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• W2087523716 abstract "The hepatitis B virus pX protein is a potent transcriptional activator of viral and cellular genes whose mechanism of action is poorly understood. Here we show that pX dramatically stimulates in vitro DNA binding of a variety of cellular proteins that contain basic region/leucine zipper (bZIP) DNA binding domains. The basis for increased DNA binding is a direct interaction between pX and the conserved bZIP basic region, which promotes bZIP dimerization and the increased concentration of the bZIP homodimer then drives the DNA binding reaction. Unexpectedly, we found that the DNA binding specificity of various pX-bZIP complexes differs from one another and from that of the bZIP itself. Thus, through recognition of the conserved basic region, pX promotes dimerization, increases DNA binding, and alters DNA recognition. These properties of pX are remarkably similar to those of the human T-cell lymphotrophic virus type I Tax protein. Although Tax and pX are not homologous, we show that the regions of the two proteins that stimulate bZIP binding contain apparent metal binding sites. Finally, consistent with thisin vitro activity, we provide evidence that both Tax and pX activate transcription in vivo, at least in part, by facilitating occupancy of bZIPs on target promoters. The hepatitis B virus pX protein is a potent transcriptional activator of viral and cellular genes whose mechanism of action is poorly understood. Here we show that pX dramatically stimulates in vitro DNA binding of a variety of cellular proteins that contain basic region/leucine zipper (bZIP) DNA binding domains. The basis for increased DNA binding is a direct interaction between pX and the conserved bZIP basic region, which promotes bZIP dimerization and the increased concentration of the bZIP homodimer then drives the DNA binding reaction. Unexpectedly, we found that the DNA binding specificity of various pX-bZIP complexes differs from one another and from that of the bZIP itself. Thus, through recognition of the conserved basic region, pX promotes dimerization, increases DNA binding, and alters DNA recognition. These properties of pX are remarkably similar to those of the human T-cell lymphotrophic virus type I Tax protein. Although Tax and pX are not homologous, we show that the regions of the two proteins that stimulate bZIP binding contain apparent metal binding sites. Finally, consistent with thisin vitro activity, we provide evidence that both Tax and pX activate transcription in vivo, at least in part, by facilitating occupancy of bZIPs on target promoters. Hepatitis B virus (HBV) 1The abbreviations used are: HBV, hepatitis B virus; aa, amino acid(s); kb, kilobase pair(s); CMV, cytomegalovirus; CREB, cyclic AMP response element-binding protein; CRE, CREB-responsive element; HTLV-I, human T-cell lymphotrophic virus, type I; IPTG, isopropyl-1-thio-β-d-galactopyranoside; PMSF, phenylmethylsulfonyl fluoride; PCR, polymerase chain reaction; GST, glutathione S-transferase; BSA, bovine serum albumin; bZIP, basic region/leucine zipper is the major cause of acute and chronic hepatitis and has been implicated in the initial stage of HBV-related hepatocarcinogenesis (1Koike K. Shirakata Y. Yaginuma K. Arii M. Takada S. Nakamura I. Hayashi Y. Kawad M. Kobayashi M. Mol. Biol. Med. 1989; 6: 151-160PubMed Google Scholar, 2Zahm P. Hofschneider P.H. Koshy R. Oncogene. 1988; 3: 169-177PubMed Google Scholar, 3Sherker A.H. Marion P.L. Annu. Rev. Microbiol. 1991; 45: 475-508Crossref PubMed Google Scholar, 4Koike K. Moriya K. Iino S. Yotsuyanagi H. Endo Y. Miyamura T. Kurokawa K. Hepatology. 