TRIM26 Negatively Regulates Interferon-β Production And Antiviral Response Through Polyubiquitination And Degradation Of Nuclear IRF3

Innate immunity is essential for the host to protect from infection of invading pathogens. Activation of the innate immune response depends on the detection and recognition of pathogen-associated molecular patterns (PAMPs) by germline DNA-encoded pattern-recognition receptors (PRRs). The well studied PRRs include Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), NOD-like receptors (NLRs) and intracellular DNA sensors.Production of type I inteferons by the innate immune cells is pivotal for the cellular antiviral immune responses. After virus infection, IFN-β transcription requires IRF3, which was activated through phosphorylation, dimerization and nuclear translocation. IRF3 activation and IFN-β production must be terminated at the appropriate time points after viral infection.Several mechanisms including dephosphorylation and polyubiquitination have been demonstrated to terminate IRF3 activation.Phosphorylated IRF3 was found to be dephosphorylated by phosphatase PP2A recruited by RACK1 adaptor protein. Phosphorylation in the C-terminal phosphor-acceptor has been reported to facilitate IRF3 proteasomal degradation after infection with SeV. But, the identity of the E3 ligase responsible for nuclear IRF3 ubiquitination and degradation was not defined.TRIM26 is a member of the tripartite motif (TRIM) protein family composed of more than 70 members in human. TRIM proteins share a similar characteristic structure, which includes a RING (R) domain, one or two B-boxes (B), and a coiled coil (CC) domain in the N-terminal and a domain in the C-terminal with variable structures. Here, we identified a novel function for TRIM26 as an E3 ubiquitin ligase for nuclear IRF3. TRIM26 was found to bind to and induce IRF3 polyubiquitination in nucleus after virus infection. TRIM26 promoted the degradation of WT IRF3 and the phosphorylation active mutant IRF3 5D, but not the phosphorylation deficient mutant IRF3 5A. Nuclear translocation of IRF3 and TRIM26 was required for IRF3 degradation. TRIM26 transgenic mice had decreased IRF3 activation, IFN-P production and antiviral immune response. Our findings demonstrated that TRIM26 is essential for the termination of nuclear IRF3 activation.Method and Results1.Virus infection induces TRIM26 expression and nuclear translocationTRIM26 is located in the MHC class I region, Western blot analysis showed that TRIM26 protein is strongly expressed in various organs including lung, thymus, liver, spleen, small intestine and brain, but not in heart and kidney. TRIM26 expression was induced upon LPS and poly(I:C) stimulation and Sendai virus (SeV) infection.To investigate the cellular localization of TRIM26, GFP-TRIM26 was constructed and transfected into HEK293 cells. TRIM26 showed diffused expression in the cytoplasm without stimulation. While, TRIM26 moved into nucleus after infection with SeV and VSV (MOI,0.1).Similarly, LPS induced TRIM26 translocation into nucleus in HEK293 cells stably expressing TLR4. All together, these data suggested that TRIM26 expression is induced upon viral infection, which could also induce the nuclear translocation of TRIM26.2.TRIM26 negatively regulates IFN-β production and antiviral responseTo explore the functions of TRIM26 in antiviral immune responses, the effect of TRIM26 on the activation of IFN-β expression downstream of various PRRs was investigated using IFN-β promoter reporter.LPS- and poly(I:C)-induced IFN-β promoter activation was attenuated in RAW264.7 macrophages transfected with TRIM26 expression plasmid, compared to that transfected with control vector. Similarly, in HEK293 cells stably expressing TLR4 and TLR3.Transfection of TRIM26 expression plasmid decreased SeV-, poly(I:C)-transfection-, ISD-and poly(dA:dT)-induced IFN-P promoter activation in HEK293 cells.These reporter assays strongly suggested that TRIM26 acts on molecules that are shared by various nucleic acid-induced signaling pathways in the innate immune system to negatively regulate IFN-P expression.To directly investigate the inhibitory roles of TRIM26 in IFN-P production, two TRIM26 specific siRNAs were designed and