| BackgroundRetinoic acid inducible gene Ⅰ(RIG-Ⅰ)-like receptors(RLRs)are one of the most important cytosolic pattern recognition receptors(PRRs).After detecting the exogenous RNA during viral replication,RLRs activate and recruit the downstream adaptor mitochondrial antiviral-signaling protein(MAVS)thus forming the MAVS-TNF receptor-associated factor 3(TRAF3)-TANK-binding kinase 1(TBK1)complex.MAVS-TRAF3-TBK1 complex activates the key transcriptional factor interferon regulatory factor 3(IRF3),subsequently induces the production of type Ⅰ interferons(type Ⅰ IFN,including IFN-α and IFN-β).By binding with membrane interferon alpha/beta receptor(IFNAR),IFNs subsequently promote the high expression of interferon-stimulated genes(ISGs)through tyrosine-protein kinase(JAK)-signal transducer and activator of transcription(STAT)pathway,leading to the establishment of antiviral state.As the key scaffold protein in RLRs pathway,DEAD box RNA helicase 3,X-linked(DDX3X)plays a vital role in the formation of MAVS-TRAF3 complex.Thus,in order to balance the activation level of RLR-mediated antiviral innate immune responses,the activation and expression of DDX3X need to be tightly regulated.Ubiquitination is one of the most important protein post-translational modifications(PTMs),participating in regulating the activation and expression level of proteins.However,whether the activation of DDX3X could be ubiquitinated by certain E3 ubiquitin ligase is still unknown.As the specific negative regulator of the tumor suppressor genes p53,E3 ubiquitin ligase murine double minute 2(MDM2)plays vital roles in the development of tumors.However,the role of MDM2 in innate immune responses,especially in macrophages and other immune cells,is remained unclear.In this study,we investigated the regulatory roles and potential mechanism of MDM2 in anti-RNA virus innate immune responses through RNA interference,high expression,as well as RNA virus infection mouse model.We show that the E3 ubiquitin ligase MDM2 promotes RLR-dependent IFN-β expression and IRF3 activation.Furthermore,MDM2 specifically interacts with DDX3X and promotes its K63-linked ubiquitin modification.Our study reveals a novel mechanism for the regulation of RLR activation by targeting DDX3X,identify MDM2 as an enhancer of RLR-dependent antiviral innate immune responses and discover a potential target for antiviral drugs.Materials and Methods1.The effects of MDM2 expression after viral infectionIsolate the mouse peritoneal macrophages and infected with SeV for 0 h,4h,8 h,12 h,24 h,36 h.Extract the whole proteins and Western blot analysis the expression of MDM2.2.The effects of MDM2 on the activation of RLR-mediated antiviral innate immune responses2.1 The specific interference RNA(siRNA)of Mdm2 is designed and synthesized.The siRNAs are transfected into mouse primary peritoneal macrophages by liposome Inteiferin.After 36 h,Western blot analyses the interference efficiency of MDM2.Mdm2 and control(Ctrl)siRNA are transfected into mouse primary peritoneal macrophages by liposome Interferin,respectively.After 36 h,cells are infected with SeV for 8 h,RNA is extracted and reverse transcribed,and the mRNA expression levels of Ifnb is detected by RT-PCR.2.2 The MDM2 expression plasmid and IFN-β reporter gene plasmid are cotransfected into HEK293T cells by liposome JetPEI.After 24 h,the cells are infected with SeV for 12 h,and the activation level of IFN-β is detected by reporter gene.The increasing amounts of MDM2 expression plasmid,RIG-Ⅰ plasmid and IFN-β reporter gene plasmid are cotransfected into HEK293T cells by liposome JetPEI.After 24 h,the activation level of IFN-β is detected by reporter gene.3.The effects of MDM2 on the RLR-mediated IRF3 activation3.1 The MDM2 expression plasmid and IRF3 reporter gene plasmid are cotransfected into HEK293T cells by liposome JetPEI.After 24 h,the cells are infected with SeV for 12 h,and the activation level of IRF3 is detected by reporter gene.Mdm2 and Ctrl siRNA are transfected into mouse primary peritoneal macrophages by Interferin,respectively.After 36 h,the cells are infected with SeV,and the phosphorylation levels of IRF3 and STAT1 are detected by Western blot.3.2 Mdm2 and control(Ctrl)siRNA are transfected into mouse primary peritoneal macrophages by liposome Interferin,respectively.After 36 h,cells are infected with VSV for 8 h,RNA is extracted and reverse transcribed,and the mRNA expression levels of Cxcl10 is detected by RT-PCR.4.The target of MDM2 in RLR signaling pathwayThe mouse primary peritoneal macrophages are