Identification of the Minimal Phosphoacceptor Site Required for in Vivo Activation of Interferon Regulatory Factor 3 in Response to Virus and Double-stranded RNA*

Marc J. ServantDagger §||, Nathalie GrandvauxDagger §**, Benjamin R. tenOeverDagger §DaggerDagger, Delphine DuguayDagger §§, Rongtuan LinDagger §¶¶, and John HiscottDagger §§§||||

From the Dagger  Terry Fox Molecular Oncology Group, Lady Davis Institute for Medical Research and the Departments of §§ Microbiology & Immunology and § Medicine, McGill University, Montreal, Quebec H3T 1E2, Canada

Received for publication, September 25, 2002, and in revised form, January 9, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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The ubiquitously expressed latent interferon regulatory factor (IRF) 3 transcription factor is activated in response to virus infection by phosphorylation events that target a cluster of Ser/Thr residues, 382GGASSLENTVDLHISNSHPLSLTSDQY408 at the C-terminal end of the protein. To delineate the minimal phosphoacceptor sites required for IRF-3 activation, several point mutations were generated and tested for transactivation potential and cAMP-response element-binding protein-binding protein/p300 coactivator association. Expression of the IRF-3 S396D mutant alone was sufficient to induce type I IFN beta , IFNalpha 1, RANTES, and the interferon-stimulated gene 561 promoters. Using SDS-PAGE and immunoblotting with a novel phosphospecific antibody, we show for the first time that, in vivo, IRF-3 is phosphorylated on Ser396 following Sendai virus infection, expression of viral nucleocapsid, and double-stranded RNA treatment. These results demonstrate that Ser396 within the C-terminal Ser/Thr cluster is targeted in vivo for phosphorylation following virus infection and plays an essential role in IRF-3 activation. The inability of the phosphospecific antibody to detect Ser396 phosphorylation in lipopolysaccharide-treated cells suggests that other major pathways may be involved in IRF-3 activation following Toll-like receptor 4 stimulation.

    INTRODUCTION
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Recognition of invading pathogens such as viruses and bacteria by host cells is known to trigger the activation of multiple latent transcription factors, such as NF-kappa B, AP-1 (ATF-2/c-Jun), and the interferon regulatory factors (IRFs)1 (1-3). Once activated, these transcription factors regulate the expression of a set of genes encoding for immunomodulatory cytokines and chemokines that are involved in the establishment of the antiviral/bacterial state, which limits the spread of infection through innate and adaptive immune mechanisms (1-3). The best characterized component of the innate host defense to virus is the family of transcriptionally activated interferon (IFN) proteins, which include type I IFN-alpha and IFN-beta and type II IFN-gamma . Type I IFNs are mainly induced in response to infection by various types of RNA and DNA viruses (2, 4), although the bacterial endotoxin lipopolysaccharide (LPS) induces production of IFN in certain cells, albeit at low levels (5, 6). Once produced, these secreted proteins act in a paracrine fashion to induce gene expression in target cells in the adjacent microenvironment through engagement of cell surface IFN receptors and the JAK-STAT signaling pathway. The ISGF3 complex (ISGF3gamma /IRF-9-STAT1-STAT2) binds to interferon-stimulated response elements (ISRE) found in numerous IFN-induced genes, such as 2'-5' oligoadenylate synthase and the double-stranded (ds) RNA-activated kinase, resulting in the induction of proteins that impair viral gene expression and replication (2, 4).

IRF-3 is part of the IRF family of transcription factors that includes nine members with distinct roles in host defense against pathogens, immunomodulation, and growth control (for review see Refs. 3, 7, and 8). Previous studies have demonstrated that the C-terminal region of IRF-3, which comprises a cluster of phosphoacceptor sites, 382GGASSLENTVDLHISNSHPLSLTSDQY408, is phosphorylated as a consequence of virus infection (9, 10). Radioactive orthophosphate labeling, peptide mapping, phosphoamino acid analysis, and decreased mobility in SDS-PAGE have shown that IRF-3 is a phosphoprotein that is further inducibly phosphorylated upon virus infection (10-15). The generation of point mutated forms of IRF-3 suggests that Ser385 and Ser386 (9), as well as Ser396, Ser398, Ser402, Ser405, and Thr404 (10) are phosphorylated following virus infection and are involved in IRF-3 activation. Indeed, C-terminal phosphorylation is thought to produce a change in protein conformation that reveals the IRF association domain and the DNA-binding domain, thus promoting dimerization and binding to IRF-3 5'-GAAA(C/G)(C/G)GAAN(T/C)-3' consensus DNA-binding site (9, 10, 16). In addition, IRF-3 C-terminal phosphorylation is required for association with the histone acetyltransferase nuclear proteins CBP and p300 (9, 10, 16) causing IRF-3, which normally shuttles into and out of the nucleus, to become predominantly nuclear (9, 10, 17). The activated form of IRF-3, bound to CBP, induces transcription through distinct positive regulatory domains (PRD) in the type I IFN promoters and through select ISRE sites found in other genes such as the chemokine RANTES, the cytokine interleukin-15, and IFN-stimulated gene (ISG) 56 (10-12, 15, 18-22). Finally, phosphorylation of IRF-3 is thought to induce its degradation by a proteasome-mediated mechanism (10, 23).

