Toll-like Receptor-dependent and -independent Viperin Gene Expression and Counter-regulation by PRDI-binding Factor-1/BLIMP1
2006; Elsevier BV; Volume: 281; Issue: 36 Linguagem: Inglês
10.1074/jbc.m604516200
ISSN1083-351X
AutoresMartina Severa, Eliana M. Coccia, Katherine A. Fitzgerald,
Tópico(s)NF-κB Signaling Pathways
ResumoHere we identify Viperin as a highly inducible gene in response to lipopolysaccharide (LPS), double-stranded RNA (poly(I-C)) or Sendai virus (SV). The only known function of Viperin relates to its ability to inhibit human Cytomegalovirus replication. Very little data are available on the regulation of this gene. In silico analysis of the promoter identified two interferon (IFN)-stimulated response elements (ISRE), which in other genes bind IRF3 or the IFN-stimulated gene factor-3 (ISGF3) complex. LPS and poly(I-C) induce very high levels of Viperin in wild type cells but not in cells deficient in TRIF, TBK1, IRF3, or the type I IFNα/βR. SV-induced Viperin gene expression was mediated independently of Toll-like receptor (TLR) signaling by retinoic acid-inducible gene (RIG-I) and the downstream adapter, mitochondrial anti-viral signaling (MAVS). Virus-induced Viperin expression was not attenuated in macrophages deficient in either TBK1 or IKKϵ alone. Moreover, IRF3-deficient, but not IFNα/βR deficient, macrophages still induced Viperin in response to SV. Promoter reporter studies combined with DNA immunoprecipitation assays identified the ISGF3 complex as the key regulator of Viperin gene expression. Moreover, positive regulatory domain I-binding factor 1 (PRDI-BF1, also called BLIMP1) binds the ISRE sites and competes with ISGF3 binding in a virus inducible manner to inhibit Viperin transcription. Collectively, these studies identify Viperin as a tightly regulated ISGF3 target gene, which is counter-regulated by PRDI-BF1. Here we identify Viperin as a highly inducible gene in response to lipopolysaccharide (LPS), double-stranded RNA (poly(I-C)) or Sendai virus (SV). The only known function of Viperin relates to its ability to inhibit human Cytomegalovirus replication. Very little data are available on the regulation of this gene. In silico analysis of the promoter identified two interferon (IFN)-stimulated response elements (ISRE), which in other genes bind IRF3 or the IFN-stimulated gene factor-3 (ISGF3) complex. LPS and poly(I-C) induce very high levels of Viperin in wild type cells but not in cells deficient in TRIF, TBK1, IRF3, or the type I IFNα/βR. SV-induced Viperin gene expression was mediated independently of Toll-like receptor (TLR) signaling by retinoic acid-inducible gene (RIG-I) and the downstream adapter, mitochondrial anti-viral signaling (MAVS). Virus-induced Viperin expression was not attenuated in macrophages deficient in either TBK1 or IKKϵ alone. Moreover, IRF3-deficient, but not IFNα/βR deficient, macrophages still induced Viperin in response to SV. Promoter reporter studies combined with DNA immunoprecipitation assays identified the ISGF3 complex as the key regulator of Viperin gene expression. Moreover, positive regulatory domain I-binding factor 1 (PRDI-BF1, also called BLIMP1) binds the ISRE sites and competes with ISGF3 binding in a virus inducible manner to inhibit Viperin transcription. Collectively, these studies identify Viperin as a tightly regulated ISGF3 target gene, which is counter-regulated by PRDI-BF1. The key to a successful anti-viral response relies on early detection of virus infection, followed by the rapid production of type I IFNs 2The abbreviations used are: IFN, interferon; Viperin, virus-inducible protein endoplasmic reticulum-associated interferon-inducible; LPS, lipopolysaccharide; ISGF3, IFN-stimulated gene factor-3; PRDI-BF1, positive