Negatively Regulates TLR3/4- and RIG-I Tripartite Motif

Tripartite Motif-Containing Protein 38 Negatively Regulates TLR3/4- and RIG-I− Mediated IFN-β Production and Antiviral Response by Targeting NAP1 This information is current as of September 27, 2021. Wei Zhao, Lijuan Wang, Meng Zhang, Peng Wang, Chao Yuan, Jianni Qi, Hong Meng and Chengjiang Gao J Immunol 2012; 188:5311-5318; Prepublished online 25 April 2012; doi: 10.4049/jimmunol.1103506 Downloaded from http://www.jimmunol.org/content/188/11/5311

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Tripartite Motif-Containing Protein 38 Negatively Regulates TLR3/4- and RIG-I–Mediated IFN-b Production and Antiviral Response by Targeting NAP1

Wei Zhao,*,1 Lijuan Wang,*,1 Meng Zhang,*,1 Peng Wang,* Chao Yuan,* Jianni Qi,* Hong Meng,† and Chengjiang Gao*

Recognition of RNA virus through TLR and RIG-I–like receptor results in rapid expression of type I IFNs, which play an essential role in host antiviral responses. However, the mechanisms to terminate the production of type I IFNs are not well deﬁned. In the current study, we identiﬁed a member of the tripartite motif (TRIM) family, TRIM38, as a negative regulator in TLR3/4- and RIG-I–mediated IFN-b signaling. Knockdown of TRIM38 expression by small interfering RNA resulted in augmented activation

of IFN regulatory factor 3 and enhanced expression of IFN-b, whereas overexpression of TRIM38 had opposite effects. Coim- Downloaded from munoprecipitation and colocalization experiments demonstrated that TRIM38 interacted with NF-kB–activating kinase- associated protein 1 (NAP1), which is required for TLR-induced IFN regulatory factor 3 activation and IFN-b production. As an E3 ligase, TRIM38 promoted K48-linked polyubiquitination and proteasomal degradation of NAP1. Thus, knockdown of TRIM38 expression resulted in higher protein level of NAP1 in primary macrophages. Consistent with the inhibitory roles in TLR3/4- and RIG-I–mediated IFN-b signaling, knockdown of TRIM38 signiﬁcantly inhibited the replication of vesicular stomatitis virus. Overexpression of TRIM38 resulted in enhanced replication of vesicular stomatitis virus. Therefore, our results http://www.jimmunol.org/ demonstrate that TRIM38 is a negative regulator for TLR and RIG-I–mediated IFN-b production by targeting NAP1 for ubiquitination and subsequent proteasome-mediated degradation. The Journal of Immunology, 2012, 188: 5311–5318.