transfected into peritoneal primary macrophages. Transfection of TRIM26 siRNAs enhanced LPS- and poly(I:C)-induced IFN-β production Similarly, SeV- and ISD-induced IFN-β production was also increased in TRIM26 siRNA-transfected primary peritoneal macrophages. These results further confirmed the above reporter data and demonstrated that TRIM26 negatively regulates IFN-P production downstream of various PRRs.To directly investigate the effect of TRIM26 on antiviral responses, vesicular stomatitis virus (VSV) was used. Transfection of TRIM26 expression plasmid into Hela cells attenuated VSV-induced IFN-β expression, while VSV RNA replicates in the cells were increased in TRIM26-transfected cells. Accordingly, plaque assays showed that overexpression of TRIM26 substantially increased viral replication compared to control vector-transfected cells. Similarly, with the transfection of TRIM26 siRNA into primary peritoneal macrophages.Taken together, these data demonstrated that TRIM26 negatively regulates IFN-P production and antiviral immune responses.3.TRIM26 targets IRF3IRF3 is the main transcription factor involved in IFN-β production downstream of TLR3/4, RLRs and intracellular DNA sensors. To investigate the effect of TRIM26 on IRF3 activation, several set of experiments were performed. Transfection with TRIM26 expression plasmid attenuated LPS-,poly(I:C)-, SeV-and ISD-induced activation of IRF3 reporter. Overexpression of TRIM26 substantially decreased TRIF-, MAVS-, STING+cGAS- and TBK1-induced IRF3 phosphorylation. In contrast, knockdown of endogenous TRIM26 expression by siRNA increased LPS-and SeV-induced IRF3 phosphorylation.To determine the molecular targets of TRIM26, the effect of TRIM26 overexpression on the activation of IFN-β promoter mediated by various molecules was examined in reporter assays. TRIF-, RIG-I-, MAVS-, TBK1- and IRF3-induced activation of IRF3 reporter was also inhibited by TRIM26 overexpression. These data indicated that TRIM26 may target IRF3 directly to regulate IFN-β production.4.TRIM26 promotes IRF3 ubiquitination and proteasomal degradationThe ability of TRIM26 to induce IRF3 polyubiquitination and degradation was investigated.Co-Immunoprecipitation (Co-IP) showed that endogenous TRIM26 formed a complex with IRF3 upon LPS stimulation and SeV infection. Co-transfection of Flag-TRIM26 and Myc-IRF3 demonstrated that Flag-TRIM26 interacted with WT IRF3 and the constitutive active mutant IRF3 5D, but not with the non-active IRF3 mutant 5A.To directly test TRIM26-mediated IRF3 ubiquitination, Myc-IRF3 was cotransfected with Flag-TRIM26 into HEK293 cells. Co-IP showed that the level of IRF3 ubiquitination was markedly increased in the presence of TRIM26 expression plasmid after treatment with MG-132.Notably, two TRIM26 mutants in the RING-finger domain (C16A and C16/36A) lost the ability to promote IRF3 polyubiquitination. in vitro pull-down and ubiquitination assays demonstrated that TRIM26 could directly interact with IRF3 and promote IRF3 ubiquitination.To further confirm TRIM26-induced IRF3 ubiquitination, primary peritoneal macrophages were transfected with TRIM26 siRNA.knockdown TRIM26 expression with siRNA substantially attenuated SeV- and LPS-induced IRF3 ubiquitination.To investigate the form of polyubiquitin chains in IRF3 induced by TRIM26, WT HA-Ubiquitin and its mutants K48 and K63 were used.We found that TRIM26 mainly conjugates K48-linked polyubiquitin chains to IRF3.Consistent with these observations, TRIM26-induced degradation of IRF3 was reversed by proteasome inhibitor MG-132, but not by lysosome inhibitor Chloroquine.Importantly, TRIM26 mutant C16A could not promote the degradation of IRF3 compared to WT TRIM26. To investigate whether TRIM26 promotes IRF3 ubiquitination through K70 and K87, IRF3 mutant K70/87A was transfected into HEK293 cells together with Flag-TRIM26.Compared to WT IRF3, TRIM26-induced IRF3 ubiquitination was decreased in IRF3 mutant K70/87A.All together, these data demonstrated that TRIM26 interacts with and promotes K48-linked polyubiqutination of IRF3 at K70/87, leading to IRF3 proteasomal degradation.5.TRIM26 promotes IRF3 degradation in nucleusTo investigate the location of TRIM26-mediated IRF3 ubiquitination and