HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 280, Issue 22, 21078-21090, June 3, 2005

This Article Abstract Full Text (PDF) All Versions of this Article: 280/22/21078    most recent M500543200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Mancl, M. E. Articles by Barnes, B. J. Search for Related Content PubMed PubMed Citation Articles by Mancl, M. E. Articles by Barnes, B. J. Social Bookmarking                      What's this?

Two Discrete Promoters Regulate the Alternatively Spliced Human Interferon Regulatory Factor-5 Isoforms

MULTIPLE ISOFORMS WITH DISTINCT CELL TYPE-SPECIFIC EXPRESSION, LOCALIZATION, REGULATION, AND FUNCTION^*

Margo E. Mancl, Guodong Hu, Niquiche Sangster-Guity, Stacey L. Olshalsky¶, Katherine Hoops, Patricia Fitzgerald-Bocarsly¶, Paula M. Pitha, Karen Pinder, and Betsy J. Barnes||

From the Division of Viral Oncology, The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21231, ^¶University of Medicine and Dentistry of New Jersey, New Jersey Medical School, and the Graduate School of Biomedical Sciences, Newark, New Jersey 07103

Received for publication, January 18, 2005 , and in revised form, March 18, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Interferon regulatory factor-5 (IRF-5) is a mediator of virus-induced immune activation and type I interferon (IFN) gene regulation. In human primary plasmacytoid dendritic cells (PDC), IRF-5 is transcribed into four distinct alternatively spliced isoforms (V1, V2, V3, and V4), whereas in human primary peripheral blood mononuclear cells two additional new isoforms (V5 and V6) were identified. The IRF-5 V1, V2, and V3 transcripts have different noncoding first exons and distinct insertion/deletion patterns in exon 6. Here we showed that V1 and V3 have distinct transcription start sites and are regulated by two discrete promoters. The V1 promoter (P-V1) is constitutively active, contains an IRF-E consensusbinding site, and is further stimulated in virus-infected cells by IRF family members. In contrast, endogenous V3 transcripts were up-regulated by type I IFNs, and the V3 promoter (P-V3) contains an IFN-stimulated responsive element-binding site that confers responsiveness to IFN through binding of the ISGF3 complex. In addition to V5 and V6, we have identified three more alternatively spliced IRF-5 isoforms (V7, V8, and V9); V5 and V6 were expressed in peripheral blood mononuclear cells from healthy donors and in immortalized B and T cell malignancies, whereas expression of V7, V8, and V9 transcripts were detected only in human cancers. The results of this study demonstrated the existence of multiple IRF-5 spliced isoforms with distinct cell type-specific expression, cellular localization, differential regulation, and dissimilar functions in virus-mediated type I IFN gene induction.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Virus infection results in the activation of a defined set of cellular genes involved in host antiviral defense. Transcription factors of the interferon (IFN)¹ regulatory factor (IRF) family have been identified as having important roles in innate immunity by participating in both the immediate-early transcriptional response to infection and the secondary response to cytokines (1–4). While playing an important role in the antiviral immune response, IRFs also participate in cell growth regulation and apoptosis (5, 6). IRF-5 is one of the most recently characterized members of the IRF family (2, 7–14). It encodes a 60–63-kDa polypeptide that was originally identified as a regulator of type I IFN gene expression (7, 9). However, recent studies have indicated that it plays a role in many aspects of host defense (11), including induction of multiple cytokines and chemokines involved in the recruitment of T lymphocytes (8). Subsequently, it has been shown that IRF-5 itself is regulated by type I IFN (8, 10), indicating an important regulatory pathway for the controlled induction of multiple immunomodulatory genes (11).

The role of IRF family members in the induction of cell arrest and cell death has been well established (5, 10, 12, 15–17). Although IRF-5 participates in the virus-induced signaling cascade, its expression is also modulated by p53 (12), and it has a role in the apoptotic signaling pathway that is p53-independent (10). Thus, IRF-5 modulates the expression of a number of factors involved in cell cycle regulation and apoptosis, independent of viral infection (10). The ability of IRF-5 to induce expression of proteins involved in the regulation of cell growth, apoptosis, and immunomodulation represents an important defense mechanism against extracellular stress, including viral infection. How significant is the overlapping cross-talk between IFN and apoptotic signaling pathways is currently unknown, yet it has been suggested recently that type I IFN can also induce p53 (6). Altogether, the current data suggest an important role for IRF-5 in both IFN- and p53-mediated signaling pathways.

To date, a large number of IRF-5 sequences have been deposited to GenBank^TM. Most interesting, the first complete coding sequence of human IRF-5, isolated from lymphocytes and deposited in 1996 to GenBank^TM (accession number U51127 [GenBank] ), was not functionally characterized. Subsequently, we had cloned two new isoforms of IRF-5, here termed variants 3 (V3; GenBank^TM accession number AY504946 [GenBank] ) and 4 (V4; GenBank^TM accession number AY504947 [GenBank] ) from a dendritic cell library (7), and we have characterized the identical polypeptide they encode (2, 7–11). In the past 2 years, two additional isoforms, termed variant 1 (V1; isoform a; NM_002200 [GenBank] ; U51127 [GenBank] ) and variant 2 (V2; isoform b; NM_032643 [GenBank] ), have been deposited to GenBank^TM. These two cDNAs encode proteins distinct from the isoforms 3 and 4 that we have identified, and their functions have yet to be characterized. Sequence analyses of these four isoforms reveal that they differ in their first exon and in the pattern of deletion(s) in exon 6. Alternative splicing of pre-mRNA has been described for other IRF family members, including IRF-1, -3, and -7, in which each isoform may be differentially expressed depending on the cell or tissue type (18–22). However, differential isoform expression and regulation of these IRFs at the promoter levels have not been addressed yet, even though the promoters of each have been isolated and partially characterized (23–25).