regulatory domain I-binding factor 1; dsRNA, double-stranded RNA; TLR, Toll-like receptor; TIR, Toll-interleukin-1 receptor; CARD, caspase recruitment and activation domain; MAVS, mitochondrial anti-viral signaling; STAT, signal transducer and activator of transcription; ISRE, IFN-stimulated response elements; HCMV, human cytomegalovirus; DC, dendritic cells; Ab, antibody; IL, interleukin; SV, Sendai virus; RT, reverse transcription; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HA, hemagglutinin; TNF, tumor necrosis factor; TNFR, TNF receptor; BM, bone marrow; ko, knockout; MEF, mouse embryonic fibroblast.2The abbreviations used are: IFN, interferon; Viperin, virus-inducible protein endoplasmic reticulum-associated interferon-inducible; LPS, lipopolysaccharide; ISGF3, IFN-stimulated gene factor-3; PRDI-BF1, positive regulatory domain I-binding factor 1; dsRNA, double-stranded RNA; TLR, Toll-like receptor; TIR, Toll-interleukin-1 receptor; CARD, caspase recruitment and activation domain; MAVS, mitochondrial anti-viral signaling; STAT, signal transducer and activator of transcription; ISRE, IFN-stimulated response elements; HCMV, human cytomegalovirus; DC, dendritic cells; Ab, antibody; IL, interleukin; SV, Sendai virus; RT, reverse transcription; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HA, hemagglutinin; TNF, tumor necrosis factor; TNFR, TNF receptor; BM, bone marrow; ko, knockout; MEF, mouse embryonic fibroblast. (IFN-αs and IFN-β) and the induction of hundreds of interferon-stimulated genes (ISGs), which limit viral replication and spread. Early detection can occur via the recognition of double-stranded RNA (dsRNA), which accumulates in virus-infected cells. Double-stranded RNA is recognized by (at least) two distinct classes of pattern recognition receptors. TLR3 (1Alexopoulou L. Holt A.C. Medzhitov R. Flavell R.A. Nature. 2001; 413: 732-738Crossref PubMed Scopus (4848) Google Scholar) is an endosomally localized membrane bound receptor which upon dsRNA recognition oligomerizes and recruits a Toll-interleukin-1 receptor (TIR) domain containing adapter molecule, TRIF (also called TICAM1). TRIF is one of four adapters involved in TLR signaling, which interacts with TRAF proteins and downstream kinase complexes (reviewed in Ref. 2Vogel S.N. Fitzgerald K.A. Fenton M.J. Mol. Interv. 2003; 3: 466-477Crossref PubMed Scopus (203) Google Scholar). Recognition of the Gram-negative bacterial product lipopolysaccharide (LPS) occurs via TLR4 and MD2, which engage all four TIR adapters: MyD88, Mal (TIRAP), TRIF, and TRAM (2Vogel S.N. Fitzgerald K.A. Fenton M.J. Mol. Interv. 2003; 3: 466-477Crossref PubMed Scopus (203) Google Scholar).A second dsRNA sensing system, localized in the cytoplasm, is mediated by the RNA helicases: retinoic acid-inducible gene (Rig-I) (3Yoneyama M. Kikuchi M. Natsukawa T. Shinobu N. Imaizumi T. Miyagishi M. Taira K. Akira S. Fujita T. Nat. Immunol. 2004; 5: 730-737Crossref PubMed Scopus (3066) Google Scholar) and melanoma differentiation associated antigen-5 (Mda-5) (4Andrejeva J. Childs K.S. Young D.F. Carlos T.S. Stock N. Goodbourn S. Randall R.E. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17264-17269Crossref PubMed Scopus (807) Google Scholar). These RNA helicases bind dsRNA and activate downstream signaling via caspase recruitment and activation domains (CARD), which engage MAVS (mitochondrial anti-viral signaling, also called IPS1, CARDif, or VISA) a CARD domain-containing adapter via homotypic interactions (5Seth R.B. Sun L. Ea C.K. Chen Z.J. Cell. 2005; 122: 669-682Abstract Full Text Full Text PDF PubMed Scopus (2419) Google Scholar, 6Kawai T. Takahashi K. Sato S. Coban C. Kumar H. Kato H. Ishii K.J. Takeuchi O. Akira S. Nat. Immunol. 