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Transcription Stimulation of the Fas-encoding Gene by Nuclear Factor for Interleukin-6 Expression upon Influenza Virus Infection

1995; Elsevier BV; Volume: 270; Issue: 30 Linguagem: Inglês

10.1074/jbc.270.30.18007

1083-351X

Naoya Wada, Miho Matsumura, Yoshiki Ohba, Nobuyuki Kobayashi, Takenori Takizawa, Yoshinobu Nakanishi,

Cell death mechanisms and regulation

We previously found that the level of Fas, a cell surface receptor for an apoptosis signal, increases at the mRNA level in influenza virus-infected HeLa cells prior to their death by apoptosis. Here we investigated the mechanism of activation of the Fas-encoding gene expression upon influenza virus infection. Nucleotide sequences for the binding of nuclear factor for interleukin-6 expression (NF-IL6), also known as CCAAT/enhancer-binding protein β, were repeated 8 times in the 5′-end region of the human FAS gene, spanning from −1360 to +320. This region directed the expression of a downstream marker gene when introduced into HeLa cells and the activity of the FAS gene promoter was stimulated about 2-fold upon influenza virus infection. Gene expression driven by the FAS promoter was activated when human NF-IL6 was overproduced in a DNA co-transfection study. Moreover, the DNA-binding activity of NF-IL6 increased after infection with the virus, whereas the amount of NF-IL6 seemed unchanged. The results suggest that NF-IL6 is activated upon influenza virus infection through post-translational modification and that the modified factor stimulates the transcription of the human FAS gene. We previously found that the level of Fas, a cell surface receptor for an apoptosis signal, increases at the mRNA level in influenza virus-infected HeLa cells prior to their death by apoptosis. Here we investigated the mechanism of activation of the Fas-encoding gene expression upon influenza virus infection. Nucleotide sequences for the binding of nuclear factor for interleukin-6 expression (NF-IL6), also known as CCAAT/enhancer-binding protein β, were repeated 8 times in the 5′-end region of the human FAS gene, spanning from −1360 to +320. This region directed the expression of a downstream marker gene when introduced into HeLa cells and the activity of the FAS gene promoter was stimulated about 2-fold upon influenza virus infection. Gene expression driven by the FAS promoter was activated when human NF-IL6 was overproduced in a DNA co-transfection study. Moreover, the DNA-binding activity of NF-IL6 increased after infection with the virus, whereas the amount of NF-IL6 seemed unchanged. The results suggest that NF-IL6 is activated upon influenza virus infection through post-translational modification and that the modified factor stimulates the transcription of the human FAS gene. Viral infection brings about a variety of effects in host cells, including changes in growth and morphology, transformation, and lytic death, all of which presumably accompany the altered expression of cellular genes. Understanding the molecular basis for virus-host interaction should lead to the development of effective therapeutics against virus-induced diseases such as cancer and AIDS. Although it has been accepted for many years that most viruses cause host cell death, the precise mechanism for this phenomenon remains unknown. Several reports have shown that host cells infected with bovine herpesvirus (1Griebel P.J. Ohmann H.B. Lawman M.J.P. Babiuk L.A. J. Gen. Virol. 1990; 71: 369-377Google Scholar), chicken anemia virus(2Jeurissen S.H.M. Wagenaar F. Pol J.M.A. van der Eb A.J. Noteborn M.H.M. J. Virol. 1992; 66: 7383-7388Google Scholar), insect baculovirus(3Lee J.-C. Chen H.-H. Wei H.-L. Chao Y.-C. J. Virol. 1993; 67: 6989-6994Google Scholar), lymphocytic choriomeningitis virus(4Razvi E.S. Welsh R.M. J. Virol. 1993; 67: 5754-5765Google Scholar), Molony murine leukemia virus(5Saha K. Yuen P.H. Wong P.K.Y. J. Virol. 1994; 68: 2735-2740Google Scholar), human immunodeficiency virus (HIV)1,2 1The abbreviations used are: HIVhuman immunodeficiency virusNF-IL6nuclear factor for interleukin-6 expressionCATchloramphenicol acetyltransferaseC/EBPCCAAT/enhancer-binding protein. 