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• W2068100440 abstract "The human T-cell leukemia virus type I (HTLV-I)-encoded Tax protein activates transcription from the viral promoter via association with the cellular basic leucine zipper factor cAMP-response element-binding protein-2. Tax is also able to induce cellular transformation of T lymphocytes probably by modulating transcriptional activity of cellular factors, including nuclear factor-κB, E2F, activator protein-1 (AP-1), and p53. Recently, we characterized in HTLV-I-infected cells the presence of a novel viral protein, HBZ, encoded by the complementary strand of the HTLV-I RNA genome (Gaudray, G., Gachon, F., Basbous, J., Biard-Piechaczyk, M., Devaux, C., and Mesnard, J.-M. (2002) J. Virol. 76, 12813–12822). HBZ is a nuclear basic leucine zipper protein that down-regulates Tax-dependent viral transcription by inhibiting the binding of cAMP-response element-binding protein-2 to the HTLV-I promoter. In searching for other cellular targets of HBZ, we identified two members of the Jun family, JunB and c-Jun. Co-immunoprecipitation and cellular colocalization confirmed that HBZ interacts in vivo with JunB and c-Jun. When transiently introduced into CEM cells with a reporter gene containing the AP-1 site from the collagenase promoter, HBZ suppressed transactivation by c-Jun. On the other hand, the combination of HBZ with Jun-B had higher transcriptional activity than JunB alone. Consistent with the structure of its basic domain, we demonstrate that HBZ decreases the DNA-binding activity of c-Jun and JunB. Last, we show that c-Jun is no longer capable of activating the basal expression of the HTLV-I promoter in the presence of HBZ in vivo. Our results support the hypothesis that HBZ could be a negative modulator of the Tax effect by controlling Tax expression at the transcriptional level and by attenuating activation of AP-1 by Tax. The human T-cell leukemia virus type I (HTLV-I)-encoded Tax protein activates transcription from the viral promoter via association with the cellular basic leucine zipper factor cAMP-response element-binding protein-2. Tax is also able to induce cellular transformation of T lymphocytes probably by modulating transcriptional activity of cellular factors, including nuclear factor-κB, E2F, activator protein-1 (AP-1), and p53. Recently, we characterized in HTLV-I-infected cells the presence of a novel viral protein, HBZ, encoded by the complementary strand of the HTLV-I RNA genome (Gaudray, G., Gachon, F., Basbous, J., Biard-Piechaczyk, M., Devaux, C., and Mesnard, J.-M. (2002) J. Virol. 76, 12813–12822). HBZ is a nuclear basic leucine zipper protein that down-regulates Tax-dependent viral transcription by inhibiting the binding of cAMP-response element-binding protein-2 to the HTLV-I promoter. In searching for other cellular targets of HBZ, we identified two members of the Jun family, JunB and c-Jun. Co-immunoprecipitation and cellular colocalization confirmed that HBZ interacts in vivo with JunB and c-Jun. When transiently introduced into CEM cells with a reporter gene containing the AP-1 site from the collagenase promoter, HBZ suppressed transactivation by c-Jun. On the other hand, the combination of HBZ with Jun-B had higher transcriptional activity than JunB alone. Consistent with the structure of its basic domain, we demonstrate that HBZ decreases the DNA-binding activity of c-Jun and JunB. Last, we show that c-Jun is no longer capable of activating the basal expression of the HTLV-I promoter in the presence of HBZ in vivo. Our results support the hypothesis that HBZ could be a negative modulator of the Tax effect by controlling Tax expression at the transcriptional level and by attenuating activation of AP-1 by Tax. The activator