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• W2017741366 abstract "Type-I interferons (IFNs), also called IFNs-α/β, are a family of cytokines induced by viral infection and are primarily involved in antiviral defense of the cells. IFNs-α/β were also reported to be produced constitutively at low levels in mouse and human cells. These so-called endogenous or constitutive IFNs are thought to exert important homeostatic functions in the uninfected host. By searching IFN genes that were not repressed by the leader protein of Theiler's virus, we identified three uncharacterized IFN-α genes that are constitutively expressed in uninfected mouse cells, in vitro and in vivo. Two of these genes corresponded to pseudogenes and were tentatively called IFN-α(ψ2) and IFN-α(ψ3). IFN-α(ψ2) transcripts are the most abundant IFN-α transcripts detected in several mouse organs in the absence of viral infection. The third gene codes for a new IFN-α subtype called IFN-α13, which exhibits acid-stable antiviral activity against Theiler's virus, Mengo virus, and vesicular stomatitis virus. IFN-α13 displays unusual characteristics, suggesting that it might have a particular function. Firstly, it is transcribed constitutively, independent of viral infection and of interferon regulatory factor-7 induction. Secondly, it contains two N-glycosylation sites, in contrast to other murine IFN-α subtypes that contain either one or no N-glycosylation site. In addition to the genes described here above, several other IFN-α subtype genes, including a new gene (IFN-α14), were expressed in tissues of uninfected mice. In contrast to IFN-α13, IFN-α14 was found to lack N-glycosylation and have its expression induced in response to viral infection. Type-I interferons (IFNs), also called IFNs-α/β, are a family of cytokines induced by viral infection and are primarily involved in antiviral defense of the cells. IFNs-α/β were also reported to be produced constitutively at low levels in mouse and human cells. These so-called endogenous or constitutive IFNs are thought to exert important homeostatic functions in the uninfected host. By searching IFN genes that were not repressed by the leader protein of Theiler's virus, we identified three uncharacterized IFN-α genes that are constitutively expressed in uninfected mouse cells, in vitro and in vivo. Two of these genes corresponded to pseudogenes and were tentatively called IFN-α(ψ2) and IFN-α(ψ3). IFN-α(ψ2) transcripts are the most abundant IFN-α transcripts detected in several mouse organs in the absence of viral infection. The third gene codes for a new IFN-α subtype called IFN-α13, which exhibits acid-stable antiviral activity against Theiler's virus, Mengo virus, and vesicular stomatitis virus. IFN-α13 displays unusual characteristics, suggesting that it might have a particular function. Firstly, it is transcribed constitutively, independent of viral infection and of interferon regulatory factor-7 induction. Secondly, it contains two N-glycosylation sites, in contrast to other murine IFN-α subtypes that contain either one or no N-glycosylation site. In addition to the genes described here above, several other IFN-α subtype genes, including a new gene (IFN-α14), were expressed in tissues of uninfected mice. In contrast to IFN-α13, IFN-α14 was found to lack N-glycosylation and have its expression induced in response to viral infection. Interferon-α/β (IFN) 1The abbreviations used are: IFNinterferonAREAU-rich elementsCMVhuman cytomegalovirusILinterleukinIRFinterferon regulatory factorMEFsmouse embryonic fibroblastsMHCmajor histocompatibility complexNDVNewcastle disease virusRTreverse transcriptionSV40simian virus 40TMEVTheiler's murine encephalomyelitis virusVREvirus-responsive elementVSVvesicular stomatitis virusWGSWhole Mouse Genome SequenceTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycineIFNARtype-I IFN receptor.1The abbreviations used are: IFNinterferonAREAU-rich elementsCMVhuman cytomegalovirusILinterleukinIRFinterferon regulatory factorMEFsmouse embryonic fibroblastsMHCmajor histocompatibility complexNDVNewcastle disease virusRTreverse transcriptionSV40simian virus 40TMEVTheiler's murine