| Innate immunity provides the first line of defense against viral infection.Activation of innate immunity requires the recognition of pathogen-associated molecular patterns through pattern-recognition receptors.Several types of pattern-recognition receptors have been identified that recognize viral nucleic acid to regulate viral infection.Toll-like receptor TLR3.TLR7-TLR8 and TLR9 detect viral RNA and DNA in the endosome.whereas the RNA helicases RIG-I and MDA-5 sense viral RNA in the cytoplasm.Several sensors that recognize cytosolic DNA,such as cGAS(cyclic GMP-AMP synthase)and II116.have been identified.These pattern-recognition receptors recognize invading viruses and initiate a series of signaling events that lead to the production of type I interferons to elicit a cellular antiviral response.MAVS(also known as IPS1,VISA or Cardif plays a very important role in RLR signaling pathway.A published study has reported that viral infection induces a conformational change in MAVS.which leads to the formation of prion-like functional aggregates that provide a sensitive trigger for antiviral signaling.However,the detailed molecular mechanisms of MAVS aggregation,especially in cells,are not clear.MAVS must be localized to mitochondria to function.We hypothesized that proteins localized to mitochondria could functionas regulators for MAVS.Here we identified a mitochondria-localized protein,the E3 ubiquitin ligase TRIM31,as a positive regulator of MAVS.TRIM31 deficiency attenuated the innate antiviral response toinfection with an RNA virus both in vitro and in vivo.TRIM31 interacted with MAVS and promoted K63-linked polyubiquitination on Lys10,Lys311 and Lys461 of MAVS after viral infection.Notably,TRIM31-mediated K63-linked polyubiquitination facilitated the formation of prion-like MAVS aggregates.Method and Results1,TRIM31 is recruited to mitochondria after infection with SeVTo investigate the possible role of TRIM31 in antiviral signaling,we studied the cellular localization of TRIM31 in HEK293T human embryonic kidney cells and mouse primary peritoneal macrophages.Fluorescence microscopy images showed that ectopically expressed TRIM31 tagged with green fluorescent protein(GFP)in HEK293T cells was distributed in the cytoplasm with very little colocalization with mitochondria before infection with Sendai virus(SeV).Infection with SeV greatly increased the colocalization of TRIM31 with mitochondria.We found,by immunoblot analysis,that TRIM31 was localized tomitochondria in primary peritoneal macrophages after infection with SeV.2,TRIM31 regulates RLR-induced IFN-? signaling.To explore the potential role of TRIM31 in antiviral signaling,we designed a small interfering RNA(siRNA)that targeted mouse Trim31 and transfected it into primary peritoneal macrophages.We found that siRNA-mediated knockdown of Trim31 expression significantly decreased the expression of Ifnbl mRNA(encoding IFN-?)and production of IFN-? protein in macrophages after infection with SeV.Similar to the data obtained with mouse macrophages,we observed that siRNA-mediated knockdown of TRIM31 expression in human THP-1 monocytic cells decreased SeV-induced expression of IFNB1.In contrast,overexpression of TRIM31 in HEK293T cells significantly increased IFNB1 expression after transfection of the TLR3 ligand poly(I:C)(polyinosinic-poly cytidylicacid)or infection with SeV.Similar to the data obtained with HEK293T cells,we observed that overexpression of Flag-tagged TRIM31 in HeLa cells greatly enhanced the expression of IFNB1 mRNA after transfection of poly(I:C)or infection with SeV.To directly investigate the effect of TRIM31 on antiviral responses,we used vesicular stomatitis virus(VSV).We found that use of a Trim31-specific siRNA significantly reduced the expression of Ifnblin primary peritoneal macrophages after infection with VSV.Consistent with that result,we found,by quantitative PCR(qPCR)and plaque assay,that the expression of VSV-specific mRNA and VSV titers were greater in macrophages in which Trim31 was knocked down than in those transfected with control siRNA.Overexpression of TRIM31 in HEK293T cells significantly increased IFNB1 expression after infection with