In addition to virus infection, other stimuli such as LPS and poly(I-C) have been shown to induce IRF-3 activation (9, 12, 15, 24-28). However, no phosphorylation of IRF-3 in response to poly(I-C) treatment has been demonstrated. In this case, IRF-3 activation has been observed through nuclear accumulation, DNA binding activity, coactivator association, and gene induction (9, 12, 15, 24). On the other hand, phosphorylation of IRF-3 in response to LPS was demonstrated (26) but not sufficiently to detail the precise phosphoacceptor sites.

The in vivo signaling pathways leading to IRF-3 phosphorylation and activation as well as the precise phosphoacceptor sites remain to be elucidated. Previous studies have demonstrated inducible N-terminal phosphorylation of IRF-3 following the activation of mitogen-activated protein kinase kinase kinase pathways (13). More recently, the activation of DNA-PK following virus infection was shown to induce Thr135 phosphorylation (29). However, no convincing physiological roles have been ascribed to these covalent modifications. In the present study, we characterized the minimal phosphoacceptor site(s) involved in the in vivo activation of IRF-3 following treatment with known inducers. Of the seven potential phosphoacceptor sites present in the C-terminal cluster, a single point mutation of Ser396 to Asp (S396D) was sufficient to generate a strong constitutively active form of IRF-3. Moreover, by using a novel phosphospecific antibody, we show for the first time that Ser396 is phosphorylated in vivo following virus infection, nucleocapsid (N) expression or dsRNA treatment.

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Reagents-- LPS (L-2654) was purchased from Sigma and dissolved in distilled water. Poly(I-C) was purchased from Amersham Biosciences and resuspended in phosphate-buffered saline. Sendai virus (SeV) was a generous gift of Dr. Ilkka Julkunen (Public Health Research Institute, Helsinki, Finland).

Plasmid Constructions and Mutagenesis-- wtIRF-3, wtIRF-3 5A, wtIRF-3 5D, wtIRF-3 3D, wtIRF-3 J2A, and wtIRF-3 J2D pFLAG constructs and the luciferase reporter plasmids IFNalpha promoter (IFNA1 pGL-3), IFNbeta promoter (IFNB pGL-3), and the RANTES promoter (RANTES pGL-3) were described previously (13, 18, 21). The expression constructs encoding different IRF-3 C-terminal point mutants pFLAG-IRF-3 3A, 2D, 2A, S396A, S396D, S398A, S398D, S398A/S402A, and S398D/S402D were generated by overlap PCR mutagenesis using Vent DNA polymerase. The pCMV-2 measles N expression construct has been described previously (30). SeV N cDNA was subcloned from pGEM SeV N plasmid (a gift from Dr. Illka Julkunen) using 5'-AATGGCTGGGTTGTTGAGCACCTTC-3' and 5'-TTAGATTCCTCCCATCCCAGCTGCT-3' as forward and reverse primers, respectively. The PCR-generated fragment was subcloned into pCMV-2.

Cell Culture-- Human embryonic kidney (HEK) 293 cells were grown in alpha -minimum essential medium. HEC-1B cells and the astrocytoma cell line U373 overexpressing the Toll-like receptor (TLR)-4 coreceptor CD14 (U373/CD14), a gift from Dr. Michael David (University of California, San Diego, CA), were cultured in Dulbecco's modified Eagle's medium. The media were supplemented with 10% fetal bovine serum and antibiotics. U937 cells were grown in RPMI supplemented with 5% fetal clone (Hyclone) and antibiotics.

Transfections, Luciferase Assays, Treatments, and Infection-- All of the transfections were carried out on subconfluent HEK 293 cells (calcium phosphate coprecipitation method) or HEC-1B cells (FuGENE method) grown in 60-mm Petri dishes or 24-well plates (for the luciferase assay). 5 µg of DNA constructs (per 60-mm dish) or 10 ng of pRLTK reporter (Renilla luciferase for internal control), 100 ng of pGL3 reporter (firefly luciferase, experimental reporter), and 250 ng of pFLAG expression plasmids (24-well plate) were introduced into HEK 293 cells. At 36 h, the cells were collected, washed in ice-cold phosphate-buffered saline, and assayed for reporter gene activities (Promega). Infections with SeV as well as treatments with poly(I-C) and LPS were accomplished in serum-free medium for the first 2 h, after which 10% fetal bovine serum was added for the rest of the incubation period. Whole cell extracts (WCE) were prepared in Nonidet P-40 lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 5 mM EDTA, 10% glycerol, 30 mM NaF, 1.0 mM Na3VO4, 40 mM beta -glycerophosphate, 0.1 mM phenylmethylsulfonyl fluoride, and 5 µg/ml of each leupeptin, pepstatin, and aprotinin) and stored at -80 °C.