1The abbreviations used are: HIVhuman immunodeficiency virusNF-IL6nuclear factor for interleukin-6 expressionCATchloramphenicol acetyltransferaseC/EBPCCAAT/enhancer-binding protein. 2N. Kobayashi and Y. Nakanishi, unpublished observations. 2N. Kobayashi and Y. Nakanishi, unpublished observations.(6Laurent-Crawford A.G. Krust B. Muller S. Rivire Y. Rey-Cuill M.-A. Bchet J.-M. Montagnier L. Hovanessian A.G. Virology. 1991; 185: 829-839Google Scholar, 7Terai C. Kornbluth R.S. Pauza C.D. Richman D.D. Carson D.A. J. Clin. Invest. 1991; 87: 1710-1715Google Scholar, 8Groux H. Torpier G. Mont D. Mouton Y. Capron A. Ameisen J.C. J. Exp. Med. 1992; 175: 331-340Google Scholar, 9Meyaard L. Otto S.A. Jonker R.R. Mijnster M.J. Keet R.P.M. Miedema F. Science. 1992; 257: 217-219Google Scholar, 10Laurent-Crawford A.G. Krust B. Rivire Y. Desgranges C. Muller S. Kieny M.P. Dauguet C. Hovanessian A.G. AIDS Res. Hum. Retroviruses. 1993; 9: 761-773Google Scholar, 11Martin S.J. Matear P.M. Vyakarnam A. J. Immunol. 1994; 152: 330-342Google Scholar), and influenza virus (12Takizawa T. Matsukawa S. Higuchi Y. Nakamura S. Nakanishi Y. Fukuda R. J. Gen. Virol. 1993; 74: 2347-2355Google Scholar, 13Hinshaw V.S. Olsen C.W. Dybdahl-Sissoko N. Evans D. J. Virol. 1994; 68: 3667-3673Google Scholar) undergo apoptotic death. The physiological meaning of this virus-induced apoptotic death is not clearly understood, but the death of virus-infected CD4-positive cells is implicated in immunodeficiency among HIV-infected individuals(14Ameisen J.C. Capron A. Immunol. Today. 1991; 12: 102-105Google Scholar, 15Ameisen J.C. Immunol. Today. 1992; 13: 388-391Google Scholar, 16Gougeon M.-L. Colizzi V. Dalgleish A. Montagnier L. AIDS Res. Hum. Retroviruses. 1993; 9: 287-289Google Scholar, 17Gougeon M.-L. Montagnier L. Science. 1993; 260: 1269-1270Google Scholar). It is thus important to study virus-induced apoptosis not only to understand the mechanism of apoptotic cell death in general but to overcome viral disease. human immunodeficiency virus nuclear factor for interleukin-6 expression chloramphenicol acetyltransferase CCAAT/enhancer-binding protein. human immunodeficiency virus nuclear factor for interleukin-6 expression chloramphenicol acetyltransferase CCAAT/enhancer-binding protein. We found that infection with influenza virus (12Takizawa T. Matsukawa S. Higuchi Y. Nakamura S. Nakanishi Y. Fukuda R. J. Gen. Virol. 1993; 74: 2347-2355Google Scholar, 18Takizawa T. Fukuda R. Miyawaki T. Ohashi K. Nakanishi Y. Virology. 1995; (in press)Google Scholar) or HIV2 augments the production of the cell surface protein Fas at the mRNA level in host cells prior to the apoptosis of virus-infected cells. Fas(19Yonehara S. Ishii A. Yonehara M. J. Exp. Med. 1989; 169: 1747-1756Google Scholar, 20Itoh N. Yonehara S. Ishii A. Yonehara M. Mizushima S. Sameshima M. Hase A. Seto Y. Nagata S. Cell. 1991; 66: 233-243Google Scholar), also called APO-1(21Trauth B.C. Klas C. Peters A.M.J. Matzku S. Moller P. Falk W. Debatin K.-M. Krammer P.H. Science. 1989; 245: 301-305Google Scholar), is a receptor for an apoptosis-mediating signal molecule termed Fas-ligand (22Suda T. Takahashi T. Golstein P. Nagata S. Cell. 1993; 75: 1169-1178Google Scholar, 23Suda T. Nagata S. J. Exp. Med. 1994; 179: 873-879Google Scholar). Fas/Fas-ligand is the most characterized signaling system in eukaryotic apoptosis, and it is believed to play important roles in a variety of biological events such as the establishment and function of the immune system(24Nagata S. Tomei L.D. Cope F.O. Apoptosis II: The Molecular Basis of Apoptosis in Disease. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1994: 313-326Google Scholar). We reported that human T cell lines become susceptible to the apoptosis trigger provided by an anti-Fas monoclonal antibody when they are chronically infected with HIV(25Kobayashi N. Hamamoto Y. Yamamoto N. Ishii A. Yonehara M. Yonehara S. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9620-9624Google Scholar). Thus, an increase in the Fas concentration on the surface of virus-infected cells might be a key cause of the induction of apoptosis. In this study, we investigated the mechanism of activation of the Fas-encoding gene upon influenza virus infection and obtained evidence for the involvement of the transcription factor, nuclear factor for interleukin-6 expression (NF-IL6). A human FAS cDNA was obtained by reverse transcription-mediated polymerase chain reaction (26Kawasaki E.S. Innis M.A. Gelfand D.H. Sninsky J.J. White T.J. PCR Protocols. Academic Press, San Diego, CA1990: 21-27Google Scholar) using RNA from the human T cell lymphoma cell line KT-3(27Shimizu S. Takiguchi T. Sugai S. Matsuoka M. Konda S. Blood. 1988; 71: 196-203Google Scholar). The nucleotide sequence of the amplified DNA was identical to that reported previously(20Itoh N. Yonehara S. Ishii A. Yonehara M. Mizushima S. Sameshima M. Hase A. Seto Y. Nagata S. Cell. 1991; 66: 233-243Google Scholar). The cDNA was labeled with 32P by nick translation and used as a probe to screen a library. A genomic DNA library (28Tomatsu S. Kobayashi Y. Fukumaki Y. Yubisui T. Orii T. Sakaki Y. Gene (Amst.). 1989; 80: 353-361Google Scholar) constructed with the EMBL3 vector and DNA from human peripheral blood cells was obtained from the Japanese Cancer Research Resources Bank. About one-million clones were screened by hybridization with the labeled probe under standard conditions, and five were selected. These clones were further hybridized with a DNA fragment corresponding to the region between nucleotide positions 196 and 346 of FAS cDNA(20Itoh N. Yonehara S. Ishii A. Yonehara M. Mizushima S. Sameshima M. Hase A. Seto Y. Nagata S. Cell. 1991; 66: 233-243Google Scholar). Three clones gave positive signals and one of them, named EhFas7, contained the most 5′-end region of FAS cDNA. HeLa S3 cells were grown in Eagle's minimal essential medium (Nissui, Japan) supplemented with 10% fetal bovine serum (Irvine Scientific). Subconfluent monolayers were infected with SP626, a wild-type strain of the influenza A/Udorn/72 (H3N2) virus at a multiplicity of infection of 10 as described previously (29Hatada E. Takizawa T. Fukuda R. J. Gen. Virol. 1992; 73: 17-25Google Scholar) or exposed to double-stranded poly(I)•poly(C) (Pharmacia Biotech, Japan) at 0.1 mg/ml. The cells were harvested for further analysis after various periods. An oligonucleotide containing the region between +171 and +187 of the FAS gene (see Fig. 2) was synthesized, labeled with 32P at the 5′-end, and used as a primer. RNA samples were mixed with the primer, and the cDNA was synthesized with a reverse transcriptase as described previously(30Jones K.A. Yamamoto K.R. Tjian R. Cell. 1985; 42: 559-572Google Scholar). The cDNA was separated on a 6% polyacrylamide gel containing 8.3 M urea together with sequence ladders constructed by sequencing a DNA with the same primer. The DNA fragment corresponding to the region between −1435 and +236 of the FAS gene was inserted 18-base pairs upstream of the translation start codon of the firefly luciferase gene in the pGV-B vector (Toyo Ink, Japan), resulting in pFLF1. Various amounts of pFLF1 were introduced into HeLa cells using DEAE-dextran(31Gorman C. Glover D.M. DNA Cloning: A Practical Approach. IRL Press, Oxford, United Kingdom1985: 143-190Google Scholar), and the cells were cultured for 24 h. The cell lysates were prepared, and luciferase was assayed using the commercially supplied reagent, PicaGene (Toyo Ink). Luciferase activity was determined using a luminometer (Lumat LB9501, Berthold, Germany). HeLa cells were also transfected with pGV-B, and luciferase activity in the cell lysates was subtracted as background from that in the lysates of the pFLF1 transfectants. A DNA containing the promoter region of the human β-actin gene fused to the coding sequence of the chloramphenicol acetyltransferase (CAT) gene (β-actin/CAT) was introduced into HeLa cells together with pFLF1 as an internal control. The expression of β-actin/CAT was determined by the CAT enzyme assay as described previously(31Gorman C. Glover D.M. DNA Cloning: A Practical Approach. IRL Press, Oxford, United Kingdom1985: 143-190Google Scholar). Nuclear “mini-extracts” were prepared from HeLa cells (0.5-1 × 106)(32Schreiber E. Matthias P. Mller M.M. Schaffner W. Nucleic Acids Res. 1989; 17: 6419Google Scholar). A double-stranded oligonucleotide with the sequence of 5′-AGATTGTGCAATCT, which contained the consensus