The Levels of Retinoic Acid-Inducible Gene I
Are Regulated by Heat Shock Protein 90- α
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J Immunol 2009; 182:2717-2725; ;
doi: 10.4049/jimmunol.0802933
http://www.jimmunol.org/content/182/5/2717
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References
Tomoh Matsumiya, Tadaatsu Imaizumi, Hidemi Yoshida,
Kei Satoh, Matthew K. Topham and Diana M. Stafforini
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*†
V
iral infection leads to the initiation of complex innate
immune responses that result from recognition of the
viral nucleic acid by cellular receptors including TLRs
(1), followed by host cell secretion of antiviral factors such as
cytokines (2, 3). Additional mechanisms are responsible for the
activation of the IFN response during viral infections (4). We recently reported that a cytoplasmic RNA helicase, retinoic acidinducible gene I (RIG-I),3 is an essential regulator of dsRNA-induced signaling (5). RIG-I is also known as Ddx58 owing to the
fact that this protein belongs to the DExH box-containing helicase
family; it harbors two caspase recruitment domains (CARD) at the
amino-terminal end and an RNA helicase motif at the carboxyl
terminus (5). The CARD domains are responsible for activating
subsequent downstream signaling events through interactions with
*Huntsman Cancer Institute and †Department of Internal Medicine, University of
Utah, Salt Lake City, UT 84112; and ‡Department of Vascular Biology, Institute of
Brain Sciences, Hirosaki University Graduate School of Medicine, Hirosaki City,
Japan
Received for publication September 4, 2008. Accepted for publication December
29, 2008.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
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 Support Grant P30 CA042014-20.
2
Address correspondence and reprint requests to Dr. Diana M. Stafforini, Huntsman
Cancer Institute, 2000 Circle of Hope, Suite 3364, University of Utah, Salt Lake City,
UT 84112. E-mail address: [email protected]
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
15; IPS-1, IFN-␤ promoter stimulator 1; poly(I:C), polyinosinic:polycytidylic acid;
HMG-1, high-mobility group protein 1.
Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.0802933
IPS-1/MAVS/VISA/Cardif, resulting in the induction of IFNs and
the activation of antiviral responses (6 –9). In previous work, we
reported that, aside from its recognized role as a viral sensor,
RIG-I has additional functions. We found that RIG-I is a transcriptional activator of the cyclooxygenase 2 gene and that its expression is enhanced following stimulation of endothelial cells with
inflammatory agents (10).
RIG-I is the subject of active investigations aimed at dissecting
its precise function and mechanism of action in viral immunity.
Factors likely to play a key role in these responses include those
that affect expression levels, location, stability, and posttranslational modifications, all of which can potentially modulate the ability of RIG-I to affect cellular functions. Zhao et al. (11) recently
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
becomes conjugated to a wide array of cellular proteins. Several of
the targets, including RIG-I, are IFN-␣/␤-induced antiviral proteins, but most are constitutively expressed proteins that function
in diverse cellular pathways, including RNA splicing, chromatin
remodeling/polymerase II transcription, cytoskeletal organization
and regulation, stress responses, and translation (11). The precise
functional consequences of ISG15 modification remain to be established but, unlike ubiquitin, ISG15 does not appear to target
proteins for proteasomal degradation (12, 13). In addition to
ISG15-mediated derivatization, RIG-I has been shown to undergo
two types of ubiquitin-dependent modifications that result in remarkably different effects on functional properties. Gack et al. (14)
recently showed that RIG-I is robustly ubiquitinated at its aminoterminal CARD domains by interacting with the E3 ubiquitin ligase TRIM, a member of the tripartite motif protein family, and
that this process is necessary for RIG-I-mediated IFN-␤ production and antiviral activity in response to infection. This finding
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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
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
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.
2718
HSP90 PROTECTS RIG-I FROM PROTEASOMAL DEGRADATION
indicates that ubiquitination is a RIG-I modification that can modulate
its signaling properties. Conversely, Arimoto et al. (15) reported that
conjugation of RIG-I with ubiquitin targets RIG-I for degradation by
the proteasome, thus effectively acting as a negative regulator of
RIG-I. These combined observations point to the existence of at least
three tightly regulated protein conjugation mechanisms that control
both the stability and signaling functions of RIG-I.
The goal of the present study was to obtain additional insights
on the mechanisms that contribute to the regulation of RIG-I function and expression levels. We found that in resting cells RIG-I is
a component of a protein complex that also includes the molecular
chaperone heat shock protein (HSP) 90-␣. In addition, our studies
showed that the association between RIG-I and HSP90 is direct
and that inhibiting the interaction between these proteins leads to
ubiquitination and proteasomal degradation of RIG-I. Importantly,
disrupting the integrity of the RIG-I-HSP90 complex also inhibits
key functional responses mediated by RIG-I. We conclude that
partnership between RIG-I and HSP90 is likely to affect the function of RIG-I in vivo.
Materials
Cycloheximide, a protease inhibitor mixture, FLAG-agarose, anti-FLAG
Abs, the expression vector p3XFLAG-CMV7.1, polyinosinic-polycytidylic
acid (poly(I:C)), His-tagged human high-mobility group protein 1 (HMG1), creatine kinase, and creatine phosphate were purchased from SigmaAldrich. We obtained a metal-affinity purification system (TALON), the
transfer vector pBacPAK8, and the pBacPAK6 viral DNA from Clontech
Laboratories. The ␤-galactosidase expression vector pSV40-␤-gal was obtained from Promega and the bacterial expression vector pGEX-5X-1 and
glutathione-Sepharose 4B were from Amersham Biosciences. Factor Xa
was from Qiagen and Lipofectamine Reagent, FBS, Sf21 insect cells,
DNase I, Platinum Pfx DNA polymerase, and TRIzol were from Invitrogen. Polyvinylidene fluoride membranes and chemiluminescence detection
reagents were from PerkinElmer Life Sciences. Protein content was assessed using a Pierce protein assay kit. Reagents for cDNA synthesis were
from Fermentas and iQ SYBR Green Supermix was from Bio-Rad. Valeant
Pharmaceuticals provided an anti-actin mouse mAb. Anti-HSP90, antirpS6, and protein A/G-agarose were purchased from Santa Cruz Biotechnology. Cell Signaling Technology provided anti-JNK1/2 Abs. We obtained
HRP-conjugated secondary Abs from BioSource International; the anti-RIG-I
antiserum was previously described (10) and the anti-ubiquitin Ab was from
Novagen. Geldanamycin was from BIOMOL and MG-132 and HSP90 were
from Calbiochem. Alexa Fluor 488-conjugated anti-rabbit and Texas Redconjugated anti-mouse IgGs were from Molecular Probes. We obtained
rabbit reticulocyte lysates from Hokudo.
