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Involvement of the IκB Kinase (IKK)-Related Kinases Tank-Binding Kinase 1/IKKi and Cullin-Based Ubiquitin Ligases in IFN Regulatory Factor-3 Degradation This information is current as of September 29, 2021. Annie Bibeau-Poirier, Simon-Pierre Gravel, Jean-François Clément, Sébastien Rolland, Geneviève Rodier, Philippe Coulombe, John Hiscott, Nathalie Grandvaux, Sylvain Meloche and Marc J. Servant

J Immunol 2006; 177:5059-5067; ; Downloaded from doi: 10.4049/jimmunol.177.8.5059 http://www.jimmunol.org/content/177/8/5059 http://www.jimmunol.org/ References This article cites 71 articles, 38 of which you can access for free at: http://www.jimmunol.org/content/177/8/5059.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Involvement of the I␬B Kinase (IKK)-Related Kinases Tank-Binding Kinase 1/IKKi and Cullin-Based Ubiquitin Ligases in IFN Regulatory Factor-3 Degradation1

Annie Bibeau-Poirier,* Simon-Pierre Gravel,* Jean-Franc¸ois Cle´ment,* Se´bastien Rolland,* Genevie`ve Rodier,† Philippe Coulombe,† John Hiscott,§ Nathalie Grandvaux,‡ Sylvain Meloche,† and Marc J. Servant2*

Activation of the innate arm of the immune system following pathogen infection relies on the recruitment of latent transcription factors involved in the induction of a subset of genes responsible for viral clearance. One of these transcription factors, IFN regulatory factor 3 (IRF-3), is targeted for proteosomal degradation following virus infection. However, the molecular mechanisms involved in this process are still unknown. In this study, we show that polyubiquitination of IRF-3 increases in response to Sendai Downloaded from virus infection. Using an E1 temperature-sensitive cell line, we demonstrate that polyubiquitination is required for the observed degradation of IRF-3. Inactivation of NEDD8-activating E1 enzyme also results in stabilization of IRF-3 suggesting the NEDDylation also plays a role in IRF-3 degradation following Sendai virus infection. In agreement with this observation, IRF-3 is recruited to Cullin1 following virus infection and overexpression of a dominant-negative mutant of Cullin1 signiﬁcantly inhibits the degradation of IRF-3 observed in infected cells. We also asked whether the C-terminal cluster of phosphoacceptor sites of IRF-3 could serve as a destabilization signal and we therefore measured the half-life of C-terminal phosphomimetic IRF-3 mutants. Interestingly, we found them to be http://www.jimmunol.org/ .short-lived in contrast to wild-type IRF-3. In addition, no degradation of IRF-3 was observed in TBK1؊/؊ mouse embryonic ﬁbroblasts All together, these data demonstrate that virus infection stimulates a host cell signaling pathway that modulates the expression level of IRF-3 through its C-terminal phosphorylation by the I␬B kinase-related kinases followed by its polyubiquitination, which is mediated in part by a Cullin-based ubiquitin ligase. The Journal of Immunology, 2006, 177: 5059–5067.

n response to pathogens, infected cells activate multiple sig- particles activate several kinases in the host including the recently naling cascades involved in the induction of latent transcrip- described I␬B kinase (IKK) homologs, IKKE (9), also called IKKi I tion factors. These transcription factors are responsible for (10), and Tank-binding kinase 1 (TBK1) (11). These kinases target by guest on September 29, 2021 the induction of a repertoire of genes known to impede pathogens’ a Ser-Thr rich cluster located in the C-terminal end of IRF-3 (3, 8, survival in the host (reviewed in Ref. 1). One of these transcription 12, 13) (for review, see Ref. 2). Phosphorylation of IRF-3 induces factors, IFN regulatory factor 3 (IRF-3)3 is essential for the normal its homodimerization and accumulation into the nucleus where it host response to pathogens (reviewed in Ref. 2). Several reports induces gene transcription through recognition of speciﬁc DNA have now documented the activation of IRF-3 following infection response elements located in the promoters of genes encoding the with RNA viruses, as well as DNA viruses (3–8). These infectious chemokines IL-15, IFN-␥-inducible protein 10 and RANTES, as well as cytokines such as the type I IFN (12, 14–16).