protein-1 (AP-1) 1The abbreviations used are: AP-1, activator protein-1; CRE, cAMP-response element; bZIP, basic leucine zipper; CREB, cAMP-response element-binding protein; HTLV-I, human T-cell leukemia virus type I; HBZ, HTLV-I bZIP factor; GST, glutathione S-transferase; C/EBP, CCAAT/enhancer-binding protein; CHOP, C/EBP-homologous protein. transcription complex is involved in a multitude of cellular processes such as proliferation, differentiation, and cell death (1Shaulian E. Karin M. Nat. Cell Biol. 2002; 4: 131-136Crossref PubMed Scopus (2251) Google Scholar). Various AP-1 components, including c-Jun (2Vogt P.K. Oncogene. 2001; 20: 2365-2377Crossref PubMed Scopus (260) Google Scholar), can induce oncogenic transformation upon chronic activation in avian and mammalian cells (3Mechta-Grigoriou F. Ferald D. Yaniv M. Oncogene. 2001; 20: 2378-2389Crossref PubMed Scopus (279) Google Scholar, 4van Dam H. Castellazzi M. Oncogene. 2001; 20: 2453-2464Crossref PubMed Scopus (380) Google Scholar). The ability to regulate such a number of biological processes is due to the formation of various dimers between the Jun (c-Jun, JunB, and JunD) and Fos (c-Fos, FosB, Fra-1, and Fra-2) family members through their leucine zipper, a structural motif involving a heptad repeat of leucine residues. Jun proteins form both homodimers and heterodimers with Fos proteins (5Gentz R. Rauscher F.J. Abate C. Curran T. Science. 1989; 243: 1695-1699Crossref PubMed Scopus (399) Google Scholar, 6Kouzarides T. Ziff E. Nature. 1988; 15: 646-651Crossref Scopus (551) Google Scholar, 7Ransone L.J. Visvader J. Sassone-Corsi P. Verma I.M. Genes Dev. 1989; 6: 770-781Crossref Scopus (104) Google Scholar), but Fos proteins are unable to homodimerize and require heterodimerization to bind to DNA (8Cohen D.R. Curran T. Oncogene. 1990; 5: 929-939PubMed Google Scholar, 9Nakabeppu Y. Nathans D. EMBO J. 1989; 8: 3833-3841Crossref PubMed Scopus (94) Google Scholar, 10Ransone L.J. Wamsley P. Morley K.L. Verma I.M. Mol. Cell. Biol. 1990; 10: 4565-4573Crossref PubMed Scopus (31) Google Scholar). Dimers formed by Fos and Jun bind with the highest affinity to the AP-1 site, but they are also able to recognize the cAMP-response element (CRE) motif (11Hai T. Curran T. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3720-3724Crossref PubMed Scopus (1119) Google Scholar, 12Nakabeppu Y. Ryder K. Nathans D. Cell. 1988; 55: 907-915Abstract Full Text PDF PubMed Scopus (531) Google Scholar, 13Ryseck R.-P. Bravo R. Oncogene. 1991; 6: 533-542PubMed Google Scholar). The AP-1 complexes are not limited to Jun and Fos dimers because certain Jun and Fos proteins have been shown to dimerize with other basic leucine zipper (bZIP) proteins, including members of the activating transcription factor/CRE-binding protein (CREB) family and the Maf transcription factors generating complexes with altered binding specificities (4van Dam H. Castellazzi M. Oncogene. 2001; 20: 2453-2464Crossref PubMed Scopus (380) Google Scholar, 14Kerppola T.K. Curran T. Oncogene. 1994; 9: 675-684PubMed Google Scholar, 15Macgregor P.F. Abate C. Curran T. Oncogene. 1990; 5: 451-458PubMed Google Scholar). The relative binding affinities of the AP-1 transcription factors depend on the specific DNA sequence, on the promoter context, and on the dimer combinations (16Chinenov Y. Kerppola T.K. Oncogene. 2001; 20: 2438-2452Crossref PubMed Scopus (579) Google Scholar). Human T-cell leukemia virus type I (HTLV-I) is an oncogenic retrovirus etiologically associated with the development of adult T-cell leukemia. HTLV-I transforms T-cells via its regulatory protein Tax (17Basbous J. Gaudray G. Devaux C. Mesnard J.