Cellular studies
HeLa and HEK293T cells were maintained in a 5% CO2 atmosphere at 37°C
in DMEM supplemented with 10% FBS. HT-29 cells were maintained in a 5%
CO2 atmosphere at 37°C in McCoys’ 5A medium supplemented with 1%
FBS. HSP90 inhibition was accomplished by treating monolayers with
geldanamycin (0 – 400 nM) for the indicated time periods. MG-132 (up to10
␮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
lysis buffer. After one cycle of freezing and thawing, the lysates were cleared
by centrifugation at 12,000 ⫻ g for 2 min at 4°C. We subjected 50 ␮g of
protein extracts to electrophoresis as described below.
Quantitative RT-PCR
We subjected DNase I-treated RNA (1 ␮g) to reverse transcription using a
cDNA synthesis kit according to the instructions provided by the manufacturer. A Chromo4 Real-Time PCR Detection System (Bio-Rad) was
used for quantitative assessment of RIG-I and GAPDH as previously described (16).
Construct generation, expression, and purification of
recombinant proteins
We extracted total RNA from HEK293T cells and subjected the samples to
reverse transcription as previously described (16). For amplification, we
used Platinum Pfx DNA polymerase and specific primers harboring a NotI
In vitro binding and ubiquitination assays
To assess whether direct interaction(s) accounted for the association between HSP90 and RIG-I, we mixed 1 pmol each of recombinant HSP90
and either His-tagged RIG-I or His-tagged HMG-1 as a control and incubated 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
added a suspension of TALON resin in PBS containing 0.5% Triton X-100
(final volume: 500 ␮l) and rocked the mixtures for 60 min at 4°C. The
beads were extensively washed with PBS containing 0.5% Triton X-100
and treated with 2⫻ SDS-PAGE sample buffer. We subjected the eluted
proteins to electrophoresis on SDS-PAGE, followed by immunoblot analyses using anti-HSP90 and anti-RIG-I Abs.
To assess geldanamycin-related effects on ubiquitination of RIG-I,
we incubated 1 pmol of His-tagged RIG-I in 50 mM Tris-HCl (pH 7.4),
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
or absence of geldanamycin (400 nM) for 60 min at 30°C. The total
volume was adjusted to 20 ␮l. His-tagged RIG-I species were isolated
by affinity chromatography using TALON beads, as above, and then
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Materials and Methods
site (shown in lowercase font) or a SalI site (shown in lowercase font), the
sequences of which were: 5⬘-GAC AAG CTT gcg gcc gcG ATG ACC
ACC GAG CAG CGA CGC-3⬘ (RIG-FL-F (sense)) and 5⬘-TCC TCT
AGA gtc gac TCA TTT GGA CAT TTC TGC TGG-3⬘ (RIG-FL-R (antisense)). The products were purified, digested with NotI and SalI, and then
cloned into a p3XFLAG-CMV7.1 expression vector. Positive clones were
subjected to automated DNA-sequencing analyses. We next generated various RIG-I deletion mutants by amplification of full-length RIG-I with the
following primers pairs: ⌬3(1– 640), RIG-FL-F (sense) and 5⬘-AGA gtc
gac CAC AAG TGC TCT GGT TTT CAC-3⬘ (antisense); RIG-CARD(1–
238), RIG-FL-F (sense) and 5⬘-AGA gtc gac GTA CAA GTT TGT ATC
AGA CAC-3⬘ (antisense); and ⌬CARD(239 –925), 5⬘-CTT gcg gcc gcG
AGC CCA TTT AAA CCA AGA AAT-3⬘ (sense) and RIG-FL-R (antisense); ⌬10(452–925), 5⬘-CTT gcg gcc gcG GTT TAT AAG CCC CAG
AAG TTT-3⬘ (sense) and RIG-I-FL-R (antisense). The PCR products were
purified by electrophoresis on agarose gels, digested with NotI and SalI,
cloned into the p3XFLAG-CMV7.1 expression vector, and analyzed by
automated sequencing.
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
BacPAK8-F (5⬘-GC ctc gag GCA GAG GCC GGC ATG ACC ACC GAG3⬘) and RIG-I-6xHis-NotI for BacPAK 8-R (5⬘-AAg cgg ccg cTC AGT
GAT GGT GAT GAT GAT GTT TGG ACA TTT CTG CTG GAT CA3⬘); the reverse primer included a 6xHis tag for purification purposes. The
product was digested with XhoI and NotI and then cloned into the
BacPAK8 expression vector to generate pBacPAK8-6xHis-RIG-I. We cotransfected pBacPAK6 viral DNA and pBacPAK8-6xHis-RIG-I into Sf21
insect cells, cultured the cells for 3 days at 27°C, collected the viruscontaining supernatant, and plated this fraction on a fresh monolayer of
Sf21 cells to generate individual plaques that were subsequently amplified
in Sf21 cells. We harvested the cellular proteins using equilibration/wash
buffer (50 mM sodium phosphate and 300 mM NaCl (pH 7.0)) containing
protease inhibitors and purified RIG-I by metal-affinity chromatography on
a TALON resin according to the manufacturer’s instructions. We eluted
6xHis-tagged RIG-I with 20 mM Tris-HCl (pH 8.0) containing 100 mM
NaCl and 75 mM imidazole, dialyzed the preparation against 10 mM TrisHCl (pH 8.0), and subjected it to immunoblot analysis.
We also expressed various domains of RIG-I and the full-length recombinant protein in the bacterial expression vector pGEX-5X-1 per the instructions provided by the manufacturer. We amplified cDNA isolated
from HeLa cells using primers BamHI-Card-F (5⬘-C GTG ggat ccc CAT
GAC CAC CGA GCA GCG A-3⬘) and XhoI-Card-R (5⬘-CG ctc gag GTG
ATG ATG ATG ATG ATG CAA GTT TGT ATC AGA CAC); EcoRIDexH-F (CC gaa ttc AGC CCA TTT AAA CCA AGA) and XhoI-DexH-R
(CG ctc gag GTG ATG ATG ATG ATG ATG AAC TTG CTC CAG TTC
CTC); EcoRI-452– 641-F (CC gaa ttc TAT AAG CCC CAG AAG) and
XhoI-452– 641-R (CG ctc gag GTG ATG ATG ATG ATG ATG CAC
AAG TGC TCT GGT TTT); and EcoRI-helicase-F (CC gaa ttc GAC GCT
TTA AAA AAT TGG) and XhoI-helicase-R (CG ctc gag GTG ATG ATG
ATG ATG ATG TTT GGA CAT TTC TGC TGG). Bacterial pellets were
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
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,
we added 5 ␮l of factor Xa and incubated the slurry for 3 h at room
temperature. The supernatants were subjected to chromatography on a
TALON resin as described above.