1994; 19: 810-819Crossref PubMed Scopus (257) Google Scholar, 5Robinson W.S. Annu. Rev. Med. 1994; 45: 297-323Crossref PubMed Scopus (167) Google Scholar). The HBV genome is 3.2 kb in length and contains four open reading frames: pre-S/S, which encodes the surface antigens; core/e, which encodes the core protein; pol, which encodes the viral reverse transcriptase; and the X protein (pX) (for review, see Ref. 6Ganem D. Varmus H.E. Annu. Rev. Biochem. 1987; 56: 651-693Crossref PubMed Scopus (821) Google Scholar). pX is required for viral infection (7Chen H.-S. Kaneko S. Girones R. Anderson R.W. Hornbuckle W.E. Tennan B.C. Cote P.J. Gerin J.L. Purcell R.H. Miller R.H. J. Virol. 1993; 67: 1218-1226Crossref PubMed Google Scholar, 8Zoulim F. Saputelli J. Seeger C. J. Virol. 1994; 68: 2026-2030Crossref PubMed Google Scholar), can transform rodent cells (3Sherker A.H. Marion P.L. Annu. Rev. Microbiol. 1991; 45: 475-508Crossref PubMed Google Scholar, 4Koike K. Moriya K. Iino S. Yotsuyanagi H. Endo Y. Miyamura T. Kurokawa K. Hepatology. 1994; 19: 810-819Crossref PubMed Scopus (257) Google Scholar,9Shirakata Y. Kawada M. Fujiki Y. Sano H. Oda L. et al.Jpn. J. Cancer Res. 1989; 80: 617-621Crossref PubMed Scopus (168) Google Scholar, 10Hohne M. Schaefer S. Seifer M. Feitelston M.A. Paul D. Gerlich W.H. EMBO J. 1990; 9: 1137-1145Crossref PubMed Scopus (230) Google Scholar), and can cause hepatocellular carcinoma in transgenic mice (11Kim C.-M. Koike K. Saito I. et al.Nature. 1991; 351: 317-320Crossref PubMed Scopus (1056) Google Scholar). HBV pX, a 154-aa protein, is a potent transcriptional activator required for efficient viral gene expression. Like several other viral activators, pX activity is promiscuous; in addition to the cognate HBV promoter, pX can activate transcription from a diverse set of cellular and viral promoters that appear to have little in common (12Twu J.-S. Schloemer R.H. J. Virol. 1987; 61: 3448-3453Crossref PubMed Google Scholar, 13Spandau D.F. Lee C.-H. J. Virol. 1988; 62: 427-434Crossref PubMed Google Scholar, 14Twu J.-S. CHu K. Robinson W.S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5168-5172Crossref PubMed Scopus (92) Google Scholar, 15Aufiero B. Schneider R.J. EMBO J. 1990; 9: 497-504Crossref PubMed Scopus (130) Google Scholar, 16Hu K.-Q. Vierling J.M. Siddiqui A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7140-7144Crossref PubMed Scopus (63) Google Scholar, 17Seto E. Mitchell P.J. Benedict Yen T.S. Nature. 1990; 344: 72-74Crossref PubMed Scopus (213) Google Scholar, 18Unger T. Shaul Y. EMBO J. 1990; 9: 1889-1895Crossref PubMed Scopus (77) Google Scholar, 19Zhou D.-X. Taraboulos A. Ou J.-H. Yen T.S.B. J. Virol. 1990; 64: 4025-4028Crossref PubMed Google Scholar, 20Lopez-Cabrera M. Letovsky J. Hu K.-Q. Siddiqui A. Virology. 1991; 183: 825-829Crossref PubMed Scopus (109) Google Scholar, 21Hu K.-Q. Yu C.-H. Vierling J.M. Proc. Natl. Acad. Sci U. S. A. 1992; 89: 11441-11445Crossref PubMed Scopus (43) Google Scholar, 22Kekule A.S. Lauer U. Weiss L. Luber B. Hofschneider P.H. Nature. 1993; 361: 742-745Crossref PubMed Scopus (336) Google Scholar, 23Natoli G. Avantaggiati M.L. Chirillo P. Costanzo A. Artini M. Balsano C. Levrero M. Mol. Cell. Biol. 1994; 14: 989-998Crossref PubMed Scopus (125) Google Scholar, 24Henkler F.F. Koshy R. J. Viral Hepat. 1996; 3: 109-121Crossref PubMed Scopus (90) Google Scholar). The mechanism by which pX activates transcription is controversial; it has been proposed that pX acts through various protein kinase signal transduction pathways (25Lucito R. Schneider R.J. J. Virol. 1992; 66: 983-991Crossref PubMed Google Scholar, 26Cross J.C. Weng P. Rutter W.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8078-8082Crossref PubMed Scopus (146) Google Scholar, 27Benn J. Schneider R.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10350-10354Crossref PubMed Scopus (404) Google Scholar, 28Natoli G. Avantaggiati M.L. Chirillo P. Puri P. Ianni A. Balsano C. Levrero M. Oncogene. 