attern recognition receptors, including TLRs and RIG-I– box RNA helicases, RIG-I, melanoma differentiation-associated like helicases (RLRs), play pivotal roles in defense against gene 5 (MDA5), and laboratory of genetics and physiology 2 (6, P viral infection (1, 2). After recognizing microbial con- 7). The helicases RIG-I and MDA5 have been found to recognize served pathogen-associated molecule patterns such as LPS, pol- viral RNAs and poly(I:C) in the cytoplasm and subsequently re- yinosinic-polycytidylic acid [poly(I:C)], and viral RNA, TLRs and cruit another antiviral signaling adaptor, mitochondrial antiviral RLRs activate immune cells to produce type I IFN (IFN-a/b) and signaling protein (MAVS, also called IPS-1, Cardif, or VISA) to by guest on September 27, 2021 proinflammatory cytokines, which are involved in the elimination initiate IFN-b signaling (8–11). Recruitment of TRIF and MAVS of viral infection (1, 2). mainly promotes the activation of TNFR-associated factor 3 TLR3 and TLR4 initiate IFN-b signaling through the recruit- (TRAF3)/NF-kB–activating kinase-associated protein (NAP1) and ment of TLR/IL-1R domain-containing adaptor protein inducing subsequent activation of TRAF family member-associated NF-kB IFN-b (TRIF) (3–5). RLRs comprise three cytoplasmic DExD–H- activator (TANK)-binding kinase 1 (TBK1; also called NAK)/ IkB kinase (IKK) ε, leading to the phosphorylation, dimerization, and nuclear translocation of IFN regulatory factor (IRF) 3 and the *Key Laboratory for Experimental Teratology of Ministry of Education, Department production of IFN-b (12, 13). Although full activation of TLR and of Immunology, Shandong University Medical School, Jinan, Shandong, 250012, RIG-I signaling and secretion of type I IFNs are important for the China; and †Institute of Basic Medicine, Shandong Academy of Medical Sciences, Jinan, Shandong, 250012, China elimination of invading microorganisms, uncontrolled expression 1W.Z., L.W., and M.Z. contributed equally to this work. of type I IFNs has manifested in diverse pathogenic autoimmune Received for publication December 5, 2011. Accepted for publication March 29, diseases, including systemic lupus erythematosus (14). Thus, 2012. TLR- and RIG-I–initiated IFN signaling must be tightly regulated. This work was supported in part by grants from the National Natural Science Foun- Tripartite motif (TRIM) family proteins are composed of .70 dation of China (81172813 and 31000407), the Taishan Scholar Program of Shandong members in humans (15). TRIM proteins are involved in a broad Province, the Shandong Provincial Nature Science Foundation for Distinguished Young Scholars (JQ201120), and the Independent Innovation Foundation of Shandong range of biological processes, including cell differentiation, apo- University (2009JQ001). ptosis, transcriptional regulation, signal transduction, and immu- Address correspondence and reprint requests to Prof. Chengjiang Gao, Department of nity (16–21). The characteristic structure of TRIM proteins is the Immunology, Shandong University School of Medicine, Jinan, Shandong, 250012, presence of a RING (R) domain, one or two B-boxes (B), and China. E-mail address: [email protected] a coiled coil (CC) domain at the N-terminal. The C-terminal re- Abbreviations used in this article: HA, hemagglutinin; IKK, IkB kinase; IP, immu- gion is variable among different TRIM proteins. Although several noprecipitation; IRF3, IFN regulatory factor 3; MAVS, mitochondrial antiviral signaling protein; MDA5, melanoma differentiation-associated gene 5; NAP1, NF-kB– TRIM proteins have been demonstrated to play very important activating kinase-associated protein 1; poly(I:C), polyinosinic-polycytidylic acid; roles in antiviral immune response, the function of the majority of RLR, RIG-I–like helicase; SeV, Sendai virus; siRNA, small interfering RNA; TANK, TNFR-associated factor family member-associated NF-kB activator; TBK1, TANK- TRIM proteins remains a mystery. binding kinase 1; TRAF, TNFR-associated factor; TRIF, TLR/IL-1R domain- In this study, we identified TRIM38 (also known as RoRet) as containing adaptor protein inducing IFN-b; TRIM, tripartite motif; VSV, vesicular a negative regulator in TLR3/4- and RIG-I–induced IFN-b sig- stomatitis virus; WT, wild-type. naling by targeting NAP1. Specifically, TRIM38 bound to NAP1 Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 and promoted K48-linked polyubiquitination and proteasomal www.jimmunol.org/cgi/doi/10.4049/jimmunol.1103506 5312 TRIM38 REGULATES IFN-b PRODUCTION degradation of NAP1. Therefore, our results outline a new manner assays were 59-CAACAAGTGTCTCCTCCAAAT-39 (sense) and 59-TCT- for the control of TLR and RLR response, and suggest TRIM38 as CCTCAGGGATGTCAAAG-39 (antisense) for IFN-b;59-GCCCTCGCT- a potential target for the intervention of autoimmune diseases with GTCATCCTCA-39 (sense) and 59-CCCGAACCCATTTCTTCTCTG-39 (antisense) for RANTES; 59-ATGGCCTCAACCACCAGC-39 (sense) and uncontrolled IFN-b production. 59-TCACCGACACTGGGGACAG-39 (antisense) for TRIM38; and 59- CAAGGTCATCCATGACAACTTTG-39 (sense) and 59-GTCCACCACC- CTGTTGCTGTAG-39 (antisense) for GAPDH. For IP, whole-cell extracts Materials and Methods were collected 36 h after transfection and were lysed in IP buffer con- Mice, cells, and reagents taining 1.0% (v/v) Nonidet P-40, 50 mM Tris-HCl (pH 7.4), 50 mM EDTA, 150 mM NaCl, and a protease inhibitor mixture (Merck). After C57BL/6J mice were obtained from Joint Ventures Sipper BK Experimental 3 Animal (Shanghai, China). All animal experiments were undertaken in centrifugation for 10 min at 14,000 g, supernatants were collected and accordance with the National Institutes of Health Guide for the Care and incubated with protein G Plus Agarose Immunoprecipitation reagent Use of Laboratory Animals, with the approval of the Scientific Investiga- (Santa Cruz Biotechnology) together with 1 mg monoclonal anti-Flag or 1 tion Board of Medical School of Shandong University (Jinan, China). mg anti-HA. After 6 h of incubation, beads were washed five times with IP Mouse macrophage cell line RAW264.7 and human HEK293 cells buffer. Immunoprecipitates were eluted by boiling with 1% (w/v) SDS were obtained from American Type Culture Collection (Manassas, VA). sample buffer. For Western blot analysis, immunoprecipitates or whole-cell HEK293-TLR3/TLR4 cell lines were obtained from Invivogen (San lysates were loaded and subjected to SDS-PAGE, transferred onto nitro- Diego, CA). Mouse primary peritoneal macrophages were prepared, as de- cellulose membranes, and then blotted, as described previously (22). scribed (22). The cells were cultured at 37˚C under 5% CO2 in DMEM Assay of luciferase activity supplemented with 10% FCS (Invitrogen-Life Technologies), 100 U/ml penicillin, and 100 mg/ml streptomycin. MG132, chloroquine, and LPS Luciferase activity was measured with the Dual-Luciferase Reporter Assay (Escherichia coli, 055:B5) were purchased from Sigma-Aldrich (St. system, according to the manufacturer’s instructions (Promega), as de- Louis, MO), and LPS was repurified, as described (22). Poly(I:C) was scribed (24). Data were normalized for transfection efficiency by division Downloaded from purchased from Invivogen. LPS and poly(I:C) were used at a final con- of firefly luciferase activity with that of Renilla luciferase. centration of 100 ng/ml and 10 mg/ml, respectively. The Abs specific for hemagglutinin (HA), Ub, b-actin, GAPDH, and protein G agarose used for Ubiquitination assays immunoprecipitation (IP) were from Santa Cruz Biotechnology (Santa For analysis of the ubiquitination of NAP1, HEK293 cells were transfected Cruz, CA). The Abs specific to Myc, IRF3, TBK1, STAT1, phospho-IRF3 396 727 with Flag-NAP1, HA-Ub (WT), or HA-Ub mutants and Myc-TRIM38 WT at Ser , and phospho-STAT1 at Ser were from Cell Signaling Tech- or Myc-TRIM38 C16A, and then whole-cell extracts were immunopreci- nology (Beverly, MA). The Ab for Flag was from Sigma-Aldrich. The Abs pitated with anti-Flag and analyzed by immunoblot with anti-HA Ab. http://www.jimmunol.org/ for TRIM38 and NAP1 were from Abcam (Cambridge, MA). Their re- spective HRP-conjugated secondary Abs were purchased from Santa Cruz Vesicular stomatitis virus plaque assay and detection of virus Biotechnology. Sendai virus was purchased from China Center for Type replication Culture Collection (Wuhan University). Vesicular stomatitis virus (VSV) plaque assay was performed, as described Sequences, plasmid constructs, and transfection (25). The HEK293 cells or macrophages (2 3 105) were transfected with the indicated plasmids or siRNA for 36 h prior to VSV infection (multi- pCMV6-Flag-TRIM38 (NM_006355) expression plasmid was purchased plicity of infection of 0.1). At 1 h postinfection, cells were washed with from OriGene (Rockville, MD). The TRIM38 C16A mutation was generated PBS for three times, and then medium was added. The supernatants were using the KOD-Plus-Mutagenesis kit (Toyobo, Osaka, Japan). TRIM38 wild- harvested at 24 h after washing. The supernatants were diluted 1:106 and type (WT) and C16A mutant cDNA were cloned in pCMV-HA and pCMV- then used to infect confluent HEK293 cells cultured on 24-well plates. At 1 by guest on September 27, 2021 Myc plasmids (Promega). TANK, IRF3, and NAP1 cDNA were amplified h postinfection, the supernatant was removed, and 3% methylcellulose was from THP1 cells by PCR and cloned in pCMV plasmid (Promega). All overlayed. At 3 d postinfection, overlay was removed; cells were fixed b constructs were confirmed by DNA sequencing. IFN- and IRF3 reporter with 4% formaldehyde for 20 min and stained with 0.2% crystal violet. plasmids and TBK1 and TRIF plasmids were gifts of X. Cao (Second Plaques were counted, averaged, and multiplied by the dilution factor Military Medical University, Shanghai, China). Expression vectors for ε to determine viral titer as log10 (PFU/ml). Total cellular RNA was ex- IKK- , HA-Ub WT, and mutant K48 and K63 were from H. Xiao (In- tracted from HEK293, or macrophages transfected with VSV and VSV stitute Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, RNA replicates were examined by quantitative RT-PCR, as described China). Flag-SINTBAD expression plasmid was a gift of F. Randow (26). Primers used for VSV replicates were as follows: 59-ACGGCG- (Medical Research Council Laboratory of Molecular Biology). For tran- TACTTCCAGATGG-39 (sense) and 59-CTCGGTTCAAGATCCAGGT-39 sient transfection of plasmids into RAW264.7 cells, jetPEI reagents were (antisense). used (Polyplus-transfection). For stable selection of cell lines over- expressing TRIM38, transfected RAW264.7 macrophages were selected Statistical analysis with G418 (600 mg/ml) and were pooled for further experiments. For transient silencing, duplexes of small interfering RNA (siRNA) were All data are presented as mean 6 SD of three or four experiments. Sta- transfected into cells with the Geneporter 2 Transfection Reagent (GTS, tistical significance was determined with the two-tailed Student t test, with San Diego, CA), according to the standard protocol. Target sequences a p value ,0.05 considered statistically significant. for transient silencing were 59-GAGGAUCGUCGGCAAACAAUU-39 (siRNA 1) and 59-GACUAAAGGUGGAAGAUUAUU-39 (siRNA 2) for Results TRIM38; scrambled control sequences were 59-UUCUCCGAACGUGU- CACGU-39. In vivo siRNA transfection to knockdown TRIM38 expression TRIM38 negatively regulates TLR3/4- and RIG-I–induced in peritoneal macrophages was performed, as described (23). Briefly, fe- IFN-b production male C57BL/6J mice (4 wk old) were i.p. injected with thioglycolate to TRIM38 is encoded within the MHC class I region, a region elicit peritoneal macrophages. After 3 d, 6 nmol siRNA was incubated with Geneporter 2 Transfection Reagent, according to manufacturer’s instruc- containing a large number of genes relevant to the immune re- tions, and then i.p. injected. After 48 h, the mice were treated with i.p. sponse (27). We previously demonstrated TRIM38 as a negative administration of LPS for 1 h, and the secretion of IFN-b in the peritoneal feedback regulator for TLR-induced production of proinflam- lavage was measured by ELISA. matory cytokines by targeting TRAF6 for ubiquitination and de- ELISA gradation (28). However, the function of TRIM38 especially in the antiviral immune response remains largely unknown. To investi- After cell stimulation, the concentrations of IFN-b in culture supernatants gate whether TRIM38 plays a role in antiviral immunity, we ini- or peritoneal lavage were measured by ELISA kits (R&D Systems, Min- neapolis, MN). tially examined the effects of TRIM38 on TLR3- and TLR4- mediated IFN-b production in macrophages. Two siRNAs were RNA quantitation, IP, and Western blot analysis transfected into primary peritoneal macrophages to suppress en- Total RNA was extracted with TRIzol reagent, according to the manu- dogenous TRIM38 expression. The expression of TRIM38 protein facturer’s instructions (Invitrogen). Specific primers used for RT-PCR was greatly decreased as measured by Western blotting with The Journal of Immunology 5313 transfection of TRIM38-specific siRNA 1 and 2 (Fig. 1A). After TNF-a in HEK293 cells. Overexpression of TRIM38 greatly at- transfection of TRIM38 siRNA, macrophages were with stimu- tenuated poly(I:C)-induced expression of IFN-b and RANTES. lated with LPS (TLR4 ligand) and poly(I:C) (TLR3 ligand), re- Collectively, these data indicate that TRIM38 negatively regulates spectively. As shown in Fig. 1B, TRIM38 knockdown significantly TLR3/4- and RIG-I–induced IFN-b signaling. increased LPS- and poly(I:C)-induced IFN-b production in mouse b peritoneal macrophages. TRIM38 siRNA 2, which has a higher TRIM38 negatively regulates LPS-induced IFN- production efficiency to knock down TRIM38 protein expression (Fig. 1A), in vivo has a greater potential to increase the TLR3- and TLR4-induced Because TRIM38-deficient mice are not available, in vivo TRIM38 IFN-b production (Fig. 1B). Therefore, TRIM38 siRNA 2 was siRNA i.p. transfection was performed to knock down TRIM38 used in the following experiments. expression in thioglycolate-elicited peritoneal cells with Gene- To further confirm the negative regulatory role of TRIM38 on porter 2 Transfection Reagent. TRIM38 siRNA i.p. transfection TLR3/4-mediated IFN-b production, RAW264.7 stable cell line successfully suppressed TRIM38 expression in i.p. cells (Fig. 2A). with TRIM38 overexpression was constructed by transfecting After i.p. transfection of TRIM38 siRNA, LPS was administrated Flag-TRIM38 expression plasmid. Overexpression of TRIM38 i.p., and the secretion of IFN-b in peritoneal lavage was measured was confirmed by Western blotting with both TRIM38 and Flag by ELISA. As shown in Fig. 2B, knockdown of TRIM38 ex- Abs (Fig. 1C). As shown in Fig. 1D, TRIM38 overexpression pression significantly increased LPS-induced IFN-b production in significantly decreased LPS- and poly(I:C)-induced IFN-b pro- peritoneal lavage. These findings demonstrate that TRIM38 could duction in RAW264.7 cells. inhibit LPS-induced IFN-b production in vivo.