encephalomyelitis virusVREvirus-responsive elementVSVvesicular stomatitis virusWGSWhole Mouse Genome SequenceTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycineIFNARtype-I IFN receptor. are inducible cytokines that play a crucial role in the early innate response to viral infection. They are encoded by an intronless multigene family clustered on murine chromosome 4. To this date, 1 IFN-β and 13 IFN-α genes have been described in the mouse, including one pseudogene (1Zwarthoff E.C. Mooren A.T. Trapman J. Nucleic Acids Res. 1985; 13: 791-804Crossref PubMed Scopus (72) Google Scholar, 2Trapman J. van Heuvel M. de Jonge P. Bosveld I.J. Klaassen P. Zwarthoff E.C. J. Gen. Virol. 1988; 69: 67-75Crossref PubMed Scopus (13) Google Scholar, 3Shaw G.D. Boll W. Taira H. Mantei N. Lengyel P. Weissmann C. Nucleic Acids Res. 1983; 11: 555-573Crossref PubMed Scopus (114) Google Scholar, 4Seif I. De Maeyer-Guignard J. Gene (Amst.). 1986; 43: 111-121Crossref PubMed Scopus (22) Google Scholar, 5Navarro S. Dion M. Vodjdani G. Berlot-Picard F. Doly J. J. Gen. Virol. 1989; 70: 1381-1389Crossref PubMed Scopus (14) Google Scholar, 6Le Roscouet D. Vodjdani G. Lemaigre-Dubreuil Y. Tovey M.G. Latta M. Doly J. Mol. Cell. 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Classically, IFN-α/β have been studied for their potent antiviral activity but they also exert various immunoregulatory functions such as regulation of major histocompatibility complex (MHC) class-I antigen expression, interleukin (IL)-12 and IL-15 production, and natural killer cell activation, which shape the adaptive immune response to an invading pathogen (12Biron C.A. Immunity. 2001; 14: 661-664Abstract Full Text Full Text PDF PubMed Scopus (599) Google Scholar, 13Samuel C.E. Clin. Microbiol. Rev. 2001; 14: 778-809Crossref PubMed Scopus (2109) Google Scholar).However, accumulating experimental evidence shows that low levels of IFN-α and IFN-β are constitutively expressed in mouse and humans (14Tovey M.G. Streuli M. Gresser I. Gugenheim J. Blanchard B. Guymarho J. Vignaux F. Gigou M. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5038-5042Crossref PubMed Scopus (112) Google Scholar, 15Haller O. Arnheiter H. Gresser I. Lindenmann J. J. Exp. Med. 1979; 149: 601-612Crossref PubMed Scopus (111) Google Scholar), notably during embryogenesis (16Khan N.U. Gibson A. Foulis A.K. Immunology. 1990; 71: 230-235PubMed Google Scholar, 17Duc-Goiran P. Robert B. Navarro S. Civas A. Cerutti I. Rudant C. Maury M. Condamine H. Doly J. Exp. Cell Res. 1994; 214: 570-583Crossref PubMed Scopus (15) Google Scholar). This finding suggests that in addition to their role as inducible inflammatory mediators, interferons exert important homeostatic functions in the uninfected host. A variety of regulatory functions have been proposed for constitutive IFN-α/β including regulation of hematopoietic cell growth (18Resnitzky D. Yarden A. Zipori D. Kimchi A. Cell. 1986; 46: 31-40Abstract Full Text PDF PubMed Scopus (153) Google Scholar), MHC-I class-I antigens expression, dendritic cell maturation (19Lee C.K. Gimeno R. Levy D.E. J. Exp. Med. 1999; 190: 1451-1464Crossref PubMed Scopus (63) Google Scholar, 20Montoya M. Schiavoni G. Mattei F. Gresser I. Belardelli F. Borrow P. Tough D.F. Blood. 2002; 99: 3263-3271Crossref PubMed Scopus (380) Google Scholar), or bone formation (21Takayanagi H. Kim S. Matsuo K. Suzuki H. Suzuki T. Sato K. Yokochi T. Oda H. Nakamura K. Ida N. Wagner E.F. Taniguchi T. Nature. 2002; 416: 744-749Crossref PubMed Scopus (584) Google Scholar). Gresser and Belardelli (22Gresser I. Belardelli F. Cytokine Growth Factor Rev. 2002; 13: 111-118Crossref PubMed Scopus (82) Google Scholar) have investigated the antitumoral role of constitutive IFN-α/β. More recently, Takaoka and co-workers (23Hata N. Sato M. Takaoka A. Asagiri M. Tanaka N. Taniguchi T. Biochem. Biophys. Res. Commun. 2001; 285: 518-525Crossref PubMed Scopus (89) Google Scholar, 24Mitani Y. Takaoka A. Kim S.H. Kato Y. Yokochi T. Tanaka N. Taniguchi T. Genes Cells. 2001; 6: 631-640Crossref PubMed Scopus (85) Google Scholar, 25Takaoka A. Mitani Y. Suemori H. Sato M. Yokochi T. Noguchi S. Tanaka N. Taniguchi T. Science. 