal the infection mechanism of SVA at the level of post-translational modification.1.Through Label-free quantitative proteomics and phosphorylation enrichment techniques of quantitative proteomics were combined with high resolution liquid chromatography-mass spectrometry,a total of 9034 phosphosites on 2794 proteins were identified before and after SVA infection on IBRS-2cells,of which 4520 sites of 2084 proteins contained quantitative information.At the threshold of 1.5times of difference ratio(t-test p-value<0.05),180 phosphorylated sites on 150 proteins were downregulated,and 63 phosphorylated sites on 58 proteins were up-regulated.Gene Ontology(GO)functional enrichment,Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment and Subcellular localization analysis showed that many of the phosphorylated proteins were involved in apoptosis and spliceosome pathway,and these proteins were mainly located in the nucleus.Motif analysis of proteins with differentially regulated phosphosites showed that proline(P),aspartic acid(D)and glutamic acid(E)were the most abundant residues in the serine(S)motif,while P,E and arginine(R)were the most abundant in the threonine(T)motif.Finally,a total of 165 kinases were predicted by kinase analysis,and the activity of 9 kinases was reversed,among which the activity of CDK2 was increased.2.40 phosphosites on previously identified 27 proteins were validated by phosphopeptide enrichment technology and targeted proteomic quantitative techniques(PRM),and 30 phosphosites on 21 proteins were verified.Excluding the sites inconsistent with the phosphoproteomics results,25 sites on17 proteins were found to be confirmed with the phosphoproteomics results.Thorugh Bioinformatics analysis and literature review,eight phosphoproteins(SRRM2,CDK13,DDX20,DDX21,BAD,ELAVL1,PBK,and STAT3)may play a role in SVA infection.Further,CDC5 L,STAT3 and CDK2 were verified by Western blot(WB),and the results were consistent with the phosphoproteomic.3.The preliminary functional study of the target proteins STAT3 and CDK2 in the process of SVA infection were conducted by their respective specific inhibitor.The cell viability test found that the cell viability of SVA infected IBRS-2 cells with inhibitor(Stattic or CVT-313)were significantly higher than SVA infected cells without inhibitor.Flow cytometry detection of cell apoptosis showed that either Stattic or CVT-313 can reduce the numbers of apoptotic cells with SVA infection.SVA-quantitative PCR and WB detection found that Stattic or CVT-313 can promote the replication of SVA.In order to preliminarily study the mechanism of these two inhibitors,WB and kinase activity detection found that Stattic can inhibit the phosphorylation of S727 and T705 of STAT3 without affecting the expression of STAT3;while CVT-313 inhibits the enzymatic activity of CDK2 without affecting its expression,indicating that Stattic works by inhibiting the phosphorylation of S727 and Y705 of STAT3,and CVT-313 works by downregulating the enzymatic activity of CDK2.In view of the results of phosphoproteomics,it is speculated that SVA may inhibit the phosphorylation of S727 site of STAT3 protein to promote its replication.The increase of CDK2 enzyme activity after SVA infection may be a strategy for host cells to resist SVA infection.