To investigate TRIM38 inhibiting LPS- and poly(I:C)-induced Downloaded from IFN-b expression at the transcriptional level, TRIM38 expression TRIM38 inhibits IRF3 activation plasmid and IFN-b promoter luciferase reporter were cotrans- IRF3 is the key transcription factor that is responsible for the fected into RAW264.7 cells. As shown in Fig. 1E, TRIM38 overexpression of IFN-b in TLR3/4 and RIG-I signaling. Activation of expression signiﬁcantly decreased LPS- and poly(I:C)-induced TLR3/4 and RIG-I led to the phosphorylation of IRF3 through IFN-b promoter activation in RAW264.7 cells. Similarly, LPS- a TBK1-dependent pathway, resulting in IRF3 dimerization and

and poly(I:C)-induced IFN-b luciferase activation was also greatly subsequent nuclear translocation and binding to the IFN-b pro- http://www.jimmunol.org/ attenuated by TRIM38 overexpression in HEK293/TLR4 and moter. To investigate the effect of TRIM38 on IRF3 activation, HEK293/TLR3 cells, which stably expressed TLR4 and TLR3, IRF3 cis-reporting plasmids were transfected into HEK293/TLR3 respectively (Fig. 1F). and HEK293/TLR4 cells. As shown in Fig. 3A, LPS and poly(I:C) Recognition of RNA virus through RIG-I may lead to the ex- greatly increased IRF3 activation in HEK293/TLR4 and HEK293/ pression of IFN-b. To investigate the function of TRIM38 on TLR3 cells, respectively. However, transfection of TRIM38 ex- RIG-I–mediated IFN-b signaling, poly(I:C) was transfected into pression plasmid substantially attenuated LPS- and poly(I:C)- HEK293 cells, which has been shown to activate IFN-b production induced IRF3 activation. through RIG-I and MDA-5 (29). As shown in Fig. 1G, transfection To investigate TRIM38 inhibition of IRF3 activation under of poly(I:C) could induce the expression of IFN-b, RANTES, and physiological conditions, endogenous TRIM38 expression was by guest on September 27, 2021

FIGURE 1. TRIM38 negatively regulates TLR3/4- and RIG-I–induced IFN-b production. (A) Western blot analysis of TRIM38 expression in mouse peritoneal macrophages transfected with control siRNA or TRIM38 siRNA 1 and siRNA 2 for 36 h. (B) ELISA of IFN-b in the supernatants of peritoneal macrophages as in (A) stimulated with LPS or poly(I:C) for 12 h. Data are shown as mean 6 SD (n = 3) of one representative experiment (**p , 0.01). (C) Western blot analysis of TRIM38 expression in RAW264.7 cells stably transfected with Flag-TRIM38 expression plasmid or control empty vector (Ctrl). (D) ELISA of IFN-b in the supernatants of RAW264.7 cells as in (C) stimulated with LPS or poly(I:C) for 12 h. Data are shown as mean 6 SD (n =3)of one representative experiment (*p , 0.05, **p , 0.01). (E) RAW264.7 cells were transiently transfected with IFN-b reporter plasmid together with TRIM38 expression plasmid or control plasmid, and luciferase activity was analyzed after treatment with LPS or poly(I:C) for 6 h. (F) The 293-TLR3/4 cells were transfected with IFN-b reporter plasmid together with TRIM38 expression plasmid or control plasmid, and luciferase activity was analyzed after treatment with poly(I:C) or LPS for 6 h, respectively. Data are shown as mean 6 SD (n = 6) of one representative experiment (**p , 0.01). (G) HEK293 cells treated with the indicated plasmids were stimulated with poly(I:C) for indicated time periods. Total RNA was prepared and analyzed for the expressions of IFN-b, RANTES, TRIM38, and GAPDH by RT-PCR. Similar results were obtained in three independent experiments. 5314 TRIM38 REGULATES IFN-b PRODUCTION