2005; 6: 981-988Crossref PubMed Scopus (1984) Google Scholar, 7Meylan E. Curran J. Hofmann K. Moradpour D. Binder M. Bartenschlager R. Tschopp J. Nature. 2005; 437: 1167-1172Crossref PubMed Scopus (1931) Google Scholar, 8Xu L.G. Wang Y.Y. Han K.J. Li L.Y. Zhai Z. Shu H.B. Mol. Cell. 2005; 19: 727-740Abstract Full Text Full Text PDF PubMed Scopus (1488) Google Scholar). Like TRIF, MAVS recruits TRAF proteins and downstream kinases to activate transcription (for a complete review, see Ref. 9Jefferies C.A. Fitzgerald K.A. Trends Mol. Med. 2005; 11: 403-411Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Signaling by TLR3, TLR4, and these helicases leads to the activation of ATF-2-c-Jun, IRF3/IRF7, and NF-κB. In resting cells, NF-κB is bound to the inhibitor protein IκBα, which, after virus infection, becomes phosphorylated by the classical IκB kinase complex (IKKα/β/γ), "marking" IκBα for ubiquitination and subsequent degradation by the proteasome. Similarly, IRF3 becomes phosphorylated, but in this case IKKϵ (IKKi) and/or TBK1 (NAK,T2K) (10Fitzgerald K.A. McWhirter S.M. Faia K.L. Rowe D.C. Latz E. Golenbock D.T. Coyle A.J. Liao S.M. Maniatis T. Nat. Immunol. 2003; 4: 491-496Crossref PubMed Scopus (2037) Google Scholar, 11Sharma S. tenOever B.R. Grandvaux N. Zhou G.P. Lin R. Hiscott J. Science. 2003; 300: 1148-1151Crossref PubMed Scopus (1342) Google Scholar) mediate this phosphorylation. The ATF-2/c-Jun complex is also rapidly activated by the c-Jun amino-terminal kinase (JNK). ATF-2-c-Jun, IRF3/IRF7, and NF-κB form a multiprotein complex, the "enhanceosome," on the IFN-β promoter to regulate IFN-B gene expression.IFN-β signals in an autocrine/paracrine manner via the heterodimeric receptor complex (IFNα/βR) 1 and IFNα/βR2 (12Mogensen K.E. Lewerenz M. Reboul J. Lutfalla G. Uze G. J. Interferon Cytokine Res. 1999; 19: 1069-1098Crossref PubMed Scopus (223) Google Scholar). Each of these receptor subunits engages a member of the Janus-activated kinase (JAK) family (13Darnell Jr., J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Crossref PubMed Scopus (4948) Google Scholar, 14Ihle J.N. Semin. Immunol. 1995; 7: 247-254Crossref PubMed Scopus (56) Google Scholar). IFNα/βR1 is constitutively associated with tyrosine kinase 2 (TYK2), whereas IFNα/βR2 associates with JAK1 (13Darnell Jr., J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Crossref PubMed Scopus (4948) Google Scholar, 14Ihle J.N. Semin. Immunol. 1995; 7: 247-254Crossref PubMed Scopus (56) Google Scholar, 15Chen J. Baig E. Fish E.N. J. Interferon Cytokine Res. 2004; 24: 687-698Crossref PubMed Scopus (72) Google Scholar, 16Platanias L.C. Pharmacol. Ther. 2003; 98: 129-142Crossref PubMed Scopus (131) Google Scholar). Ligand-induced rearrangement and dimerization of the receptor subunits causes the receptor-associated JAKs to become activated by autophosphorylation resulting in the tyrosine phosphorylation and heterodimerization of signal transducer and activator of transcription (STAT) 1 and STAT2, which associate in the cytoplasm with IRF9 (also called p48). The trimeric STAT1/2/IRF9 complex, also called the IFN-stimulated gene factor 3 (ISGF3) complex, translocates to the nucleus and initiates transcription by binding IFN-stimulated response elements (ISRE) in the promoters of multiple ISGs. Although some anti-viral genes can be induced directly by IRF3, the main workhorses of the anti-viral response are the diverse ISGs, many of which are of unknown function (17Doly J. Civas A. Navarro S. Uze G. Cell Mol. Life Sci. 1998; 54: 1109-1121Crossref PubMed Scopus (67) Google Scholar).Viperin (virus inhibitory protein, endoplasmic reticulum-associated, interferon-γ-inducible) is an anti-viral gene whose function is still unclear. Viperin was initially identified as a