-M. Recent Res. Dev. Mol. Cell. Biol. 2002; 3: 155-166Google Scholar), which interferes with cell growth control pathways through activation of cellular transcription factors, including nuclear factor-κB (18Xiao G. Cvijic M.E. Fong A. Harhaj E.W. Yuhlik M.T. Waterfield M. Sun S.-C. EMBO J. 2001; 20: 6805-6815Crossref PubMed Scopus (254) Google Scholar), E2F (19Lemasson I. Thébault S. Sardet C. Devaux C. Mesnard J.-M. J. Biol. Chem. 1998; 273: 23598-23604Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), and AP-1 (20Iwai K. Mori N. Oie M. Yamamoto N. Fujii M. Virology. 2001; 279: 38-46Crossref PubMed Scopus (60) Google Scholar), and inactivation of p53 (21Suzuki T. Uchida-Toita M. Yoshida M. Oncogene. 1999; 18: 4137-4143Crossref PubMed Scopus (88) Google Scholar). Previous studies have shown that T-cell lines transformed by HTLV-I express high levels of AP-1 activity (20Iwai K. Mori N. Oie M. Yamamoto N. Fujii M. Virology. 2001; 279: 38-46Crossref PubMed Scopus (60) Google Scholar, 22Mori N. Fujii M. Iwai K. Ikeda S. Yamasaki Y. Hata T. Yamada Y. Tanaka Y. Tomonaga M. Yamamoto N. Blood. 2000; 95: 3915-3921PubMed Google Scholar) with increased levels of mRNAs coding for c-Jun, JunB, JunD, c-Fos, and Fra-1 (23Fujii M. Niki T. Mori T. Matsuda T. Matsui M. Nomura N. Seiki M. Oncogene. 1991; 6: 1023-1029PubMed Google Scholar, 24Hooper W.C. Rudolph D.L. Lairmore M.D. Lal R.B. Biochem. Biophys. Res. Commun. 1991; 181: 976-980Crossref PubMed Scopus (16) Google Scholar). It has been demonstrated that Tax induces expression of genes coding for c-Fos (25Alexandre C. Verrier B. Oncogene. 1991; 6: 543-551PubMed Google Scholar, 26Fujii M. Sassone-Corsi P. Verma I.M. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8526-8530Crossref PubMed Scopus (285) Google Scholar, 27Nagata K. Ohtani K. Nakamura M. Sugamura K. J. Virol. 1989; 63: 3220-3226Crossref PubMed Google Scholar), Fra-1 (23Fujii M. Niki T. Mori T. Matsuda T. Matsui M. Nomura N. Seiki M. Oncogene. 1991; 6: 1023-1029PubMed Google Scholar, 28Tsuchiya H. Fujii M. Niki T. Tokuhara M. Matsui M. Seiki M. J. Virol. 1993; 67: 7001-7007Crossref PubMed Google Scholar), c-Jun (23Fujii M. Niki T. Mori T. Matsuda T. Matsui M. Nomura N. Seiki M. Oncogene. 1991; 6: 1023-1029PubMed Google Scholar), and JunD (23Fujii M. Niki T. Mori T. Matsuda T. Matsui M. Nomura N. Seiki M. Oncogene. 1991; 6: 1023-1029PubMed Google Scholar). Moreover, AP-1 sites have been characterized as responsive elements for Tax in different cellular genes, including fra-1 (28Tsuchiya H. Fujii M. Niki T. Tokuhara M. Matsui M. Seiki M. J. Virol. 1993; 67: 7001-7007Crossref PubMed Google Scholar), interleukin-2 (29Curtiss V.E. Smilde R. McGuire K.L. Mol. Cell. Biol. 1996; 16: 3567-3575Crossref PubMed Scopus (28) Google Scholar), interleukin-5 (30Yamagata T. Mitani K. Ueno H. Kanda Y. Yazaki Y. Hirai H. Mol. Cell. Biol. 1997; 17: 4272-4281Crossref PubMed Scopus (46) Google Scholar), interleukin-8 (31Mori N. Mukaida N. Ballard D.W. Matsushima K. Yamamoto N. Cancer Res. 1998; 58: 3993-4000PubMed Google Scholar), TR3/nur77 (32Liu X. Chen X. Zachar V. Chang C. Ebbesen P. J. Gen. Virol. 1999; 80: 3073-3081Crossref PubMed Scopus (28) Google Scholar), and TIMP-1 (33Uchijima M. Sato H. Fujii M. Seiki M. J. Biol. Chem. 1994; 269: 14946-14950Abstract Full Text PDF PubMed Google Scholar). It has been suggested that Tax could be involved in AP-1 regulation at a post-transcriptional level (20Iwai K. Mori N. Oie M. Yamamoto N. Fujii M. Virology. 2001; 279: 38-46Crossref PubMed Scopus (60) Google Scholar, 22Mori N. Fujii M. Iwai K. Ikeda S. Yamasaki Y. Hata T. Yamada Y. Tanaka Y. Tomonaga M. Yamamoto N. Blood. 2000; 95: 3915-3921PubMed Google Scholar), but the exact mechanism of activation remains misunderstood. Tax is also involved in the transcription control of the HTLV-I genome itself (34Thébault S. Gachon F. Gaudray G. Mesnard J.-M. Recent Res. Dev. Virol. 