The Levels of Retinoic Acid-Inducible Gene I Are Regulated by Heat Shock Protein 90- α Tomoh Matsumiya, Tadaatsu Imaizumi, Hidemi Yoshida, Kei Satoh, Matthew K. Topham and Diana M. Stafforini This information is current as of October 2, 2021. J Immunol 2009; 182:2717-2725; ; doi: 10.4049/jimmunol.0802933 http://www.jimmunol.org/content/182/5/2717 Downloaded from References This article cites 44 articles, 19 of which you can access for free at: http://www.jimmunol.org/content/182/5/2717.full#ref-list-1 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 2, 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 © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology The Levels of Retinoic Acid-Inducible Gene I Are Regulated by Heat Shock Protein 90-␣1 Tomoh Matsumiya,*‡ Tadaatsu Imaizumi,‡ Hidemi Yoshida,‡ Kei Satoh,‡ Matthew K. Topham,*† and Diana M. Stafforini2*† Retinoic acid-inducible gene I (RIG-I) is an intracellular pattern recognition receptor that plays important roles during innate immune responses to viral dsRNAs. The mechanisms and signaling molecules that participate in the downstream events that follow activation of RIG-I are incompletely characterized. In addition, the factors that define intracellular availability of RIG-I and determine its steady-state levels are only partially understood but are likely to play a major role during innate immune responses. It was recently reported that the antiviral activity of RIG-I is negatively regulated by specific E3 ubiquitin ligases, suggesting participation of the proteasome in the regulation of RIG-I levels. In this study, we used immunoprecipitation combined with mass spectrometry to identify RIG-I-interacting proteins and found that RIG-I Downloaded from forms part of a protein complex that includes heat shock protein 90-␣ (HSP90-␣), a molecular chaperone. Biochemical studies using purified systems demonstrated that the association between RIG-I and HSP90-␣ is direct but does not involve participation of the CARD domain. Inhibition of HSP90 activity leads to the dissociation of the RIG-I-HSP90 complex, followed by ubiquitination and proteasomal degradation of RIG-I. In contrast, the levels of RIG-I mRNA are unaffected. Our studies also show that the ability of RIG-I to respond to stimulation with polyinosinic:polycytidylic acid is abolished when its interaction with HSP90 is inhibited. These novel findings point to HSP90-␣ as a chaperone that shields RIG-I from http://www.jimmunol.org/ proteasomal degradation and modulates its activity. These studies identify a new mechanism whose dysregulation may seriously compromise innate antiviral responses in mammals. The Journal of Immunology, 2009, 182: 2717–2725. iral infection leads to the initiation of complex innate IPS-1/MAVS/VISA/Cardif, resulting in the induction of IFNs and immune responses that result from recognition of the the activation of antiviral responses (6–9). In previous work, we V viral nucleic acid by cellular receptors including TLRs reported that, aside from its recognized role as a viral sensor, (1), followed by host cell secretion of antiviral factors such as RIG-I has additional functions. We found that RIG-I is a transcrip- cytokines (2, 3). Additional mechanisms are responsible for the tional activator of the cyclooxygenase 2 gene and that its expres- activation of the IFN response during viral infections (4). We re- sion is enhanced following stimulation of endothelial cells with by guest on October 2, 2021 cently reported that a cytoplasmic RNA helicase, retinoic acid- inflammatory agents (10). inducible gene I (RIG-I),3 is an essential regulator of dsRNA-in- RIG-I is the subject of active investigations aimed at dissecting duced signaling (5). RIG-I is also known as Ddx58 owing to the its precise function and mechanism of action in viral immunity. fact that this protein belongs to the DExH box-containing helicase Factors likely to play a key role in these responses include those family; it harbors two caspase recruitment domains (CARD) at the that affect expression levels, location, stability, and posttransla- amino-terminal end and an RNA helicase motif at the carboxyl tional modifications, all of which can potentially modulate the abil- terminus (5). The CARD domains are responsible for activating ity of RIG-I to affect cellular functions. Zhao et al. (11) recently subsequent downstream signaling events through interactions with reported that RIG-I becomes conjugated to IFN-regulated gene 15 (ISG15)/ubiquitin cross-reacting protein, a 15-kDa ubiquitin-like protein expressed following