human cytomegalovirus (HCMV)- and IFNγ-inducible protein in fibroblasts (18Chin K.C. Cresswell P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 15125-15130Crossref PubMed Scopus (315) Google Scholar) and was shown to inhibit productive HCMV infection by down-regulating several HCMV structural proteins critical for viral assembly and maturation. Viperin (known as cig5 in humans, vig1 in mouse, and also RSAD2 in both human and mouse) has also been shown to be induced after infection with vesicular stomatitis virus, Yellow Fever virus, human polyomavirus JC, hepatitis C virus (19Boudinot P. Riffault S. Salhi S. Carrat C. Sedlik C. Mahmoudi N. Charley B. Benmansour A. J. Gen. Virol. 2000; 81: 2675-2682Crossref PubMed Scopus (60) Google Scholar, 20Khaiboullina S.F. Rizvanov A.A. Holbrook M.R. St Jeor S. Virology. 2005; 342: 167-176Crossref PubMed Scopus (40) Google Scholar, 21Verma S. Ziegler K. Ananthula P. Co J.K. Frisque R.J. Yanagihara R. Nerurkar V.R. Virology. 2006; 345: 457-467Crossref PubMed Scopus (41) Google Scholar, 22Helbig K.J. Lau D.T. Semendric L. Harley H.A. Beard M.R. Hepatology. 2005; 42: 702-710Crossref PubMed Scopus (199) Google Scholar), or after the lipofection-mediated delivery of bacterial or viral DNA into the cytoplasm (23Ishii K.J. Coban C. Kato H. Takahashi K. Torii Y. Takeshita F. Ludwig H. Sutter G. Suzuki K. Hemmi H. Sato S. Yamamoto M. Uematsu S. Kawai T. Takeuchi O. Akira S. Nat. Immunol. 2006; 7: 40-48Crossref PubMed Scopus (637) Google Scholar). The rapid and robust induction of the Viperin gene by a range of different viruses and microbial products such as LPS and DNA suggests that it is an important component of innate immunity to diverse pathogens.In this study, we have examined the molecular mechanisms regulating Viperin gene expression. We demonstrate that Viperin is induced via IRF3-dependent as well as IRF3-independent pathways all of which rely on type I IFN signaling. The key factor regulating Viperin promoter activity appears to be the ISGF3 complex and not IRF3 itself. Moreover, positive regulatory domain I-binding factor 1 (PRDI-BF1, also called BLIMP1) blocks Viperin expression. Collectively these studies identify Viperin as a highy inducible ISGF3 target gene and define the molecular mechanisms controlling its expression.EXPERIMENTAL PROCEDURESGeneration of Human Dendritic Cells (DC) and Mouse Macrophages—DC were generated and their maturation state examined as described (24Coccia E.M. Severa M. Giacomini E. Monneron D. Remoli M.E. Julkunen I. Cella M. Lande R. Uze G. Eur. J. Immunol. 2004; 34: 796-805Crossref PubMed Scopus (418) Google Scholar, 25Lande R. Giacomini E. Grassi T. Remoli M.E. Iona E. Miettinen M. Julkunen I. Coccia E.M. J. Immunol. 2003; 170: 1174-1182Crossref PubMed Scopus (125) Google Scholar). Bone marrow-derived macrophages were generated as described (26Fitzgerald K.A. Rowe D.C. Barnes B.J. Caffrey D.R. Visintin A. Latz E. Monks B. Pitha P.M. Golenbock D.T. J. Exp. Med. 2003; 198: 1043-1055Crossref PubMed Scopus (919) Google Scholar) and their differentiation state confirmed by staining for F4/80 (Caltag, Burlingame, CA) and CD11b (Pharmingen) using pure Abs or as direct conjugates.Mice, Cell Lines, Viruses, and Reagents—MyD88-/-, TRIF-/-, TRAM-/-, and Mal-/- mice were from S. Akira (Osaka University, Osaka, Japan). IRF3-/- mice were from T. Taniguchi (University of Tokyo, Tokyo, Japan). IFNα/βR-/- mice were from J. Sprent (Scripps Research Institute, San Diego, CA). TBK1-/-TNFRI-/- mice were from T. Mak and W.