2001; 3: 151-164Google Scholar). The elements that impart Tax responsiveness to the promoter consist of three 21-bp repeats containing an imperfect CRE sequence. Because Tax does not bind CRE by itself, previous studies have focused on identifying cellular proteins that bind to the viral CRE in conjunction with Tax. Using the yeast two-hybrid approach, the cellular bZIP transcription factor CREB-2 has been identified as a protein that interacts with Tax (35Gachon F. Péléraux A. Thébault S. Dick J. Lemasson I. Devaux C. Mesnard J.-M. J. Virol. 1998; 72: 8332-8337Crossref PubMed Google Scholar, 36Reddy T.R. Tang H. Li X. Wong-Staal F. Oncogene. 1997; 14: 2785-2792Crossref PubMed Scopus (63) Google Scholar). It has also been shown that Tax enhances CREB-2 binding to the 21-bp repeats (37Gachon F. Thebault S. Peleraux A. Devaux C. Mesnard J.-M. Mol. Cell. Biol. 2000; 20: 3470-3481Crossref PubMed Scopus (60) Google Scholar), probably by promoting homodimerization of the CREB-2 bZIP domain (38Gachon F. Gaudray G. Thebault S. Basbous J. Koffi A.J. Devaux C. Mesnard J.-M. FEBS Lett. 2001; 502: 57-62Crossref PubMed Scopus (34) Google Scholar, 39Wagner S. Green M.R. Science. 1993; 262: 395-399Crossref PubMed Scopus (290) Google Scholar). Then, the promoter-bound Tax molecule recruits the transcriptional coactivator CREB-binding protein, which is involved in the acetylation of the core histone tails (40Gachon F. Devaux C. Mesnard J.-M. Virology. 2002; 299: 271-278Crossref PubMed Scopus (23) Google Scholar, 41Georges S.A. Kraus W.L. Luger K. Nyborg J.K. Laybourn P.J. Mol. Cell. Biol. 2002; 22: 127-137Crossref PubMed Scopus (51) Google Scholar). AP-1 is also able to transactivate the HTLV-I promoter in vivo (42Jeang K.-T. Chiu R. Santos E. Kim S.-J. Virology. 1991; 181: 218-227Crossref PubMed Scopus (56) Google Scholar, 43Lemasson I. Polakowski N.J. Laybourn P.J. Nyborg J.K. J. Biol. Chem. 2002; 277: 49459-49465Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). However, no direct interactions between the AP-1 factors and Tax have been characterized, suggesting that AP-1 is obviously involved in basal transcription of the HTLV-I genome. Recently, we characterized a novel HTLV-I protein encoded by the complementary strand of the viral RNA genome (44Larocca D. Chao L.A. Seto M.H. Brunck T.K. Biochem. Biophys. Res. Commun. 1989; 163: 1006-1013Crossref PubMed Scopus (126) Google Scholar) and named by us HBZ for HTLV-I bZIP factor (45Gaudray G. Gachon F. Basbous J. Biard-Piechaczyk M. Devaux C. Mesnard J.-M. J. Virol. 2002; 76: 12813-12822Crossref PubMed Scopus (395) Google Scholar). The structure of HBZ resembles that of a prototypical bZIP transcription factor, with an N-terminal transcriptional activation domain and a C-terminal bZIP domain. HBZ acts as a repressor of viral transcription by forming heterodimers with CREB-2 that are no longer able to bind to the viral CRE (45Gaudray G. Gachon F. Basbous J. Biard-Piechaczyk M. Devaux C. Mesnard J.-M. J. Virol. 2002; 76: 12813-12822Crossref PubMed Scopus (395) Google Scholar). Here, through a combination of in vitro and in vivo experiments, we demonstrate that HBZ also interacts with two other bZIP factors, JunB and c-Jun. Our analysis reveals that HBZ decreases the DNA-binding activity of JunB and c-Jun, as is the case for CREB-2. In addition, we show that c-Jun is no longer capable of activating the basal expression of the HTLV-I promoter in the presence of HBZ in vivo. Our results support the hypothesis that HBZ could be a negative modulator of the Tax effect by controlling Tax expression at the transcriptional level and by attenuating activation of AP-1 by Tax. Yeast Two-hybrid Screen—MT2 cDNA fused to the Gal4 activation domain of the pGAD10 vector (35Gachon F. Péléraux A. Thébault S. Dick J. Lemasson I. Devaux C. Mesnard J.