Family That Binds to the Interferon-Stimulated Response Element and Activates Expression of Interferon-Induced Genes WEI-CHUN AU*T, PAUL A

Proc. Natl. Acad. Sci. USA Vol. 92, pp. 11657-11661, December 1995 Biochemistry

Identification of a member of the interferon regulatory factor family that binds to the interferon-stimulated response element and activates expression of interferon-induced genes WEI-CHUN AU*t, PAUL A. MOOREtt, WILLIAM LOWTHER*, YUANG-TAUNG JUANG*, AND PAULA M. PITHA*§¶ *Oncology Center and §Department of Molecular Biology and Genetics, School of Medicine, The Johns Hopkins University, Baltimore, MD 21231; and tHuman Genome Sciences, Inc., Rockville, MD 20850 Communicated by Hamilton 0. Smith, The Johns Hopkins University, Baltimore, MD, August 17, 1995 (received for review June 21, 1995)

ABSTRACT A family of interferon (IFN) regulatory fac- IRF-1 was sufficient to induce transcription from the IFN-a tors (IRFs) have been shown to play a role in transcription of promoters: however, it induced expression of both virus- IFN genes as well as IFN-stimulated genes. We report the inducible and -uninducible IFN-a promoters (6). Further, the identification of a member of the IRF family which we have binding of IRF-1 to the respective IRF-1 binding sites in named IRF-3. The IRF-3 gene is present in a single copy in murine and human IFN-a promoters was very weak, suggest- human genomic DNA. It is expressed constitutively in a variety ing that if IRF-1 plays a role in induction of IFN-a genes, it of tissues and no increase in the relative steady-state levels of cooperates with another binding protein(s) (7). The strongest IRF-3 mRNA was observed in virus-infected or IFN-treated argument against the limiting role of IRF-1 and IRF-2 is that cells. The IRF-3 gene encodes a 50-kDa protein that binds the virus-mediated induction of IFN-a or -3 genes was not specifically to the IFN-stimulated response element (ISRE) altered in IRF-1 null mice or in cells with the IRF-1 gene but not to the IRF-1 binding site PRD-I. Overexpression of deleted (8, 9), suggesting that IRF-1 function can be replaced IRF-3 stimulates expression of the IFN-stimulated gene 15 by other transactivators, which may bind to the same domain. (ISG15) promoter, an ISRE-containing promoter. The murine IRF-1 was also shown to bind to the interferon-stimulated IFNA4 promoter, which can be induced by IRF-1 or viral response element (ISRE) present in promoters of genes infection, is not induced by IRF-3. Expression of IRF-3 as a activated by IFN (10) and to have a direct role in regulation of Gal4 fusion protein does not activate expression of a chlor- expression of several IFN-induced genes (11-13). In addition, amphenicol acetyltransferase reporter gene containing re- two other proteins [IFN consensus sequence-binding protein peats of.the Gal4 binding sites, indicating that this protein (ICSBP) and IFN-stimulated gene factor 3 'y polypeptide does not contain the transcription transactivation domain. (ISGF3'y)] show similarity to IRF-1 and IRF-2 at the amino The high amino acid homology between IRF-3 and ISG factor acid level and bind to the ISRE, indicating the existence of a 3 'y polypeptide (ISGF3y) and their similar binding properties family of IRF-1-like transcription factors (14, 15). All of these indicate that, like ISGF3'y, IRF-3 may activate transcription factors share a high degree of homology in the N-terminal by complex formation with other transcriptional factors, DNA-binding domain but have diversity in the C-terminal possibly members of the Stat family. Identification of this region, where IRF-1 contains the activation ISRE-binding protein may help us to understand the speci- transcription in domain. ICSBP, which, unlike IRF-1 and -2, is expressed only ficity the various Stat pathways. in cells of lymphoid origin, was shown to bind the ISRE of many ISGs and repress the IFN-mediated activation of IFN- The virus-induced expression of interferon (IFN) genes in inducible promoters (16). The ISGF3'y (p48) present in the infected cells involves interplay of several constitutively ex- cytoplasm is assembled, in cells treated with IFN-a, together pressed and virus-activated transcriptional factors (1). Two of with the Statl and Stat2 proteins into the ISGF3 complex and these factors binding to the virus-inducible element of the mediates binding of this complex to the ISRE (14, 17). c-Myb IFN-3 gene have been proposed to play a crucial role in the also belongs to the family of IRF-1-like proteins, although its regulation of expression of IFN-a and IFN-,3 genes. Interferon relationship to the IFN system is unclear (14). regulatory factor 1 (IRF-1) was shown to act as an activator, The aim of this study was to find a transcriptional factor that and the closely related IRF-2 was a repressor (2). It was can replace the function of IRF-1 and stimulate transcription proposed that induction of the IFN-,3 gene was the result of the of the IFN-a We have identified and removal of repressor IRF-2 and the subsequent binding of the promoter. characterized activator IRF-1 (3). Several observations supported this a member of the IRF-1 family, IRF-3, that may function as a model. Expression of IRF-1 was found to be upregulated in regulatory component in virus-infected cells.11 virus-infected cells. The IRF-1 binding sites were identified in the IFN-3 gene promoter region and reporter plasmids with MATERIALS AND METHODS multiple repeats of the AAGTGA hexanucleotides (which are the strong IRF-1 binding site) were inducible by overexpres- Isolation of a cDNA Showing Homology to the IRF Family sion of IRF-1. This transactivation could be repressed by IRF-2 Members. An expressed sequence tag (EST) cDNA database (2). In embryonal carcinoma cells, overexpression of IRF-1 induced both the transfected and the endogenous IFN-a and Abbreviations: IFN, interferon; ICSBP, IFN consensus sequence- -,B genes (4). Moreover, a decrease in IFN-,B induction was binding protein; IRF, IFN regulatory factor; ISG, IFN-stimulated gene; ISGF3, ISG factor 3; ISRE, IFN-stimulated response element; observed in cells expressing the IRF-1 antisense mRNA (5). CAT, chloramphenicol acetyltransferase; EST, expressed sequence In contrast, studies of IFN-a promoters did not support the tag; GST, glutathione S-transferase; NDV, Newcastle disease virus; role of IRF-1 as the limiting factor in the virus-mediated PRD, positive regulatory domain. induction of these genes. In mouse cells, overexpression of tW.-C.A. and P.A.M. contributed equally to this project. ITo whom reprint requests should be addressed at: Oncology Center, The Johns Hopkins University, 418 North Bond Street, Baltimore, The publication costs of this article were defrayed in part by page charge MD 21231-1001. payment. This article must therefore be hereby marked "advertisement" in lThe sequence reported in this paper has been deposited in the accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank database (accession no. Z56281). 11657 Downloaded by guest on September 30, 2021 11658 Biochemistry: Au et aL Proc. Natl. Acad. Sci. USA 92 (1995)