1994; 9: 2837-2843PubMed Google Scholar), is itself a protein kinase (29Wu J.Y. Zhou Z.Y. Judd A. Cartwright C.A. Robinson W.S. Cell. 1990; 16: 687-695Abstract Full Text PDF Scopus (89) Google Scholar), has a ribo/deoxy ATPase activity (30De-Medina T.I. Haviv N.S. Shaul Y. Virology. 1994; 202: 401-407Crossref PubMed Scopus (32) Google Scholar), and is a protease inhibitor (31Takada S. Kido H. Fukutomi A. Mori T. Koike K. Oncogene. 1994; 9: 341-348PubMed Google Scholar). We have noted several similarities between HBV pX and the Tax protein of human T-cell lymphotrophic virus, type I (HTLV-I). First, like pX, Tax activates transcription from its own promoter as well as heterologous promoters (for review, see Ref. 32Yoshida M. Trends Microbiol. 1993; 1: 131-135Abstract Full Text PDF PubMed Scopus (77) Google Scholar). Second, the HTLV-I long terminal repeat and the HBV enhancer contain binding sites for ATF proteins (ATFs) (33Maguire H.F. Hoeffler J.P. Siddiqui A. Science. 1991; 252: 842-844Crossref PubMed Scopus (379) Google Scholar, 34Beimling P. Moelling K. Oncogene. 1992; 7: 257-262PubMed Google Scholar, 35Xu X. Kang S.H. Heidenreich O. Brown D.A. Neremberg M.I. Virology. 1996; 218: 362-371Crossref PubMed Scopus (13) Google Scholar), an extensive family of cellular transcription factors that contain homologous basic region/leucine zipper (bZIP) DNA binding domains (36Hai T. Liu F. Coukos W.J. Green M.R. Genes Dev. 1989; 3: 2083-2090Crossref PubMed Scopus (760) Google Scholar). These viral ATF binding sites have been directly implicated in the Tax (reviewed in Ref. 37Feuer G. Chen I.S. Biochim. Biophys. Acta. 1992; 1114: 223-233PubMed Google Scholar) and pX (33Maguire H.F. Hoeffler J.P. Siddiqui A. Science. 1991; 252: 842-844Crossref PubMed Scopus (379) Google Scholar) transcriptional responses. Third, like Tax, pX is present in the nucleus of expressing cells (10Hohne M. Schaefer S. Seifer M. Feitelston M.A. Paul D. Gerlich W.H. EMBO J. 1990; 9: 1137-1145Crossref PubMed Scopus (230) Google Scholar, 38Doria M. Klein N. Lucito R. Schneider R.J. EMBO J. 1995; 14: 4747-4757Crossref PubMed Scopus (275) Google Scholar). Finally, and of particular significance, Tax can substitute for pX in transcriptional activation of the HBV enhancer (2Zahm P. Hofschneider P.H. Koshy R. Oncogene. 1988; 3: 169-177PubMed Google Scholar,39Faktor O. Shaul Y. Oncogene. 1990; 5: 867-872PubMed Google Scholar, 40Marriot S.J. Lee T.H. Slagle B.L. Butel J.S. Virology. 1996; 224: 206-213Crossref PubMed Scopus (5) Google Scholar). Taken together, these observations suggest that pX and Tax may stimulate transcription by a common mechanism. Recent studies have helped clarify the mechanism by which Tax functions. Tax can dramatically increase the in vitro DNA binding of proteins containing bZIP DNA binding domain (41Armstrong A.P. Franklin A.A. Uittenbogaard M.N. Giebler H.A. Nyborg J.K. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7303-7307Crossref PubMed Scopus (104) Google Scholar, 42Franklin A.A. Kubik M.F. Uittenbogaard M.N. Brauweiler A. Utaisincharoen P. Matthews M.-A.H. Dynan W.S. Hoeffler J.P. Nyborg J.K. J. Biol. Chem. 1993; 268: 21225-21231Abstract Full Text PDF PubMed Google Scholar, 43Wagner S. Green M.R. Science. 1993; 262: 395-399Crossref PubMed Scopus (290) Google Scholar, 44Baranger A.M. Palmer C.R. Hamm M.K. Gleber H.A. Brauweiler A. Nyborg J.K. Schepartz A. Nature. 1995; 376: 606-608Crossref PubMed Scopus (162) Google Scholar, 45Perini G. Wagner S. Green M.R. Nature. 1995; 376: 602-605Crossref PubMed Scopus (135) Google Scholar). bZIP domains comprise a leucine-rich dimerization motif and a basic region that mediates DNA contact (46Blatter E.E. Ebright Y.W. Ebright R.H. Nature. 