Factors During Viral Infection Factor (IRF)-3 and IRF-5 Transcription

The Journal of Immunology

Differential Activation of IFN Regulatory Factor (IRF)-3 and IRF-5 Transcription Factors during Viral Infection1

Tsu-Fan Cheng, Sabrina Brzostek, Osamu Ando,2 Sarah Van Scoy, K. Prasanna Kumar,3 and Nancy C. Reich4

Members of the IFN regulatory factor (IRF) family regulate gene expression critical to immune response, hemopoiesis, and proliferation. Although related by homology at their N-terminal DNA-binding domain, they display individual functional properties. The distinct properties result from differences in regulated expression, response to activating signals, and interaction with DNA regulatory elements. IRF-3 is expressed ubiquitously and is activated by serine phosphorylation in response to viral infection or TLR signaling. Evidence indicates that the kinases TANK-binding kinase 1 and inhibitor of NF-␬B kinase-␧ specifically phosphorylate and thereby activate IRF-3. We evaluated the contribution of another member of the IRF family, IRF-5, during viral infection since prior studies provided varied results. Analysis of phosphorylation, nuclear translocation, dimerization, binding to CREB-binding protein, recognition of DNA, and induction of gene expression were used comparatively with IRF-3 as a measure of IRF-5 activation. IRF-5 was not activated by viral infection; however, expression of TANK-binding kinase 1 or inhibitor of NF-␬B kinase-␧ did provide clear activation of IRF-5. IRF-5 is therefore distinct in its activation profile from IRF-3. However, similar to the biological effects of IRF-3 activation, a constitutively active mutation of IRF-5 promoted apoptosis. The apoptosis was inhibited by expression of Bcl-xL but not a dominant-negative mutation of the Fas-associated death domain. These studies support the distinct activation profiles of IRF-3 in comparison to IRF-5, but reveal a potential shared biological effect. The Journal of Immunology, 2006, 176: 7462–7470.

successful defense response to microbial infection re- the IFN-␤-positive regulatory domain (17, 19). The C regions of quires the activation of specific latent transcription fac- the IRFs are clearly different, and this diversity promotes their A tors. These factors serve as molecular switches to induce interaction with distinct transcription factors and results in their a subset of genes that are critical for host defense. Certain mem- ability to regulate both unique and common target genes. bers of the IFN regulatory factor (IRF)5 family are activated in The cellular localization of transcription factors that serve as sig- response to infection, and consequently regulate the expression of naling molecules is understandably critical to their function. One genes that function in innate immunity (1–10). There are nine member of the IRF family, IRF-3, plays a key role in response to viral members of the mammalian IRF family that function in microbial infection and is necessary for the transcriptional induction of the type defense, cellular survival, and hemopoietic development (11, 12). I IFN genes and a subset of IFN-stimulated genes (ISGs) (20). IRF-3 Some of the IRFs are expressed ubiquitously, some are expressed pre-exists in a latent state in the cytoplasm of all cells, but accumu- in a tissue-specific manner, and some are expressed conditionally lates in the nucleus following activation in response to viral infection following gene induction. The members share a homologous or TLR-3 and -4 signaling (21–23). IRF-3 is activated by specific DNA-binding domain at their N termini that allows recognition of serine phosphorylation, and this modification leads to a conforma- a core DNA sequence GAAA (13–15). This core target sequence tional change that allows it to homodimerize, accumulate in the nu- is usually embedded in a more complex DNA regulatory element cleus by tight association with histone acetyl transferases (CREB- such as the IFN-stimulated response element (ISRE) (2, 16, 18) or binding protein (CBP) and p300), and to bind to specific DNA targets (3–6, 8, 24). Only in response to direct serine phosphorylation does Department of Molecular Genetics and Microbiology, Stony Brook University, Stony IRF-3 accumulate in the nucleus, and two kinases have been identified Brook, NY 11794 that are capable of this activation, NF-␬B-activating kinase/TANK- Received for publication December 5, 2005. Accepted for publication March 31, 2006. binding kinase 1 (NAK/TBK1) and inhibitor of NF-␬B kinase-␧ The costs of publication of this article were defrayed in part by the payment of page (IKK␧)/IKK inducible (25, 26). Targeted gene knockout studies have charges. This article must therefore be hereby marked advertisement in accordance ␧ with 18 U.S.C. Section 1734 solely to indicate this fact. supported an essential role of NAK and IKK for a successful innate 1 This work was supported by grants from the National Institutes of Health immune response to viral infection (27, 28). (PO1CA2814 and PO1AI0555621). A less well-characterized IRF family member, IRF-5, was demon- 2 Current address: Sankyo, Shinagawa-ku, Tokyo, Japan. strated recently to play an intrinsic part in innate immunity. IRF-5 is 3 Current address: Rheogene, Norristown, Philadelphia, PA. expressed primarily in lymphocytes and dendritic cells, but it is in- 4 Address correspondence and reprint requests to Dr. Nancy C. Reich, Department of duced in other cells in response to type I IFN (29). Animals with a Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY targeted gene disruption of IRF-5 were found to have defective re- 11794. E-mail address: [email protected] sponses to TLR signaling and specifically survived challenge with 5 Abbreviations used in this paper: IRF, IFN regulatory factor; ISRE, IFN-stimulated lethal doses of LPS (TLR-4) or CpG oligodeoxynucleotide (TLR-9) response element; ISG, IFN-stimulated gene; NAK, NF-␬B-activating kinase; TBK, TANK-binding kinase; IKK, inhibitor of NF-␬B; NDV, Newcastle’s disease virus; (30). Additional studies have demonstrated activation of IRF-5 in the LMB, leptomycin B; FADD, Fas-associated death domain; DNFADD, dominant- response to an imiquimod derivative R-848 (TLR-7/8), and also to negative form of FADD; HA, hemagglutinin; CBP, CREB-binding protein; PARP, poly(ADP-ribose) polymerase; DAPI, 6-diamidino-2-phenylindole; NLS, nuclear lo- viral infection with the paramyxovirus Newcastle’s disease virus calization sequence; NES, nuclear export signal. (NDV) (31, 32).