The Journal of Immunology
2719
analyzed by electrophoresis on SDS-PAGE, followed by immunoblot
analyses using anti-ubiquitin and anti-RIG-I Abs.
Results
Immunoprecipitation studies
Our initial goal was to identify protein partners that associated
with RIG-I under basal conditions. We used HT-29, a human
colonic adenocarcinoma cell line that expresses high levels of
RIG-I (our unpublished observations). We subjected HT-29 lysates to immunoprecipitation using anti-RIG-I polyclonal Abs
or control IgG. After electrophoretic separation of the immunoprecipitated proteins, we noted the presence of multiple species that specifically associated with RIG-I (Fig. 1A). We initially focused our attention on the identification of RIG-Iinteracting proteins that ranged in size between 70 and 110 kDa,
as these represented a considerable proportion of the immunoprecipitated material (Fig. 1A). We analyzed various species
using mass spectrometry and identified the two largest proteins
(100 and 110 kDa) as RIG-I (data not shown). These results
validated the specificity of our immunoprecipitation approach
and suggested that the smaller, less abundant 100-kDa species
was a proteolytic fragment derived from full-length RIG-I (Mr
⫽ 110 kDa). We next analyzed the composition of the coimmunoprecipitated 90- and 70-kDa proteins (Fig. 1A, arrow and
asterisk, respectively). We found that the 90-kDa RIG-I-interacting protein harbored peptides that precisely matched sequences present in human HSP90-␣ (Table I). Our studies also
revealed the presence of a 70-kDa protein in immunoprecipitates generated using both control IgG and anti-RIG-I Abs (Fig.
1A). The amount of 70-kDa protein in the latter appeared to be
much higher than that observed in the control lane, suggesting
that RIG-I may specifically interact with this protein.
Additional studies revealed that the 70-kDa protein was heat
shock 70-kDa protein 8 (also known as HSPA8 and HSP70;
Table I). This conclusion was drawn based on the sequence of
seven peptides identified by mass spectrometric analyses and
that precisely matched sequences found in the 70-kDa human
chaperone (Table I). Our data supported the existence of in
vivo partnerships between RIG-I and select heat shock
proteins.
Immunoblot analyses
We subjected the protein extracts indicated in each case to electrophoresis
on 8.5% SDS-PAGE gels and then transferred the proteins to polyvinylidene difluoride membranes that were blocked for 60 min at room temperature in 1⫻ TBST buffer (50 mM Tris-HCl (pH 7.5), 250 mM NaCl, and
0.1% Tween 20) containing either 3% nonfat dry milk or 3% BSA. The
membranes were incubated for 60 min at room temperature with the primary Ab indicated in each case. After five washes with blocking solution,
we added a HRP-conjugated secondary Ab, incubated the membranes for
60 min at room temperature, repeated the washes using TBST, and then
visualized the immunoreactive bands using chemiluminescence detection
reagents.
Immunofluorescence analyses
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
PBS. The cells were then incubated for 1 h with a polyclonal anti-RIG-I Ab
(1/250-fold dilution) and a monoclonal anti-HSP90 Ab (1/100). After a
washing step, we incubated the coverslips with Alexa Fluor 488 – conjugated anti-rabbit and Texas Red-conjugated anti-mouse IgGs. Excess reagents were washed with PBS and the expression levels of RIG-I and
HSP90 were analyzed by confocal microscopy (Olympus FV300).
Mass spectrometry
HeLa cell proteins that coimmunoprecipitated with RIG-I were resolved on
SDS-PAGE and identified by staining, and individual lanes were subjected
to in-gel trypsin digestion at the Mass Spectrometry Core Facility (University of Utah). The tryptic peptides were identified by mass spectrometry
(MS)/MS studies and analyzed using MASCOT software (Matrix Science).
Reporter assays
To generate an IFN-␤ reporter vector that spanned nt ⫺299 to ⫹19, we
amplified HeLa cell genomic DNA with a sense primer harboring a 5⬘-XhoI
site (XhoI-IFN-␤-299, 5⬘-GG ctc gag AGT GTT TTG AGG TTC TTG
A-3⬘) and an antisense primer designed with a 5⬘-HindIII site (HindIIIIFN-␤⫹19, 5⬘-GCC aag ctt AAG GTT GCA GTT AGA ATG T-3⬘). We
cloned the product in the pGL3-basic vector and cotransfected 300 ng of
the plasmid with 50 ng of pSV40-␤-gal and 100 ng of a p3XFLAG7.1
expression vector (RIG-I full length or RIG-CARD) using Lipofectamine.
The following day we transfected the cells with 200 ng of poly(I:C) using
Lipofectamine 2000 and incubated the cells in the presence or absence of
200 nMf geldanamycin for 24 h. We harvested the cells in 200 ␮l of
reporter lysis buffer and assessed luciferase and ␤-galactosidase activities
in the extracts (n ⫽ 4).
Molecular characterization of RIG-I-HSP90 interaction
We next focused on the characterization of the interaction between HSP90 and RIG-I. To address this issue, we used molecular, immunohistological, and biochemical approaches.
First, transfection of HEK293T cells with FLAG-tagged RIG-I
followed by immunoprecipitation using anti-FLAG or antiHSP90 Abs revealed that endogenous HSP90 interacted with
exogenous RIG-I (Fig. 1, B and C). We next considered whether
the interactions observed in transfected cells were the consequence of forced expression of RIG-I and did not represent a
naturally occurring association between endogenous proteins.
To address this issue, we tested the ability of endogenous RIG-I
and HSP90, both of which have been reported to be expressed
in the cytoplasm, to colocalize under basal conditions (14, 17).
Although no particular cellular structure was highlighted, we
observed that in HeLa cells HSP90 and RIG-I were expressed in
the same cytoplasmic region, suggesting that the proteins
formed part of the same complex (Fig. 1D). In addition, immunoprecipitation studies confirmed that endogenous RIG-I interacted with endogenous HSP90 (Fig. 1E), indicating that the
association between these proteins occurs in intact cells and is
not an artifact of overexpression systems.