*Faculty of Pharmacy, †Institute of Research in Immunology and Cancer, ‡Centre It has been suggested that following virus infection, IRF-3 is Hospitalier de l’Universite´de Montreál and Department of Biochemistry, University targeted for degradation by the proteasome based on the observa- § of Montreal, Montreal, Canada; and Lady Davis Institute for Medical Research, tion that MG-132, a proteasome inhibitor, abrogates its degrada- McGill University, Montreal, Canada tion (17, 18). The ubiquitin-proteasome proteolytic pathway plays Received for publication February 23, 2006. Accepted for publication July 28, 2006. key roles in regulating the levels of many proteins involved in 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 diverse cellular processes. Proteins targeted for degradation are with 18 U.S.C. Section 1734 solely to indicate this fact. first tagged with a polyubiquitin chain in a three-step cascade re- 1 This work was supported by research grants from the Canadian Institutes of Health action involving ubiquitin activation (catalyzed by the ubiquitin- Research (CIHR) to M.J.S. (MOP-53282) and S.M. (MOP-14168). M.S. is a recipient activating enzyme (E1)), ubiquitin transfer (catalyzed by a ubiq- of a Rx&D/CIHR Health Research Foundation Career Award in Health Sciences. S.M. and N.G. are both recipients of Canada Research Chairs. J.H. is supported by a uitin carrier protein or conjugating enzyme (E2)), and ubiquitin CIHR Senior Scientist award. P.C. is a recipient of a studentship from the CIHR. ligation (catalyzed by a ubiquitin-protein ligase enzyme (E3)) (see J.-F.C and S.-P.G are both recipients of a studentship from the Fonds de la Recherche Refs. 19 and 20 for reviews). There are two major groups of E3s en Sante´ du Que´bec. G.R. holds a fellowship from the American Association for Cancer Research. classified according to a common motif shared by one of the en- 2 Address correspondence and reprint requests to Dr. Marc J. Servant, Faculte´de zyme components: the homology to the E6-associated protein C Pharmacie, Universite´ de Montreál, C.P. 6128, succursale Centre-Ville, Montreál, terminus domain-containing E3s and the really interesting new Que´bec, Canada H3C 3J7. E-mail address: [email protected] gene (RING) domain-containing E3s (reviewed in Ref. 19). Other 3 Abbreviations used in this paper: IRF-3, IFN regulatory factor-3; IKK, I␬B kinase; RING-like domain-containing E3 ligases have also been identified TBK1, Tank-binding kinase 1; RING, really interesting new gene; Cul1, Cullin1; SCF, Skp1-Cul1-F-box; TPEN, N,N,NЈ,NЈ,-tetrakis(2-pyridylmethyl)ethylenedia- and include the protein inhibitors of activated STATs family small mine; MEF, murine embryonic fibroblast; SeV, Sendai virus; HA, hemagglutinin; ubiquitin-related modifier ligases, the plant homeodomain domain- HCMV, human CMV; RIPA, radioimmunoprecipitation assay; APP-BPI, amyloid percursor protein-binding protein 1; NEDD8, neural precursor cell expressed devel- containing E3s and the U-box E3s. The RING family represents opmentally down-regulated protein 8. the largest class of E3s, which are found in single subunits or

Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 5060 UBIQUITIN-DEPENDENT DEGRADATION OF IRF-3 multicomponent protein complexes (see Refs. 21 and 22 for re- N252) was a gift from M. Pagano (New York University School of Med- views). The best characterized of these multisubunit complexes icine) and cultured in DMEM containing 10% FBS tetracycline-free FBS. consist of the three invariable subunits Skp1, Cullin1 (Cul1), and Human diploid fibroblasts (Hel 299), HeLa, and human embryonic Kinney 293T cell lines (293T) were obtained from American Type Culture Col- the RING finger protein Rbx1/Roc1 and a variable component lection (ATCC) and cultured in DMEM containing 10% FBS. 293T cells known as the F-box protein. Together, they formed a ubiquitin- were transfected with the calcium phosphate coprecipitation method. HeLa protein ligase complex termed SCF (Skp1-Cul1-F-box) (21, 22). cells were transfected with Lipofectamine 2000. When specified, cells were Whereas Rbx1/Roc1 proteins are thought to provide a docking site also transfected with 12.5 ␮g of poly(I:C) in 30 ␮l of Lipofectamine 2000. Human CMV (HCMV) Towne strain was obtained from ATCC and prop- for the E2 enzyme, the F-box proteins act as receptors and are agated as previously described (3). Sendai virus (SeV) was obtained from responsible for substrates recognition and specificity. Members of specific pathogen-free avian supply. the SCF E3 ligases family generally polyubiquitinate substrates phosphorylated at specific sites such as I␬B␣, p27Kip1, and c-Myc Infection and therefore play a key role in the regulation of the cell cycle, Cells were infected with HCMV Towne strain at a multiplicity of infection signal transduction and transcriptional activation (23–26). Neural of 1.0 PFU/cell or with SeV at 100 HA units/106 cells for2hinserum-free precursor cell expressed developmentally down-regulated protein medium. Then the serum-free medium was replaced with complete medium for the rest of the kinetic. 8 (NEDD8) is a mammalian member of ubiquitin-like proteins, which modify proteins in a manner similar to ubiquitin (reviewed Immunoprecipitation, immunoblot analysis, and native-PAGE in Ref. 27). The ability of SCF E3 ligases to ubiquitinate their Preparation of whole cell extracts, immunoprecipitation, native-PAGE, and substrates is enhanced by covalent modification of Cul1 proteins immunoblot analysis were performed as described previously (3, 42). with NEDD8. This NEDDylation of Cul1 on arginine 720 is Downloaded from thought to result in an increased affinity of SCF E3 ligases for In vivo ubiquitination assays some E2 enzymes (28). The NEDDylation reaction requires the To monitor the polyubiquitination of IRF-3, cells were lysed in a radio- coordinated action of amyloid precursor protein-binding protein 1 immunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, pH 7.4, 100 ␤ (APP-BPI)/Uba3 (a heterodimeric E1-like enzyme) and UBC12 mM NaCl, 5 mM EDTA, 50 mM sodium fluoride, 40 mM -glycerophos- phate, 1 mM sodium orthovanadate, 1% Triton X-100, 0.1% SDS, 1% (an E2-like enzyme) (29, 30). sodium deoxycholate, 2 mM N-ethylmaleimide, and protease inhibitors