-C. Yeh, (University of Toronto, Canada) and IKKϵ-/- mice were from Millennium Pharmaceuticals (Cambridge, MA). HEK293-TLR2, TLR3 and TLR4/MD2 cells were generated as described (26Fitzgerald K.A. Rowe D.C. Barnes B.J. Caffrey D.R. Visintin A. Latz E. Monks B. Pitha P.M. Golenbock D.T. J. Exp. Med. 2003; 198: 1043-1055Crossref PubMed Scopus (919) Google Scholar). Mouse embryonic fibroblasts from BLIMP1fl/fl mice and MEFs targeted by Cre-recombinase (BLIMP1-/-) were prepared by J. Ye and T. Manaitis (Harvard University, Cambridge, MA) from mice provided by K. Calame (Columbia University). Sendai virus, Cantrell strain was from Charles River Laboratories (Boston, MA). Polyinosinic-polycytidylic acid (poly(I-C)) was from Amersham Biosciences. Re-extracted LPS was generated as described (27Hirschfeld M. Ma Y. Weis J.H. Vogel S.N. Weis J.J. J. Immunol. 2000; 165: 618-622Crossref PubMed Scopus (960) Google Scholar). IFN-β was from Biogen Inc. (Cambridge, MA). TNF-α and IL1-β were from R&D Systems (Minneapolis, MN), and Pam2cysk4 was from EMC Microcollections (Tuebingen, Germany). Sheep antiserum raised against human leukocyte IFN (28Mogensen K.E. Pyhala L. Cantell K. Acta Pathol. Microbiol. Scand. B. 1975; 83: 443-450PubMed Google Scholar) was used at a dilution 1:100.Plasmid Constructs—The human and mouse Viperin promoters were cloned from human THP-1 and mouse ES cell (129SV) genomic DNA, respectively, and inserted into pGL3-basic. pEF-BOS-RIG-I, Mda-5, and the IFN-β luciferase reporter (p125Luc) construct were from T. Fujita (Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan) (3Yoneyama M. Kikuchi M. Natsukawa T. Shinobu N. Imaizumi T. Miyagishi M. Taira K. Akira S. Fujita T. Nat. Immunol. 2004; 5: 730-737Crossref PubMed Scopus (3066) Google Scholar). pEF-Bos MAVS was cloned from human 293T total cDNA. pEF-BOS-Lgp2 and the NF-κB luciferase reporter were as described previously (29Rothenfusser S. Goutagny N. Diperna G. Gong M. Monks B.G. Schoenemeyer A. Yamamoto M. Akira S. Fitzgerald K.A. J. Immunol. 2005; 175: 5260-5268Crossref PubMed Scopus (481) Google Scholar). The ISG54-ISRE was purchased from Stratagene (La Jolla, CA). pCDNA3-FLAG-PRDI-BF1, pCDNA3-FLAG-PRD1-BF1Δ398-789, and pCMV-FLAG-p65 were from T. Maniatis (Harvard University). pRc-CMV-FLAG-STAT1 and pRc-CMV-FLAG-STAT1-Y701 were from G. Stark (Cleveland Clinic, Cleveland, OH). pCDNA3-HA-IRF9 and pCMV-FLAG-IRF3 were from Paula M. Pitha (John Hopkins University, Baltimore, MD); and pRK-HA-IRF1 was from H-B Shu (National Jewish Medical and Research Center, Denver, CO). pCDNA3-FLAG-IKKϵ and pCDNA3-FLAG-IKKβ have been described (30Peters R.T. Liao S.M. Maniatis T. Mol. Cell. 2000; 5: 513-522Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). pCDNA3-FLAG-TBK1 was from M. Nakanishi (Nagoya, Japan).Reporter Assays—For reporter assays, HEK293 (seeded 105 cells/ml in 96-well plates) were transfected with 40 ng of the indicated luciferase reporter gene together with 40 ng of thymidine kinase Renilla luciferase reporter gene (Promega, Madison, WI) and the indicated amount of expression plasmids using Genejuice (Novagen, San Diego, CA). Where indicated SV (300 hemagglutination units/ml) or IFN-β (200 pm) were added for 16 h, and luciferase activity was measured as described previously (10Fitzgerald K.A. McWhirter S.M. Faia K.L. Rowe D.C. Latz E. Golenbock D.T. Coyle A.J. Liao S.M. Maniatis T. Nat. Immunol. 2003; 4: 491-496Crossref PubMed Scopus (2037) Google Scholar). In all experiments, data were normalized for transfection efficiency with Rotylenchulus reniformis luciferase.Quantitative Real-time PCR—RNA from human monocyte-derived DC and murine bone marrow macrophages was extracted with RNeasy kit (Qiagen Inc., Valencia, CA) according to the manufacturer's instructions. cDNA was synthesized, and quantitative RT-PCR analysis was performed on a DNA engine Opticon 2 cycler (MJ Research, Watertown MA) using the SuperScript III two-step qRT-PCR kit with SYBR Green (Invitrogen). The