We next asked whether RIG-I and HSP90 interacted directly
or whether participation of an additional protein(s) was required
for association. To test this, we conducted in vitro experiments
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HeLa cells subjected to various treatments were washed twice with icecold PBS and then lysed in immunoprecipitation buffer (20 mM Tris-HCl
(pH 7.4), 100 mM NaCl, 1.5 mM MgCl2, and 1% Triton X-100) containing
0.5% protease inhibitor mixture for 30 min on ice. The lysates were centrifuged at 12,000 ⫻ g for 10 min at 4°C, and the pellets were discarded.
The supernatant fractions were precleared by incubation with normal IgG
and protein A/G-plus for 2 h. Immunoprecipitations were performed by
overnight rocking of the precleared supernatants with 1 ␮l of the primary
Ab indicated in each case at 4°C. The Ag-Ab complexes were harvested by
incubation with protein A/G-agarose for 1 h at 4°C. FLAG-tagged proteins
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
min in 2⫻ SDS-PAGE sample buffer to dissociate the immunoprecipitated
proteins. These fractions were analyzed by electrophoresis on SDS-PAGE
and immunoblot analyses as described below. Studies involving coimmunoprecipitation of endogenous proteins were conducted on the human colon cancer cell line HT-29 using essentially the same approach, except that
immunoprecipitations were performed by overnight rocking of the precleared supernatants with 1 ␮l of an anti-RIG-I antiserum at 4°C. The
complexes were harvested by incubation with protein A/G-plus for 1 h at
4°C. After extensive washing with ice-cold PBS containing 0.5% Triton
X-100, the beads were boiled for 5 min in 2⫻ SDS-PAGE sample buffer
and the eluted proteins were subjected to SDS-PAGE and immunoblot
analyses.
Identification of heat shock proteins as partners of RIG-I
2720
HSP90 PROTECTS RIG-I FROM PROTEASOMAL DEGRADATION
with purified recombinant proteins. We found that incubation of
purified, His-tagged RIG-I with recombinant HSP90 resulted in
the association between the proteins, as shown by the binding of
both species to a His-tag purification resin (Fig. 1F). Control
experiments revealed no binding of HSP90 to the beads in the
absence or presence of a control His-tagged protein (HMG-1;
Fig. 1F). These combined studies provide evidence indicating
that the association between RIG-I and HSP90 involves direct
interaction(s) between the proteins.
Identification of domains required for interaction between
HSP90 and RIG-I
Our next goal was to identify RIG-I domains involved in its
interaction with HSP90. In previous studies, it was demonstrated that the ability of RIG-I to interact with the adaptor
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,
it was recently shown that the CARD domain is the primary
binding site of HSP90-␤ on Apaf-1 (18). To systematically analyze individual contributions, we generated four FLAG-tagged
deletion mutants harboring various domains of RIG-I (Fig. 2A).
We transfected HEK293T cells with each deletion construct and
found robust expression of all species (Fig. 2B, left panel).
Next, we subjected cell lysates from transfected cells to immunoprecipitation using anti-HSP90 Abs and found that the CARD
domain lacked the ability to associate with HSP90 (Fig. 2B,
right panel). The remaining deletion constructs interacted with
HSP90 in an efficient manner, suggesting that a domain common to these constructs was necessary for the interaction. An
examination of the deletion mutants tested in this study allowed
us to conclude that the region comprised between aa 452– 641
was involved in the association between RIG-I and HSP90.
To further explore this issue and to assess whether additional
RIG-I domains also contributed to its interaction with HSP90,
we expressed His-tagged constructs harboring various regions
of RIG-I (Fig. 3A). We found that the constructs were expressed
to various extents (Fig. 3B, bottom panel). The CARD domain,
which was efficiently expressed, lacked the ability to interact
with HSP90. Moreover, deletion of the CARD domain did not
affect association with HSP90. These combined studies are in
complete agreement with our results using FLAG-tagged constructs (Fig. 2B) and they firmly establish that the CARD domain does not participate in the interaction between RIG-I and
HSP90. Interestingly, our studies also showed that all of the
remaining RIG-I domains retained the ability to bind HSP90
(Fig. 3B), suggesting that more than one domain participates in
the interaction between the proteins.
HSP90 affects the levels of RIG-I protein
HSP90 is primarily known as a molecular chaperone owing to
its ability to ensure the stability and correct conformation, activity, intracellular localization, and proteolytic cleavage of a
range of proteins involved in cell growth, differentiation, and
survival (19). These include key signaling molecules such as
ERBB2, AKT, Raf, hypoxia inducible factor 1␣ and steroid
hormone receptors (20). To investigate the functional consequences of HSP90/RIG-I association, we destabilized the interaction using pharmacological approaches. Geldanamycin is a
naturally occurring ansamycin antibiotic and antitumor agent
that binds tightly to the HSP90 ATP/ADP pocket and prevents
association of client proteins (20, 21). Treatment with geldanamycin prevented association between transfected RIG-I and
HSP90 (Fig. 4A) and significantly reduced the levels of endogenous RIG-I protein in a time-dependent manner (Fig. 4B). To
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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 sequential 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
with protein A/G-plus and eluted from the beads with 2⫻
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
purification, and the bound proteins were eluted and subjected
to immunoblot analyses using anti-HSP90 and anti-His-tag
Abs.
The Journal of Immunology
2721
Table I. Mass spectrometric identification of HSP90-␣ and HSP70 protein 8
Protein
Size
Tryptic Peptide Sequences
Identified by MS/MS
Identified Protein
Location in protein
ADLINNLGTIAKVILHLK
HSP90-␣ (HSP90AA1, HSP90AA2,
EDQTEYLEERNPDDITNE
HSP90A, HSP90N, HSPC1,
EYGEFYKGVVDSEDLPLN
HSPCA, HSPCAL1, HSPCAL3,
ISR
HSPCAL4, HSPN, HSP86,
HSP89, HSP90,LAP2)
70 kDa
TTPSYVAFTDTERSFYPE
HSP70 protein 8 (HSPA8, HSC54,
EVSSMVLTKTVTNAVVTV
HSC70, HSC71, HSP71,
PAYFNDSQRDAGTIAGL
HSPA10. LAP1, NIP71)
NVLRIINEPTAAAIAYGL
DKKFEELNADLFRSQI
HDIVLVGGSTR
assess the efficacy of geldanamycin treatment, we took advantage of a study reported by Piatelli et al. (22) who showed that
geldanamycin selectively depletes cellular Raf-1, thus interrupting activation of MEK1/2 and ERK. In addition, these investigators showed that geldanamycin does not affect the levels
of total ERK. We found that geldanamycin decreased basal levels of phospho-ERK in cells grown in the presence of serum,
thus confirming the efficacy of the treatment in our studies (Fig.
