Chimeric Proteins Composed of Jun and CREB Define Domains

JOURNAL OF VIROLOGY, Oct. 1995, p. 6209–6218 Vol. 69, No. 10 0022-538X/95/$04.00ϩ0 Copyright ᭧ 1995, American Society for Microbiology

Chimeric Proteins Composed of Jun and CREB Deﬁne Domains Required for Interaction with the Human T-Cell Leukemia Virus Type 1 Tax Protein

MIN JEAN YIN, EVYIND PAULSSEN, JACOB SEELER,† AND RICHARD B. GAYNOR* Departments of Internal Medicine and Microbiology, Divisions of Molecular Virology and Hematology-Oncology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-8594

Received 5 April 1995/Accepted 26 June 1995

The regulation of human T-cell leukemia virus type 1 (HTLV-1) long terminal repeat gene expression is dependent on three cis-acting elements known as 21-bp repeats and the transactivator protein Tax. Mutagen- esis has demonstrated that sequences in each of the 21-bp repeats can be divided into three domains designated A, B, and C. Tax stimulates the binding of CREB to the B domain, which is essential for Tax activation of HTLV-1 gene expression. In this study, we demonstrate that Tax will stimulate the binding of CREB to the HTLV-1 21-bp repeats but does not stimulate CREB binding to the consensus cyclic AMP response element (CRE) element found in the somatostatin promoter. However, Tax stimulates CREB binding to a consensus CRE in the context of the 21-bp repeats, indicating the importance of these sequences in stimulating CREB binding. To determine the mechanism by which Tax stimulates CREB binding and determine potential interactions between Tax and CREB, we used the mammalian two-hybrid system in conjunction with in vitro binding and gel retardation assays. Two-hybrid analysis indicated that mutations in either the basic or leucine zipper region of CREB prevented interactions with Tax. Since several studies have demonstrated that Tax will also stimulate the binding of a variety of different basic region-leucine zipper proteins to their cognate binding sites, we assayed whether chimeric proteins composed of portions of CREB and another basic region-leucine zipper protein, Jun, could be used to map domains required for interactions with Tax. These studies were possible because we did not detect in vivo or in vitro interactions between Tax and Jun. The amino acid sequence of the CREB basic region and a portion of its leucine zipper were required for both in vivo and in vitro interactions with Tax and increased binding of CREB to the 21-bp repeats in response to Tax. These studies deﬁne the domains in CREB required for both in vivo and in vitro interactions by the HTLV-1 Tax protein.

Human T-cell leukemia virus type 1 (HTLV-1) is a human strated to bind members of the ATF/CREB family of transcrip- retrovirus which is the etiologic agent of adult T-cell leukemia/ tion factors (4, 5, 59, 62, 65–67). The B motif mediates tran- lymphoma (33, 47, 63) and a degenerative neurologic syn- scriptional activation of HTLV-1 gene expression in response drome designated tropical spastic paraparesis (28, 44). to both Tax (27, 29, 41, 51, 60) and increases in cAMP (36, 48) HTLV-1 encodes a 40-kDa transactivator protein, Tax, which levels, indicating that cellular factors that bind to the CRE is critical for modulating HTLV-1 gene expression (9, 13, 16, such as CREB are likely critical for regulating gene expression 24, 58) and is also involved in the cellular transformation by from the 21-bp repeats. However, Tax activation of other viral HTLV-1 (32, 61). In addition, Tax also activates the expression and cellular promoters is mediated by factors that bind to sites of several other viral and cellular genes, including interleu- that are different from that of the 21-bp repeats. For example, kin-2, interleukin-2 receptor ␣,c-fos, and those of human im- Tax activation of the interleukin-2 receptor ␣ (12, 35, 38) and munodeficiency type 1 (20, 21, 22, 35, 38), and also suppresses the human immunodeficiency virus LTR (7, 53, 56, 58) is the expression of the DNA polymerase ␤ gene (37). Thus, Tax mediated by NF-␬B sites (14, 16) and Tax activation of the is a relatively permissive viral transactivator which can modu- c-fos promoter is mediated by serum response factor sites (20, late the expression of both viral and cellular genes. 21). Thus, Tax may either directly or indirectly interact with a The HTLV-1 long terminal repeat (LTR) contains three variety of different transcription factors to alter the level of cis-acting regulatory elements designated 21-bp repeats which gene expression from tax-responsive genes. are necessary for transactivation by Tax (8, 25, 46, 55). The Tax activation of HTLV-1 gene expression is not due to its 21-bp repeats have been further subdivided into three motifs direct binding to the HTLV-1 LTR (5, 26, 29, 40). Instead, Tax designated A, B, and C (27, 41, 45). The B motif in each of the has been demonstrated to interact with cellular factors which 21-bp repeats contains the core sequence TGACG, which is are able to bind to the HTLV-1 LTR, including members of designated the tax response element and is very homologous to the ATF/CREB family (2, 4, 19, 45, 59, 62, 66, 67), HEB1 (6), sequences found in cellular genes which contain cyclic AMP and TFIID (10). Tax stimulates the binding of CREB to the B (cAMP) response elements (CREs) and have been demon- motif within the 21-bp repeats (19, 62, 67) in gel retardation analysis, though results have differed on whether this association is stable during electrophoresis (2, 4, 19, 45, 62, 64, 66, 67). * Corresponding author. Mailing address: Division of Molecular Other studies have demonstrated that Tax is able to stimulate Virology, Department of Internal Medicine, Southwestern Medical Center, Harry Hines Blvd., Dallas, TX 75235-8594. Phone: (214) 648- the binding of a variety of different basic region-leucine zipper 7570. Fax: (214) 648-8862. (bZIP) proteins to their cognate binding sites, using gel retar- † Present address: Unite´ de Recombinaison et Expression dation assays (4, 19, 62). It is unclear how this permissive effect Geńe´tique, Institut Pasteur, Paris, France. of Tax on the binding of multiple bZIP proteins can be corre-

