| BackgroundInnate immunity(also known as natural immunity)is the first line of host to defense against the invasion of various foreign pathogenic microorganisms.The host identifies pathogen associated molecule patterns(PAMPs)from different pathogenic microorganisms through various pattern recognition receptors(PRRs).Retinoic acid-inducible gene-I(RIG-I)-like receptors(RLRs),as important nucleic acid recognition receptors,are responsible for the recognition of viral RNA in the cytoplasm and recruits mitochondrial antiviral signaling protein(MAVS),and then promotes TBK1(tank-binding kinase 1)activation and IRF3 phosphorylation to initiate the antiviral immune response.At present,a variety of molecules have been identified as regulators specific to the RLR pathway that regulate the innate immune response.However,the regulatory mechanism of RLR-mediated antiviral innate immune signaling is still far from being clarified.Ubiquitination of protein is an important post-translation modification(PTM)in the host,which can regulate protein stability and intermolecular interaction.A variety of RING-domain E3 ubiquitin ligases organized in a tight cluster within the major histocompatibility complex(MHC)class I region have been implicated in the regulation of innate immunity.RING finger protein 39(RNF39,also called HZFW),which contains a RING domain and possesses potential E3 ubiquitin ligase activity,is also encoded within the MHC-I region.Here we show that the E3 ubiquitin ligase RNF39 inhibits RLR pathway through mediating K48-linked ubiquitination and proteasomal degradation of DDX3X.At the same time,we confirm that RNF39 regulates DDX3X ubiquitination modification at sites 55,138 and 162 of lysine.Concordantly,Rnf39 deficiency enhances RNA viruses-triggered innate immune responses and attenuates viral replication.Thus,our results identify an important regulatory role of E3 ubiquitin ligase RNF39 in innate immunity;this study reveal the molecular mechanism of RNF39 inhibiting RLR signaling pathway by mediating DDX3X ubiquitination modification and protein degradation;our results suggest that RNF39 may be a potential target for viral diseases and autoimmune diseases related to abnormal RLR activation.ObjectiveThis study aims to analyze the effects and regulatory mechanism of E3 ubiquitin ligase RNF39 in innate antiviral immune signaling pathway;to elucidate the the important role of DDX3X ubiquitination in the activation of RLR pathway and the initiation of antiviral innate immunity during virus infection;to further enrich the understanding of the interaction between virus and innate immunity;to provides theoretical basis for the prevention and control of viral diseases.Methods and results1.RNF39 inhibits RLR-induced innate immune response(1)Viral infection induces RNF39 expressionThe expression of RNF39 in mouse primary peritoneal macrophages(PMs)and mouse embryonic fibroblasts(MEFs)were detected by western blot during infection with vesicular stomatitis virus(VSV)and sendai virus(SeV)for the indicated time periods(0h,4h,8h,12h,24h,36h,48h).The expression of RNF39 in mouse PMs and MEFs were detected by western blot during stimulation with interferon-?(IFN-?)for the indicated time periods(0h,2h,4h,8h,12h,24h,36h).These results showed that SeV and VSV infection induced the expression of RNF39 in PMs and MEFs.Meanwhile,RNF39 expression was considerably enhanced following IFN-? stimulation.Taken together,these data indicate that RNF39 expression is induced during viral infection,suggesting that RNF39 is involved in host antiviral responses.(2)RNF39 inhibits RNA viruses-induced cytokine expressionThe CRISPER-Cas9 technique was used to construct Rnf39 knockout mice,and the expression of RNF39 in protein and mRNA levels of mice was detected by western blot and PCR.We detected the secretion and mRNA expression of IFN-?,tumor necrosis factor-?