4B). We next investigated whether these effects were the consequence of altered rates of mRNA transcription or decreased
mRNA stability, as was shown for other proteins (21). However, quantitative assessment of RIG-I mRNA levels demonstrated that transcriptional effects did not account for the
geldanamycin-mediated decrease in RIG-I protein levels (Fig.
4C). Higher concentrations of geldanamycin (⬎2 ␮M) were
avoided because these resulted in generalized cytotoxic effects
(data not shown). Our results indicate that the chaperone activity of HSP90 protects RIG-I from proteolytic degradation, suggesting that this mechanism contributes to the definition of
steady-state levels of RIG-I.
HSP90 protects RIG-I from ubiquitination and proteasomal
degradation
Our next goal was to identify the protease system that participates in the degradation of RIG-I following disruption of the
RIG-I-HSP90 complex. We hypothesized that degradation of
RIG-I might involve the proteasome because most proteins known
to be stabilized by HSP90 are degraded through this mechanism
MPEETQTQDQPMEEEEVETFAFQAEIAQLMSLIINTFYSNK
EIFLRELISNSSDALDKIRYESLTDPSKLDSGKELHINL
IPNKQDRTLTIVDTGIGMTKADLINNLGTIAKSGTKAFM
EALQAGADISMIGQFGVGFYSAYLVAEKVTVITKHNDDE
QYAWESSAGGSFTVRTDTGEPMGRGTKVILHLKEDQTEY
LEERRIKEIVKKHSQFIGYPITLFVEKERDKEVSDDEAE
EKEDKEEEKEKEEKESEDKPEIEDVGSDEEEEKKDGDKK
KKKKIKEKYIDQEELNKTKPIWTRNPDDITNEEYGEFYK
SLTNDWEDHLAVKHFSVEGQLEFRALLFVPRRAPFDLFE
NRKKKNNIKLYVRRVFIMDNCEELIPEYLNFIRGVVDSE
DLPLNISREMLQQSKILKVIRKNLVKKCLELFTELAEDK
ENYKKFYEQFSKNIKLGIHEDSQNRKKLSELLRYYTSAS
GDEMVSLKDYCTRMKENQKHIYYITGETKDQVANSAFVE
RLRKHGLEVIYMIEPIDEYCVQQLKEFEGKTLVSVTKEG
LELPEDEEEKKKQEEKKTKFENLCKIMKDILEKKVEKVV
VSNRLVTSPCCIVTSTYGWTANMERIMKAQALRDNSTMG
YMAAKKHLEINPDHSIIETLRQKAEADKNDKSVKDLVIL
LYETALLSSGFSLEDPQTHANRIYRMIKLGLGIDEDDPT
ADDTSAAVTEEMPPLEGDDDTSRMEEVD
MSKGPAVGIDLGTTYSCVGVFQHGKVEIIANDQGNRTTPSY
VAFTDTERLIGDAAKNQVAMNPTNTVFDAKRLIGRRFDD
AVVQSDMKHWPFMVVNDAGRPKVQVEYKGETKSFYPEEV
SSMVLTKMKEIAEAYLGKTVTNAVVTVPAYFNDSQRQAT
KDAGTIAGLNVLRIINEPTAAAIAYGLDKKVGAERNVLI
FDLGGGTFDVSILTIEDGIFEVKSTAGDTHLGGEDFDNR
MVNHFIAEFKRKHKKDISENKRAVRRLRTACERAKRTLS
SSTQASIEIDSLYEGIDFYTSITRARFEELNADLFRGTL
DPVEKALRDAKLDKSQIHDIVLVGGSTRIPKIQKLLQDF
FNGKELNKSINPDEAVAYGAAVQAAILSGDKSENVQDLL
LLDVTPLSLGIETAGGVMTVLIKRNTTIPTKQTQTFTTY
SDNQPGVLIQVYEGERAMTKDNNLLGKFELTGIPPAPRG
VPQIEVTFDIDANGILNVSAVDKSTGKENKITITNDKGR
LSKEDIERMVQEAEKYKAEDEKQRDKVSSKNSLESYAFN
MKATVEDEKLQGKINDEDKQKILDKCNEIINWLDKNQTA
EKEEFEHQQKELEKVCNPIITKLYQSAGGMPGGMPGGFP
GGGAPPSGGASSGPTIEEVD
once the interaction is inhibited (23). In control experiments, we
found that treatment with MG-132, an agent known to specifically
inhibit proteasomal activity, led to substantial accumulation of
RIG-I (Fig. 5A). To specifically assess the contribution of HSP90
to the stability of RIG-I, we investigated whether the effects of
geldanamycin on RIG-I levels were affected when proteasomal
activity was inhibited with MG-132. We found (Fig. 5A) that MG132 prevented geldanamycin-mediated RIG-I proteolysis, thus
providing evidence for a role of HSP90 activity in the protection of
RIG-I from proteasomal degradation.
Protein degradation through the proteasome commonly involves conjugation with ubiquitin, and HSP90 has been shown
to utilize this mechanism in the stabilization of a variety of
proteins. In addition, the degradation of RIG-I requires derivatization with ubiquitin (14, 15). To assess whether HSP90-mediated effects on RIG-I involved the ubiquitin pathway, we developed an in vitro assay that allowed us to assess changes in
the extent of ubiquitination of RIG-I. We incubated purified,
His-tagged RIG-I with a ubiquitination-competent rabbit reticulocyte system, isolated RIG-I using affinity chromatography,
and then assessed the extent of ubiquitination by immunoblot
analyses. We found that under basal conditions, a fraction of
RIG-I was present as a high molecular mass ubiquitinated species (Fig. 5B, top left panel). In addition, dissociation of the
RIG-I-HSP90 complex with geldanamycin stimulated RIG-I
ubiquitination, as demonstrated by robust increases in the extent
of RIG-I ubiquitination and by attenuated expression of underivatized RIG-I (Fig. 5B). We did not detect high-molecular mass
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
90 kDa
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
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
FIGURE 3. Several RIG-I domains can mediate binding to HSP90. A,
Schematic representation of purified RIG-I mutant constructs used in this
portion of the study. B, Purified recombinant HSP90 and the constructs
indicated were incubated with purified, full-length His-tagged RIG-I, or
various purified His-tagged RIG-I domains. The resulting mixtures were
subjected to affinity purification, and the bound proteins were eluted and
subjected to immunoblot analyses using anti-HSP90 and anti-His-tag Abs.