The aim of the present study was to examine the regulated transcription of the human IRF-5 gene. The transcription analyses demonstrate that two distinct promoter regions differentially regulate exon 1 of V1-associated transcripts and exon 1 of V3 transcripts, where the P-V1 promoter responds to viral infection and the P-V3 promoter responds to IFN stimulation. However, the transcription pattern of the IRF-5 gene is more complex, and we have identified nine distinct alternatively spliced IRF-5 mRNAs (V1–V9). Here we describe the alternative splice patterns of the new IRF-5 isoforms, their cell type-specific expression, localization, inducibility, and function in virus-mediated type I IFN gene induction.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cell Culture and Reagents
Human HeLa, 293T, A549, and Madin-Darby bovine kidney cells were obtained from the American Type Tissue Collection (ATCC). Human fibroblasts (2fTGH) were from G. Stark (Cleveland Clinic), and primary human fibroblasts were from G. Hayward (Johns Hopkins University). These adherent cell lines were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Daudi, Namalwa, and BJAB B cell lymphomas, THP-1 acute monocytic leukemia, U937 monocytic lymphoma, and BC-3 and BCBL-1 primary effusion lymphomas (PEL) were from ATCC and cultivated in RPMI with 10% fetal bovine serum. Peripheral blood mononuclear cells (PBMC), plasmacytoid dendritic cells (PDC), monocytes (MO), natural killer (NK), and B and T cells were isolated and purified as described previously (26–28) from fresh heparinized peripheral blood obtained with informed consent from healthy volunteers. PDC (Lin^–, CD123⁺, CD11c^–, and human leukocyte antigen-DR⁺ cells) were isolated by using BDCA-4 microbeads, MO by using CD14 beads, NK by using CD16 beads, T cells by using CD3 beads, and B cells by using CD19 beads (Miltenyi Biotec). Cell purities were all greater than 95%, as determined by flow cytometry. The human studies were approved by the Institutional Review Board of the New Jersey Medical School. Newcastle Disease virus (NDV) was obtained from the ATCC (VR-699); vesicular stomatitis virus was from P. Marcus (University of Connecticut); recombinant IFN-2B was purchased from Schering Plough (Kenilworth, NJ), and IFN- was from R&D Systems, Inc. Herpes simplex virus type I (HSV-1) strain 2931 was originally obtained from Carlos Lopez then of the Sloan-Kettering Institute for Cancer Research (New York, NY); CpG (ODN 2336) was from the Coley Pharmaceutical Group (Wellesly, MA); Resiquimod (R848) was from GLSynthesis Inc. (Worcester, MA).

5'-Rapid Amplification of cDNA Ends (RACE)
The 5' ends of the IRF-5 mRNAs were determined by using RNA from unstimulated or IFN2-stimulated (500 units/ml for 16 h) BJAB cells using the 5'/3'-RACE Kit, 2nd Generation (Roche Applied Science), as described by the manufacturer. For amplification from each first exon (Ex1), the RACEV1R primer was used for Ex1V1, RACEV2R for Ex1V2, and RACEV3R for Ex1V3 (primers 1–3, Table I). RNA was reverse-transcribed using these three primers that are specific for each first exon, with subsequent RNase I degradation of the RNA template. Following the addition of a homopolymeric A-tail to the antisense strand of the 5' end of cDNAs, the tailed cDNA was amplified using a sense oligo(dT)-anchor primer and the same antisense primers specific for each first exon as named above. A second nested PCR was then performed using the oligo(dT)-anchor primer and a nested antisense primer specific for each first exon (primers 4–6, Table I). PCRs were carried out using Pfu proofreading polymerase for the generation of blunt-ended products, and an initial 2-min denaturation step at 94 °C followed by 10 cycles of successive incubations at 94 °C (15 s), 70 °C (30 s), and 72 °C (1 min) and 15 cycles of successive incubations at 94 °C (15 s), 70 °C (30 s), and 72 °C (initially 40 s with an increase of 20 s in each successive cycle), and a final 7-min elongation at 72 °C. PCR products were either electrophoresed on a 1.5% agarose gel or cloned in the pCR 4Blunt-TOPO vector using the Zero Blunt TOPO PCR cloning kit for sequencing (Invitrogen) and sequenced.

Reverse Transcription (RT)-PCR and IRF-5 Isoform Expression Analysis
Total RNA was isolated using the Qiagen RNeasy mini kit, and 1 µg was reverse-transcribed to cDNA using oligo(dT) primers. From this mixture of cDNAs, IRF-5, IRF-7, IFNA, IFNB, and -actin cDNA were amplified by PCR as described (7, 29). The IRF-5 primers (7) (primers 19 and 20, Table I) used to detect all known IRF-5 isoforms bind to a region in the carboxyl terminus spanning exons 7–9. For screening of IRF-5 isoform expression associated with a specific exon 1, sense primers that were specific for each first exon and a common antisense primer that results in amplification through exon 4 were used (Fig. 1B). IRF-5 V1 cDNA was amplified with PIRF5V1-F77 (specific to Ex1V1), V2 cDNA with PIRF5-F13 (specific to Ex1V2), or V3 cDNA with PIRF5V3-F25 (specific to Ex1V3) and PIRF5-R7 (exon 4 specific) primers (primers 21–24, Table I). Optimal PCR cycling conditions for the V1, V2, and V3 transcripts were as follows: 1 cycle at 94 °C (4 min); 37 cycles at 94 °C (1 min), 63.9 °C (1 min), and 72 °C (1 min 30 s); and 1 cycle at 72 °C (5 min). For the determination of exon 6 patterning in various cell types, primers that amplify exon 5 (primer 25) through exon 7 (primer 26) were used. Optimal PCR conditions were as follows: 1 cycle at 94 °C (4 min); 40 cycles at 94 °C (1 min), 63.9 °C (1 min), and 72 °C (1 min 30 s); and 1 cycle 72 °C (5 min).

In order to distinguish between V1- and V4-specific expression and to determine expression of new IRF-5 variants associated with their specific deletion/insertion pattern(s) in exons 5–7, exons 1–7 were amplified with each specific exon 1 sense primer (primers 21–23, Table I and Fig. 1B) and a common antisense primer (primer 26, Table I and Fig. 1B). Optimal PCR conditions were identical to the exon 1–4 PCR amplification, except 37 cycles were used. PCR products were either electrophoresed on a 1.5% agarose gel and/or cloned to the pCR®2.1-TOPO vector using the TOPO TA cloning kit (Invitrogen) and sequenced.

Expression Plasmids, SuperScript One-step RT-PCR, and Cloning of Full-length IRF-5 Isoforms
The IRF-1, IRF-3, IRF-5 (V3 and V4), and IRF-7 expression vectors were described (7, 30–32). Full-length IRF-5 V2 was subcloned from the pOTB7 plasmid (Open Biosystems) to pCMV-tag2b (Stratagene Inc.) at EcoRI and XhoI restriction sites. For the cloning of new IRF-5 isoforms, total RNA was isolated from PBMC or THP-1 cells. Superscript one-step PCR was performed using a Pfu proofreading polymerase with sense primers specific to each first exon (primers 21–23, Table I) and a common antisense primer that results in amplification through exon 9 (primer 27, Table I). Briefly, cDNA was synthesized at 45 °C (30 min) and 94 °C (2 min). PCR cycling conditions were as follows: 35 cycles 94 °C (30 s), 57 °C (30 s), and 68 °C (2 min 30 s); 1 cycle 72 °C (10 min). PCR products were electrophoresed on a 1.0% agarose gel, purified, and either cloned directly to the pCR 4Blunt-TOPO vector (Invitrogen) and sequenced or end conversion was performed, and the PCR product was cloned directly to the pSTBlue-1 vector (Novagen) and sequenced. Once full-length positive clones were identified through sequencing, each IRF-5 isoform cDNA was then subcloned to the pCMV-Tag2b mammalian FLAG-tagged expression vector (Stratagene). IRF-5 isoforms were also cloned to pEGFP-C1 vector (Clontech) at EcoRI and SalI sites. Nucleotide sequences (V5, accession number AY693665 [GenBank] ; V6, accession number AY693666 [GenBank] ; V7, accession number AY693667 [GenBank] ; V8, accession number AY693668 [GenBank] ; and V9, accession number AY693669 [GenBank] ) were deposited to GenBank^TM.

