The Structural Protein VP0 Of Foot-and-mouth Disease Virus Inhibits Type Ⅰ Interferon Signaling Pathway

Objective: Foot-and-mouth disease virus can induce the generation of natural immune response in the process of infecting host cells.There are related literature reported that foot and mouth disease virus non-structural protein(L and 3C)on the natural immune system activation has a direct blocking effect,but the mechanism of action of structural protein VP0 in inhibiting natural immune response is not clear.This study will research the effect on interferon signaling pathway of FMDV structural protein VP0 on type I.Methods: VP0 eukaryotic expression vector was constructed by reverse transcription PCR,the expression of VP0 protein was transfected into HEK-293 T cells by western blot.Real-time PCR was used to detect the effects of VP0 protein and PVR protein on BHK cells,and the expression of VP0 protein and PVR protein on the expression of RIG-I,IRF3,IFN-β and ISG15 and ISG20 induced by SeV were detected.The expression of IFN-β and NF-κB promoters induced by VP0 protein and PVR protein were detected by double luciferase reporter assay.and the effect of RIG-I-like receptors(RIG-I receptors,RLRs)signaling pathway activating IFN promoter;the immunoprecipitation was used to detect the interaction between VP0 protein and PVR protein and key molecules in RLRs signaling pathway,the indirect immunofluorescence was used to detect the localization of VP0 protein and PVR protein in the cells with the key molecule IRF3.Results: The expression of pCAGGs-VP0 eukaryotic expression vector in HEK-293 T cells was successfully constructed.VP0 protein could significantly increase the replication of FMDV on BHK cells(P <0.01 or P <0.05),PVR protein could significantly inhibite the replication of FMDV(P <0.01 or P <0.05).The expression of VP0 protein on the downstream stimulating gene of interferon was significantly inhibited by VP0 protein(P <0.05),PVR protein could promote the expression of interferon downstream stimulated gene(P <0.05).In the double luciferase reporter assay,VP0 inhibited the activation of IFN-β and NF-κB in a dose-dependent manner(P <0.01 or P <0.05),and mediated by RIG-I,MDA5,VISA,TBK1 and IRF3 of IFN-β production has inhibitory effect(P <0.05),but no significant effect on IRF7.PVR protein can promote the activation of SeV-mediated IFN-β and NF-κB(P <0.01 or P <0.05)and promote the production of RIG-I,MDA5,VISA,TBK1 and IRF3-mediated IFN-β(P <0.05),but had no significant effect on IRF7.Immunoprecipitation showed VP0 protein and PVR protein could interact with IRF3,indirect immunofluorescence confirmed the co-localization of VP0 and PVR proteins with IRF3 in cells.Conclusion: VP0 protein can significantly inhibit the activation of IFN-β and NF-κB by fluorescence quantitative PCR and double luciferase reporter assay.PVR protein can significantly promote the activation of IFN-β and NF-κB,and thus,we suggesting that FMDV structural protein VP0 has inhibition in signaling pathway on type Ⅰ interferon,PVR protein has a positive effect on type Ⅰ interferon signaling pathway.Immunoprecipitation and laser confocal experiments further confirmed the VP0 protein,PVR protein can interact with IRF3 to inhibit and promote the activation of type Ⅰ interferon signaling pathway,and VP0 protein and PVR protein can also interact,We predict that these three proteins may form complexes that will have a role in the innate immune system.