Reverse Transcriptase-PCR-- Total RNA was harvested using Trizol reagent (Invitrogen) as recommended by the manufacturer. RNA was reverse transcribed with Superscript (Invitrogen), and the resulting cDNA were used in a PCR with the primers described for the original cloning of virus N cDNAs (see above) at an annealing temperature of 60 °C using Taq DNA polymerase (Amersham Biosciences).

Peptide and Antibody Production-- To generate the polyclonal antibody specific for IRF-3 phosphorylated at Ser396, termed HIS5033, a phosphopeptide corresponding to residues 388-402 of human IRF-3, KENTVDLHIS(PO<UP><SUB>4</SUB><SUP>−</SUP></UP>)NSHPLS, was synthetized (W. M. Keck Biotechnology Resource Center), coupled to keyhole limpet hemocyanin, and used to immunize rabbits (Pocono Rabbit Farms & Laboratory, Inc, Canadensis, PA). Briefly, 2 mg of peptide in phosphate-buffered saline was coupled to 5 mg of keyhole limpet hemocyanin (Sigma) in 0.2% glutaraldehyde. After neutralization with glycine, the coupled peptide was dialyzed overnight against cold phosphate-buffered saline and resuspended at a concentration of 2 mg/ml for immunization.

Immunoblot Analysis-- To analyze the state of IRF-3 phosphorylation and to confirm the expression of the transgenes, WCE (30-60 µg) were subjected to electrophoresis on 7.5% or 10% acrylamide gels. The proteins were electrophoretically transferred to Hybond-C nitrocellulose membranes (Nycomed Amersham, Inc.) in 25 mM Tris, 192 mM glycine, and 20% methanol. The membranes were blocked in Tris-buffered saline containing 5% nonfat dry milk and 0.1% Tween 20 for 1 h at 25 °C before incubation for 2 h at 25 °C with anti-IRF-3 (Santa Cruz; SC-9082), anti-FLAG M2 (Sigma), anti-Ikappa Balpha (MAD 10) (1 µg/ml), anti-MYC (9E10) (1 µg/ml), or anti-ISG56 (a gift from Dr. G. Sen (Cleveland, OH) (1:1000) in blocking solution. For the phosphospecific antibodies anti-Ikappa Balpha Ser32 phosphospecific antibody (1:2000) (New England Biolab) and HIS5033 (1:10000), the membranes were incubated in blocking solution for 1 h at 25 °C followed by incubation in Tris-buffered saline containing 5% bovine serum albumin and 0.1% Tween 20 for overnight at 4 °C. After washing four times in Tris-buffered saline, 0.1% Tween 20, the membranes were incubated for 1 h with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (1:10000) in blocking solution. Immunoreactive bands were visualized by enhanced chemiluminescence (PerkinElmer Life Sciences). For coimmunoprecipitation studies, WCE (500 µg) were incubated with 1 µg of anti-CBP antibody A-22 (Santa Cruz) or 1 µg of anti-FLAG antibody M2 linked to 30 µl of protein A- or protein G-Sepharose beads, respectively, for 3 h at 4 °C (Amersham Biosciences). The beads were washed five times with Nonidet P-40 lysis buffer and resuspended in denaturating sample buffer, and the eluted IRF-3 proteins associated with CBP were analyzed by immunoblotting.

    RESULTS
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INTRODUCTION
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IRF-3 S396D Phosphomimetic Is a Strong Transactivator of PRD I-III- and ISRE-containing Promoters-- To delineate the minimal residues required for IRF-3 activation, the effects of various phosphomimetic point mutations (Fig. 1A) on the transactivating potential of IRF-3 were analyzed using reporter gene assays with the IRF-3-responsive promoters IFNalpha 1, IFNbeta , and RANTES (Fig. 1B). Overexpression of wtIRF-3 alone minimally induced IFNalpha 1, IFNbeta , and RANTES promoter activities, as demonstrated previously (10, 21, 31), whereas introduction of the C-terminal point mutation, S396D, enhanced IRF-3 transactivating potential over wtIRF-3 by 13-, 14-, and 11-fold for the IFNalpha 1, IFNbeta , and RANTES promoters, respectively (Fig. 1B). The IFNalpha 1, IFNbeta , and RANTES promoters were also activated by the double point mutant 2D (13-, 12-, and 12-fold over wtIRF-3, respectively) and, as previously reported (10, 16, 18, 21), the multiple point mutant 5D (9-, 5.5-, and 8-fold induction, respectively). However, the point mutants S398D, S398D/S402D, and S402D/S404D/S405D exhibited only intermediate effects, and the S385D/S386D mutant did not induce these promoters. Our initial result thus demonstrates that substitution of Ser396 with the phosphomimetic Asp is sufficient to generate a constitutively active form of IRF-3 that functions as a strong activator of promoters containing PRD I-III or ISRE regulatory elements.