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
nM) for the indicated time periods and cell lysates were then subjected to
SDS-PAGE and immunoblotting using Abs against RIG-I, phospho-ERK,
HSP90, and actin. The numbers above this panel depict quantitative assessments of band intensity relative to actin controls performed using
Image-J. C, HeLa cells were treated with geldanamycin (100 nM) for the
indicated time periods. Total RNA was then extracted and the levels of
RIG-I were assessed by quantitative RT-PCR.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 2. The CARD domain is not required for association of RIG-I
with HSP90. A, Schematic representation of RIG-I mutant constructs used
in this portion of the study. B, HEK293T cells were transfected with the
constructs indicated. Twenty-four hours after transfection, the cells were
harvested and the levels of expression of each construct were assessed by
immunoblot analyses (left panel). In addition, aliquots of the lysates were
subjected to immunoprecipitation using anti-HSP90 Abs, followed by immunoblotting with an anti-FLAG Ab (right panel).
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 ⫺299 to ⫹19 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
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
for this response. In addition, we found robust basal IFN-␤ promoter activation mediated by the CARD domain of RIG-I that was
not affected by geldanamycin treatment (Fig. 6). These results indicate that blocking the ability of RIG-I to interact with HSP90
inhibits its biological activity. The fact that IFN-␤ promoter activation mediated by the CARD domain was not affected by
The Journal of Immunology
geldanamycin is consistent with our studies (Figs. 2 and 3) showing that the CARD domain is not required for association of RIG-I
with HSP90.
Discussion
RIG-I has been described as a pattern recognition receptor that
detects intracellular dsRNA through its helicase domain in a TLRindependent fashion (5, 6). A series of well-characterized downstream signaling events that require the CARD domains of RIG-I
lead to the activation of a family of IFN-induced genes, such as
IFN regulatory factor 3, IFN regulatory factor 7, and NF-␬B,
through the signaling adaptor protein IPS-1 (6). The ability of
RIG-I to elicit downstream signaling events requires the formation
of a macromolecular protein complex necessary for gene activation (26). This complex is most likely in dynamic equilibrium depending on the state of cellular activation, viral stimulation, and
other factors. To identify novel proteins that associated with RIG-I
under basal conditions, we used biochemical approaches that,
when combined with mass spectrometric measurements, resulted
in the discovery of HSP90, and perhaps HSP70, as partners of
RIG-I. HSP90 is a ubiquitous, constitutively expressed, highly
conserved molecular chaperone involved in the folding, activation
and assembly of key mediators of signal transduction, cell cycle
control, and transcriptional regulation (27). This chaperone interacts with native and denatured partners such as protein kinases,
transcription factors, viral replication proteins, and a range of intracellular receptors, affecting their turnover, trafficking, cellular
localization, and activity (28, 29). Recent studies by Kim et al. (23)
demonstrated that HSP90 associates with 40S ribosomal components and prevents ubiquitin-dependent ribosomal protein degradation. Our data point to a similar mechanism in which partnership
between HSP90 and RIG-I serves to stabilize this protein. In addition, we provide novel evidence showing that the interaction
between HSP90 and RIG-I is direct and independent of the CARD
domain. Interestingly, we found that the DexH Box, the helicase,
and other domains of RIG-I efficiently associated with HSP90.
These results suggest that the interaction between these proteins
may involve multiple domains. Similar results were reported by
Kim et al. (23) who showed that interaction of HSP90 with ribosomal protein S3 involves interaction with two independent do-
FIGURE 6. Inhibition of HSP90/RIG-I association abolishes RIG-I-mediated transcriptional activation of the IFN-␤ promoter. HEK293T cells
were cotransfected with a reporter construct harboring nt ⫺299/⫹19 of the
IFN-␤ gene, a ␤-galactosidase expression vector, and either full-length
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 ⫽ 4).
mains. Thus, it is apparent that HSP90 frequently associates with
client proteins through multiple domains.
Our studies also provide evidence suggesting that one of the
functional consequences of the interaction between HSP90 and
RIG-I is the shielding of the latter from ubiquitin-dependent proteasomal degradation. The mechanisms that regulate the levels of
RIG-I protein under various conditions have been partially characterized and clearly require ubiquitin-mediated functions at different levels. Arimoto et al. (15) reported that the ubiquitin ligases
RNF125 and UbcH8 are key determinants of RIG-I levels through
their ability to catalyze the conjugation of RIG-I to ubiquitin or
ISG15, respectively. These authors (30) described participation of
the E2 and E3 ubiquitin ligases in a regulatory circuit, the function
of which is to control the levels of RIG-I protein. In addition, Lin
et al. (31) found that A20, a virus-inducible protein that harbors
two ubiquitin-editing domains, functions as a negative regulator of
RIG-I through indirect mechanisms that require degradation of an
as yet unidentified adapter of the RIG-I pathway. These combined
observations reveal the existence of several control points to ensure that appropriate levels of RIG-I are expressed in response to
changing cellular needs.
In humans, two distinct genes, HSP90-␣ and HSP90-␤, encode
closely related cytoplasmic proteins that display minor sequence
differences at the amino termini (32–34). The isoforms differ in
their ability to activate certain client proteins and regulate cellular
functions of immune cells (34 –38). The ␣ and ␤ isoforms of
HSP90 often cooperate with other proteins to generate a cytosolic
multichaperone machinery that includes HSP70, peptidyl-prolyl
isomerases, and other cochaperones to achieve adequate protein
folding (39). Certain cytosolic client polypeptides have been
shown to first bind HSP70 and then interact with dimeric HSP90
(39 – 42). Interestingly, our studies suggest that HSP70 may be a
second chaperone that interacts with RIG-I under basal conditions.
Our work did not directly address whether HSP70 facilitates the
formation or stability of the RIG-I-HSP90 complex, but it is tempting to speculate that HSP70 contributes to this process and that a
cochaperone(s) may be involved as well.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
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
at 30°C and then isolated RIG-I using affinity chromatography on TALON
beads. We eluted the bound proteins and subjected them to electrophoresis
on SDS-PAGE, followed by immunoblot analyses using anti-ubiquitin and
anti-RIG-I Abs.