binding-sequence for NF-IL6, 5′-T(T/G)NNGNAA(T/G)(33Akira S. Isshiki H. Sugita T. Tanabe O. Kinoshita S. Nishio Y. Nakajima T. Hirano T. Kishimoto T. EMBO J. 1990; 9: 1897-1906Google Scholar), was labeled with 32P at the 5′-ends and used as a probe. Nuclear extracts (2 μl) were incubated on ice for 10 min in a reaction mixture consisting of 12 mM Hepes (pH 7.9), 40 mM KCl, 120 mM NaCl, 0.2 mM EDTA, 0.2 mM EGTA, 0.4 mM dithiothreitol, 0.4 mM phenylmethylsulfonyl fluoride, 8% glycerol, and 0.1 mg/ml of poly[d(I•C)]•poly[d(I•C)] (Sigma). The probe (0.05 pmol) was then added to the mixture, and the binding reaction proceeded for 10 min on ice. The mixture was loaded onto a 6% polyacrylamide gel containing 5% glycerol, and resolved by electrophoresis at 12.5 V/cm at 4°C in 25 mM Tris borate (pH 8.3) containing 0.5 mM EDTA. Oligonucleotide competitors (25-fold excess over the probe) were incubated with nuclear extracts before the probe was added. Oligo-AP4, 5′-CCAGCTGTGGAATG, contained the AP4-binding site of simian virus 40 DNA. Antibodies raised against synthetic peptides of the rat CCAAT/enhancer-binding protein (C/EBP) (34Williams S.C. Cantwell C.A. Johnson P.F. Genes & Dev. 1991; 6: 1553-1567Google Scholar) (Santa Cruz Biotechnology), which cross-react with the human homolog, were added after the binding reaction was completed, and the mixture was further incubated for 1 h on ice. The intensity of the specific signal was determined using an image analyzer (BA100, Fuji Photo Film, Japan). A gel shift assay for nuclear factor I was similarly performed using an oligonucleotide with the sequence 5′-TATACCTTATACTGGACTAGTGCCAATATTAAAATG as a probe as described previously(35Kawamura H. Wada N. Makino Y. Tamura T. Koikeda S. Shiroki K. Masamune Y. Nakanishi Y. J. Virol. 1994; 68: 5056-5062Google Scholar). Nuclear extracts were separated on a 10% polyacrylamide gel containing SDS and electrophoretically transferred onto a polyvinylidene difluoride membrane (Millipore, Corp.). The membrane was blocked with 1% bovine serum albumin and incubated with 0.1 μg/ml of anti-rat C/EBPβ immunoglobulin G in a buffer consisting of 10 mM Tris-HCl (pH 8), 0.15 M NaCl, and 0.5% Tween 20. The membrane was washed and reacted with anti-rabbit immunoglobulin G conjugated with horseradish peroxidase (Amersham, Corp.). Signals were then visualized using the ECL System (Amersham, Corp.). To determine the specificity of the reaction, the first antibody was initially incubated for 30 min with 0.2 μg/ml of either the peptide used to raise the antibody or that containing the region between amino acid positions 144 and 157 of human Fas(20Itoh N. Yonehara S. Ishii A. Yonehara M. Mizushima S. Sameshima M. Hase A. Seto Y. Nagata S. Cell. 1991; 66: 233-243Google Scholar). The nucleotide sequence was determined for both strands by dideoxy chain termination(36Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Google Scholar). Total cellular RNA was extracted according to the method of Chomczynski and Sacchi (37Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Google Scholar) and poly(A)+ RNA was enriched by means of oligo(dT)-cellulose chromatography. Northern and Southern blots were performed under standard conditions. To obtain a DNA containing the transcription promoter of the human FAS gene, a genomic DNA library constructed with DNA from human peripheral blood cells was screened, using the human FAS cDNA as a probe. Five positive clones were selected from one million independent clones. One of them, named EhFas7, was further analyzed since it contained the most 5′-end region of the FAS cDNA. A restriction map of EhFas7 is shown in Fig. 1A. Various DNA regions of EhFas7 were used as probes against the RNA from KT-3 cells in a Northern blot. The 0.8-kilobase pair XhoI-HindIII fragment detected Fas mRNA, whereas more upstream regions failed to do so (data not shown, see Fig. 1A). We then searched for the transcription initiation site (+1) within this DNA segment. An oligo-DNA primer was constructed corresponding to a sequence within the region, and primer extension proceeded using the RNA from the human T cell line MOLT-4, which contains a relatively large amount of Fas mRNA (data not shown). A single