1992; 359: 650-652Crossref PubMed Scopus (71) Google Scholar, 47Ellenberger T.E. Brandl C.J. Struhl K. Harrison S.C. Cell. 1992; 71: 1223-1237Abstract Full Text PDF PubMed Scopus (825) Google Scholar, 48Kim J. Tzamarias D. Ellenberger T. Harrison S.C. Struhl K. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4513-4517Crossref PubMed Scopus (71) Google Scholar, 49Vinson C.R. Hai T. Boyd S.M. Genes Dev. 1993; 7: 1047-1058Crossref PubMed Scopus (294) Google Scholar). This DNA binding increase occurs by a mechanism in which Tax promotes dimerization of the bZIP domain in the absence of DNA and the elevated concentration of the bZIP homodimer then drives the DNA binding reaction (43Wagner S. Green M.R. Science. 1993; 262: 395-399Crossref PubMed Scopus (290) Google Scholar, 44Baranger A.M. Palmer C.R. Hamm M.K. Gleber H.A. Brauweiler A. Nyborg J.K. Schepartz A. Nature. 1995; 376: 606-608Crossref PubMed Scopus (162) Google Scholar). Here we show that pX functions in a manner remarkably similar to Tax. Sequences were as follows: collagenase TRE, 5′-AGCTTTGACTCATCCGGA-3′; CRE, 5′-TCCTAAGTGACGTCAGTGGAA-3′; C/EBP binding site, 5′-TGCAGATTGCGCAAT CTGCA-3′; OR1 site of the λ phage PRM/PR promoter, 5′-TATCACCGCCAGAGGTA-3′. pX cDNA was cloned in the pRSET-C vector (Invitrogen) and expressed as a His-tagged derivative in the BL21(pLysE) Escherichia coli strain. BL21 cells were grown at 30 °C in the presence of 50 μmZnCl2, and pX expression was induced with 0.5 mm IPTG for 2 h. Cells were harvested and resuspended in lysis buffer (1 m NaCl, 20% glycerol, 0.1% Tween 20, 20 mm Tris-HCl, pH 8, 20 μmZnCl2, 1 mm PMSF, 10 mmβ-mercaptoethanol, 40 mm imidazole), frozen on dry ice, and then thawed on ice and sonicated. The bacterial lysate was centrifuged for 30 min at 12,000 rpm. The supernatant was incubated with NTA-Ni2+-agarose beads (Qiagen) for 4 h. Beads were washed four times with lysis buffer. pX protein was eluted with 0.5 m imidazole, pH 7.5, and dialyzed twice against buffer X (0.2 m NaCl, 20% glycerol, 20 mm Hepes, pH 8, 20 mm ZnCl2, 5 mmβ-mercaptoethanol, 0.1% Tween 20, 1 mm PMSF). pX was first cloned in the pGEX-CS vector (50Parks T.D. Leuther K.K. Howard E.D. Johnston S.A. Dougherty W.G. Anal. Biochem. 1994; 216: 413-417Crossref PubMed Scopus (258) Google Scholar) and subsequently digested with the appropriate enzymes to generate CΔ1, CΔ2, and CΔ3 mutants. To generate CΔ1, pX vector was digested with HincII andHindIII, treated with Klenow, and self-ligated. CΔ2 and CΔ3 were obtained in the same way except that CSP1/HindIII and AatII/HindIII were used, respectively. Ligation products were transformed in the E. coli BL21 (pLysE) strain. CΔ4 was instead obtained by PCR amplification of the region between aa 1 and 36. The primers were oligo X5′ (CAT GCC ATG GCT GCT CGG GTG TGC TGC) and oligo XΔ4 (CGG AAT TCT TAT TAG AGA GTC CCA ACC GGC CCG CA). The PCR product was then digested with NcoI andEcoRI and cloned in pGEX-CS vector. pX N-terminal deletions were also obtained by PCR amplification. NΔ1 sequences were oligo X N1 (CAT GCC ATG CGT CCC GTC GGC GCT GAA TCC) and oligo X3′ (CGG AAT TCT TAT TTA GGC AGA GGT GAA AAA CAA AC). NΔ2 sequences were oligo X N2 (CAT GCC ATG CTC TCT TTA CGC GGT CTC CCC) and oligo X3. PCR products were digested with NcoI/EcoRI and cloned in the pGEX-CS vector. Protein expression was induced with 0.5 mmIPTG for 2 h at 30 °C. Protein mutants were purified on a glutathione affinity column and then dialyzed against buffer X. The GST moiety was removed by cleaving the GST fusion proteins with TEV protease (Life Technologies, Inc.) and by incubating the protein with fresh glutathione-agarose beads. Beads were spun down, and the supernatant containing the pX proteins was dialyzed against buffer X. Tax mutants were obtained by PCR