2723
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HSP90 PROTECTS RIG-I FROM PROTEASOMAL DEGRADATION
Acknowledgments
We are indebted to Dr. Stephen M. Prescott for support, encouragement,
and excellent advice
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
Disclosures
28.
The authors have no financial conflict of interest.
29.
References
30.
1. Hiscott, J. 2004. Another detour on the Toll road to the interferon antiviral response. Nat. Struct. Mol. Biol. 11: 1028 –1030.
2. Akira, S., K. Takeda, and T. Kaisho. 2001. Toll-like receptors: critical proteins
linking innate and acquired immunity. Nat. Immunol. 2: 675– 680.
3. Janeway, C. A., Jr., and R. Medzhitov. 2002. Innate immune recognition. Annu.
Rev. Immunol. 20: 197–216.
4. Diebold, S. S., M. Montoya, H. Unger, L. Alexopoulou, P. Roy, L. E. Haswell,
A. Al-Shamkhani, R. Flavell, P. Borrow, and C. Reis e Sousa. 2003. Viral in-
31.
32.
fection switches non-plasmacytoid dendritic cells into high interferon producers.
Nature 424: 324 –328.
Yoneyama, M., M. Kikuchi, T. Natsukawa, N. Shinobu, T. Imaizumi,
M. Miyagishi, K. Taira, S. Akira, and T. Fujita. 2004. The RNA helicase RIG-I
has an essential function in double-stranded RNA-induced innate antiviral responses. Nat. Immunol. 5: 730 –737.
Kawai, T., K. Takahashi, S. Sato, C. Coban, H. Kumar, H. Kato, K. J. Ishii,
O. Takeuchi, and S. Akira. 2005. IPS-1, an adaptor triggering RIG-I- and Mda5mediated type I interferon induction. Nat. Immunol. 6: 981–988.
Sen, G. C., and S. N. Sarkar. 2005. Hitching RIG to action. Nat. Immunol. 6:
1074 –1076.
Xu, L. G., Y. Y. Wang, K. J. Han, L. Y. Li, Z. Zhai, and H. B. Shu. 2005. VISA
is an adapter protein required for virus-triggered IFN-␤ signaling. Mol. Cell. 19:
727–740.
Meylan, E., J. Curran, K. Hofmann, D. Moradpour, M. Binder, R. Bartenschlager,
and J. Tschopp. 2005. Cardif is an adaptor protein in the RIG-I antiviral pathway
and is targeted by hepatitis C virus. Nature 437: 1167–1172.
Imaizumi, T., S. Aratani, T. Nakajima, M. Carlson, T. Matsumiya, K. Tanji,
K. Ookawa, H. Yoshida, S. Tsuchida, T. M. McIntyre, et al. 2002. Retinoic
acid-inducible gene-I is induced in endothelial cells by LPS and regulates expression of COX-2. Biochem. Biophys. Res. Commun. 292: 274 –279.
Zhao, C., C. Denison, J. M. Huibregtse, S. Gygi, and R. M. Krug. 2005. Human
ISG15 conjugation targets both IFN-induced and constitutively expressed proteins functioning in diverse cellular pathways. Proc. Natl. Acad. Sci. USA 102:
10200 –10205.
Malakhov, M. P., K. I. Kim, O. A. Malakhova, B. S. Jacobs, E. C. Borden, and
D. E. Zhang. 2003. High-throughput immunoblotting: ubiquitin-like protein
ISG15 modifies key regulators of signal transduction. J. Biol. Chem. 278:
16608 –16613.
Schwartz, D. C., and M. Hochstrasser. 2003. A superfamily of protein tags: ubiquitin, SUMO and related modifiers. Trends Biochem. Sci. 28: 321–328.
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
ubiquitin ligase is essential for RIG-I-mediated antiviral activity. Nature 446:
916 –920.
Arimoto, K., H. Takahashi, T. Hishiki, H. Konishi, T. Fujita, and K. Shimotohno.
2007. Negative regulation of the RIG-I signaling by the ubiquitin ligase RNF125.
Proc. Natl. Acad. Sci. USA 104: 7500 –7505.
Matsumiya, T., S. M. Prescott, and D. M. Stafforini. 2007. IFN-epsilon mediates
TNF-␣-induced STAT1 phosphorylation and induction of retinoic acid-inducible
gene-I in human cervical cancer cells. J. Immunol. 179: 4542– 4549.
Pratt, W. B., and D. O. Toft. 2003. Regulation of signaling protein function and
trafficking by the hsp90/hsp70-based chaperone machinery. Exp. Biol. Med. 228:
111–133.
Kurokawa, M., C. Zhao, T. Reya, and S. Kornbluth. 2008. Inhibition of apoptosome formation by suppression of Hsp90␤ phosphorylation in tyrosine kinaseinduced leukemias. Mol. Cell. Biol. 28: 5494 –5506.
Whitesell, L., and S. L. Lindquist. 2005. HSP90 and the chaperoning of cancer.
Nat. Rev. Cancer 5: 761–772.
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.
Wax, S., M. Piecyk, B. Maritim, and P. Anderson. 2003. Geldanamycin inhibits
the production of inflammatory cytokines in activated macrophages by reducing
the stability and translation of cytokine transcripts. Arthritis Rheum. 48: 541–550.
Piatelli, M. J., C. Doughty, and T. C. Chiles. 2002. Requirement for a hsp90
chaperone-dependent MEK1/2-ERK pathway for B cell antigen receptor-induced
cyclin D2 expression in mature B lymphocytes. J. Biol. Chem. 277:
12144 –12150.
Kim, T. S., C. Y. Jang, H. D. Kim, J. Y. Lee, B. Y. Ahn, and J. Kim. 2006.
Interaction of Hsp90 with ribosomal proteins protects from ubiquitination and
proteasome-dependent degradation. Mol. Biol. Cell 17: 824 – 833.
Fensterl, V., D. Grotheer, I. Berk, S. Schlemminger, A. Vallbracht, and
A. Dotzauer. 2005. Hepatitis A virus suppresses RIG-I-mediated IRF-3 activation
to block induction of ␤ interferon. J. Virol. 79: 10968 –10977.
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
recognition of double-stranded ribonucleic acids by retinoic acid-inducible
gene-I and melanoma differentiation-associated gene 5. J. Exp. Med. 205:
1601–1610.
Johnson, C. L., and M. Gale, Jr. 2006. CARD games between virus and host get
a new player. Trends Immunol. 27: 1– 4.
Kamal, A., M. F. Boehm, and F. J. Burrows. 2004. Therapeutic and diagnostic
implications of Hsp90 activation. Trends Mol. Med. 10: 283–290.
