High-Throughput Quantitative Proteomic Analysis of Coxsackievirus A16-infected 16HBE Cells Based on tandem mass tag (TMT)-labeled Quantitative Proteomics Running title: Proteome profiling of Coxsackievirus A16 in 16HBE cells

Abstract Hand, foot and mouth disease (HFMD) caused by Coxsackievirus A16 (CV-A16) is a global health concern worldwide. There are no vaccines or antiviral compounds available to either prevent or treat CV-A16 infection which may trigger severe neurological complications, and even lead to death. Moreover, its pathogenic mechanisms and pathophysiology are still poorly elucidated. To increase our understanding of the interaction of CV-A16 with the host cell, we analyzed changes in the proteome of 16HBE cells in response to CV-A16 infection using tandem mass tag (TMT) in combination with LC-MS/MS. It was identified and quantified 6615 proteins and there were 172 proteins showed a significant alteration during CV-A16 infection. To validate the proteomics data, 3 randomly selected proteins exhibited consistent changes in protein expression with the TMT results using a Western blotting and immunofluorescence method. Then, functional enrichment analysis showed that these differentially expressed proteins mainly involved in various biological processes and signaling pathways, such as metabolic process, Cytokine-cytokine receptor interaction, B cell receptor signaling pathway, Neuroactive ligand-receptor interaction, etc. And further bioinformatics analysis revealed that these differentially expressed proteins contained distinct domains, localized in different subcellular components, and established a complex network. In conclusion, results from this study have helped elucidate the molecular pathogenesis of CV-A16 and may facilitate the development of new antiviral therapies as well as innovative diagnostic methods.