Α Are Regulated by Heat Shock Protein 90

Home , Hsp90, HSP90AA2

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 September 24, 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 September 24, 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- by guest on September 24, 2021 activation of the IFN response during viral infections (4). We re- sion is enhanced following stimulation of endothelial cells with 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. Importantly, automated sequencing. disrupting the integrity of the RIG-I-HSP90 complex also inhibits For studies that required the use of purified, full-length RIG-I, we amplified cDNA isolated from HeLa cells using primers RIG-I-XhoI for key functional responses mediated by RIG-I. We conclude that BacPAK8-F (5Ј-GC ctc gag GCA GAG GCC GGC ATG ACC ACC GAG- partnership between RIG-I and HSP90 is likely to affect the func- 3Ј) and RIG-I-6xHis-NotI for BacPAK 8-R (5Ј-AAg cgg ccg cTC AGT tion of RIG-I in vivo. GAT GGT GAT GAT GAT GTT TGG ACA TTT CTG CTG GAT CA- Downloaded from 3Ј); the reverse primer included a 6xHis tag for purification purposes. The Materials and Methods product was digested with XhoI and NotI and then cloned into the BacPAK8 expression vector to generate pBacPAK8-6xHis-RIG-I. We co- Materials transfected pBacPAK6 viral DNA and pBacPAK8-6xHis-RIG-I into Sf21 Cycloheximide, a protease inhibitor mixture, FLAG-agarose, anti-FLAG insect cells, cultured the cells for 3 days at 27°C, collected the virus- Abs, the expression vector p3XFLAG-CMV7.1, polyinosinic-polycytidylic containing supernatant, and plated this fraction on a fresh monolayer of Sf21 cells to generate individual plaques that were subsequently amplified acid (poly(I:C)), His-tagged human high-mobility group protein 1 (HMG- http://www.jimmunol.org/ 1), creatine kinase, and creatine phosphate were purchased from Sigma- in Sf21 cells. We harvested the cellular proteins using equilibration/wash Aldrich. We obtained a metal-affinity purification system (TALON), the buffer (50 mM sodium phosphate and 300 mM NaCl (pH 7.0)) containing transfer vector pBacPAK8, and the pBacPAK6 viral DNA from Clontech protease inhibitors and purified RIG-I by metal-affinity chromatography on Laboratories. The ␤-galactosidase expression vector pSV40-␤-gal was ob- a TALON resin according to the manufacturer’s instructions. We eluted tained from Promega and the bacterial expression vector pGEX-5X-1 and 6xHis-tagged RIG-I with 20 mM Tris-HCl (pH 8.0) containing 100 mM glutathione-Sepharose 4B were from Amersham Biosciences. Factor Xa NaCl and 75 mM imidazole, dialyzed the preparation against 10 mM Tris- was from Qiagen and Lipofectamine Reagent, FBS, Sf21 insect cells, HCl (pH 8.0), and subjected it to immunoblot analysis. DNase I, Platinum Pfx DNA polymerase, and TRIzol were from Invitro- We also expressed various domains of RIG-I and the full-length recom- gen. Polyvinylidene fluoride membranes and chemiluminescence detection binant protein in the bacterial expression vector pGEX-5X-1 per the in- reagents were from PerkinElmer Life Sciences. Protein content was as- structions provided by the manufacturer. We amplified cDNA isolated

Ј by guest on September 24, 2021 sessed using a Pierce protein assay kit. Reagents for cDNA synthesis were from HeLa cells using primers BamHI-Card-F (5 -C GTG ggat ccc CAT Ј Ј from Fermentas and iQ SYBR Green Supermix was from Bio-Rad. Valeant GAC CAC CGA GCA GCG A-3 ) and XhoI-Card-R (5 -CG ctc gag GTG Pharmaceuticals provided an anti-actin mouse mAb. Anti-HSP90, anti- ATG ATG ATG ATG ATG CAA GTT TGT ATC AGA CAC); EcoRI- rpS6, and protein A/G-agarose were purchased from Santa Cruz Biotechnol- DexH-F (CC gaa ttc AGC CCA TTT AAA CCA AGA) and XhoI-DexH-R ogy. Cell Signaling Technology provided anti-JNK1/2 Abs. We obtained (CG ctc gag GTG ATG ATG ATG ATG ATG AAC TTG CTC CAG TTC HRP-conjugated secondary Abs from BioSource International; the anti-RIG-I CTC); EcoRI-452–641-F (CC gaa ttc TAT AAG CCC CAG AAG) and antiserum was previously described (10) and the anti-ubiquitin Ab was from XhoI-452–641-R (CG ctc gag GTG ATG ATG ATG ATG ATG CAC Novagen. Geldanamycin was from BIOMOL and MG-132 and HSP90 were AAG TGC TCT GGT TTT); and EcoRI-helicase-F (CC gaa ttc GAC GCT from Calbiochem. Alexa Fluor 488-conjugated anti-rabbit and Texas Red- TTA AAA AAT TGG) and XhoI-helicase-R (CG ctc gag GTG ATG ATG conjugated anti-mouse IgGs were from Molecular Probes. We obtained ATG ATG ATG TTT GGA CAT TTC TGC TGG). Bacterial pellets were rabbit reticulocyte lysates from Hokudo. resuspended in PBS containing 0.5% Triton X-100 and then incubated with glutathione-Sepharose 4B beads for 30 min at 4°C. The beads were washed Cellular studies twice with PBS and twice with factor Xa buffer (20 mM Tris (pH 7.4), 50 ␮ mM NaCl, 1 mM CaCl2). After resuspension in 500 l of factor Xa buffer, HeLa and HEK293T cells were maintained in a 5% CO2 atmosphere at 37°C we added 5 ␮l of factor Xa and incubated the slurry for3hatroom in DMEM supplemented with 10% FBS. HT-29 cells were maintained in a 5% temperature. The supernatants were subjected to chromatography on a CO2 atmosphere at 37°C in McCoys’ 5A medium supplemented with 1% TALON resin as described above. FBS. HSP90 inhibition was accomplished by treating monolayers with geldanamycin (0–400 nM) for the indicated time periods. MG-132 (up to10 In vitro binding and ubiquitination assays ␮M, 6 h) was used to inhibit proteasomal activity. Unless otherwise stated, we washed treated cells twice with PBS and solubilized the monolayers in cell To assess whether direct interaction(s) accounted for the association be- lysis buffer. After one cycle of freezing and thawing, the lysates were cleared tween HSP90 and RIG-I, we mixed 1 pmol each of recombinant HSP90 by centrifugation at 12,000 ϫ g for 2 min at 4°C. We subjected 50 ␮gof and either His-tagged RIG-I or His-tagged HMG-1 as a control and incu- protein extracts to electrophoresis as described below. bated the mixtures in binding buffer (200 mM NaCl, 50 mM HEPES, 1% BSA (pH 7.4)) for 30 min at 30°C in a total volume of 20 ␮l. We then Quantitative RT-PCR added a suspension of TALON resin in PBS containing 0.5% Triton X-100 ␮ We subjected DNase I-treated RNA (1 ␮g) to reverse transcription using a (final volume: 500 l) and rocked the mixtures for 60 min at 4°C. The cDNA synthesis kit according to the instructions provided by the manu- beads were extensively washed with PBS containing 0.5% Triton X-100 ϫ facturer. A Chromo4 Real-Time PCR Detection System (Bio-Rad) was and treated with 2 SDS-PAGE sample buffer. We subjected the eluted used for quantitative assessment of RIG-I and GAPDH as previously de- proteins to electrophoresis on SDS-PAGE, followed by immunoblot anal- scribed (16). yses using anti-HSP90 and anti-RIG-I Abs. To assess geldanamycin-related effects on ubiquitination of RIG-I, Construct generation, expression, and purification of we incubated 1 pmol of His-tagged RIG-I in 50 mM Tris-HCl (pH 7.4), recombinant proteins 20 mM MgCl2, 2 mM ATP, 10 mM creatine phosphate, and 10 IU/ml creatine kinase with a rabbit reticulocyte lysate (33.3%) in the presence We extracted total RNA from HEK293T cells and subjected the samples to or absence of geldanamycin (400 nM) for 60 min at 30°C. The total reverse transcription as previously described (16). For amplification, we volume was adjusted to 20 ␮l. His-tagged RIG-I species were isolated used Platinum Pfx DNA polymerase and specific primers harboring a NotI by affinity chromatography using TALON beads, as above, and then The Journal of Immunology 2719 analyzed by electrophoresis on SDS-PAGE, followed by immunoblot Results analyses using anti-ubiquitin and anti-RIG-I Abs. Identification of heat shock proteins as partners of RIG-I Immunoprecipitation studies Our initial goal was to identify protein partners that associated HeLa cells subjected to various treatments were washed twice with ice- with RIG-I under basal conditions. We used HT-29, a human cold PBS and then lysed in immunoprecipitation buffer (20 mM Tris-HCl colonic adenocarcinoma cell line that expresses high levels of

