Research On The Mechanism Of EV71 Immune Evasion Through Inhibiting Host RIG-Ⅰ Ubiquitination

Enteroviruses 71(EV71),as one of the Enterovirus A species under the Enterovirus genus of the Picornaviridae family,has emerged as a severely neurotropic virus.EV71 is the primary causative agent of hand,foot,and mouth disease(HFMD)which mainly occurs among young children and infants.In the past few decades,EV71 was found circulating all over the world,and became a serious threat to the public health.During EV71 invading host cell,dsRNA is generated as by-products of EV71 genome replication.The intracellular RIG-Ⅰ-like receptors(RLRs)sense viral dsRNA,then one of the first events following is robust ubiquitination of RIG-Ⅰ.This is mediated by the ubiquitin ligase.Ultimately,type I IFN signal pathway will be activated.Our previous study found that EV71 inhibits the RIG-Ⅰ ubiquitination,but whether this event eventually mediates EV71 evading innate immune remains elusive.Therefore,in this study,on the basis of the new founding as the breakthrough point that EV71 inhibits host RIG-Ⅰ ubiquitination,we will elucidate the molecular mechanisms of EV71 antagonizing type I interferon production by inhibiting host RIG-Ⅰ ubiquitination,which will add newly important theoretical theory basis about EV71 escaping from host innate immune response.In order to examined the replication of EV71 virus in RD cells,cytopathic effect(CPE)were observed at indicated times using light microscope and fluorescent microscope after RD cells were infected by EV71 virus for 24 hours.The result showed that large amount of viral protein were detected and the infected RD cells showed evident CPE in 6 hours post-infection.The effect of EV71 virus on the activity of IFN-β promoter in RD cells was detected by Dual-Luciferase Reporter Assay System after transfection of RD cells with IFN-β-Luc plasmid.The result showed that EV71(MOI=20)effectively reduced the activity of IFN-β promoter in RD cells.In order to confirm the inhibition of EV71 virus on IFN-β,we examined the effects of EV71 virus on the transcription of IFN-β by RT-PCR.The result showed that EV71 virus significantly inhibited transcription of IFN-β.Interferon regulatory factors 3(IRF3)transport to the nucleus is essential to activation of host innate immunity.To understand if EV71 virus antagonize type I interferon pathway through inhibiting nuclear import of IRF3,nuclear localization of IRF3 was detected by immunoflourescence after transfection of RD cells with pEGFP-IRF3 plasmid.The result showed that EV71 virus significantly inhibited the nuclear import of IRF3.Interferon-stimulated gene(ISG)is the final respondent to antiviral activity.In order to explore the effect of EV71 virus on ISGs,ISG54,ISG56 and IFN-β transcription were determined by RT-PCR after RD cells were infected with EV71 virus.The result showed that EV71 virus infection significantly down-regulated the mRNA levels of IFN-β and ISGs in RD cells.We next examined the effects of EV71 infection on the endogenous ubiquitination of RIG-Ⅰ by Western Blot.The results showed that ubiquitination of endogenous RIG-Ⅰ decreased in a time-dependent manner during EV71 infection.To further prove this effect,we transfected RD cells with both HA-Ub and Flag-RIG-Ⅰ,and then infected RD cells with EV71.Pull-down and Western Blot showed that EV71 infection inhibited polyubiquitination of exogenous expressed RIG-Ⅰ remarkably.To further determine which type of RIG-Ⅰ ubiquitination is inhibited,we transfected RD cells with both exogenous Flag-RIG-Ⅰ and HA-Ub,HA-Ub K63 or HA-Ub K48 and then infected RD cells with EV71.Pull-down showed that EV71 infection inhibited K63-linked polyubiquitination of RIG-Ⅰ remarkably,but not K48-linked polyubiquitination.Finally,increase of RIG-Ⅰ ubiquitination level by simultaneously transfection of HA-Ub and Flag-RIG-Ⅰ in RD cells increased IFN-β and ISGs mRNA level remarkably.Our results above showed that EV71 inhibited K63-linked ubiquitination of RIG-Ⅰ,blocking the nuclear import of IRF3,suppressing the IFN-β expression,and blocking transcription and expression of innate immune effector molecules ISG54 and ISG56 during EV71 infected of RD cell.Thereby,EV71 evades elimination of host by innate immune.This study not only contributes to enriching theoretical theory basis about EV71 escaping from host innate immune response,but also contributes to revealing a new potential antiviral replication strategy.