VSV relative to its expression in cells expressingthe control vector.Consistent with those data,the expression of VSV mRNA and VSV titers were decreased after overexpression of TRIM31 in HEK293T cells.We also infected HEK293Tcells that ectopically expressed TRIM31 with VSV expressing GFP(VSV-GFP).Fluorescence microscopy of HEK293T cells showed that overexpression of TRIM31 substantially inhibited viral replication relative to such replication in cells expressing the control vector.Together these data suggested that TRIM31 positively regulated RLR mediated IFN-P signaling.3,TRIM31 deficiency impairs cellular antiviral response.To investigate the physiological role of TRIM31,we generated Trim31-/-mice through TALEN('transcription-activation-like effector nuclease')technology.We prepared peritoneal macrophages from Trim31 and Trim31-/-mice;we then stimulated them with lipopolysaccharide(LPS),poly(I:C)or RNA mimics,or infected them with SeV or herpes simplex virustype 1(HSV-1).We observed that Trim3-/-macrophages had lower Ifnbl expression after infection with SeV or stimulation with RNA mimics(5 '-pppRNA)than that of their Trim31-/-counterparts.The expression of Cc15 and Cxc110 downstream genes in IFN-? signaling,was also lower in Trim31-/-macrophages than in Irim31-/-macrophages.In contrast,Ifnbl expression-induced by LPS,poly(I:C)or HSV-1 was not impaired in Trim31-/-macrophages.IFN-P secretion was also lower in Trim31-/-macrophages after infection with SeV than in their Trim31-/-counter parts.Infection with SeV or treatment with 5'-pppRNA resulted in much lower expression of Ifnbl,Ccl5 and Cxc110 in bone-marrow-derived macrophages(BMDMs)prepared from Trim31-/-mice than in those from Trm31-/-mice,whereas LPS-,poly(I:C)-or HSV-1-induced expression of Ifnbl was not impaired.To further confirm the function of TRIM31,we prepared MEFs from Trim31+/+ and Trim3-/-mice.Ifnbl expression induced by SeV or 5'-pppRNA,but not that induced by LPS,poly(I:C)or HSV-1,was lower in Trim31-/-MEFs than in Trim31+/+ ' MEFs.Trim31-/-macrophages expressed less Ifnbl in response to infection with VSV than did Trim31+/+ macrophages.VSV replication was significantly enhanced in Trim31-/-macrophages,whereas replication of the DNA virus HSV-1 in Trim31-/-macrophages was not affected.Together these data demonstrated that TRIM31 specifically regulated IFN-? signaling and antiviral innate immune responses mediated by RLRs but not those mediated by TLRs or DNA sensors.To assess the relevance of TRIM31 in vivo,we challenged Trim31+/+ and Trim31-/-mice with VSV and found,by qPCR analysis,that theexpression of Ifnbl in the spleen,liver and lungs of Trim31-/-mice was significantly lower than that in those organs from Trim31 +/+mice,after infection with VSV.ELISA also showed less IFN-?in serum from Trim31-/-mice than in serum from Trim31+/+ mice.In contrast,the production of IFN-? after challenge with HSV-1 was barely affected in Trim31-/-mice relative to that in Trim31+/+ mice.Consistent with less production of IFN-?,we found that VSV titers and replication were significantly greater in the spleen,liver and lungs of Trim31-/-mice than in those from Trim31-/-mice.However,we did not observe any difference in the copy number of HSV-1 genomic DNA and viral titer of HSV-1 in the brains of Trim31-/-mice versus those of Trim31-/-mice.Moreover,Trim31-/-mice were less resistant than Trim31-/-' mice to infection with VSV but not to infection with HSV-1.Hematoxylinand-eosin staining showed greater infiltration of immune cells and injury in the lungs of Trim31-/-mice.relative to that in the lungs of Trim31-/-mice,after infection with VSV.These data demonstrated that Trim31-/-mice were more susceptible than wild-typemice to infection with an RNA virus.4,TRIM31 promotes RLR-mediated innate immune signaling.To investigate the effect of TRIM31 on RLR-mediated signaling,we performed several experiments.We transfected a TRIM31-expression plasmid into HEK293T cells and saw enhanced SeV-induced activation of an IFNB1 promoter reporter in a TRIM31-dose-dependent manner.Notably,overexpression of TRIM31 increased SeV-induced activation of a IFNB1 reporter containing only PRD ?