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Fig. 1.   IRF-3 S396D phosphomimetic is a strong transactivator of PRD I-III- and ISRE-containing promoters. A, schematic representation of IRF-3 and location of the point mutants used in this study. The DNA-binding domain, the nuclear export sequence element, the proline-rich region, and the C-terminal IRF association domain are indicated. The region between amino acids 382 and 414 is expanded below the schematic. The amino acids targeted for alanine or aspartic acid substitutions are shown in large letters. The point mutations are indicated below the sequence: S396D; S396A; S398D; S398A; 2D, S396D and S398D; 2A, S396A and S398A; S398D/S402D; S398A/S402A; 3D, S402D, T404D, and S405D; 3A, S402A, T404A, and S405A; 5D, S396D, S398D, S402D, T404D, and S405D; 5A, S396A, S398A, S402A, T404A, and S405A; J2D, S385D and S386D; and J2A, S385A and S386A. B, HEK 293 cells were transiently transfected with luciferase reporter constructs containing the IFN-alpha promoter (left panel), the IFN-beta enhancer (middle panel), and the RANTES promoter (right panel) and different IRF-3 point mutants as indicated below the bar graphs. At 36 h post-transfection, the luciferase activity was measured. The relative luciferase activity was measured as fold activation over the transfection of pFLAG-CMV2 alone. Each value represents the mean ± S.E. of triplicate determinations. The data are representative of at least four different experiments with similar results.

The Minimal Phosphoacceptor Site Required for IRF-3 Association with CBP Coactivator Maps to Ser396-- Formation of the IRF-3 holocomplex, which consists of an IRF-3 dimer and CBP or p300 coactivators, is a critical step in the activation of the transcription factor (32). Association with CBP/p300 is strictly localized to the nucleus and tethers IRF-3 in the nucleus after induction with viruses (16, 17, 32). Therefore, the relationship between the different IRF-3 point mutants and CBP association was evaluated using coimmunoprecipitation assays. As shown in Fig. 2, infection with SeV stimulated wtIRF-3 and CBP association (Fig. 2, A and B, lanes 1 and 2). Mutation of Ser396 to Ala completely abrogated virus-induced CBP association (Fig. 2A, lanes 3 and 4), whereas the phosphomimetic point mutant S396D constitutively associated with CBP (Fig. 2B, compare lanes 1 and 3), an association that was enhanced following virus infection (Fig. 2B, lane 4). Similar results were obtained with the other mutants IRF-3 2A (Fig. 2A, lanes 7 and 8) and IRF-3 5A (Fig. 2A, lanes 13 and 14), where no inducible association with CBP was observed. Conversely, strong constitutive binding to CBP was present with IRF-3 2D (Fig. 2B, lane 7) and IRF-3 5D (Fig. 2B, lane 13). As a control for these results, the S398A form of IRF-3 was still able to associate with CBP following virus infection (Fig. 2A, lanes 5 and 6), but only a basal association of the phosphomimetic counterpart IRF-3 S398D with CBP was observed in the absence of infection (Fig. 2B, compare lanes 1 and 5). As previously reported (16), coactivator association was not observed with either the IRF-3 J2A or J2D mutants (Fig. 2, A and B, lanes 15 and 16). As discussed previously (30), Ser385/386 phosphorylation may be required for the sequential phosphorylation of the Ser/Thr cluster at amino acids 396-405. Together these data indicate that Ser396 is a critical phosphoacceptor site for coactivator CBP/p300 association and that a clear correlation exists between the capacity of the point mutants to induce promoter activity and to associate with the CBP coactivator.


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Fig. 2.   Ser396 is the minimal phosphoacceptor site required for IRF-3 association with CBP coactivator. HEK 293 cells were transiently transfected with different FLAG-tagged IRF-3 constructs as indicated and 24 h post-transfection were left untreated or infected with SeV (40 HAU/106 cells) for 12 h. WCE (500 µg) were immunoprecipitated (IP) with anti-CBP antibody A22 absorbed to protein A-Sepharose beads. The immunoprecipitated proteins were resolved by SDS gel electrophoresis on 7.5% acrylamide gel and transferred to nitrocellulose membrane. The membranes were probed with anti-FLAG antibody. WCE (25 µg; 5% input) run on a 7.5% acrylamide gel, transferred to membrane, and immunoblotted with anti-FLAG antibody are also shown for both set of mutants: alanine (A) and aspartic acid substitutions (B).