expression could not inhibit IKKε-induced IRF3 activation (Fig. 4B). Therefore, we conclude that TRIM38 targets molecules up- stream of TBK1 to inhibit the signal transduction. Recently, Sasai et al. (30, 31) reported that NAP1 is a subunit of the kinase complex IKKε/TBK1 and can synergize with MAVS, TRIF, and TBK1 to increase IRF3 activation. Consistent with their data, we found cotransfection of NAP1 with TRIF or TBK1 greatly increased TRIF- and TBK1-induced IFN-b promoter ac- FIGURE 2. Function of TRIM38 in vivo. (A) Western blot analysis of tivation (Fig. 4C). However, this activation was significantly in- TRIM38 expression in thioglycolate-elicited peritoneal cells transfected hibited with TRIM38 overexpression in a dose-dependent manner i.p. with TRIM38 siRNA for 48 h. (B) ELISA of IFN-b in the peritoneal (Fig. 4C). Transfection of NAP1 alone could slightly induce lavage of mice as treated in (A) after i.p. administration with PBS or LPS IFN-b promoter activation, and this activation was also inhibited for 1 h. Data are shown as mean 6 SD (n =5;**p , 0.01). Similar results with TRIM38 overexpression (Fig. 4D). TANK and SINTBAD were obtained in three independent experiments. (similar to NAP1 TBK1 adaptor) are another two adaptors that can bind TBK1 and IKKε. Consistent with reported data, we found silenced with siRNA transfection in mouse peritoneal macro- cotransfection of TANK or SINTBAD with TBK1 substantially phages and IRF3 phosphorylation was measured upon LPS stim- increased TBK1-induced IFN-b promoter activation (Fig. 4E). In ulation. Knockdown of endogenous TRIM38 expression substan- contrast to NAP1, this activation was not inhibited with TRIM38 tially enhanced LPS-induced phosphorylation of IRF3 (Fig. 3B). overexpression (Fig. 4E). Taking all these reporter data together, Downloaded from Consistently, TRIM38 overexpression significantly decreased LPS- we speculate NAP1 may be a TRIM38 target. induced phosphorylation of IRF3 in RAW264.7 cells (Fig. 3C). To confirm that TRIM38 targets NAP1, the function of TRIM38 Virus-induced IFN-b production can activate the transcription on the degradation of the molecules in TLR3/4 and RIG-I signaling factor STAT1. Phosphorylated STAT1 conjugates with STAT2 and was investigated. As shown in Fig. 5A and 5B, TRIM38 promoted IRFs to generate the IFN-stimulated gene factor 3 transcription degradation of NAP1 in a dose-dependent manner. In contrast,

complex to regulate the expression of IFN-stimulated genes. Con- TRIM38 overexpression had no effects on TBK1, TANK, IKKε, http://www.jimmunol.org/ sistent with the inhibitory function of TRIM38 on IFN-b produc- SINTBAD, STING, and IRF3 expression (Fig. 5B, 5C). To further tion, knockdown of endogenous TRIM38 expression greatly conﬁrm TRIM38-mediated NAP1 degradation under physiological increased LPS-induced STAT1 phosphorylation in mouse perito- condition, TRIM38 expression was silenced by TRIM38 siRNA neal macrophages (Fig. 3B). In contrast, TRIM38 overexpression transfection. TRIM38 knockdown greatly increased NAP1 protein inhibited LPS-induced STAT1 phosphorylation (Fig. 3C). All to- level in peritoneal macrophages (Fig. 5D). As a control, TBK1, gether, these data suggest that TRIM38 inhibits TLR-induced IRF3, and STAT1 protein levels were not impaired (Fig. 5D). All IRF3 activation and subsequent IFN-b signaling. together, these data indicate that TRIM38 targets NAP1 for degradation to inhibit IFN-b production.

TRIM38 targets NAP1 by guest on September 27, 2021 TLR3/4 and RIG-I recognize the microbial patterns and recruit TRIM38 promotes K48-linked polyubiquitination and adaptors TRIF and MAVS, respectively. TRIF and MAVS mediate proteasomal degradation of NAP1 IRF3 activation and IFN-b production through a complex cascade To understand the mechanisms by which TRIM38 degrades NAP1 composed of various molecules, including TRIF, MAVS, TBK1, expression, we first investigated the interaction between TRIM38 IKKε, and IRF3. To determine the molecular order and molec- and NAP1. HA-TRIM38 and Flag-NAP1 were cotransfected into ular targets of TRIM38 in TLR- and RIG-I–induced IFN-b sig- HEK293 cells; IP experiments were performed with HA or Flag naling, the effects of TRIM38 overexpression on IFN-b promoter Abs. Flag-NAP1 was coprecipitated with HA-TRIM38 and vice activation mediated by various molecules were examined in re- versa (Fig. 6A, 6B). porter assays. As shown in Fig. 4A and 4B, TRIF-, RIG-I–, and TRIM38 contains a cluster of domains composed of a RING MAVS-induced IFN-b promoter activation and IRF3 activation finger, two B-boxes, and a C-terminal PRY/SPRY (16). The pres- were significantly inhibited by TRIM38 overexpression in a dose- ence of RING-finger domain indicates TRIM38 may function as dependent manner. However, TBK1 was still able to activate an E3 ligase. To test the role of TRIM38 in NAP1 ubiquitination, IFN-b promoter activation and IRF3 activation in the presence of NAP1 was cotransfected with HA-ubiquitin and WT TRIM38 TRIM38 expression plasmid (Fig. 4A, 4B). Similarly, TRIM38 into HEK293 cells. Without MG-132, NAP1 ubiquitination was

FIGURE 3. TRIM38 inhibits IRF3 activation. (A) The 293-TLR3/4 cells were transfected with IRF3 reporter plasmid together with TRIM38 expression plasmid or control plasmid, and luciferase activity was analyzed after treatment with poly(I:C) and LPS or for 6 h, respectively. Data are shown as mean 6 SD (n = 6) of one representative experiment (**p , 0.01). (B) Western blot analysis of phosphorylated IRF3, phosphorylated STAT1, and total IRF3; STAT1 in mouse peritoneal macrophages transfected with control siRNA (Ctrl) or TRIM38 siRNA (siRNA) stimulated with LPS. (C) Western blot analysis of phosphorylated IRF3, phosphorylated STAT1, and total IRF3; STAT1 in RAW264.7 cells as in Fig. 1C stimulated with LPS. Similar results were obtained in three independent experiments. The Journal of Immunology 5315 Downloaded from