-M. J. Virol. 1998; 72: 8332-8337Crossref PubMed Google Scholar) was screened using the HBZ bZIP domain as a bait fused to the Gal4 DNA-binding domain of the pGBT9 vector. The two-hybrid screen was performed with the Saccharomyces cerevisiae HF7c reporter strain (3 × 106 clones were screened) as already described (35Gachon F. Péléraux A. Thébault S. Dick J. Lemasson I. Devaux C. Mesnard J.-M. J. Virol. 1998; 72: 8332-8337Crossref PubMed Google Scholar). Immunoprecipitation and Western Blotting—For immunoprecipitation of HBZ, plasmid pCI-HBZ, pcDNA-JunB, or pcDNA-c-Jun was cotransfected into 293T cells using the FuGENE 6 transfection reagent (Roche Applied Science) as recommended by the manufacturer. Cultures were grown to saturation in Dulbecco's modified Eagle's medium supplemented with 1% penicillin and streptomycin antibiotic mixture and 10% fetal calf serum. After centrifugation, cells were lysed in 50 mm Tris-HCl (pH 8), 100 mm NaCl, 1 mm EDTA, 1 mm MgCl2, and 1% Triton X-100, and proteins were immunoprecipitated from protein extracts (2.5 mg of total proteins) by incubation at 4 °C for 2 h using rabbit anti-HBZ antiserum (45Gaudray G. Gachon F. Basbous J. Biard-Piechaczyk M. Devaux C. Mesnard J.-M. J. Virol. 2002; 76: 12813-12822Crossref PubMed Scopus (395) Google Scholar), followed by another incubation with protein A/G-agarose for 1 h. Bound fractions were washed six times and then electrophoresed on SDS-10% polyacrylamide gel and analyzed by immunoblotting as described (46Thébault S. Gachon F. Lemasson I. Devaux C. Mesnard J.-M. J. Biol. Chem. 2000; 275: 4848-4857Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Mouse anti-JunB and anti-c-Jun antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA) and Oncogene Research Products (San Diego, CA), respectively. Fluorescence Microscopy Analysis—COS-7 cells were transfected with 1 μg of plasmid pEGFP-HBZ, expressing a green fluorescent protein-HBZ fusion protein (45Gaudray G. Gachon F. Basbous J. Biard-Piechaczyk M. Devaux C. Mesnard J.-M. J. Virol. 2002; 76: 12813-12822Crossref PubMed Scopus (395) Google Scholar), and 1 μg of pcDNA-JunB or pcDNA-c-Jun. Cells were cultured on glass slides and then analyzed by fluorescence 24 h after transfection as described (35Gachon F. Péléraux A. Thébault S. Dick J. Lemasson I. Devaux C. Mesnard J.-M. J. Virol. 1998; 72: 8332-8337Crossref PubMed Google Scholar, 38Gachon F. Gaudray G. Thebault S. Basbous J. Koffi A.J. Devaux C. Mesnard J.-M. FEBS Lett. 2001; 502: 57-62Crossref PubMed Scopus (34) Google Scholar). Jun proteins were detected using the above-cited mouse antibodies and secondary goat anti-mouse IgG antibody coupled to rhodamine (Pierce). Analysis of the green, red, and merged fluorescence was carried out using a fluorescence microscope. Transfections and Luciferase Assays—CEM cells were transiently cotransfected according to a previously published procedure (46Thébault S. Gachon F. Lemasson I. Devaux C. Mesnard J.-M. J. Biol. Chem. 2000; 275: 4848-4857Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). 5 μgof pcDNA3.1-lacZ (β-galactosidase-containing reference plasmid) was included in each transfection to control transfection efficiency. The total amount of DNA in each transfection was the same, the balance being made up with empty plasmids. Cell extracts equalized for protein content were used for luciferase and β-galactosidase assays. For the assays with the Gal4-binding site promoter-reporter plasmid, JunB or c-Jun fused in-frame with the DNA-binding domain of Gal4 into vector pBIND was cotransfected into CEM cells in the presence of the luciferase reporter plasmid pG5luc, containing five Gal4-binding sites upstream of a minimal TATA box. Microwell Colorimetric