IRF-3 S396D Expression Induces the Endogenous Interferon-stimulated Gene, ISG56-- Recent studies have demonstrated that the 561 gene encoding the ISG56 protein is strongly induced in response to virus infection, type I IFN, dsRNA (33) or direct expression of IRF-3 5D (20). The effect of IRF-3 on 561 gene induction is direct, because the promoter contains two tandem ISRE sites, mutation of which results in the complete loss of promoter activation by IRF-3 5D (20). To determine the effect of transient expression of the different phosphomimetic IRF-3 forms on the induction of the endogenous ISG56 in the IFN-unresponsive HEC-1B cells, induction of ISG56 was analyzed by immunoblot (Fig. 3). Sendai virus infection resulted in 25-fold induction of ISG56 (Fig. 3, lane 10), whereas wtIRF-3 expression alone resulted in a weak induction of ISG56 protein of 2.6-fold (Fig. 3, lane 2). Transfection of S396D as well as 2D and 5D resulted in a stronger induction of the protein of 5.2-, 6.4-, and 10-fold, respectively (Fig. 3, lanes 3, 5, and 8). However, only weak inductions between 2.2- and 3.5-fold were observed with the other point mutants (Fig. 3, lanes 4, 6, 7, and 9). Thus, mutation at position 396 is sufficient to create a phosphomimetic form of IRF-3 that allows stable binding to CBP/p300 coactivator, transactivation of reporter genes controlled by PRD and ISRE response elements, and, importantly, enhancement of endogenous expression of ISG56.


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Fig. 3.   Expression of IRF-3 S396D induces the endogenous gene encoding ISG56. HEC 1B cells were transiently transfected with the indicated FLAG-tagged IRF-3 phosphomimetic point mutants. In one condition, HEC 1B cells were also infected for 15 h with SeV (40 HAU/106 cells). At 48 h post-transfection, WCE (30 µg) were resolved by SDS gel electrophoresis on 10% acrylamide gel and transferred to nitrocellulose membrane. The membrane was probed with anti-ISG56 antibody (upper panel), stripped and reprobed with anti-actin (bottom panel), and stripped and finally reprobed with an anti-FLAG antibody (middle panel). Fold induction is expressed as a ratio between the densitometric values for ISG56 and actin expression level. The values were then normalized to pFLAG-CMV2.

In Vivo Phosphorylation of Ser396 of IRF-3 following Virus Infection-- To verify whether in vivo phosphorylation of IRF-3 occurred at Ser396, an antibody directed against a phosphopeptide spanning Ser396, named HIS5033, was raised (see "Materials and Methods"). Fig. 4A shows that the antibody reacted only toward the transfected FLAG-wtIRF-3 transgene when cells were infected with SeV; indeed, no signal was observed in IRF-3 S396A-overexpressing cells infected with SeV. In contrast, Fig. 4B shows a specific endogenous signal when extracts derived from SeV-infected HEK 293 cells were analyzed by immunoblot using the phosphospecific 396-P antibody HIS5033 (Fig. 4B, lanes 2 and 3). Reblotting of the stripped membrane with an anti-IRF-3 antibody (SC-9082) showed that the specific signal observed with HIS5033 antibody corresponded to the slowly migrating form of IRF-3, previously shown to represent the activated form of IRF-3 (Fig. 4B, compare lanes 4-6) (10, 13). Although IRF-3 degradation had already reduced the amount of IRF-3 in these cells at 6 h post-infection (Fig. 4B, lane 5) and the IRF-3 signal was difficult to detect with anti-IRF-3 antibody (SC-9082), the phosphospecific antibody HIS5033 reacted strongly under these conditions (Fig. 4B, compare lanes 2 and 5 and lanes 3 and 6).