was screened for homology by using the BLAST network service RESULTS provided by the National Center for Biotechnology Informa- tion. Several overlapping ESTs showing homology to IRF-1 Identification and Characterization of the IRF-3 Clone. A and IRF-2 were identified, and one that appeared to be database of ESTs (21) generated from multiple human tissues full-length was sequenced and designated IRF-3. The first at Human Genome Sciences, Inc., and the Institute of designated methionine codon in the IRF-3 clone is likely to be Genomic Research was searched for homologs of IRF-1 and the start codon because its context (ACC-AUG-G) fits the IRF-2 by using the BLAST algorithm (22). A cDNA with an Kozak consensus sequence for translation (RCC-AUG-G). open reading frame of 427 aa was identified (Fig. 1), desig- The IRF-3 clone was used for subsequent studies. nated IRF-3, characterized, and used for further studies. Plasmid Constructs. The IRF-3 expression plasmid was In addition to IRF-1 and IRF-2, IRF-3 is also homologous prepared by cloning the BamHI-Xho I fragment containing to ICSBP and ISGF3,y (Fig. 2). The homology to IRF-1 and the IRF-3 cDNA from the pSKIRF-3 plasmid behind the IRF-2 is restricted to the N-terminal 110 aa containing the cytomegalovirus promoter in the pCDNAINEO vector (In- characteristic tryptophan repeats, whereas the homology to vitrogen). The IRF-1 expression plasmid has been described ICSBP and ISGF3,y extends through the whole coding region. (18). The Gal4-IRF fusion plasmids were constructed by first In addition, IRF-3, ISGF-y, and ICSBP contain an identical amplifying full-length IRF-1, IRF-2, and IRF-3 cDNAs by domain of 7 aa (positions 34-40) from which 6 aa are also PCR. After digestion of the amplified fragments with Xho I preserved in IRF-1 and IRF-2. In the N-terminal region, IRF-3 andXba I (for IRF-1 and IRF-3) or Sal I andXba I (for IRF-2), is 34% and 37% identical with IRF-1 and IRF-2 respectively, these fragments were cloned in-frame with the GAL4 sequence whereas the values for ICSBP and ISGF3,y are 39.8% and corresponding to aa 1-147 of the Gal4 DNA binding domain 35.2%, respectively. In the C-terminal region, IRF-3 is 25.3% in pSG424 (19). The indicator plasmids containing the chlor- identical to ICSBP and 18.6% identical to ISGF3-y. amphenicol acetyltransferase (CAT) gene inserted behind the Expression of IRF-3 Gene in Various Tissues and Cells. virus-inducible 452-nt IFNA4 promoter region (IFNA4/CAT) Southern hybridization of human genomic DNA (HeLa cells) or the IFN-inducible promoter of ISG15 (ISG15/CAT) or showed a single copy of the IRF-3 gene (Fig. 3A). Northern three copies of ISRE (from ISG15) inserted in front of the hybridization detected a 1.6-kb band in all tissues examined, -119 human immunodeficiency virus promoter-CAT plasmid indicating that this gene is expressed constitutively (Fig. 3B). have been described (7). For construction of glutathione To determine whether IRF-3 gene expression is further stim- S-transferase (GST)-IRF and GST-ISGF3,y fusion plasmids, ulated in virus-infected or IFN-treated cells, total RNA iso- the EcoRI-Xho I fragment of the IRF-3 cDNA or the PCR- lated from either NDV-infected or IFN-treated cells at various amplified ISGF3,y cDNA were cloned into pGEX-4T-2 vector times postinduction was analyzed by Northern hybridization (Pharmacia Biotech) digested with the same enzymes. For and S1 nuclease mapping. From 1 to 24 hr after infection, the construction of the GST-IRF-1 fusion plasmid, murine IRF-1 relative levels of IRF-3 mRNA in NDV-infected HeLa cells cDNA isolated as a Pst I fragment from pIRF-1AS (18) was did not change significantly, nor were there changes in the cloned into pGEX-4T-2 digested with Sal I. In Vitro Translation of IRF-3 mRNA and Expression of GST M GT P K was GGTTCCAGCTGCCCGCACGCCCCGACCTTCCATCGTAGGCCGGACCATGGGAACCCCAAA Fusion Protein. IRF-3 mRNA translated in a rabbit P R I L P W L V S Q L D L G Q L E G V A reticulocyte lysate (Promega). The GST fusion proteins were GCCACGGATCCTGCCCTGGCTGGTGTCGCAGCTGGACCTGGGGCAACTGGAGGGCGTGGC W V N K S RT R F R I P W K H G L R Q D purified from bacterial lysates by affinity chromatography on CTGGGTGAACAAGAGCCGCACGCGCTTCCGCATCCCTTGGAAGCACGGCCTACGGCAGGA a glutathione-agarose column (Sigma). To remove the GST A Q Q E D F G I F Q A W A E A T G A Y V bound to TGCACAGCAGGAGGATTTCGGAATCTTCCAGGCCTGGGCCGAGGCCACTGGTGCATATGT portion, fusion protein glutathione-agarose beads P G P DK P D L P T W K R N F R S A L N was treated with thrombin (20%, wt/wt) for 2 hr and recom- TCCCGGGAGGGATAAGCCAGACCTGCCAACCTGGAAGAGGAATTTCCGCTCTGCCCTC'A binant IRF-3 was eluted with phosphate-buffered saline. R K E G L RL A E D R S K D P H D P H K CCGCAAAGAAGGGTTGCGTTTAGCAGAGGACCGGAGCAAGGACCCTCACGACCCACATAA Transfection and CAT Assay. Transfections were done by I Y E F V N S G V G D F S Q P D T S P D calcium phosphate precipitation (6, 7). Treatment with type I AATCTACGAGTTTGTGAACTCAGGAGTTGGGGACTTTTCCCAGCCAGACACCTCTCCGGA T N G G G S T S D T Q E D I L DEL L G IFN (100 units/ml) or infection with Newcastle disease virus CACCAATGGTGGAGGCAGTACTTCTGATACCCAGGAAGACATTCTGGATGAGTTACTGGG (NDV) (multiplicity of infection, 5) was done 16 hr after N M V L A P L P D P G P P S L A V A P E transfection for 8 hr. IFN or virus inoculum was then TAACATGGTGTTGGCCCCACTCCCAGATCCGGGACCCCCAAGCCTGGCTGTAGCCCCTGA removed, P C P Q P L R S P S L D N P T P F P N L and cells were washed and incubated in medium for 16 hr GCCCTGCCCTCAGCCCCTGCGGAGCCCCAGCTTGGACAATCCCACTCCCTTCCCAAACCT before harvest for the CAT assay. G P S E N P L K R L L V P G E E W E F E GGGGCCCTCTGAGAACCCACTGAAGCGGCTGTTGGTGCCGGGGGAAGAGTGGGAGTTCGA Gel Mobility-Shift Assay. The purified GST-IRF fusion V T A F Y R G R Q V F Q Q T I S C P E G proteins (100 ng) were incubated with labeled probes (1-10 pg) GGTGACAGCCTTCTACCGGGGCCGCCAAGTCTTCCAGCAGACCATCTCCTGCCCGGAGGG L R L V G S E V G D R T L P G W P V T L in the presence of nonspecific competitor poly(dIdC) (1 ,ug) CCTGCGGCTGGTGGGGTCCGAAGTGGGAGACAGGACGCTGCCTGGATGGCCAGTCACACT as described (17) and the protein-DNA complexes were P D P G M S L T D R G V M S Y V R H V L resolved in a GCCAGACCCTGGCATGTCCCTGACAGACAGGGGAGTGATGAGCTACGTGAGGCATGTGCT nondenaturing 4% polyacrylamide gel. The S C L G G G L A L W R A G Q W L W A Q R following oligonucleotides were used for DNA-binding studies GAGCTGCCTGGGTGGGGGACTGGCTCTCTGGCGGGCCGGGCAGTGGCTCTGGGCCCAGCG or as competitors: ISRE, 5'-CAGTTTCGGTTTCCCTTT-3'; L G H C H T Y W A V S E E LL P N S G H GCTGGGGCACTGCCACACATACTGGGCAGTGAGCGAGGAGCTGCTCCCCAACAGCGGGCA positive regulatory domain I (PRD-I), 5'-GAGAAGTGAA- G P D G E V P K D K E G G V F D L G P F AGTGGGAACCCTCTCCTT-3' (the underlined sequence TGGGCCTGATGGCGAGGTCCCCAAGGACAAGGAAGGAGGCGTGTTTGACCTGGGGCCCTT I V D L I T F T E G S G R S P R Y A L W was used for annealing of primer 5'-AAGGAGAGGG-3' and CATTGTAGATCTGATTACCTTCACGGAAGGAAGCGGACGCTCACCACGCTATGCCCTCTG synthesis of a complementary strand). F C V G E S W P Q D Q P W T K R L V M V Southern and GTTCTGTGTGGGGGAGTCATGgCCCCAGGACCAGCCGTGGACCAAGAGGCTCGTGATGGT Northern Hybridizations and Si Nuclease K V V P TC L R A L V E M A R V G G A S Analysis. Southern hybridization used 32P-labeled IRF-3 CAAGGTTGTGCCCACGTGCCTCAGGGCCTTGGTAGAAATGGCCCGGGTAGGGGGTGCCTC cDNA as a probe. Tissue distribution of IRF-3 mRNA was S L E N T V D L H I S N S H P L S L T S CTCCCTGGAGAATACTGTGGACCTGCACATTTCCAACAGCCACCCACTCTCCCTCACCTC determined with a multiple-tissue Northern blot of poly(A)+ D Q Y K A Y L Q D L V E G M DF Q G P G RNA (Stratagene) probed with 32P-labeled IRF-3 cDNA by CGACCAGTACAAGGCCTACCTGCAGGACTTGGTGGAGGGCATGGATTTCCAGGGCCCTGG E S * modified hybridization (20). Total RNA isolated from NDV- GGAGAGCTGAGCCCTCGCTCCTCATGGTGTGCCTCCAACCCCCCTGTTCCCCACCACCTC infected HeLa cells was analyzed by hybridization with 32p- AACCAATAAACTGGTTCCTGCTATGAAAAAAAAAAAAAAAAAAAAA labeled IRF-3 cDNA probe as described (7). For S1 nuclease analysis, total RNA was hybridized with 32P-labeled IRF-3 and FIG. 1. Nucleotide and predicted amino acid sequence of the IRF-3 ,3-actin RNA probes. cDNA. Asterisk marks termination codon. Downloaded by guest on September 30, 2021 Biochemistry: Au et al. Proc. Natl. Acad. Sci. USA 92 (1995) 11659 A