AP-1 Assay—15 μg of nuclear cell extracts was incubated with 30 μl of binding buffer (10 mm HEPES (pH 7.5), 8 mm NaCl, 12% glycerol, 0.2 mm EDTA, and 0.1% bovine serum albumin) in microwells coated with probes containing the AP-1 site (Trans-AM™ AP-1, Active Motif Europe, Rixensart, Belgium). After a 1-h incubation at room temperature, microwells were washed three times with phosphate-buffered saline and 0.1% Tween 20. The AP-1-bound complexes were detected with mouse anti-c-Jun or anti-JunB antibody and peroxidase-conjugated goat antibodies. For colorimetric detection, tetramethylbenzidine was incubated at room temperature before addition of stop solution. Optical density was read at 450 nm using a 620-nm reference wavelength with a Tecan microplate reader. Streptavidin-Biotin Complex Assay—A biotinylated oligonucleotide containing the AP-1 site (5′-cgcttgaTGAGTCAgccggaa-3′) was annealed with its complementary oligonucleotide to form a double-stranded DNA. Biotinylated double-stranded DNA was incubated with bacterially produced proteins in 200 μl of binding buffer containing 50 mm Tris (pH 7.5), 500 mm NaCl, 1 mm EDTA, 1 mm dithiothreitol, 5 mm MgCl2, 0.1% Triton, 5% glycerol, and 10 mg/ml bovine serum albumin at room temperature for 2 h before addition of streptavidin beads (Pierce). After a 1-h incubation at 4 °C, the beads were extensively washed with binding buffer without bovine serum albumin. The proteins that remained bound to the beads were eluted in SDS loading buffer and analyzed by Western blotting. Binding of HBZ to JunB and c-Jun—To identify proteins that interact with HBZ, a two-hybrid approach in yeast was performed with the bZIP domain of HBZ (amino acids 123–209) as a bait. The yeast strain expressing the HBZ bZIP domain fused to the DNA-binding domain of Gal4 was transformed with a library of cDNA derived from the HTLV-I-infected MT2 cell line fused to the transcriptional activation domain of Gal4 (35Gachon F. Péléraux A. Thébault S. Dick J. Lemasson I. Devaux C. Mesnard J.-M. J. Virol. 1998; 72: 8332-8337Crossref PubMed Google Scholar). Several positive clones were found to correspond to two members of the Jun family, JunB and c-Jun (Fig. 1, A and B). The leucine zipper domain was, as expected, necessary for the interaction between both cellular factors and HBZ because truncated mutants of JunB (amino acids 1–273) and c-Jun (amino acids 1–287) lacking the leucine zipper did not bind to HBZ (Fig. 1C). To be sure of the specificity of the test in yeast, JunB and c-Jun were also tested in the presence of an unrelated protein, lamin, which was unable to interact with either of the two proteins (Fig. 1C). Moreover, HBZ was unable to form homodimers in yeast, as shown in Fig. 1C. To eliminate the possibility that the binding of HBZ to JunB or c-Jun may be indirect and dependent on yeast components, fusion proteins of either JunB or c-Jun with glutathione S-transferase (GST) were produced in Escherichia coli, and their binding to [35S]methionine-labeled HBZ, produced in rabbit reticulocyte lysate, was analyzed. As shown in Fig. 2A, HBZ bound to JunB (lane 3) or c-Jun (lane 6) fused to GST, but not to GST alone (lanes 2 and 5). Taken together, our results show that HBZ interacts with JunB and c-Jun in vitro.Fig. 2HBZ interacts with JunB and c-Jun. A, in vitro binding assays carried out with HBZ and JunB or c-Jun. Equal amounts of GST (lane 2) and GST-JunB (lane 3) immobilized on glutathione-Sepharose beads were incubated with 35S-labeled HBZ (lane 1), and bound proteins were analyzed by SDS-PAGE and autoradiography. The same assay was carried out with GST-c-Jun (lane 6). B, HBZ-JunB association in vivo. 293T cells were transfected with HBZ expression vector pCI-HBZ (lanes 1–3) or the corresponding empty vector (lanes 4 and 5) and plasmid pcDNA-JunB (lanes 1–5). Proteins from total lysates were directly probed with mouse anti-JunB antibody (lanes 1 and 4) or immunoprecipitated with rabbit anti-HBZ antibody (lanes 2 and 5) or preimmune serum (PI; lane 3), followed by Western analysis with anti-JunB antibody (lanes 2, 3, and 5). Proteins from the total lysate of the HTLV-I-infected C8166 cells (lanes 8 and 9) or uninfected CEM cells (lane 7) were also immunoprecipitated with anti-HBZ antibody (lane 7 and 8) or preimmune serum (lane 9), and immunoprecipitated proteins were analyzed with anti-JunB antibody (lanes 7–9). Lane 6 corresponds to C8166 proteins directly probed with anti-JunB antibody. C, HBZ-c-Jun association in vivo. The experiment was performed as described for HBZ and JunB, but with mouse anti-c-Jun antibody.View Large Image Figure ViewerDownload Hi-res image Download (PPT) HBZ Co-immunoprecipitates with JunB and c-Jun in Vivo—To examine the in vivo interaction between HBZ and JunB, we coexpressed both proteins in 293T cells. Cell extracts were then immunoprecipitated with either rabbit anti-HBZ antiserum or preimmune serum from the same rabbit (45Gaudray G. Gachon F. Basbous J. Biard-Piechaczyk M. Devaux C. Mesnard J.-M. J. Virol. 2002; 76: 12813-12822Crossref PubMed Scopus (395) Google Scholar), followed by Western analysis using anti-JunB monoclonal antibody. By this approach, JunB was found in the immunoprecipitate with anti-HBZ antiserum (Fig. 2B, lane 2), but not in the control immunoprecipitate (lane 3). When the same experiment was performed with extracts of 293T cells transfected only with JunB, no protein was found in the immunoprecipitate with anti-HBZ antiserum (Fig. 2B, lane 5), confirming the specificity of the interaction between HBZ and JunB. To confirm that both endogenous HBZ and JunB could also associate in infected cells, immunoprecipitation from a cell lysate of the HTLV-I-infected C8166 cell line expressing HBZ (45Gaudray G. Gachon F. Basbous J. Biard-Piechaczyk M. Devaux C. Mesnard J.-M. J. Virol. 2002; 76: 12813-12822Crossref PubMed Scopus (395) Google Scholar) was performed with anti-HBZ antiserum and probed with anti-JunB antibody. As expected, this approach revealed the presence of a complex between HBZ and JunB in C8166 extracts (Fig. 2B, lane 8). By the same approach, such a complex was also detected in MT4 cells (data not shown), another HTLV-I-infected cell line, but not in the uninfected CEM cell line (Fig. 2B, lane 7). Altogether, these results clearly demonstrate that HBZ and JunB interact in vivo. As shown in Fig. 2C, the same co-immunoprecipitation experiments were carried out with c-Jun. The data obtained show that HBZ also bound to c-Jun in vivo. HBZ Colocalizes with JunB and c-Jun in the Nucleus—The HBZ-JunB interaction was further investigated in COS-7 cells by confirming the co-distribution of the proteins by immunofluorescence microscopy. In transient transfection assays, we found that HBZ tagged with green fluorescent protein exhibited a granular distribution in the nucleus (Fig. 3a), as described previously (45Gaudray G. Gachon F. Basbous J. Biard-Piechaczyk M. Devaux C. Mesnard J.-M. J. Virol. 2002; 76: 12813-12822Crossref PubMed Scopus (395) Google Scholar). On the other hand, although JunB was also localized in the nucleus, here it showed a diffuse pattern (Fig. 3b). However, when JunB was cotransfected with HBZ, both proteins colocalized in the nuclear spots formed by the viral protein (Fig. 3, d–f). The same approach was also performed with c-Jun in COS-7 cells. Unlike JunB, c-Jun was distributed in either a diffuse or punctate staining pattern throughout the nucleus (Fig. 3c). Although we have already observed the existence of these two different staining patterns for other cellular proteins (47Thébault S. Basbous J. Gay B. Devaux C. Mesnard J.