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Fig. 4.   In vivo phosphorylation of Ser396 of IRF-3 following virus infection. A, HEK 293 cells were transiently transfected with FLAG-tagged constructs as indicated. At 24 h post-transfection, the cells were left untreated or infected with SeV (40 HAU/106 cells) for 7h. WCE (500 µg) were immunoprecipitated with anti-FLAG antibody linked to protein G-Sepharose beads. The immunoprecipitated proteins were resolved by SDS gel electrophoresis on a 7.5% acrylamide gel and transferred to nitrocellulose membrane. The membranes were probed with HIS5033 antibody. Following stripping, the membrane was reprobed with an antibody that reacts against whole IRF-3 (Santa Cruz; SC-9082). B, endogenous phosphorylation of IRF-3 on Ser396. WCE (50 µg) prepared from HEK 293 cells uninfected (-) or infected with SeV (40 HAU/106 cells) for the indicated times were resolved by 10% SDS-PAGE and transferred to nitrocellulose. IRF-3 was analyzed by immunoblotting (IB) for the presence of Ser396 phosphorylation with HIS5033 antibody (lanes 1-3). The membranes were stripped and reprobed with the anti-IRF-3 antibody SC-9082 (lanes 4-6). C, HEK 293 cells were transiently transfected with expression plasmids encoding either SeV or measles virus N. At 36 h post-transfection WCE (50 µg) were analyzed as detailed above using IRF-3 396 phosphospecific HIS5033 (upper panel) and anti-IRF-3 antibody SC-9082 (middle panel). Expression of SeV and measles virus N was analyzed by reverse transcriptase-PCR using the full-length primers described under "Materials and Methods."

To investigate whether the phosphorylation of Ser396 could be mimicked by expression of viral N protein (30), transfection experiments were performed with constructs expressing measles virus and SeV N for 36 h post-transfection. As shown in Fig. 4C, expression of either SeV or measles virus N induced higher migrating forms of IRF-3 (middle panel). Use of HIS5033 antibody revealed that Ser396 was phosphorylated under these conditions (Fig. 4C, top panel). Taken together, these results demonstrate that virus infection or expression of viral nucleocapsid protein is sufficient to induce Ser396 phosphorylation.

IRF-3 Ser396 Is Targeted by Virus and dsRNA in U373/CD14-- To study the kinetics of Ser396 phosphorylation in response to virus, dsRNA, or LPS, the LPS-sensitive human U373/CD14 astrocytoma cell line was used as previously described (34). Detailed kinetics of SeV, LPS, and poly(I-C) induction was performed, and the phosphorylation state of Ser396 was analyzed by immunoblot using the phosphospecific antibody HIS5033. As shown in Fig. 5 (A and B), both SeV and poly(I-C) induced Ser396 phosphorylation of IRF-3 with maximum signals detected at 4 and 2 h, respectively (Fig. 5, A, lanes 4-7, and B, lanes 3 and 4). Specific phosphoserine 396 signal was detected when IRF-3 first displayed a retarded mobility in SDS-PAGE, as observed by the use of the SC-9082 antibody (Fig. 5, A, lanes 12-15, and B, lanes 11-13). However, no signal was detected when cells were treated with LPS (Fig. 5C, lanes 1-8). Furthermore, qualitative and temporal differences in the kinetics of IRF-3 Ser396 phosphorylation by SeV and poly(I-C) were identified: 1) Although phosphorylation of IRF-3 in response to SeV infection, first detected at 4 h post-infection, was followed by degradation (Fig. 5A, lanes 12-16), phosphorylation in response to poly(I-C) was detected at 2 h and did not lead to degradation. Rather, IRF-3 appeared to be dephosphorylated over time and returned to basal forms (Fig. 5B, lanes 12-16). 2) Although Ser396 phosphorylation was not detected after LPS stimulation, a transient shift from form I to form II (Fig. 5C, lanes 13-15) was observed. This transition was previously shown to correlate with an N-terminal phosphorylation possibly mediated through a mitogen-activated protein kinase kinase kinase-related pathway (13). To verify that LPS treatment was efficient, phosphorylation of Ser32 of Ikappa Balpha by the Ikappa B kinase complex was measured using either the Ser32 phosphospecific antibody (Fig. 5F, lanes 2-8) or change of mobility in SDS gel observed by the use of the Ikappa Balpha -specific MAD 10 antibody (Fig. 5F, lanes 11-16). Together, these data demonstrate that Ser396 phosphorylation of IRF-3 occurs in vivo following treatment with inducers such as SeV and poly(I-C), but not LPS.


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Fig. 5.   IRF-3 Ser396 is targeted by virus and dsRNA in U373/CD14. WCE (45 µg) prepared from the U373 astrocytoma cell line that overexpressed the TLR4 coreceptor CD14 (U373/CD14) untreated (-) or treated with SeV (40 HAU/106 cells) (A and D), poly(I-C) (100 µg/ml) (B and E), and LPS (10 µg/ml) (C and F) for the indicated time points were resolved on 7.5% acrylamide gels and transferred to nitrocellulose membranes. Ser396 phosphorylation of IRF-3 was detected with HIS5033 antibody in immunoblot analysis (A-C, lanes 1-8) and with anti-IRF-3 antibody SC-9082 (A-C, lanes 9-16). The membranes were stripped and reprobed with a phosphospecific antibody against Ikappa Balpha (Ikappa Balpha -P) (D-F, lanes 1-8) and an antibody that recognized nonphosphorylated and phosphorylated Ikappa Balpha (MAD 10) (D-F, lanes 9-16). The same results were observed in three separate experiments.