To elucidate the mechanisms that stimulate IRF-5, we have Native PAGE for detecting protein dimers were prepared without SDS characterized the response of IRF-5 to viral infection with NDV or and the gel was prerun for 30 min at 4°C with 0.5% deoxycholate in the to overexpression of the kinases TBK1 or IKK␧. The function of cathode chamber (34). A total of 0.5% deoxycholate was added to lysate ␧ samples before addition of sample buffer lacking SDS and 2-ME. Follow- TBK1 or IKK has been shown to be essential for the phosphor- ing electrophoresis at 4°C, the proteins were transferred to Immobilon be- ylation of IRF-3 during viral infection (25, 26). Unexpectedly, we fore Western blot. find that IRF-5 is not activated in response to NDV infection, but Anti-CBP Abs (A-22), anti-p300 Abs (N-15), and normal rabbit IgG it is activated by the TBK1 or IKK␧ kinases. In addition, the re- were purchased from Santa Cruz Biotechnology. Anti-GFP Ab was pur- ␧ chased from Roche. Ab to the T7 epitope was purchased from Novagen. sponse of IRF-5 to phosphorylation by TBK1 or IKK promotes Anti-FLAG M2 mAb and anti-␣-tubulin were obtained from Sigma- apoptosis. Aldrich. Anti-poly(ADP-ribose) polymerase (PARP) p85 fragment Ab was obtained from Promega, anti-HA Ab (12CA5) was obtained from Roche Materials and Methods Applied Science. Polyclonal anti-IRF-5 Ab was generated by immunizing Cell culture, virus, reagents rabbits with GST fusion protein of polypeptide 119–212 of human IRF-5. Polyclonal anti-IRF-3 Ab was used as described (4). Human HEC-1B, HT1080, HeLa Cl2, and THP1 cells were obtained from American Type Culture Collection. Mouse embryo fibroblasts from IRF-3 Fluorescence imaging knockout animals (irf3Ϫ/Ϫ) and wild-type siblings and were gifts from Dr. Cells seeded on coverslips were fixed in 4% paraformaldehyde and either T. Taniguchi (University of Tokyo, Tokyo, Japan). Human Huh7 cells visualized directly for GFP with a Zeiss Axioskop or processed for immu- were obtained from Dr. P. Sarnow (Stanford University, Stanford, CA). nofluorescence before visualization. For immunofluorescence, cells were Cells were grown in DMEM with 8% FBS with the exception of THP1 permeabilized in 0.2% Triton X-100 in PBS, blocked in 10% goat serum/ cells maintained in RPMI 1640 medium with 8% FBS and 20 ␮M 2-ME. PBS, and incubated in anti-HA Ab in blocking solution in 1/200 dilution at NDV (NJ-LaSota-1946), a gift from Dr. P. M. Pitha-Rowe (Johns Hopkins room temperature for 1 h. Secondary Abs conjugated to rhodamine (Jack- University, Baltimore, MD), was propagated in embryonated hen eggs and son ImmunoResearch Laboratories) were applied at 1/200 dilution for 1 h titers were determined by hemagglutination assay. Infections were per- at room temperature. Coverslips were washed with PBS and mounted in formed at 100 hemagglutination units/ml or with allantoic fluid from mock- antifade solution (Vectashield; Vector Laboratories). infected hen eggs for 6 h. Leptomycin B (LMB) was a gift from B. Wolff- Winiski (Novartis Research Institute, Vienna, Austria). Metabolic [32P] incorporation Plasmid constructs, mRNA analyses, transfections Cells were incubated with 250 ␮Ci of [32P]orthophosphate (DuPont NEN) The human gene encoding IRF-5 variant 5 was amplified from THP1 per milliliter in phosphate-free medium for 1 h before NDV infection, and cDNA prepared by RT-PCR with primers using Pfu Turbo polymerase were maintained in radiolabel during 6 h of infection (4). Uninfected controls (Stratagene). AflII and XbaI restriction sites were constructed in the primers were radiolabeled in a similar manner. Cell lysates were prepared, normalized as the cloning sites (5Ј-ACTTAAGATGAACCAGTCCATCCCAGTG-3Ј, for equivalent trichloroacetic acid-precipitable radioactivity, and subjected to 5Ј-ATCTAGATTATTGCATGCCAGCTGGGTG-3Ј). The cDNA was immunoprecipitation with either anti-GFP or anti-T7 Abs. Proteins were sep- cloned into pcDNA3 containing a T7 epitope tag (T7-IRF-5) at the N arated by SDS-PAGE, and visualized by autoradiography. terminus, GFP-IRF-5 was generated by cloning into the pcDNA-GFP as described (33). IRF-5 mutants were constructed by Quick Change mu- DNA-binding assay tagenesis kit (Stratagene) and verified by sequencing. The EMSA was performed as described (4). The probe used was a dsDNA For analyses of IRF-5 variants, human B and T lymphocytes obtained oligonucleotide containing the ISG15 ISRE: 5Ј-GGGAAAGGGAAAC from two different healthy donors (Long Island Blood Services, Westbury, CGAAACTGAAG-3Ј, end-labeled