amplification. Oligonucleotides used to generate Tax constructs were as follows: Tax wt: oligo T5′ (TCC AAC AAC ATG GCC CAC TCC CCA GGG TTT GGA) and oligo T3′ (AAA GGG GGA TCC TCA GAC TTC TGT TTC TCG GAA ATG); Tax CΔ1:oligo T5′ and oligo TD31 (AAA GGG GGA TCC TCA ATG AAA GGA AGA GTA CTG TAT GAG); Tax CΔ2: oligo T5′ and oligo TD32 (AAA GGG GGA TCC TCA GCC ATC GGT AAA TGT CCA AAT AAG); Tax CΔ3: oligo T5′ and oligo TD33 (AAA GGG GGA TCC TCA CCC TGT GGT GAG GGA AAT TTT ATA); Tax CΔ4: oligo T5′ and oligo TD34 (AAA GGG GGA TCC TCA GCA GAC AAC GGA GCC TCC CCA GAG); Tax CΔ5: oligo T5′ and oligo TD35 (AAA GGG GGA TCC TCA GGG TGG AAT GTT GGG GGT TGT ATG); Tax CΔ6: oligo T5′ and oligo TD36 (AAA GGG GGA TCC TCA CTG ATG CTC TGG ACA GGT GGC CAG). PCR products were digested with NcoI and BamHI and cloned in pGEX-CS vector. Proteins were expressed and purified as described previously (43Wagner S. Green M.R. Science. 1993; 262: 395-399Crossref PubMed Scopus (290) Google Scholar). cDNA sequences of CREB (aa 23–341), ATF-1 (aa 57–271), and ATF-2 (aa 144–505) were cloned in pGEX vectors and expressed as GST fusion proteins in E. coli. The C/EBP Δ1–2 derivative (a gift from P. Rorth) was expressed by IPTG induction. After sonication, cell debris was removed by centrifugation and the supernatant used in gel-shift assays. Histidine-tagged JUN (aa 225–334) was obtained from T. Curran. Epstein-Barr virus ZTA peptide (aa 175–229) containing the DNA binding domain only was a gift from M. Carey. The FOS-ZTA chimera was obtained from D. S. Hayward. The construct containing the c-Fos basic region (aa 133–162) and the ZTA coiled-coil domain (aa 197–229) was PCR-amplified and cloned in the pGEX-2T vector. λ-ZIP protein was obtained by IPTG induction of JH372(pJH370) E. coli (a gift from R. T. Sauer). Crude extracts were prepared and dialyzed against 1× buffer D. The synthetic peptide BR-CC (NH-ALKRARNTEAARRSRARKLQRMKQLEDVKELEEKLKALEEKLKALEEKLKALG-COOH; Ref. 51O'Neil K.T. Hoess R.H. DeGrado W.F. Science. 1990; 249: 774-778Crossref PubMed Scopus (241) Google Scholar) was kindly provided by W. DeGrado, and the basic region peptide br1s (NH4-ALKRARNTEA-ARRSRARKLQRMKQGGC; Ref. 52Talanian R.V. McKnight C.J. Kim P.S. Science. 1990; 249: 769-771Crossref PubMed Scopus (267) Google Scholar) was a generous gift of P. Kim. Each reaction mixture contained 1 μg of BSA, 1 μg of poly(dI-dC), 0.5× buffer D, and 0.1 ng of a32P-labeled CRE oligoduplex. The amount of DNA-binding protein and pX used is indicated in the figure legends. The reaction products were analyzed on a 5% polyacrylamide, 0.5× TBE gel except where otherwise noted. The indicated amounts of GCN4 polypeptide were incubated with 200 ng of BSA or 100 ng of pX. Cross-linking was performed by adding glutaraldehyde (Fisher) to a final concentration of 0.02% and incubated for 30 min at room temperature. Reactions were terminated by addition of 0.5 μl of 0.5m Tris, pH 8.0. Monomers and dimers were separated on a 15% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. The filter was probed with an anti-GCN4 polyclonal serum (kindly provided by Peter Kim). 20 ng of H6-T7tag-pX were incubated for 10 min with glutaraldehyde to a final concentration of 0.01%. Multimers were separated on a 10% SDS-polyacrylamide gel, transferred to a nitrocellulose membrane, and probed with an anti-T7tag monoclonal antibody (Novagen). Purified proteins were separated on an SDS-polyacrylamide gel and transferred to an Immobilon membrane (Millipore). The filter was preincubated in far-Western buffer (150 mm KCl, 20 mm Hepes, pH 7.5, 5 mmMgCl2 0.5 mm EDTA, 0.05% Nonidet P-40, 1 mm dithiothreitol, 1 mm PMSF, and 5% BSA). After 2 h of incubation, [35S]methionine-labeled pX was added and incubation continued for 4 h. The membrane was washed twice with