2000; 288: 2357-2360Crossref PubMed Scopus (269) Google Scholar, 26Taniguchi T. Takaoka A. Nat. Rev. Mol. Cell. Biol. 2001; 2: 378-386Crossref PubMed Scopus (398) Google Scholar) have elegantly shown that low levels of constitutive IFN-α/β are essential for effective IFN-γ and IL-6 signaling and for maintaining cells ready to switch on the antiviral response. However, the precise nature of the constitutive IFN-α in murine cells has never been determined.According to current models, induction of interferon transcription occurs as a two-step mechanism. In the mouse, IFN-α4 and IFN-β, the so-called “immediate-early interferons,” can be produced by naive cells upon viral infection. Transcription of these immediate-early interferons involves phosphorylation, dimerization, and subsequent nuclear translocation of a constitutively expressed transcription factor called interferon regulatory factor-3 (IRF-3) (27Servant M.J. Tenoever B. Lin R. J. Interferon Cytokine Res. 2002; 22: 49-58Crossref PubMed Scopus (81) Google Scholar). In contrast to IFN-α4 and IFN-β, the other IFN subtypes can only be produced in cells that have been “primed” through the binding of IFNs-α/β on their surface receptor. Priming induces the production of IRF-7, a transcription factor which upon viral infection will participate in the transcriptional activation of late IFN genes along with IRF-3 (28Marié I. Durbin J.E. Levy D.E. EMBO J. 1998; 17: 6660-6669Crossref PubMed Google Scholar).We have previously shown that in murine fibroblasts infected with Theiler's murine encephalomyelitis virus (TMEV or Theiler's virus), IFN-α was transcribed despite the inhibition of the immediate-early IFN production by the leader peptide encoded by this virus (29van Pesch V. van Eyll O. Michiels T. J. Virol. 2001; 75: 7811-7817Crossref PubMed Scopus (103) Google Scholar). This result suggested that contrary to the predictions of the model, certain IFN-α subtype genes could be transcribed independently of the autocrine or paracrine loop mediated by the immediate-early IFNs.We report here the identification and the characterization of such genes. Two of these genes correspond to pseudogenes. The third gene encodes a novel IFN-α subtype that we tentatively called IFN-α13. This gene appears to be constitutively transcribed in vitro as well as in several mouse tissues, suggesting that it might correspond to one of the constitutive IFN-α gene. Interestingly, IFN-α13 displayed an unusual glycosylation pattern, suggesting that it could play a particular role as a homeostatic cytokine produced at very low levels by various cell types. We also investigated the nature of other IFN-α subtypes expressed in vivo in the absence of viral infection. Among these subtypes, we identified another undescribed murine IFN-α subtype that we called IFN-α14.EXPERIMENTAL PROCEDURESViruses and Cell Culture—Viruses used in this study were Theiler's virus DA1 and its Lcys mutant, TM598, carrying two Cys to Arg mutations, disrupting the zinc finger of the L protein (29van Pesch V. van Eyll O. Michiels T. J. Virol. 2001; 75: 7811-7817Crossref PubMed Scopus (103) Google Scholar, 30Michiels T. Dejong V. Rodrigus R. Shaw-Jackson C. J. Virol. 1997; 71: 9549-9556Crossref PubMed Google Scholar). The KJ6 and TM659 are the corresponding wild-type and Lcys-mutant viruses carrying a capsid adapted to infect L929 cells (29van Pesch V. van Eyll O. Michiels T. J. Virol. 2001; 75: 7811-7817Crossref PubMed Scopus (103) Google Scholar, 31Jnaoui K. Michiels T. Virology. 1998; 244: 397-404Crossref PubMed Scopus (43) Google Scholar). These viruses were produced as described previously by transfection of BHK-21 cells with viral RNAs transcribed in vitro from the corresponding infectious cDNA clones, pTMDA1, pTM598, pKJ6, and pTM659 (29van Pesch V. van Eyll O. Michiels T. J. Virol. 2001; 75: 7811-7817Crossref PubMed Scopus (103) Google Scholar). An attenuated strain of Mengo virus was produced in a similar way from the pMC16 infectious clone (32Duke G.M. Palmenberg A.C. J. Virol. 