The Role And Regulatory Mechanism Of The E3 Ligase RNF128 In Innate Antiviral Immunity

ObjectiveInnate immunity is the first line of host defense against microbial invasion,which is critical for eliciting immediate antiviral responses to eradicate virus infection.Activation of the innate immune response requires the pattern recognition receptors(PRRs)to recognize pathogen-associated molecular patterns(PAMPs).Viral nucleic acid including DNA,double stranded RNA(dsRNA)and single stranded RNA(ssRNA)are efficient PAMPs to initiate the innate antiviral immunity.The well identified PRRs that recognize these viral nucleic acids includes Toll-like receptor 3(TILR3).retinoic acidinducible gene-I(RIG-Ⅰ),melanoma differentiation-associated gene 5(MDA5)and some DNA sensors such as cyclic GMP-AMP synthase(cGAS).Upon recognition of the nucleic acids,these PRRs recruit different adaptors including TLR/IL-1R domain-containing adaptor protein inducing IFN-(3(TRIF),mitochondrial antiviral signaling protein(MAVS)and stimulator of IFN genes(STING)to activate TANK-binding kinase 1(TBK1).Activated TBK1 phosphorylates IRF3 and IRF7,triggers their dimerization,nuclear translocation and binding to IFN stimulation response elements(ISRE)to activate type Ⅰ IFN expression.Insufficient IFN production causes chronic infection,whereas excessive IFN causes autoimmune and/or inflammatory diseases.Thus,precise control of IFN production is critical for efficient viral clearance without harmful immunopathology.RNF128 is an important gatekeeper of T-cell unresponsiveness.So far,almost all studies related to RNF128 focused on CD4+ T cells and adaptive immunity.RNF128 controls T cells anergy and tolerance through K48-linked ubiquitination and degradation of several molecules involved in T cell activation.RNF128 was demonstrated to interact with and degrades TCR-CD3,CD40L,CD83 and CD151 through K48-linked ubiquitination.Several other E3 ligases such as Cbl-b,c-Cbl and Itch that regulate T cell anergy have been demonstrated to play important roles in the regulation of innate immunity.However,the function of RNF128 in other immune responses is not known.Thus,we investigated the function of RNF128 in innate immunity,and uncovered the molecular mechanism involved.Methods1.We measured RNF128 expression in macrophages and THP1 cells stimulated with nucleic acid mimics poly(I:C)and interferon-stimulatory DNA(ISD)or infected with RN A virus Sendai virus(SeV)and DNA virus HS V-1;2.We first designed three mouse RNF128 siRNAs and transfected into primary peritoneal macrophages and THPI cells,upon simulation with poly(I:C)and ISD or infection with SeV and HSV-1,Real time quantitative PCR were utilized to detect expression of IFNB1,CXCL10,MX1.CCL5,IL6 and TNFA;3.IFNB1 mRNA expression and luciferase activity were detected to identify the targets of RNF128.Then we investigated the interaction between RNF128 and TBK1 through Co-Immunoprecipitation(Co-IP)and Immunofluorescence(IF);4.We investigated the effects of RNF128 on the ubiquitination of TBK1 through in Coimmunoprecipitation analysis in vivo and in vitro.Besides,we used TBK1 mutants to study the sites ubiquinated by RNF128.Then we detected TBK1 ubiquitination in the animal models.5.To test the function of RNF128 in vivo,VSV and HSV-1 were used to challenge Rnf128-/-and WT mice.ELISA assay were performed to detect the amount of IFN-βprotein induced by VSV and HSV-1 infection in sera.VSV titer and replication in the lung and liver and HSV-1 genomic DNA copy number and viral titer were measured.Then,we measured survival rate in WT and Rnfl 28-/-mice after infection with VSV and HSV-1.Results1.Virus infection induces RNF128 expressionIn order to explore the function of RNF128 in innate immunity,we first measured RNF128 expression in macrophages and THP1 cells stimulated with poly(I:C)and ISD or infected with SeV and HSV-1.We found the level of RNF128 mRNA and protein was increased after stimulations.We also found IFN-β stimulation increased RNF128 protein expression in primary