| Type I IFNs (IFN-a/(3) possess strong anti-viral activity and play pivotal roles in defense against viral infection. During viral infection, pattern-recognition receptors (PRRs), including TLRs and retinoic acid-inducible gene-Ⅰ (RIG-I)-like helicases (RLRs), activate immune cells to produce type IIFN and proinflammatory cytokines, which are involved in the elimination of virus.Protein ubiquitination plays an essential role in the regulation of RIG-I activation and antiviral immune response. However, the function of the opposite process deubiquitination in RIG-Ⅰ activation remains elusive. In this study, we have identified the deubiquitinating enzyme Ubiquitin-specific protease4(USP4) as a new regulator for RIG-I activation through deubiquitination and stabilization of RIG-Ⅰ. USP4expression was attenuated after virus-induced RIG-Ⅰ activation. Overexpression of USP4significantly enhanced RIG-Ⅰ protein expression and RIG-Ⅰ-triggered IFN-β signaling, at the same time, inhibited VSV replication. siRNA knockdown of USP4expression has an opposite effect. Furthermore, USP4was found to interact with RIG-Ⅰ and remove K48-linked polyubiquitinattion chains from RIG-Ⅰ.Therefore, our study identified USP4as a new positive regulator for RIG-Ⅰ through deubiquitinating K48-linked ubiquitin chains and stabilizing RIG-Ⅰ. Objectives:1. To investigate the function of USP4in RIG-I signaling;2. To determine the molecular mechanisms and targets of USP4;3. To investigate the effect of USP4on antiviral response.Methods:1. Identification differentially expressed USP4upon RIG-I activation in different cell lines.1.1Identification differentially expressed USP4upon RIG-I activation in Hela cells.a. I RT-PCR analysis of USP4expression in Mela cells transfected with poly(I:C) for0,2,4,8,12h (mRNA level);II Western blot analysis of USP4expression in Mela cells transfected with poly(I:C) for0,12,24,36h (protein level).b. I RT-PCR analysis of USP4expression in Hela cells infected with SeV (Sendai virus) for0,8,12,24h (mRNA level);II Western blot analysis of USP4expression in Mela cells infected with SeV for0,8,12,24,36h (protein level).1.2Identification differentially expressed USP4upon RIG-I activation in mouse peritoneal macrophages.After bone marrow derived macrophages (BMDM) were infected with SeV for0,4,8,12,24h, RT-PCR analysis of USP4expression (mRNA level); infected with SeV for0,12,24,36h. Western blot analysis of USP4expression (protein level).2. Confirmation of whether the RIG-I-induced IFN-β production was regulated by USP4.2.1RT-PCR analysis whether the RIG-I-induced IFN-β production was regulated by USP4on mRNA level.a. HEK293cells transfected with the indicated plasmids were transfected with poly(I:C) or poly(dA:dT) for12h. Total RNA was prepared and analyzed for the expressions of IFN-β, USP4and GAPDH by RT-PCR.b.ⅠHEK293or Hela cells were transfected with control siRNA or USP4siRNA for36h, and then transfected with poly(I:C) or for0,4h,8h, Total RNA was prepared and analyzed for the expressions of IFN-β and GAPDH by RT-PCR.Ⅱ HEK293or Hela cells were transfected with control siRNA or USP4siRNA for36h, and then transfected with poly(dA:dT) or for0,16h, Total RNA was prepared and analyzed for the expressions of IFN-β and GAPDH by RT-PCR.Ⅲ Hela cells were transfected with control siRNA or USP4siRNA for36h, and then infected with SeV for12h. Total RNA was prepared and analyzed for the expressions of IFN-β and GAPDH by RT-PCR.2.2Luciferase assay analysis whether the RIG-I-induced IFN-P promoter and IRF3activation was regulated by USP4.a. Hela cells were transiently transfected with IFN-β or IRF3reporter plasmid together with USP4expression plasmid or control plasmid, analyzed luciferase activity after SeV infection for12h.b. Hela cells were transfected with control siRNA or USP4siRNA, and then transfected with IFN-P or IRF3reporter plasmid, analyzed luciferase activity after SeV infection for12h.3、To determine the molecular targets of USP4.3.1RT-PCR analysis the IFN-β production induced by RIG-I or adaptors in RIG-I pathway on mRNA level modulated upon differentially expression of USP4.a. HEK293cells were transfected with RIG-I, MAVS, TBK1, IRF35D, TRIF, IKK-e, MDA5along with USP4plasmid, and24h later IFN-P mRNA were analyzed by RT-PCR.b. HEK293cells were transfected with RIG-I, MAVS, TBK1, IRF35D, MDA5along with USP4specific siRNA or control siRNA, and IFN-P mRNA were analyzed by RT-PCR. c. Huh7.5cells were transfected with RIG-I, MAVS, IRF35D along with USP4plasmid, and24h