1989; 63: 1822-1826Crossref PubMed Google Scholar). VSV (Strain Indiana) was a gift from Eliane Meurs (Pasteur Institute, Paris, France). Viruses were titrated on BHK-21 cells by a standard plaque assay. BHK-21, COS-7, BALB/3T3, and L929 cells were cultured as described previously (29van Pesch V. van Eyll O. Michiels T. J. Virol. 2001; 75: 7811-7817Crossref PubMed Scopus (103) Google Scholar).RNA Extraction and RT-PCR—For the detection of cytokine mRNA, total RNA was extracted from cells using either the technique of Chomczynski and Sacchi (33Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62975) Google Scholar) or the Microprep® kit (Stratagene). Because the interferon genes are intronless, RNA samples (5–10 μg) were additionally treated once or twice with 20 units of DNase I (fast protein liquid chromatography-pure, Amersham Biosciences) prior to RT-PCR as described previously (34Shaw-Jackson C. Michiels T. J. Virol. 1999; 73: 2729-2738Crossref PubMed Google Scholar). RT-PCR reactions were performed with and without reverse transcriptase (Superscript-II, Invitrogen) to rule out that PCR products were amplified from genomic DNA contamination. For cloning purposes, enzymes used for amplification were Pfu polymerase (Promega) or Expand high fidelity (Roche Applied Science). For analytical purposes, Taq polymerase (Promega) or Dynazyme (Finnzymes, Thermo-life Science) were used. Primers used for PCR are shown in Supplemental Tables A–C. Primers used for IRF-7 amplification were as described by Marié et al. (28Marié I. Durbin J.E. Levy D.E. EMBO J. 1998; 17: 6660-6669Crossref PubMed Google Scholar). Primers for total IFN-α and IFN-β were as described by Chinsangaram et al. (35Chinsangaram J. Piccone M.E. Grubman M.J. J. Virol. 1999; 73: 9891-9898Crossref PubMed Google Scholar), and primers for IFN-α4 were as described by Deonarain et al. (36Deonarain R. Alcami A. Alexiou M. Dallman M.J. Gewert D.R. Porter A.C. J. Virol. 2000; 74: 3404-3409Crossref PubMed Scopus (143) Google Scholar).IFN-α Subtype Expression Profiling—Total IFN-α was amplified by RT-PCR using consensus primers for IFN-α (TM235–236). The fragment amplified with Dynazyme (Finnzymes, Thermo-life Science) was gel-purified and subsequently cloned by TOPO-TA cloning (Invitrogen). A series of clones containing an insert of the expected size was then sequenced with the CEQ sequencing kit (Beckman) using a Beckman CEQ2000 8-capillary sequencer. The identity of the cloned IFN fragments was then determined by BLAST homology search.Determination of New Murine Gene Sequences and Multiple Alignments—Using the BLAST algorithm (37Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (59084) Google Scholar), we identified fragments from the Whole Mouse Genome Sequence (WGS) data base that aligned perfectly with our clones. The WGS fragments were further assembled using the MULTIALIN software (38Corpet F. Nucleic Acids Res. 1988; 16: 10881-10890Crossref PubMed Scopus (4255) Google Scholar) into four new IFN-α gene sequences tentatively named IFN-α(ψ2), IFN-α(ψ3), IFN-α13, and IFN-α14.Construction of Luciferase Reporter Constructs—The putative promoters of IFN-α4, IFN-α5, IFN-α13, IFN-α(ψ2), and IFN-β were cloned in the pGL3 basic reporter plasmid (Promega) to drive the transcription of the firefly luciferase gene. Fragments cloned as putative promoter sequences were the 480 nucleotides preceding the IFN gene initiation codon and comprising the putative virus-responsive elements (39Braganca J. Civas A. Biochimie (Paris). 1998; 80: 673-687Crossref PubMed Scopus (20) Google Scholar). These fragments were amplified by PCR from L929 cells genomic DNA using Pfu polymerase (Promega) and cloned in pGL3-basic using Asp-718 and HindIII restriction sites introduced in the sense and antisense PCR primers, respectively (see Supplemental Tables A–C). Constructed plasmids were called pVV21 (IFN-α4), pVV31 (IFN-α5), pVV33 (IFN-α13), pVV30 (IFN-α(ψ2)), and pVV19 (IFN-β). All of the cloned fragments were sequenced to confirm that no unexpected mutation occurred during the PCR amplification and cloning steps.Cloning of Constructs Expressing IFN-α Subtypes—The coding sequence of IFN-α4, IFN-α5, IFN-α6T, IFN-α13, and IFN-α14 were amplified by PCR from 129/Sv mouse genomic DNA and cloned into the pcDNA3 vector (Invitrogen) downstream