Smurf2 Negatively Modulates RIG-I− dependent Antiviral Response by Targeting VISA/MAVS for Ubiquitination and Degradation This information is current as of October 1, 2021. Yu Pan, Rui Li, Jun-Ling Meng, He-Ting Mao, Yu Zhang and Jun Zhang J Immunol published online 11 April 2014 http://www.jimmunol.org/content/early/2014/04/11/jimmun ol.1302632 Downloaded from Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on October 1, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published April 11, 2014, doi:10.4049/jimmunol.1302632 The Journal of Immunology Smurf2 Negatively Modulates RIG-I–dependent Antiviral Response by Targeting VISA/MAVS for Ubiquitination and Degradation Yu Pan, Rui Li, Jun-Ling Meng, He-Ting Mao, Yu Zhang, and Jun Zhang VISA (also known as MAVS, Cardif, IPS-1) is the essential adaptor protein for virus-induced activation of IFN regulatory factors 3 and 7 and production of type I IFNs. Understanding the regulatory mechanisms for VISA will provide detailed insights into the positive or negative regulation of innate immune responses. In this study, we identified Smad ubiquitin regulatory factor (Smurf) 2, one of the Smad ubiquitin regulator factor proteins, as an important negative regulator of virus-triggered type I IFN signaling, which targets at the VISA level. Overexpression of Smurf2 inhibits virus-induced IFN-b and IFN-stimulated response element activation. The E3 ligase defective mutant Smurf2/C716A loses the ability to suppress virus-induced type I IFN signaling, suggesting that the negative regulation is dependent on the ubiquitin E3 ligase activity of Smurf2. Further studies demonstrated Downloaded from that Smurf2 interacted with VISA and targeted VISA for K48-linked ubiquitination, which promoted the degradation of VISA. Consistently, knockout or knockdown of Smurf2 expression therefore promoted antiviral signaling, which was correlated with the increase in protein stability of VISA. Our findings suggest that Smurf2 is an important nonredundant negative regulator of virus- triggered type I IFN signaling by targeting VISA for K48-linked ubiquitination and degradation. The Journal of Immunology, 2014, 192: 000–000. http://www.jimmunol.org/ hen invaded by viruses, host pattern recognition Antiviral innate immunity is positively or negatively regulated at receptors such as the TLRs and the retinoic acid–in- multiple levels to ensure appropriate response is initiated (10). W duced gene I (RIG-I)–like receptors (RLRs) sense Ubiquitination is an important posttranscriptional modification in viral nucleic acids and initiate a series of signaling events that lead this process. The cascade involves the stepwise action of three to production of type I IFNs and proinflammatory cytokines (1). enzymes: ubiquitin activating enzyme (E1), ubiquitin-conjugating RIG-I and melanoma differentiation–associated gene 5 (MDA-5), enzyme (E2), and a ubiquitin ligase (E3) (11). E3s provide sub- members of the RIG-I–like receptors, are cytoplasmic RNA sen- strate specificity and are broadly categorized into two families: sors that recognize distinct types of RNA viruses. RIG-I recog- homologous to the E6-accessory protein C terminus (HECT) do- by guest on October 1, 2021 nizes short dsRNA and 59-triphosphate panhandle RNA, whereas main E3 ubiquitin ligases and RING-type E3 ligases (12). Lysine MDA-5 binds to long dsRNA (2–4). Upon binding to viral RNA, 48 (K48)–linked polyubiquitination usually targets proteins for both RIG-I and MDA-5 signal through the common mitochondrial proteasomal degradation, which is essential in constraint of sig- adaptor protein VISA (also known as MAVS, IPS-1, and Cardif) naling to prevent the detrimental effects of excessive responses on (5–8). VISA further activates the noncanonical IkB kinase (IKK) the host. Both HECT and RING domain E3 ubiquitin ligases have family members TANK-binding kinase 1 (TBK1) and IKKε, been reported to function as important negative regulators in an- which phosphorylate and activate IFN regulatory factor (IRF) 3 or/ tiviral immune responses. The RING-type E3 ligase RNF125 can and IRF7 for type I IFN responses. It also activates canonical IKK ubiquitinate and induce degradation of MDA-5, RIG-I, and VISA to molecules for NF-kB activation for production of proinflammatory inhibit RIG-I–like helicase–mediated responses (13). RNF5 reg- cytokines (5–9). ulates