Oligonucleotide Pull-down and Immunoblot Assays
Biotinylated antisense oligonucleotides were annealed with the corresponding sense oligonucleotides (primers 28–31, Table I). Briefly, 4 µg of biotinylated DNA was incubated with 100 µg of DynaBeads M-280 streptavidin (Dynal Inc.) for 16 h in 200 µl of TEN buffer (20 mM Tris, pH 8.0, 1 mM EDTA, 0.1 M NaCl), and unbound DNA was removed by extensive washing. Cell extracts were prepared as described previously (29) from control untreated BJAB cells or cells stimulated with 500 units/ml IFN2 for 16 h. Extracts (300 µg of protein) were incubated with bound DNA (7, 24) and washed, and the bound proteins were identified by immunoblotting. Rabbit polyclonal antibodies against ISGF3p48, STAT1, and STAT2 were purchased from Santa Cruz Biotechnologies. Signals were visualized using the ECL detection reagents (Amersham Biosciences).

Treatments, Transient Transfections, Infections, and Reporter Assays
Purified PBMC, PDC, MO, NK, and B and T cells were stimulated with either IFN-2B (10,000 IU), HSV-1 (multiplicity of infection of 1), or CpG DNA (40 µg/ml) for 6 h at 37°C with 5% CO₂. BJAB and Daudi were stimulated with IFN-2 (1000 units/ml) for 16 h; Daudi and THP-1 were infected with NDV (240 plaque-forming units) for 16 h; THP-1 was stimulated with VSV (multiplicity of infection of 2) or R848 (10 µM) for 16 h. 2fTGH cells were transiently transfected with gfp-IRF-5 variant expression plasmids using the Superfect transfection reagent (Qiagen) for 30 h and then examined by fluorescent microscopy using a Nikon TE-200 and DXM12000F at x40 magnification.

Dual Luciferase Assay—293T, 2fTGH, or HeLa cells were transfected in 60-mm dishes using SuperFect. 2 µg of the reporter plasmid DNA and 0.1 µg of pRL-CMV (internal control; Promega) were used for each transfection. In co-transfection experiments a 1:1 ratio of the reporter and expression plasmid (2 µg each) were used. The final concentration of transfected DNA was kept constant in all co-transfection assays. Cells were harvested 24 h post-transfection unless additional treatments were performed. For further treatments, cells were split 12–16 h post-transfection into 6-well plates and incubated for another 8 h, after which medium was replaced either with Dulbecco's modified Eagle's medium containing recombinant IFN-2 (500 units/ml), IFN- (1000 units/ml), or NDV (50 µl/ml). Treatments/infections were incubated an additional 16 h, and cells were then harvested and lysed with 1x Passive Lysis Buffer (Promega). Protein concentrations were determined using the Bio-Rad Protein Assay according to manufacturer's instructions. Luciferase assays were carried out using the Dual Luciferase Assay kit according to the manufacturer's specifications (Promega). Experiments were repeated at least three times in duplicate.

SAP Assay—2 x 10⁵ 2fTGH or 2fTGH/IRF-5 V3 (7) stable expressing cells were transfected with a constant amount of DNA (2 µg/6-well plate) by using the Superfect transfection reagent (Qiagen). Equal amounts of the indicated SAP reporter plasmids and IRF5-expressing plasmids were co-transfected with the -galactosidase expression plasmid (50 ng). Transfected cells were split 16 h later, incubated for an additional 6 h, and either left uninfected or infected with NDV for another 16 h. The SAP was determined as described (29, 33). Each experiment was repeated at least three times; -galactosidase expression levels were used to normalize the difference in transfection efficiency.

IFN Cytopathic Effect Assay—2fTGH cells (0.5 x 10⁵ cells/well of a 6-well plate) were transiently transfected with 50 ng of each FLAG-tagged IRF-5 full-length variant expression vector. 16 h later, cells were split in duplicate to a 24-well plate, incubated for 8 h, and either left uninfected or infected with NDV for an additional 16 h. The levels of biologically active type I IFN or IFN- were determined in the cell culture supernatants by the viral cytopathic effect assay (34). VSV was used as the challenging virus, and the cytopathic effect was determined in human fibroblasts (type I IFN) or Madin-Darby bovine kidney cells (IFN-).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Structural Analysis of Isoform-specific Exons of the Human IRF-5 Gene—Although cDNA sequences for human IRF-5 isoform 1 (V1) and 2 (V2) have been deposited to GenBank^TM, the proteins encoded by the cDNAs have not been functionally characterized. We had identified previously two new mRNA isoforms (V3 and V4), and we functionally characterized the identical polypeptide encoded by the two cDNAs (2, 7–11). Here we examine the transcriptional regulation of these four isoforms (Fig. 1).

View larger version (23K): [in this window] [in a new window]   FIG. 1. A schematic of the genomic organization of the human IRF-5 gene. A, the exons are indicated by boxes or lines depending on the size of the exon: a small exon is diagrammed as a line and a larger exon by a box. The three 5'-noncoding first exons are shown as a dark gray box (Ex1V1), a line (Ex1V2), and a white box (Ex1V3). Exon 2, containing the translational start codon ATG, is indicated with a black box, as are the remaining common exons 3–9. Exon 9 contains the translational stop codon TAA. Shown in boldface are the labeled promoter regions P-V1, P-V2, P-V3, and P-Ex2. B, multiple alignments of the 5'-UTRs (exons 1) and exon 6 define four isoforms of IRF-5 transcripts resulting from alternative splicing of noncoding first exons. The four isoforms are here referred to as variant 1 (V1), variant 2 (V2), variant 3 (V3), and variant 4 (V4). Primers used to amplify the isoforms are shown in boldface (pr#) and correspond to the primer list in Table I. C, characterization of the 5'-UTR of the IRF-5 mRNA. 5'-RACE was performed using RNA isolated from either unstimulated or IFN-stimulated BJAB cells. Three 5'-RACE experiments were performed, one for each first exon of IRF-5; the two successful experiments are shown. Electrophoretic analysis of Ex1V1 5'-RACE products from unstimulated BJAB cells shows a single band, resulting from nested PCR amplification of the three original bands. The identification of a nested PCR product from IRF-5 Ex1V3 following 5'-RACE was possible in IFN2-stimulated BJAB cells. D, sequencing of 5'-RACE products demonstrated that the V1 and V3 transcription start sites match the consensus sequence for transcription initiation. The A residue in each site has been assigned as position +1 of the transcripts.