from the immediate-early promoter of the human cytomegalovirus (CMV). Cloning was performed using BamHI and XhoI restriction sites introduced in the PCR primers. Constructs were done in such a way that all of the clones contained identical sequences upstream and downstream from the IFN-coding sequences. The sequence from the BamHI restriction site to the second codon of the IFN reading frame was 5′-GGATCCACCATGGCT-3′. In this sequence, the CCACCATGG motif and in particular the underlined nucleotides (purine at position –3 and G at position +4) match a bona fide consensus defined by Kozak for efficient translation initiation. For IFN-α13, a second construct called pcDNA3-IFN-α13or was made in which the original translation initiation context of the gene was retained. In this clone, the sequence from the BamHI site to the second codon is 5′-GGATCCTTTTTAATGGCT-3′. In that sequence, the nucleotide in position –3 is a T instead of a purine. All of the clones were sequenced to confirm that no unexpected mutation occurred in the cloned fragment during the PCR amplification and cloning steps.Metabolic Labeling and Glycosylation Analysis—COS-7 cells seeded in 6-well plates were transfected with the different IFN-α-expressing constructs using FuGENE 6 reagent (Roche Applied Science). 24 h after transfection, cells were washed twice and incubated for 24 h in 1 ml of methionine- and cysteine-deficient Dulbecco's modified Eagle's medium (ICN) containing one-tenth of the normal methionine concentration (3 mg/liter) and 70 or 80 μCi/ml 35S-labeled methionine-cysteine mixture (Redivue™ Pro-mix™, Amersham Biosciences). After a 24-h incubation at 37 °C, supernatants were collected, centrifuged at 15,000 × g to remove cell debris, and stored at –70 °C.Crude or glycosidase-treated (protein deglycosylation kit, Calbiochem) supernatants were diluted in sample buffer (62.5 mm Tris, pH 6.8, 2% β-mercaptoethanol, 3% sodium dodecyl sulfate, 10% glycerol, 0.1% bromphenol blue) and run on Tris-Tricine and SDS-11% PAGE. Gels were dried and exposed.Antiviral Assay—Interferon antiviral activity was quantified by a cytopathic effect reduction assay adapted from Meager (40Meager A. J. Immunol. Methods. 2002; 261: 21-36Crossref PubMed Scopus (110) Google Scholar), performed in 96-well plates on BALB/3T3 cells. Cells were seeded in 96-well plates at a density of 1.5 104 cells/well. After 24 h, cells were incubated for an additional 24 h with 2-fold serial dilutions of the IFN-containing supernatants to be tested. Cells were then infected with Mengo virus at a multiplicity of infection of 0.5 pfu/cell or with VSV at 100 pfu/well. Cytopathic effect was monitored by optical microscopy, and samples were compared at the time cells incubated without IFN were lysed. The well in which cytopathic effect was the closest to 50% was used to compare the relative antiviral activities of the IFN-α subtypes. IFNs to be compared were always tested in parallel. When necessary, a commercially available IFN-β preparation (PBL Biomedical Laboratories) of known titer was added to the series to calculate IFN activity.Accession Numbers—Sequences were deposited in GenBank™ or in the third party annotation database subset of GenBank™ under the following accession numbers: IFN-α13 (AY220461), IFN-α14 (AY220462), IFN-α4 (AY220463), IFN-α5 (AY220464), IFN-α6T (AY220465), IFN-α(ψ2) (BK001229), IFN-α(ψ3) (BK001230), IFN-α4 promoter cloned in pVV21 (AY226994), IFN-α5 promoter cloned in pVV31 (AY226995), IFN-α13 promoter cloned in pVV33 (AY226996), and IFN-α(ψ2) promoter cloned in pVV30 (AY226997).RESULTSIdentification of IFN-α Genes Transcribed Independently of the Immediate-early IFNs—We observed previously that in murine L929 fibroblasts infected with Theiler's virus, total IFN-α was transcribed despite the inhibition of immediate-early IFN production by the leader protein (L protein) of this virus (29van Pesch V. van Eyll O. Michiels T. J. Virol. 