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A CRM1-dependent Nuclear Export Pathway Is Involved in the Regulation of IRF-5 Subcellular Localization*

Interferon regulatory factors (IRFs) are involved in gene regulation in many biological processes including the antiviral, growth regulatory, and immune modula-tory functions of the interferon system. Several studies have demonstrated that IRF-3, IRF-5, and IRF-7 specif-ically contribute to the innate antiviral response to virus infection. It has been reported that virus-specific phosphorylation leads to IRF-5 nuclear localization and up-regulation of interferon, cytokine, and chemokine gene expression. Two nuclear localization signals have been identified in IRF-5, both of which are sufficient for nuclear translocation and retention in virus-infected cells. In the present study, we demonstrate that a CRM1-dependent nuclear export pathway is involved in the regulation of IRF-5 subcellular localization. IRF-5 possesses a functional nuclear export signal (NES) that controls dynamic shuttling between the cytoplasm and the nucleus. The NES element is dominant in unstimulated cells and results in the predominant cytoplasmic localization of IRF-5. Mutation of two leucine residues in the NES motif to alanine, or three adjacent Ser/Thr residues to the phosphomimetic Asp, results in constitutively nuclear IRF-5 and suggests that phosphorylation of adjacent Ser/Thr

The success of the innate host defense to viral and bacterial infections is dependent on the ability of the cell to detect the presence of the invading pathogen. In response to the recognition of components specific to viruses and bacteria, the host cell activates several signal transduction cascades that produce protein messengers in the form of cytokines and chemokines that impede viral/bacterial replication and spread through innate and adaptive immune mechanisms (1,2). Type I interferons secreted by virus-infected cells activate the innate immune machinery, modulate adaptive immune responses, promote apoptosis of infected cells, and induce an antiviral program in uninfected cells (3,4).
Molecular regulation of IFN 1 gene expression is tightly regulated by extra-and intracellular signals generated during primary infection, culminating in the activation of NF-B, AP-1, and interferon regulatory factor (IRF) transcription factors that trigger an immediate early IFN response characterized by the release of IFN␤ and IFN␣1 (5)(6)(7). Once produced, secreted IFN acts in a paracrine fashion to induce gene expression in neighboring cells through engagement of cell surface IFN receptors. Activation of the JAK-STAT signaling pathway leads to the formation of STAT1/2 heterodimers, which in conjunction with IRF-9 (or interferon-stimulated gene factor 3␥) bind to interferon-stimulated response elements found in hundreds of IFN-induced genes including 2Ј-5Ј oligoadenylate synthase, Mx, double-stranded RNA-activated kinase, and major histocompatibility complex class I, resulting in the induction of proteins that impair viral gene expression and replication (1,8). In addition, IFNs have other pleiotropic effects in the host, with important roles in apoptosis, growth inhibition, and development of protective immune responses via increased expression of major histocompatibility complex class I proteins (9) and other components of adaptive immunity. IFNs thus link the innate immune responses to adaptive immunity (5,10).
Biochemical, molecular biological, and gene knock-out studies have demonstrated that the members of the interferon regulatory factor family play important roles in pathogen response, cytokine signaling, hematopoietic differentiation, regulation of cell cycle, and apoptosis (reviewed in Refs. 3, 7, and 11-14). Among the members of the IRF family, IRF-3 and IRF-7 play essential roles in the virus-induced type I IFN gene expression (15)(16)(17)(18)(19)(20)(21)(22). Phosphorylation of the C-terminal serines of both IRF-3 and IRF-7 is essential for nuclear localization and transactivation, and both proteins play complementary rather than redundant roles in the regulation of IFN and chemokine gene expression (15,16,18,19,(22)(23)(24)(25)(26)(27). The net result is the generation of the antiviral activity of IFNs including induction of apoptosis, inhibition of cell growth, and immune response modulation. Recently, the IKK-related kinases, IKK⑀ (28) and * This work was supported in part by grants from the Cancer Research Society Inc. and Canadian Institutes of Health Research. 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.