1m3 MGTPKPRILPWLVSQIDLGQLEGVAlVNIKSRTREMIPAGLRQDAQQE-DF)GI}OANAEATGAYVPGRDXCPDLPTM5N^RSANRKEGLRLAEDRSK-DPHDPHKIY 108 ICSBP MCDRNGGGRLRQI@LIEQIDSSMYPGL VENEEKSHMFR PAGKQDYNQEVDASIFKAVFKGKFKEG-DXAEPATWIICTRRCALNKS PDFEEVTDRSQLDI SEPYKVY 111 ISGF3y MASGRARCTRKIRNIvVEQVESGQFPGVCWV DTAKTQFMPG TAGKQDFRED EDAQFDKAUFKGKYKErG-DTGGPAVNKTRLACALNKSSEFKEVPERGRMDVAEPYKVY 113 IRF1 MPITREMMQPEMINSNQI PGLIlfINltEEMIFQI" AKHGWDINKDACLFRSlEI HTGRYKAGEKEPDPKllMMCAMNSLPDIEEVKDQSRNKGSSAVRVY 110 IRF2 MPVERMERMEEQINSNTIPPGLK2MEKKIFQIPW2IAARHGWDVZKDAPLFRNRAIHTGKHQPGVDDPKTOKANlCAMNSLPDIEEVKDKSIKKGNNAFRVY 110 B