later IFN-β mRNA were analyzed by RT-PCR.3.2Luciferase assay analysis the RIG-I or other adaptors in RIG-I pathway induced IFN-β promoter and IRF3activation was regulated upon overexpression of USP4.HEK293cells were transfected with TRIF, RIG-I, MAVS, TBK1, IRF35D along with IFN-β or IRF3reporter plasmid and USP4plasmid, and24h later luciferase activity was analyzed.3.3Western blot analysis of expression of adaptors in RIG-I pathway upon differentially expression of USP4.a. Western blot analysis of the lysates from HEK293cells transfected with Myc-RIG-I, MAVS, STING, TBK1, IRF3, IKK-ε, MDA5together with increasing concentration of Flag-USP4expression plasmid.b. Western blot analysis of the expression of RIG-I, MAVS, and TBK1in Hela cells transfected with control siRNA or USP4siRNA and36h later infected with SeV for indicated time periods.4. To clarify the molecular mechanisms of USP4.4.1To investigate the interaction between USP4and the target molecules by immunopreicpitation.a. Lysates from HEK293cells transiently cotransfected with Flag-tag target molecules and Myc-USP4expression plasmids were subjected to immunoprecipitation with anti-Myc or anti-Flag antibody followed by western blot analysis with anti-Flag or anti-Myc antibody.b. Hela cells transfected with poly(I:C) for0,8,12h were subjected to immunoprecipitation with anti-USP4or control IgG followed by western blot analysis with specific antibody. Proteins in whole-cell lysate were used as positive control (Input).c. Lysates from HEK293cells transiently cotransfected with Flag-tagged target molecule and HA-tagged USP4mutants were subjected to immunoprecipitation with anti-Flag antibody followed by western blot analysis with anti-HA antibody.4.2To test the role of USP4in ubiquitination of the target molecular.a. Lysates from HEK293cells transiently cotransfected with Flag-target molecule, Myc-USP4and HA-Ub plasmids were subjected to immunoprecipitation with anti-Flag antibody followed by western blot analysis with anti-HA antibody.b. Lysates from HEK293cells transiently cotransfected with Flag-target molecule, Myc-USP4or vector control and HA-Ub (WT), HA-Ub (K48) or HA-Ub (K63) plasmids were subjected to immunoprecipitation with anti-Flag antibody followed by western blot analysis with anti-HA antibody.c. Lysates from HEK293cells transfected with control siRNA or USP4siRNA and Flag-target molecule and HA-Ub (WT), HA-Ub (K48) or HA-Ub (K63) plasmids were subjected to immunoprecipitation with anti-Flag antibody followed by western blot analysis with anti-HA antibody.d. Lysates from Hela cells transfected with control siRNA or USP4siRNA and infected with SeV for4h were subjected to immunoprecipitation with specific antibody followed by western blot analysis with anti-Ub or anti-Ub(K48) antibody.5. To investigate the effect of USP4on antiviral response.a. HEK293cells (2×105) transfected with the indicated plasmids (1μ g each).24h later, cells were further transfected with poly(I:C)(0.1μ g) or left untreated.18h after poly(I:C) transfection, cells were infected with VSV (MO1,0.1), and the supernatants were harvested at12h post-infection. Supernatants were analyzed for VSV titers with standard plaque assays. Intracellular VSV RNA replicates were measured by QRT-PCR.b. HEK293cells (2×105) were transfected with control siRNA or USP4siRNA and then treated as in a. Results:1. USP4expression is inhibited by RIG-Ⅰ activation.a. Transfection of poly(I:C) or infection of SeV, which has been shown to activate RIG-I signaling substantially attenuated USP4mRNA and protein expression in Hela cells.b. SeV infection could also attenuate USP4expression in murine peritoneal macrophages.2. USP4enhances RIG-Ⅰ-induced IFN-β production.a. In HEK293cells, overexpression of USP4further increased poly(I:C)-induced expression of IFN-β.b. USP4knockdown through siRNA transfection significantly inhibited poly(I:C) or poly(dA:dT)-induced IFN-β expression in HEK293cells. Similarly, poly(I:C) or poly(dA:dT)-induced IFN-β production was also greatly attenuated by USP4knockdown in Hela cells.c. SeV-induced IFN-β production was substantially attenuated after knockdown of USP4expression in Hela cells.d. USP4overexpression significantly enhanced SeV-induced IFN-β promoter and IRF3activation in Hela cells. In contrast, USP4knockdown significantly inhibited SeV-induced IFN-β promoter and IRF3activation.3. USP4targets RIG-I.3.1USP4specifically enhanced RIG-Ⅰ-induced IFN-β mRNA expression.a. In HEK293cells, USP4enhanced RIG-Ⅰ-induced IFN-β mRNA expression, while, MDA5-, MAVS-, TBK1-, IKK-ε-, TRIF-and IRF35D-induced IFN-β mRNA expression was not impaired by USP4overexpression. Similarly, RIG-Ⅰ-induced IFN-β mRNA expression was also attenuated by USP4overexpression in Huh7.5cells.b. In HEK293cells, USP4siRNA transfection substantially decreased RIG-induced IFN-β mRNA expression, but not MDA5-, MAVS-, TBK1-, IKK-ε-, TRIF and IRF35D-induced IFN-β mRNA expression. 