Many viruses execute multiple immune-evasive activities in in- mixture (Sigma)). Cells lysates were transferred to microcentrifuge tubes http://www.jimmunol.org/ fected cells by targeting the type 1 IFN signaling pathway (5, 31). and passed through a 25-gauge needle five times and then centrifuged at Notably, it was recently reported that a viral product from rotavi- 14,000 rpm for 20 min at 4°C. Immunoprecipitated IRF-3 (epitope tagged rus, the nonstructural protein 1, induces a rapid degradation of or endogenous) was washed 5 times with RIPA buffer before electrophoresis on 7.5% acrylamide gels. In some experiments, cell lysates were boiled IRF-3 through a proteasome-dependent pathway (32). In contrast, for 7 min at 90°C in 1% SDS for removal of noncovalently attached pro- uncontrolled IRF-3 activation is detrimental for the host since re- teins prior to immmunoprecipitation. Proteins were electrophoretically ports have demonstrated a role of activated IRF-3 in septic shock transferred to Hybound-C nitrocellulose membranes and polyubiquitinated syndrome, ischemia-reperfusion injury of the liver, as well as ap- IRF-3 was detected by immunoblotting using monoclonal anti-HA or anti-ubiquitin Abs. optosis (33–37). Thus, IRF-3 activity needs to be strictly controlled. In this context, the host cell-mediated degradation of IRF-3 Biosynthetic-labeling experiments by guest on September 29, 2021 following virus infection may play an important role in the termi- To examine the stability of the different IRF-3 phosphomimetic point mu- nation of an IRF-3-mediated response. This study was undertaken tants, transiently transfected HeLa cells in 60-mm petri dishes were pulse- to characterize the cellular mechanisms involved in IRF-3 degra- labeled for 2 h with 170 ␮Ci/ml [35S]methionine and [35S]cysteine and dation following virus infection. then chased for the indicated times in complete medium containing excess methionine and cysteine. The cells were then washed twice with ice-cold PBS and lysed in Triton X-100 lysis buffer (50 mM Tris-HCl (pH 7.4), 100 Materials and Methods mM NaCl, 5 mM EDTA, 50 mM sodium fluoride, 40 mM ␤-glycerophos- Reagent, Abs, and plasmids phate, 1 mM sodium orthovanadate, 1% Triton X-100, and protease inhibitor mixture (Sigma-Aldrich)). Lysates (300 ␮g of protein) were precleared MG-132 and lactacystin were purchased from Boston Biochem. Doxycy- for 1 h with 2 ␮g of normal mouse serum and the resulting supernatants clin was obtained from Sigma-Aldrich. Poly(I:C) (Amersham Pharmacia) were incubated with protein G-Sepharose beads preabsorbed with 2 ␮gof was reconstituted in PBS at 2 mg/ml, denatured at 55°C for 30 min, and anti-Flag for 16 h at 4°C. Immune complexes were washed five times with allowed to anneal to room temperature before use. N,N,NЈ,NЈ,-tetrakis(2- Triton X-100 lysis buffer. Proteins were eluted by heating at 95°C for 5 min pyridylmethyl)ethylenediamine (TPEN) and N-ethylmaleimide were ob- in denaturing sample buffer and analyzed by SDS-gel electrophoresis on tained from Sigma-Aldrich. Commercial Abs were from the following sup- 10% acrylamide gels. The IRF-3 proteins were detected by fluorography pliers: anti-IRF-3 Abs specific for human and rodent species were from and visualized using a gel documentation device (Typhoon scanner 9410; Immuno-Biological Laboratories (IBL) and Zymed Laboratories, respec- Amersham Biosciences) for densitometric analysis. For qualitative mea- tively; anti-IKKE Ab (IMG-270A) (that recognize as well TBK1) was from surement of the stability of the different IRF-3 phosphomimetic point mu- Imgenex; anti-ubiquitin mAb (clone P4D1) and mAb to MYC were from tants, transiently transfected HeLa cells in 60 mm were treated with 100 Santa Cruz Biotechnology; mAbs to hemagglutinin (HA) (clone HA-7) and ␮g/ml cycloheximide (cycloheximide chase) for up to 8 h. Cell extracts Flag epitopes and ␤-actin (clone AC-74) were from Sigma-Aldrich. Plas- were prepared and subjected to immunoblotting analysis. mids encoding for Flag-wtIRF-3, Flag-IRF3 5D, Flag-IRF-3 J2D, Flag- IRF-3 7D, Flag-IRF-3 5A, and Myc-wtIRF-3 have been described (17, 38). Plasmids encoding for Flag-IKKiwt and the dominant-negative version Results Flag-IKKi K38A were gifts of Dr. R. Lin (McGill University, Montreal, Virus infection results in proteasome-dependent degradation of Quebec, Canada). Flag-Cul1 was a gift from Dr. M. Pagano (New York IRF-3 University School of Medicine, New York, NY). pMT123-HA-ubiquine has been described (39). IRF-3 activation has been observed with both DNA and RNA enveloped viruses. In addition to its activation, a number of studies Cell types, transfection, and virus strains have shown that IRF-3 is degraded following infection with RNA The ts20 and ts41 were used as previously described (39, 40). TBK1 wild- viruses such as vesicular stomatitis virus, Newcastle disease virus, type and knockout murine embryonic fibroblasts (MEFs) have been de- measles virus, and SeV (6–8, 17, 18, 38, 42, 43). In contrast, DNA scribed (8) and were immortalized using the 3T3 protocol (41). Immortal- ized MEFs were maintained in MEM containing 10% FBS, 2 mM viruses such as the Herpesvirus family member HSV-1 do not glutamine, 0.1 mM nonessential amino acids. The Tet-inducible cell line induce IRF-3 degradation (6). Because HCMV, another Herpesvi- HEK 293 expressing a dominant-negative version of Cul1 (Flag-Cul1 rus family member, was recently shown to induce IRF-3 activation The Journal of Immunology 5061