(TNF-?)and interleukin-6(IL-6)in Rnf39+/+ or Rnf39-/-mouse PMs by enzyme linked immunosorbent assay(ELISA)and real-time Quantitative polymerase chain reaction(qPCR).RNF39/Rnf39 small interfering RNA(siRNA)of human and mouse origin was constructed.After transfection of RNF39/Rnf39 siRNA in human myeloid leukemia mononuclear cells THP1 and mouse PMs for 48h,the RNA interference efficiency of RNF39 were determined by western blot.We detected the expression of IFN-? mRNA level in knockdown mouse PMs and RNF39 knockdown THP-1 by qPCR.We detected the expression of IFN-? in Rnf39-/-mouse bone marrow-derived dendritic cells(BMDCs)by ELISA and qPCR.These results showed that Rnf39 deficiency enhance RNA viruses-induced IFN-?,TNF-? and IL-6 expression in mouse PMs.Meanwhile,RNF39/Rnf39 knockdown enhanced VSV induced IFN-? expression in THP-/-cells and mouse PMs.In addition,Rnf39 deficiency enhanced RNA viruses-induced IFN-? expression in BMDCs.These results indicate that RNF39 specifically inhibits RNA viruses-induced cytokine expression.(3)RNF39 inhibits the RLR pathway via its E3 ubiquitin ligase activityWe transfected RNF39 and its E3 ubiquitin ligase disrupted mutant C108S expression plasmids together with IFN-? reporter gene plasmid into HEK293T cells.We detected the regulatory effects of RNF39 and its mutant C108S on VSV-induced IFN-? reporter gene activity by reporter gene assay.We detected the regulation effects of Rnf39 deficiency on both polyriboinosinic acid-polyribocytidylic acid[poly(I:C)]high molecular weight(HMW)and low molecular weight(LMW)transfection-induced Ifnb mRNA level by qPCR.We detected the rescue effects of RNF39 and its mutant C108S in Rnf39-/-MEFs on both poly(I:C)HMW and LMW transfection-induced Ifnb mRNA level by qPCR.These results showed that RNF39 significantly inhibited VSV-induced activation of IFN-? promoter,while the mutant C108S lost its inhibitory effect.Rnf39 deficiency greatly promoted Ifnb mRNA level induced by transfection of both poly(I:C)HMW and LMW.In addition,in this rescue experiments,the transfection of RNF39 wild type plasmid,but not C108S mutant.could restore the inhibitory effects on Ifnb mRNA level.These results indicate that RNF39 inhibites the RLR pathway via its E3 ubiquitin ligase activity.(4)RNF 39 inhibits RLR-induced IRF3 activationWe detected RNA viruses-induced phosphorylation of Interferon regulatory factor 3(IRF3)in Rnf39 knockout and knockdown mouse PMs by western blot.We transfected RNF39,IRF3 reporter gene and indicated adaptor plasmids into HEK293T cells,and then detected the regulatory effects of RNF39 on RIG-I-and melanoma differentiation associated gene 5(MDA5)-medicated IFN-? reporter gene activity by reporter gene assay.These results showed that Rnf39 deficiency and knockdown both enhanced RNA viruses-induced phosphorylation of IRF3.Furthermore,RNF39 overexpression inhibited RIG-I-and MDA5-induced IRF3 reporter gene activation.These results indicate that RNF39 attenuates RLR-medicated IRF3 activation,leading to the inhibition of downstream signaling.2.Rnf39 deficiency enhances anti-RNA viral responses(1)Rnf39 deficiency enhances cellular anti-RNA viral immune responseWe detected RNA viruses-induced phosphorylation of signal transducerand activator of transcription 1(STAT1)in Rnf39-/-mouse PMs by western blot.We detected subsequent interferon stimulated genes(ISGs)expression,including Ifit1,Ifit2,Isg15,Mx1 and Rantes in macrophages by qPCR.These results showed that Rnf39 deficiency promoted RNA viruses-induced phosphorylation of STAT1 and subsequent ISGs expression.These results indicate that RNF39 inhibits cellular anti-RNA viral immune responses.(2)Rnf39 deficiency inhibits RNA virus replicationAfter the mouse PMs of Rnf39+/+ and Rnf39-/-were infected with VSV(1 MOI)which containing green fluorescent protein(GFP)for 12 hours,the fluorescence intensity of GFP was detected by fluorescence microscopy to qualitatively analyze the replication amount of VSV.Meanwhile,the replication of VSV virus in mouse PMs of Rnf39+/+ and Rnf39-/-were quantitatively analyzed by qPCR.These results indicate that Rnf39 deficiency significantly inhibits RNA virus replication.(3)Rnf39 deficiency has no effect on IFN-?-triggered signaling.We detected IFN-?-induced phosphorylation of STAT1 in Rnf39-/-mouse PMs by western blot.We detected IFN-?-triggered downstream ISGs expression,including Ifit1,Ifit2,Isg15,Mxl and Rantes in macrophages by qPCR.These results showed that Rnf39 deficiency had no effects on IFN-?