infected with SeV and the MDM2 antibody is used for immunoprecipitation.The endogenous binding of MDM2 with DDX3X and TBK1 are detected by Western blot.The THP-1 cells are infected with SeV and the MDM2 antibody is used for immunoprecipitation.The endogenous binding of MDM2 with DDX3X and TBK1 are detected by Western blot.5.The expression of DDX3X affected by MDM2Different concentrations of MDM2 and DDX3X plasmids are cotransfected into HEK293T cells by Lipo2000.After 24 h,the expression of DDX3X is detected by Western blot.6.The ubiquitination of DDX3X affected by MDM2Mdm2 and Ctrl siRNA are transfected into mouse primary peritoneal macrophages by Interferin,respectively.After 36 h,the cells are infected with SeV,and lysates are subjected to immunoprecipitation with anti-DDX3X followed by Western blot to analyze the ubiquitination of DDX3X.The MDM2,DDX3X and Ub-K63 expression plasmids are cotransfected into HEK293T cells by liposome Lipo2000.After 24 h,the lysates are subjected to immunoprecipitation with the tag antibody of DDX3X followed by Western blot to analyze the ubiquitination of DDX3X.Results1.SeV promotes the expression of MDM2After infected with dsRNA virus SeV,the protein expression level of MDM2 is increased,indicating the participation of MDM2 in the regulation of antiviral innate immune resposnes.2.MDM2 promotes the expression of IFN-βMdm2 knockdown obviously inhibited SeV-induced Ifnb mRNA level in mouse primary peritoneal macrophages.Meanwhile,luciferase reporter gene assay was analyzed the activation level of IFN-β in HEK293T cells.As expected,MDM2 overexpression significantly promoted SeV-and RIG-Ⅰ-induced IFN-β gene promoter activation.In summary,these data indicate that MDM2 increases RNA virus mediated innate immune responses and IFN-β expression.3.MDM2 promotes IRF3 activation and antiviral abilityMdm2 knockdown greatly inhibited the SeV infection induced phosphorylation of IRF3 in mouse primary peritoneal macrophages.In addition,MDM2 overexpression greatly increased the SeV infection induced the activation of IRF3 luciferase reporter in HEK293T cells.Furthermore,Mdm2 knockdown significantly attenuated the phosphorylation level of IFN-β downstream signaling pathway--STAT1 after SeV infection.The expression of downstream ISG--Cxcl10 was dramatically decreased in Mdm2 knockdown mouse primary peritoneal macrophages after VSV infection.In a word,these data reveal that MDM2 enhances RNA virus mediated IRF3 activation and antiviral ability through downstream IFN-β-mediated antiviral signaling pathway.4.MDM2 interacts with DDX3XTo detect the molecular mechanism of MDM2 underlying the regulation on IFN-β expression,we next measured the potential target of MDM2.Immunoprecipitation revealed that both in mouse primary peritoneal macrophages and human THP-1 cells,MDM2 was interacted with DDX3X,but not TBK1,with or without SeV infection.These results suggested DDX3X as a potential substrate of MDM2.5.MDM2 does not affect the expression of DDX3XWe next detected whether the expression level of DDX3X could be affected by MDM2.However,Western blot displayed that there is no effect on DDX3X expression under the present of MDM2,indicating that the positive regulation of RLR signaling pathway by MDM2 does not work through the expression of DDX3X.6.MDM2 enhances K63-linked ubiquitination of DDX3XWe further tested the ubiquitin modification of DDX3X promoted by MDM2.After SeV infection,the ubiquitin modification of DDX3X was dramatically enhanced,while Mdm2 knockdown aborted this modification in mouse primary peritoneal macrophages.Besides,MDM2 promoted the K63-linked polyubiquitination of DDX3X in HEK293T cells.In summary,all the results demonstrated that MDM2 selectively interacts to DDX3X,and promotes its K63-linked ubiquitin modification,which is irreplaceable for the activation of DDX3X in RLR pathway.ConclusionE3 ubiquitin ligase MDM2 promotes the RLR-mediated the activation of IRF3 and the production of IFN-β by specifically binding with DDX3X and subsequently catalyzing the K63-linked ubiquitin modification of DDX3X.Innovation and significance1.In this study,we discover that MDM2 specifically promotes RLR-mediated IFN-β production through catalyzing the K63-linked ubiquitination of DDX3X.Our study reveals a new regulatory role of MDM2 in antiviral innate immune responses and supplement our understanding of antivrial innate immune regulation.2.In this study,we identify that DDX3X activity controlled by MDM2 may be potential targets for the prevention and treatment of viral diseases. |