w3 210 YRGRVFISCPZG--IRSVGSEGDTL-PGWPVTLPDPGMSLTDRGVMSYVRHVLSCGGGLLWRAGaIJ WAQRLGHCHTYWAVSEELLPNSGH 305 ICSBP 211 YGGKL GCRLSLSQPGLPGTKLYGP30LELVRFPPADTI6PSERQRQVFGHGV-LHSSR GRVF-CSGNAVCK--- 306 ISGF3y 226 YNGRVVGEAQVQSLDC--RLVAEPSGSESSM ---Z--QVLFPKP------GPLEPTQRLLQLZGILVASNPRGP7VgRLPIPISWNAPQ ---APPGP 309 306 GPDGEVPKDKEGGVFDLGPFIVDLITFTEGSRSPRYALWFCVGSWPQDQPWTKRLVMVKVVPTCLRALV-EMARVGGASSL 387 307 GRPNKLERDZVVQV!DTSQFFEELQQTYNSQGRLPDGRVVLCIYZFPDMAPLRSKLILVQIEQLYVRQLAEZAGKSCGAGSV 389 310 GPH-LLPSNZCVELFRTAYFCRDLVRYFQGLGPPPKFQVTLNFWZSHGSSHTPQNLITVKISQAFARYLL-EQTPEQQAAIL 390

FIG. 2. (A) N-terminal amino acid homology among members of the IRF family. Conserved amino acids are shown in bold type. (B) C-terminal amino acid homology among IRF-3, ISGF3-y, and ICSBP.