peritoneal macrophages.Furthermore,blockage of IFN-β signaling by antibody against IFN-β receptor 1(IFNRA1)attenuated SeV-and HSV-1-induced RNF128 expression.Transfection of STAT1 and STAT2 specific siRNA also attenuated SeV-and HSV-1-induced RNF128 expression.2.RNF128 positively regulates IRF3 activation and IFN-β productionTo explore the function of RNF128 in innate antiviral response,we found Ifnb1 mRNA expression was greatly decreased in macrophages transfected with mRNF128 siRNA upon simulation with poly(I:C)and ISD or infection with SeV and HSV-1.The expression of the genes downstream of IFN-β signaling Cxcl10,Mx1 and Ccl5 were also decreased.Similarly,knockdown of human RNF128 expression in THP-1 cells also decreased poly(I:C)-,SeV-,ISD-and HSV-1-induced expression of IFNB1,CXCL10,MX1 and CCL5.After stimulation with poly(I:C)and ISD or infection with SeV and HSV-1,macrophages from Rnfl28-/-mice showed less IFN-β expression and secretion.Transfection of TRIF,cGAS+STING and RIG-IN induced IFN-βpromoter activation and Ifnb1 mRNA expression in HEK293 cells.While,siRNA knockdown of RNF128 expression greatly decreased TRIF-,cGAS+STING-and RIG-IN-induced activation of IFN-β reporter and Ifnbl mRNA expression.In contrast,ectopic expression of RNF128 markedly increased TRIF-,RIG-IN-,cGAS+STING induced IFN-P promoter activation and Ifnbl mRNA expression.RNF128 overexpression increased TRIF-,MAVS-and cGAS+STING-induced activation of full length IFN-β promoter reporter and reporter containing IFN-β promoter PRD Ⅰ-Ⅲin HEK293 cells.Overexpression of RNF 128 increased TRIF-,MAVS-and STING+cGAS-induced IRF3 phosphorylation,dimerization and nuclear translocation.Taken together,these data demonstrated that RNF 128 positively regulates IRF3 activation to regulate IFN-β production.3.RNF128 targets TBK1We next sought to determine the molecular mechanisms by which RNF 128 enhances type I IFN signaling.We found TRIF-,cGAS+STING-,RIG-IN-,MAVS-and TBK1-induced IFN-P mRNA and promoter activity was greatly increased in the presence of RNF128 expression plasmid,whereas IRF3 5D-induced IFN-PmRNA and promoter activity was not increased by RNF128 overexpression.These data suggested that RNF128 may enhance type I interferon signaling by targeting TBK1.To confirm RNF128 targets TBK1,the interaction between RNF128 and TBK1 was confirmed by Co-IP.Notably,the interaction between endogenous RNF 128 and TBK1 was enhanced upon SeV or HSV-1 infection.Those interactions were further supported by the colocalization studies.A low degree of colocalization between endogenous RNF 128 and TBK1 was also detected in mouse primary peritoneal macrophages.The colocalization between endogenous RNF 128 and TBK1 was greatly enhanced after infection with SeV or HSV-1.4.RNF 128 promotes K63-linked ubiquitination of TBK1To investigate the E3 ligase activity of RNF 128 was involved in the regulation of TBK1 activation,Myc-TBK1 was transfected into HEK293 cells together with V5-RNF128.Co-IP experiments showed that the level of TBK1 ubiquitination was markedly increased in the presence of RNF 128 expression plasmid.In vitro ubiquitination assays demonstrated that RNF128 could directly ubiquitinate TBK1 in the presence of E1 and E2.To investigate the form of RNF128-mediated TBK1 polyubiquitination,HA-ubiquitin mutants K48 and K63 were used in the transfection assays.RNF128-mediated polyubiquitination of TBK1 was significantly increased in the presence of both WT and K63 plasmids,but not in the presence of K48 plasmid.In vitro ubiquitination assays with mutant ubiquitin K48 and K63 also confirmed RNF128 conjugated K63-linked ubiquitin chains to TBK1.To directly confirm RNF128-induced TBK1 ubiquitination,TBK1 ubiquitination was measured in primary macrophages from WT and Rnfl28 mice after SeV or HSV-1 infection.SeV or HSV-1 infection greatly increased K63-linked TBK1 ubiquitination in WT macrophages.However,SeV and HSV-1-induced TBK1 K63-linked ubiquitination was substantially attenuated in Rnfl28 macrophages.To investigate whether