2001; 75: 7811-7817Crossref PubMed Scopus (103) Google Scholar). This observation suggested that, contrary to the predictions of actual models, some IFN-α subtypes could be expressed in the absence of autocrine or paracrine activation by immediate-early IFNs.To identify the nature of such IFN-α subtypes, total RNA was extracted from L929 or BALB/3T3 cells infected for 7 h with Theiler's virus expressing the wild-type L protein. Total IFN-α sequences were then amplified by RT-PCR, cloned, and sequenced. To our surprise, a majority of clones did not correspond to any of the known IFN-α sequences deposited in GenBank™ but their nucleotide sequences shared approximately 90% identity with that of IFN-α1. We identified fragments from the WGS data base that aligned perfectly with our clones. The WGS fragments were further assembled into three new IFN-α gene sequences that subsequently appeared in the supercontig of chromosome 4 (GenBank™ accession NT_039262).Two of these sequences corresponded to pseudogenes and were tentatively named IFN-α(ψ2) and IFN-α(ψ3). The third gene showed a complete open reading frame coding for a new IFN-α subtype that we tentatively named IFN-α13.Structure of the IFN-α13, IFN- α(ψ2), and IFN- α(ψ3) Genes—The IFN-α(ψ2) region lacks the AUG codon and contains multiple deletions and one insertion, disrupting the potential coding sequence (Fig. 1A). The IFN-α(ψ3) sequence contains one G to A mutation at nucleotide 50 that interrupts the open reading frame at codon 17. It contains two additional point deletions that create further frameshifts.The IFN-α13 gene codes a putative 189 amino acid-long protein containing a predicted signal sequence of 23 amino acids. The amino acid sequence aligns perfectly with that of other IFN-α subtypes. It shares 87% identity with IFN-α1 (Table I).Table IIdentity of IFN-α13 and IFN-α14 to other murine IFN-α subtypesα1α2α4α5α6α6Tα7α8α9α11αAαBα12α13α14α138780808784808384848484878810084α148780788388848788828386878984100 Open table in a new tab The putative promoter sequences of the IFN-α13, IFN- α(ψ2), and IFN- α(ψ3) genes contain potential virus-responsive element (VRE) in similar positions relative to those of classical IFN-α genes. However, in the three promoters, point mutations occurred in the modules known to regulate virus inducibility (Fig. 1B). No TATA box aligning with that of other IFN-α genes was detected in the putative promoters of the two pseudogenes, and no polyadenylation signal was found in their 3′-non-coding region. In contrast, the IFN-α13 sequence contains a TATA box and a polyadenylation signal aligning with those of other IFN genes. In contrast to all of the IFN-α genes sequenced so far, the nucleotide sequence preceding the AUG codon of IFN-α13 does not match the optimal Kozak consensus. In particular, the nucleotide in position –3 is a T, whereas it would be expected to be a purine. None of the three genes possesses the consensus AU-rich elements (ARE) sequence dictating mRNA degradation that is found in some of the other IFN-α sequences (Fig. 1C) (41Bakheet T. Frevel M. Williams B.R. Greer W. Khabar K.S. Nucleic Acids Res. 2001; 29: 246-254Crossref PubMed Scopus (341) Google Scholar).Expression of IFN-α Subtypes in L929 Cells—To determine the conditions in which IFN-α13 and the two pseudogenes are expressed, we compared the proportions of the different IFN-α subtypes transcribed in infected and uninfected L929 cells (Fig. 2).Fig. 2Relative expression of IFN-α subtypes in infected and uninfected, primed or unprimed L929 cells. Histograms show the percentage of expressed IFN-α subtypes identified by the RT-PCR-cloning-sequencing procedure. A–E, L929 cells were left uninfected (C) or were infected with either the wild-type (WT) KJ6 virus (A and D) or the l-mutant TM659 virus (Lcys) (B and E). Infections were performed on naive cells (A and B) or on cells primed for 24 h with a cell culture supernatant containing IFN-α and IFN-β (D and E). RNA was extracted from cells 7 h after infection. Total IFN-α RNA was amplified by RT-PCR with consensus primers and cloned. A number (n) of individual clones were sequenced to identify the proportion of the different IFN-α subtypes expressed. Only the six IFN subtypes shown on the graph were found to be expressed in the various conditions used in this experiment. Note that the graphs do not show the level of IFN-α expression but the relative proportion of the expressed IFN subtypes.