with [␥-32P]ATP. Nuclear extracts NY) were isolated by Lymphoprep (Invitrogen Life Technologies). Cyto- were prepared and incubated with the radiolabeled probe for 20 min at 8 plasmic RNA was isolated from 2 ϫ 10 cells using the Qiagen RNeasy kit room temperature. Specific Abs were incubated with nuclear extracts at and selection on Qiagen Oligotex mini columns. cDNA was generated with 4°C for 60 min before the addition of the probe. Complexes were separated random hexamer primers and SuperScriptII (Invitrogen Life Technologies) on 4.5% native gels at 4°C and visualized by autoradiography. according to the manufacturer’s protocol. IRF5 cDNA was amplified by PCR using primers for full-length IRF-5 and nested primers to amplify the Gene expression assays most variable region. Full-length IRF-5 was amplified using Pfu Turbo polymerase and primers as described previously. The internal domain was RNA was isolated 36 h posttransfection by RNeasy mini kit (Qiagen) ac- amplified with Platinum Taq polymerase (Invitrogen Life Technologies) cording to manufacturer’s protocol, cDNA was synthesized by oligo-dT with primers 5Ј-CCCAGCCCCCTGAGGATT-3Ј and 5Ј-AGGGGGCT primer (Invitrogen Life Technologies) and was subject to real-time PCR GGGGTCTGGA-3Ј. The PCR products were cloned and sequenced. with primers for murine ISG54 (5Ј-GGGCTTCATCCAGCAACAGC-3Ј; FLAG-TBK1 and hemagglutinin (HA)-IKK␧ were gifts from Dr. M. 5Ј-CCTCCTCACAGTCAAGAGCAGG-3Ј) and IL-6 (5Ј-TCTACTCG Karin (University of California, San Diego, CA). The ISRE-luciferase re- GCAAACCTAGT-3Ј;5Ј-CCAAGAAACCATCTGGC-3Ј) with a Light porter was described in Ref. 33. The IFN-␤-luciferase was constructed by Cycler (Roche) based on manufacturer’s specifications. Each PCR ampli- cloning the IFN-␤ promoter by PCR with primers, 5Ј-TCAGGTCGTTT fication was normalized to actin RNA (5Ј-ATGCTCTCCCTCACGC GCTTTCCTT-3Ј and 5Ј-TTGACAACACGAACAGTGTCG-3Ј upstream CATC-3Ј;5Ј-CGCACGATTTCCCTCTCAGC-3Ј). of the firefly luciferase gene in pGL3-Basic vector (Promega). Bcl-xL and Luciferase assays were performed with T7-IRF-5, T7-IRF-5/4A, T7- the dominant-negative form of the Fas-associated death domain (DN- IRF-5/4D, T7-IRF-3, or pGEM (Promega) and ISRE luciferase gene or FADD) were gifts from Dr. Colin Duckett (University of Michigan, Ann IFN-␤ luciferase gene. Five hundred nanograms of HA-IKK␧ and 100 ng Arbor, MI). Fugene6 (Roche) was used as the transfection reagent. of ␤-galactosidase gene as the internal control were cotransfected into each sample. Firefly luciferase was recorded using a Lumat model LB 9507 Immunoprecipitation, Western blot, and Abs luminometer and normalized to ␤-galactosidase expression. The data shown were averaged from three independent experiments. Cells were lysed in 50 mM Tris (pH 7.6), 400 mM NaCl, 0.5% Nonidet P-40, 5 mM EDTA, 50 mM sodium fluoride, 2 mM sodium vanadate, Apoptosis assays protease inhibitor mixture (Sigma-Aldrich), and 1 mM PMSF (Sigma- Aldrich). The insoluble material was removed by centrifugation at Cells on coverslips were fixed in 4% paraformaldehyde/PBS, permeabil- 12,000 ϫ g and 4°C. For immunoprecipitation, lysates were prepared from ized with 0.5% Triton X-100/PBS, washed and stained with 6-diamidino- 10-cm plates and the NaCl concentration was reduced to 250 mM. Lysates 2-phenylindole (DAPI) at 0.01 ␮g/ml (33). TUNEL assay was performed were precleared with protein G-agarose slurry (Invitrogen Life Technolo- following DAPI staining by In situ Cell Death Detection kit TMR Red gies), and incubated with specific Abs at 4°C overnight. Immunocomplexes (Roche) according to manufacturer’s directions. For PARP-1 cleavage as- were collected on protein G beads, the complexes were washed with lysis say, cells were lysed in isotonic lysis buffer containing 140 mM NaCl, 50 buffer, and eluted from the beads with SDS-sample buffer before separation mM Tris-HCl (pH 8.0), 5 mM EDTA, 0.5% Nonidet P-40, 1 mM PMSF, on SDS-PAGE. Prestained protein standards were obtained from Crystal- 1 mM DTT, 1ϫ protease inhibitor mixture (Sigma-Aldrich). Nuclei were gen. Western blotting was performed following transfer to Immobilon-P separated by centrifugation at 12,000 ϫ g, 4°C. Cytoplasmic protein was (Millipore), and incubation with primary Ab (1/3000) and secondary Abs quantified by Bio-Rad assay and the corresponding ratio of nuclear protein (Amersham) and visualized by ECL. was separated on SDS-PAGE and processed for Western blotting. 7464 DISTINCT ACTIVATION PATHWAY OF IRF-5 AND IRF-3