Characterization of the 5'-UTR of IRF-5 mRNA—To investigate the transcriptional mechanism(s) underlying the regulation of V1, V2, V3, and V4 transcript expression, we identified and characterized the IRF-5 gene promoter sequences. To this effect, we first determined the transcription start site(s) of the IRF-5 gene by 5'-RACE. Because BJAB cells contain a functional/inducible IRF-5 promoter, as demonstrated by the stimulation of IRF-5 gene expression after IFN treatment (8, 10), we used RNA extracted from BJAB cells for the 5'-RACE experiments. Based on the IRF-5 cDNA sequences deposited to GenBank^TM, we performed three 5'-RACE experiments, one for each of the known first exons (Fig. 1A). As shown in Fig. 1C, when unstimulated BJAB cells were subjected to 5'-RACE using an antisense primer specific for Ex1V1, three distinct products were visualized. Following nested PCR, one distinct band was detected (Fig. 1C, left panel), indicating the presence of a transcription start site for IRF-5 isoforms containing Ex1V1 in unstimulated BJAB cells. When we used the same approach to identify the 5'-ends of IRF-5 V2 and V3 transcripts, we were unable to amplify either of the two 5'-UTRs in unstimulated BJAB cells. This is likely because of the very low levels of general IRF-5 transcripts and each of these isoforms present in unstimulated BJAB cells (8, 10). Only when RNA was isolated from IFN-treated BJAB cells could the IRF-5 V3 5'-UTR be amplified (Fig. 1C, right panel). However, amplification of the IRF-5 V2 5'-UTR was not detected in either IFN-stimulated or unstimulated BJAB cells.

View larger version (17K): [in this window] [in a new window]   FIG. 2. The P-V1 promoter is regulated by virus and IRF. A, schematic representation of IRF-5 P-V1 promoter constructs. Shown are the locations of the consensus IRFE and TATA box elements, all numbered in relation to the location of the 5'-RACE-determined transcription start site of IRF-5 V1. The IRFE site mutation (P-V1-IRFE-M) is also shown (indicated by a star) as inserted upstream of a luciferase (Luc) reporter gene. B, the P-V1 IRFE site closely matches the consensus sequences. C, activation of the P-V1 promoter in HeLa cells following co-transfection with IRF family members and infection with NDV. The P-V1 and P-V1-IRFE-M constructs were co-transfected with pRL-CMV and IRF-1, -3, -5, or -7, and cells were infected with NDV. In all transfection assays the levels of reporter firefly luciferase activity were normalized to a constant level of pRL-CMV Renilla luciferase activity that served as an internal control. The results are presented as a percentage of the luciferase activity of the P-V1 construct in uninfected cells that is considered 100%. The error bars represent the S.D. of three independent experiments repeated in duplicate. D, a mutated IRFE site results in the complete ablation of IRF and virus-mediated P-V1 promoter activity.

Isolation and Cloning of Two Alternative Promoters of IRF-5—As depicted in Fig. 1A, there are four 5'-flanking regions of the IRF-5 gene that have the potential to control/regulate IRF-5 gene transcription; these regions are located as follows: (i) upstream of the Ex1V1 (P-V1); (ii) upstream of the Ex1V2 (P-V2); (iii) upstream of the Ex1V3 (P-V3); and (iv) upstream of exon 2 (P-Ex2), which contains the translation initiation ATG codon of human IRF-5. By using the TRANSFAC data base (www.gene-regulation.com), we have identified a TATA box upstream of Ex1V1 and Ex1V3 but not upstream of Ex1V2 or exon 2. This analysis, combined with the 5'-RACE data, suggests that the IRF-5 gene contains only two promoters, as defined by the presence of the TATA boxes and transcription start sites upstream of Ex1V1 and Ex1V3. However, it is also possible that the Ex1V2 and exon 2 5'-flanking regions may be functioning TATA-less promoters. It has been shown previously that mRNA transcription initiating from multiple sites, as 5'-RACE demonstrates, is usually driven by TATA-less promoters, and thus, we cannot exclude the possibility that additional transcripts initiating from these two 5'-flanking regions will be identified (36).

In order to test whether all of these 5'-flanking regions (Fig. 1A, P-V1, P-V2, P-V3, and P-Ex2) function as constitutive or inducible promoters, DNA fragments corresponding to P-V1, P-V2, P-V3, and P-Ex2 (Fig. 1A) were amplified from BJAB genomic DNA and cloned into the promoter-less luciferase reporter pGL3-Basic. The putative promoter constructs were named according to the exon they flank. These four putative promoter constructs were transiently transfected to HeLa, 293T, or 2fTGH cells and assayed for luciferase activity. Of the four constructs, only the P-V1 and P-V3 promoters displayed basal activity, suggesting that only these two 5' regions of the IRF-5 genome can function as constitutive promoters in the cell types examined (data not shown). Because the P-V1 promoter contains an IRF-E consensus site and the P-V3 contains an ISRE site, we have focused on the role of these two sites in the regulation of IRF-5 gene expression in virus-infected cells (8, 10).

Virus-mediated Activation of the IRF-5 Isoform 1 Promoter (P-V1); Regulation by IRFs—We first investigated the function of the IRF-E site, a known recognition site for members of the IRF family. Reporter constructs containing the entire wild type 0.64-kb P-V1 region or this region in which the IRF-E site was mutated were both inserted upstream of the luciferase gene in the pGL3-Basic vector (Fig. 2, A and B). The activities of these constructs were analyzed in transient transfection assays in 293T, HeLa, and 2fTGH cell lines (Fig. 2, C and D, depict data from HeLa cells, however all cell lines gave similar results). Transfection of the full-length wild type P-V1 promoter resulted in significant luciferase reporter activity that was 100 times higher than the activity of the promoterless pGL3-Basic control vector (Fig. 2C), indicating that in the cell types examined, the P-V1 promoter is constitutively active. Infection with NDV enhanced promoter activity by 3-fold. When the P-V1 promoter construct was co-transfected to HeLa cells with IRF-1, -3, -5 (V3/4), or -7 expression plasmids, only IRF-1 and IRF-5 significantly enhanced promoter activity in NDV-infected cells (Fig. 2C). Co-transfection of IRF-7 had no effect on promoter activity, and IRF-3 only slightly enhanced P-V1 reporter activity in NDV-infected cells. No activation was detected when the IRFs were co-transfected with the full-length P-V1 promoter carrying a mutated IRF-E site (Fig. 2D, P-V1-IRFE-M), indicating that the IRF-E site is essential both for the constitutive and virus-induced activation. This result suggests that in vitro the IRF-E site may be required for the constitutive activation of the IRF-5 P-V1 promoter and constitutive expression of the IRF-5 V1 and V4 isoforms.