extended product of about 190 bases was detected with the RNA that bound oligo(dT)-cellulose (Fig. 1B). The position of +1 was G, according to the sequence ladders. To eliminate the possibility that the EhFas7 clone was derived from a pseudogene, genomic Southern hybridization was performed using DNA from human peripheral blood mononuclear cells and a small fragment within the first exon as a probe (see Fig. 1A). The results in Fig. 1C show a single hybridizing band of the predicted size. This indicates that EhFas7 is not a pseudogene and that it contains the 5′-end region of the human FAS gene. The 2.2-kilobase pair HindIII-HindIII DNA fragment was then sequenced, and we found that this DNA contained the sequence corresponding to the region between nucleotide positions 1 and 226 of the FAS cDNA, which was flanked by an additional 132-base pair transcribed sequence (Fig. 2). The first exon (boxed) and part of the first intron were included in this clone. The proximal promoter region, 20-40-base pairs upstream of +1, did not possess TATA, GC, or CCAAT boxes, but there were sequences for the binding of AP-1, Ets, and NF-IL6 (also called C/EBPβ) in the more upstream region. AP-1- and Ets-binding sites were present at around −490, and there were six binding sequences for NF-IL6 in the region spanning from −130 to −1360. The first exon contained two more sequences for NF-IL6 binding. We examined whether the 5′-upstream region of the cloned FAS gene can direct the transcription of a downstream sequence. The region between −1435 and +236, which includes seven out of eight NF-IL6-binding sites, was fused to the coding sequence of the firefly luciferase gene, and various amounts of the resulting pFLF1 DNA were introduced into HeLa cells. The lysates from transfectants contained luciferase activity, and 5 μg of the DNA gave maximal expression (data not shown). The effect of influenza virus infection on the activity of the FAS promoter was then examined. HeLa cells were transfected with pFLF1 and cultured for 24 h. The cells were then infected with influenza virus, and lysates were prepared from the infected cells after various periods to determine the luciferase activity. The expression of the luciferase gene began to increase soon after infection, reaching a maximal stimulation of about 2-fold at 2 h postinfection, and then it gradually descended to the control level at 6 h postinfection (Fig. 3A). Transient stimulation of the FAS promoter coincides with an increase of the Fas mRNA in influenza virus-infected HeLa cells; the amount of Fas mRNA increases about 3-fold with a sharp peak at 3 h postinfection (18Takizawa T. Fukuda R. Miyawaki T. Ohashi K. Nakanishi Y. Virology. 1995; (in press)Google Scholar). Moreover, the specificity of gene expression in virus-infected cells (18Takizawa T. Fukuda R. Miyawaki T. Ohashi K. Nakanishi Y. Virology. 1995; (in press)Google Scholar) was reproduced in a DNA transfection assay since the human β-actin promoter used as an internal negative control was little affected by influenza virus infection (Fig. 3B). Since the presence of dispersed multiple binding sites in transcription regulatory regions is typical of NF-IL6-inducible genes, we considered that NF-IL6 might be a transcription factor responsible for the activation of the FAS promoter in cells infected with influenza virus. We first established a gel shift assay to determine the DNA-binding activity of NF-IL6 using an oligo-DNA having the consensus sequence for NF-IL6 binding as a probe. Nuclear extracts of uninfected HeLa cells showed a shifted band, which disappeared only in the presence of an excess of the unlabeled probe (Fig. 4A, lanes 1-3). The formation of this specific complex was almost completely inhibited by an anti-C/EBPβ antibody, but it was not affected by an antibody raised against either C/EBPα or C/EBPδ (lanes 4-7). These results indicate that the binding activity in uninfected HeLa cells contains C/EBPβ, but not C/EBPα and C/EBPδ, suggesting that it consists of the homodimer of C/EBPβ (NF-IL6). NF-IL6 activity in HeLa cells markedly increased when HeLa cells were transfected with pEF-NFIL6 that expresses human NF-IL6(38Matsusaka T. Fujikawa K. Nishio Y. Mukaida N. Matsushima