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In L929 cells infected with the virus expressing the wild-type L protein (Fig. 2A), no “classical” IFN subtype transcript was detected from the 66 clones sequenced, in agreement with the reported inhibition of immediate-early IFN production by the leader protein. 87% of the transcripts corresponded to IFN-α13, and 13% corresponded to IFN-α(ψ3). In cells infected with the Lcys mutant virus (Fig. 2B), IFN-α13 and IFN-a(ψ3) transcripts were the most abundant (52.2 and 33.3%, respectively) but transcripts corresponding to IFN-α4, IFN-α5, or IFN-α6 were also detected (7.2, 2.9, and 2.9%, respectively).Interestingly, IFN-α13 and IFN-a(ψ3) transcripts were found in similar proportions in uninfected cells (Fig. 2C) and in cells infected with the wild-type virus (Fig. 2A), suggesting that the IFNs produced in the presence of the leader protein corresponded to constitutively expressed IFNs.We next analyzed the profile of IFN-α subtypes expressed in cells primed with IFN-α/β to evaluate the influence of IRF-7 activation on the transcription of these genes (Figs. 2 and 3). RT-PCR analysis confirmed transcriptional activation of IRF-7 in response to priming (Fig. 3). In unprimed cells, IFN-α4 and IFN-β but not IFN-α13 were repressed by the virus expressing the wild-type L protein. In primed cells, IFN expression with the exception of IFN-α13 was highly induced by infection with either the wild-type or the mutant virus, showing that the presence of IRF-7 overrides the inhibition of IFN production by the leader protein. The proportion of the different IFN-α subtypes expressed in primed cells was examined (Fig. 2, D and E). Following IRF-7 induction, a panel of classical IFN-α subtypes was expressed. IFN-α4 was the predominant subtype induced in primed cells following infection with both the wild-type and the l-mutant viruses. The proportion of IFN-α13 or of IFN pseudogenes transcripts strongly decreased after priming, showing that these genes are unresponsive to IRF-7 contrary to the other IFN-α subtype genes.Fig. 3IFN-α13 expression is independent of IRF-7 induction. L929 cells, either unprimed or primed for 24 h with IFN-α/β, were infected with virus KJ6 expressing the wild-type L protein (WT) with virus TM659 expressing the mutant L protein (Lcys) or were left uninfected (–). RT-PCR was performed 7 h after infection to detect IRF-7, total IFN-α, and specific IFN subtype mRNA. Viral RNA and β-actin mRNA were amplified as controls.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Constitutive Activity of the IFN-α13 and IFN-α(ψ2) Promoters—Promoter activity of the IFN-α13 and IFN-α(ψ2) genes was compared with that of other known type-I IFN promoters (IFN-α4, IFN-α5, and IFN-β) by using a luciferase reporter assay in BALB/3T3 fibroblasts.In uninfected cells, IFN-α13 and IFN-α(ψ2) promoters did not show any activity above base line compared with a promoterless control (data not shown), suggesting very low constitutive expression of these promoters.To evaluate virus inducibility of the promoters, luciferase activity was measured 24 h after infection with either wild-type or mutant viruses (Fig. 4A). IFN-α4 promoter activity increased at least 10-fold when the cells were infected with the Lcys virus but not with the wild-type virus, encoding a functional leader peptide. The mutant virus also induced the transcription from the IFN-α5 and the IFN-β promoters more than the wild-type virus did. However, the activation of these promoters was of lower magnitude (2–4-fold), possibly because these promoters had a higher basal transcription rate than the IFN-α4 promoter in uninfected cells. Inhibition of transcription by the leader prote" @default.
• W2017741366 created "2016-06-24" @default.
• W2017741366 creator A5002824370 @default.
• W2017741366 creator A5041933832 @default.
• W2017741366 date "2003-11-01" @default.
• W2017741366 modified "2023-10-16" @default.
• W2017741366 title "Characterization of Interferon-α 13, a Novel Constitutive Murine Interferon-α Subtype" @default.
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