IRF-5 is an additional direct transducer of virus-mediated signaling that plays a role in the expression of multiple cytokine/chemokines (34 -36). The sequence of human IRF-5 cDNA was first published in the NCBI GenBank TM by T. Mak's group (accession number U51127), and the transcript encoded 504 amino acids (isoform a). Three IRF-5 transcript variants have been reported: IRF-5 transcript variant 2 spliced out 48 nucleotides corresponding to the 5Ј portion of exon 6 and contained an additional 30 nucleotides (encoded 498 aa, isoform b, accession number NM_032643); IRF-5 transcript variant 3 and 4 spliced out 48 nucleotides corresponding to the 5Ј portion of exon 6 encoding an identical protein of 488 aa (accession numbers AY504946 and AY504947). Mori et al. (37) also identified two transcript variants: transcript b spliced out 48 nucleotides corresponding to the 5Ј portion of exon 6, and transcript c spliced out 34 nucleotides corresponding to exon 5. Using cell line overexpression of the 488 aa (encoded by variant 3 or 4) IRF-5, Barnes et al. (34) demonstrated that IRF-5 was specifically activated by NDV but not by Sendai virus and that IRF-5 induction also up-regulates expression of IFNA genes. Furthermore, overexpression of IRF-5 induced multiple cytokines and chemokines in infected BJAB cells (35).
IRF-5 can act as both an activator and a repressor of IFN gene expression depending on the IRF-interacting partner: IRF-5 cooperates with IRF-3 in the stimulation of IFNA gene transcription and suppresses IRF-7-mediated IFN gene expression (36). As a direct p53 target gene product, IRF-5 also inhibits the growth of tumor cells both in vitro and in vivo (37,38). IRF-5-mediated growth inhibition is associated with a p53-independent G 2 -M cell cycle arrest and with the stimulation of multiple cell cycle regulatory and proapoptotic genes including Bak, caspase 8, Bax, and p21 (38).
Nucleocytoplasmic trafficking of protein and RNA molecules plays an important role in eukaryotic cell function (39). A related family of shuttling transport factors, importins and exportins, recognizes nuclear localization sequence (NLS)-containing or nuclear export sequence (NES)-containing proteins and coordinates trafficking between the nucleus and the cytoplasm. CRM1 (exportin 1) has been identified as an export receptor that recognizes NES sequences directly and is responsible for the export of NES-containing proteins (reviewed in Refs. 39 and 40). The pharmacological compound leptomycin B (LMB) directly interacts with CRM1 and blocks NES-mediated protein export (41).
Like IRF-3 and IRF-7, IRF-5 is localized to the cytoplasm in unstimulated cells and accumulates in the nucleus following virus infection (15,16,22,23,25,27,34). IRF-3 contains a functional NES (15,16,42) and a functional NLS (42). Two NLSs have been identified in the N-and C-terminal regions of IRF-5, and both of these NLS elements are necessary for virusinduced nuclear translocation (35,36). In this report, we demonstrate that in addition to the two NLS elements, IRF-5 possesses a functional NES that controls dynamic shuttling between the cytoplasm and the nucleus. In unstimulated cells, both NLS and NES are active, but the NES element is dominant, resulting in the predominant cytoplasmic localization of IRF-5. Although IRF-5 is phosphorylated by IKK⑀ and TBK1 in co-transfected cells, phosphorylation of IRF-5 did not promote cytoplasmic to nuclear translocation, stimulation of IRF-5 transactivation potential, or induction of IFN promoters.
Cell Culture, Transfections, and Luciferase Assays-Transfections for luciferase assay were carried out in either human embryonic kidney 293 cells grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum, glutamine, and antibiotics or BJAB B-cell line grown in RPMI 1640 (Wisent) supplemented with 10%, heat-inactivated fetal bovine serum. Subconfluent 293 cells were transfected with 50 ng of pRLTK reporter (Renilla luciferase for internal control), 100 ng of pGL-3 reporter (firefly luciferase, experimental reporter), and 200 ng of expression plasmids by calcium phosphate co-precipitation method. Electroporation was performed at 950 microfarads and 250 V for BJAB cells. Luciferase assays were performed following a transfection of 2.5 g of the reporter gene, 2.5 g of the Renilla internal control (pRLTK), and 5 g of expression plasmids into 10 ϫ 10 6 BJAB cells. The reporter plasmids were: RANTES pGL-3, IFNB pGL3, and IFNA14 pGL-3 reporter genes; the transfection procedures were described previously (43). At 24 h after transfections, the reporter gene activities were measured by dual-luciferase reporter assay, according to the manufacturer's instructions (Promega).

FIG. 1. Leptomycin B induced cytoplasmic-nuclear translocation of IRF-5.