K. Kishimoto T. Akira S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10193-10197Google Scholar), while a control vector showed no effect (lanes 8-13). To assess whether NF-IL6 is involved in the transcription regulation of the human FAS gene, we transfected HeLa cells with pFLF1 and pEF-NFIL6, and luciferase activity in the lysates from transfectants was determined. Gene expression driven by the FAS promoter was significantly augmented in the presence of pEF-NFIL6, whereas the β-actin promoter, whose DNA was simultaneously introduced into HeLa cells, was not affected (Fig. 4B). These results indicate that NF-IL6 is a positive transcription factor for the human FAS gene. We then examined whether NF-IL6 activity changes upon influenza virus infection using a gel shift assay (Fig. 5, A and B). NF-IL6 activity significantly increased soon after virus infection, reaching a maximal stimulation of about 3-fold at 2 h postinfection. It then rapidly descended to below the control level. This profile coincided with the change in the FAS promoter activity that occurs upon influenza virus infection (see Fig. 3A). On the other hand, poly(I)•poly(C), which also augments the accumulation of the Fas mRNA in HeLa cells(18Takizawa T. Fukuda R. Miyawaki T. Ohashi K. Nakanishi Y. Virology. 1995; (in press)Google Scholar), somewhat differently affected NF-IL6. An increase of NF-IL6 activity was first detectable at 2 h, and maximal 3-fold stimulation occurred 6 h after the RNA was added. In contrast to the experiment with virus infection, NF-IL6 activity did not decrease and maintained this level even after 10 h. This profile again paralleled the change in the Fas mRNA in HeLa cells exposed to poly(I)•poly(C) (18Takizawa T. Fukuda R. Miyawaki T. Ohashi K. Nakanishi Y. Virology. 1995; (in press)Google Scholar). NF-IL6 activity did not change when HeLa cells were simply cultured with no added reagents. The activity of nuclear factor I was examined in the nuclear extracts prepared from HeLa cells exposed to poly(I)•poly(C), but no significant change was found (data not shown). This indicates that poly(I)•poly(C) does not affect the activity of DNA-binding nuclear factors in general. The increased binding activity in the extracts of the cells exposed to either influenza virus or poly(I)•poly(C), was sensitive specifically to an anti-NF-IL6 antibody (Fig. 5C), suggesting that only the activity of NF-IL6 increased. These results indicate that the DNA-binding activity of NF-IL6 is augmented by either influenza virus infection or by poly(I)•poly(C). Changes in the NF-IL6 concentrations in HeLa nuclear extracts caused by either influenza virus infection or poly(I)•poly(C) were analyzed by immunoblotting. The antibody detected discrete doublet proteins with apparent molecular masses of 46 and 44 kDa (Fig. 6A), in line with the data of Nishio et al.(39Nishio Y. Isshiki H. Kishimoto T. Akira S. Mol. Cell. Biol. 1993; 13: 1854-1862Google Scholar). Both signals significantly decreased when the antibody was first incubated with an excess of the antigen peptide, whereas a peptide with an unrelated sequence caused little effect (Fig. 6A). This indicates that the antibody specifically detected NF-IL6 in HeLa cells. Three peptides, of which calculated molecular masses are 36, 34, and 16 kDa, are presumably translated from a single mRNA of human NF-IL6 (33Akira S. Isshiki H. Sugita T. Tanabe O. Kinoshita S. Nishio Y. Nakajima T. Hirano T. Kishimoto T. EMBO J. 1990; 9: 1897-1906Google Scholar) and are thought to be the human homolog of the rat proteins: liver-enriched activator protein*, liver-enriched activator protein, and liver-enriched inhibitory protein(40Descombes P. Schibler U. Cell. 1991; 67: 569-579Google Scholar), respectively(41Tesmer V.M. Rajadhyaksha A. Babin J. Bina M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7298-7302Google Scholar). Judging from the molecular masses of the two proteins detected by immunoblotting, they are likely to be the products from the first two AUG codons(33Akira S. Isshiki H. Sugita T. Tanabe O. Kinoshita S. Nishio Y. Nakajima T. Hirano T. Kishimoto T. EMBO J. 1990; 9: 1897-1906Google Scholar). A discrepancy in the molecular masses of these proteins