and degradation (3, 44), we wanted to revisit the possibility that lane 4). Reciprocally, in the presence of HA-tagged ubiquitin, both RNA as well as DNA viruses target IRF-3 to the proteasome. a subpopulation of IRF-3 is also represented by a significant high Infection of Hel 299 fibroblasts with SeV resulted in a dramatic molecular mass Flag-signal (Fig. 2B, lane 4). Importantly, polyu- reduction in the steady-state level of IRF-3 (Fig. 1A, lanes 3–5). biquitination of endogenous IRF-3 increased following SeV infec- Native gel electrophoresis demonstrated that it is the activated tion and was only observed in the presence of MG-132 (Fig. 2C, form of IRF-3 that is subjected to degradation as observed by the upper panel). This increase in polyubiquitination clearly correlated reduction in the activated dimeric forms of IRF-3 (Fig. 1B, lanes with the stabilization of the hyperphosphorylated forms of IRF-3 3–5). As shown in Fig. 1A, treatment of cells with small concen- (Fig. 2C, lower panel). Boiling the cell extracts prior to immuno- tration (1 ␮M) of MG-132 significantly diminished the degrada- precipitation resulted in the same signal in Western blot analysis tion of IRF-3 following virus infection and induced the accumu- confirming polyubiquination of IRF-3 and not coimmunoprecipi- lation of C-terminal hyperphosphorylated forms of IRF-3 tated proteins (data not shown). To address the role of ubiquitina- (compare lanes 3–5 with lanes 8–10), which migrate more slowly tion in the modulation of the steady state abundance of IRF-3 on SDS-gel (17, 38). Higher concentrations of MG-132 totally following SeV infection, we used the temperature-sensitive cell blocked IRF-3 degradation but also inhibited the activation of the line ts20, in which the ubiquitin-activating enzyme E1 is active at latter following virus infection (data not shown). The concentra- 34°C but inactive at 39°C (48). Fig. 2D shows that the expression tion of MG-132 used in this study did not affect IRF-3 activation level of IRF-3 declined significantly at 8 and 12 h postinfection as observed by its dimerization state (Fig. 1B). Treatment with when cells were infected at the permissive temperature (see lanes lactacystin, another structurally unrelated proteasome inhibitor, 3 and 4, a). However, shifting the cells at the nonpermissive tem-

also resulted in the accumulation and stabilization of activated perature resulted in the stabilization of IRF-3 (Fig. 2Da, lanes Downloaded from forms of IRF-3 following virus infection (Fig. 1). Similar re- 7–8). This effect was not related to a lower infectability of the cells sults were observed in HCMV-infected cells (data not shown). at 39°C since the expression of viral proteins was comparable be- These results further substantiate the importance of the protea- tween the two conditions of infection (Fig. 2Dc). These data dem- some in IRF-3 degradation following infection by with both onstrate that ubiquitination of IRF-3 is involved in its degradation DNA and RNA viruses and also reiterate a potential role for the following SeV infection.

C-terminal phosphorylation of IRF-3 as a destabilization signal (17). http://www.jimmunol.org/ A Cullin-based ubiquitin ligase pathway is involved in host A ubiquitin-dependent process is involved in the degradation of cell-mediated IRF-3 degradation following SeV infection IRF-3 following SeV infection Because we observed the stabilization of phosphorylated forms of Because previous reports have suggested that some proteins may IRF-3 in the presence of proteasome inhibitors (Fig. 1), we then be targeted to the proteasome without ubiquitination (45–47), we asked whether a SCF complex was involved in IRF-3 degradation. ϩ first verified whether IRF-3 was polyubiquitinated following in- Rbx1/Roc1 is a RING finger protein that binds Zn2 . TPEN is a ϩ fection with SeV. Cotransfection of 293T cells with a HA-tagged Zn2 chelator that has been shown to inhibit the activity of purified ubiquitin construct together with a Flag-tagged-IRF-3 construct RING domain-containing E3 ligases, presumably by removing the

ϩ by guest on September 29, 2021 revealed that IRF-3 is polyubiquitinated in intact cells as noted Zn2 that is normally complexed with the RING domain (49–51). by the appearance of a high molecular mass HA signal (Fig. 2A, However, high concentrations of TPEN are often used, which can lead to nonspeciﬁc effects. Interestingly, the use of only 5 ␮M TPEN on Hel 299 ﬁbroblasts did not prevent IRF-3 activation as judged by the accumulation of the dimeric forms, but totally blocked its degradation following virus infection (Fig. 3A). Addi-

tion of ZnCl2 completely antagonized the effect of TPEN. Next, given that Cul1 protein is one of the invariable components of SCF complexes, we specifically targeted this subunit to inactivate the complex. First, we used the temperature-sensitive cell line ts41,in which the APP-BPI subunit of the NEDD8-activating enzyme E1 is inactivated at the nonpermissive temperature (39°C) (52), thus enabling the use of these cells as a model for study of Cul1-dependent protein degradation (53). Infection of ts41 cells at the permissive temperature (34°C) resulted in a significant loss of IRF-3 at 12 h postinfection, whereas no degradation of IRF-3 was observed when cells were infected at the nonpermissive temperature (Fig. 3Ba, compare lanes 6 and 12) under similar conditions of infection (Fig. 3Bc). Infection of the parental cell lines at the nonpermissive temperature (39°C) did not affect the degradation of IRF-3 following virus infection (data not shown). We also tested whether IRF-3 was recruited to Cul1 following virus infection. Coimmunoprecipitation experiments in 293T cells overexpressing FIGURE 1. Proteasome-dependent degradation of IRF-3 in virus-in- IRF-3 and Cul1 clearly demonstrated the recruitment of IRF-3 to ␮ fected cells. A, Hel 299 fibroblasts were pretreated for 30 min with 1 M Cul1 following SeV infection (Fig. 3C). To further substantiate the MG-132, 10 ␮M lactacystin, or vehicle (0.1% DMSO). Then, cells were hypothesis of a possible involvement of Cul1 in host cell-mediated either uninfected (Ϫ) or infected with SeV for the indicated periods of time in the continuous presence of the drug inhibitors. Cell lysates were ana- IRF-3 degradation following SeV infection, we took advantage of lyzed by immunoblotting with anti-IRF-3 Ab. When indicated, membranes a deletion mutant version of Cul1 (Cul1-N252), which lacks the were stripped and reprobed with anti-␤-actin Ab. B, Cell lysates from docking sites for Rbx1/Roc1 but is able to bind and sequestrate above were used in a native-PAGE assay to verify the dimerization status Skp1 (54). When overexpressed in cells, it acts in a dominant- of IRF-3. negative fashion to prevent degradation of known SCF E3 ligases 5062 UBIQUITIN-DEPENDENT DEGRADATION OF IRF-3