levels of IRF-3 mRNA in these cells after treatment with IRF-3 protein formed with the ISRE probe one major, fast- IFN-a (data not shown). To determine whether IRF-3 is moving complex (Fig. 4A, lane 2) and two minor, slowly induced by IFN in cells of lymphoid origin (as observed for moving complexes (seen after a prolonged exposure; Fig. 4A, ICSBP), we compared by Si analysis the levels of IRF-3 lane 4). Addition of antibodies to the GST fusion protein mRNA with those of the constitutively expressed ,B-actin completely eliminated the complex formation, indicating that message in IFN-a-treated Jurkat cells (T cells) and Namalwa the multiple complexes formed represented binding of the cells (B cells). All samples showed a single 268-nt protected GST-IRF-3 fusion protein and were not the result of its band, the intensity of which did not change (after normaliza- degradation (data not shown). Removal of GST fusion protein tion to the actin mRNA levels) during 16 hr of IFN treatment from IRF-3 by cleavage with thrombin did not change the (Fig. 3C). These data indicate that expression of the IRF-3 binding pattern (data not shown). Complex formation was gene is not stimulated in infected or IFN-treated cells. specific; it was inhibited by a 50-fold excess of nonradioactive Identification of a 50-kDa IRF-3 Peptide and DNA-Binding ISRE oligonucleotide but not by a nonspecific polynucleotide Properties of IRF-3 Synthesized in Bacteria. Translation of (Fig. 4B). The binding of GST-IRF-1 fusion protein to ISRE IRF-3 mRNA in the rabbit reticulocyte system yielded a showed only one DNA-protein complex (Fig. 4A, lanes 1 and 50-kDa polypeptide (data not shown), indicating that IRF-3 is 3), suggesting that the binding properties of IRF-1 and IRF-3 very similar in size to ISGFy (48 kDa) and IRF-1 (45 kDa) (2, are not identical. Since IRF-1 binds very effectively to the 14). IRF-3 expressed as a GST fusion protein in bacteria was PRD-I domain present in the IFN-,B gene promoter region, we two to purified on a glutathione-agarose beads column and used in compared the binding of these proteins the PRD-I IRF-1 very to this a DNA-binding assays. The GST protein alone did not bind to probe. bound strongly probe and formed single DNA-protein complex (Fig. lane but no any of the probes used. In a mobility-shift assay, the GST- 4A, 5), complex formation was detected between IRF-3 and PRD-I (lane 6). B Thus, IRF-3 is a DNA-binding protein that can bind specifically to ISRE, but not to the IRF-1 binding site. Since ISGF3,y .A,k -Mo. binds only to the ISRE, but not to PRD-I (17), we compared A I"pw 0 the binding of ISGF3,y and IRF-3 GST fusion proteins with the

1 2 3 4 5 6 7 8 A B - I I?- 7 W ( ISRE poly dl.dC 0 1 10 50 100 1 10 50 100 C

-..... --. _~~~~

1 2 3 4 5 6 7 8

1 2 1 2 3 4 5 FIG. 4. Gel mobility-shift assay. (A) An ISRE oligodeoxynucleotide was used as probe for binding of the recombinant GST-IRF-1 FIG. 3. (A) Southern blot hybridization of human (HeLa cell) (lanes 1 and 3), GST-IRF-3 (lanes 2,4, and 8), and GST-ISGF3'y (lane DNA digested with HindlIl (lane 1) or EcoRI (lane 2) and probed with 7) fusion proteins. Equal amounts (100 ng) of fusion proteins were IRF-3 cDNA. (B) Northern blot analysis of poly(A)+ RNA from used in all binding reactions. Lanes 1 and 2 show a 4-hr exposure; lanes human tissues. Lanes: 1, spleen; 2, thymus; 3, prostate; 4, testis; 5, 3 and 4 show a 16-hr exposure; and lanes 7 and 8 show a 24-hr exposure, ovary; 6, small intestine; 7, colon; 8, peripheral blood leukocytes. (C) respectively. A PRD-I oligodeoxynucleotide was used as probe for S1 analysis of total RNA (5 jig) isolated from HeLa cells treated with binding of IRF-1 and IRF-3 GST fusion proteins (lanes 5 and 6, IFN-a and IFN--y for various times. Lane 1, untreated cells; lanes 2 and respectively; 24-hr exposure). (B) Specificity of GST-IRF-3 fusion 3, cells treated with IFN-a (500 units/ml) for 5 and 16 hr, respectively; protein binding to the ISRE. Binding reaction mixtures contained lanes 4 and 5, cells treated with IFN--y (100 units/ml) for 5 and 16 hr, unlabeled ISRE oligodeoxynucleotide (specific competitor) or respectively. The lower band (145 nt) represents ,B-actin mRNA; the poly(dIdC) (nonspecific competitor) at the indicated molar ratio upper band (268 nt) represents IRF-3 mRNA. (0-100) relative to the radioactive ISRE probe. Downloaded by guest on September 30, 2021 11660 Biochemistry: Au et al. Proc. Natl. Acad. Sci. USA 92 (1995)