6209 6210 YIN ET AL. J. VIROL. lated with the restricted ability of Tax to activate viral and The bacterial expression vectors pGEX-2T and pQE60 with either Tax or CREB cellular promoters (27, 45). Previously, we characterized the cDNA inserts were transformed into Escherichia coli M15; 400-ml cultures of E. coli were grown to an optical density at 600 nm of 0.6 to 0.8 and induced with 0.1 determinants of Tax interaction with members of the ATF/ mM isopropylthiogalactopyranoside (IPTG) for 3 h. Cells were pelleted, resus- CREB family, using both in vivo and in vitro assays, and dem- pended in buffer A (50 mM Tris-HCl [pH 8.0], 100 mM NaCl, 0.1 mM phenyl- onstrated that Tax specifically interacted with CREB but not methylsulfonyl fluoride), and mildly sonicated, and the debris was pelleted. For another member of the ATF/CREB family, CRE-BP1, nor the GST fusion proteins, the supernatant was incubated with 1 ml of glutathione- Sepharose beads (Sigma) for 60 min at 4ЊC. The beads were then washed five bZIP protein Jun (64). In the current study, we further char- times with 10 ml of buffer A containing 1% Triton X-100. The proteins were acterize domains within CREB which are required for the eluted by incubation with elution buffer (50 mM Tris-HCl [pH 8.0], 100 mM interaction with Tax, using both site-directed mutagenesis and NaCl, 1 mM dithiothreitol, 1 mM EDTA) containing 10 mM glutathione at 4ЊC. chimeric proteins composed of chimeric CREB and different Proteins were dialyzed against protein storage buffer (10 mM Tris-HCl [pH 8.0], 50 mM NaCl, 10% glycerol, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl domains of the bZIP protein Jun. These CREB proteins were fluoride) and were stored at Ϫ80ЊC. For 6ϫHis-tagged proteins, bacterial cells assayed by using gel retardation in the presence and absence of were cultured and fractionated under the same conditions except that the cell Tax, in in vitro binding assays with Tax, and in two-hybrid lysate was incubated with 1 ml of Ni-nitrilotriacetate agarose (Qiagen) for 60 min analysis. Our results suggest that the primary amino acid se- at 4ЊC. The proteins were eluted with elution buffer containing from 150 to 500 mM imidazole. The proteins were dialyzed against protein storage buffer and quence of both the CREB basic domain and a portion of the kept at Ϫ80ЊC. leucine zipper region and the ability of CREB to form protein Gel retardation analysis. For gel retardation assays, 50 to 100 ng each of dimers are critical for Tax interaction and Tax stimulation of bacterially produced CREB-6ϫHis and the CREB-6ϫHis mutants in either the CREB binding to the HTLV-1 21-bp repeats. presence or absence of bacterially produced Tax-6ϫHis proteins was incubated with 1 ␮g of poly(dI-dC) (Pharmacia) and 50,000 cpm of labeled probe in binding buffer (10 mM Tris-HCl [pH 7.4], 1 mM EDTA, 1 mM dithiothreitol, 5% MATERIALS AND METHODS glycerol, 50 mM NaCl). The reaction time was 20 min at room temperature in a 40-␮l final volume. Synthetic double-stranded oligonucleotides contained the Plasmid constructs. The 5ϫGAL-E1B TATA-CAT reporter plasmid contain- somatostatin CRE (5Ј-GGTTCCTCCTTGGCTGACGTCAGAGAGAGA-3Ј), ing five GAL4 DNA binding sites and the E1B TATA box linked to the chlor- HTLV-1 LTR 21-bp repeat III (5Ј-TCGACGTCCTCAGGCGTTGACGACAA amphenicol acetyltransferase (CAT) gene has previously been described (50). CCCCTCAC-3Ј), and the CRE in the HTLV-1 background (5Ј-TCGACGTCC The Tax-GAL eukaryotic expression vector was constructed by fusing an NcoI- TCAGGCGCTGACGTCAGCCCTCAC-3Ј). The protein-DNA complexes were cut Tax cDNA fragment (51) in frame with the GAL4(1-147) coding sequence in subjected to electrophoresis and applied to a 5% acrylamide gel in 0.25ϫ Tris- the