Table I. Alignment of nine IRF-5 variantsa

Variant Sequences

v1 142EEEEEEEELQRMLPSLSLTDAVQSGPHMTPYSLLKEDVKW––––––––––PPTLQPPTLQ191… v2/6 142EEEEEEEELQRMLPSLSLT––––––––––––––––EDVKWPPTLQPPTLRPPTLQPPTLQ185… 142 175 v3/4 EEEEEEEELQRMLPSLSLT––––––––––––––––EDVKW––––––––––PPTLQPPTLQ … v5 142EEEEEEEELQRMLPSLSLTDAVQSGPHMTPYSLLKEDVKWPPTLQPPTLRPPTLQPPTLQ201… v7 142EEEEEEEELQRMLPSLSLT––––––––––––––––EDVKWPPTLQPPTLRPPTLQPPTLQ186… v8 142EEEEEEEELQRMLPSLSLTVTDLEIKK168… v9 142EEEEEEEELQCSLAPT157

a Partial protein sequence of IRF-5 variants is aligned from the N-terminal glutamate at position 142. Arrowheads above v5 note junctions of exons 3/4 and exons 4/5. Missing amino acids are designated by a dash (–).

Results isoforms of IRF-3 serve as an indicator of a productive viral in- IRF-5 isoforms fection. Although IRF-5 was not phosphorylated in response to viral infection, it did appear to be a substrate of TBK1/IKK␧. Co- Various isoforms of human IRF-5 have been described, and these transfection with IKK␧ resulted in an increase of phosphorylation likely result in part from differential splicing of the nine exons for both IRF-5 and IRF-3 (lanes 3 and 6). These results indicate encoding IRF-5 (35). We generated an IRF-5 cDNA by RT-PCR that IRF-5 does not respond to viral infection, but is phosphory- from mRNA encoded by the human monocyte cell line THP1. This lated in response to IKK␧ expression. cDNA is now designated variant 5 (v5) and is the longest of the characterized variants encoding a protein of 514 aa. A partial se- Nuclear accumulation of IRF-5 quence comparison of some of these variants is provided in Table The nuclear/cytoplasmic redistribution of proteins is regulated by I. To ensure the appropriate use of v5, we evaluated the IRF-5 the function of nuclear localization sequences (NLS) or nuclear mRNA species from a mixed pool of B and T lymphocytes from export signals (NES) on proteins destined for transport (36–39). several healthy donors. To eliminate the presence of inaccurate To evaluate the localization of IRF-5, we visualized the behavior splice forms, nuclear and cytoplasmic cell fractions were prepared, of GFP-IRF-5 by fluorescent microscopy (Fig. 2A). IRF-5 clearly and RNA was used only from the cytoplasmic pool to examine resides in the cytoplasm of cells in the absence of any activating IRF-5 mRNA species by RT-PCR. We identified the presence of stimulus, similar to latent IRF-3. To determine whether IRF-5 is IRF-5 variants v1, v3/4, and v5, in relatively equal proportion (data restricted to the cytoplasm or whether it shuttles between nucleus not shown). Because most prior studies used total cellular RNA to and cytoplasm, we tested the effect of an inhibitor of nuclear ex- generate IRF-5 cDNA, the existence of many variants may repre- port, LMB. LMB blocks export mediated by the exportin carrier, sent inaccurate or incomplete splice forms that do not accumulate CRM1 (40, 41). Treatment of cells with LMB for1hintheab- in the cytoplasmic mRNA pool. Variant v9 encodes only the IRF-5 sence of any stimulus was found to cause IRF-5 to accumulate in DNA-binding domain (1–157 aa), whereas v7 lacks the DNA- the nucleus. This result indicates that IRF-5 dynamically shuttles binding domain and is predicted to encode a truncated protein in and out of the nucleus, but nuclear export is dominant. This is (110–389 aa). These variants may function as negative interfering similar to the trafficking behavior of IRF-3 (42). molecules. However, the encoded proteins of these variants have To determine whether IRF-5 redistributes from the cytoplasm to not been identified, and so activity cannot be confirmed. Because the nucleus in response to viral infection, cells expressing GFP- the IRF-5 variant v5 was well-represented in the cytoplasmic IRF-5 were infected with NDV for 6 h (Fig. 2). During the course mRNA from primary lymphocytes, we focused our studies on this isoform.

In vivo phosphorylation of IRF-5 Cells respond to viral infection with the activation of serine kinases that phosphorylate latent IRF-3, a modification that is required for its transcriptional ability. Viral infection has been reported to activate IRF-5, and so to determine whether IRF-5 is also modified by phosphorylation during viral infection, we evaluated phosphorylation by metabolic radiolabeling of cells in the presence of [32P]orthophosphate (Fig. 1). HEC-1B cells that cannot respond to autocrine IFN during the course of infection were cotransfected with expression plasmids encoding GFP-tagged IRF-5 (GFP- IRF-5) or T7-tagged IRF-3 (T7-IRF-3). The cells were cultured in the presence of [32P]orthophosphate, and infected with NDV for FIGURE 1. IRF-5 phosphorylation by coexpression of IKK␧, but not by 6 h. The NDV paramyxovirus was used because it produces sig- NDV infection. HEC-1B cells were cotransfected with plasmids encoding nificant viral dsRNA and is a potent activator of IRF-3 and type I GFP-IRF-5 and T7-IRF-3 (lanes 1, 2, 4, and 5), or with GFP-IRF-5, T7- ␧ IFN production. Cells were lysed, the IRF-5 or IRF-3 proteins IRF-3, and IKK (lanes 3 and 6). Cultures were incubated in the presence of [32P]orthophosphate and left untreated or infected with NDV for 6 h were immunoprecipitated with specific Abs, and phosphate mod- (lanes 2 and 5). Top panel, Abs to GFP or T7 were used to immunopre- ification was evaluated by autoradiography following SDS-PAGE. cipitate GFP-IRF-5 or T7-IRF-3. Proteins were separated by SDS-PAGE, A comparative analysis clearly demonstrated that following viral and [32P] modification was detected by autoradiography. Bottom panel,50 infection there was no increase in phosphorylation of IRF-5 (lanes mg of each lysate was analyzed by Western blot with anti-GFP (lanes 1–3) 1 and 2), whereas there was a significant increase in IRF-3 phos- or anti-T7 (lanes 4–6) to evaluate protein levels of IRF-5 and IRF-3, re- phorylation (lanes 4 and 5). The slower mobility phosphorylated spectively. Results are representative of three experiments. The Journal of Immunology 7465