ISRE probe. The gel mobility-shift assay showed weak binding 10 of GST-ISGF3-y fusion protein and only one DNA-protein ,3p3. 8 complex was detected (Fig. 4A, lane 7), indicating that recom- .... binant IRF-3 has a much stronger affinity for the ISRE than .60

does recombinant ISGF3y. 0 IRF-3 Activates the ISG15 Promoter Region and Minimal z Promoter Containing Multiple Copies of the ISRE in a U4, 2 Transient Expression Assay. To determine whether IRF-3 can 0~~ U- C)4 m activate promoters containing ISREs or promoters of IFN 0-- M E:a.a genes containing the virus-inducible element, the IRF-3 o6 > z 4 (N M > z LL IL 0 cDNA was placed under the control of the cytomegalovirus z cc cc z promoter. 4 U- !-J e C'X LL -J J -j uL cc Cotransfection of IRF-3 expression plasmid with a reporter cc < < A (9 4 ( ( 4 plasmid containing 2000 nt of the ISG15 promoter region < (9 < (9 inserted 5' of the CAT gene resulted in a dose-dependent (9 (9 increase in CAT activity (Fig. 5A). This promoter region is activated by IFN-a (17, 23, 24). When IFN induction was done FIG. 6. Gal4-IRF-3 fusion protein does not activate transcription when bound to a Gal4 DNA-binding domain upstream from the under suboptimal conditions (8-hr treatment) leading to only transcription start site. Gal4-IRF-1, -2, or -3 expression plasmid (5 jig) a small increase in CAT activity, transfection with IRF-3, was transfected into L929 cells together with an indicator plasmid that followed by 8 hr of IFN treatment, showed a highly synergistic contained four Gal4 DNA binding sites cloned 5' of the minimal activation (25-fold increase, compared with the 6-fold increase thymidine kinase promoter-CAT plasmid. CAT activity was assayed 48 hr after the transfection. Where indicated, transfected cells were 80 A treated with murine IFN (500 units/ml) or infected with NDV (multiplicity of infection, 5) for 8 hr. r 60 .2 (a by IRF-3 and 3-fold increase by IFN). While treatment with 0C> 40 IFN for 24 hr led to a significant increase in CAT activity, the synergy between IFN and IRF-3 was less obvious (20-fold U - 20 increase by IFN and IRF-3 and 10-fold increase by IFN alone). ...,.- In contrast, cotransfection of IRF-3 expression plasmid with ... the reporter plasmid containing the IFNA4 promoter region in co trm front of the CAT gene did not lead to increased CAT activity, CN C(N z z tL z z indicating that the IFNA4 promoter is not activated by IRF-3 U- U- (Fig. SB). This promoter is, however, activated by cotransfec- 0 r tion with the IRF-1 expression plasmid or by viral infection (NDV). Although IRF-3 did not affect IRF-1-mediated stim- ISG 15/CAT:IRF-3 ulation (data not shown), infection of IRF-3-transfected cells led to an -3-fold increase in virus-mediated induction of the 16 B 14 IFNA4 promoter. Thus, whereas overexpression of IRF-3

c alone was unable to induce this promoter, it could enhance the 0 12 .) 10 NDV-mediated induction of the IFNA4 promoter. 8 Gal4-IRF-1, but Not Gal4-IRF-3, Fusion Protein Can 0 6 Activate Transcription. To determine whether IRF-3 contains 4 an activation region similar to that identified in IRF-1 (25), we