-induced STAT1 phosphorylation and ISGs expression.These results indicate that Rnf39 deficiency has no effect on IFN-?-triggered signaling.(4)Rnf39 deficiency enhances anti-RNA viral responses in vivoWe established an in vivo model of viral infection by intraperitoneally injecting VSV into Rnf39+/+ and Rnf39-/-mice.The secretions of IFN-?,TNF-? and IL-6 of mice sera were detected by ELISA after infecting with VSV for 12h.The VSV viral burden of lung and spleen tissues were detected by qPCR after infecting with VSV for 18h.We detected the inflammatory infiltration of lung tissues by hematoxylin-eosin(HE)staining after infecting with VSV for 48h.These results showed that following VSV infection,Rnf39 deficiency produced significantly more IFN-?,TNF-? and IL-6 in sera than Rnf39+/+ mice.Concordantly,the VSV viral burden was significantly decreased in lung and spleen of Rnf39-/-mice.Less infiltration of inflammatory cells was observed in the lungs of Rnft39-/-mice.Collectively,these data indicate that Rnf39 deficiency enhances RNA-dependent antiviral responses and suppresses RNA viral replication.(5)Rnf39-deficient mice are more resistant in survival assays upon VSV infectionTo explore the survival assays of mice,we established an in vivo model of viral infection by intraperitoneally injecting VSV into Rnf39+/+ and Rnf39-/-mice.Collectively,these data indicate that Rnf39 deficiency enhances RNA-dependent antiviral responses and suppresses RNA viral replication in vivo.3.RNF39 promotes proteasomal degradation of DDX3X(1)RNF39 targets DDX3XWe co-transfected RNF39 expression plasmid together with DDX3X,RIG-I,mitochondrial antiviral signaling protein(MAVS),TNF receptor-related factor 3(TRAF3)and tank-binding kinase 1(TBK1)plasmids into HEK293T cells.And then we detected the regulatory effects of RNF39 on the expression of DDX3X,RIG-I,MAVS,TRAF3 and TBK1 in HEK293T cells by western blot.We detected the regulatory effects of Rnf39 knockdown on the expression of DDX3X,TBK1,TRAF3 and STAT1 in mouse PMs by western blot.These results showed that RNF39 significantly inhibited the expression of DDX3X but has no effect on the expression of other adaptor molecules in the RLR pathway,suggesting that DDX3X may be a target of RNF39 in regulating RLR pathway.(2)RNF39 interacts with DDX3XWe co-transfected RNF39 expression plasmid together with DDX3X,TBK1,TRAF3,IRF3,MDA5,RIG-I and MAVS plasmids into HEK293T cells.We detected the binding of RNF39 to adaptors by western blot.We detected the binding of RNF39 to DDX3X in mouse PMs by western blot.We detected the co-localization of RNF39 and DDX3X in MEFs by immunofluorescence.We detected the binding of DDX3X which was transcripted and translated in vitro to RNF39 recombinant protein by in vitro binding assay.These results showed that RNF39 coprecipitated with DDX3X.Furthermore,RNF39 interacted with DDX3X in SeV infected mouse PMs.Consistently,confocal analysis showed the colocalization between RNF39 and DDX3X in MEFs.In vitro binding assays demonstrated that RNF39 could directly interact with DDX3X.Taken together,these data suggest that RNF39 directly interacts with DDX3X.(3)RNF39 interacts with DDX3X via its N-terminal regionWe constructed a series of RNF39 truncated mutants by point mutation kit,including RING domain deletion mutant(?RING),N209 and C210.We co-tranfected DDX3X expression plasmid together with ?RING,N209 and C210 plasmids into HEK293T cells and then peformed coprecipitation process.We detected the binding domain of RNF39 which combined with DDX3X by western blot.These results showed that DDX3X was coprecipitated with RNF39 RING domain deletion mutant(?RING)and N209,but not with C210.These results indicate that RNF39 binds to DDX3X through its N-terminal region.(4)DDX3X interacts with RNF39 via its N-terminal regionWe constructed a series of DDX3X truncated mutants by point mutation kit,including RecA-like domain 1 of N-terminal and RecA-like domain 2 together with RS-like domain of C-terminal.We co-tranfected RNF39 expression plasmid together with N413 and C414 plasmids into HEK293T cells and then peformed coprecipitation process.We detected the binding domain of DDX3X which combined with RNF39 by western blot.These results showed that RNF39 was coprecipitated with DDX3X N413 but not with C414,indicating that DDX3X binds to RNF39 through its N-terminal region.