far-Western buffer for 15 min, dried, and autoradiographed. HepG2 cell were maintained in Dulbecco's modified Eagle's medium containing nonessential amino acids (Life Technologies, Inc.), 2 mml-glutamine (Life Technologies, Inc.), antibiotics, and 10% FBS (HyClone). Cell transfections were carried out by using Opti-MEM reduced serum medium and LipofectAMINE reagent as described by the manufacturer (Life Technologies, Inc.). To investigate the mechanism of pX action a series of recombinant bZIP proteins were tested for DNA binding in the presence and absence of pX. pX was expressed in E. coli as a histidine-tagged derivative and purified according to a nondenaturing procedure (see “Materials and Methods”). For reasons described below, DNA binding was performed at several protein concentrations, which, in the absence of pX, gave rise to a low level of DNA binding. Fig.1 shows that, under these conditions, addition of purified pX greatly increased DNA binding of proteins of the ATF family (CREB, ATF-1, ATF-2), c-Jun family (c-Jun, GCN4), and C/EBP family (C/EBP). Significantly, however, pX did not stimulate DNA binding of all bZIPs (e.g. ATF-4; Fig. 5 B). pX cannot enhance the DNA binding activity of the ATF-4 bZIP transcription factor (36Hai T. Liu F. Coukos W.J. Green M.R. Genes Dev. 1989; 3: 2083-2090Crossref PubMed Scopus (760) Google Scholar). Neither an E. coli crude extract nor unrelated recombinant proteins stimulated DNA binding (data not shown; and see Ref. 43Wagner S. Green M.R. Science. 1993; 262: 395-399Crossref PubMed Scopus (290) Google Scholar). pX comparably stimulated DNA binding of bZIP derivatives containing or lacking a GST moiety. Consistent with previous studies (33Maguire H.F. Hoeffler J.P. Siddiqui A. Science. 1991; 252: 842-844Crossref PubMed Scopus (379) Google Scholar, 39Faktor O. Shaul Y. Oncogene. 1990; 5: 867-872PubMed Google Scholar), we could not detect an interaction between purified pX and DNA (data not shown).Figure 5pX requires the bZIP basic region to stimulate DNA binding. A, λ-ZIP, a chimera containing the λ repressor DNA binding domain (aa 1–101) fused to the GCN4 leucine zipper (aa 250–281) was tested for binding to the OR1 site of the λ phage PRM/PR promoter in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of pX. A schematic diagram of the λ-ZIP protein is shown below. B, several concentrations of GST-ATF4 protein were tested for binding to the ATF-C site (see “Materials and Methods”) in the absence or presence of pX. Comparison of the ATF4 and GCN4 basic region is shown below. Non-conserved amino acids are shaded. C,upper panel, equal amounts of proteins were fractionated on a SDS-polyacrylamide gel and stained with Coomassie Blue for protein quantitation. Lower panel, far-Western analysis; purified GST-bZIP derivatives were fractionated on an SDS-polyacrylamide gel and transferred to an Immobilon membrane. The filter was probed with 35S-labeled pX. Excess probe was removed and bound pX visualized by autoradiography.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To investigate the possible role of bZIP concentration in the pX-mediated DNA binding increase, we measured the effect of pX at GCN4 concentrations ranging from 0.05 to 20 nm. Fig. 2 shows that maximal stimulation of DNA binding was observed at peptide concentrations below 4 nm and, at higher protein concentrations, pX did not significantly increase DNA binding. The failure to increase DNA binding at higher bZIP concentrations was not due to a limiting amount of pX; DNA binding was again not affected when the concentration of pX was increased (data not shown). These results indicate that pX overcomes a concentration-dependent step that normally limits the extent of DNA binding. Dimerization of bZIP proteins occurs in the absence of DNA and is a prerequisite for DNA binding (51O'Neil K.T. Hoess R.H. DeGrado W.F. Science. 