2 i, -r tested the ability of IRF-3 to stimulate transcription and fused

0 IRF-3 cDNA in-frame to the coding sequence for the DNA- C') 0 binding domain of Gal4. The reporter contained five Gal4 cc z z z binding sites inserted upstream of a minimal thymidine kinase 0 CL) LL promoter. Gal4-IRF-1, but not Gal4-IRF-3 or Gal4-IRF-2, cm cc stimulated the transcription of the CAT gene (Fig. 6). It has FIG. 5. IRF-3 induces expression from the ISG15 promoter but not been shown by others that IRF-2, in contrast to IRF-1, does not the IFNA4 promoter. (A) Hybrid plasmid (5 ,g) containing 2000 nt of contain the transactivation domain (J. Hiscott, personal com- the ISG15 promoter region inserted in front of the CAT gene was munication). In cotransfection studies, we have observed transfected into murine L929 cells either alone or with increasing enhancement of IRF-3-mediated transactivation in IFN- amounts of IRF-3 expression plasmid. CAT activity was assayed 24 hr treated or virus-infected cells. Therefore, we tested whether after transfection. All transfections were done in the presence of a virus or IFN can also modulate the activity of the Gal4-IRF-3 reference plasmid, pCH1 10 (1 jig), encoding ,B-galactosidase. Where fusion protein. Neither IFN treatment nor viral infection indicated, transfected cells were treated with murine IFN (100 units/ or ml) for 8 hr or 24 hr. (B) Hybrid plasmid (5 ,ug) containing 452 nt of activated the inducing potential of Gal4-IRF-3 Gal4-IRF-1 the murine IFNA4 promoter region inserted in front of the CAT gene (Fig. 6). These results indicate that IRF-3 does not contain a (6) was cotransfected with either IRF-I or IRF-3 expression plasmid transactivation domain and that the observed activation of the (5 ,ug) and pCHI 10 (1 ,ug). Where indicated, the transfected cells were ISRE may be mediated by coassembly of IRF-3 with another infected with NDV. Percent chloramphenicol conversion was calcu- transcriptional activator(s). lated by dividing the radioactivity (cpm) present in 3-acetylchloramphenicol and 1-acetylchloramphenicol fractions by the sum of radioactivity in unconverted chloramphenicol and these two fractions. The DISCUSSION levels of CAT activity were normalized to the constant level of /3-galactosidase. The maximal difference in 3-galactosidase activity in We have isolated and expressed a cDNA clone designated a single experiment was 2-fold. An average value from three inde- IRF-3 that shows a high degree of similarity to members of the pendent experiments is given. There was 15% variability between IRF family. The IRF-3 gene is present as a single copy in individual experiments. genomic DNA and is expressed constitutively at the mRNA Downloaded by guest on September 30, 2021 Biochemistry: Au et al. Proc. Natl. Acad. Sci. USA 92 (1995) 11661 level in various human tissues. Its expression is not further to the ISRE was identified in cells infected with vesicular stimulated by virus infection or IFN treatment. stomatitis virus (31). Whether this protein is IRF-3 is un- The predicted amino acid sequence in the N-terminal part known. However, the existence of another DNA-binding pro- of IRF-3 shows 34-40% identity with other members of the tein of the IRF family that can function as a regulatory IRF family, including five tryptophan residues preserved in the component in infected or IFN-treated cells could provide first 90 aa of all IRF-like proteins. The DNA-binding domain further complexity and specificity to the regulatory network of IRF-1, IRF-2, and ISGF3,y is localized in the N-terminal mediating the responses to viral infection. 120-180 aa (refs. 1 and 14; J. Hiscott, personal communica- tion). Thus, although the binding domain of IRF-3 has not The contribution of the Human Genome Sciences and the Institute been determined, we assume that it is also localized in the for Genomic Research sequencing facilities in sequencing the initial N-terminal region. ESTs displaying homology to IRF-1 and IRF-2 is appreciated. This IRF-3 shows DNA-binding specificity similar to that of study was supported by an American Foundation for AIDS Research fails to bind the scholarship (P.A.M.) and by Grant A119713 from the National Insti- ISGF3y. IRF-3 binds the ISRE sequence but tutes of Health (P.M.P.). W.L. is a student in the predoctoral training PRD-I oligodeoxynucleotide that serves as a strong binding program in Human Genetics (T32GM07814). site for IRF-1. However, in a mobility-shift assay, the complexes formed between IRF-3 or ISGF3y and the ISRE did not 1. Tjian, R. & Maniatis, T. (1994) Cell 77, 5-8. show identical mobility. Whereas the binding of ISGF3y to the 2. Fujita, T., Kimura, Y., Miyamoto, M., Barsoumian, E. L. & Taniguchi, ISRE is rather weak and yields a single complex, IRF-3 showed T. (1989) Nature (London) 337, 270-272. a stronger affinity for the ISRE and complexes with three 3. Harada, H., Fujita, T., Miyamoto, M., Kimura, Y., Maruyama, M., were detected. Since purified IRF-3 pro- Furia, A., Miyata, T. & Taniguchi, T. (1989) Cell 58, 729-739. different mobilities 4. Harada, H., Willison, K., Sakakibara, J., Miyamoto, M., Fujita, T. & tein was used, these data suggest that IRF-3 may be dimerized. Taniguchi, T. (1990) Cell 63, 303-312. Interestingly, the central part of IRF-3 is proline-rich and 5. Reis, L. F., Harada, H., Wolchok, J. D., Taniguchi, T. & Vilcek, J. contains one cluster (aa 150-197) with prolines making up (1992) EMBO J. 11, 185-193. 34% of the amino acids. Mutational analysis is needed to 6. Au, W.