pDP18 expression vector, which contains a Rous sarcoma virus promoter and borate-EDTA; then the gel was dried and autoradiography was performed. In vitro protein-protein binding. The bacterially produced Tax-E6K protein a simian virus 40 polyadenylation site. CREB-VP16 fusions were constructed by 32 insertion of cDNA fragment in frame upstream of the VP16 activation domain was labeled with [␥- P]ATP at the introduced protein kinase A substrate site by (positions 412 to 490) in the pDP18 expression vector. addition of the protein kinase A catalytic subunit for 15 min at 30ЊC; 20 to 50 ␮g To construct point mutations in CREB (34), single-stranded M13 DNA was of either wild-type or mutant GST-CREB or GST-CREB-Jun fusion protein was isolated and site-directed in vitro mutagenesis was performed by using the incubated with glutathione-agarose beads for1hat4ЊC. Approximately 1 ␮gor oligonucleotide-directed in vitro mutagenesis system (Amersham). These mu- 300,000 cpm of Tax-E6K labeled proteins was then added in TIB buffer (10% tant CREB cDNAs were subcloned upstream of VP16(412–490) in the pDP18 glycerol, 20 mM N-2-hydroxyethylpiperazine-NЈ-2-ethanesulfonic acid [HEPES; vector and confirmed by sequencing. The oligonucleotide primers used to pH 7.5], 50 mM KCl, 50 nM ZnCl2, 2.5 mM MgCl2, 0.1% Nonidet P-40, 1 mM construct the point mutations of CREB were as follows: for CREB(284–286RKRT dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride) for1hat4ЊC. The protein- TG), 5Ј-GCTGAAGAAGCAGGATCTAGAGAGGTCCGTCTAATG-3Ј; for protein complexes were washed five times at 4ЊC with 10 volumes of TIB buffer. CREB(301–305RRKKK3GTQQE), 5Ј-TCTAGAAAAGAATATGTGAAATG The Tax proteins which remained attached to the beads were resolved on sodium TTTAG-3Ј; for CREB(282–284AAR3PQL), 5Ј-CCTGCTGAAGAACCACAAC dodecyl sulfate (SDS)–10% polyacrylamide gels, the gels were dried, and auto- TAAAGAGAGAG-3Ј; for CREB(304K3E), 5Ј-GTGTCGTAGAAAGGAGAA radiography was performed. AGAATATGTG-3Ј; for CREB leucine zipper 1 (CREBL1) (L-3113V), 5Ј-TA In vitro translation and protein dimerization assay. Wild-type and mutant TGTGAAATGTGTAGAAAACAGAGTG-3Ј; for CREBL2 (L-3183V), 5Ј-A CREB DNAs were cloned into pGEM-3Z and used in in vitro transcription and translation, using the TNT T7 polymerase-coupled reticulocyte lysate system GAGTGGCAGTGGTTGAAAATCAAAAC-3Ј; for CREBL3 (L-3253V), 5Ј- 35 CAAAACAAGACAGTGATTGAGGAGC-3Ј; and for CREBL4 (L-3323V), (Promega), by adding 40 ␮Ci of [ S]methionine in a total volume of 50 ␮l. 5Ј-GAGCTAAAAGCAGTTAAGGACCTTTAC-3Ј. The resulting DNA frag- Approximately 2 ␮l of in vitro translation products was incubated with 0.02% ments were digested with KpnI and BamHI and cloned into a KpnI-BamHI- glutaraldehyde at 30ЊC for 30 min, and samples were immediately subjected to digested CREB-VP16 vector. SDS-polyacrylamide gel electrophoresis (PAGE) and autoradiography. The CREB-Jun chimeric fusion proteins were constructed by creating EcoRV Analysis of wild-type and mutant CREB protein expression. To analyze the or SpeI restriction enzyme sites upstream of the basic domain or leucine zipper expression of the CREB-VP16 constructs, each of the constructs was cloned region in both CREB-VP16 and Jun-VP16 constructs (3). The resulting Jun- downstream of the T7 promoter in a modified pTM1 expression construct which VP16 constructs were cut with EcoRV and PstI or with SpeI and PstI, and the contains an in-frame carboxy-terminal influenza virus hemagglutinin epitope Jun-VP16 fragments were then ligated into EcoRV-PstI- or SpeI-PstI-digested (49). The resulting constructs express CREB linked to VP16(412-435) and a CREB-VP16 vectors. All mutant clones constructed by site-directed mutagenesis hemagglutinin virus epitope on the carboxyl