1990; 249: 774-778Crossref PubMed Scopus (241) Google Scholar, 52Talanian R.V. McKnight C.J. Kim P.S. Science. 1990; 249: 769-771Crossref PubMed Scopus (267) Google Scholar, 53O'Shea E.K. Rutkowski R. Kim P.S. Science. 1989; 243: 538-542Crossref PubMed Scopus (698) Google Scholar, 54Weiss M.A. Ellenberger T. Wobbe C.R. Lee J.P. Harrison S.C. Struhl K. Nature. 1990; 347: 575-578Crossref PubMed Scopus (338) Google Scholar). pX could therefore stimulate DNA binding by increasing either dimerization or the subsequent interaction between the bZIP homodimer and DNA. We measured the effect of pX on bZIP dimerization in the absence of DNA using a chemical cross-linking assay. Previous studies have shown that the two subunits of bZIP dimers can be cross-linked to one another with glutaraldehyde, a bifunctional cross-linking reagent (53O'Shea E.K. Rutkowski R. Kim P.S. Science. 1989; 243: 538-542Crossref PubMed Scopus (698) Google Scholar). In Fig.3 A, GCN4 was incubated in the presence or absence of pX, and following addition of glutaraldehyde the products were fractionated on an SDS-polyacrylamide gel and analyzed by immunoblotting. At a concentration of 0.1 μm, GCN4 is predominantly a monomer (lane 2) and as the concentration of GCN4 was raised, homodimer formation increased (lane 5). Significantly, addition of pX dramatically increased the amount of GCN4 homodimer (comparelanes 2 to 3 and 5 to6). An additional band with an approximate size of 46 kDa (arrow) appeared only when pX and GCN4 were both present. We suspect that this product results from a pX dimer cross-linked to a GCN4 dimer, but further experimentation would be required to confirm this supposition. To confirm that bZIP dimerization was required for the pX-mediated DNA binding increase, we analyzed the ability of pX to stimulate DNA binding of a “pre-dimerized” protein. The synthetic peptide br1s (52Talanian R.V. McKnight C.J. Kim P.S. Science. 1990; 249: 769-771Crossref PubMed Scopus (267) Google Scholar) contains two GCN4 basic regions joined by a disulfide linkage. Fig. 3 B shows that pX did not stimulate DNA binding of br1s, under the same conditions in which DNA binding of GCN4 was markedly enhanced. Thus, pX function requires a protein with an appropriate dimerization domain, consistent with the ability of pX to promote dimerization. The fact that pX can stimulate DNA binding of proteins containing only a bZIP (GST-JUN bZIP, GST-ATF2 bZIP, GCN4) implies that this domain is the target of pX action. To delineate the portion of the bZIP required for pX recognition, we analyzed a series of bZIP derivatives. Because pX increases dimerization, we began by examining the role of the leucine zipper. The synthetic peptide BR-CC contains the basic region of the yeast GCN4 bZIP protein attached to an idealized coiled-coil dimerization motif (51O'Neil K.T. Hoess R.H. DeGrado W.F. Science. 1990; 249: 774-778Crossref PubMed Scopus (241) Google Scholar). BR-CC contains the canonical leucine repeat, but otherwise the dimerization domains of GCN4 and BR-CC bear no similarity (Fig. 4, bottom). BR-CC dimerizes and binds an AP-1 site with an affinity similar to GCN4 and Fig. 4 shows that pX stimulated DNA binding of BR-CC and GCN4 comparably. We next asked whether the leucines were important for pX recognition. The DNA binding domain of Zta, an Epstein-Barr virus protein, is a highly divergent member of the bZIP family (55Chang Y.-N. Dong D.L.-Y. Hayward G.S. Hayward S.D. J. Virol. 1990; 64: 3358-3369Crossref PubMed Google Scholar, 56Lieberman P.M. Hardwick J.M. Sample J. Hayward G.S. Hayward S.D. J. Virol. 