The Mechanism Of Enterovirus Type A Cleavage Hyperosmotic Stress Protein NFAT5

HFMD(Hand,Foot and Mouth Disease)is a prevalent disease mainly affecting children.In the past decade,the incidence of HFMD has been increasing,posing a serious threat to children's health and public health security.Enterovirus type A(EV-A)is the main pathogen of hand,foot and mouth disease(HFMD).There are more than 20serotypes of EV-A causing HFMD,among which coxsackievirus A16(CV-A16)and enterovirus 71(EV-A71)are the most common ones.NFAT5,a member of the protein family of activated T nuclear factors(NFATs),was found to be a hypertonicity-responsive factor of stress protein.Recent studies have revealed the critical roles of NFAT5 in viral infection and immune response.For instance,NFAT5 is capable of binding to the p65 subunit of NF-?B,enhancing the NF-?B pathway and downstream innate immune response.However,the roles of NFAT5 in the infection of EV-A have not been reported.This study aims to investigate the change of NFAT5 protein in the infection of CV-A16 and EV-A71 as well as the influence of NFAT5 on the infection of the two viruses.Our results suggested that the intact NFAT5protein(?170 k Da)was cleaved in the infection of CV-A16 and EV-A71,generating an N-terminal product of 70 k Da.By bioinformatic prediction and point-mutation verification,we proved that this cleavage is mediated by the viral protease 2A^pro on the peptide bond before the 596^th glycine residue.Our further studies showed that the overexpression of NFAT5 inhibited the replication of CV-A16 and EV-A71 while the cleavage products of NFAT5 could not inhibit the viral replication any more.To uncover the underlying mechanism,we assessed the effects of NFAT5 cleavage on NF-?B and IFN-?pathways by dual-luciferase-reporter assay.Our results showed that the overexpression of NF AT5could enhance the activation of both pathways while the cleavage products of NFAT5could not,indicating that NFAT5 inhibits EV-A replication via elevating NF-?B-and IFN-?-dependent innate immune response but such mechanism is impaired when NFAT5is cleaved by the viral protease.In summary,our studies have revealed the preliminary mechanism of EV-A inhibiting host immune response via cleaving NFAT5.This finding indicates the potential of NFAT5 as the drug target of anti-EV-A therapy.