(pH 7.4), 100 mM NaCl, 1.5 mM MgCl2, and 1% Triton X-100) containing RIG-I (our unpublished observations). We subjected HT-29 ly- 0.5% protease inhibitor mixture for 30 min on ice. The lysates were cen- sates to immunoprecipitation using anti-RIG-I polyclonal Abs ϫ trifuged at 12,000 g for 10 min at 4°C, and the pellets were discarded. or control IgG. After electrophoretic separation of the immu- The supernatant fractions were precleared by incubation with normal IgG and protein A/G-plus for 2 h. Immunoprecipitations were performed by noprecipitated proteins, we noted the presence of multiple spe- overnight rocking of the precleared supernatants with 1 ␮l of the primary cies that specifically associated with RIG-I (Fig. 1A). We ini- Ab indicated in each case at 4°C. The Ag-Ab complexes were harvested by tially focused our attention on the identification of RIG-I- incubation with protein A/G-agarose for1hat4°C. FLAG-tagged proteins interacting proteins that ranged in size between 70 and 110 kDa, were harvested by overnight incubation with FLAG-agarose beads at 4°C. After extensive washing with ice-cold PBS, the beads were boiled for 5 as these represented a considerable proportion of the immuno- min in 2ϫ SDS-PAGE sample buffer to dissociate the immunoprecipitated precipitated material (Fig. 1A). We analyzed various species proteins. These fractions were analyzed by electrophoresis on SDS-PAGE using mass spectrometry and identified the two largest proteins and immunoblot analyses as described below. Studies involving coimmu- (100 and 110 kDa) as RIG-I (data not shown). These results noprecipitation of endogenous proteins were conducted on the human co- validated the specificity of our immunoprecipitation approach lon cancer cell line HT-29 using essentially the same approach, except that and suggested that the smaller, less abundant 100-kDa species immunoprecipitations were performed by overnight rocking of the pre- Downloaded from cleared supernatants with 1 ␮l of an anti-RIG-I antiserum at 4°C. The was a proteolytic fragment derived from full-length RIG-I (Mr complexes were harvested by incubation with protein A/G-plus for1hat ϭ 110 kDa). We next analyzed the composition of the coim- 4°C. After extensive washing with ice-cold PBS containing 0.5% Triton munoprecipitated 90- and 70-kDa proteins (Fig. 1A, arrow and X-100, the beads were boiled for 5 min in 2ϫ SDS-PAGE sample buffer and the eluted proteins were subjected to SDS-PAGE and immunoblot asterisk, respectively). We found that the 90-kDa RIG-I-inter- analyses. acting protein harbored peptides that precisely matched sequences present in human HSP90-␣ (Table I). Our studies also Immunoblot analyses revealed the presence of a 70-kDa protein in immunoprecipi- http://www.jimmunol.org/ We subjected the protein extracts indicated in each case to electrophoresis tates generated using both control IgG and anti-RIG-I Abs (Fig. on 8.5% SDS-PAGE gels and then transferred the proteins to polyvinyli- 1A). The amount of 70-kDa protein in the latter appeared to be dene difluoride membranes that were blocked for 60 min at room temper- much higher than that observed in the control lane, suggesting ature in 1ϫ TBST buffer (50 mM Tris-HCl (pH 7.5), 250 mM NaCl, and that RIG-I may specifically interact with this protein. 0.1% Tween 20) containing either 3% nonfat dry milk or 3% BSA. The Additional studies revealed that the 70-kDa protein was heat membranes were incubated for 60 min at room temperature with the primary Ab indicated in each case. After five washes with blocking solution, shock 70-kDa protein 8 (also known as HSPA8 and HSP70; we added a HRP-conjugated secondary Ab, incubated the membranes for Table I). This conclusion was drawn based on the sequence of 60 min at room temperature, repeated the washes using TBST, and then seven peptides identified by mass spectrometric analyses and by guest on September 24, 2021 visualized the immunoreactive bands using chemiluminescence detection that precisely matched sequences found in the 70-kDa human reagents. chaperone (Table I). Our data supported the existence of in Immunofluorescence analyses vivo partnerships between RIG-I and select heat shock proteins. HeLa cells grown on glass coverslips were rinsed in PBS, fixed with 4% formaldehyde for 20 min, permeabilized with 0.1% Triton X-100 for 10 min, and blocked with 5% BSA for 1 h. All solutions were prepared in Molecular characterization of RIG-I-HSP90 interaction PBS. The cells were then incubated for 1 h with a polyclonal anti-RIG-I Ab We next focused on the characterization of the interaction be- (1/250-fold dilution) and a monoclonal anti-HSP90 Ab (1/100). After a washing step, we incubated the coverslips with Alexa Fluor 488–conju- tween HSP90 and RIG-I. To address this issue, we used mo- gated anti-rabbit and Texas Red-conjugated anti-mouse IgGs. Excess re- lecular, immunohistological, and biochemical approaches. agents were washed with PBS and the expression levels of RIG-I and First, transfection of HEK293T cells with FLAG-tagged RIG-I HSP90 were analyzed by confocal microscopy (Olympus FV300). followed by immunoprecipitation using anti-FLAG or anti- HSP90 Abs revealed that endogenous HSP90 interacted with Mass spectrometry exogenous RIG-I (Fig. 1, B and C). We next considered whether HeLa cell proteins that coimmunoprecipitated with RIG-I were resolved on the interactions observed in transfected cells were the conse- SDS-PAGE and identified by staining, and individual lanes were subjected quence of forced expression of RIG-I and did not represent a to in-gel trypsin digestion at the Mass Spectrometry Core Facility (Uni- versity of Utah). The tryptic peptides were identified by mass spectrometry naturally occurring association between endogenous proteins. (MS)/MS studies and analyzed using MASCOT software (Matrix Science). To address this issue, we tested the ability of endogenous RIG-I and HSP90, both of which have been reported to be expressed Reporter assays in the cytoplasm, to colocalize under basal conditions (14, 17). To generate an IFN-␤ reporter vector that spanned nt Ϫ299 to ϩ19, we Although no particular cellular structure was highlighted, we amplified HeLa cell genomic DNA with a sense primer harboring a 5Ј-XhoI observed that in HeLa cells HSP90 and RIG-I were expressed in site (XhoI-IFN-␤-299, 5Ј-GG ctc gag AGT GTT TTG AGG TTC TTG the same cytoplasmic region, suggesting that the proteins Ј Ј A-3 ) and an antisense primer designed with a 5 -HindIII site (HindIII- formed part of the same complex (Fig. 1D). In addition, immu- IFN-␤ϩ19, 5Ј-GCC aag ctt AAG GTT GCA GTT AGA ATG T-3Ј). We cloned the product in the pGL3-basic vector and cotransfected 300 ng of noprecipitation studies confirmed that endogenous RIG-I inter- the plasmid with 50 ng of pSV40-␤-gal and 100 ng of a p3XFLAG7.1 acted with endogenous HSP90 (Fig. 1E), indicating that the expression vector (RIG-I full length or RIG-CARD) using Lipofectamine. association between these proteins occurs in intact cells and is The following day we transfected the cells with 200 ng of poly(I:C) using not an artifact of overexpression systems. Lipofectamine 2000 and incubated the cells in the presence or absence of 200 nMf geldanamycin for 24 h. We harvested the cells in 200 ␮lof We next asked whether RIG-I and HSP90 interacted directly reporter lysis buffer and assessed luciferase and ␤-galactosidase activities or whether participation of an additional protein(s) was required in the extracts (n ϭ 4). for association. To test this, we conducted in vitro experiments 2720 HSP90 PROTECTS RIG-I FROM PROTEASOMAL DEGRADATION