Pearl, L. H., C. Prodromou, and P. Workman. 2008. The Hsp90 molecular chaperone: an open and shut case for treatment. Biochem. J. 410: 439 – 453.
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.
Arimoto, K., H. Konishi, and K. Shimotohno. 2008. UbcH8 regulates ubiquitin
and ISG15 conjugation to RIG-I. Mol. Immunol. 45: 1078 –1084.
Lin, R., L. Yang, P. Nakhaei, Q. Sun, E. Sharif-Askari, I. Julkunen, and
J. Hiscott. 2006. Negative regulation of the retinoic acid-inducible gene I-induced
antiviral state by the ubiquitin-editing protein A20. J. Biol. Chem. 281:
2095–2103.
Hickey, E., S. E. Brandon, G. Smale, D. Lloyd, and L. A. Weber. 1989. Sequence
and regulation of a gene encoding a human 89-kilodalton heat shock protein. Mol.
Cell. Biol. 9: 2615–2626.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
Pratt and Toft (17) have pointed out that many of the client
proteins of HSP90 are involved in signal transduction and speculated that HSP90 may facilitate access of its client proteins to
diverse intracellular sites and/or ligands (17), thus affecting their
ability to elicit biological activities. We hypothesized that the formation of a complex between RIG-I and HSP90 had important
functional consequences additional to protecting RIG-I from proteasomal degradation. Indeed, we found that interfering with the
ability of HSP90 to associate with RIG-I prevented RIG-I-mediated transcriptional activation of the IFN-␤ promoter. An interesting observation resulting from our studies was the finding that the
CARD domain of RIG-I, which does not bind to HSP90, transcriptionally activated the IFN-␤ promoter in a robust, dsRNA-independent manner. A number of mechanisms could account for this
effect, but one possibility is that stimulation of the HSP90-RIG-I
complex with dsRNA results in conformational changes that facilitate CARD domain-mediated functions that are normally silent
in the absence of stimulation. Additional possibilities include
HSP90-mediated regulation of the ability of RIG-I to respond
and/or bind to ligands or to undergo protein modification.
Although HSP90 seems to play beneficial physiological roles,
this chaperone has been shown to stabilize oncogenic proteins,
thus contributing to cancer progression (27). Increased expression
of HSP90 is associated with tumorigenesis and small molecule
HSP90 inhibitors that include geldanamycin and its derivatives
selectively kill certain types of cancer cells by promoting apoptosis
(43). In addition, virus-mediated sequestration of HSP90 facilitates
viral assembly and replication (27, 44). Thus, our results, combined with those of Kim et al. (23), suggest that the ability of
HSP90 to protect endogenous proteins from degradation may represent a physiological regulatory mechanism and that this function
may be hijacked in settings of viral infection or oncogenic transformation, resulting in pathological consequences.
In summary, we have shown that RIG-I exists as a complex
with the chaperone HSP90. The association is direct and stabilizes RIG-I by inhibiting its ubiquitination and proteasomal
degradation. In addition, the formation of an HSP90-RIG-I
complex is essential for expression of functional activities. Our
study, combined with other recent reports (15, 30) demonstrate
that ubiquitination plays dual roles in the regulation of RIG-I
activity and protein levels, thus providing sophisticated mechanisms to control its function and expression in temporally and
spatially regulated manners. The function of HSP90 may be
compromised in settings of infection owing to virus-mediated
chaperone sequestration, a process thought to facilitate viral
assembly and replication (27, 44). These findings provide new
insights into the physiological mechanisms that define expression of a key protein thought to profoundly impact innate antiviral responses in mammals.
The Journal of Immunology
33. Rebbe, N. F., J. Ware, R. M. Bertina, P. Modrich, and D. W. Stafford. 1987.
Nucleotide sequence of a cDNA for a member of the human 90-kDa heat-shock
protein family. Gene 53: 235–245.
34. Chadli, A., S. J. Felts, and D. O. Toft. 2008. GCUNC45 is the first Hsp90 cochaperone to show ␣/␤ isoform specificity. J. Biol. Chem. 283: 9509 –9512.
35. Zhang, S. L., J. Yu, X. K. Cheng, L. Ding, F. Y. Heng, N. H. Wu, and Y. F. Shen.
1999. Regulation of human hsp90alpha gene expression. FEBS Lett. 444:
130 –135.
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.
37. Millson, S. H., A. W. Truman, A. Racz, B. Hu, B. Panaretou, J. Nuttall,
M. Mollapour, C. Soti, and P. W. Piper. 2007. Expressed as the sole Hsp90 of
yeast, the ␣ and ␤ isoforms of human Hsp90 differ with regard to their capacities
for activation of certain client proteins, whereas only Hsp90␤ generates sensitivity to the Hsp90 inhibitor radicicol. FEBS J. 274: 4453– 4463.
38. Kuo, C. C., C. M. Liang, C. Y. Lai, and S. M. Liang. 2007. Involvement of heat
shock protein (Hsp)90 ␤ but not Hsp90 ␣ in antiapoptotic effect of CpG-B oligodeoxynucleotide. J. Immunol. 178: 6100 – 6108.
2725
39. Young, J. C., I. Moarefi, and F. U. Hartl. 2001. Hsp90: a specialized but essential
protein-folding tool. J. Cell Biol. 154: 267–273.
40. Hutchison, K. A., K. D. Dittmar, M. J. Czar, and W. B. Pratt. 1994. Proof that
hsp70 is required for assembly of the glucocorticoid receptor into a heterocomplex with hsp90. J. Biol. Chem. 269: 5043–5049.
41. Smith, D. F. 1993. Dynamics of heat shock protein 90-progesterone receptor
binding and the disactivation loop model for steroid receptor complexes. Mol.
Endocrinol. 7: 1418 –1429.
42. Smith, D. F., B. A. Stensgard, W. J. Welch, and D. O. Toft. 1992. Assembly of
progesterone receptor with heat shock proteins and receptor activation are ATP
mediated events. J. Biol. Chem. 267: 1350 –1356.
43. Isaacs, J. S., W. Xu, and L. Neckers. 2003. Heat shock protein 90 as a molecular
target for cancer therapeutics. Cancer Cells 3: 213–217.
44. An, W. G., T. W. Schulte, and L. M. Neckers. 2000. The heat shock protein 90
antagonist geldanamycin alters chaperone association with p210bcr-abl and v-src
proteins before their degradation by the proteasome. Cell Growth Differ. 11:
355–360.
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