A, schematic illustration of isoforms a and b of IRF-5 proteins. The DNA binding domain and two NLSs are indicated. Subcellular localization of IRF-5a and b is summarized on the right: C, predominantly cytoplasmic; N, predominantly nuclear. Shown in the lower part of the panel are the amino acid sequence differences between isoforms a and b. B, representative green fluorescence images of living cells expressing IRF-5a and IRF-5b fused to GFP. The subcellular localization of the GFP-IRF-5a and GFP-IRF-5b was analyzed in untreated and leptomycin B-treated COS-7 cells at 15, 30, and 60 min as indicated. GFP fluorescence was analyzed in living cells with a Olympus BX51 fluorescence microscope using a ϫ40 objective. fluent COS-7 or Hela cells by Lipofectamine (Invitrogen). At 24 h after transfection, cells were left untreated or treated with 10 ng/ml LMB (a gift from Dr. Minoru Yoshida) for 60 min or the indicated times. GFP fluorescence was analyzed in living cells with an Olympus BX51 fluorescence microscope and photographed at ϫ400.
Western Blot Analysis-For Western blotting, 2.0 g of pEGFP-C1, GFP-IKK⑀, or GFP-TBK1 with FLAG-IRF-3 or FLAG-IRF-5 was cotransfected into HEK293 cells. At 24 h after transfection, whole cell extracts (20 g) were prepared and subjected to SDS-PAGE in a 10% polyacrylamide gel. After electrophoresis, proteins were transferred to Hybond transfer membrane (Amersham Biosciences) in a buffer containing 30 mM Tris, 200 mM glycine, and 20% methanol for 1 h. The membrane was blocked by incubation in phosphate-buffered saline containing 5% dried milk for 1 h and then probed with monoclonal FLAG antibody M2 (Sigma) in 5% milk/phosphate-buffered saline, at a dilution of 1:3000. The incubation was done at 4°C overnight. After four 10-min washes with phosphate-buffered saline, membranes were reacted with a peroxidase-conjugated secondary goat anti-mouse antibody (Amersham Biosciences) at a dilution of 1:2500. The reaction was then visualized with ECL as recommended by the manufacturer (Amersham Biosciences).
Immunoprecipitation and Immunoblot Analysis of Protein-Protein Interactions-293 cells were co-transfected with expression plasmids as indicated. Whole cell extracts (300 g) were prepared from co-transfected cells and precleared with 5 l of preimmune rabbit serum and 20 l of protein A-Sepharose beads (Amersham Biosciences) for 1 h at 4°C. The extract was incubated with 1 l of anti-Myc antibody 9E10 and 30 l of protein A-Sepharose beads for 1-2 h at 4°C. Precipitates were washed five times with lysis buffer and then eluted by boiling the beads for 3 min in 1ϫ SDS sample buffer. Eluted proteins or 5% input whole cell extracts were subjected to SDS-PAGE in a 7.5% polyacrylamide gel. After electrophoresis, proteins were transferred to Hybond transfer membrane (Amersham Biosciences) in a buffer containing 30 mM Tris, 200 mM glycine, and 20% methanol for 1 h. The membrane was blocked by incubation in phosphate-buffered saline containing 5% dried milk for 1 h and then probed with anti-FLAG, anti-GFP, or anti-Myc antibody (1:1000 -1:3000). Immunocomplexes were detected by using ECL (15).

Cytoplasmic Localization of IRF-5 Is Sensitive to Treatment with LMB-Two
NLSs have been identified in N-and C-terminal regions of IRF-5, and both NLS are necessary for virusinduced nuclear translocation (35,36). However, in unstimulated cells, IRF-5 resides in the cytoplasm (34), and only following virus infection does IRF-5 accumulate in the nucleus (34). To determine whether a CRM1-dependent nuclear export pathway is involved in IRF-5 subcellular localization, isoform a and b of IRF-5 were linked to GFP, transfected into COS-7 cells, and examined for LMB-induced changes in subcellular localization (Fig. 1). Fluorescence microscopy analysis of these two GFP fusion proteins revealed that both IRF-5 isoforms localized exclusively to the cytoplasm in untreated cells; upon LMB treatment, both fusion proteins accumulated in the nucleus within 15 min (Fig. 1B). Since LMB is a CRM1-specific inhibitor (41, 44 -46), this initial observation suggested that the subcellular localization of IRF-5 may be regulated by CRM1.
