| Innate immunity is the first defense line of host against pathogens.When the pathogens infected host cell,host pathogen-recognition receptors(PRRs)firstly recognize pathogen-associated molecular patterns(PAMPs)of the pathogens,thereby eliciting a series of immune responses to clear the pathogens.RIG-I-like receptors(RLRs)is one of the major pathogen-recognition receptors of cells.When RLRs recognize PAMPs,RLRs-mediated signaling pathways activate and induce the expression of interferon(IFN)and other genes,which in turn trigger a series of antiviral responses.Autophagy is a self-protection mechanism of cells,by which the lysosomes degrade and clear the organelles and macromolecules of damaged cells and maintain cell stability.Autophagy is regulated by autophagy-related gene(ATG)-encoded proteins.A number of autophagy-related proteins have been reported in autophagy,and ATG13 plays a key role in the initial stage of autophagy.Currently,the mechanism of ATG13 in activating the innate immune RLRs signaling pathway has not been reported.In this study,the mechanism of ATG13 protein in the activation of RLRs signaling pathway and mediating cellular antiviral response were explored by dual luciferase reporter system,gene overexpression,RNA interference,protein interaction and viral infection inhibition assays.The results are as follows:1.ATG13 significantly activated ISRE report gene activity in a dose-dependent manner.2.ATG13 significantly induced IFN-β production with the stimulation of RIG-IN,and the mRNA levels of downstream ISGs,such as CCL5,ISG56,CXCL10 and OASL increased obviously.3.The ATG13 protein and the adaptor protein in the RLRs signaling pathway were co-transfected into HEK 293 T cells.It was confirmed that ATG13 interact with MAVS,TRAF3 and TRAF6 protein by immunoprecipitation method.4.HEK 293 T cells were infected with Encephalomyocarditis virus(EMCV),Vesicular Stomatitis virus(VSV),or treated with exogenous interferon to test the antiviral effect of ATG13 gene.The results showed that viral infection and exogenous IFN-α treatment induced the transcription of ATG13 gene.When the ATG13 protein was overexpressed the replication of the virus significantly reduced,while it was knocked down,the replication of the virus increased.The cell culture supernatant of ATG13 overexpression and exogenous interferon IFN-α treatment were incubated with fresh cells which were infected with EMCV and VSV,respectively,and the virus replication level decreased.5.JAK inhibitors was used to block the JAK-STAT signaling pathway,the results showed that with different concentrations of JAK inhibitor in tested cells,EMCV and VSV replication level gradually increase.In summary,these results indicated that ATG13 inhibit the virus replication by interacting with MAVS,TRAF3,or TRAF6 to activate the cellular RLRs signaling pathway mediated IFN production which activated JAK-STAT signaling pathways and induced ISGs that exert antiviral activity. |
