TRAF-interacting Protein (TRIP) Negatively Regulates IFN-βproduction And Antiviral Response By Promoting Proteasomal Degradation Of TANK-binding Kinase1

Host cells express various pattern recognition receptors (PRRs), including Toll-like receptors (TLRs) and retinoic acid-inducible gene I (RIG-I)-like helicases(RLRs), which sense invading pathogens. Once recognition of microbial components, such as LPS, polyinosinic:polycytidylic acid (poly(I:C)), and viral RNA, TLRs. and RLRs, are activated, they initiate a series of signaling events leading to production of type I IFN (IFN-β) and proinfl ammatory cytokines. Although full activation of TLR and RIG-I signaling and secretion of type I IFNs are important for the elimination of invading microorganisms, inappropriate production of IFN-β and proinflammatory cytokines might promote the development of immunopathological conditions.TBK1is essential for both TLR and RLR signaling and is required for activation of IRF3and subsequent induction of IFN-β. The active state of TBK1is tightly regulated by posttranslational modifications such as phosphorylation and ubiquitination. GSK-3β promoted TBK1dimerization and autophos phorylation in virus-triggered type I IFN induction and cellular antiviral response. Nrdp1mediates K63-linked polyubiquitination and promotes TBK1kinase activity. The mindbomb E3ligases (MIB1and MIB2) have been proposed to activate TBK1by catalyzing its K63-linked polyubiquitylation. During the revision of the manuscript, Cui et al.(2012) reported that NLRP4could recruit the E3ubiquitin ligase DTX4to TBK1for Lys48(K48)-linked polyubiquitination, which led to degradation of TBK1. However, how TBK1activity is negatively regulated remains largely unknown.TRAF-interacting protein (TRIP, or TRAIP) was initially identified as a TRAF1-and2-interacting protein that functions to inhibit NF-k B activation. TRIP contains an N-terminal RING finger and coiled-coil and leucine zipper regions that bind TRAF-family proteins and a C-terminal region reported to interact with CYLD that facilitate the inhibition of TNF-mediated NF-kB activation. TRIP interacts with the protein tyrosine kinase Syk and sensitizes to TNF-induced apoptosis. TRIP plays important roles in the regulation of cell cycle progression and keratinocyte proliferation (Almeida et al.,2011). In addition, TRIP-deficient mouse embryos died shortly after implantation as a result of proliferation defects and excessive cell death (Park et al.,2007). TRIP possesses RING-dependent Ub ligase activity, but its substrate and physiological functions remain largely unclear. In the present study, we identifi ed TRIP as a new regulator in TLR3/4-and RIG-I-mediated IFN-β signaling and antiviral responses by mediating ubiquitination and degradation of TBK1through the N-terminal RING domain. The identification of a physiological suppressor of TBK1provides an explanation on the inability of the kinase to activate the IFN pathways under physiological conditions.Method and Results1.TRIP expression is induced by TLR stimulation in macrophagesTo determine the potential role of TRIP in TLR signaling, we analyzed its expression upon stimulation with TLR4agonists. TRIP expression was up-regulated after4h of stimulation and reached the peak level of expression at8h. After24h, FRIP expression declined to the basal level as in untreated cells. Collectively, these data indicate that TRIP was up-regulated after TLR activation and Sev infection in macrophages, raising a possibility that TRIP may be involved in this signaling transduction. To investigate the subcellular localization of TRIP, GFP-TRIP was transfected into HeLa cells, followed by immunofluorescence staining and microscopic analysis. We found that TRIP was presented by small speckled structures diff used throughout the cell and that its expression pattern remains unchanged after TLR4activation (unpublished data). Collectively, these data indicate that TRIP diff used in the whole cytoplasm and nucleus.2.TRIP negatively regulates TLR3/4-and RIG-I-induced