Localization of the IRF-5 NES Element-To investigate the sequences of IRF-5 that may exhibit nuclear export activity, the subcellular localization of a series of deletion mutants encompassing amino acids 1-450, 1-400, 1-200, 1-250, 1-150, and 161-504 of isoform a fused to GFP was examined ( Fig. 2A). As shown in Fig. 2B, mutants 1-450, 1-400, 1-250, and 1-200 were predominantly cytoplasmic in untreated cells, and following LMB treatment, these IRF-5 forms accumulated in the nucleus (Fig. 2B), indicating that the putative NES element was localized to the N-terminal 200 aa. The IRF-5 peptide of aa 1-150 (deleted of the C-terminal residues 151-504) was constitutively localized to the nucleus (Fig. 2B). IRF-5 with a deletion of N-terminal residues 1-160 (aa 161-504) was also predominantly nuclear in untreated and LMB-treated cells (Fig. 2B). Western blotting analysis with anti-GFP antibody indicated that all deletion mutants were correctly expressed (data not shown). These results therefore localize a putative NES element to an N-terminal region of IRF-5 between aa 150 and 160; NES activity is LMB-sensitive, and its association with the CRM1-dependent export machinery may determine the cytoplasmic localization of IRF-5 in unstimulated cells.
Mutational Analysis of the IRF-5 NES-Sequence inspection revealed that residues 150 -160 contain a stretch of hydrophobic amino acids resembling the NES consensus (LXXXLXX-LXL) (Fig. 3A). Since CRM1 is known to recognize leucine-rich or other hydrophobic motifs (47), residues 150 -160 may constitute a functional NES. To test this possibility, point mutations within the putative NES motif in which two leucine residues were replaced with alanine (L157A/L159A, IRF-5-NES) (Fig. 3A) were generated. Replacement of these two residues within the context of full-length IRF-5 isoform a leads to nuclear accumulation of the mutated IRF-5 protein (Fig. 3B), indicating that these two leucine residues are important for nuclear export of IRF-5. Interestingly, these two leucine residues are flanked by two serine residues and one threonine residue. To determine whether these Ser/Thr residues are involved in the regulation of IRF-5 subcellular localization, the three residues were substituted with the phosphomimetic Asp or Ala in GFP-IRF-5 (Fig. 3A) and examined for subcellular localization. As shown in Fig. 3B, the mutation of these residues to phosphomimetic Asp (D156/158/160, IRF-5-3D) resulted in constitutively nuclear IRF-5, whereas the alanine substitution (A156/158/160, IRF-5-3A) did not change the subcellular localization. Western blotting analysis with anti-GFP antibody indicated that all point mutants were correctly expressed (data not shown). This result suggested that the phosphorylation of adjacent Ser/Thr residues may contribute to IRF-5 nuclear accumulation in virus-induced cells.
The putative NES peptide and the A157/159 peptide (mut-NES) were fused to the C-terminal end of GFP (Fig. 4A), and as shown in Fig. 4B, the NES peptide but not mutNES peptide was capable of directing GFP to the cytoplasm; furthermore, GFP-NES export from the nucleus was sensitive to leptomycin (Fig. 4B), thus demonstrating that the IRF-5 NES is sufficient to direct GFP to the cytoplasm. Taken together, these data indicate that residues 150 -160 of IRF-5 function as an authentic NES that is responsible for the cytoplasmic localization of IRF-5 in unstimulated cells.
Physical Interaction between IRF-5 and CRM1-To examine the possibility that IRF-5 functionally associated with the CRM1 in vivo, interactions between IRF-5 and CRM1 were investigated by co-immunoprecipitation using 293 cells cotransfected with CRM1 and wild type or mutated forms of IRF-5 expression plasmids (Fig. 5). After immunoprecipitation of Myc-tagged CRM1 from cell extracts with anti-Myc antibody, immunoblot analysis revealed that FLAG-tagged wild type IRF-5 co-precipitated with Myc-tagged CRM1 (Fig. 5, lane 2) but not with preimmune serum (data not shown) or with anti-Myc antibody in the absence of Myc-tagged CRM1 (Fig. 5, lane  1). Interaction required functional NES since the IRF-5 3D and IRF-5 NES mutants did not immunoprecipitate with Myc tag CRM1 (Fig. 5, lane 3, 4, and 5); also, co-transfected FLAGtagged IRF-7 did not co-precipitate with the Myc-tagged CRM1 (Fig. 5A, lane 6).
