Effect Of TRIM13 On EMCV Proliferation In Vitro And Preliminary Study Of Mechanism

Encephalomyocarditis virus(EMCV)is a member of the gene family Picornaviridae,which can lead to one of important zoonotic infectious diseases and a large number of livestock deaths.EMCV is widely used as models to study virus-mediated immune mechanisms,viral myocarditis and insulin-dependent diabetes.Studies have found RLR-mediated IFN signaling pathway plays an important role in the replication of EMCV.Innate immunity responds to viral infections by activating the production of interferons and proinflammatory cytokines.Among them,the TRIM family proteins of produced can regulate the infection of various viruses.In this study,TRIM13 with its own polyubiquitination-modified E3 ubiquitin ligase was used as the research object.By up-regulating and down-regulating protein expression levels,the effects on EMCV were observed,and the mechanism was preliminary explored.The results show:1.After HEK293 cells were infected with EMCV,the mRNA level of TRIM13 gradually increased with the increase of the virus infection time.Compared with the control,TRIM13 protein was significantly up-regulated at 36 hpi and 48 hpi,suggested that TRIM13 was related to EMCV infection in vitro.2.After transient overexpressed or downregulated TRIM13 in HEK293 cells,the EMCV infection test was did.The results showed that the EMCV replication increased significantly when expression of TRIM13 promoted(P<0.05);similarly,downregulated TRIM13 using specific RNA interference inhibited the proliferation of EMCV in HEK293 cells significantly(P<0.05).3.Results of EMCV adsorption and invasion tests suggested that TRIM13 is related to the invasion process of EMCV,and has nothing to do with virus adsorption.4.The expressions of IFN signaling pathway molecules and inflammatory factors were detected by qPCR and ELISA experiments after TRIM13-overexpressed HEK293 cells were infected with EMCV.The results suggested that TRIM13 inhibited inhibited the production of IFN-β and IL-6,TNF-α which triggered by EMCV.5.Next,different concentrations TRIM13 recombinant-plasmids was co-transfected with key adapter molecules MDA5,MAVS and TBK1 into HEK293 cells,qPCR results suggested that TRIM13 inhibited the production of IFN-β by the downregulated adapter molecules MDA5,MAVS and TBK1 and was concentration-dependent.WB results is consistant with the qRT-PCR,TRIM13 could directly inhibit the expression of MAVS and TBK1 and the effects was reversed when TRIM13 was down-regulated.Co-immunoprecipitation and immunofluorescence experiments showed that TRIM13 interacted with MAVS and TBK1 and was co-localized in the cytoplasm.Collectively,the above results showed that TRIM13 increased EMCV replication in HEK293 cells,and participated in the process of virus invasion.In addition,TRIM13 negatively regulates the IFN-I signaling pathway and the expression of inflammatory factors inhibits the expression of ISGs,which in turn promotes the EMCV replication in host cells in vitro.