antiviral responses by mediating ubiquitination and degra- dation of the adaptor protein MITA (also known as STING, ERIS) (14). The HECT domain containing ubiquitin ligase AIP4 (Itch in Department of Immunology, School of Basic Medical Sciences, Key Laboratory of mice) promotes the ubiquitination and degradation of VISA in Medical Immunology (Ministry of Health), Peking University Health Science Center, Beijing 100191, People’s Republic of China collaboration with poly(rC) binding protein (PCBP) 2 (15). Received for publication September 30, 2013. Accepted for publication March 5, Smad ubiquitin regulatory factor (Smurf) 1 and Smurf2 belong to 2014. HECT type E3 ubiquitin ligases. Smurfs contain an N-terminal C2 This work was supported by National Basic Research Program of China Grants domain for membrane binding, a central region containing two or 2011CB946103 and 2010CB945300, as well as by the Program for New Century three WW domains for protein–protein interaction, and a C-ter- Excellent Talents in University (to J.Z.). minal HECT domain for ubiquitin protein ligation (16). They were Address correspondence and reprint requests to Dr. Jun Zhang, Peking University Health Science Center, 38 Xue Yuan Road, Hai Dian District, Beijing 100191, first implicated in Smad degradation. Smurf1 and Smurf2 can People’s Republic of China. E-mail address: [email protected] bind to TGF-b family receptors via the inhibitory Smads, Smad6 Abbreviations used in this article: HA, hemagglutinin; HECT, homologous to the and Smad7, to induce their ubiquitin-dependent degradation (17). E6-accessory protein C terminus; IKK, IkB kinase; IRF, IFN regulatory factor; ISRE, Recently, diverse substrates were reported for Smurf2, including IFN-stimulated response element; MDA-5, melanoma differentiation–associated gene 5; PCBP, poly(rC) binding protein; poly(I:C), polyinosinic–polycytidylic acid; Runx2, Rap1B, Smurf1, and Id1, suggesting that Smurf2 may RIG-I, retinoic acid–induced gene I; RLR, RIG-I–like receptor; SeV, Sendai virus; be involved in many biological processes (18–20). siRNA, small interfering RNA; Smurf, Smad ubiquitin regulatory factor; TBK1, In this study, we identified Smurf2 as a novel negative regulator TANK-binding kinase 1; WT, wild-type. in RIG-I–mediated antiviral signaling. Overexpression of Smurf2 Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 inhibited Sendai virus (SeV) or polyinosinic–polycytidylic acid www.jimmunol.org/cgi/doi/10.4049/jimmunol.1302632 2 NEGATIVE REGULATION OF VIRUS-INDUCED SIGNALING BY Smurf2 [poly(I:C)]–induced IFN-stimulated response element (ISRE) and 59-TGGAAAGGGTAAGACCGTCCT-39,reverse,59-GGTGTCCGTGACTAA- IFN-b activation, whereas knockout or knockdown of Smurf2 CTCCAT-39;mouseIfnb1,forward,59-GGCAGTGTAACTCTTCTGCAT-39, 9 9 exerted the opposite effects and promoted SeV-induced signaling. reverse, 5 -CAGCTCCAAGAAAGGACGAAC-3 . Further studies demonstrated that Smurf2 physically interacted Coimmunoprecipitation and immunoblot analysis with VISA and promoted the K48-linked ubiquitination and For transient transfection and coimmunoprecipitation experiments, degradation of Smurf2. HEK293T cells were transfected for 18 h. Then cells were lysed in lysis buffer containing proteinase inhibitor mixture (Roche) and PMSF. Cell lysates were incubated with 1 mg/ml anti-HA Ab or anti-Flag or control Ig Materials and Methods (IgG) and protein A-Sepharose (GE Healthcare) and resolved by SDS- Cells and reagents PAGE. The blot was then probed with anti-Flag or anti-HA Ab. IRDye HEK293T, HeLa, and MEF cells were cultured with high-glucose DMEM 700–conjugated anti-IgG or HRP-conjugated anti-IgG was used as a sec- (Life Technologies) medium plus 10% heat-inactivated FBS (Life Tech- ondary Ab, and proteins were identified using the Odyssey imaging system nologies) and supplemented with antibiotics (100 U/ml peniclillin, 100 mg/ml or detected by ECL assay. For endogenous coimmunoprecipitation, HeLa streptomycin; Life Technologies). Immortalized Smurf2-deficient and cells were left infected or uninfected by SeV, then the cells were lysed and wild-type (WT) MEF cells were provided by Dr. Hong Zhang (University immunoprecipitated by IgG control or anti-Smurf2 Ab, and then the blots of Massachusetts Medical School). Cells were grown at 37˚C in a humid- were detected by anti-VISA Ab. ified atmosphere with 5%