Transactivation of IFNA and IFNB Promoters by IRF-5-Next, the capacity of IRF-5 to regulate gene expression was analyzed by transient transfection in human 293 HEK cells and BJAB cells using the IFNA14 and IFNB promoters in reporter gene assays. Expression of wild type IRF-5 did not enhance IFNA14 promoter activity and only slightly increased IFNB promoter activity (Fig. 6, A and B). Co-transfection of IRF-5-3D, the constitutively nuclear IRF-5, minimally induced IFNA14 and IFNB promoter activity between 3-and 4-fold (Fig. 6, A and B), indicating that nuclear accumulation alone is not sufficient for full activation of IRF-5. It has been reported that IRF-5 protein contains potential serine phosphorylation sites in the C-terminal region between aa 471 and 486 and mutational analysis indicated that two serine residues are phosphorylated in NDV-infected cells and play a critical role in IRF-5-mediated activation of IFNA promoters (35). The substitution of the Ser cluster at aa 437-446 (S437/441/443/446) in IRF-5 with the phosphomimetic Asp (IRF-5-4D) stimulated the IFNA14 promoter 5-fold in 293 cells (Fig. 5A) and 11-fold in BJAB cells (Fig. 6B). However, the introduction of the phos-phomimetic Asp in this region did not significantly change the subcellular localization of IRF-5 protein (data not shown). These data suggest that phosphorylation of the Ser/Thr residues within NES motif may lead to IRF-5 nuclear translocation and that phosphorylation of C-terminal Ser residues may contribute to IRF-5 transactivation.

IRF-5 Only Weakly Stimulates Type I IFN Promoter
Activities-It was reported that IRF-5 was able to enhance the transcription from IFNB, IFNA1, IFNA2, and IFNA14 promoter in virus-infected cells (34,35). To assess the role of IRF-3, IRF-5, and IRF-7 in the activation of type I IFN gene expression, wild type and constitutively active forms of IRF-3, IRF-5, and IRF-7 were co-transfected with the luciferase reporter gene driven by the IFNB and IFNA14 promoters. As shown in Fig. 8A, IFNB promoter was strongly activated by both IRF-3 and IRF-7. The constitutively active form of IRF-3 (IRF-3(5D)) activated the IFNB luciferase reporter gene 250-fold, the constitutively active form of IRF-7 (IRF-7(4D)) activated this reporter gene 125-fold, whereas phosphomimetic forms of IRF-5 (IRF-5(4D) and IRF-5(7D)) only resulted in 7-8-fold stimulation of IFNB promoter activity. The IFNA14 promoter was activated 1000fold by the constitutively active form of IRF-7 (IRF-7(4D)) ( Fig.  8B) but was only weakly stimulated by phosphomimetic forms of IRF-3 and IRF-5 (between 4-and 6-fold, Fig. 8B). Similar results were obtained with IFNA1, IFNA2, IFNA4, and IFNA7 promoters (data not shown). These results demonstrate that IRF-5 is a weak activator of type I IFN gene expression. DISCUSSION The results of the present study demonstrate that in addition to the two functional NLSs in the N-and C-terminal regions, IRF-5 possesses an authentic NES that is necessary for cytoplasmic retention in unstimulated cells. Following treatment with leptomycin B, a known CRM1-specific inhibitor (41, 44 -46), IRF-5 rapidly accumulated in the nucleus (Fig. 1B), indicating that IRF-5 is subject to active nuclear export. Deletion  and point mutational analyses revealed that a hydrophobic motif (LQRMLPSLSLT) located between residues 150 -160 of IRF-5 functions as an NES element. Alanine substitution of two critical leucine residues, as indicated in bold letters (LQRMLPSASAT) resulted in the nuclear accumulation of the mutated protein in unstimulated cells. Furthermore, the addition of a single copy of the wild type but not mutated NES of IRF-5 downstream of GFP confers the property of LMB-sensitive cytoplasmic localization to the fusion protein. Our findings suggest that IRF-5, like the IRF-3 protein, actively shuttles between the nuclear and cytoplasmic compartments in uninduced cells.
The IRF-5 protein possesses two traditional monopartite NLS elements that are solely responsible for nuclear localization of IRF-5 protein in virus-infected cells (35). Mutation of these two NLS elements led to the cytoplasmic localization of the resulting protein in both infected and uninfected cells. It has been suggested that the 3Ј NLS is exposed and responsible for the transactivation activity of IRF-5 in uninfected cells, whereas the 5Ј NLS is masked either by an intramolecular interaction or by association with another protein. Phosphorylation of serine residues in the C-terminal region of IRF-5 results in the exposure of the 5Ј NLS and accumulation of IRF-5 in the nucleus (35). Both NLSs and NES in IRF-5 are constitutively active, but nuclear export is dominant in unstimulated cells (Fig. 1).
