Proteomics Analysis Of CV-A10 Infection In 16HBE Cell And The Preliminary Study On The Mechanism Of HMGB1 Regulating Virus Replication

Hand-food-mouth disease(HFMD)has been a serious public health problem in the world,especially in the Asia-Pacific region.Previous studies reported that the main pathogens causing HFMD were Enterovirus 71(EV-71)and Coxsackievirus A16(CV-A16).In recent years,the epidemic data showed that the pathogen spectrum of HFMD had changed.Coxsackievirus A6(CV-A6)and Coxsackievirus A10(CV-A10)are the main pathogens of the current HFMD outbreak.And it seems that the CV-A10 and CV-A6 infections are more severe in the Asia-Pacific region.However,there has been no effective vaccine or antiviral drug for CV-A10 treatment,so it is urgent to explore the pathogenesis of CV-A10 infection,so as to formulate prevention and treatment measures related to HFMD.A large number of studies have reported that quantitative proteomics techniques can be used to observe the differential expression of proteins after viral infection from a global perspective in order to explore potential protein targets.However,there has been no proteomic study of CV-A10 infection at home and abroad.Based on these discoveries,this study selected the human bronchial epithelial cells(16HBE),the primary entry point of enterovirus through respiratory pathway.Using liquid chromatography tandem mass spectrometry(LC-MS/MS)proteomics techniques based on Tandem mass tags(TMT),we identified 293 differentially expressed proteins in 16HBE cells infected with CV-A10,includeing 104 up-regulated proteins and 189 down-regulated proteins.The Gene Ontology(GO)enrichment analysis of differential protein showed that:in up-regulated proteins,the main biological process(BP)is metabolic process,cellular component(CC)is BBsome and molecular function(MF)is catalytic activity.In down-regulated proteins,BP is mainly Nitrogen compound metabolic process,CC is intracellular metabolic process,and MF is structural constiuent of ribosome,structural molecule activity and DNA binding.The enrichment analysis of differential protein Kyoto Encyclopedia of Genes and Genomes pathway(KEGG pathway)showed that the proteasome is the most significant pathways in the up-regulated and the ribosome is the most significant pathways in down-regulated proteins.Subcellular localization and protein domain enrichment analysis of differential proteins showed that differential proteins were mainly concentrated in domains related to DNA-binding proteins and ribosomal protein L44e in the nucleus.Based on the above analysis,we screened out the target protein high mobility group protein 1(HMGB1).Subsequently,western blot was used to detect changes in the expression level of target protein HMGB1 after CV-A10 infection,we found that CV-A10 infection decreased the protein expression level of HMGB1.We also observed that silencing of HMGB1 remarkably promote viral replication,while overexpressing of HMGB1 markedly inhibitviral replication.Finally,western blot results also showed that CV-A10 infection would activate p38 MAPK signaling pathway.However,after silencing and overexpression of HMGB1,it was observed that there was no correlation between HMGB1 and p38 MAPK signaling pathway in CV-A10 replication.In summary,after infecting 16HBE cells,CV-A10 mainly affects proteins related to DNA binding and ribosome binding in the nucleus,and has significant effects on intracellular metabolic processes,synthesis of organic compounds,especially nitrogen compounds,and catalytic activity,as well as on neural-related signaling pathways,MAPK and oxidative phosphorylation related signaling pathways.CV-A10 infection also activates the p38 MAPK signaling pathway and down-regulates the intracellular DNA-binding protein HMGB1,while HMGB1 inhibits the replication of CV-A10,suggesting that CV-A10 promotes self-replication by down-regulating host protein HMGB1,and HMGB1 can regulate the replication and proliferation of CV-A10.