FIGURE 1. RIG-I associates with HSP90.A, Cell lysates from HT-29 cells were immunoprecipitated with control IgG (left lane) or with an anti-RIG-I Ab (right lane) and proteins associated with the RIG-I immune complex were resolved by SDS-PAGE (7.5%). The arrow and asterisk indicate the relative mobilities of HSP90 and HSP70, respectively. B and C, A plasmid encoding FLAG-tagged RIG-I was transfected into HEK293T cells. Protein-protein interactions were monitored by coimmunoprecipitation using anti-FLAG-conjugated agarose followed by immunoblotting with anti-HSP90 Ab (B) and by coimmunoprecipitation using anti-HSP90 Abs followed by immunoblotting with an anti-FLAG Ab (C). D, HeLa cells were immunostained with anti-RIG-I or control rabbit IgG (green; left) or with anti-HSP90 or control mouse IgG (red; center). The left and center panels represent sequen- tial confocal images of the same ﬁeld. The right panel depicts an overlay of the images. E, Interaction between endogenous RIG-I and HSP90 was assessed by subjecting lysates from HT-29 cells to immunoprecipitation with anti-RIG-I or control IgG. The immune complexes were isolated by incubation Downloaded from with protein A/G-plus and eluted from the beads with 2ϫ SDS-PAGE buffer. The eluted proteins were subjected to electrophoresis on SDS-PAGE and immunoblot analyses using anti-RIG-I and anti-HSP90 Abs. F, Puriﬁed recombinant HSP90 was incubated with buffer (left panel), His-tagged RIG-I (center panel), or His-tagged HMG-1 as a control

(right panel). The resulting mixtures were subjected to afﬁnity http://www.jimmunol.org/ puriﬁcation, and the bound proteins were eluted and subjected to immunoblot analyses using anti-HSP90 and anti-His-tag Abs.