Dimerization of activated IRF-5 Site-specific phosphorylation has been shown to alter the conformation of proteins such as the IRF-3 molecule and promote their homodimerization (43, 44). Because IKK␧ phosphorylated IRF-5 and stimulated its nuclear accumulation, we evaluated the possi- bility that activated IRF-5 could dimerize. Two assays were performed to assess IRF-5 self-interaction, coimmunoprecipitation and altered migration in nondenaturing gel electrophoresis. If IRF-5 gains the ability to dimerize or oligomerize following activation by phosphorylation, protein association could be detected by coimmunoprecipitation of IRF-5 molecules with distinct epitope tags. For this reason, cells were cotransfected with plasmids encoding GFP-IRF-5 and T7-IRF-5, and were untreated or infected with NDV for 6 h (Fig. 3A). In addition, cells were cotransfected with GFP-IRF-5, T7-IRF-5, and IKK␧. Detergent cell lysates were prepared and T7-IRF-5 was immunoprecipitated with anti-T7 Abs. Proteins in the immunocomplexes were resolved on SDS-PAGE, and the presence of T7-IRF-5 or GFP-IRF-5 was detected by Western blot with anti-IRF-5 polyclonal Ab. Because the GFP-tagged IRF-5 migrates more slowly than T7-IRF-5, the presence of each species is easily evaluated. The Western blot detected FIGURE 2. Nuclear trafficking of IRF-5. A, HT1080 cells were trans- a significant induction of protein-protein association of T7-IRF-5 fected with GFP-IRF-5 or GFP-IRF3. Localization of GFP-IRF-5 (top with GFP-IRF-5 in the presence of coexpressed IKK␧ kinase. panel) or GFP-IRF-3 (bottom panel) was evaluated in the absence of any There was only minimal basal association detected in the untreated treatment, following the addition of LMB for1htotheculture, following or NDV-infected lysates, and this was not always apparent. These infection with NDV for 6 h, or following cotransfection with an HA- results indicate that a significant dimerization of IRF-5 occurs fol- ␧ ␧ epitope tagged IKK gene. The expression of the IKK was confirmed by lowing specific phosphorylation by the IKK␧ kinase. To determine immunofluorescence of the same cells with anti-HA Ab as indicated. Im- whether IRF-5 heterodimerizes with activated IRF-3, similar co- ages represent 70–100% of the cells in the cultures. B, Localization of GFP-IRF-5 encoding 1–150 aa, or 1–173 aa or full-length IRF-5 wild type immunoprecipitation assays were performed, but in contrast to (wt) or with substitutions of L150A or LL150,154AA. other reports, no association was detectable with IRF-3 (data not shown). Thus, IRF-5 and IRF-3 appear to function in distinct transcription complexes.

of infection GFP-IRF-5 remained predominant in the cytoplasm of the cell. In contrast, GFP-IRF-3 localized to the nucleus during the same viral infection. Because expression of IKK␧ was found to increase the phosphorylation of IRF-5 as shown in Fig. 1, the effect of IKK␧ on IRF-5 nuclear accumulation was examined. Coexpres- sion of IKK␧ clearly stimulated the nuclear accumulation of GFP- IRF-5, and, similarly, induced nuclear accumulation of GFP- IRF-3. Nuclear accumulation of IRFs was only present in cells expressing HA-tagged IKK␧ as detected by immunofluorescence. The dominant localization of latent IRF-5 in the cytoplasm, and its nuclear accumulation in the presence of the export inhibitor LMB indicate that IRF-5 has a constitutive NES. To identify the position of the NES within the protein, the cellular distribution of various deletion mutations was evaluated. A truncation containing 1–173 aa showed significant cytoplasmic expression in a majority of cells; however, a truncation containing 1–150 aa showed only nuclear accumulation (Fig. 2B). These results indicated that a region critical for NES function was present within aa 150–173. The amino acid sequence in this region contains a leucine-rich hydro- phobic domain between 150 and 160 aa, similar to the NES rec- ognized by CRM1. To more precisely identify the NES, site-directed mutagenesis was used to evaluate the contribution of leucine residues within this sequence. A single amino acid substitution of leucine at position 150 to alanine (L150A) disrupted the FIGURE 3. Dimerization of activated IRF-5. A, HEC-1B cells were co- Ϫ NES function within the native IRF-5 molecule. An additional transfected with GFP-IRF-5 and T7-IRF-5, and were untreated ( )orin- fected with NDV. In addition, cells were cotransfected with GFP-IRF-5, substitution of leucine at position 154 (L150A/L154A) generated T7-IRF-5, and IKK␧. T7-IRF-5 was immunoprecipitated with anti-T7 Abs, even more apparent nuclear accumulation. Based on these obser- and proteins in the immunocomplexes were detected by Western blot with vations, this region of IRF-5 serves as a constitutive NES to po- anti-IRF-5 Ab. B, Lysates from cells expressing T7-IRF-5 or T7-IRF-3 sition the protein in the cytoplasm until an activating signal allows transfected or infected as in A and electrophoresed in nondenaturing gels, it to accumulate in the nucleus. and proteins were identified by Western blot with anti-T7 Ab. 7466 DISTINCT ACTIVATION PATHWAY OF IRF-5 AND IRF-3