degradation, cytoplasmic and nuclear fractions were prepared from RAW264.7 macrophages after SeV infection.Co-IP and IRF3 ubiquitination showed that TRIM26 mainly interacts with and promotes ubiquitination of IRF3 in the nucleus.To investigate whether TRIM26 targets active IRF3 in nucleus, IRF3 WT and mutants 5D and 5A were transfected into HEK293 cells together with Flag-TRIM26.TRIM26 was found to degrade WT IRF3 and IRF35D in a dose dependent manner, but not IRF35A even under the condition of infection with SeV and VSV.To investigate where IRF3 were degraded by TRIM26, cytoplasmic and nuclear fractions were prepared from IRF3 and TRIM26 or control vector transfected cells after infection with SeV or left uninfected. Overexpression of TRIM26 promoted the degradation of nuclear IRF3, but not the cytoplasmic IRF3. Similar to WT IRF3, Overexpression of TRIM26 also promoted the degradation of IRF35D in nucleus, but not in cytoplasm. IRF35A was not degraded in both cytoplasm and nucleus.These data suggested that TRIM26 mainly promotes the degradation of active form IRF3 in nucleus.6.Nuclear translocation promotes TRIM26-mediated IRF3 degradationTo investigate TRIM26-mediated IRF3 degradation is really a nuclear event, IRF3 and IRF3 mutant KR77/78NG were transfected into HEK293 cells together with TRIM26 expression plasmid. IRF3 WT and 5DKR77/78NG mutant could not be degraded by TRIM26.Consistent with the inability to degrade IRF3 KR77/78NG, Flag-TRIM26 was not interacted with IRF3 KR77/78NG, and 5D KR77/78NG.To further confirm IRF3 was degraded in the nucleus, IRF3 NES mutant IL139/140MM was used.WB analysis of cytoplasmic and nuclear fractions showed that IRF3 WT nad 5D IL139/140MM, but not the 5A IL139/140MM was efficiently degraded by TRIM26 in nucleus, but not in the cytoplasm.These data demonstrated that IRF3 nuclear translocation is required for TRIM26-induced degradation.To investigate whether this NLS is responsible for TRIM26 nuclear translocation, the conserved amino acids (RKK and KKK) were mutated to arginine to give rise TRIM26 NLS mutant TRIM26-M-NLS. Fluorescent microscopy showed that TRIM26-M-NLS lost the ability to translocate into the nucleus after SeV and VSV infection. GFP-TRIM26-M-NLS lost the ability to degrade both WT IRF3 and IRF3 5D, indicating TRIM26 nuclear localization is required for IRF3 degradation.To directly confirm nuclear translocaion of IRF3 and TRIM26 is required for IRF3 degradation, protein nuclear importer inhibitor Ivermectin was used. Inhibition of IRF3 nuclear translocation by Ivermectin abolished TRIM26-induced IRF3 degradation in SeV-infected cells and uninfected cells. Taken together, these results suggested that TRIM26 mediates ubiquitination and degradation of active IRF3 in the nucleus.7.Impaired IFN-β signaling and antiviral responses in TRIM26 transgenic miceTo investigate the physiological function of TRIM26, TRIM26-transgenic mice (TRIM26-Tg mice) was established.Consistent with the data of TRIM26-mediated IRF3 degradation and ubiquitination,SeV-induced IRF3 phsophorylation was reduced and more IRF3 ubiquitination was detected in macrophages from TRIM26-Tg mice.Primary peritoneal macrophages from TRIM26-Tg and WT mice were prepared and stimulated with LPS and poly(I:C) or infected with SeV and VSV, the expression of IFN-β mRNA and secretion of IFN-β protein was reduced in TRIM26-Tg mice, compared to the macrophages from WT mice.VSV replication in macrophages from TRIM26-Tg mice was greatly increased compared to that from WT mice.To test the importance of TRIM26 in vivo, TRIM26-Tg mice and control WT littermates were infected with VSV, and the antiviral immune responses were examined.The amount of IFN-β protein induced by VSV infection was much less in sera, lung and liver of TRIM26-Tg mice, VSV replication in the lungs and livers of TRIM26-Tg mice was higher than WT controls. HE staining showed that severe infiltration of immune cells and injury were observed in the lungs of TRIM26-Tg mice. Moreover, TRIM26-Tg mice were more susceptible to VSV infection than WT mice. These data suggested that TRIM26 is an important negative regulator of IFN-β production and antiviral immune responses, therefore TRIM26-transgenic mice have impaired antiviral response.