Regulation Of Cellular Antiviral Innate Immune Response To EV71 Infection By Deubiquitinating Enzymes

Being a member of Human Enterovirus A,Picornaviridae,enterovirus 71(EV71)causes hand,foot,and mouth disease(HFMD)and even fatal central nervous system infections,including encephalitis,aseptic meningitis,acute flaccid paralysis,and neurogenic pulmonary edema,in children under 5 years old.EV71 has been listed as a virus in the Third Type Management Regulation since the prevalence of HFMD at China in 2008.However,there are yet no effective vaccines and therapeutic drugs available to control EV71 infection.In this context,it seems significant to enhance host innate immunity for defending against EV71 infection.Innate immunity represents the first defending line for hosts to combat viralinfections.Upon infections,recognition of relatively conserved components of pathogens (known as pathogen-associated molecular patterns,PAMPs) by pattern recognition receptors (PRRs) of host cells provokes host innate immune responses,resulting in the induction of type I interferon (IFN-?)and a variety of pro-inflammatory cytokines and finally controlling the spread of infections.As obligate intracellular parasites,viral infections are recognized by cytosolic PRRs to activate host antiviral responses;these PRRs include Toll-like receptor(TLR)-3,-7/8,-9,and retinoic acid-inducible gene I (RIG-?)-like receptors(RLRs).As one of the best-characterized post-translational modifications,protein ubiquitination controls the fate and functions of a great number of proteins.Similar to protein phosphorylation,protein ubiquitination is reversible and can be regulated by a variety of protein hydrolase known as deubiquitinating enzymes (DUBs),which means DUBs remove ubiquitin molecules from target proteins to reverse the modification of ubiquitination,and thusly function in important roles in signal transduction,DNA damage repair,the maintenance of immune functions,and many other core respects of cellular activities.It has been revealed that activation of antiviral innate immunity requires not only the recognition of viral PAMPs,ubiquitination of signaling adaptor molecules is also crucial to provoke antiviral responses;and lysine 63(K63)-linked poly-ubiquitination of tumor necrosis factor associated factor 3(TRAF3)is essential for the activation of IFN regulatory factor 3(IRF3)and sequential induction of IFN-?.DUBs have also been studied to regulate antiviral innate immune responses.However,whether or not DUBs are involved in EV71 infection and the potential regulatory mechanisms are still little known.To explore whether or not DUBs regulate EV71-induced type I interferon signaling,we performed a PCR array analysis to screen differentially-expressed DUBs in EV71-infected Rhabdomyosarcoma(RD)cells.Through further analysis and confirmation,we found that the expression of USP19 gene was up-regulated and negatively correlated with EV71-induced phosphorylation of IRF3 during viral infection.Knockdown of USP19 notably enhanced both EV71 and Poly(I:C)-induced activation of type ? interferon signaling.We then performed immunoprecipitation experiments combined with mass spectrometry analysis to further explore the molecular mechanism that underlines the regulatory roles of USP19 in EV71-induced cellular IFN-? signaling.It revealed that USP19 interacts with TRAF3 via both its N-terminal domain and its peptidase C19 domain.Further,USP19 decreases TRAF3 poly-ubiquitination of K63-linkage,which is dependent of the peptidase C19 domain of USP19,but not the N-terminal domain.Additionally,knockdown of USP19 notably enhanced K63-linked poly-ubiquitination of TRAF3,which is essential to provoke cellular IFN-? antiviral immune response and thusly attenuates EV71 propagation in host cel s significantly.Taken together,our findings demonstrate that USP19 negatively regulates cellular IFN-? antiviral immune response to contribute to EV71 propagation in host cells through the decrease of K63-linked poly-ubiquitination of TRAF3.This is also a previously undescribed mechanism employed by EV71 to escape from host defenses in the context of host DUBs.