specificity of amplification was assessed for each sample by melting curve analysis. Relative quantification was performed using standard curve analysis. The human quantifications data are presented as a ratio to the GAPDH level and the murine ones were normalized with β-actin. The standard errors (95% confidence limits) were calculated using the Student's t test. All gene expression data are presented as a ratio of gene copy number per 100 copies of GAPDH or β-actin (as indicated) ± S.D.The following primer pairs have been used. For human: GAPDH, 5-ACAGTCCATGCCATCACTGCC-3 (forward) and 5-GCCTGCTTCACCACCTTCTTG-3 (reverse); cig5, 5-CTTTGTGCTGCCCCTTGAGGAA-3 (forward) and 5-CTCTCCCGGATCAGGCTTCCA-3 (reverse). For mouse: β-actin, 5-TTGAACATGGCATTGTTACCAA-3 (forward) and 5-TGGCATAGAGGTCTTTACGGA-3 (reverse); vig1, 5-AACCCCCGTGAGTGTCAACTA (forward) and 5-AACCAGCCTGTTTGAGCAGAA (reverse).Cell Extracts—293T were transiently transfected with 4 μg (or the indicated amounts) of the HA-tagged IRF9, FLAG-tagged STAT1 or FLAG-tagged PRDI-BF1 expression vectors using Genejuice (Novagen) in 10-cm plates. Where indicated, the cells were treated for 16 h with SV. Cell pellets (1 × 107 cells) were resuspended in 400 μl of Buffer I (10 mm of Tris, pH 7.8, 5 mm MgCl2, 10 mm KCl, 1 mm EGTA, pH 7, 0.3 m sucrose, 1 mm dithiothreitol, 1 mm sodium vanadate, 1 mm phenylmethylsulfonyl fluoride, and mixture protease inhibitors) and then incubated on ice for 15 min. Nuclei were sedimented by centrifuging at 1,500 × g for 5 min. The nuclear pellets were then resuspended in 80 μl of Buffer II (20 mm of Tris, pH 7.8, 5 mm MgCl2, 300 mm KCl, 0.2 mm EGTA, pH 7, and 25% glycerol, 1 mm dithiothreitol, 1 mm sodium vanadate, 1 mm phenylmethylsulfonyl fluoride, and mixture protease inhibitors). The suspensions were clarified by centrifuging at 15,000 × g for 15 min. The nuclear extracts were stored at -20 °C.DNA Affinity Purification Assay—Biotinylated oligonucleotide ISRE (5′-GTTTCACATGTGGAAAAATCGAAACTCTAAC-3′) was annealed with the corresponding antisense oligonucleotide in 1× STE buffer, containing 10 mm Tris-HCl, pH 8, 50 mm NaCl, and 2 mm EDTA. Biotinylated DNA oligonucleotides were mixed with 500 μg of nuclear extracts in 500 μl of binding buffer containing 20 mm Tris-HCl, pH 7.5, 75 mm KCl, 1 mm dithiothreitol, and 5 mg/ml bovine serum albumin in presence of 13% glycerol and 20 μg of poly(dI-dC) and incubated for 25 min at room temperature. Then streptavidin magnetic beads (Promega), washed three times with 400 μl of 1× binding buffer, were added to the reaction mixture and incubated for 30 min at 4 °C and for 10 min at room temperature with mixing by rotation. The beads were collected with a magnet and washed three times with 500 μl of binding buffer. The bound proteins were eluted from the beads by boiling in sample buffer and were resolved on 7.5% SDS-PAGE followed by immunoblotting with the indicated specific Abs: anti-HA-horseradish peroxidase (Roche Applied Science), anti-FLAG-horseradish peroxidase (Sigma), anti-STAT1 (Santa Cruz Biotechnology, Santa Cruz, CA), anti-PRDI-BF1 (Novus Biologicals Inc., Littleton, CO). The level of the nuclear lysates used in the assay was verified by immunoblotting with the anti-USF2 (Santa Cruz Biotechnology).Western Blot Analysis—Nuclear cell extracts were separated by 7.5% SDS-PAGE gel and blotted onto nitrocellulose membranes. Blots were incubated with rabbit polyclonal or mouse monoclonal Abs, as requested, against the specified Abs using an ECL system (Amersham Biosciences).RESULTSWe first examined cig-5/Viperin gene expression in human monocyte-derived DC in response to various stimuli. DC were treated for 3 h with the synthetic mycobacterial lipoprotein, Pam2Cysk4, poly(dI-dC), and LPS, ligands