(5)RNF39 inhibits DDX3X expressionWe detected the expression of DDX3X in Rnf39 deficiency mouse PMs/BMDCs and RNF39 knowdown THP-1 cells by western blot.We detected the mRNA expression of Ddx3x in Rnf39 deficiency mouse PMs by qPCR.We co-transfected DDX3X expression plasmid and RNF39 plasmids which at different dose concentrations(0.5?g,1?g,2?g).And then we detected the inhibition of RNF39 to DDX3X expression by western blotThese results showed that Rnf39 deficiency increased DDX3X protein expression in both resting and viral infected mouse PMs and BMDCs.Consistently,RNF39 knockdown enhanced DDX3X protein expression in THP-1.RNF39 overexpression dose-dependently inhibited DDX3X expression in HEK293T cells.But Rnf39 deficiency had no influence on Ddx3x mRNA level,suggesting that RNF39 inhibits the expression of DDX3X by affecting its post-translational modification process.(6)TRIM15,TRIM31 and TRIM40 have no effects on DDX3X expressionIn the MHC-I region encoded a series of E3 ubiquitin ligase expressed genes,such as TRIM15,TRIM31,TRIM40.We transfected DDX3X expression plasmid together with RNF39,TRIM 15,TRIM31 and TRIM40 plasmids into HEK293T cells,and then we detected the effects of other E3 ubiquitin ligases on DDX3X expression by western blot.These rusults showed that TRIM 15,TRIM31 and TRIM40 had no effects on DDX3X expression.Taken together,these data suggest that RNF39 inhibites DDX3X expression don't depend on the assistance of other E3 ubiquitin ligases.(7)RNF39 promotes the degradation of DDX3XCycloheximide(CHX)chase assay was conducted in PMs of Rnf39+/+ and Rnf39-/-mice.To prove whether RNF39 regulates DDX3X expression by affecting its degradation.These results showed that when the protein synthesis process was inhibited,the degradation rate of DDX3X in PMs of Rnf39-/-mice were significantly reduced,suggesting that RNF39 regulates the degradation of DDX3X.(8)RNF39 promotes DDX3X degradation in ubiquitin-proteasome pathwayHEK293T cells were treated with proteasome inhibitor MG132,lysosome/autophagy inhibitor chloroquine and 3-MA,and the pathway through which RNF39 degrade DDX3X was detected by western blot.The regulation of DDX3X expression by C108S mutant which disruption of E3 ubiquitin ligase activity was detected by western blot.These results showed that RNF39-mediated inhibition of DDX3X expression could be reversed by the proteasome inhibitor MG132,but not by lysosome/autophagy inhibitors chloroquine and 3-MA.In addition,the RNF39 point mutation C108S lost the ability to promote DDX3X degradation.Collectively,these data indicate that RNF3 9 promotes DDX3X protein degradation in proteasome via its E3 ubiquitin ligase activity.4.RNF39 promotes K48-linked ubiquitination of DDX3X(1)RNF39 enhances ubiquitination of DDX3XWe co-transfected DDX3X expression plasmid together with ubiquitin(Ub),RNF39 and E3 ubiquitin ligase disrupted mutant C108S plasmids into HEK293T cells.We detected the effect of RNF39 on DDX3X ubiquitination in HEK293T cells by western blot.Given that DDX3X or RNF39 perhaps form a complex with other nonspecific proteins,we performed a two-step immunoprecipitation(Re-IP)assay to reduce the presence of nonspecific associated proteins.We detected the ubiquitination of DDX3X in Rnf39-/-mouse PMs and MEFs by western blot.These results showed that the ubiquitination of DDX3X was substantially increased in the presence of RNF39 expression plasmid in HEK293T cells.Importantly,the RNF39 C108S mutant lost the ability to enhance the ubiquitination of DDX3X.Meanwhile,the same results were obtained in Rnf39-/-mouse PMs and MEFs.These results indicate that RNF39,as an E3 ubiquitin ligase,directly catalyses the ubiquitination of DDX3X.