-M. Eur. J. Cell Biol. 2000; 79: 834-838Crossref PubMed Scopus (17) Google Scholar), we do not know the exact mechanisms of their formation. However, like JunB, after cotransfection with HBZ, c-Jun localized with HBZ in the nuclear spots (Fig. 3, g–i). These observations support the notion that HBZ and either JunB or c-Jun colocalize in the nucleus, but also show that HBZ entails an intranuclear redistribution of both JunB and c-Jun. Taken together with co-immunoprecipitation, our results strongly suggest that HBZ and either JunB or c-Jun can interact with each other in vivo. HBZ Decreases the DNA-binding Activity of c-Jun—We next examined the effect of HBZ on transcription from the collagenase promoter containing a canonical AP-1 element. The reporter plasmid was first cotransfected into CEM cells with pcDNA-c-Jun in the presence of increasing amounts of the HBZ expression vector pCI-HBZ. As shown in Fig. 4A, c-Jun alone activated expression of the luciferase reporter gene by 34-fold; but this stimulation was inhibited in the presence of HBZ, and this inhibitory effect was proportional to the quantity of transfected HBZ plasmid. Moreover, we checked that the level of c-Jun expressed from pcDNA-c-Jun was not significantly decreased by the expression of HBZ (data not shown). Thus, HBZ negatively modulates c-Jun activity on a promoter containing the AP-1 element. Two simple mechanisms may explain the repression of c-Jun activity by HBZ. First, HBZ and c-Jun may form heterodimers that may not bind to the AP-1 site. If the heterodimer does not bind to the AP-1 motif, obviously it cannot stimulate transcription. Second, for unknown reasons, the complex between HBZ and c-Jun bound to the AP-1 site is much less active than the c-Jun homodimers. To discriminate between these two possibilities, we fused the full-length c-Jun protein to the DNA-binding domain of the yeast transcription factor Gal4 and used this chimera to analyze the effect of HBZ on c-Jun directly bound to a promoter bearing Gal4-binding sites. The Gal4-c-Jun chimera was assayed using the reporter plasmid pG5luc, encoding luciferase under the control of five Gal4-binding sites upstream of a minimal TATA box. After cotransfection of CEM cells with pG5luc and Gal4-c-Jun, luciferase expression was stimulated by 5-fold (Fig. 4B). When increasing amounts of HBZ were added, luciferase expression was stimulated by 23-fold (Fig. 4B). These results show that HBZ is no longer capable of inhibiting c-Jun activity when c-Jun is stably bound to the promoter. Consequently, they also suggest that HBZ could negatively regulate c-Jun activity by forming heterodimers that are unable to form stable complexes with the AP-1 site. To confirm this hypothesis, we compared c-Jun DNA-binding activity in the presence and absence of HBZ. The first series of experiments were based on the analysis of c-Jun DNA-binding activity by the microwell colorimetric assay from Active Motif Europe (48Renard P. Ernest I. Houbion A. Art M. Le Calvez H. Raes M. Remacle J. Nucleic Acids Res. 2001; 29: E21Crossref PubMed Scopus (343) Google Scholar). Nuclear cell extracts of 293T cells transfected with c-Jun alone or associated with HB" @default.
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• W2068100440 date "2003-10-01" @default.
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• W2068100440 title "The HBZ Factor of Human T-cell Leukemia Virus Type I Dimerizes with Transcription Factors JunB and c-Jun and Modulates Their Transcriptional Activity" @default.
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• W2068100440 doi "https://doi.org/10.1074/jbc.m307275200" @default.
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