P120-Catenin Promotes Innate Antiviral Immunity Through Stabilizing TBK1-IRF3 Complex

Objective:TBK1-IRF3 complex,which is essential for the production of type I IFN,plays vital roles in antiviral immune responses,but its regulatory mechanisms are currently incompletely understood.p120-catenin(p120)is an armadillo-repeat protein that mainly regulates the stability of classical cadherins and the development of epithelial-to-mesenchymal transitions(EMTs),and is essential for inhibiting epidermal proliferation and promoting epidermal differentiation.It also plays important role in WNT signaling pathway,brain development regulation,tumor invasion and so on.However,the biological functions of p120 in innate antiviral immunity is unknown.Thus,we investigated the function of p120 in production of IFN-β and the molecular mechanism involed.Methods:1.In vitro experiment:1.1 To explore the role of p120 in antiviral immunityP120 siRNA of human and mouse origin was constructed and transfected into THP-1 cells and mouse primary peritoneal macrophages 48 hours,then infected with HSV/VSV/SeV.The mRNA expression of VSV and IFN-β was detected by qPCR,the secretion level of IFN-β was detected by ELISA,and the protein expression of p-IRF3 and VSV-G was detected by WB.Transfected p120 plasmid into HEK293T and infected with VSV for 6h,VSV-G protein expression was detected by WB and mRNA expression of VSV and IFN-β were detected by qPCR.The replication of the virus was observed by fluorescence microscope.1.2 To determine the targets and mechanism of p120The expression plasmids of p120,TBK1,IRF3 and STING were transfected into HEK293T cells.The interaction between p120 and other proteins and the influence of p120 on the interaction between TBK1 and IRF3 were detected by immunoprecipitation,the activation of interferon gene was detected by qPCR.Macrophages and HEK293T cells transfected with p120 and TBK1 plasmids were infected with VSV.The co-localization of p120 with TBK1 and mitochondria was detected by immunofluorescence.Macrophages transfected with p120 siRNA or Control siRNA and infected with VSV and HSV-1,the effect of p120 on the interaction between TBK1 and IRF3 was detected by immunoprecipitation.2.In vivo experiment:p120KD mice and WT mice were constructed by caudal vein injection of p120 siRNA or Control siRNA and infected by intraperitoneal injection of VSV.The secretion of interferon in serum was detected by ELISA,the expression of VSV mRNA in lung tissue was detected by qPCR,inflammatory cell infiltration and alveolar damage in lung tissue were observed by HE stainingResults:1.In vitro experiments1.1 p120 positively regulates antiviral immunitysiRNA-mediated knockdown of p120 significantly reduced the phosphorylation of IRF3,the expression of IFN-β mRNA and the secretion of IFN-β induced by viral infection but increased the protein and mRNA expressions of VSV.In HEK293T cells,overexpression of p120 significantly increased the expression of IFN-β mRNA and reduced the expression of VSV-G protein and mRNA,inhibited viral replication.1.2 p120 targets the TBK1-IRF3 complex and enhances its stabilityImmunoprecipitation and immunofluorescence showed that p120 combined with TBK1,IRF3 and STING,and colocalized with TBK1 on mitochondria.Overexpression of p120 enhanced the binding of TBK1 to IRF3 and IFN-β reporter activation induced TBK1,what’s more,p120 enhanced the IFNB1 mRNA expression induced by TBK1 and IRF3,while downregulation of p120 inhibited their binding.2.In vivo experimentp120 KD mice are more susceptible to VSV infection as indicated by higher tissue viral titers,less IFN-I production and greater infiltration of immune cells.Conclusion:p120 is an important positive regulator of innate antiviral immunity,which promotes IRF3 phosphorylation by stabilizing TBK1-IRF3 complex,promotes viral infection-induced type Ⅰinterferon production,and promotes antiviral immunity.