View larger version (20K): [in this window] [in a new window]   FIG. 3. Identification of a functional ISRE site that confers IFN2 inducibility to the IRF-5 gene. A, schematic representation of IRF-5 P-V3 promoter constructs. Shown are the location of the consensus TATA box and ISRE elements numbered in relation to the location of the 5'-RACE-determined transcription start site of IRF-5 variant 3. The 1.2-kb P-V3 promoter construct containing the ISRE site mutation (P-V3-ISRE-M) was inserted into pGL-3 basic vector upstream of a luciferase (Luc) reporter gene. The mutation sites are indicated by a star. B, the P-V3 ISRE site closely matches the consensus sequence. C, activation of the P-V3 promoter in 293T or HeLa cells following treatment with IFN-2 or IFN-. The P-V3 and P-V3-ISRE-M constructs were co-transfected with pRL-CMV, and cells were treated with IFNs for 16 h. In all transfection assays, the levels of reporter firefly luciferase activity were normalized to a constant level of pRL-CMV Renilla luciferase activity that served as an internal control. The results are presented as a percentage of the luciferase activity of the P-V3 construct in untreated cells that is considered as 100%. The error bars represent the S.D. of three independent experiments repeated in duplicate. D, analyses of the binding profile of proteins to the ISRE consensus site and the IRF-5 P-V3 promoter ISRE site. Biotinylated oligodeoxynucleotides containing consensus ISRE and IRF-5-ISRE sites were incubated with extracts of IFN2-stimulated and unstimulated BJAB cells. The bound proteins were eluted from the beads and analyzed by immunoblot (IB) with antibodies against ISGF3p48, STAT-1, and STAT-2. The signals were visualized by the ECL kit.

Interferon-mediated Activation of the IRF-5 Isoform 3 Promoter (P-V3); Regulation by IFN—We have observed previously that transcription of the IRF-5 gene is stimulated by human IFN-2 (8, 10) (Fig. 1C). Because 5'-RACE from the IRF-5 Ex1V3 was only successful in BJAB cells stimulated with IFN-2 and the P-V3 promoter contains an ISRE site (Fig. 3A), suggesting its role in the IFN2-mediated induction of IRF-5 V3 transcription (Fig. 1C), we examined whether the ISRE site was active. Reporter plasmids containing the entire 1.2-kb P-V3 region or its analogue with a mutated ISRE site (P-V3-ISRE-M) were analyzed for reporter activity by transient transfection to 293T, HeLa, or 2fTGH cells (Fig. 3, B and C). Transfection of the full-length wild type putative P-V3 promoter resulted in significant luciferase reporter activity that was 100 times higher than when the promoterless vector pGL3-Basic control was transfected alone (Fig. 3C), indicating that the P-V3 promoter is constitutively active in these cells. Treatment of the P-V3-transfected cells with IFN-2 for 16 h resulted in about a 3-fold induction above the basal level activity (Fig. 3C), whereas IFN- treatment led to only a 2-fold increase. Mutation of the ISRE element (Fig. 3, A–C, P-V3-ISRE-M) abolished the response to IFN-2 without affecting the base-line activity of the P-V3 reporter (Fig. 3C). These results indicate that the 5'-flanking region of IRF-5 V3 can function as a promoter and that the ISRE site in this promoter confers the IFN-2-mediated stimulation of the IRF-5 gene.

The induction of IFN-stimulated gene (ISG) promoters by IFN- is generally mediated by binding of the ISGF3 complex, which consists of p48 (IRF-9), STAT1, and STAT2 proteins, to an ISRE consensus site (37). We have therefore used the oligonucleotide pull-down assay to examine whether the ISGF3 complex binds to the ISRE present in the IRF-5 P-V3 promoter. Because this ISRE differs slightly from the consensus ISRE sequence (Fig. 3B), we analyzed and compared the binding of the ISGF3 complex from whole cell extracts of unstimulated and IFN-2-stimulated BJAB cells to the biotin-labeled P-V3 ISRE and the consensus ISRE oligonucleotides immobilized on magnetic beads. The bound proteins separated on SDS gels were identified by immunoblotting. Results show that both the P-V3 ISRE and the consensus ISRE bound p48, STAT1, and STAT2 from the extracts of IFN-treated cells, whereas only low levels of STAT2 bound in uninfected cells (Fig. 3D). These data demonstrate that the P-V3 ISRE is functional and that the ISGF3 complex contributes to the IFN2-mediated activation of the IRF-5 P-V3 promoter.

View larger version (36K): [in this window] [in a new window]   FIG. 4. RT-PCR analysis of IRF-5 isoform expression in primary purified subpopulations of peripheral blood mononuclear cells or human immortalized hematological malignancies. A, optimization of IRF-5 Ex1-specific PCR amplification using V1/4, V2, and V3 control cDNAs, as indicated at the top; primer sets are shown at the bottom, and each was amplified from the specific exon 1 to the conserved exon 4. Lanes 0 depict the water controls for each indicated primer set. B, the expression of endogenous IRF-5 transcripts was determined in purified PBMC, PDC, MO, NK, and T and B cells from a single donor. RNA was isolated from each of the purified cell populations after stimulation with HSV-1, IFN-, CpG, or from unstimulated cells. RT-PCR was performed with exon 1 isoform-specific primers that amplify through exon 4. IFNA and IRF-7 transcripts were examined in PBMC and PDC following stimulation with CpG (right panel). The control lane (cont) indicates amplification for contaminating DNA in each of the examined primer sets. C, IRF-5 Ex1-specific isoform expression was examined in unstimulated Daudi, Daudi infected with NDV, or stimulated with IFN-, as indicated. Unstimulated BC-3 and BCBL-1 PEL cells, unstimulated THP-1, and THP-1 infected with NDV or VSV, and THP-1 stimulated with R848 were examined, along with unstimulated U937 and Namalwa, as indicated. The levels of -actin were determined as a control for RNA integrity.