FIGURE 4. TRIM38 targets NAP1. (A) HEK293 cells were transfected with TRIF, RIG-I, MAVS, TBK1, along with IFN-b reporter plasmid and increasing amount of TRIM38 plasmid, and luciferase activity was analyzed. (B) HEK293 cells were transfected with TRIF, RIG-I, MAVS, TBK1, IKKε, http://www.jimmunol.org/ along with IRF3 reporter plasmid and increasing amount of TRIM38 plasmid, and luciferase activity was analyzed. (C) HEK293 cells were transfected with 100 ng IFN-b luciferase reporter plasmid, 50 ng pTK-Renilla luciferase, 100 ng NAP1-expressing plasmid, together with 10 ng TIRF or TBK1 plasmid and increasing amount of TRIM38 plasmid, and luciferase activity was measured. Total amounts of plasmid DNA were equalized with empty control vector. (D) HEK293 cells were transfected with NAP1, along with IFN-b reporter plasmid and increasing amount of TRIM38 plasmid, and luciferase activity was analyzed. (E) HEK293 cells were transfected with 100 ng IFN-b luciferase reporter plasmid, 50 ng pTK-Renilla luciferase, 100 ng SINTBAD or TANK- expressing plasmid, together with 10 ng TBK1 plasmid and increasing amount of TRIM38 plasmid, and luciferase activity was measured. Total amounts of plasmid DNA were equalized with empty control vector. Data are shown as mean 6 SD (n = 6) of one representative experiment (**p , 0.01). by guest on September 27, 2021 hardly detectable (data not shown). After MG-132 treatment, the ubiquitination could be detected in the presence of WT and K48 level of NAP1 ubiquitination was markedly increased in the plasmid (lanes 2 and 4), but not with K63 plasmid (lane 6), in- presence of TRIM38 expression plasmid (Fig. 6C, lane 3). Im- dicating TRIM38 medicates K48-linked polyubiquitination of portantly, the TRIM38 point mutation (C16A) with substitution NAP1. K48-linked protein ubiquitination leads to the degradation of the cysteine residue at position 16 within the RING domain of the corresponding protein by 26S proteasome. Consistently, with alanine lost the ability to promote polyubiquitination of TRIM38-induced degradation of NAP1 protein could be reversed NAP1 (Fig. 6C, lane 4), indicating TRIM38 could promote the by proteasome inhibitor MG-132, but not by lysosome inhibitor ubiquitination of NAP1 though the RING-ﬁnger domain. To chloroquine (Fig. 6E). Accordingly, the TRIM38 mutant C16A study the forms of TRIM38-mediated NAP1 polyubiquitination, lost the ability to inhibit TRIF-induced IFN-b activation, com- ubiquitin mutant vectors K48 and K63, which contain arginine pared with WT TRIM38 (Fig. 6F). All together, these data substitutions of all of its lysine residues except the one at posi- demonstrate that TRIM38 interacts with NAP1 and promotes tions 48 and 63, respectively, were used in the transfection K48-linked polyubiquitination and proteasomal degradation of assays. As shown in Fig. 6D, TRIM38-mediated NAP1 poly- NAP1.

FIGURE 5. TRIM38 promotes the degradation of NAP1. (A) Western blot analysis of Flag-NAP1 in HEK293 cells transfected with Flag-NAP1 together with increasing concentration of HA-TRIM38 expression plasmid. (B and C) Western blot analysis of the lysates from HEK293 cells transfected with various tagged molecules with Myc-tagged TRIM38 for 24 h. (D) Western blot analysis of the expression of NAP1, IRF3, TBK1, and STAT1 in peritoneal macrophages transfected with control siRNA (Ctrl) or TRIM38 siRNA (siRNA). Similar results were obtained in three independent experiments. 5316 TRIM38 REGULATES IFN-b PRODUCTION Downloaded from