Another method used to detect IRF dimerization was altered protein migration in nondenaturing gels (34). For this assay, cells were transfected with T7-IRF-5 and were untreated or infected with NDV for 6 h, or were cotransfected with T7-IRF-5 and TBK1 or T7-IRF-5 and IKK␧ (Fig. 3B). As a positive control, cells were transfected with T7-IRF-3 and infected with NDV. Cell lysates were prepared, proteins were separated by electrophoresis in nondenaturing gels, and the T7-tagged IRFs were identified by West- ern blot. There was no change in IRF-5 migration following NDV FIGURE 5. Binding of IRF-5 to the ISRE. EMSAs were evaluated with extracts from HEC1B cells transfected with T7-IRF-5 or T7-IRF-3 with or infection, but cotransfection with TBK1 or IKK␧ generated a dis- without cotransfection with IKK␧ or infection by NDV. Either no Abs (Ab) tinct increase in a slow mobility complex apparently representing were added to the DNA binding reactions (Ϫ) or normal rabbit Ab (c), an activated IRF-5 dimer. This result indicates an alteration in IRF-3 Ab, or IRF-5 Ab as indicated. The position of the IRF-3 and IRF-5 protein conformation occurs following specific phosphorylation. complexes are noted by an arrow. Active IRF-5 binds to CBP/p300 acetylases It is known that phosphorylated IRF-3 forms a strong association onstrated a detectable binding to the ISRE although not as robust with the histone acetylases CBP and p300 in the nucleus. In fact, as the IRF-3 complex. The complex was eliminated with specific this association appears to be responsible for nuclear retention of IRF-5 Ab or with Ab to CBP or p300 (data not shown) indicating IRF-3 (42). To determine whether activated IRF-5 binds to CBP/ IRF-5 in association with CBP/p300 can weakly bind the ISRE. p300 in the nucleus, we performed coimmunoprecipitation assays IRF-5 effect on gene expression (Fig. 4). Endogenous CBP/p300 were immunoprecipitated from cells transfected with T7-IRF-5 or T7-IRF-3 and associated pro- The ability of IRF-5 to weakly bind to the ISRE in response to teins were identified by Western blot with anti-T7 Ab. IRF-3 phosphorylation by IKK␧ suggested that it might be able to induce served as a positive control because it strongly associates with expression of genes regulated by the ISRE. For this reason, we CBP/p300 only following activation by phosphorylation in re- tested the effect of activated IRF-3 or IRF-5 on endogenous ex- sponse to either NDV infection or IKK␧ expression. The results pression of an IFN-induced gene, ISG54, or a proinflammatory clearly indicate that IRF-5 activated in response to IKK␧ can bind gene, IL-6. ISG54 has a characterized ISRE, and the promoter of to CBP/p300, but NDV infection does not stimulate this associa- the IL-6 gene has several putative IRF-binding sites. To eliminate tion. The ability of phosphorylated IRF-5 to bind to CBP/p300 the contribution of endogenous IRF-3 protein, murine fibroblasts Ϫ Ϫ may confer transcriptional activity to the complex. from cells lacking IRF-3 (irf-3 / ) were evaluated (20). Cells were transfected with the IKK␧ gene with or without IRF-3 or Interaction of IRF-5 with the ISRE IRF-5 and mRNA corresponding to ISG54 or IL-6 was quantitated Because the IRF proteins share a similar DNA-binding domain by real-time PCR (Fig. 6A). The results show that there is an over- that can recognize a core target site within the ISRE, we tested the lapping but differential effect of IRF-3 and IRF-5 on these genes. ability of activated IRF-5 to bind to the ISRE. EMSAs were per- ISG54 is induced preferentially by IRF-3, whereas IL-6 is induced formed with radiolabeled oligonucleotide corresponding to the primarily by IRF-5. Differences in IRF binding or interaction with ISRE and lysates from HEC-1B cells expressing T7-IRF-5 or T7- other transcription factors may influence their distinct effects on IRF-3 with or without IKK␧ cotransfection or infection by NDV gene targets. We also tested the ability of IRF-5 to effect expres- (Fig. 5). HEC-1B cells lack an autocrine response to IFNs and sion of a luciferase reporter gene regulated by an ISRE (33) (Fig. therefore eliminate this signal pathway to ISRE-binding proteins 6B). The IRF-3-deficient cells were transfected with the reporter (4). Specific Abs were included in the DNA-binding reactions to gene with or without IRF-5 or IRF-3 expression plasmids and the identify specific IRF complexes by elimination of the IRF-DNA effect of cotransfection with IKK␧ was tested. IRF-3 served as a complex. IRF-3 activated in response to NDV infection or IKK␧ positive control because activation by coexpression of IKK␧ in- expression clearly formed DNA-binding complexes. IRF-5 phos- duced a clear response. Gene expression was compared with the phorylated in response to IKK␧, but not by NDV infection, dem- mean level induced by IRF-3 with IKK␧ coexpression in this representative experiment. IRF-5 coexpression with IKK␧ induced expression of the reporter gene by nearly 20-fold. Although IRF-5 does not induce ISRE-regulated gene expression to the same extent as IRF-3, it clearly can regulate genes with this DNA target as has been suggested in the recent study of IRF-5null animals (30) Because the IRF-3 protein is modified by serine phosphorylation at the C terminus in response to viral infection, we tested IRF-5 mutations that were modified in a similar region of the molecule. Site-directed mutagenesis was used to replace the serine residues at aa 451, 453, 456, and 462 to either alanine (IRF-5/4A) or aspartic acid (IRF-5/4D). The alanine substitution was expected to eliminate phosphorylation effects in this region, and the aspartic acid substitutions would possibly function as phosphoserine mi- metics similar to the effect observed with a constitutively active FIGURE 4. Association of IRF-5 with CBP/p300. HEC-1B cells were IRF-3 mutant (8). Cells were transfected with IRF-5/4A, or IRF- ␧ transfected with T7-IRF-3 or T7-IRF-5 and were untreated (Ϫ) or infected 5/4D, or IRF-3 as a positive control, with or without IKK coex- with NDV (ϩ). Cells were also cotransfected as indicated with IKK␧ (ϩ). pression. The IRF-5/4A mutation had negligible effects on ISRE- CBP/p300 Abs were used for immunoprecipitations, and the immunocom- dependent gene expression; however, the IRF-5/4D substitution plexes were subjected to SDS-PAGE and Western blot with anti-T7 Abs. functioned as a constitutively active molecule and significantly The Journal of Immunology 7467

FIGURE 6. Effect of IRF-3 or IRF-5 on gene expression. A, Differential induction of ISG54 and IL-6 genes by IRF-3 and IRF-5. Murine embryo ﬁbroblasts derived from IRF-3 knockout animals were transfected with the IKK␧ gene or cotransfected with the IKK␧ gene and either the IRF-3 or IRF-5 gene. Induction of endogenous ISG54 or IL-6 RNA was measured 36 h posttransfection by quantitative real-time PCR. B, IRF-3Ϫ/Ϫ cells were transfected with an ISRE-dependent luciferase reporter gene and the ␤-galactosidase gene as transfection reference. Cells were transfected with plasmid vector control, IRF-3, or IRF-5 with or without IKK␧ as indicated. Results are expressed as relative luciferase activity. C, Cells were transfected with IRF-3, IRF-5/4A, or IRF-5/4D and treated as described in A. Results are expressed as relative luciferase activity. D, HT1080 cells were transfected with GFP-IRF-5, GFP-IRF-5/4A or GFP-IRF-5/4D, and cellular localization was evaluated by ﬂuorescence microscopy.