with purified recombinant proteins. We found that incubation of To further explore this issue and to assess whether additional purified, His-tagged RIG-I with recombinant HSP90 resulted in RIG-I domains also contributed to its interaction with HSP90, the association between the proteins, as shown by the binding of we expressed His-tagged constructs harboring various regions by guest on September 24, 2021 both species to a His-tag purification resin (Fig. 1F). Control of RIG-I (Fig. 3A). We found that the constructs were expressed experiments revealed no binding of HSP90 to the beads in the to various extents (Fig. 3B, bottom panel). The CARD domain, absence or presence of a control His-tagged protein (HMG-1; which was efficiently expressed, lacked the ability to interact Fig. 1F). These combined studies provide evidence indicating with HSP90. Moreover, deletion of the CARD domain did not that the association between RIG-I and HSP90 involves direct affect association with HSP90. These combined studies are in interaction(s) between the proteins. complete agreement with our results using FLAG-tagged constructs (Fig. 2B) and they firmly establish that the CARD do- Identification of domains required for interaction between main does not participate in the interaction between RIG-I and HSP90 and RIG-I HSP90. Interestingly, our studies also showed that all of the Our next goal was to identify RIG-I domains involved in its remaining RIG-I domains retained the ability to bind HSP90 interaction with HSP90. In previous studies, it was demon- (Fig. 3B), suggesting that more than one domain participates in strated that the ability of RIG-I to interact with the adaptor the interaction between the proteins. protein IFN-␤ promoter stimulator 1 (IPS-1) and the ubiquitin ligases RNF125 and TRIM25 depended on the presence of the amino-terminal CARD domain of RIG-I (5, 6, 14, 15). Second, HSP90 affects the levels of RIG-I protein it was recently shown that the CARD domain is the primary HSP90 is primarily known as a molecular chaperone owing to binding site of HSP90-␤ on Apaf-1 (18). To systematically an- its ability to ensure the stability and correct conformation, ac- alyze individual contributions, we generated four FLAG-tagged tivity, intracellular localization, and proteolytic cleavage of a deletion mutants harboring various domains of RIG-I (Fig. 2A). range of proteins involved in cell growth, differentiation, and We transfected HEK293T cells with each deletion construct and survival (19). These include key signaling molecules such as found robust expression of all species (Fig. 2B, left panel). ERBB2, AKT, Raf, hypoxia inducible factor 1␣ and steroid Next, we subjected cell lysates from transfected cells to immu- hormone receptors (20). To investigate the functional conse- noprecipitation using anti-HSP90 Abs and found that the CARD quences of HSP90/RIG-I association, we destabilized the inter- domain lacked the ability to associate with HSP90 (Fig. 2B, action using pharmacological approaches. Geldanamycin is a right panel). The remaining deletion constructs interacted with naturally occurring ansamycin antibiotic and antitumor agent HSP90 in an efficient manner, suggesting that a domain com- that binds tightly to the HSP90 ATP/ADP pocket and prevents mon to these constructs was necessary for the interaction. An association of client proteins (20, 21). Treatment with geldana- examination of the deletion mutants tested in this study allowed mycin prevented association between transfected RIG-I and us to conclude that the region comprised between aa 452–641 HSP90 (Fig. 4A) and significantly reduced the levels of endog- was involved in the association between RIG-I and HSP90. enous RIG-I protein in a time-dependent manner (Fig. 4B). To The Journal of Immunology 2721

Table I. Mass spectrometric identiﬁcation of HSP90-␣ and HSP70 protein 8

Protein Tryptic Peptide Sequences Size Identiﬁed by MS/MS Identiﬁed Protein Location in protein

90 kDa ADLINNLGTIAKVILHLK HSP90-␣ (HSP90AA1, HSP90AA2, MPEETQTQDQPMEEEEVETFAFQAEIAQLMSLIINTFYSNK EDQTEYLEERNPDDITNE HSP90A, HSP90N, HSPC1, EIFLRELISNSSDALDKIRYESLTDPSKLDSGKELHINL EYGEFYKGVVDSEDLPLN HSPCA, HSPCAL1, HSPCAL3, IPNKQDRTLTIVDTGIGMTKADLINNLGTIAKSGTKAFM ISR HSPCAL4, HSPN, HSP86, EALQAGADISMIGQFGVGFYSAYLVAEKVTVITKHNDDE HSP89, HSP90,LAP2) QYAWESSAGGSFTVRTDTGEPMGRGTKVILHLKEDQTEY LEERRIKEIVKKHSQFIGYPITLFVEKERDKEVSDDEAE EKEDKEEEKEKEEKESEDKPEIEDVGSDEEEEKKDGDKK KKKKIKEKYIDQEELNKTKPIWTRNPDDITNEEYGEFYK SLTNDWEDHLAVKHFSVEGQLEFRALLFVPRRAPFDLFE NRKKKNNIKLYVRRVFIMDNCEELIPEYLNFIRGVVDSE DLPLNISREMLQQSKILKVIRKNLVKKCLELFTELAEDK ENYKKFYEQFSKNIKLGIHEDSQNRKKLSELLRYYTSAS GDEMVSLKDYCTRMKENQKHIYYITGETKDQVANSAFVE RLRKHGLEVIYMIEPIDEYCVQQLKEFEGKTLVSVTKEG LELPEDEEEKKKQEEKKTKFENLCKIMKDILEKKVEKVV VSNRLVTSPCCIVTSTYGWTANMERIMKAQALRDNSTMG YMAAKKHLEINPDHSIIETLRQKAEADKNDKSVKDLVIL LYETALLSSGFSLEDPQTHANRIYRMIKLGLGIDEDDPT Downloaded from ADDTSAAVTEEMPPLEGDDDTSRMEEVD 70 kDa TTPSYVAFTDTERSFYPE HSP70 protein 8 (HSPA8, HSC54, MSKGPAVGIDLGTTYSCVGVFQHGKVEIIANDQGNRTTPSY EVSSMVLTKTVTNAVVTV HSC70, HSC71, HSP71, VAFTDTERLIGDAAKNQVAMNPTNTVFDAKRLIGRRFDD PAYFNDSQRDAGTIAGL HSPA10. LAP1, NIP71) AVVQSDMKHWPFMVVNDAGRPKVQVEYKGETKSFYPEEV NVLRIINEPTAAAIAYGL SSMVLTKMKEIAEAYLGKTVTNAVVTVPAYFNDSQRQAT DKKFEELNADLFRSQI KDAGTIAGLNVLRIINEPTAAAIAYGLDKKVGAERNVLI

HDIVLVGGSTR FDLGGGTFDVSILTIEDGIFEVKSTAGDTHLGGEDFDNR http://www.jimmunol.org/ MVNHFIAEFKRKHKKDISENKRAVRRLRTACERAKRTLS SSTQASIEIDSLYEGIDFYTSITRARFEELNADLFRGTL DPVEKALRDAKLDKSQIHDIVLVGGSTRIPKIQKLLQDF FNGKELNKSINPDEAVAYGAAVQAAILSGDKSENVQDLL LLDVTPLSLGIETAGGVMTVLIKRNTTIPTKQTQTFTTY SDNQPGVLIQVYEGERAMTKDNNLLGKFELTGIPPAPRG VPQIEVTFDIDANGILNVSAVDKSTGKENKITITNDKGR LSKEDIERMVQEAEKYKAEDEKQRDKVSSKNSLESYAFN MKATVEDEKLQGKINDEDKQKILDKCNEIINWLDKNQTA EKEEFEHQQKELEKVCNPIITKLYQSAGGMPGGMPGGFP

