Transcriptional Regulation Of USP25 And Mechanisms Of INKIT-mediated Regulation Of Innate Antiviral Signaling

Viral infection or lipopolysaccharide(LPS)treatment induces expression of a large array of cytokines and inflammatory factors such as type I interferons,the products of which play a critical role in host antipathogen immunity and inflammation.The type I interferons bind to the type I interferon receptors(Interferon alpha receptor 1/2,IFNAR1/2)in an autocrine or paracrine manner to induce the activation of JAK-STAT signaling pathways.The phosphorylated STAT1 and STAT2 interact with IRF9 to form a transcription complex ISGF3 which translocates to the nucleus to mediate the expression of IFN-stimulated genes(ISGs).We have previously reported that the expression of ubiquitin specific protease 25(USP25)is significantly upregulated after viral infection or LPS treatment which is essential for innate immune signaling.However,the mechanism behind this phenomenon is unclear.In this study,we find that viral infection-induced up-regulation of USP25 is diminished in cells lacking interferon regulatory factor 7(IRF7)or interferon-receptor 1(IFNAR1)but not p65.Sendai virus(SeV)-or type I interferon-induced upregulation of USP25 requires de novo protein synthesis of the transcription factor IRF7,but not the transcription complex ISGF3.Furthermore,IRF7 directly binds to the two conserved IRF binding sites on the USP25 promoter to drive transcription of USP25,and mutation of these two sites abolished SeV-induced IRF7-mediated activation of the USP25 promoter.Our study has uncovered a previously unknown mechanism by which viral infection or LPS induces upregulation of USP25.The transcription factors p65 and IRF3 play key roles in the induction of cellular antiviral responses.The classical and the non-classical IKKs,namely IKKa/IKK?/IKK? complex and TBK1/IKKs complex mediates virus-tirggered phosphorylation of p65 and IRF3,respectively.However,how the phosphorylation is regulated after viral infection remains largely unknown.In this study,we find that viral infection induces upregulation of INKIT(Inhibitor for NF-?B and IRF3),in a manner dependent on the transcription factor p65.Overexpression of INKIT inhibits virus-induced phosphorylation of p65 and IRF3 and expression of downstream genes as well as innate antiviral responses.In contrast,knockout of INKIT had the opposite effect.Inkit-/-cells or mice produced elevated levels of type ? interferons and proinflammatory cytokines after viral infection and the Inkit-/-mice were more resistant to lethal viral infection compared to wild-type controls.INKIT interacted with IKK?/? and TBK1/IKK?,impairing the recruitment and phosphorylation of p65 and IRF3.Viral infection induced IKK-mediated phosphorylation of INKIT at Ser58,resulting in its dissociation from the IKKs.Our findings thus uncover INKIT as a new regulator of innate antiviral responses.