IFN-β productionTo investigate whether TRIP plays a role in TLR signaling, we initially examined the effects of TRIP on LPS and poly(I:C) induced IFN-β production in macrophages. TRIP expression was significantly decreased with transfection of TRIP specific small interference RNAs. TRIP knockdown substantially increased LPS and poly(I:C) induced IFN-β production in mouse peritoneal macrophages at both mRNA and protein levels. TRIP overexpression significantly decreased LPS and poly(I:C) induced IFN-β reporter activation in mouse peritoneal macrophages.Consistently, TRIP overexpression also significantly decreased poly(I:C) induced IFN-β reporter activation in HEK293-TLR3cells. To address the physiological function of TRIP in RIG-I induced IFN-β production, HEK293cells were transfected with poly(I:C) to activate RIG-I signaling. RIG-I-induced up-regulation of IFN-β and TNF was attenuated in TRIP-overexpression HEK293cells. To investigate whether TRIP specifically inhibited TLR and RLR pathways, we observed the effect of TRIP on IFN-β induced activation of GAS and iNOS promoter reporter in RAW264.7cells. TRIP could not inhibit IFN-β induced activation of GAS reporter and iNOS reporter. As a parallelcontrol, TRIP overexpression was significantly decreased LPS-and poly(I:C)-induced IFN-β reporter activation in RAW264.7cells. Collectively, these data indicate that TRIP specifically repress TLR3/4-and RIG-I-induced IFN-β signaling.3.TRIP inhibits IRF3activation1RF3is the key transcription factor that mediates the expression of IFN-β in TLR3/4-and RIG-I-mediated signal transduction. We then observed the effect of TRIP on IRF3activation. TRIP significantly inhibited LPS-and poly(I:C)-induced IRF3reporter activation in mouse peritoneal macrophages. To address the function of TRIP in RIG-I-induced IRF3activation, HEK293cells were transfected with poly(I:C) to activated RIG-I signaling. TRIP significantly attenuated RIG-I-induced IRF3reporter activation. TRIP also significantly inhibited RIG-I-, MAVS-, and TBK1-dependent IRF3activation. Furthermore, TRIP over-expression inhibited RIG-I induced phosphorylation of IRF3in HEK293cells. To investigate the physiological function of TRIP in IRF3activation, mouse peritoneal macrophages were transfected with TRIP siRNA. TRIP knockdown substantially enhanced poly(I:C) induced phosphorylation of IRF3. All together, these data suggest that TRIP negatively regulates TLR-and RIG-I-induced IRF3activation.4.TRIP targets TBK1for degradationTo determine the molecular targets of TRIP in TLR-and RIG-I induced IFN-β signaling, the effects of TRIP on the IFN-β expression mediated by various adaptors were examined. TRIP inhibited RIG-I-,MAVS-, TBK1-, TRIF-, and MDA5-induced IFN-β mRNA expression. Consistently, TRIF-, RIG-I-,MAVS-and TBK1-induced IFN-β promoter activation were also significantly inhibited by TRIP over-expression. IRF3-induced IFN-β promoter activation remains unchanged by TRIP over-expression. Therefore, we conclude that TRIP targets molecules upstream of IRF3.To clarify TRIP target, the function of TRIP on the degradation of the molecules involved in TLR3/4and RIG-I signaling was investigated. TRIP knockdown greatly increased TBK1protein level in peritoneal macrophages. IRF3, STAT1, and TRAF3protein levels were not impaired. Consistently, TBK1protein level in TRIP stable transfection RAW264.7cells was greatly decreased. TRAF3and TRAF6protein levels were not changed. To confirm, TRIP targets TBK1, HA-TBK1, and Flag-TRIP were co-transfected into HEK293cells. TRIP promoted degradation of TBK.1. As a control, TRIP over-expression had no effects on TRAF3expression. To determine whether TRIP inhibits TBK1activity, in vitro kinase assay of TBK1was assessed with myelin basic protein (MBP) as substrate. As expected, LPS stimulation could increase TBK1kinase activity; remarkably, TBK1kinase activity was further increased in TRIP siRNA-transfected macrophages after LPS stimulation. All together, these data indicate that TRIP targets TBK1for degradation to inhibit TLR-and