FIGURE 2. IRF-3 is polyubiquitinated in vivo. A and B, 293T cells were cotransfected with the indicated constructs. After 48 h, cell lysates were divided in two groups that were both subjected to immunoprecipitation Downloaded from using anti-Flag Ab. The immunoprecipitated material was washed several times in RIPA buffer and simulta- neously analyzed by immunoblotting with anti-HA (A) or anti-Flag (B) Abs. The membrane was stripped and reprobed with anti-Flag Ab (A, lower panel). The proteasome inhibitor MG-132 was added for the last 16 h of transfection. C, Hel 299 ﬁbroblasts were pretreated for http://www.jimmunol.org/ 30 min with 1 ␮M MG-132 and then uninfected (Ϫ)or infected with SeV for the indicated periods of time in the continuous presence of the drug inhibitor. Cell lysates were prepared in RIPA buffer and endogenous IRF-3 was immunoprecipitated. The immunoprecipitated material was washed several times in RIPA buffer and analyzed by immunoblotting with anti-ubiquitin Ab. D, Ubiquitination of IRF-3 is required for its degrada-

tion following SeV infection. E1-thermosensitive mu- by guest on September 29, 2021 tant (ts20) cells were preincubated at the permissive temperature (34°C) or shifted to the restrictive temperature (39°C) for 16 h. Then, cells were left uninfected (Ϫ) or infected with SeV for the indicated times and temperatures. Cell lysates were analyzed by IRF-3 immunoblotting (a). Membranes were stripped and reprobed with an anti-␤ actin Ab (b) to control for the amount of extract proteins. Membrane was then revealed with an anti-paramyxovirus Ab (c).

substrates such as p27Kip1, cyclin E, ␤-catenin, p105, and I␬B␣ in the stability of the hyperphosphorylated forms of IRF-3 was also (Ref. 54 and data not shown). Induction of Cul1 mutant by doxy- associated with a sustained activation of IRF-3 as veriﬁed by the pres- cycline resulted in a net increase in the stability of phosphorylated ence of dimers or its association to CREB coactivator after infection forms of IRF-3 following SeV infection of HEK 293 cells (Fig. with SeV (Fig. 3E). These data strongly suggest that upon C-terminal 3Da, compare lanes 3–5 with lanes 8–10). Interestingly, this increase phosphorylation, IRF-3 is recognized by a Cullin-based ubiquitin The Journal of Immunology 5063 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 3. A Cullin-based ubiquitin ligase is involved in host cell-mediated IRF-3 degradation following SeV infection. A, A RING domain-containing ␮ ␮ E3 ligase is involved in IRF-3 degradation. Hel 299 cells were preincubated for 30 min with 5 M TPEN in presence of 10 M ZnCl2 when indicated. Then cells were left uninfected (Ϫ) or infected with SeV for8hinthecontinuous presence of the Zn2ϩ chelator. Cell lysates were analyzed by immunoblotting with an anti-IRF-3 Ab. The membrane was stripped and reprobed with an anti-paramyxovirus Ab. The same cellular extracts were also used in a native PAGE assay to verify the dimerization status of IRF-3 (lower panel). B, IRF-3 requires a functional NEDD8 conjugation pathway for its efﬁcient degradation. ts41 cells were preincubated at the permissive temperature (34°C) or shifted to the restrictive temperature (39°C) for 16 h. Then, cells were left uninfected (Ϫ) or infected with SeV for the indicated times and temperatures. Cell lysates were analyzed by IRF-3 immunoblotting (a). Membranes were stripped and reprobed with an anti-␤ actin Ab (b) to show equal amount of cell extracts. Membrane was then revealed with an anti-paramyxovirus Ab (c). C, IRF-3 is recruited to Cul1 following virus infection. 293 T cells were cotransfected with the indicated constructs. Twenty- two hours posttransfection, cells were left uninfected (Ϫ) or infected with SeV for 8 h. Then, cell lysates were prepared and subjected to immunoprecipitation using anti-Flag Ab. The immunoprecipitated material was analyzed by immunoblotting with anti-Myc Ab. The membrane was stripped and reprobed with anti-Flag Ab. D, Expression of a Cul1 dominant-negative mutant increases IRF-3 stability following SeV infection. The tetracycline-inducible cell line HEK 293-Cul1 N252 was either left untreated (-dox) or incubated with 1 ␮g/ml doxycycline (ϩdox) for 20 h. Then, the cells were left uninfected (Ϫ) or infected with SeV for the indicated periods of time in the continuous presence of doxycycline. Cell lysates were analyzed by immunoblotting with anti-IRF-3 Ab (a), anti-Flag Ab (b), anti-paramyxovirus Ab (c), and anti-␤ actin Ab (d) to show equal amount of cell extracts. E, The increase in IRF-3 stability by Cul1N252 results in a prolonged activation of the transcription factor. The tetracycline-inducible cell line HEK 293-Cul1 N252 was treated and infected as above. Cell lysates where prepared and analyzed by immunoblotting with anti-IRF-3 Ab, anti-Flag Ab, anti-paramyxovirus Ab, and anti-␤-actin Ab as indicated. The same cellular extracts were also used in native-PAGE analysis (Native gel) or in coimmunoprecipitation experiments with an anti-CBP Ab (IP CBP). Following the electrophoresis, gels were transferred to nitrocellulose membranes which were probed with an anti-IRF-3 Ab.