FIGURE 6. TRIM38 promotes K48-linked ubiquitination and proteasomal degradation of NAP1. (A and B) Lysates from HEK293 cells transiently cotransfected with Flag-NAP1 and HA-TRIM38 expression plasmids were subjected to IP with anti-HA or anti-Flag Ab, followed by Western blot analysis http://www.jimmunol.org/ with anti-Flag or anti-HA Ab, respectively. (C) Lysates from HEK293 cells transiently cotransfected with Flag-NAP1, Myc-TRIM38 WT, or TRIM38 C16A, and HA-Ub plasmids were subjected to IP with anti-Flag Ab, followed by Western blot analysis with anti-HA Ab. (D) Lysates from HEK293 cells transiently cotransfected with Flag-NAP1, Myc-TRIM38, or vector control, and HA-Ub (WT), HA-Ub (K48), or HA-Ub (K63) plasmids were subjected to IP with anti-Flag Ab, followed by Western blot analysis with anti-HA Ab. (E) Western blot analysis of Flag-NAP1 expression in HEK293 cells cotransfected with Flag-NAP1 and HA-TRIM38 or vector control and then treated with chloroquine or MG132 for 4 h. (F) HEK293 cells were transfected with IFN-b reporter plasmid and TRIF expression plasmid together with TRIM38 WT and C16A. Twenty-four hours later, luciferase activity was measured. Data are shown as mean 6 SD (n = 6) of one typical experiment (**p , 0.01). by guest on September 27, 2021 TRIM38 negatively regulates cellular antiviral response TRIM38 expression was silenced by TRIM38 siRNA transfection Type I IFNs play critical roles in the innate immune responses in mouse peritoneal macrophages, and then the macrophages against viral infection. The fact that TRIM38 negatively regulates were infected with VSV. Plaque assay showed that transfection IFN-b production prompted us to investigate the function of of TRIM38 siRNA greatly decreased VSV viral replication in TRIM38 in antiviral immunity. TRIM38 protein expression in macrophages in the presence or absence of poly(I:C) (Fig. 7D). primary macrophages was measured postinfection with Sendai Accordingly, knockdown of TRIM38 signiﬁcantly decreased in- virus (SeV). SeV infection substantially induced the expression of tracellular VSV RNA replicates, compared with control siRNA- TRIM38 in macrophages at different time points after SeV in- transfected macrophages (Fig. 7D). Taken together, these data fection (Fig. 7A), suggesting TRIM38 expression was modulated indicate that virus-induced TRIM38 negatively regulates produc- by virus infection. To investigate the function of SeV-induced tion of IFN-b and antiviral immune responses, thus facilitating TRIM38 expression, production of IFN-b and TNF-a was mea- viral invasion of the immune system. sured by ELISA, followed by SeV infection in TRIM38 siRNA- transfected macrophages. As shown in Fig. 7B, SeV infection Discussion greatly induced the production of IFN-b, whereas production Innate immunity plays an essential role in the control and elimi- of IFN-b was further increased after TRIM38 siRNA tranfec- nation of viral infection through the production of type I IFNs tion. Similarly, TNF-a production was also greatly increased in (7). Production of type I IFNs requires the recognition of viral TRIM38 siRNA-transfected macrophages upon SeV infection pathogen-associated molecule patterns by TLRs and RIG-I–like (Fig. 7B). To directly investigate the effect of TRIM38 on antiviral receptors. TLR3/4 and RIG-I use TRIF and MAVS (also known as responses, VSV, a kind of ssRNA virus recognized by RIG-I, was Cardif, IPS-1, or VISA) to mediate IFN-b signaling, respectively. used to infect HEK293 cells and macrophages. Plaque assay of Downstream of TRIF and MAVS, both TLR3/4 and RIG-I could HEK293 cells infected with VSV showed that overexpression of activate a similar pathway that involves NAP1, TBK1, and IRF3 to TRIM38 substantially increased viral replication in the presence induce the production of IFN-b (6, 7). Although production of or absence of poly(I:C) (Fig. 7C). In sharp contrast, TRIM38 type I IFNs is essential for the antiviral immune responses, un- mutant C16A, which lost the ability to promote ubiquitination controlled production is harmful and has been found in diverse and degradation of NAP1, could not increase viral replication phathogenic autoimmune diseases, including systemic lupus erythe- (Fig. 7C). Similarly, VSV RNA replicates in HEK293 cells were matosus (14). Therefore, TLR3/4- and RIG-I–mediated IFN-b sig- greatly increased in TRIM38-transfected cells, compared with naling must be tightly controlled. control vector- or TRIM38 C16A-transfected cells, as measured TRIM family proteins are composed of .70 members in hu- by quantitative RT-PCR (Fig. 7C). To further conﬁrm the function mans (15). Several TRIM proteins have been reported to play key of TRIM38 on VSV replication under physiological conditions, roles either positively or negatively in TLR- or other pattern The Journal of Immunology 5317 Downloaded from

FIGURE 7. TRIM38 negatively regulates cellular antiviral response. (A) Western blot analysis of TRIM38 expression in mouse peritoneal macrophages infected with SeV for indicated time periods. (B) Mouse peritoneal macrophages were transfected with control siRNA or TRIM38 siRNA for 36 h. ELISA of IFN-b in the supernatants of peritoneal macrophages infected with SeV for 12 h. Data are shown as mean 6 SD (n = 3) of one representative experiment , C 3 5

(**p 0.01). ( ) The 293 cells (2 10 ) were transfected with the indicated plasmids (1 mg each). Twenty-four hours later, cells were further transfected http://www.jimmunol.org/ with poly(I:C) (0.1 mg) or left untreated. Eighteen hours after poly(I:C) transfection, cells were infected with VSV (multiplicity of infection, 0.1), and the supernatants were harvested at 12 h postinfection. Supernatants were analyzed for VSV titers with standard plaque assays. Intracellular VSV RNA replicates were measured by quantitative RT-PCR. Data are shown as mean 6 SD of three independent experiments (**p , 0.01). (D) Mouse peritoneal macrophages (4 3 105) were transfected with control siRNA (Ctrl) or TRIM38 siRNA (siRNA). VSV titers and intracellular VSV RNA replicates were measured as in (C). recognition receptor-mediated signaling and antiviral immunity In this study, we provided evidence to demonstrate that TRIM38 through ubiquitination (17–21). TRIM25 interacts with the could also act as an E3 ligase to promote the ubiquitination and caspase-recruitment domain of RIG-I through its PRY/SPRY do- proteasomal degradation of NAP1. NAP1 is an adaptor protein for by guest on September 27, 2021 main, which results in the K63-linked ubiquitylation of RIG-I and, the kinase IKKε/TBK1 and plays a very important role in TLR3/4- consequently, the induction of type I IFN production and NF-kB and RLR-induced IRF3 activation and IFN-b production (30, 31, activity (32). TRIM56 was required for dsDNA virus-triggered 38, 39). Activation of TLR7/8/9 could also lead to the production signaling through K63-linked polyubiquitination of STING (33). of type I IFNs. We found that siRNA knockdown of TRIM38 TRIM23 has been shown to catalyze K27-linked ubiquitination of expression in macrophages greatly increased R848-induced IFN-b NEMO, which is crucial for IRF3 and NF-kB activation down- production (data not shown), indicating TRIM38 also negatively stream of TLR3 and RIG-I (34). TRIM21 has been shown to regulates IFN signaling in the TLR7/8/9 pathway. Given the fact mediate K48-linked ubiquitination of various IRFs, and thereby that TLR7/8/9-mediated production of type I IFNs requires directly inhibit production of type I IFNs (35–37). TRAF6 (40), we concluded that TRIM38-mediated ubiquitination In the current study, we have identified TRIM38 as a new and degradation of TRAF6 may account for the inhibitory effect regulator in TLR3/4- and RIG-I–mediated IFN-b signaling. Over- of TRIM38 on TLR7/8/9-mediated IFN signaling. Thus, TRIM38- expression of TRIM38 inhibited TLR- and RIG-I–induced acti- mediated ubiquitination and degradation of NAP1 and TRAF6 vation of IRF3 and production of IFN-b and downstream STAT1 represent a new mechanism to terminate the excessive produc- phosphorylation. Moreover, siRNA knockdown of TRIM38 ex- tion of type I IFNs. pression potentiated TLR- and RIG-I–induced activation of IRF3 TANK and SINTBAD are another two adaptor proteins that and production of IFN-b. Relevant to the function of IFN-b in were described to specifically bind TBK1/IKKε and participate in antiviral immunity, siRNA knockdown of TRIM38 or over- TLR/RLR-induced IFN production (41, 42). In contrast to NAP1, expression significantly inhibited or enhanced the replication of we found that TANK- and SINTBAD-induced IFN-b activation VSV, respectively. Therefore, to our knowledge, our study pro- was not inhibited by TRIM38. Importantly, TRIM38 could not vided the first evidence to demonstrate that TRIM38 negatively promote the degradation of TANK and SINTBAD. Therefore, regulates both TLR3/4- and RIG-I–induced IFN-b production, TRIM38 specifically targets NAP1 for degradation to regulate the thus facilitating the viral immune evasion. TLR3/4- and RLR-mediated IFN signaling. NAP1 as well as TRIM38, a member of TRIM family, is encoded within the SINTBAD and TANK have been reported to bind TBK1 and MHC class I region (27). The characteristic structure of TRIM IKKε via a TBK1/IKKε binding domain (42). Another conserved proteins is the presence of a RING (R) domain, one or two B- domain among these three adaptors is coiled coils-forming do- boxes (B), and a coiled coil (CC) domain (16). The presence of main (CC). The different substrate specificity for TRIM38 to- the RING domain indicates TRIM proteins may work as E3 ward NAP1, TANK, and SINTBAD may arise from the different ligases. Indeed, we have demonstrated that TRIM38 targets binding activity between TRIM38 and the three adaptors. We have TRAF6 for ubiquitination and degradation to negatively regu- confirmed the binding between TRIM38 and NAP1. However, late TLR-induced production of proinflammatory cytokines (28). whether the interaction between TRIM38 and TANK and SINT- 5318 TRIM38 REGULATES IFN-b PRODUCTION