Primer sets that specifically recognize exon 1 of each isoform (Fig. 1B, Ex1V1, Ex1V2, and Ex1V3) and a common region in exon 4 of IRF-5 were optimized using cloned IRF-5 V1/4, V2, and V3 cDNAs. As shown in Fig. 4A, PCR amplification using the exon 1-specific sense primers was isoform-specific. Because we have detected previously high levels of general IRF-5 transcripts in PBMC and PDC (26), we first examined the levels of constitutive and inducible exon 1-specific IRF-5 isoform expression in similar purified cell populations. The isolated cell populations were greater than 95% pure as determined by flow cytometry. Cells (PBMC, PDC, MO, NK, and T and B cells) were either left unstimulated or stimulated with IFN-, HSV-1, or CpG as described under "Materials and Methods." The effect of CpG DNA on IRF-5 gene transcription was examined because the stimulation of PDC with oligodeoxyribonucleotides containing unmethylated CpG motifs has been shown to produce high levels of IFN (38–40). Results in Fig. 4B show that the Ex1V1 transcripts were detected in all types of unstimulated cells except T cells; the highest levels were detected in unstimulated MO and B cells (top panel). Although treatment with IFN- or CpG had either no effect (PDC and B cells) or decreased (PBMC, MO, NK, and T cells) IRF-5 Ex1V1 transcript levels, stimulation with HSV-1 enhanced the levels of Ex1V1-associated transcripts in PBMC, NK, and T cells. Unstimulated and stimulated (with HSV or IFN-) B cells expressed the highest levels of IRF-5 Ex1V1-associated transcripts that were unaffected by these inducers; expression was nearly identical in all three samples even at lower cycle numbers where amplification was not saturated but was in the linear range (data not shown). In comparison, transcripts associated with Ex1V2 were either very low (MO, NK, and B cells) or nearly undetectable (PBMC, PDC, and T cells) at 37 amplification cycles (Fig. 4B, middle panel). However, transcripts were detected in all cell types examined by increasing the amount of amplified cDNA and by using a higher number of amplification cycles compared with that required for the amplification of Ex1V1 or Ex1V3 transcripts. These results suggest that IRF-5 V2 transcripts, although present, were expressed at significantly lower levels than V1 or V3 transcripts (Fig. 4B). Furthermore, even at the higher number of amplification cycles, levels of the Ex1V2 transcripts were unaffected by stimulation with HSV-1, IFN-, or CpG (data not shown). Although Ex1V3 transcripts were detected in unstimulated PDC, MO, and B cells, stimulation with IFN- significantly up-regulated the levels of Ex1V3 transcripts in all cell types examined (Fig. 4B, lower panel). The origin of the multiple amplification bands for Ex1V3 PCR in the IFN--stimulated PBMC, PDC, and MO, and all B cell samples except for the CpG-stimulated B cells is presently unclear. However, these data may suggest the presence of multiple Ex1V3-associated IRF-5 isoforms. Although stimulation with HSV-1 did not affect Ex1V3 transcripts in most of the cells, transcript levels were dramatically decreased in MO and increased in NK, T, and B cells. CpG stimulation had either no effect or slightly decreased Ex1V3 transcript levels in all cell types except NK cells, where levels were up-regulated compared with unstimulated cells. However, stimulation with CpG did induce high levels of IFNA transcripts in PBMC and PDC but not in B cells and IRF-7 transcripts, and although dramatically up-regulated in PBMC, induction was less dramatic in PDC, and levels were down-regulated by CpG in B cells (Fig. 4B, right panel).

We next examined IRF-5 isoform expression in human immortalized cancer cell lines, Daudi, BC-3 and BCBL-1, THP-1, U937, and Namalwa. For these studies, Daudi cells were either left unstimulated, infected with NDV, or stimulated with IFN-2. As shown in Fig. 4C, Ex1V1-associated transcript levels were up-regulated after infection with NDV, whereas V2 and V3 transcript levels were nearly undetectable in either the uninfected or infected samples. In comparison, IRF-5 V3 transcript levels were specifically induced after treatment with IFN-2. Constitutive isoform expression was then examined in the two PEL cell lines: where Ex1V1-associated transcripts were expressed at high levels in BCBL-1 and not BC-3 cells, and both cell lines expressed low levels of constitutive IRF-5 V3. On the other hand, THP-1 cells expressed high constitutive levels of both Ex1V1 and Ex1V3 transcripts and low levels of Ex1V2 transcripts. Upon further stimulation with either NDV or VSV, only Ex1V1-associated transcript levels were up-regulated (Fig. 4C). With the recent finding that IRF-5 is critical for TLR7/8-mediated induction of type I IFNs in THP-1 cells, we examined the inducible expression of IRF-5 isoforms by R848 (41). We found that although THP-1 cells indeed expressed the Ex1V1-, V2-, and V3-associated transcripts constitutively, albeit at distinct levels, only the IRF-5 V3 transcripts were up-regulated by R848. Furthermore, the examination of constitutive IRF-5 expression in U937 and Namalwa revealed the overwhelming presence of Ex1V1-associated transcripts, and low levels of V3 transcripts were only detected in U937 cells. Levels of human -actin transcripts are shown as a control for RNA levels.

Taken together, results from the 5'-RACE experiments, reporter assays and RT-PCR indicate that only the P-V3 promoter and its Ex1V3-associated transcripts are stimulated by IFN treatment (Fig. 3C and Fig. 4, B and C). Furthermore, these data support the finding that IRF-5 Ex1V1-associated transcript expression is regulated by the virus-mediated activation of P-V1 (Fig. 4, B and C). The differences in the levels of constitutive and inducible IRF-5 isoform expression in purified immune cell subpopulations from healthy donors and in tumor cell lines of different origin suggest that expression of the IRF-5 isoforms is cell type-specific.

Isolation and Cloning of Five New Alternatively Spliced IRF-5 Isoforms—Whereas our previous screening method for IRF-5 isoform expression yielded valuable information with regard to expression and induction of exon 1-associated transcripts, we were unable to distinguish between variants 1 and 4 because they both utilize the same exon 1 (Ex1V1). Moreover, the previous screening method did not allow for the identification of potential new alternatively spliced isoforms. In order to distinguish between V1 and V4 transcripts, we optimized primer sets that amplified from exon 1 to exon 7 that includes the region where the different deletion patterns occur (Fig. 1B). PCR fragments amplified with each of the three primer sets (primers 21–23 and 26, Table I) from THP-1, PDC, PBMC, Namalwa, U937, and/or BCBL1 were purified, cloned, and sequenced. At least 75 positive clones from each ligation reaction were isolated and sequenced. From the sequences of these clones, we identified nine new IRF-5 isoforms, most of which were alternatively spliced from Ex1V1 (Table II). Five of these new IRF-5 isoforms were re-cloned in order to obtain full-length IRF-5 cDNAs (Fig. 5A). Expression of the IRF-5 isoforms 5 and 6 was detected in PBMC and THP-1 cells, whereas expression of IRF-5 isoforms 7–9 was identified only in THP-1 cells. In addition, we were able to clone the full-length IRF-5 isoform 1 from THP-1 cells. All of the new IRF-5 isoforms share Ex1V1 except for V9 that contains Ex1V2. IRF-5 V5 contains the full genomic sequence of IRF-5 that does not show any deletions in exon 6 (Fig. 5A). IRF-5 V6 is a spliced isoform from Ex1V1 with a coding region identical to V2. IRF-5 V7 is lacking the majority of the DNA binding domain and thus may function as an endogenous dominant negative (DN) mutant of full-length IRF-5, it has the same deletion pattern as V2. IRF-5 V8 has a large single deletion that spans most of exons 6 and 7. For IRF-5 V9, although we obtained the full-length mRNA, alternative splicing led to the introduction of an early termination codon, and the protein is therefore missing its carboxyl terminus. By using both RT-PCR and sequencing methods, the levels of the IRF-5 isoforms appear to be distinctly expressed. Purified PDC expressed only IRF-5 V1, V2, V3, and V4 transcripts, with V3 and V4 being the most predominant transcripts expressed in unstimulated PDC. In contrast, the most abundant transcripts constitutively expressed in the other cell types/lines examined were IRF-5 V5, V6, and V1, respectively (data not shown).