induced gene expression in the absence of IKK␧. In fact, coex- were stained with DAPI and assessed microscopically for DNA pression of IKK␧ did not increase the activity of IRF-5/4D. To damage by the TUNEL assay, either 42 or 66 h following trans- evaluate the cellular localization of the constitutively active IRF- fection (Fig. 7A). Transfections with GFP served as negative con- 5/4D, expression of GFP-IRF-5/4D was examined by ﬂuorescent trols, whereas expression of IRF-3 with IKK␧ or the constitutively microscopy (Fig. 6C). In comparison to IRF-5 or IRF-5/4A, the active IRF-3/5D served as positive controls. Cells that expressed active mutant IRF-5/4D accumulated constitutively in the nucleus GFP-IRF-5 alone elicited a low level of apoptosis; however, co- in the absence of any stimulus. This mutation was also found to transfection with the IKK␧ promoted a signiﬁcant apoptotic re- cause IRF-5 dimerization and binding to CBP (data not shown). sponse. Expression of the constitutively active mutant of IRF-5, The results suggest that a C-terminal phosphorylation event is crit- GFP-IRF-5/4D, promoted an even more apparent apoptotic effect. ical for IRF-5 to function as a transcription factor. Phosphorylation of GFP-IRF-3 by IKK␧ stimulated the apoptotic Activation of IRF-5 promotes apoptosis potential of IRF-3, as expected, and the constitutively active IRF-3/5D elicited high levels of apoptosis independent of IKK␧ Several reports indicate IRF-5 plays a role in innate immunity, (33, 46). but some studies provide evidence that IRF-5 overexpression As an independent measure of apoptosis, we also evaluated the blocks cell cycle progression or sensitizes cells to DNA damage caspase cleavage of PARP-1, an enzyme required for DNA repair effects (29, 45). For this reason, we evaluated the contribution of IRF-5 to programmed cell death, and we used several assays (47). Caspase activation results in the cleavage of PARP-1 into two to access apoptosis. fragments of 24 and 89 kDa, and this cleavage has been established Fig. 7 displays the results of representative experiments access- as a characteristic of apoptosis. For this assay, the same transfected ing IRF-5 effects both on a single-cell basis, and on the cell pop- cultures that were analyzed morphologically for apoptosis were ulation. Cells were transfected with GFP-tagged constructs with or lysed, and PARP-1 cleavage was detected by Western blot with an without cotransfection with IKK␧ as indicated. Cells on coverslips Ab that detects the p85 cleavage product (Fig. 7B). There was a 7468 DISTINCT ACTIVATION PATHWAY OF IRF-5 AND IRF-3

FIGURE 7. Activation of IRF-5 promotes apoptosis. A, HeLa cells on coverslips were transfected with GFP- tagged constructs as indicated with or without IKK␧ cotransfection (Ϫ/ϩ). Nuclei were visualized by DAPI staining, and apoptosis was scored by determining the percentage of GFP-positive cells that were TUNEL positive at either 44- or 66-h posttransfection. Five visual fields were microscopically examined, and the average number of TUNEL-positive and green fluorescent cells as a percentage of total green fluorescent cells is provided. B, Cells were harvested from the same transfected cultures in A, and protein lysates were prepared and evaluated by Western blot 66 h following transfection for PARP-1 cleavage. C, Huh-7 cells were transfected with GFP-IRF-5, GFP-IRF-5/4D, GFP-IRF-3, or

GFP-IRF-3/5D with or without Bcl-xL or DNFADD as indicated. Lysates were prepared 66 h following transfection, and Western blots were used to detect PARP-1 cleavage. Results are representative of multiple independent experiments.

low level of background PARP-1 cleavage in the cultures trans- optosis by either IRF molecule. These results suggest that apopto- fected with IRF-5, but cotransfection of IKK␧ with IRF-5 in- sis by the IRF factors does not depend on paracrine or autocrine creased the levels of PARP-1 cleavage. A similar effect of PARP-1 ligands that promote death through the TNFR molecule. cleavage was produced by the constitutive active IRF-5/4D mutation, whereas the IRF-5/4A mutation did not induce PARP cleav- Discussion age. GFP-IRF-3 was used as a positive control, and similar to Characterization of members of the IRF family indicates they have IRF-5, it promoted apoptosis only after activation by IKK␧,oras distinct contributions to innate or acquired immunity. Although the constitutively active mutant, IRF-3/5D. These results clearly they share a similar DNA-binding domain at their N terminus, they demonstrate that IRF-5 has the potential to induce apoptosis either possess unique sequences that result in specific protein-protein and following activation by IKK␧ phosphorylation or expressed as the protein-DNA interactions. These distinctive interactions are re- constitutively active mutant IRF-5/4D. sponsible for both specificity of activation and individual function. To investigate the mechanisms by which IRF-5 promotes apo- IRF-3 is phosphorylated in response to viral infection or TLR-3 ptosis, we evaluated the effects of characterized negative interfer- stimulation leading to its DNA-binding capability and transcrip- ing molecules (Fig. 7C). The Bcl-2 family of proteins consists of tional activity. The targeted gene disruption of IRF-3 demonstrated antiapoptotic and proapoptotic members that act at cellular or- its critical role in IFN-␤ production and survival of viral infections ganelles such as mitochondria (48). Overexpression of members of (20). Previous studies with IRF-5 have reported that it plays a role the antiapoptotic subclass, including Bcl-xL, is able to prevent pro- in the response to viral infection (32, 50, 51), and more recently grammed cell death induced by various agents. Because these mol- that it responds to stimulation of TLR-7, -8, and -9 (30, 31). In this ecules function in intracellular organelles, the apoptotic mecha- report, we evaluated comparatively the activation of IRF-3 and nism is often referred to as intrinsic, or mitochondrial dependent. IRF-5 in response to viral infection or specific kinase expression. In contrast, extrinsic or mitochondrial-independent apoptosis often We did not find IRF-5 activation in response to NDV, a virus refers to the response of cells to extracellular ligands that bind to previously described to activate IRF-5. Intriguingly, we found cell surface receptors such as members of the TNFR superfamily IRF-5 to be activated in response to expression of kinases TBK1 or (49). These receptors do not exclusively induce extrinsic signal IKK␧. These kinases are associated in complexes with multiple pathways, but they possess an intracellular death domain region components and appear to respond to distinct pathways of activa- that binds to an adaptor called the FADD, and a DNFADD has tion by viral infection or TLR signaling (52, 53). It is possible that been shown to block apoptosis stimulated by this pathway. To the signal pathway of activation is linked to downstream target compare the inhibitory effects of Bcl-xL or DNFADD on IRF-5- specificity in vivo which would be coordinate with our findings. induced apoptosis, we cotransfected these modulators with the Activation of IRF-3 can be detected in several ways, by serine constitutively active form of IRF-5 or IRF-3, and evaluated phosphorylation, dimerization, cytoplasmic to nuclear redistribu- PARP-1 cleavage as the assay to detect apoptosis (Fig. 7C). Cells tion, binding to CBP or p300, binding to an ISRE-containing DNA were transfected with GFP-tagged forms of IRF-5, IRF-5/4D, target, and induction of reporter gene activity. We used these same