BAD is present needs to be confirmed. Another possibility for the 21. Jefferies, C., C. Wynne, and R. Higgs. 2011. Antiviral TRIMs: friend or foe in autoimmune and autoinflammatory disease? Nat. Rev. Immunol. 11: 617–625. different substrate specificity may be the ubiquitin-receiving ly- 22. Zhao, W., L. Wang, L. Zhang, C. Yuan, P. C. Kuo, and C. Gao. 2010. Differential sine residues that are different among these three adaptors. There- expression of intracellular and secreted osteopontin isoforms by murine mac- fore, the exact mechanism deserves further investigation. rophages in response to Toll-like receptor agonists. J. Biol. Chem. 285: 20452– 20461. In conclusion, we identified TRIM38 as a critical negative 23. An, H., W. Zhao, J. Hou, Y. Zhang, Y. Xie, Y. Zheng, H. Xu, C. Qian, J. Zhou, regulator of TLR3/4- and RIG-I–induced IFN-b production by Y. Yu, et al. 2006. SHP-2 phosphatase negatively regulates the TRIF adaptor targeting NAP1 for ubiquitination and degradation. Given the protein-dependent type I interferon and proinflammatory cytokine production. Immunity 25: 919–928. important roles of IFN-b in host antiviral immunity, virus may 24. Zhao, W., L. Wang, M. Zhang, P. Wang, L. Zhang, C. Yuan, J. Qi, Y. Qiao, modulate TRIM38 expression to escape immune surveillance and P. C. Kuo, and C. Gao. 2011. Peroxisome proliferator-activated receptor g negatively regulates IFN-b production in Toll-like receptor (TLR) 3- and TLR4- facilitate the viral replication. Indeed, we found poly(I:C) treat- stimulated macrophages by preventing interferon regulatory factor 3 binding to ment or SeV infection greatly increases TRIM38 expression. In the IFN-b promoter. J. Biol. Chem. 286: 5519–5528. contrast, excessive IFN-b production has manifested in diverse 25. Mao, A. P., S. Li, B. Zhong, Y. Li, J. Yan, Q. Li, C. Teng, and H. B. Shu. 2010. Virus-triggered ubiquitination of TRAF3/6 by cIAP1/2 is essential for induction pathogenic autoimmune diseases (14). Our results provide a strat- of interferon-b (IFN-b) and cellular antiviral response. J. Biol. Chem. 285: egy to downregulate IFN-b production and suggest that TRIM38 9470–9476. may have therapeutic potential for the intervention of autoimmune 26. Zheng, Y., H. An, M. Yao, J. Hou, Y. Yu, G. Feng, and X. Cao. 2010. Scaffolding adaptor protein Gab1 is required for TLR3/4- and RIG-I-mediated production of diseases with uncontrolled IFN-b production. proinflammatory cytokines and type I IFN in macrophages. J. Immunol. 184: 6447–6456. 27. Meyer, M., S. Gaudieri, D. A. Rhodes, and J. Trowsdale. 2003. Cluster of TRIM Acknowledgments genes in the human MHC class I region sharing the B30.2 domain. Tissue We thank Drs. Xuetao Cao, Felix Randow, and Hui Xiao for providing Antigens 61: 63–71. Downloaded from plasmids. 28. Zhao, W., L. Wang, M. Zhang, C. Yuan, and C. Gao. 2012. E3 ubiquitin ligase tripartite motif 38 negatively regulates TLR-mediated immune responses by proteasomal degradation of TNF receptor-associated factor 6 in macrophages. J. Disclosures Immunol. 188: 2567–2574. The authors have no financial conflicts of interest. 29. Kato, H., O. Takeuchi, S. Sato, M. Yoneyama, M. Yamamoto, K. Matsui, S. Uematsu, A. Jung, T. Kawai, K. J. Ishii, et al. 2006. Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses. Nature 441: 101–

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