-? elements,which represent the binding site for IRF3.SeV-induced phosphorylation of TBK1 and IRF3 was greater in HEK293T cells transfected with the TRIM31-expression plasmid than in those transfected with empty vector.Phosphorylation of TBK1 and IRF3 after infection with SeV was lower in Trim31-/-macrophages than in Trim31+/+macrophages.The dimerization of IRF3 was also lower in macrophages from Trim3l-/-mice infected with SeV than in their Trim31+/+ counterparts.Trim31-/-macrophages had less translocation of IRF3 to the nucleus than did Trim31 macrophages in response to infection with SeV.In contrast,translocation of IRF3 to thenucleus was not affected in Trim31-/-macrophages after infection with HSV-1.Together these data demonstrated that TRIM31 positively regulated RLR-mediated innate antiviral signaling.5,TRIM31 targets MAVS.To identify the molecules regulated by TRIM31,we first assessed the effects of TRIM31 on IFNB1 expression in HEK293T cells mediated by the molecules in the RLR signaling pathway.We knocked down TRIM31 expression and found that the expression of IFNB1 Mrna mediated by RIG-I,MDA-5 or MAVS was attenuated.whereas that mediated by TBK1 or IRF3-5D was not impaired.We overexpressed TRIM31 and found increased expression of IFNB1 mRNA mediated by RIG-1,MDA-5 or MAVS,but not that mediated by IKK?,TBK1 or IRF3-5D.We also observed that TRIM31 substantially increased activation of IFNB1 reporter induced by MDA-5.RIG-I or MAVS.but it had no effect on such activation induced by IKK?,TBK1 or IRF3-5D.siRNA-mediated knockdown of TRIM31 expression in HEK293T cells inhibited activation of IFNB1 reporter and the interferon-stimulated response element(ISRE)reporter mediated by RIG-I or MAVS but not that mediated by TBK1.These data suggested that TRIM31 might specifically target MAVS to regulate the RLR signaling.To confirm that TRIM31 targets MAVS,we investigated their interaction and colocalization.Co-immunoprecipitation experiments in HEK293T cells showed that TRIM31 associated with MAVS but not with RIG-I,TBK1,IRF3 or STING.We performed confocal microscopy and found that TRIM31 colocalized with MAVS.To further verify that TRIM31 associated with MAVS,we expressed Flag-tagged TRIM31 and Myc-tagged MAVS in a in vitro protein-expression system,mixed the recombinant proteins together and monitored them in immunoprecipitation assays.Myc-MAVS co-imunoprecipitated with Flag-TRIM31,which indicated that TRIM31 interacted with MAVS in vitro.Furthermore,in co-immunoprecipitation experiments,we found that endogenous TRIM31 and MAVS formeda complex in macrophages after infection with SeV.To determine which domain of TRIM31 was necessary for interaction with MAVS,we constructed several TRIM31 deletion mutants and assessed them in co-immunoprecipitation experiments.We found that TRIM31-AC-C(in which the C-C motif was deleted)lost the ability to interact with MAVS.MAVS is composed of a CARD(amino acids 1-90),a prolinerich domain(amino acids 91-172)and a transmembrane domain(amino acids 513-540).Deletion of the MAVS proline-rich domain ablated the interaction between MAVS and TRIM31.Together these data demonstrated that TRIM31 physically interacted with MAVS through the C-C motif of TRIM31 and the proline-rich domain of MAVS.6,TRIM31 catalyzes the K63-linked polyubiquitination of MAVS.To investigate whether TRIM31 regulates MAVS signaling through its E3 ligase activity,we assessed TRIM31-mediated ubiquitination of MAVS.TRIM31-mediated polyubiquitination of MAVS was readily detectable in HEK293T cells transfected with plasmids expressing Myc-tagged MAVS and hemagglutinin(HA)-tagged ubiquitin in the presence of a plasmid expressing Flag-tagged TRIM31.TRIM31(C53A,C56A)lost the ability toincrease the polyubiquitination of MAVS.which indicating that the E3 ligase activity of TRIM31 was required for the polyubiquitination of MAVS.To investigate the type of TRIM31-mediated polyubiquitination of MAVS,we used vectors expressing HA-tagged mutants ubiquitin(K48)and ubiquitin(K63),which contain substitution of arginine for alllysine residues except the lysine at position 48 or position 63,respectively.TRIM31 catalyzed polyubiquitination of MAVS in the