3.2USP4specifically enhanced RIG-Ⅰ-induced activation of IFN-β promoter and IRF3reporter.RIG-Ⅰ-induced activation of IFN-β promoter and IRF3reporter were also significantly enhanced by USP4overexpression. But, MAVS-, TBK1, TRIF and IRF3-induced activation of IFN-β promoter and IRF3reporter was not affected by USP4ovexpression.3.3USP4specifically enhanced RIG-Ⅰ expression.a. USP4expression significantly enhanced RIG-Ⅰ expression in a dose dependent manner. In contrast, USP4overexpression had no effects on MDA5, MAVS, STING, TBK1, IKK-ε and IRF3expression.b. Knockdown of USP4expression significantly decreased RIG-Ⅰ protein expression especially. As a control, MAVS and TBK1protein levels were not impaired.4. USP4specifically interacts with RIG-Ⅰ.4.1USP4interacts with RIG-Ⅰ.a. Myc-USP4and Flag-RIG-I were cotransfected into HEK293cells,24h after transfection, immunoprecipitation experiments were performed with Myc or Flag antibodies. Flag-RIG-I was coprecipitated with Myc-USP4using anti-Myc antibody and vice versa.b. Endogenous interaction between RIG-Ⅰ and USP4was examined in Hela cells transfected with poly(I:C) for various times by immunopreicpitation with USP4antibody and western blotting with RIG-Ⅰ antibody. USP4was found constitutively to interact with RIG-Ⅰ. As a control, the interaction could not be detected with normal IgG.4.2USP4interacts with the N-terminal domain of RIG-Ⅰ.Two RIG-Ⅰ truncations (N-terminal domain composed of two CARD motifs and a C-terminal domain) were used in the immunoprecipitation assays. USP4was found to interact with the N-terminal domain of RIG-Ⅰ. Consistently, overexpression of USP4could also increase the protein expression of the RIG-Ⅰ mutant RIG-Ⅰ-N harboring the N-terminal domain. 4.3USP4and RIG-Ⅰ form a complex through the DUSP domain of USP4and the N-terminal domain of RIG-Ⅰ.Coimmunoprecipitation experiments showed that RIG-Ⅰ interacted with WT USP4and the N-terminal DUSP domain, but not the C-terminal USP domain.5. USP4removes K48-linked polyubiquitination conjugate from RIG-I.5.1USP4overexpression remarkably decreased RIG-Ⅰ K48-linked ubiquitination.a. RIG-Ⅰ was co-expressed with HA-ubiquitin and USP4or vector control. Cotransfection of USP4expression plasmid remarkably decreased RIG-Ⅰ ubiquitination.b. Ubiquitin mutant vectors K48and K63, which contain arginine substitutions of all of its lysine residues except the one at position48and63respectively, were used in the transfection assays. Cotransfection of USP4greatly decreased the polyubiqutination of RIG-Ⅰ in the setting of ubiquitin WT and K48transfected cells, but not in the K63-transfected cells. In contrast, neither K48-linked nor K63-linked ubiquitination of MAVS was impaired by USP4overexpression.5.2K-48-linked polyubiqutination of RIG-I was greatly increased after USP4knockdown.a. The level of RIG-Ⅰ polyubiqutination was greatly increased after transfection of USP4siRNA in the setting of WT ubiquitin and ubiquitin mutant K.48transfected cells compared to control siRNA transfected cells, but not in mutant K63transfected cells.b. Transfection of USP4siRNA further increased K48-linked polyubiqutination of RIG-Ⅰ in SeV-transfected Mela cells.6. USP4is involved in cellular antiviral response.Plaque assays of HEK.293cells infected with VSV showed that overexpression of USP4substantially decreased viral replication compared to control vector-transfected cells. And transfection of USP4siRNA greatly increased VSV viral replication in HEK.293cells. Conclusion:1、USP4expression is inhibited by RIG-I activation.2、USP4enhances RIG-I-induced IFN-β production.3、USP4positively regulates RIG-I pathway by interacting with RIG-I and removing K48-linked polyubiquitinattion chains from RIG-I.4、USP4is involved in cellular antiviral response.Innovation and significances:1. We identified USP4as a new positive regulator for RIG-I through deubiquitinating K48-linked ubiquitin chains and stabilizing RIG-I.2. Our study provided new experimental evidence for negative regulation anti-virus research. |
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