ligase, belonging to the SCF complex family, thereby leading to its 2C). Therefore, we next addressed whether C-terminal phosphor- polyubiquitination and targeting to the proteasome. ylation of IRF-3 is essential for its degradation. To determine the effect of the C-terminal Ser/Thr cluster on the rate of IRF-3 turn- Degradation of IRF-3 is dependent of the TBK1/IKKi-signaling over, pulse-chase experiments were conducted in HeLa cells trans- pathway fected with different phosphomimetic point mutants (Fig. 4A). Phosphorylation of IRF-3 always precedes its degradation (see Whereas wild-type IRF-3 and the phosphomimetic J2D were very Refs. 3, 6, 17, 38, 42, and 43 and this study). In addition, phos- stable over a 12 h period, the phosphomimetics IRF-3 5D and 7D phorylation of IRF-3 correlates with its polyubiquitination (Fig. were unstable (Fig. 4B, a and b, and c). IRF-3 5D behaves as 5064 UBIQUITIN-DEPENDENT DEGRADATION OF IRF-3

wild-type IRF-3 and IRF-3 J2D (15.5 and 17.5 h, respectively). Similar results were obtained using cycloheximide chase experiments (data not shown). We next directly examined the contribution of the IKK-related kinases in IRF-3 degradation by ﬁrst using RNA interference silencing technology. Upon transfection of siRNA duplexes directed against IKKi and TBK1, the expression levels of both kinase iso- forms were down-regulated by ϳ70%. However, under these conditions, IRF-3 was still phosphorylated and degraded upon SeV infection (data not shown). Therefore, we switched to TBK1Ϫ/Ϫ MEFs, in which the activation of IRF-3 was reported to be dra- matically reduced in response to LPS, dsRNA and viral infection (15, 16), and observed a complete stabilization of IRF-3 following SeV infection (Fig. 5A). The Ab used to verify the expression level of TBK1 also detected a lower migrating band below TBK1, which is likely the other isoform IKKi. However, IKKi was not signiﬁ- cantly involved in IRF-3 activation as neither dimerization nor Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 4. Role of the C-terminal cluster of phosphoacceptor sites in the degradation of IRF-3. A, Schematic representation of the different phosphomimetic point mutants of IRF-3 used in this study. B, Analysis of IRF-3 turnover using [35S]methionine/cysteine biosynthetic labeling methodol- ogy. Transiently transfected HeLa cells were pulse-labeled with [35S]methionine/[35S]cysteine for 120 min and chased for the times indicated in complete medium containing excess methionine and cysteine. Cell extracts were subjected to immunoprecipitation with anti-Flag Ab (a and b)or anti-Myc Ab (c). After extensive washing, the immunoprecipitated proteins were separated by SDS-gel electrophoresis and analyzed by ﬂuorography using a gel documentation device (Typhoon 9410). C, Quantitative analysis of the data shown in B. Data points represent the densitometric analysis of IRF-3 degradation rate. ࡗ, IRF-3 J2D (r ϭ 0.994); f, wtIRF-3 (r ϭ 0.959); F, IRF-3 5D (r ϭ 0.996); Œ, IRF-3 7D (r ϭ 0.995); Ⅺ, wtIRF- 3ϩIKKi (r ϭ 0.957). The data points represent mean Ϯ SEM of three separate experiments.

FIGURE 5. The IKK-related kinase TBK1 is essential for the degrada- ϩ/ϩ constitutive activated forms of IRF-3 when overexpressed in target tion of IRF-3 following infection of fibroblasts with SeV. A, TBK1 and TBK1Ϫ/Ϫ MEFs were infected with SeV for the indicated times. Whole cells (reviewed in Refs. 2 and 5) and the cluster ranging form 396 405 cell extracts were prepared and analyzed for IRF-3, viral proteins, and Ser to Ser is thought to be directly phosphorylated by TBK1 TBK1/IKKi expression by immunoblotting. B, Cell extracts from above and IKKi (3, 8, 13). As suspected, in the presence of IKKi, IRF-3 were also subjected to native-PAGE analysis. C, TBK1ϩ/ϩ and TBK1Ϫ/Ϫ was significantly phosphorylated but also very unstable (Fig. 4Bc MEFs were transfected with 5 ␮g/ml poly I:C for the indicated time. Whole and C). Quantification of the data revealed that the half-lives of cell extracts were prepared and analyzed for IRF-3 and TBK1/IKKi ex- IRF-3 5D, IRF-3 7D, and IRF-3 in the presence of IKKi were pression by immunoblotting. D, Cell extracts from C were also subjected reduced to 7.2, 5.9, and 5.5 h, respectively, compared with that of to native-PAGE analysis. The Journal of Immunology 5065 degradation was observed in native-PAGE assay (Fig. 5, B and D). Transfection of poly I:C into TBK1ϩ/ϩ MEFs also resulted in IRF-3 degradation (Fig. 5C) and activation (Fig. 5D) whereas both of these biochemical process were completely abolished in TBK1- deficient MEFs. All together, our data suggest that the phosphorylation of the phosphoacceptor sites (between amino acid 396– 405) by the IKK-related kinases TBK1/IKKi create a signal that leads to the destabilization of IRF-3.

