Research On The Antagonistic Effect Of Encephalomyocarditis Virus Structural Protein VP2 On The Activation Of IFN-? Signaling Pathway

Encephalomyocarditis virus(EMCV)is a single strand non-enveloped small RNA virus,which can cause encephalitis,myocarditis,nervous system diseases and reproductive disorders after infection in humans and animals,and it is an important zoonotic pathogen and has important public health significance to human health.The viruses envolve a variety of ways and strategies to invades from host immune system.Many viruses encoding different kinds of viral proteins to antagonize or inhibit the host innate immune process,so as to achieve the effective proliferation of itself in host cells.In the early stage of this study,the structural protein VP2,which can significantly promoted the proliferation of EMCV in vitro,was screened by real-time fluorescence quantitative PCR,then further explore the mechanism of VP2 in promoting the proliferation of EMCV and the regulatory role of VP2 in the production of IFN-? and RLRs signaling pathway.The target molecules of RLRs signaling pathway regulated by VP2 protein and the possible mechanism of VP2 inhibiting RLRs signaling pathway were screened and verified by immunoblotting,co-immunoprecipitation and laser confocal experiments.The results were listed as follows:1.EMCV structural proteins VP1,VP2 and VP3 significantly promoted the proliferation of EMCV in vitro,among which VP2 had the most obvious effect.In addition,VP2 promoted the proliferation of EMCV was closely related to the activation of RLRs signaling pathway and the production of IFN-?.2.The structural protein VP2 significantly inhibited the production of IFN-? and the expression of RLRs signaling pathway proteins mediated by EMCV,Poly(I:C)and key proteins in RLRs pathway in a dose-dependent manner.In addition,VP2 significantly inhibited the phosphorylation and nuclear translocate levels of IRF3 and STAT1,indicating that VP2 reduced the production of IFN-? by inhibiting RLRs signaling pathway.3.The results of Confocal and co-immunoprecipitation experiments showed that VP2 protein was co-located and had specific interaction with MDA5,MAVS andTBK1,the key proteins of RLRs signaling pathway.4.MG132,an inhibitor of proteasome pathway,could significantly reduce the inhibition of VP2 on MDA5,MAVS,TBK1 and IRF3 expression,which indicated that VP2 exploited or promoted proteasome pathway to degradate MDA5,MAVS,TBK1 and IRF3 expression and inhibit the activation of RLRs signaling pathway.In conclusion,this study is the first to confirm that EMCV VP2 protein can significantly inhibit IFN-? production and RLRs signaling pathway activation by down-regulating the expression of key RLRs proteins MDA5,MAVS,TBK1 and IRF3,and interacting with MDA5,MAVS and TBK1 proteins.Moreover,VP2 protein target to inhibit the expression of MDA5,MAVS,TBK1 and IRF3 protein through proteasome pathway,and ultimately promote the proliferation of the virus by reducing the production of IFN-?.These results lay a foundation for further elucidating the innate immune mechanism mediated by EMCV structural proteins,provide important information for further elucidating the pathogenic mechanism of EMCV infection,and also provide a new insight for preventing and controlling of EMCV infection.