presence of HA-tagged wild-type ubiquitin(HA-ubiquitin(WT)and HA-ubiquitin(K63)but not in the presence of HA-ubiquitin(K48).Furthermore,we performed in vitro ubiquitination assaysto confirm that TRIM31 promoted K63-linked polyubiquitination of MAVS.TRIM31 was found to catalyze the polyubiquitination of MAVS in the presence of ubiquitin(WT)and ubiquitin(K63)but not in the presence of ubiquitin(K48)in these in vitro ubiquitination assay.We further performed in vitro ubiquitination assays using the mutants ubiquitin(K48R)and ubiquitin(K63R),which contain a single lysine-to-arginine substitution at position 48 or 63,respectively.MAVS was ubiquitinated by TRIM31 in the presence of ubiquitin(WT)and ubiquitin(K48R)but not in the presence of ubiquitin(K63R).To confirm the TRIM31-induced ubiquitination of endogenous MAVS,we measured the polyubiquitination of MAVS in SeV-infected primary macrophages prepared from Trim31-/-and Trim31-/-mice and found that endogenous MAVS was robustly ubiquitinated with both K48-linked and K63-linked chains.However,the total amount of K63-linked ubiquitination of MAVS was much lower in Trim31-/-macrophages than in Trim31-/-macrophages,whereas there was little difference between Trim3-/-macrophages and Trim31-/-macrophages in the amount of K48-linked ubiquitination of MAVS.In a control experiment,we found that SeV-induced polyubiquitination of MAVS was not impaired in Trirn26-/-macrophages,as TRIM26 has been reported to target IRF3.K63-linked polyubiquitination has often been reported to activate signaling via the innate immune system.Consistent with that,TRIM31 expression increased SeV-induced activation of the IFNB1 and ISRE reporters in HEK293T cells,inhibited VSV replication and increased SeV-induced phosphorylation of IRF3.However.TRIM31(C53A.C56A)lost the ability to regulate IFNB1 expression or an antiviral response.MAVS has 14 lysine residues present in different domains.To identify the lysine residues responsible for TRIM31-mediated polyubiquitination,we first generated the mutant MAVS-K0,in which all of the lysine residues in MAVS were replaced with arginine.Then.we reintroduced individual lysine residues into MAVS-KO to generate the single-lysine mutants.Cotransfection and co-immunoprecipitation analyses of HEK293T cells showed that TRIM31 induced polyubiquitination of MAVS(WT)and the mutants MAVS(KIO),MAVS(K311)and MAVS(K461).We further constructed the mutants MAVS(K10R),MAVS(K10R,K311R)and MAVS(K10R,K311R,K461R),in which the lysine residues at positions 10,311 and 461 were replaced with arginine.Co-immunoprecipitation analysis of HEK293T cells showed that TRIM31-induced polyubiquitination of the MAVS mutants was gradually decreased with an increase in the number of lysine-to-arginine substitutions relative to that of cells expressing MAVS(WT).Notably,theK63-linked ubiquitination of MAVS(K10R,K311R,K461R)decreased to a level similar to that of MAVS(KO).Together these data demonstrated that TRIM31 catalyzeds the K63-linked polyubiquitination of MAVS on Lys 10,Lys311 and Lys461.7,TRIM31 promotes the formation of MAVS aggregates.MAVS forms prion-like aggregates that potently propagate downstream signaling after infection with an RNA virus.Infection with SeV induced the formation of MAVS aggregates inperitoneal macrophages,as measured by semi-denaturing detergent agarose gel electrophoresis(SDD-AGE).whereas the amount of SeV-induced aggregation of MAVS was much lower in Trim31-/-macrophages than in Trim31+/+ macrophages.Similarly,the amount of MAVS aggregation induced by infection with SeV was lower in Trim3]-/-MEFs than in Trim31+ MEFs.In contrast,overexpression of TRIM31 in HEK293T cells greatly increased the formation of MAVS aggregates relative to their formation in cells transfected with control vector.To confirm that the decreased amount of MAVS aggregation in Trim3-/-MEFs was due to the deletion of TRIM31,we reintroduced an expression plasmid encoding Flag-tagged mouse TRIM31(mTRIM31)into Trim31-/-MEFs.This reintroduction restored the SeV-induced formation of MAVS aggregates.Notably,reintroduction of mTRIM31(C52A,C55A)in Trim31-/-MEFs did not restore SeV-induced formation of MAVS