GGGAPPSGGASSGPTIEEVD by guest on September 24, 2021 assess the efficacy of geldanamycin treatment, we took advan- once the interaction is inhibited (23). In control experiments, we tage of a study reported by Piatelli et al. (22) who showed that found that treatment with MG-132, an agent known to specifically geldanamycin selectively depletes cellular Raf-1, thus inter- inhibit proteasomal activity, led to substantial accumulation of rupting activation of MEK1/2 and ERK. In addition, these in- RIG-I (Fig. 5A). To specifically assess the contribution of HSP90 vestigators showed that geldanamycin does not affect the levels to the stability of RIG-I, we investigated whether the effects of of total ERK. We found that geldanamycin decreased basal lev- geldanamycin on RIG-I levels were affected when proteasomal els of phospho-ERK in cells grown in the presence of serum, activity was inhibited with MG-132. We found (Fig. 5A) that MG- thus confirming the efficacy of the treatment in our studies (Fig. 132 prevented geldanamycin-mediated RIG-I proteolysis, thus 4B). We next investigated whether these effects were the con- providing evidence for a role of HSP90 activity in the protection of sequence of altered rates of mRNA transcription or decreased RIG-I from proteasomal degradation. mRNA stability, as was shown for other proteins (21). How- Protein degradation through the proteasome commonly in- ever, quantitative assessment of RIG-I mRNA levels demon- volves conjugation with ubiquitin, and HSP90 has been shown strated that transcriptional effects did not account for the to utilize this mechanism in the stabilization of a variety of geldanamycin-mediated decrease in RIG-I protein levels (Fig. proteins. In addition, the degradation of RIG-I requires deriva- 4C). Higher concentrations of geldanamycin (Ͼ2 ␮M) were tization with ubiquitin (14, 15). To assess whether HSP90-me- avoided because these resulted in generalized cytotoxic effects diated effects on RIG-I involved the ubiquitin pathway, we de- (data not shown). Our results indicate that the chaperone activ- veloped an in vitro assay that allowed us to assess changes in ity of HSP90 protects RIG-I from proteolytic degradation, sug- the extent of ubiquitination of RIG-I. We incubated purified, gesting that this mechanism contributes to the definition of His-tagged RIG-I with a ubiquitination-competent rabbit reticu- steady-state levels of RIG-I. locyte system, isolated RIG-I using affinity chromatography, and then assessed the extent of ubiquitination by immunoblot HSP90 protects RIG-I from ubiquitination and proteasomal analyses. We found that under basal conditions, a fraction of degradation RIG-I was present as a high molecular mass ubiquitinated spe- Our next goal was to identify the protease system that partici- cies (Fig. 5B, top left panel). In addition, dissociation of the pates in the degradation of RIG-I following disruption of the RIG-I-HSP90 complex with geldanamycin stimulated RIG-I RIG-I-HSP90 complex. We hypothesized that degradation of ubiquitination, as demonstrated by robust increases in the extent RIG-I might involve the proteasome because most proteins known of RIG-I ubiquitination and by attenuated expression of underi- to be stabilized by HSP90 are degraded through this mechanism vatized RIG-I (Fig. 5B). We did not detect high-molecular mass 2722 HSP90 PROTECTS RIG-I FROM PROTEASOMAL DEGRADATION

data are consistent with a model whereby blockade of RIG-I-HSP90 interactions results in ubiquitination and proteasomal degradation of RIG-I.

The interaction between HSP90 and RIG-I is functionally important Our ﬁnal goal was to assess whether the interaction between HSP90 and RIG-I had functional consequences on known biological activities mediated by RIG-I. It was previously shown that RIG-I acts as an intracellular dsRNA receptor that regulates IFN regulatory factor 3 activation and IFN-␤ induction at the transcriptional level (24). Thus, we investigated whether the ability of RIG-I to transcriptionally activate the IFN-␤ gene required association with HSP90. We transfected 293T cells with a reporter construct harboring nt Ϫ299 to ϩ19 of the IFN-␤ gene combined with either empty vector, a FLAG-tagged construct harboring full-length RIG-I, or a FLAG-tagged CARD construct. We stimulated the cells with poly(I:C) which has been shown to be recognized by RIG-I (25) in the absence and presence of geldanamycin. We Downloaded from found that geldanamycin signiﬁcantly inhibited both basal and poly(I:C)-stimulated IFN-␤ promoter activation (Fig. 6), suggesting that the association between RIG-I and HSP90 was necessary FIGURE 2. The CARD domain is not required for association of RIG-I for this response. In addition, we found robust basal IFN-␤ pro- with HSP90. A, Schematic representation of RIG-I mutant constructs used moter activation mediated by the CARD domain of RIG-I that was in this portion of the study. B, HEK293T cells were transfected with the not affected by geldanamycin treatment (Fig. 6). These results in- http://www.jimmunol.org/ constructs indicated. Twenty-four hours after transfection, the cells were dicate that blocking the ability of RIG-I to interact with HSP90 harvested and the levels of expression of each construct were assessed by inhibits its biological activity. The fact that IFN-␤ promoter acti- immunoblot analyses (left panel). In addition, aliquots of the lysates were subjected to immunoprecipitation using anti-HSP90 Abs, followed by im- vation mediated by the CARD domain was not affected by munoblotting with an anti-FLAG Ab (right panel). forms of RIG-I using anti-RIG-I Abs, even after treatment with geldanamycin, suggesting that our RIG-I Abs were unable to recognize ubiquitinated forms of the protein. Our combined by guest on September 24, 2021

FIGURE 4. HSP90 stabilizes RIG-I. A, HEK293T cells were transfected with a plasmid encoding FLAG-tagged RIG-I. Twenty-four hours after transfection, we supplemented vehicle or geldanamycin (100 nM) for 2 h. Protein-protein interactions were monitored by immunoprecipitation using anti-FLAG-conjugated agarose beads, followed by immunoblotting with anti-HSP90 Abs. B, HeLa cells were treated with geldanamycin (100 FIGURE 3. Several RIG-I domains can mediate binding to HSP90. A, nM) for the indicated time periods and cell lysates were then subjected to Schematic representation of purified RIG-I mutant constructs used in this SDS-PAGE and immunoblotting using Abs against RIG-I, phospho-ERK, portion of the study. B, Purified recombinant HSP90 and the constructs HSP90, and actin. The numbers above this panel depict quantitative as- indicated were incubated with purified, full-length His-tagged RIG-I, or sessments of band intensity relative to actin controls performed using various purified His-tagged RIG-I domains. The resulting mixtures were Image-J. C, HeLa cells were treated with geldanamycin (100 nM) for the subjected to affinity purification, and the bound proteins were eluted and indicated time periods. Total RNA was then extracted and the levels of subjected to immunoblot analyses using anti-HSP90 and anti-His-tag Abs. RIG-I were assessed by quantitative RT-PCR. The Journal of Immunology 2723