View larger version (24K): [in this window] [in a new window]   FIG. 5. Identification of new alternatively spliced IRF-5 isoforms. A, multiple alignments of five new full-length IRF-5 isoforms amplified from human primary PBMC and/or human cancer cells. The five isoforms are here referred to as variant 5 (V5), variant 6 (V6), variant 7 (V7), variant 8 (V8), and variant 9 (V9). The exon 1 of each new isoform is shown, as are the deletion(s) in exon 5–7, and the deletion of exon 2 in V7. B, expression of each FLAG-tagged IRF-5 isoform in 2fTGH was determined by immunoblot analysis with M2 anti-FLAG antibodies (Sigma). 20 µg of whole cell lysates were run on 10% SDS-PAGE. Levels of actin are shown as a control for loading and protein integrity. C, subcellular localization of gfp-IRF-5 fusion proteins in uninfected cells.

Distinct Roles for IRF-5 Isoforms in Type I IFN Gene Induction—We have shown that cDNA encoding the IRF-5 V3 or V4 polypeptides can regulate the virus-mediated expression of distinct IFN- subtypes, which are encoded by 13 human IFNA genes (2, 7). In a transient co-transfection assay, IRF-5 V3/4 stimulated IFNA1, A2, and A14 SAP reporter activity in both uninfected and NDV-infected cells (7). We have also shown recently that the polypeptides encoded by V3 and V4 cDNAs are important for the virus-mediated induction of IFNB and other immune response genes (11). Given that the predominant sources of type I IFN in human blood are the PDC, and PDC constitutively express IRF-5 isoforms 1–4, we evaluated first their contribution to virus-mediated type I IFN gene induction. The IFNA subtype promoter reporters (IFNA1, A2, A4, and A14) and the IFNB promoter reporter plasmids were transiently co-transfected to 2fTGH cells (which do not express endogenous IRF-5 or IRF-7) with IRF-5 V1, V2, or V3/V4 expression plasmids (Fig. 5B). Transfected cells were either left uninfected or infected with NDV. As shown in Fig. 6A, whereas ectopic expression of V1 stimulated low levels of each of the examined reporter activities (IFNA2 > IFNB > A4 > A14 > A1) in NDV-infected cells, transactivation was significantly lower than that observed for V3/4 proteins. Ectopic expression of V2 stimulated IFNB > A14 > A4 reporter activities but not IFNA1 or A2 after infection with NDV. The polypeptide encoded by V3/4 cDNAs stimulated the IFNB promoter reporter most effectively, whereas the IFNA promoters were stimulated less effectively.

By having shown that IRF-5 V1, V2, V3, and V4 enabled transactivation of type I IFN promoter reporters after infection with NDV, we next measured the ability of the other IRF-5 isoforms to induce IFNA1 or IFNB reporter gene activity. Each variant was co-transfected with the indicated IFN SAP reporter and infected with NDV. Of note, whereas V2 and V6 cDNA encode for the identical polypeptide (Fig. 1B and Fig. 5, A and C), we have cloned V2 with the first exon (Ex1V2) deleted, whereas the first exon (Ex1V1) of V6 is present. The noncoding first exon in V6 may affect its translational efficiency (Fig. 5B). The activation of the IFNA1 reporter by the V3/4 polypeptide was used as a positive control; this reporter was also activated by V6 and V9 polypeptides and to a lesser degree by V5, V7, and V8 (Fig. 6B). In contrast, IFNB reporter gene activity was only stimulated by V3/4, V6, and to a lower extent by V7, after NDV infection (Fig. 6C). To evaluate further the role of IRF-5 isoforms in mediating type I IFN gene responses, we measured the synthesis of endogenous biologically active type I IFN in NDV-infected 2fTGH cells transiently expressing each isoform. Infection of 2fTGH cells with NDV resulted in only low levels of endogenous type I IFN (Fig. 7A). In contrast, ectopic expression of FLAG-tagged IRF-5 isoforms (V2, V3, V4, and V6) conferred on these cells the ability to induce type I IFN upon NDV infection, albeit low levels by V2 (Fig. 7A). Most importantly, only IRF-5 V3 and V4 induced measurable levels of synthesized IFN- assayed in Madin-Darby bovine kidney cells, indicating that the low levels of type I IFN induced by V2 and V6 are most likely IFN-. We also measured the endogenous IFNA and IFNB transcript levels in these cells by semiquantitative RT-PCR. Fig. 7B shows the relative intensity of IFNA and IFNB transcripts induced by each isoform after normalization with -actin levels. Again, measurable levels of IFNA transcripts could only be detected in NDV-infected 2fTGH cells expressing the IRF-5 V3/V4 polypeptide. Altogether, these data indicate that whereas IRF-5 V6 is an efficient inducer of IFNB in infected cells, IRF-5 V3 and V4 are the primary inducers of type I IFN (Figs. 5, 6, 7).