aggregates.Consistent with the restoration of MAVS polymerization after the reintroduction of mTRIM31 in Trim31-/-MEFs.SeV-induced expression of Ifnbl,Ccl5 and Cxc11O was also restored in these MEFs.whereas expression of mTRIM31(C52A,C55A)in Trim31-/-MEFs had no such effect.To confirm that the aggregation of MAVS was directly regulated by TRIM31-induced polyubiquitination of MAVS,we isolated crude mitochondria(P5)from macrophages that were either infected with SeV or left uninfected.The mitochondria were incubated in an in vitro ubiquitination assay buffer containing ubiquitin,the ubiquitin-activating enzyme El,the ubiquitin-conjugating enzyme UbcH5a and TRIM31.In SDD-AGE experiments,we found no formation of MAVS aggregates in mitochondria before infection with SeV,whereas infection with SeV induced MAVS aggregation.In the presence of El,E2 and TRIM31,SeV-induced aggregation of MAVS was greatly increased.Of note,we found that TRIM31 promoted the polyubiquitination of mitochondrial MAVS with or without infection with SeV,in the in vitro ubiquitinaton system.Furthermore,we used ubiquitin(K63)and ubiquitin(K48)in the in vitro ubiquitination system and demonstrated that TRIM31-mediated K63-linked ubquitination,but not K48-linked ubquitination,was required for the formation of MAVS aggregates following infection with SeV.To directly address the function of TRIM31-induced ubiquitination in the activation and aggregation of MAVS,we studied Mavs-MEFs reconstituted with wild-type MAVS or its ubiquitinationsite mutants MAVS(K10),MAVS(K311).MAVS(K461),MAVS(K10R),MAVS(K10R,K311R)and MAVS(K10R,K311 R,X461 R).Infectionwith SeV did not stimulate the production of IFN-? by Mavs-/-MEFs,whereas transfection of wild-type MAVS-expression plasmids into Mavs-/-MEFs restored the expression of Ifnbl.The overexpression of TRIM31 further increased the SeV induced activation of the IFNBI and ISRE reporters in Mavs-/-MEFs reconstituted with wild-type MAVS.However.SeV-induced activation of the IFNB1 and ISRE reporters in Mavs-/-MEFs reconstituted with the mutant MAVS(K10R),MAVS(K10R,K311R)or MAVS(K10R,K311R,K461R)was gradually decreased with an increase in the number of lysine-to-arginine substitutions in MAVS in the presence of TRIM31.Notably,MAVS(K10R,K311R,K461R)-mediated activation of the IFNB1 and ISRE reporters in the presence of TRIM31 decreased to a level similar to that seen after transfection of a construct expressing MAVS alone.Consistent with the observation that TRIM31 did not increase the MAVS(K10R,K311R,K461R)-induced activation of IFN-?,we found that expression of TRIM31 accelerated MAVS aggregation only in Mavs-/-MEFs transfected with the MAVS(WT)-expressing plasmid,not in transfected with the MAVS(K10R,K311R,K461R)-expressing plasmid.Consistent with the MAVS-aggregation data.SeV-induced expression of Ifnbl,Cc15 and CxcllO was enhanced only in the Mavs-/-MEF stransfected with MAVS(WT)-expressing plasmid.not in those transfected with MAVS(K10R,K311R,K461R)-expressing plasmid in thepresence of TRIM31.To investigate whether TRIM31 interacted with MAVS independently of MAVS polymerization,we studied the polymerization-deficient mutant MAVS(W56R).As expected,aggregation of MAVS(W56R)was abolished,relative to the aggregation of MAVS(WT),when overexpressed in Mavs-/-MEFs in the presence or absence of TRIM31.Consistent with the loss of MAVS aggregation,MAVS(W56R)was unable to induce Ifnbl expression after infection with SeV,which indicated that the action of TRIM31 on MAVS was dependent exclusively on the abilityof MAVS to polymerize.However,in co-immunoprecipitation experiments,we found that,similarly to MAVS(WT),MAVS(W56R)interacted with TRIM31,Notably,we found that MAVS(W56R)was ubiquitinated by TRIM31 in a manner similar to the ubiquitination of MAVS(WT).These data suggested that TRIM31 interacted with MAVS independently of MAVS polymerization.Together these data indicated that TRIM31 bound to inactive MAVS after viral infection and catalyzed K63-linked polyubiquitination on Lys10,Lys311 and Lys461 to further promote aggregation of MAVSand thus enhance IFN-? production and antiviral signaling. |
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