-C., Raj, N. B. K., Pine, R. & Pitha, P. M. (1992) Nucleic Acids determine whether this proline-rich region is involved in the Res. 20, 2877-2884. 7. Raj, N. B. K., Au, W.-C. & Pitha, P. M. (1991) J. Biol. Chem. 266, interaction of IRF-3 with other proteins or its dimerization. In 11360-11365. ISGF3y, the region required for interaction with the Statl and 8. Ruffner, H., Reis, L. H., Naf, D. & Weissmann, C. (1994) EMBO J. Stat2 proteins lies between aa 271 and 377 (26). IRF-3 and 13, 4798-4806. ISGF3y share some homology in this region; however, a proline- 9. Matsuyama, T., Kimura, T., Kitagawa, M., Watanabe, N., Kundig, T., rich cluster (aa 285-309) is found only in ISGF3,y, and not in Amakawa, R., Kishihara, K., Wakeham, A., Potter, J., Furlonger, C., IRF-3 or the other IRF family members. It is possible that this Narendran, A., Suzuki, H., Ohashi, P., Paige, C., Taniguchi, T. & Mak, T. (1993) Cell 75, 83-97. region is involved in the interaction of ISGF3,y with the Stat 10. Pine, R., Decker, T., Kessler, D. S., Levy, D. E. & Darnell, J. E., Jr. proteins. After this work was completed, another member of the (1990) Mol. Cell. Biol. 10, 2448-2457. IRF-I family, Pip (27), was described that is closely related to 11. Briken, V., Ruffner, H., Schultz, U., Schwarz, A., Reis, L. F. L., ICSBP. IRF-3 and Pip are not identical proteins. Strehlow, I., Decker, T. & Staeheli, P. (1995) Mol. Cell. Biol. 15, Several observations indicate that IRF-3 may act through 975-982. an association with other proteins. (i) IRF-3 fused to the 12. Neish, A. S., Read, M. A., Thanos, D., Pine, R., Maniatis, T. & Collins, T. (1995) Mol. Cell. Biol. 15, 2558-2569. DNA-binding domain of Gal4 is unable to activate transcrip- 13. Kamijo, R., Harada, H., Matsuyama, T., Bosland, M., Gerecitano, J., tion of a reporter gene containing the Gal4 binding site. In Shapiro, D., Le, J., Koh, S. I., Kimura, T., Greene, S. J., Mak, T. W., contrast, IRF-1, which contains a transactivation domain in the Taniguchi, T. & Vilcek, J. (1994) Science 263, 1612-1615. C-terminal part of the molecule, is a very efficient transacti- 14. Veals, S. A., Schindler, C., Leonard, D., Fu, X.-Y., Aebersold, R., vator in the same system. (ii) IRF-3-mediated transactivation Darnell, J. E., Jr., & Levy, D. E. (1992) Mol. Cell. Biol. 12, 3315-3324. of the ISG15 promoter is significantly more effective in cells 15. Driggers, P. H., Ennist, D. L., Gleanson, S. L., Mak, W.-H., Marks, M. S., Levi, B.-Z., Flanagan, J. R., Appella, E. & Ozato, K. (1990) treated with IFN for a short time (that alone does not result Proc. Natl. Acad. Sci. USA 87, 3743-3747. in an effective transactivation), indicating that enhancement is 16. Nelson, N., Marks, M. S., Driggers, P. H. & Ozato, K. (1993) Mol. Cell. the result of an IFN-induced modification of a cellular pro- Biol. 13, 588-599. tein(s) that interacts with IRF-3. This enhancement of IRF-3 17. Kessler, D. S., Veals, S. A., Fu, X.-Y. & Levy, D. E. (1990) Genes Dev. activity by IFN can be demonstrated only when IRF-3 binds to 4, 1754-1765. and not when IRF-3 binds to DNA through the Gal4 18. Au, W.-C., Su, Y., Raj, N. B. K. & Pitha, P. M. (1993) J. Biol. Chem. the ISRE 268, 24032-24040. binding site. This result indicates that interaction with other 19. Sadowski, I. & Ptashne, M. (1989) Nucleic Acids Res. 17, 7539. proteins may depend on the conformation of IRF-3 and/or 20. Church, G. M. & Gilbert, W. (1984) Proc. Natl. Acad. Sci. USA 81, that the protein interacting with IRF-3 also binds to the ISRE 1991-1995. or the adjacent sequences. Similarly, in the ISGF3 complex, 21. Adams, M. D., Dubnick, M., Kervlavage, A. R., Moreno, R., Kelley, not only ISGF3y but also Statl and Stat2 interact with the J. M., Utterback, T. R., Nagle, J. W., Fields, C. & Venter, J. C. (1991) does not activate the IFNA4 Nature (London) 355, 632-634. ISRE (28). (iii) Although IRF-3 22. Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. promoter, it enhances the virus-mediated induction of that (1990) J. Mol. Biol. 215, 403-410. promoter. This activation may result from the association of 23. Stark, G. R. & Kerr, I. M. (1992) J. Interferon Res. 12, 147-151. IRF-3 with virus-modified cellular factors and a consequent 24. Su, Y., Popik, W. & Pitha, P. M. (1995) J. Virol. 69, 110-121. increase in the IRF-3 binding to the virus-inducible element in 25. Lin, R., Mustafa, H., Nguyen, N. & Hiscott, J. (1994) J. Biol. Chem. the IFNA4 promoter. Modification of the DNA-binding do- 269, 17542-17549. 26. Veals, S. A., Santa Maria, T. & Levy, D. E. (1993) Mol. Cell. Biol. 13, main through interaction of the DNA-binding protein with a 196-206. protein unable to bind DNA was reported for the Oct-I 27. Eisenberg, C. S., Singh, H. & Storb, U. (1995) Genes Dev. 9, 1377- protein, which recognized a G+A-rich binding site upon 1387. interaction with the viral protein VP16 (29, 30). 28. Qureshi, S. A., Salditt-Georgieff, M. & Darnell, J. E., Jr. (1995) Proc. In conclusion, our data suggest the existence of a DNA- Natl. Acad. Sci. USA 92, 3829-3833. binding protein, IRF-3, that by association with cellular pro- 29. Kristie, T. M., LeBowitz, J. H. & Sharp, P. A. (1989) EMBO J. 8, 4429-4238. teins activated by viral infection or by IFN, increases tran- 30. Stern, S. & Herr, W. (1991) Genes Dev. 5, 2555-2566. scriptional activity of targeted promoters. The proteins that 31. Bovolenta, C., Lou, J., Kanno, Y., Park, B.-K., Thornton, A. M., interact with IRF-3 and the target gene that these complexes Coligan, J. E., Schubert, M. & Ozato, K. (1995) J. Virol. 69, 4173- activate are unknown. Recently, a novel nuclear factor binding 4181. Downloaded by guest on September 30, 2021