As shown in Fig. 3, the NES motif of IRF-5 contains two serine residues and one threonine residue. The substitution of these three residues to phosphomimetic Asp (D156/158/160, IRF-5-3D) resulted in nuclear retention of IRF-5. Therefore, one mechanism by which virus-induced nuclear localization of IRF-5 may operate is through the phosphorylation of these serine/threonine residues, which would then block IRF-5 from binding to CRM1 (Fig. 5). By analogy, IRF-3 protein also contains a functional NES located downstream of the DNA binding domain between aa 140 and 150 (15,16), and the primary sequence of the NES element of IRF-5 is highly conserved with the NES of IRF-3. The NES peptide of IRF-3 has been shown to possess strong nuclear export activity and is responsible for the cytoplasmic localization of IRF-3 in uninduced cells (15,16); IRF-3 nuclear localization following virus infection is due to the association of IRF-3 with CBP/p300, a strictly nuclear co-activator that sequesters IRF-3 in the nucleus (42). A similar mechanism may likewise apply to nuclear retention of IRF-5.
Following viral infection, IRF-3, IRF-5, and IRF-7 are posttranslationally modified and activated by phosphorylation of specific serine-threonine residues located within their C-terminal regions (15,16,20,23,24,34,35,48). The replacement of the serine-threonine residues in IRF-3 and IRF-7 with phosphomimetic aspartic acid generated the constitutively active forms of IRF-3 and IRF-7 (IRF-3(5D) and IRF-7(D475-479), respectively), which behaved like virus-activated IRF-3 and IRF-7, with the capacity to activate the transcription of target genes in the absence of viral infection (Fig. 8) (15,19,26). Overexpression of IRF-5 has also been reported to stimulate expression of reporter genes driven by type I IFN promoters including IFNA1, IFNA2, IFNA14, and IFNB in unstimulated cells; furthermore, all of these IFN promoter-driven constructs were activated more efficiently by IRF-5 in NDV-infected cells than in Sendai virus-infected cells (34). Although the data showed that co-transfection of IRF-5 activated IFNA1-, IFNA2-, and IFNA14-SAP (soluble alkaline phosphatase) reporter constructs 5-10-fold, further activation in NDV-infected cells was less than 2-fold (34). In the present studies, activation of IFNA reporter constructs in HEK293 and BJAB by wild type IRF-5 is negligible (less than 2-fold), a significant discrepancy that may be due to differences in the IRF-5 isoforms. The expression plasmids of IRF-5 used in this study encode 504 aa (isoform a) and 498 aa (isoform b), although the IRF-5 used in the study of Barnes et al. (35) was variant 3. Like IRF-3 and IRF-7, mutation of the C-terminal serine residues of IRF-5 to phosphomimetic aspartic acid created a form of IRF-5 (IRF-5(4D)) that activated IFNA and IFNB promoter activities between 4-and 8-fold (Fig. 8), a level of activation that was extremely weak as compared with the over 100-fold stimulation of IFNB promoter by constitutively active forms of IRF-3 and IRF-7 and over 1000-fold stimulation of the IFNA promoter by constitutively active forms of IRF-7 (Fig. 8).
Previous experiments furthermore suggested that, unlike IRF-3 and IRF-7, IRF-5 is selectively phosphorylated and activated in NDV-and vesicular stomatitis virus-infected cells but not in Sendai virus-infected cells (34 -36). Recent studies have identified IKK⑀/TBK1 as components of the virus-activated kinase responsible for IRF-3 and IRF-7 phosphorylation in response to virus infection (32,33). Although co-expression of IKK⑀ or TBK1 resulted in the phosphorylation (Fig. 7A) and dimerization (Fig. 7B) of IRF-5, IKK⑀/TBK1 did not induce IRF-5 nuclear accumulation (Fig. 7C) or stimulate IRF-5 transactivation activity (Table I). This result suggests either that a distinct signaling pathway is involved in the activation of IRF-5, or alternatively, that IRF-5 simply is not an activator of IFN gene expression. This possibility has been suggested by recent experiments indicating that heterodimerization of IRF-5 with IRF-3 or IRF-7 confers transactivation capacity (36). Based on recent observations that IRF-5 is a p53-targeted gene (37), the primary physiological role of IRF-5 may be to regulate gene expression during cell growth or as a response to DNA damage, as recently reported (38).