for TLR2, TLR3, and TLR4, respectively. As seen in Fig. 1A, quantitative real-time RT-PCR measurements demonstrate that both poly(dI-dC) and LPS induced a robust induction of Viperin. In contrast Pam2Csk4 or the mixture of pro-inflammatory cytokines TNF-α and IL1-β did not induce this gene. Sendai virus (SV) also induced a strong expression of Viperin in DC. This differential inducibility of Viperin prompted us to examine its promoter region for transcription factor-binding sites. Using the transcription factor-binding site prediction program MatInspector (31Cartharius K. Frech K. Grote K. Klocke B. Haltmeier M. Klingenhoff A. Frisch M. Bayerlein M. Werner T. Bioinformatics. 2005; 21: 2933-2942Crossref PubMed Scopus (1627) Google Scholar), a region spanning 1,000 bp upstream of the transcription starting site and 500 bp downstream was analyzed. Several transcription factor-binding sites for IFN-regulatory factors (IRF1, IRF2, IRF3, and IRF4), as well as two ISREs, which can bind IRFs and the IFN-stimulated gene factor-3 (ISGF3) complex, were identified within 500 bp of the transcription starting site. An NF-κB-binding site in close proximity to the transcription starting site was also detected (Fig. 1B). Interestingly, we also found binding sites for PRDI-BF1 (see below).We next examined the activation of both human and mouse Viperin promoters generated using luciferase-based reporter constructs. HEK293 cells expressing TLR3 induced the human Viperin promoter following treatment with poly(dI-dC). HEK293 cells expressing TLR4, MD2, and the TLR4-specific adapter TRAM also induced this reporter in response to LPS (of note, we did not observe either Viperin or IFNB reporter gene activation without TRAM coexpression). Consistent with our data in human DC, neither the TLR2 ligand nor IL-1β/TNF-α treatment induced the reporters (Fig. 1C). Importantly, these latter treatments strongly induced an NF-κB-driven reporter gene (data not shown). SV infection also resulted in a strong activation of the reporters in the parental cells in the absence of exogenous TLR expression. Similar results were obtained using the murine promoter (data not shown).We next examined Viperin induction in bone marrow (BM) macrophages derived from MyD88-, Mal-, TRIF-, and TRAM-deficient mice following LPS treatment to probe the adapter molecule requirements for this response. Macrophages from these mice were treated for 3 h with LPS and the level of Viperin mRNA was examined by quantitative RT-PCR (Fig. 2A). Viperin gene expression was completely abrogated in LPS-treated macrophages from TRIF- and TRAM-deficient mice. In contrast, the response in both Mal- or MyD88-deficient mice was not impaired, in fact these two strains showed an even stronger induction compared with C57BL/6 wild type mice highlighting the possible involvement of a MyD88/Mal-dependent repressor of Viperin gene expression (see "Discussion"). These findings support a key role for the TRIF/TRAM pathway in the induction of Viperin following LPS treatment.FIGURE 2TIR and CARD domain-containing adapters are differentially required for Viperin gene transcription. A, BM macrophages from C57BL/6, MyD88-/-, Mal-/-, TRIF-/-, and TRAM-/- mice were stimulated with LPS (3 h). mRNA expression of the Viperin gene was measured by quantitative RT-PCR. B, HEK293 cells were transfected with the Viperin reporter gene and then cotransfected with full-length RIG-I, MDA-5, and Lgp2. Cells were infected with SV for 16 h, and luciferase reporter activity was measured. In all cases, data are expressed as fold induction relative to the reporter-only control and are the mean ± S.D. C, Viperin reporter was transfected in Huh7 and Huh7.5 cell lines with or without 40 ng of full-length RIG-I. Luciferase reporter activity was measured in nonstimulated (ns) or SV-infected cells. D, 40 ng of an expression vector for MAVS was transfected in HEK293 together with the Viperin reporter and luciferase reporter activity measured by assay in SV-infected or untreated cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We have previously shown that the RNA helicase RIG-I is important in sensing of SV (29Rothenfusser S. Goutagny N. Diperna G. Gong M. Monks B.G. Schoenemeyer A. Yamamoto M. Akira S. Fitzgerald K.A. J. Immunol. 