(2)RNF39 enhances K48-linked ubiquitination of DDX3XTo analyze the polyubiquitin chains on DDX3X catalyzed by RNF39,a panel of ubiquitin mutants which contain arginine substitutions of all its lysine(K)residues except the one as indicated were used in the transfection assays,including K6,K11,K27,K29,K33,K48 and K63.We transfected DDX3X expression plasmid together with RNF39,ubiquitin and its mutant plasmids into HEK293T cells,and then performed Re-IP assay.The form of RNF39 regulating DDX3X ubiquitination was detected by Western blot.We transfected DDX3X expression plasmid together with RNF39,ubiquitin,K48 and K63 plasmids into HEK293T cells,and then performed coprecipitation assay.We detected the polyubiquitin chain linkage of DDX3X in Rnf39-/-mouse PMs by western blot.DDX3X polyubiquitination could be detected in the presence of K48 or K63 plasmid,but not with other mutants.Moreover,RNF39 markedly enhanced K48-linked polyubiquitination of DDX3X,with no effect on K63-linked polyubiquitination of DDX3X.In addition,Rnf39 deficiency selectively inhibited K48-linked polyubiquitination of DDX3X,whereas K63-linked polyubiquitination of DDX3X was not affected.Collectively,these data indicate that RNF39 selectively promotes K48-linked polyubiquitination of DDX3X.(3)K55,K138,and K162 of DDX3X are essential residues for its ubiquitinationWe predicted the possible ubiquitination sites of DDX3X by the UbPred program and replaced each of the eight DDX3X lysine(K)residues individually with arginine(R)to create K50R,K55R,K64R,K66R,K130R,K138R,K162R and K581 mutants of DDX3X,respectively.In HEK293T cells,a two-step immunoprecipitation method was carried out to detect the ubiquitination sites of RNF39 on DDX3X by western blot.We detected whether RNF39-mediated ubiquitination and protein degradation of DDX3X was completely abolished in DDX3X K55/K138/K162R mutant.Re-IP assay revealed that only K55R,K138R,and K162R partly blocked the ubiquitination of DDX3X mediated by RNF39.RNF39-mediated ubiquitination and the degradation of DDX3X were completely abolished in DDX3X K55/K138/K162R mutant.Thus,these results suggest that K55,K138,and K162 of DDX3X are essential residues for its ubiquitination and degradation.(4)DDX3X K55/K138/K162R mutant enhances RLR pathway activationWe co-transfected DDX3X K55/K138/K162R mutant into HEK293T cells to block the effect of RNF39.We detected the regulatory effect of DDX3X K55/K138/K162R on VSV-induced IFN-? promoter activation by reporter gene assay.These results showed that,compared to DDX3X wild-type,K55/K138/K162R significantly enhanced VSV-induced IFN-? promoter activation.These results suggest that RNF39 promotes the activation of RLR pathway by applying to the K55/K138/K162 sites of DDX3X.Conclusions1.E3 ubiquitin ligase RNF39 participates in and negatively regulates anti-RNA viral innate immune response.2.E3 ubiquitin ligase RNF39 specifically promotes K48-linked ubiquitination of DDX3X.3.E3 ubiquitin ligase RNF39 promotes proteasomal degradation of DDX3X.4.The lysine 55,138 and 162(K55,K138 and K162)of DDX3X are essential residues for its ubiquitination mediated by RNF39.Novelty and significance1.Here,we demonstrate that DDX3X can be modified by ubiquitination,and identify the E3 ubiquitin ligase RNF39,which regulates DDX3X ubiquitination in the RLR pathway.2.We reveals the mechanism that RNA virus-induces RNF39 expression and then RNF39 promotes the ubiquitination and proteasome degradation of DDX3X,thereby inhibiting RLR-mediated antiviral immunity and realizing the immune escape of the virus.We elucidate a novel mechanism that RNF39 inhibits RLR-dependent innate immune pathway by regulating the expression of DDX3X.3.This study elucidates the roles of RNF39 in viral diseases and innate immune regulation.It is suggesting that RNF39 may be used as a potential therapeutic target for the intervention of viral diseases and autoimmune diseases caused by innate immune abnormalities.LimitationsHSV-1 infection can also induce RNF39 expression,suggesting that RNF39 may also play a regulatory role in anti-DNA virus immunity.The regulatory role of RNF39 in the innate immune response mediated by DNA-recognized receptor cGAS and its possible molecular mechanism need to be further explored. |
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