1990; 64: 1143-1155Crossref PubMed Google Scholar). Although the Zta basic region is similar to that of other bZIP proteins, the dimerization domain is not a typical leucine zipper. Nevertheless, this domain assumes a coiled-coil structure that supports dimerization and DNA binding. Fig. 4 (right) shows that pX efficiently stimulated binding of ZTA to the collagenase promoter AP-1 site. Similarly, pX increased DNA binding of a chimeric protein containing the human c-Fos basic region fused to the ZTA dimerization domain (Fig.4, right). The combined data of Fig. 4 indicate that no specific sequence of the leucine zipper is required for pX function. To test whether the conserved basic region is the target for pX, we first analyzed a hybrid protein, λ-ZIP, in which the DNA binding domain of bacteriophage λ repressor cI was fused in-frame to the GCN4 leucine zipper (57Hu J.C. O'Shea E.K. Kim P.S. Sauer R.T. Science. 1990; 250: 1400-1403Crossref PubMed Scopus (317) Google Scholar). λ-ZIP efficiently dimerizes through the leucine zipper and binds to the OR1 site of bacteriophage phage PRM/PRpromoter. Fig. 5 A shows that pX failed to stimulate DNA binding of λ-ZIP, indicating that the leucine zipper is not sufficient for pX responsiveness and suggesting an essential role for the basic region. Likewise, ATF4 (36Hai T. Liu F. Coukos W.J. Green M.R. Genes Dev. 1989; 3: 2083-2090Crossref PubMed Scopus (760) Google Scholar), a bZIP protein with an atypical basic region that diverges at several residues from the consensus (Fig. 5 B, bottom), was also not pX-responsive (Fig. 5 B, top). To confirm that pX interacted directly with the basic region, we performed a far-Western blotting experiment. bZIP derivatives were fractionated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and probed with 35S-labeled pX. The results show that pX efficiently bound to proteins that contain a complete bZIP (Fig. 5 C). In addition, pX interacted with a GST-ATF2 derivative containing the ATF2 basic region alone but not with ATF2 or c-Jun derivatives containing only the leucine zipper. Moreover, pX did not interact with ATF-4, consistent with its inability to stimulate ATF-4 DNA binding. These results provide strong evidence that pX binds to the bZIP basic region. The fact that pX interacts with the basic region, which mediates DNA contact, prompted us to investigate the DNA binding specificity of pX-bZIP complexes. We analyzed the ability of pX to increase DNA binding of several bZIP proteins to four different DNA binding sites. These DNA probes contained an AP1 or an ATF consensus flanked by sequences derived from either the collagenase promoter (AP1-C, ATF-C) or a synthetic polylinker (AP1-S, ATF-S) (Fig.6, bottom). Remarkably, the requisite combination of consensus binding site and flanking sequence differed for each bZIP protein tested. For example, pX stimulated DNA binding of CREB only to the AP1-C, ATF-C, and ATF-S sites. For GCN4, the pX-mediated DNA binding increase occurred with AP1-C, AP1-S, and ATF-C but was very modest with ATF-S. The effect of pX on FOS-ZTA DNA binding was maximal only with collagenase flanking sequences and independent of the core binding sites. In contrast, pX-mediated stimulation of ATF2 DNA binding depended only on the ATF core site. To exclude that the diverse bZIP response to pX was n" @default.
• W2087523716 created "2016-06-24" @default.
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• W2087523716 date "1999-05-01" @default.
• W2087523716 modified "2023-09-26" @default.
• W2087523716 title "The Hepatitis B pX Protein Promotes Dimerization and DNA Binding of Cellular Basic Region/Leucine Zipper Proteins by Targeting the Conserved Basic Region" @default.
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• W2087523716 doi "https://doi.org/10.1074/jbc.274.20.13970" @default.
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