Discussion Over the last ﬁve years, the regulation of the innate immune response through activation of IRF-3 has been the subject of several reports. These studies have led to the characterization of new in- ducers of IRF-3 activity as well as the intracellular signaling pathways leading to IRF-3 activation (38, 42, 43, 55, 56). Notably, two IKK homologs, IKKi and TBK1, were shown to be involved in IRF-3 phosphorylation and activation (12–16, 57). Another important issue, which remains poorly understood, is the molecular mechanisms that trigger the degradation of IRF-3 following virus Downloaded from infection. Using a combination of pharmacological, biochemical, and genetic approaches, the results presented here strongly suggest that phosphorylation of the C-terminal phosphoacceptor sites by the IKK-related kinases TBK1/IKKi create a signal that leads to the recognition of IRF-3 by a Cullin-based ubiquitin ligase path-

way, which then induces the polyubiquitination-dependent degra- http://www.jimmunol.org/ dation of IRF-3. This conclusion is based on several observations. First, the two unrelated protease inhibitors MG-132 and lactacystin stabilized the hyperphosphorylated-activated forms of IRF-3. Sec- FIGURE 6. Proposed model for IRF-3 degradation. Following virus in- ond, the accumulation of high molecular mass ubiquitin conjugates fection, replicative intermediates such as dsRNA are recognized by intra- in MG-132-treated cells following viral infection. Third, the sta- cellular sensors like the RNA helicases RIGi and MDA5. These intracel- bilization of IRF-3 protein at the nonpermissive temperature in lular sensors transmit a signal to the mitochondrial-associated protein cells bearing a thermolabile allele of E1 or APP-BP1. Fourth, the VISA (also termed IPS-1, Cardif of MAVS). This adaptor protein activates ability of TPEN to stabilize IRF-3 following virus infection, sug- IRF-3 through the IKK-related kinases TBK1/IKKi. Phosphorylated IRF-3 gesting the implication of a RING domain-containing E3 in the dimerizes and accumulates into the nuclear compartment where it induces by guest on September 29, 2021 polyubiquitination-dependent degradation of IRF-3. Fifth, the re- antiviral genes. Phosphorylated IRF-3 is recognized by a Cullin-based E3 ligase such as a SCF complex that induces its polyubiquitination. The use cruitment of IRF-3 by Cul1 following virus infection and its sta- of leptomycin B, an antibiotic that specifically bind and inhibits chromo- bilization in cells overexpressing the Cul1-N252 mutant. Finally, some region maintenance/exportin 1 (CRM1), resulted in nuclear trapping the half-lives of C-terminal phosphomimetic IRF-3 mutants were of IRF-3 but did not prevent its degradation (data not shown) suggesting shorter than that of wild-type IRF-3. Overexpression of IKKi re- that the degradation of IRF-3 occurs in this cellular compartment. sulted in a net decrease in the half-life of IRF-3 and reciprocally, no degradation of IRF-3 was observed in TBK1Ϫ/Ϫ mouse embryonic fibroblasts. Given that Cul1 is one of the invariable components of SCF complexes our data thus suggest an important role modifier SUMO to IRF-3 might also be involved in the regulation for this family of E3 ligases in the control of IRF-3 stability (see of its transcriptional activity (66). Fig. 6). Overexpression of Cul1-N252 does not completely prevent Analysis of the human genome suggests that there are Ͼ70 IRF-3 degradation following SeV infection (Fig. 3, D and E), genes encoding for F-box proteins in mammals (reviewed in Ref. therefore suggesting that ubiquitin ligases other than the SCF com- 58). So far, only four SCFs have been characterized in details: plex are also likely to be involved in IRF-3 degradation following SCF␤TrCP1/Fbw1A, SCF␤TrCP2/Fbw1B, SCFSkp2, and SCFhCDC4/Fbw7 virus infection. The nature of these other E3 ligases is presently (reviewed in Refs. 59–61). SCF␤TrCP1/Fbw1A specifically recog- unknown. However, because chelating the Zn2ϩ and blocking the nizes I␬B␣,I␬B␤,I␬B␧, and ␤-catenin as substrates only when NEDDylation pathway completely abrogated the degradation of they are phosphorylated at both serine residues in the conserved IRF-3 following virus infection (see Fig. 3, A and B), this suggests DSGXXS motif (reviewed in Ref. 19). This motif is not found in that RING-type E3 ligases or more specifically members of Cullin- IRF-3. SCF␤TrCP2/Fbw1B contributes also to I␬B␣ ubiquitination RING ubiquitin ligase family may be involved in IRF-3 degrada- (62), whereas SCFSkp2 ubiquitinates various cell cycle regulators tion (21, 22). including p27Kip1 (23), p21Cip1 (63), and p130 (64). SCF hCDC4/Fbw7 To circumvent the innate immune response, several RNA and was shown to target cyclin E (65). Interestingly, both SCFSkp2 and DNA viruses express viral proteins with antagonistic activities to- SCFhCDC4/Fbw7 interact and promote degradation of c-Myc tran- ward essential components of the innate immune system. For ex- scription factor (25, 26). In addition to promoting c-Myc degra- ample, a recent study has shown that the immediate-early tran- dation, SCFSkp2 increases its transactivation activity, suggesting scription factor RTA from HHV8 has an unconventional intrinsic that SCFSkp2 is a transcriptional cofactor (25, 26). Given these Ub E3 ligase activity that targets both IRF-7 and IRF-3 for pro- interesting observations, we speculate that SCF complexes might teasome-mediated degradation. These authors also observed that also regulate IRF-3 transcriptional activity. Other posttranslational RTA was associated with a homology to the E6-associated protein modifications such as the conjugation of small ubiquitin-related C terminus domain E3 ligase protein that also catalyzes poly-Ub 5066 UBIQUITIN-DEPENDENT DEGRADATION OF IRF-3

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unique pathways. Genes Cells 6: 375–388. isomerase Pin1. Nat. Immunol. 7: 598–605. by guest on September 29, 2021 8878 CORRECTIONS

Yang, M.-L., H. A. Doyle, R. J. Gee, J. D. Lowenson, S. Clarke, B. R. Lawson, D. W. Aswad, and M. J. Mamula. 2006. Intracellular protein modiﬁcation associated with altered T cell functions in autoimmunity. J. Immunol. 177: 4541–4549.