View larger version (18K): [in this window] [in a new window]   FIG. 6. Distinct IRF-5 isoforms regulate the expression of virus-induced type I IFNs. A, IRF-5 isoforms (V1, V2, V3, and V4) differentially transactivate the IFNA and IFNB gene promoters. 2fTGH cells were co-transfected with each IRF-5 expression plasmid and the indicated IFN SAP reporter plasmids. Relative SAP activity is shown with and without virus infection. Data are representative of three independent experiments. B and C, 2fTGH cells were co-transfected with IRF-5 expression plasmids and the IFNA1 SAP reporter (B) or IFNB SAP reporter (C). Error bars represent the S.D. of three independent experiments repeated in duplicate.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The family of IRFs was originally identified as regulators and/or mediators of type I and II IFN signaling. IRF-3, IRF-5, and IRF-7 are central mediators controlling the expression of human type I IFNs (2). Whereas IRF-3 is constitutively expressed in all cell types, expression of IRF-5 and IRF-7 has been primarily detected in lymphoid cells and can be further stimulated by type I IFNs (2). It was shown that expression of IRF-3 is sufficient to support induction of IFNB, whereas IRF-5 or IRF-7 were sufficient for stimulation of IFNA gene expression in virus-infected cells (7, 29). Consistent with these observations, PDCs express high constitutive levels of IRF-3, IRF-5, and IRF-7 and are thus uniquely preprogrammed to respond rapidly to a range of viral pathogens with high levels of IFN / production (26). IRF-5, like IRF-3 and IRF-7, requires phosphorylation of carboxyl-terminal serines for the nuclear localization and transactivation (8). Most interesting is the fact that unlike IRF-3 or IRF-7, IRF-5 is activated in a virus-specific manner (7), and not by double-stranded RNA, suggesting that IRF-5 may be activated by distinct signaling pathways leading to virus-mediated type I IFN gene expression (8). With the recent finding that IRF-5 exists as multiple isoforms that are expressed in a cell type-specific manner, and the fact that at least two alternative promoters were found to differentially regulate expression of these isoforms, it seems likely that they would have distinct functions. Data from Lin et al. (13), examining for the first time the dissimilar polypeptides encoded by V1 and V2 cDNAs, support this hypothesis. Since it had been shown recently that the IKK-related kinases TBK1 and IKK phosphorylate and activate IRF-3 and IRF-7 leading to the production of type I IFNs (43–44), they examined the in vitro phosphorylation and activation of IRF-5 V1 and V2 by TBK1 and IKK and showed that although these two isoforms were indeed phosphorylated, phosphorylation did not lead to nuclear localization or activation (13). Whereas Lin et al. (13) did not examine localization/activation of these two isoforms by NDV, or their ability to induce type I IFNs after NDV infection, they concluded that IRF-5 might simply not be an activator of IFN gene expression. Important to note, Lin et al. (13) only examined the ability of Sendai virus to induce IRF-5-mediated transactivation of IFN promoter reporters even though it has been shown previously that Sendai virus is not an activator of IRF-5 (7, 41).

View larger version (21K): [in this window] [in a new window]   FIG. 7. IRF-5 isoforms 3 and 4 are the primary transducers of endogenous type I IFNs in virus-infected cells. A, levels of biologically active endogenous type I IFNs (IFN- and/or IFN-) synthesized in 2fTGH cells that were transiently transfected with the individual IRF-5 expression plasmids and infected with NDV were determined by the cytopathic assay. Levels of IFN are expressed in units per ml. B, IFNA and IFNB transcript levels were determined in the same cells described above, by RT-PCR, and expressed quantitatively as a ratio of IFN transcript intensity divided by the intensity of amplified B-actin transcripts. C, 2fTGH/IRF-5 V3 stable overexpressing cells were co-transfected with the IFNB SAP promoter reporter and increasing amounts of the IRF-5 V7 expression plasmid (100, 200, 400, 600, and 800 ng), as indicated. Relative SAP activity is shown after infection with NDV for 16 h.

The presence of multiple IRF-5 isoforms suggests that their functions may not be redundant; instead, these isoforms may have distinct expression, regulation, and/or function, as has been shown for the IRF-3 isoforms (21, 22). Alternative splicing is a mechanism that allows for individual genes to express multiple mRNAs that encode proteins with diverse and even antagonistic function. This process generates segments of mRNA variability that can insert or remove amino acids, shift reading frame, or introduce a termination codon. It also affects gene expression by removing or inserting elements controlling translation, mRNA stability, and/or localization. To this effect, we demonstrate that the IRF-5 alternatively spliced isoforms are expressed in a cell type-specific manner and are differentially regulated by at least two alternative promoters. In addition, the subcellular localization of IRF-5 isoforms was dependent on alternative splicing events because the isoforms gave distinct localization patterns, which likely lead to alternate functions (Figs. 5D, 6, and 7). For example, analysis of the IRF-5 V7 (IRF-5 DN) function indicated that in the absence of endogenous or ectopically expressed IRF-5, i.e. 2fTGH cells (Fig. 6), V7 has a low level of transactivation ability. However, in cells that express IRF-5 V3, the IRF-5 V7 acts as a DN mutant and inhibits IRF-5 V3-mediated IFN induction. Most interestingly, because the IRF-5 V7 mRNA contains the noncoding exon 1 of V1 (Ex1V1), which is associated with P-V1, expression of this alternatively spliced isoform in virus-infected cells may provide an alternate mechanism for the virus to shut down type I IFN gene expression mediated by IRF-5 V3/4. We are currently examining this possibility. Altogether, these results imply that transcription of the IRF-5 isoforms is under multiple levels of control and that the alternatively spliced mRNAs encode for IRF-5 isoforms with potentially unique biological functions. A preliminary examination of the expression profile of mouse IRF-5 isoforms by gel electrophoresis of amplified exons 2–7 in RNA from peripheral blood lymphocytes of wild type C57Bl/6 mice or from the A20 murine B cell lymphoma cell line revealed a similar phenomenon of multiple banding patterns, indicating that murine IRF-5 may also exist as multiple isoforms (data not shown). A more detailed analysis is currently underway.

We have previously shown that the expression of IRF-5 is stimulated by IFN (8, 10), and results from the present study demonstrate that this induction is isoform-specific (Figs. 1C, 3C, and 4). As summarized in Fig. 8, only the Ex1V3-specific transcripts were up-regulated by IFN- stimulation or treatment with the TLR7/8 ligand R848. Although stimulation with either of these two inducers had no significant effect on Ex1-associated transcripts, stimulation with HSV-1 primarily up-regulated the Ex1V1-associated transcripts. In addition, NDV-infected Daudi or THP-1 cells specifically up-regulated Ex1V1 transcripts and not Ex1V2 or Ex1V3 (Fig. 4C). Infection of IRF-5 expressing cells with virus or treatment with R848 leads to the phosphorylation and translocation of IRF-5 to the nucleus (7–8, 41). All of these data correlate well with that obtained by promoter reporter assays (Fig. 8). However, in contrast to the data reported by Takaoka et al. (45), we were unable to detect up-regulation of the human IRF-5 isoforms by CpG stimulation. This may reflect differences in the amount and time of CpG ODN stimulation where Takaoka et al. (45) examined IRF-5 transcript levels after only 2 h of treatment at 1 µM, and here we examined levels after 6 h at 40 µg/ml. Although they observed an up-regulation of murine IRF-5 transcripts by CpG, they found that IRF-5 was not a mediator of CpG-induced IFN- (45). TLR7 and TLR9 signaling are both MyD88-dependent, and are potent inducers of IFN, yet the ability of either R848 or CpG to up-regulate IRF-5 transcripts levels may not be a prerequisite for its involvement in either signaling pathway since unstimulated PBMC and PDC already express IRF-5. Both R848 and CpG are capable of inducing IRF-5 nuclear translocation (41, 45). A distinct difference between these two signaling pathways could be in the mechanism of IRF-