2005; 175: 5260-5268Crossref PubMed Scopus (481) Google Scholar). To determine whether RIG-I contributes to SV-induced Viperin gene expression, HEK293 cells were transfected with a reporter gene encoding the human Viperin promoter and cotransfected with an expression vector for RIG-I or Mda-5 (Fig. 2B). Transfection of the full-length RIG-I alone activated the Viperin reporter and this effect was dramatically enhanced upon SV infection. Mda-5 overexpression slightly induced Viperin promoter; however, no enhancement was observed with SV (29Rothenfusser S. Goutagny N. Diperna G. Gong M. Monks B.G. Schoenemeyer A. Yamamoto M. Akira S. Fitzgerald K.A. J. Immunol. 2005; 175: 5260-5268Crossref PubMed Scopus (481) Google Scholar). Lgp2, a protein structurally related to RIG-I (32Cui Y. Li M. Walton K.D. Sun K. Hanover J.A. Furth P.A. Hennighausen L. Genomics. 2001; 78: 129-134Crossref PubMed Scopus (49) Google Scholar), which seems to be a post-induction feedback inhibitor of RIG-I signaling (29Rothenfusser S. Goutagny N. Diperna G. Gong M. Monks B.G. Schoenemeyer A. Yamamoto M. Akira S. Fitzgerald K.A. J. Immunol. 2005; 175: 5260-5268Crossref PubMed Scopus (481) Google Scholar, 33Yoneyama M. Kikuchi M. Matsumoto K. Imaizumi T. Miyagishi M. Taira K. Foy E. Loo Y.M. Gale Jr., M. Akira S. Yonehara S. Kato A. Fujita T. J. Immunol. 2005; 175: 2851-2858Crossref PubMed Scopus (1262) Google Scholar), decreased SV-induced Viperin reporter activity (Fig. 2B). To clearly demonstrate a role for RIG-I we used the human Huh7.5 hepatoma cell line that has a single point mutation within the CARD domain of RIG-I, which impairs its signaling. Consistent with the previous data, the Viperin promoter was induced in the parental Huh7 cell line upon SV stimulation. However, we observed an induction in Huh7.5 upon SV infection only after their complementation with wild type RIG-I (Fig. 2C). Moreover, overexpression of MAVS in HEK293 cells potently activated the Viperin promoter, and this response was enhanced by SV infection (Fig. 2D). SV-induced Viperin reporter activity was blocked by the Hepatitis C virus NS3/4A protease (data now shown), in fact it has been recently shown that NS3/4A abrogates signaling through RIG-I by cleaving MAVS from the mitochondrial membrane (34Li K. Foy E. Ferreon J.C. Nakamura M. Ferreon A.C. Ikeda M. Ray S.C. Gale Jr., M. Lemon S.M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 2992-2997Crossref PubMed Scopus (912) Google Scholar). Collectively these data demonstrate that RIG-I and MAVS regulate Viperin gene expression in the SV pathway.We next examined Viperin reporter gene activity upon overexpression of the IKK kinases, which act downstream of TRIF and MAVS to regulate either IRF3 or NF-κB. Overexpression of TBK1 and IKKϵ but not the related kinase IKKβ induced the reporter gene (Fig. 3A). TBK1, but neither IKKϵ nor IKKβ, greatly enhanced SV-induced reporter activity suggesting that TBK1 may be the main kinase regulating the Viperin response. The lack of induction by IKKβ suggests that the NF-κB site may not be as important. We also compared the induction of Viperin in macrophages from C57BL/6 wild type, IKKϵ-deficient, and TBK1
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