IRF-3 Ser396 Phosphorylation Is Not Detected in LPS-treated U937 Cells-- To further analyze the LPS effect, IRF-3 Ser396 phosphorylation was also examined in the monocytic cell line U937. Fig. 6A demonstrates that infection of U937 cells with SeV resulted in IRF-3 Ser396 phosphorylation beginning as early as 2 h post-infection, whereas no signal with HIS5033 antibody was detected with cell extracts derived from LPS-stimulated cells (Fig. 6B). Both SeV and LPS were able to induce the phosphorylation of Ikappa Balpha (Fig. 6, C and D).


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Fig. 6.   IRF-3 Ser396 phosphorylation is not detected in LPS treated U937 cells. WCE (45 µg) prepared from the U937 cell line untreated (-) or treated with SeV (40 HAU/106 cells) (A and C) and LPS (10 µg/ml) (B and D) for the indicated times were resolved on SDS gels. The phosphorylation of IRF-3 and Ikappa Balpha in U937 cells was measured as described in Fig. 5. These data are representative of at least three experiments with similar observations.


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The rate-limiting step in the activation of IRF-3 is its phosphorylation by at least one unidentified kinase, namely virus-activated kinase, activated following virus infection or dsRNA treatment (9, 10, 12-15, 24). The major phosphoacceptor sites described to date are clustered in the C-terminal end of the protein. Yoneyama et al. (9) identified two sites, Ser385 and Ser386, in which substitution to alanine residues completely abrogates inducible phosphorylation as diagnosed by mobility change on SDS-PAGE, nuclear localization, association with p300 coactivator, and gene transactivation. On the other hand, our group identified five other phosphoacceptor sites, namely Ser396, Ser398, Ser402, Thr404, and Ser405 (10). Mutation of these sites to aspartic acid generates a strong constitutive active form of IRF-3, IRF-3 5D, in which slow migration in SDS-PAGE, dimerization, CBP association, DNA binding, and gene transactivation occur without the need of virus infection. Conversely, generation of an IRF-3 mutant in which the five phosphoacceptor sites are replaced with alanine completely inhibits virus-induced dimerization, coactivator association, and transactivation activity (10, 13, 16, 18, 21).

Despite these molecular observations, the contribution of different phosphorylation sites has not been addressed in vivo. In a previous study (21), we showed that the IRF-3 2A mutant behaves like the IRF-3 5A mutant. In fact, the IRF-3 2A mutant is not phosphorylated in response to virus infection. Moreover, as determined through immunofluorescence analysis, the IRF-3 2A-GFP protein does not translocate to the nucleus. These results point out the importance of these two residues in the IRF-3 activation process. In the present study, we demonstrate that mutation of Ser396 to Asp is the minimal mutation required to produce a strong phosphomimetic form of IRF-3. This observation was confirmed using a phosphospecific antibody raised against a peptide containing the phosphorylated Ser residue at position 396. We show for the first time that this site is phosphorylated in vivo when cells are exposed to SeV, nucleocapsid, or poly(I-C), well characterized inducers of IRF-3. Together these results demonstrate that the Ser396 residue is critical for IRF-3 activation.

Recent studies also suggested LPS as another IRF-3 inducer (25-28). Indeed, treatment of murine macrophages with LPS was shown to induce the production of IFN, suggesting the activation of some IRF members (5, 6). In this context, Navarro and David (25) reported that LPS treatment of human U373 astrocytoma cells resulted in IRF-3 activation (nuclear translocation and DNA binding activity) via a TLR- and p38-dependent pathway. This observation was supported by recent findings demonstrating that IRF-3 mediates a TLR-3/TLR-4-specific antiviral gene program when murine B cells are exposed to dsRNA and LPS, respectively (35). In addition, it has recently been shown through gene disruption targeting studies that neither the universal adaptor protein MyD88 nor the novel adaptor TIRAP/Mal (36) were involved in IRF-3 activation following LPS stimulation of peritoneal macrophages (26, 37). The adaptor protein involved in LPS-induced IRF-3 activation has yet to be identified. However, the newly identified adaptor protein TRIF appears to be involved in dsRNA-induced IRF-3 activation by TLR-3 (Ref. 38 and references therein), which might also be the case for IRF-3 activation by TLR-4. Our data suggest that LPS-induced activation of IRF-3 does not lead to phosphorylation of Ser396. How LPS activates IRF-3 remains to be determined; treatment of target cells with LPS generates multiple signaling pathways in addition to IKK/JNK/p38, such as protein kinase C, Src-type tyrosine kinases, and the phosphatidylinositol 3-kinase-protein kinase B pathway (1). Phosphorylation of IRF-3 in response to LPS was shown by slower mobility in one-dimensional immunoblot (26). However, the analysis of IRF-3 phosphorylation was not resolved sufficiently to delineate the different IRF-3 phosphorylated forms. As described previously, stress inducers induce N-terminal IRF-3 phosphorylation, which is characterized by a shift from form I to form II in immunoblot analysis (13), an effect also observed with LPS in the U373 astrocytoma cell line (Fig. 5C, lanes 13 and 14) and B16 melanoma cells.2 Therefore, N-terminal phosphorylation or C-terminal phosphorylation at other Ser or Thr residues may be induced by LPS and therefore involve other signaling pathways in the activation of IRF-3. However, a limited sensitivity of the phosphospecific antibody cannot be ruled out, and a weak but significant phosphorylation of IRF-3 at Ser396 might occur following TLR-4 activation.