RIG-I -induced expression of IFN-β.5.TRIP promotes proteasomal degradation and K48-linked ubiquitination of TBK1Protein ubiquitination is a crucial posttranscriptional modification to provide specificity and regulate the intensity of TLR signaling. K48-linked protein ubiquitination leads to the degradation of the corresponding protein by26S proteasome.. TRIP is a RING-dependent E3ubiquitin ligase, Therefore, we speculated that TRIP promoted TBK1degradation through the ubiquitin-proteasome pathway. First, we investigated whether TRIP interacted with TBK1. Myc-TBK1, Myc-IRF3, or Myc-TRAF3and Flag-TRIP were cotransfected into HEK293cells, and immunoprecipitation experiments were performed with Flag antibody. TBK1was coprecipitated with TRIP. Endogenous interaction was examined in mouse peritoneal macrophages stimulated with LPS for indicated time periods, followed by immunoprecipitation with TBK1antibody. TBK1interacted with TRIP in both LPS-stimulated and nonstimulated cells. The interaction could not be detected with control IgG. Collectively, these results show that TRIP binds directly to TBK1.To test the role of TRIP in TBK1ubiquitination and degradation, we constructed various TRIP mutants. The TRIP CA lost the ability to promote polyubiquitination of TBK1, indicating that the RING-finger domain is required for the TRIP-mediated ubiquitination of TBK1. To investigate the forms of TRIP-mediated polyubiquitination of TBK1, ubiquitin mutant vectors K48and K63, which contain arginine substitutions of all of its lysine residues except the one at position48and63, respectively, were used in the transiection assays. TRIP-mediated polyubiquitination of TBK1was significantly increased in the presence of both WT and K48mutant ubiquitin plasmids, but not in the presence of K63mutant ubiquitin plasmid, indicating that TRIP mediates K48-linked ubiquitination of TBK1. K48-linked polyubiquitination leads to the degradation of target proteins through26S proteasome. Consistently, TRIP-induced degradation of TBK1could be reversed by proteasome inhibitor MG-132. Furthermore, TRIP mutant C51lost the ability to promote degradation of TBK1. 6.TRIP negatively regulates virus-mediated signalingType I IFNs play critical roles in the immune responses against viral infection. We further investigated the potential role of TRIP in cellular antiviral responses. TRIP inhibited IFN-β and RNATES expression in HEK293cells infected with Sev. Consistently, TRIP inhibited Sev-induced IFN-β and IRF3promoter activation and TRIP CA mutant lost the inhibitory effects. Importantly, Sev-induced IRF3dimerization was significantly attenuated by transfection of TRIP expression plasmid in HEK293cells. Furthermore, Sev-induced IFN-β and RNATES expression was significantly enhanced by TRIP knockdown in peritoneal macrophages.TBK1protein level was greatly decreased in Sev-infected HEK293cells with TRIP overexpression. Consistent with the observation of TRIP-mediated decrease of TBK1expression, TRIP knockdown greatly increased TBK1kinase activity after Sev infection.7.TRIP enhanced the VSV replication abilityTo directly investigate the effects of TRIP on antiviral responses, VSV, a kind of single-stranded RNA virus recognized by RIG-I, was used to infect HEK293cells and macrophages. Plaque assay of HEK293cells infected with VSV showed that TRIP overexpression substantially increased viral replication in the presence of poly(I:C). In sharp contrast, TRIP mutant CA could not increase viral replication. Similarly, VSV RNA replicates were greatly increased in TRIP-transfected cells compared with control vector-or TRIP CA-transfected cells. To further confirm the function of TRIP on VSV replication under physiological condition, TRIP expression was silenced by TRIP siRNA in mouse peritoneal macrophages, and then the macrophages were infected with VSV. TRIP knockdown greatly decreased VSV viral replication in macrophages in the presence or absence of poly(I:C). Accordingly, TRIP knockdown significantly decreased intracellular VSV RNA replicates.