FIGURE 5. HSP90 protects RIG-I from proteasomal degradation and ubiquitination. A, HeLa cells were treated with geldanamycin (400 nM) in the absence and presence of MG-132 (0–10 ␮M) for 16 h. The cells were harvested and the lysates were subjected to SDS-PAGE and immunoblot analyses using Abs against RIG-I, HSP90, and actin. The numbers below this panel depict quantitative assessments of band intensity. B, We incubated His-tagged RIG-I with a rabbit reticulocyte lysate in the presence (right panel) or absence (left panel) of geldanamycin (400 nM) for 60 min FIGURE 6. Inhibition of HSP90/RIG-I association abolishes RIG-I-me- at 30°C and then isolated RIG-I using afﬁnity chromatography on TALON ␤

diated transcriptional activation of the IFN- promoter. HEK293T cells Downloaded from beads. We eluted the bound proteins and subjected them to electrophoresis were cotransfected with a reporter construct harboring nt Ϫ299/ϩ19 of the on SDS-PAGE, followed by immunoblot analyses using anti-ubiquitin and IFN-␤ gene, a ␤-galactosidase expression vector, and either full-length anti-RIG-I Abs. RIG-I or the CARD domain. The following day we transfected one-half of the cells with poly(I:C) in the presence or absence of geldanamycin (GA) for 24 h; the remaining cells were treated with vehicle or geldanamycin. We assessed luciferase and ␤-galactosidase activity in the extracts (n ϭ 4).

geldanamycin is consistent with our studies (Figs. 2 and 3) show- http://www.jimmunol.org/ ing that the CARD domain is not required for association of RIG-I with HSP90. mains. Thus, it is apparent that HSP90 frequently associates with Discussion client proteins through multiple domains. RIG-I has been described as a pattern recognition receptor that Our studies also provide evidence suggesting that one of the detects intracellular dsRNA through its helicase domain in a TLR- functional consequences of the interaction between HSP90 and independent fashion (5, 6). A series of well-characterized down- RIG-I is the shielding of the latter from ubiquitin-dependent pro- stream signaling events that require the CARD domains of RIG-I teasomal degradation. The mechanisms that regulate the levels of lead to the activation of a family of IFN-induced genes, such as RIG-I protein under various conditions have been partially char- by guest on September 24, 2021 IFN regulatory factor 3, IFN regulatory factor 7, and NF-␬B, acterized and clearly require ubiquitin-mediated functions at dif- through the signaling adaptor protein IPS-1 (6). The ability of ferent levels. Arimoto et al. (15) reported that the ubiquitin ligases RIG-I to elicit downstream signaling events requires the formation RNF125 and UbcH8 are key determinants of RIG-I levels through of a macromolecular protein complex necessary for gene activa- their ability to catalyze the conjugation of RIG-I to ubiquitin or tion (26). This complex is most likely in dynamic equilibrium de- ISG15, respectively. These authors (30) described participation of pending on the state of cellular activation, viral stimulation, and the E2 and E3 ubiquitin ligases in a regulatory circuit, the function other factors. To identify novel proteins that associated with RIG-I of which is to control the levels of RIG-I protein. In addition, Lin under basal conditions, we used biochemical approaches that, et al. (31) found that A20, a virus-inducible protein that harbors when combined with mass spectrometric measurements, resulted two ubiquitin-editing domains, functions as a negative regulator of in the discovery of HSP90, and perhaps HSP70, as partners of RIG-I through indirect mechanisms that require degradation of an RIG-I. HSP90 is a ubiquitous, constitutively expressed, highly as yet unidentified adapter of the RIG-I pathway. These combined conserved molecular chaperone involved in the folding, activation observations reveal the existence of several control points to en- and assembly of key mediators of signal transduction, cell cycle sure that appropriate levels of RIG-I are expressed in response to control, and transcriptional regulation (27). This chaperone inter- changing cellular needs. acts with native and denatured partners such as protein kinases, In humans, two distinct genes, HSP90-␣ and HSP90-␤, encode transcription factors, viral replication proteins, and a range of in- closely related cytoplasmic proteins that display minor sequence tracellular receptors, affecting their turnover, trafficking, cellular differences at the amino termini (32–34). The isoforms differ in localization, and activity (28, 29). Recent studies by Kim et al. (23) their ability to activate certain client proteins and regulate cellular demonstrated that HSP90 associates with 40S ribosomal compo- functions of immune cells (34–38). The ␣ and ␤ isoforms of nents and prevents ubiquitin-dependent ribosomal protein degra- HSP90 often cooperate with other proteins to generate a cytosolic dation. Our data point to a similar mechanism in which partnership multichaperone machinery that includes HSP70, peptidyl-prolyl between HSP90 and RIG-I serves to stabilize this protein. In ad- isomerases, and other cochaperones to achieve adequate protein dition, we provide novel evidence showing that the interaction folding (39). Certain cytosolic client polypeptides have been between HSP90 and RIG-I is direct and independent of the CARD shown to first bind HSP70 and then interact with dimeric HSP90 domain. Interestingly, we found that the DexH Box, the helicase, (39–42). Interestingly, our studies suggest that HSP70 may be a and other domains of RIG-I efficiently associated with HSP90. second chaperone that interacts with RIG-I under basal conditions. These results suggest that the interaction between these proteins Our work did not directly address whether HSP70 facilitates the may involve multiple domains. Similar results were reported by formation or stability of the RIG-I-HSP90 complex, but it is tempt- Kim et al. (23) who showed that interaction of HSP90 with ribo- ing to speculate that HSP70 contributes to this process and that a somal protein S3 involves interaction with two independent do- cochaperone(s) may be involved as well. 2724 HSP90 PROTECTS RIG-I FROM PROTEASOMAL DEGRADATION