cellular stimulation with IFN. ISG15 *Huntsman Cancer Institute and †Department of Internal Medicine, University of becomes conjugated to a wide array of cellular proteins. Several of Utah, Salt Lake City, UT 84112; and ‡Department of Vascular Biology, Institute of ␣ ␤ Brain Sciences, Hirosaki University Graduate School of Medicine, Hirosaki City, the targets, including RIG-I, are IFN- / -induced antiviral pro- Japan teins, but most are constitutively expressed proteins that function Received for publication September 4, 2008. Accepted for publication December in diverse cellular pathways, including RNA splicing, chromatin 29, 2008. remodeling/polymerase II transcription, cytoskeletal organization The costs of publication of this article were defrayed in part by the payment of page and regulation, stress responses, and translation (11). The precise charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. functional consequences of ISG15 modification remain to be es- tablished but, unlike ubiquitin, ISG15 does not appear to target 1 This work was supported by the Huntsman Cancer Foundation, by a grant from the National Institutes of Health (P01-CA73992) to D.M.S., and by Cancer Center Sup- proteins for proteasomal degradation (12, 13). In addition to port Grant P30 CA042014-20. ISG15-mediated derivatization, RIG-I has been shown to undergo 2 Address correspondence and reprint requests to Dr. Diana M. Stafforini, Huntsman two types of ubiquitin-dependent modifications that result in re- Cancer Institute, 2000 Circle of Hope, Suite 3364, University of Utah, Salt Lake City, UT 84112. E-mail address: [email protected] markably different effects on functional properties. Gack et al. (14) recently showed that RIG-I is robustly ubiquitinated at its amino- 3 Abbreviations used in this paper: RIG-I, retinoic acid-inducible gene I; CARD, caspase recruitment domain; HSP, heat shock protein 70; ISG15, IFN-regulated gene terminal CARD domains by interacting with the E3 ubiquitin li- 15; IPS-1, IFN-␤ promoter stimulator 1; poly(I:C), polyinosinic:polycytidylic acid; gase TRIM, a member of the tripartite motif protein family, and HMG-1, high-mobility group protein 1. that this process is necessary for RIG-I-mediated IFN-␤ produc- Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 tion and antiviral activity in response to infection. This finding www.jimmunol.org/cgi/doi/10.4049/jimmunol.0802933 2718 HSP90 PROTECTS RIG-I FROM PROTEASOMAL DEGRADATION indicates that ubiquitination is a RIG-I modification that can modulate site (shown in lowercase font) or a SalI site (shown in lowercase font), the its signaling properties. Conversely, Arimoto et al. (15) reported that sequences of which were: 5Ј-GAC AAG CTT gcg gcc gcG ATG ACC Ј Ј conjugation of RIG-I with ubiquitin targets RIG-I for degradation by ACC GAG CAG CGA CGC-3 (RIG-FL-F (sense)) and 5 -TCC TCT AGA gtc gac TCA TTT GGA CAT TTC TGC TGG-3Ј (RIG-FL-R (anti- the proteasome, thus effectively acting as a negative regulator of sense)). The products were purified, digested with NotI and SalI, and then RIG-I. These combined observations point to the existence of at least cloned into a p3XFLAG-CMV7.1 expression vector. Positive clones were three tightly regulated protein conjugation mechanisms that control subjected to automated DNA-sequencing analyses. We next generated var- both the stability and signaling functions of RIG-I. ious RIG-I deletion mutants by amplification of full-length RIG-I with the following primers pairs: ⌬3(1–640), RIG-FL-F (sense) and 5Ј-AGA gtc The goal of the present study was to obtain additional insights gac CAC AAG TGC TCT GGT TTT CAC-3Ј (antisense); RIG-CARD(1– on the mechanisms that contribute to the regulation of RIG-I func- 238), RIG-FL-F (sense) and 5Ј-AGA gtc gac GTA CAA GTT TGT ATC tion and expression levels. We found that in resting cells RIG-I is AGA CAC-3Ј (antisense); and ⌬CARD(239–925), 5Ј-CTT gcg gcc gcG a component of a protein complex that also includes the molecular AGC CCA TTT AAA CCA AGA AAT-3Ј (sense) and RIG-FL-R (anti- ⌬ Ј chaperone heat shock protein (HSP) 90-␣. In addition, our studies sense); 10(452–925), 5 -CTT gcg gcc gcG GTT TAT AAG CCC CAG AAG TTT-3Ј (sense) and RIG-I-FL-R (antisense). The PCR products were showed that the association between RIG-I and HSP90 is direct purified by electrophoresis on agarose gels, digested with NotI and SalI, and that inhibiting the interaction between these proteins leads to cloned into the p3XFLAG-CMV7.1 expression vector, and analyzed by ubiquitination and proteasomal degradation of RIG-I.