In Table III, the second column heading was inadvertently duplicated in the ﬁfth column. “MRL/lpr mouse strain (10-wk-old)” is the correct heading. The corrected table is shown below.

Table III. isoAsp content is elevated in spontaneously activated T cells from MRL micea

Percentages (ϮSD) isoAsp Residue (pmol/mg protein Ϯ SD)

B10.BR MRL/1pr B10.BR MRL/lpr CD4ϩ T Cell mouse strain mouse strain mouse strain mouse strain Subsets (10-wk-old) (10-wk-old) (10-wk-old) (10-wk-old)

Naive 77.9 Ϯ 3.7% 23.5 Ϯ 7.8% 71.75 Ϯ 2.89 68.75 Ϯ 2.19 Activated 3.5 Ϯ 0.1% 28.1 Ϯ 2.3% ND 102.95 Ϯ 14.21 Memory 10.9 Ϯ 0.9% 42.7 Ϯ 5.6% ND 79.17 Ϯ 0.24

a Cell percentages were obtained via FACS analysis for the naive (CD44low CD62Lhigh), activated (CD44highCD62Lhigh), and memory (CD44highCD62Llow) subsets as shown in Fig. 5. After sorting by the expression proﬁle of CD44 and CD62L, cell lysates from naive, activated, and memory CD4ϩ T cells were measured for intracellular isoAsp content. n ϭ 3.

Leslie, A., D. A. Price, P. Mkhize, K. Bishop, A. Rathod, C. Day, H. Crawford, I. Honeyborne, T. E. Asher, G. Luzzi, A. Edwards, C. M. Rosseau, J. I. Mullins, G. Tudor-Williams, V. Novelli, C. Brander, D. C. Douek, P. Kiepiela, B. D. Walker, and P. J. R. Goulder. 2006. Differential selection pressure exerted on HIV by CTL targeting identical epitopes but restricted by distinct HLA alleles from the same HLA supertype. J. Immunol. 177: 4699–4708.

The twelfth author’s last name is misspelled. The correct name is Christine M. Rousseau.

Bibeau-Poirier, A., S.-P. Gravel, J.-F. Cle´ment, S. Rolland, G. Rodier, P. Coulombe, J. Hiscott, N. Grandvaux, S. Meloche, and M. J. Servant. 2006. Involvement of the I␬B kinase (IKK)-related kinases tank-binding kinase 1/IKKi and cullin-based ubiquitin ligases in IFN regulatory factor-3 degradation. J. Immunol. 177: 5059–5067.

In the first paragraph, fifth sentence of the Introduction and Materials and Methods, and in References, IKKE and IKKe should be IKK␧. The corrected sentences and Reference 9 are shown below. These infectious particles activate several kinases in the host including the recently described I␬B kinase (IKK) homologs, IKK␧ (9), also called IKKi (10), and Tank-binding kinase 1 (TBK1) (11). Commercial Abs were from the following suppliers: anti-IRF-3 Abs specific for human and rodent species were from Immuno-Biological Laboratories (IBL) and Zymed Laboratories, respectively; anti-IKK␧ Ab (IMG-270A) (that recognize as well TBK1) was from Imgenex; anti-ubiquitin mAb (clone P4D1) and mAb to MYC were from Santa Cruz Biotechnology; mAbs to hemagglutinin (HA) (clone HA-7) and Flag epitopes and ␤-actin (clone AC-74) were from Sigma-Aldrich.

9. Peters, R., S. M. Liao, and T. Maniatis. 2000. IKK␧ is part of a novel PMA-inducible I␬B kinase complex. Mol. Cell 5: 513–522. The Journal of Immunology 8879

In Results, in sentence 16 under the heading A Cullin-based ubiquitin ligase pathway is involved in host cell-mediated IRF-3 degradation following SeV infection, reference to CREB coactivator is incorrect. In sentence ten, under the heading Degradation of IRF-3 is dependent of the TBK1/IKKi-signaling pathway; reference to RNA interference silencing technology is incorrect. The corrected sentences are shown below. Interestingly, this increase in the stability of the hyperphosphorylated forms of IRF-3 was also associated with a sustained activation of IRF-3 as veriﬁed by the presence of dimers or its association to CREB binding protein (CBP) coactivator after infection with SeV (Fig. 3E). We next directly examined the contribution of the IKK-related kinases in IRF-3 degradation by ﬁrst using RNA interference (RNAi) technology.

Saidi, H., J. Eslaphazir, C. Carbonneil, L. Carthagena, M. Requena, N. Nassreddine, and L. Belec. 2006. Differential modulation of human lactoferrin activity against both R5 and X4-HIV-1 adsorption on epithelial cells and dendritic cells by natural antibodies. J. Immunol. 177: 5540–5549.

The second author’s last name is misspelled. The correct name is Jobin Eslahpazir.