Pratt and Toft (17) have pointed out that many of the client fection switches non-plasmacytoid dendritic cells into high interferon producers. proteins of HSP90 are involved in signal transduction and specu- Nature 424: 324–328. 5. Yoneyama, M., M. Kikuchi, T. Natsukawa, N. Shinobu, T. Imaizumi, lated that HSP90 may facilitate access of its client proteins to M. Miyagishi, K. Taira, S. Akira, and T. Fujita. 2004. The RNA helicase RIG-I diverse intracellular sites and/or ligands (17), thus affecting their has an essential function in double-stranded RNA-induced innate antiviral responses. Nat. Immunol. 5: 730–737. ability to elicit biological activities. We hypothesized that the for- 6. Kawai, T., K. Takahashi, S. Sato, C. Coban, H. Kumar, H. Kato, K. J. Ishii, mation of a complex between RIG-I and HSP90 had important O. Takeuchi, and S. Akira. 2005. IPS-1, an adaptor triggering RIG-I- and Mda5- functional consequences additional to protecting RIG-I from pro- mediated type I interferon induction. Nat. Immunol. 6: 981–988. 7. Sen, G. C., and S. N. Sarkar. 2005. Hitching RIG to action. Nat. Immunol. 6: teasomal degradation. Indeed, we found that interfering with the 1074–1076. ability of HSP90 to associate with RIG-I prevented RIG-I-medi- 8. Xu, L. G., Y. Y. Wang, K. J. Han, L. Y. Li, Z. Zhai, and H. B. Shu. 2005. VISA ated transcriptional activation of the IFN-␤ promoter. An interest- is an adapter protein required for virus-triggered IFN-␤ signaling. Mol. Cell. 19: 727–740. ing observation resulting from our studies was the finding that the 9. Meylan, E., J. Curran, K. Hofmann, D. Moradpour, M. Binder, R. Bartenschlager, CARD domain of RIG-I, which does not bind to HSP90, transcrip- and J. Tschopp. 2005. Cardif is an adaptor protein in the RIG-I antiviral pathway tionally activated the IFN-␤ promoter in a robust, dsRNA-inde- and is targeted by hepatitis C virus. Nature 437: 1167–1172. 10. Imaizumi, T., S. Aratani, T. Nakajima, M. Carlson, T. Matsumiya, K. Tanji, pendent manner. A number of mechanisms could account for this K. Ookawa, H. Yoshida, S. Tsuchida, T. M. McIntyre, et al. 2002. Retinoic effect, but one possibility is that stimulation of the HSP90-RIG-I acid-inducible gene-I is induced in endothelial cells by LPS and regulates expression of COX-2. Biochem. Biophys. Res. Commun. 292: 274–279. complex with dsRNA results in conformational changes that fa- 11. Zhao, C., C. Denison, J. M. Huibregtse, S. Gygi, and R. M. Krug. 2005. Human cilitate CARD domain-mediated functions that are normally silent ISG15 conjugation targets both IFN-induced and constitutively expressed pro- in the absence of stimulation. Additional possibilities include teins functioning in diverse cellular pathways. Proc. Natl. Acad. Sci. USA 102: 10200–10205. HSP90-mediated regulation of the ability of RIG-I to respond 12. Malakhov, M. P., K. I. Kim, O. A. Malakhova, B. S. Jacobs, E. C. Borden, and Downloaded from and/or bind to ligands or to undergo protein modification. D. E. Zhang. 2003. High-throughput immunoblotting: ubiquitin-like protein Although HSP90 seems to play beneficial physiological roles, ISG15 modifies key regulators of signal transduction. J. Biol. Chem. 278: 16608–16613. this chaperone has been shown to stabilize oncogenic proteins, 13. Schwartz, D. C., and M. Hochstrasser. 2003. A superfamily of protein tags: ubiq- thus contributing to cancer progression (27). Increased expression uitin, SUMO and related modifiers. Trends Biochem. Sci. 28: 321–328. of HSP90 is associated with tumorigenesis and small molecule 14. Gack, M. U., Y. C. Shin, C. H. Joo, T. Urano, C. Liang, L. Sun, O. Takeuchi, S. Akira, Z. Chen, S. Inoue, and J. U. Jung. 2007. TRIM25 RING-finger E3