Based on the capacity of bacterially produced IRF-3 to bind the ISRE in vitro (22, 39), it was concluded that mutation of Ser396 to Asp in recombinant IRF-3 eliminated DNA binding (39). This observation is in apparent contradiction with the results of the present study demonstrating that ectopically expressed IRF-3 S396D acts as a strong transactivator of ISRE containing promoters (Figs. 1 and 3). However, a recent study demonstrated that CBP/p300 was required for the DNA binding activity of the holocomplex (32). This observation may explain why no stable binding to DNA was observed with recombinant IRF-3 S396D in the absence of coactivators (39) and strengthens the observation that IRF-3 S396D is a strong IRF-3 phosphomimetic in part because of its capacity to bind CBP (Fig. 2).

Another surprising finding was the relative stability of the phosphomimetic point mutants (data not shown). Indeed, whereas inactive IRF-3 is very stable (40), virus infection results in a rapid degradation of IRF-3 via a proteasome-dependent pathway (10, 13, 23). Based on the observation that virus-induced degradation of IRF-3 always follows phosphorylation (10, 13), we assumed that the destabilization of IRF-3 might be the consequence of phosphorylation of residues in the C-terminal cluster. Based on two observations, this is unlikely to be the signal for degradation of IRF-3 in vivo: 1) introduction of phosphomimetic point mutations did not lead to increased degradation of IRF-3 when the mutants were overexpressed in HEK 293 cells (data not shown) and 2) treatment of U373/CD14 with poly(I-C) clearly induced IRF-3 Ser396 phosphorylation (Fig. 5B, lane 4). However, this phosphorylation, which induces a shift in the migration profile of IRF-3, was not followed by degradation (Fig. 5B, lanes 11-16). IRF-3 instability may therefore be specific to a product of the virus life cycle that recognizes activated IRF-3 and targets it for degradation by the proteasome pathway. Finally, the critical role of Ser396 phosphorylation in IRF-3 activation following virus infection and the development of a specific and sensitive phosphospecific antibody against Ser396 may be useful as a research and diagnostic tool as a marker of virus infection.

    ACKNOWLEDGEMENTS

We thank members of the Molecular Oncology Group at the Lady Davis Institute, McGill University for helpful discussions.

    FOOTNOTES

* This work was supported by research grants from the Canadian Institutes of Health Research, the National Cancer Institute of Canada, and the Canadian Network for Vaccines and Immunotherapeutics.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

These authors contributed equally to this work.

|| Supported by a Canadian Institutes of Health Research post-doctoral fellowship.

** Supported by a post-doctoral Fonds de la Recherche en Santé du Quebec (FRSQ) fellowship.

Dagger Dagger Supported by an National Science and Engineering Research Council studentship.

¶¶ Supported by a FRSQ Chercheur Boursier.

|||| Supporte by a Canadian Institutes of Health Research Senior Scientist award. To whom correspondence should be addressed: Lady Davis Institute-Jewish General Hospital, McGill University, 3755 Cote Ste. Catherine, Montreal, PQ H3T 1E2, Canada. Tel.: 514-340-8222 (ext. 5265); Fax: 514-340-7576.

Published, JBC Papers in Press, January 10, 2003, DOI 10.1074/jbc.M209851200

2 D. Duguay and J. Hiscott, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: IRF, interferon regulatory factor; IFN, interferon; LPS, lipopolysaccharide; STAT, signal transducers and activators of transcription; ISRE, interferon-stimulated response element(s); ds, double-stranded; CBP, cAMP-response element-binding protein-binding protein; PRD, positive regulatory domain(s); RANTES, regulated on activation normal T cell expressed and secreted; ISG, IFN-stimulated gene; N, nucleocapsid; SeV, Sendai virus; HEK, human embryonic kidney; TLR, Toll-like receptor; WCE, whole cell extracts; HAU, hemagglutination units.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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