is probably due to abundant Pro residues(33Akira S. Isshiki H. Sugita T. Tanabe O. Kinoshita S. Nishio Y. Nakajima T. Hirano T. Kishimoto T. EMBO J. 1990; 9: 1897-1906Google Scholar), which could cause improper migration of the peptides on an SDS gel. The intensity of the two signals did not significantly change at any time after exposure to either reagent (Fig. 6B). This suggests that the DNA-binding activity of NF-IL6 is stimulated in those cells by posttranslational modification with no change in the concentration of the transcription factor. Such modification seemed to be rapidly reversed in cells infected with influenza virus but not in those exposed to poly(I)•poly(C), since NF-IL6 activity in virus-infected cells sharply peaked at 2 h postinfection (see Fig. 5). We previously found that infection with influenza virus(12Takizawa T. Matsukawa S. Higuchi Y. Nakamura S. Nakanishi Y. Fukuda R. J. Gen. Virol. 1993; 74: 2347-2355Google Scholar, 18Takizawa T. Fukuda R. Miyawaki T. Ohashi K. Nakanishi Y. Virology. 1995; (in press)Google Scholar) or HIV2 augmented the expression of the Fas-encoding gene in host cells before the onset of apoptosis. The expression of the FAS gene is also enhanced by murine leukemia virus in lymphocytes(42Hiromatsu K. Aoki Y. Makino M. Matsumoto Y. Mizuochi T. Gotoh Y. Nomoto K. Ogasawara J. Nagata S. Yoshikai Y. Eur. J. Immunol. 1994; 24: 2446-2451Google Scholar), by ischemia in the brain(43Matsuyama T. Hata R. Tagaya M. Yamamoto Y. Nakajima T. Furuyama J.-I. Wanaka A. Sugita M. Brain Res. 1994; 657: 342-346Google Scholar), by hypoxia in cultured cardiomyocytes(44Tanaka M. Itoh H. Adachi S. Akimoto H. Nishikawa T. Kasajima T. Marumo F. Hiroe M. Circ. Res. 1994; 75: 426-433Google Scholar), by interferons in various cultured cell lines(18Takizawa T. Fukuda R. Miyawaki T. Ohashi K. Nakanishi Y. Virology. 1995; (in press)Google Scholar, 20Itoh N. Yonehara S. Ishii A. Yonehara M. Mizushima S. Sameshima M. Hase A. Seto Y. Nagata S. Cell. 1991; 66: 233-243Google Scholar, 45Watanabe-Fukunaga R. Brannan C.I. Itoh N. Yonehara S. Copeland N.G. Jenkins N.A. Nagata S. J. Immunol. 1992; 148: 1274-1279Google Scholar), and by activation with various reagents in human peripheral lymphocytes(46Miyawaki T. Uehara T. Nibu R. Tsuji T. Yachie A. Yonehara S. Taniguchi N. J. Immunol. 1992; 149: 3753-3758Google Scholar, 47Owen-Schaub L.B. Yonehara S. Crump III, W.L. Grimm E.A. Cell Immunol. 1992; 140: 197-205Google Scholar, 48Drappa J. Brot N. Elkon K.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10340-10344Google Scholar). It is thus probable that apoptotic cell death is regulated, at least in part, through changes in the amounts of Fas. Structural analysis of the 5′-end region of the human FAS gene revealed that the gene possesses a TATA-less promoter and that its 5′-upstream region contains putative binding sequences for a limited number of transcription factors. In this study, we focused upon one of these factors, termed NF-IL6, of which eight recognizable sequences are dispersed in a 1.7-kilobase pair DNA at the 5′-end region of the FAS gene. We obtained evidence that NF-IL6 is a positive transcription factor for the human FAS gene. The production of a large amount of Fas mRNA in the cell line KT-3(20Itoh N. Yonehara S. Ishii A. Yonehara M. Mizushima S. Sameshima M. Hase A. Seto Y. Nagata S. Cell. 1991; 66: 233-243Google Scholar), which is interleukin-6 dependent(27Shimizu S. Takiguchi T. Sugai S. Matsuoka M. Konda S. Blood. 1988; 71: 196-203Google Scholar), can also be attributed to the function of NF-IL6, since NF-IL6 participates in signal transduction in response to interleukin-6(49Kishimoto T. Taga T. Akira S. Cell. 1994; 76: 253-262Google Scholar). We found that the DNA-binding activity of NF-IL6 increases without a change in its molecular number upon infection with influenza virus or exposure to poly(I)•poly(C). All circumstantial evidence supports the notion that the increased activity of NF-IL6 is responsible for the stimulation of FAS gene transcription in virus-infected cells. NF-IL6 is considered to be post-translationally modified upon virus infection. Phosphorylation is the most likely candidate for such modification, since a protein kinase inhibitor shuts down an increase of the Fas mRNA in poly(I)•poly(C)-treated HeLa cells(18Takizawa T. Fukuda R. Miyawaki T. Ohashi K. Nakanishi Y. Virology. 