HSP90 inhibitors that include geldanamycin and its derivatives ubiquitin ligase is essential for RIG-I-mediated antiviral activity. Nature 446: http://www.jimmunol.org/ selectively kill certain types of cancer cells by promoting apoptosis 916–920. 15. Arimoto, K., H. Takahashi, T. Hishiki, H. Konishi, T. Fujita, and K. Shimotohno. (43). In addition, virus-mediated sequestration of HSP90 facilitates 2007. Negative regulation of the RIG-I signaling by the ubiquitin ligase RNF125. viral assembly and replication (27, 44). Thus, our results, com- Proc. Natl. Acad. Sci. USA 104: 7500–7505. bined with those of Kim et al. (23), suggest that the ability of 16. Matsumiya, T., S. M. Prescott, and D. M. Stafforini. 2007. IFN-epsilon mediates TNF-␣-induced STAT1 phosphorylation and induction of retinoic acid-inducible HSP90 to protect endogenous proteins from degradation may rep- gene-I in human cervical cancer cells. J. Immunol. 179: 4542–4549. resent a physiological regulatory mechanism and that this function 17. Pratt, W. B., and D. O. Toft. 2003. Regulation of signaling protein function and may be hijacked in settings of viral infection or oncogenic trans- trafficking by the hsp90/hsp70-based chaperone machinery. Exp. Biol. Med. 228: 111–133. formation, resulting in pathological consequences. 18. Kurokawa, M., C. Zhao, T. Reya, and S. Kornbluth. 2008. Inhibition of apopto- In summary, we have shown that RIG-I exists as a complex some formation by suppression of Hsp90␤ phosphorylation in tyrosine kinase- by guest on September 24, 2021 with the chaperone HSP90. The association is direct and stabi- induced leukemias. Mol. Cell. Biol. 28: 5494–5506. 19. Whitesell, L., and S. L. Lindquist. 2005. HSP90 and the chaperoning of cancer. lizes RIG-I by inhibiting its ubiquitination and proteasomal Nat. Rev. Cancer 5: 761–772. degradation. In addition, the formation of an HSP90-RIG-I 20. Maloney, A., and P. Workman. 2002. HSP90 as a new therapeutic target for cancer therapy: the story unfolds. Expert Opin. Biol. Ther. 2: 3–24. complex is essential for expression of functional activities. Our 21. Wax, S., M. Piecyk, B. Maritim, and P. Anderson. 2003. Geldanamycin inhibits study, combined with other recent reports (15, 30) demonstrate the production of inflammatory cytokines in activated macrophages by reducing that ubiquitination plays dual roles in the regulation of RIG-I the stability and translation of cytokine transcripts. Arthritis Rheum. 48: 541–550. 22. Piatelli, M. J., C. Doughty, and T. C. Chiles. 2002. Requirement for a hsp90 activity and protein levels, thus providing sophisticated mech- chaperone-dependent MEK1/2-ERK pathway for B cell antigen receptor-induced anisms to control its function and expression in temporally and cyclin D2 expression in mature B lymphocytes. J. Biol. Chem. 277: spatially regulated manners. The function of HSP90 may be 12144–12150. 23. Kim, T. S., C. Y. Jang, H. D. Kim, J. Y. Lee, B. Y. Ahn, and J. Kim. 2006. compromised in settings of infection owing to virus-mediated Interaction of Hsp90 with ribosomal proteins protects from ubiquitination and chaperone sequestration, a process thought to facilitate viral proteasome-dependent degradation. Mol. Biol. Cell 17: 824–833. assembly and replication (27, 44). These findings provide new 24. Fensterl, V., D. Grotheer, I. Berk, S. Schlemminger, A. Vallbracht, and A. Dotzauer. 2005. Hepatitis A virus suppresses RIG-I-mediated IRF-3 activation insights into the physiological mechanisms that define expres- to block induction of ␤ interferon. J. Virol. 79: 10968–10977. sion of a key protein thought to profoundly impact innate an- 25. Kato, H., O. Takeuchi, E. Mikamo-Satoh, R. Hirai, T. Kawai, K. Matsushita, A. Hiiragi, T. S. Dermody, T. Fujita, and S. Akira. 2008. Length-dependent tiviral responses in mammals. recognition of double-stranded ribonucleic acids by retinoic acid-inducible gene-I and melanoma differentiation-associated gene 5. J. Exp. Med. 205: Acknowledgments 1601–1610. 26. Johnson, C. L., and M. Gale, Jr. 2006. CARD games between virus and host get We are indebted to Dr. Stephen M. Prescott for support, encouragement, a new player. Trends Immunol. 27: 1–4. and excellent advice 27. Kamal, A., M. F. Boehm, and F. J. Burrows. 2004. Therapeutic and diagnostic implications of Hsp90 activation. Trends Mol. Med. 10: 283–290. 28. Pearl, L. H., C. Prodromou, and P. Workman. 2008. The Hsp90 molecular chap- Disclosures erone: an open and shut case for treatment. Biochem. J. 410: 439–453. The authors have no financial conflict of interest. 29. Wu, J. M., L. Xiao, X. K. Cheng, L. X. Cui, N. H. Wu, and Y. F. Shen. 2003. PKC epsilon is a unique regulator for hsp90 ␤ gene in heat shock response. J. Biol. Chem. 278: 51143–51149. References 30. Arimoto, K., H. Konishi, and K. Shimotohno. 2008. UbcH8 regulates ubiquitin 1. Hiscott, J. 2004. Another detour on the Toll road to the interferon antiviral re- and ISG15 conjugation to RIG-I. Mol. Immunol. 45: 1078–1084. sponse. Nat. Struct. Mol. Biol. 11: 1028–1030. 31. Lin, R., L. Yang, P. Nakhaei, Q. Sun, E. Sharif-Askari, I. Julkunen, and 2. Akira, S., K. Takeda, and T. Kaisho. 2001. Toll-like receptors: critical proteins J. Hiscott. 2006. Negative regulation of the retinoic acid-inducible gene I-induced linking innate and acquired immunity. Nat. Immunol. 2: 675–680. antiviral state by the ubiquitin-editing protein A20. J. Biol. Chem. 281: 3. Janeway, C. A., Jr., and R. Medzhitov. 2002. Innate immune recognition. Annu. 2095–2103. Rev. Immunol. 20: 197–216. 32. Hickey, E., S. E. Brandon, G. Smale, D. Lloyd, and L. A. Weber. 1989. Sequence 4. Diebold, S. S., M. Montoya, H. Unger, L. Alexopoulou, P. Roy, L. E. Haswell, and regulation of a gene encoding a human 89-kilodalton heat shock protein. Mol. A. Al-Shamkhani, R. Flavell, P. Borrow, and C. Reis e Sousa. 2003. Viral in- Cell. Biol. 9: 2615–2626. The Journal of Immunology 2725

33. Rebbe, N. F., J. Ware, R. M. Bertina, P. Modrich, and D. W. Stafford. 1987. 39. Young, J. C., I. Moarefi, and F. U. Hartl. 2001. Hsp90: a specialized but essential Nucleotide sequence of a cDNA for a member of the human 90-kDa heat-shock protein-folding tool. J. Cell Biol. 154: 267–273. protein family. Gene 53: 235–245. 40. Hutchison, K. A., K. D. Dittmar, M. J. Czar, and W. B. Pratt. 1994. Proof that 34. Chadli, A., S. J. Felts, and D. O. Toft. 2008. GCUNC45 is the first Hsp90 co- hsp70 is required for assembly of the glucocorticoid receptor into a heterocom- chaperone to show ␣/␤ isoform specificity. J. Biol. Chem. 283: 9509–9512. plex with hsp90. J. Biol. Chem. 269: 5043–5049. 35. Zhang, S. L., J. Yu, X. K. Cheng, L. Ding, F. Y. Heng, N. H. Wu, and Y. F. Shen. 41. Smith, D. F. 1993. Dynamics of heat shock protein 90-progesterone receptor 1999. Regulation of human hsp90alpha gene expression. FEBS Lett. 444: binding and the disactivation loop model for steroid receptor complexes. Mol. 130–135. Endocrinol. 7: 1418–1429. 36. Shen, Y., J. Liu, X. Wang, X. Cheng, Y. Wang, and N. Wu. 1997. Essential role of the first intron in the transcription of hsp90␤ gene. FEBS Lett. 413: 92–98. 42. Smith, D. F., B. A. Stensgard, W. J. Welch, and D. O. Toft. 1992. Assembly of 37. Millson, S. H., A. W. Truman, A. Racz, B. Hu, B. Panaretou, J. Nuttall, progesterone receptor with heat shock proteins and receptor activation are ATP M. Mollapour, C. Soti, and P. W. Piper. 2007. Expressed as the sole Hsp90 of mediated events. J. Biol. Chem. 267: 1350–1356. yeast, the ␣ and ␤ isoforms of human Hsp90 differ with regard to their capacities 43. Isaacs, J. S., W. Xu, and L. Neckers. 2003. Heat shock protein 90 as a molecular for activation of certain client proteins, whereas only Hsp90␤ generates sensi- target for cancer therapeutics. Cancer Cells 3: 213–217. tivity to the Hsp90 inhibitor radicicol. FEBS J. 274: 4453–4463. 44. An, W. G., T. W. Schulte, and L. M. Neckers. 2000. The heat shock protein 90 38. Kuo, C. C., C. M. Liang, C. Y. Lai, and S. M. Liang. 2007. Involvement of heat antagonist geldanamycin alters chaperone association with p210bcr-abl and v-src shock protein (Hsp)90 ␤ but not Hsp90 ␣ in antiapoptotic effect of CpG-B oli- proteins before their degradation by the proteasome. Cell Growth Differ. 11: godeoxynucleotide. J. Immunol. 178: 6100–6108. 355–360. Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021