IRF-3, or IRF-3/5D in the absence or presence of Bcl-xL or parameters to evaluate the comparative activation of IRF-5 fol- DNFADD. The results clearly indicate that expression of Bcl-xL lowing either expression of kinases or viral infection. In a latent blocks caspase-induced cleavage of PARP-1 in response to either state, IRF-5 shuttles in and out of the nucleus and therefore it has active IRF-5 or IRF-3, but the DNFADD is not able to block ap- active NLS and NES sequences (42). We identiﬁed an NES by The Journal of Immunology 7469

mutational analysis of leucine residues at positions 150 and 154 Bcl-xL was found to inhibit the apoptotic effects of both IRF-5/4D (Fig. 2B). A previous report noted the requirement of leucine res- and IRF-3/5D; however, the DNFADD molecule did not block idues 157 and 159 for export activity, and together the results apoptosis. FADD has been reported to be required for the upstream indicate the NES sequence spans the region of aa 150–160 activation of IRF-3 in response to intracellular viral dsRNA (55). (LQRMLPSLSLT) (54). It is not clear why the previous study did Our analyses indicate that FADD is not required for the down- not detect nuclear accumulation following phosphorylation by stream proapoptotic effects of IRF-3 or IRF-5. There is a delicate TBK1 or IKK␧. We clearly found kinase expression increased balance in the cellular response to stimuli that activate both pro- IRF-5 phosphorylation and stimulated its nuclear accumulation apoptotic and antiapoptotic signal pathways. The strength of (Figs. 1 and 2A). Our results with IRF-5/4D indicated that C-ter- distinct pathways may dictate the final outcome of the cell. In minal phosphorylation may be a regulatory switch for nuclear ac- response to viral infection or TLR signaling, there are phosphor- cumulation. In addition, we showed that modification of IRF-5 in ylation events that lead to activation of antiapoptotic molecules response to kinase activity promoted its dimerization, binding to including transcription factors such as NF-␬B as well as IRFs. We CBP/p300, and binding to DNA. Dimerization indicates that like have tipped the balance and analyzed primarily the effects of ac- IRF-3, there is a conformational change of IRF-5 following phos- tivated IRF-3 or IRF-5 to identify their ability to promote apopto- phorylation. The altered conformation leads to the gain of its abil- sis. In addition, in the context of viral infection our study clearly ity to bind CBP/p300 in the nucleus. This binding to CBP/p300 by indicates that IRF-3 and not IRF-5 appears to be a primary IRF IRF-5 may be responsible for its retention in the nucleus and abil- mediator. ity to bind DNA, as is the case for IRF-3 (42). Note added in proof: We have analyzed the behavior or IRF-5 v1 The ability of IRF-5 to induce gene expression was tested by and find that it also responds to TBK1 or IKK⑀ with nuclear evaluating its effect on endogenous genes as well as a reporter gene accumulation. regulated by an ISRE in response to either TBK1 or IKK␧. The IRF-5null animals appear to be deficient in the production of IL-6 Acknowledgments and IL-12, and the ISRE elements in these genes have been pro- We thank all the members of the laboratory for their helpful discussions posed to respond to IRF-5 (30). We performed the studies in cells and support. Special thanks to Dr. Gregg Banninger for advice on protein derived from an IRF-3 knockout animal so that the contribution of localization studies, and to Jermel Watkins for propagation of virus. The endogenous IRF-3 could be eliminated (20). The results show that gift of irf-3Ϫ/Ϫ cells from Dr. Tadatsugu Taniguchi is much appreciated. activation of IRF-3 or IRF-5 can induce expression of endogenous ISG54 and IL-6 genes, but with differential effects (Fig. 6A). IRF-3 Disclosures is a more effective inducer of ISG54, whereas IRF-5 has a greater The authors have no financial conflict of interest. stimulatory effect on IL-6. A reporter gene regulated by an ISRE was also tested. IRF-5 activated by IKK␧ could detectably induce References the reporter; however, the induction is much less than that induced 1. Akira, S., and K. Takeda. 2004. Functions of Toll-like receptors: lessons from by phosphorylated IRF-3 (Fig. 6B). It is possible that modifications KO mice. C. R. Biol. 327: 581–589. 2. 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