RNF128 promotes TBK1 ubiquitination through K30 and K401,TBK1 point mutants(K30R,K401R,K30/401R)were constructed and transfected into HEK293 cells together with RNF128.Compared to WT TBK1,both individual point mutants K30R and K401R and double mutant K30R/K401R exhibited decreased TBK1 ubiquitination induced by RNF128.5.RNF128 promotes TBK1 activationTo determine whether RNF128-induced TBK1 ubiquitination affects TBK1 activation,TBK1 was immunoprecipitated with anti-TBK1 antibody from the cell lysates,followed by in vitro TBK1 kinase assays.TBK1 kinase activity was increased in peritoneal macrophages upon SeV and HSV-1 infection.But,SeV and HSV-1 inf’ection-induced TBK1 kinase activity was greatly decreased in RNF128 deficient macrophages,compared to that in WT macrophages.Consistent with the kinase activity,SeV-and HSV-1-induced TBK1 phosphorylation was also decreased in RNF128 deficient macrophages.Transfection of WT TBK1 into TBK1-deficient MEFs restored TBK1 activation as indicated by TBK1 phosphorylation and IFN-β expression.RNF 128 expression further increased TBK1 phosphorylation and TBK1-induced IFN-β expression.However,phosphorylation of TBK1 mutants K30R,K401R and K30/401R and IFN-βexpression was greatly decreased in the presence of RNF128 expression plasmid.We also found binding of NEMO to TBK1 was decreased in RNF128-deficient cells after SeV and HSV-1-infection.RNF128 showed equal binding to WT TBK1 and TBK1 S172A mutant,and RNF 128-induced ubiquitination in S172A was not impaired,suggesting that RNF128-mediated TBK1 ubiquitination is upstream of TBK1 phosphorylation and activation.6.RNF128 is a general regulator for TBK1 activationTo evaluate the function of MI31/2 and Nrdp1 in TBK1 activation in the context of RNF128,siRNA against MIB1/2 and Nrdpl were transfected into RNF128 deficient macrophages.RNF128 deficiency decreased LPS-,SeV-and HSV-1-induced IFN-β expression,IRF3 phosphorylation and TBK1 phosphorylation in peritoneal macrophages.siRNA knockdown of MIB 1/2 in RNF128 KO macrophages further attenuated SeV-induced IFN-β expression,IRF3 phosphorylation and TBK1 phosphorylation,while LPS-and HSV-1-induced IFN-β expression,IRF3 phosphorylation and TBK1 phosphorylation was not impaired.siRNA knockdown of Nrdpl in RNF128 KO macrophages further decreased LPS-induced IFN-β expression,IRF3 phosphorylation and TBK1 phosphorylation,but,SeV-and HSV-1-induced IFN-β expression,IRF3 phosphorylation and TBK1 phosphorylation was not impaired.7.RNF128 positively regulates antiviral responseTo test the function of RNF128 in vivo,VSV and HSV-1 were used to challenge Rnfl28-/-and WT mice,the amount of IFN-β protein induced by VSV and HSV-1 infection was much less in the sera of Rnfl28-/-mice than that of WT mice.VSV titer and replication in the lung and liver of Rnfl28 mice were significantly increased compared to WT controls.Similarly.HSV-1 genomic DNA copy number and viral titer were significantly increased in the brain of Rnfl28-/-mice in comparison to their WT counterparts.Severe injuries were observed in the lungs of Rnf128-/-mice after infection with VSV and HSV-1.Furthermore,Rnfl28-/-mice were more susceptible to infection with HSV-1 in brains than WT mice.Conclusion1.Virus infection induces RNF128 expression2.RNF128 positively regulates IRF3 activation and IFN-β production3.RNF128 targets TBK14.RNF128 promotes K63-linked ubiquitination of TBK15.RNF128 promotes TBK1 activation6.RNF128 is a general regulator for TBK1 activation7.RNF128 positively regulates antiviral responseInnovation and significance1.Our study defined RNF128 as a new positive regulator of innate antiviral immunity except for its function in the regulation of T cells.2.Here,we demonstrated that the ubiquitin E3 ligase RNF128 interacted with TBK1 and promoted K63-linked ubiquitination of TBK1 after RNA and DNA virus infection.3.In this study,we demonstrated RNF128 is a general regulator for TBK1 ubiquitination and IFN-β expression downstream of LPS-,RNA virus-and DNA virus-induced innate signaling.