1995; (in press)Google Scholar). Moreover, other investigators have reported that NF-IL6 is activated through phosphorylation by calcium/calmodulin-dependent protein kinase(50Wegner M. Cao Z. Rosenfeld M.G. Science. 1992; 256: 370-373Google Scholar), by mitogen-activated kinase(51Nakajima T. Kinoshita S. Sasagawa T. Sasaki K. Naruto M. Kishimoto T. Akira S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2207-2211Google Scholar), by protein kinase C(52Trautwein C. Caelles C. van der Geer P. Hunter T. Karin M. Chojkier M. Nature. 1993; 364: 544-547Google Scholar, 53Trautwein C. van der Geer P. Karin M. Hunter T. Chojkier M. J. Clin. Invest. 1994; 93: 2554-2561Google Scholar), and by protein kinase A(53Trautwein C. van der Geer P. Karin M. Hunter T. Chojkier M. J. Clin. Invest. 1994; 93: 2554-2561Google Scholar). These enzymes phosphorylate Ser and/or Thr residues at various positions within NF-IL6. Another protein kinase, termed double-stranded RNA-activated protein kinase(54Hovanessian A.G. J. Interferon Res. 1989; 9: 641-647Google Scholar), could be involved in NF-IL6 phosphorylation upon influenza virus infection because it is activated in virus-infected cells. This kinase has to be autophosphorylated to become functional, which only occurs in the presence of double-stranded RNA(55Galabru J. Hovanessian A.G. Cell. 1985; 43: 685-694Google Scholar). There is no doubt that double-stranded RNA-activated protein kinase is active in poly(I)•poly(C)-treated HeLa cells, where the DNA-binding activity of NF-IL6 increased. Moreover, our preliminary experiments indicated that an increase of Fas protein upon influenza virus infection was abolished in the presence of a DNA that expresses a dominant negative form of double-stranded RNA-activated protein kinase.3 3T. Takizawa, unpublished observations. We thus suggest that autophosphorylated double-stranded RNA-activated protein kinase phosphorylates NF-IL6 and that activated NF-IL6 stimulates FAS gene transcription in influenza virus-infected or poly(I)•poly(C)-treated cells. The activation of NF-IL6 occurred only transiently upon virus infection. This might be due to an inhibitor of double-stranded RNA-activated protein kinase that is induced in influenza virus-infected cells(56Lee T.G. Tomita J. Hovanessian A.G. Katze M.G. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6208-6212Google Scholar). Lee and Esteban (57Lee S.B. Esteban M. Virology. 1994; 199: 491-496Google Scholar) have reported that the ectopic expression of double-stranded RNA-activated protein kinase using the vaccinia virus vector brought about the apoptosis of HeLa cells. Although they maintain that HeLa cells undergo apoptotic death because of an increase in the protein kinase, we speculate that vaccinia virus is also involved. We showed that poly(I)•poly(C), which probably leads to the activation of double-stranded RNA-activated protein kinase, did not cause apoptosis of HeLa cells despite an increase in the amount of Fas (18Takizawa T. Fukuda R. Miyawaki T. Ohashi K. Nakanishi Y. Virology. 1995; (in press)Google Scholar). Fas must be bound by a specific signaling molecule, called Fas-ligand (22Suda T. Takahashi T. Golstein P. Nagata S. Cell. 1993; 75: 1169-1178Google Scholar, 23Suda T. Nagata S. J. Exp. Med. 1994; 179: 873-879Google Scholar), to transmit an apoptosis signal. Fas-ligand should thus participate in influenza virus-induced apoptosis. It is unlikely that HeLa cells constitutively produce Fas-ligand, since poly(I)•poly(C)-treated cells do not undergo apoptosis despite the presence of a sufficient amount of Fas(18Takizawa T. Fukuda R. Miyawaki T. Ohashi K. Nakanishi Y. Virology. 1995; (in press)Google Scholar). We speculate that the synthesis of Fas-ligand as well as of its receptor, Fas, increases in virus-infected cells prior to cell death. We are currently assessing this issue. We thank Yukito Masamune for discussion and encouragement throughout the study. We also thank Shizuo Akira for pEF-NFIL6 and suggestions, Masaaki Tsuda for suggestions, and Shigekazu Nagata for pEF-BOS.