terminus. HeLa cell plates (60-mm and domain switching were confirmed by DNA sequence analysis. diameter) were transfected by using the calcium phosphate transfection proce- The full-length CREB cDNA, the CREB point mutations, and the CREB-Jun dure with 10 ␮g of each pTM1 expression construct. Protein expression was fusion constructs were cloned as BamHI-EcoRI fragments downstream of the induced by infection at a multiplicity of infection with a T7 RNA polymerase glutathione S-transferase (GST) protein in pGEX-2T vector (57). The CREB encoding vaccinia virus helper virus as previously described (15, 52). Cells were point mutation and CREB-Jun fusion constructs were made by digestion with harvested 24 h later and lysed in 500 ␮l of protein gel loading dye, and 10 ␮lof KpnI and SmaI and cloning these fragments into KpnI-SmaI-digested GST- this cell lysate was used in Western blot (immunoblot) analysis with monoclonal CREB vectors. For the six-histidine (6ϫHis)-tagged protein expression system, antibody 12CA5, which is directed against the 12-amino-acid influenza virus PCR was performed, using as template CREB-VP16, CREB(304K3E)-VP16, hemagglutinin virus epitope (49). CREB(311L318L3311V318V)-VP16, CREBL1-Jun, CREBL2-Jun, CREBL3-Jun, Transfection and CAT assays. Jurkat cells was maintained in RPMI containing 10% fetal bovine serum with penicillin and streptomycin. Cells were diluted 1:2 or CREBL4-Jun along with the 5Ј oligonucleotide primer 5Ј-GTTGCCATGGC 6 ATCCTCCCCAGC-3Ј and the 3Ј oligonucleotide primer 5Ј-ATTCCCCATGG in fresh medium on the day prior to transfection, and 5 ϫ 10 cells were AATCTGATTTGTGGCAGTAAAGG-3Ј, which created the in-frame NcoI transfected by using 0.2 mg of DEAE-dextran. Cells were harvested after 48 h, site. The resulting PCR products which contained amino acids 271 to 341 of and one-quarter of the cell extract was used for CAT assays. CREB were digested with NcoI and cloned into the NcoI-digested pQE60 vector (Qiagen). For in vitro translation, CREB-VP16, CREB-VP16 mutants, and RESULTS CREB-Jun-VP16 constructs were each digested with EcoRI and HindIII, and the resulting fragments were cloned into the EcoRI-HindIII-digested pGEM-3Z vector (Promega). For the pTM expression system, PCR was performed on Tax stimulates CREB binding to the HTLV-1 21-bp repeat. wild-type and mutant CREB-VP16 templates, using the 5Ј primer 5Ј-ATGAGC The HTLV-1 LTR contains three 21-bp repeats, each of which ATGCAATCTGAAGCCGAGAACCAGC-3Ј and the 3Ј primer 5Ј-AAGCTTG contain sequences homologous to similar motifs found in CCTGCAGGTCGACTC-3Ј. The resulting PCR products were digested with CREs which bind members of the ATF/CREB family (25, 45, SphI and SacI and then cloned into SphI-SacI-digested pTM vector, which left an in-frame influenza virus hemagglutinin epitope in their carboxyl termini. 46, 55). CREB has been demonstrated to bind to both consen- Expression and purification of bacterially produced CREB and Tax proteins. sus and nonconsensus CRE sites, and its transcriptional acti- VOL. 69, 1995 HTLV-1 Tax AND CREB PROTEIN INTERACTIONS 6211

bind to the somatostatin CRE in the absence of Tax and that the addition of Tax was not able to further increase CREB binding activity (Fig. 1A, lanes 6 to 10). These results suggested either that Tax was not able to increase CREB binding in the presence of a consensus CRE or that the A and C motifs flanking the CRE were critical for Tax stimulation. To better characterize the DNA sequences required for Tax stimulation of CREB binding, we used an oligonucleotide which contained the palindromic somatostatin CRE site flanked by the 21-bp repeat A and C sequences. CREB binding to this DNA sequence was higher than that seen with the 21-bp repeat but lower than that seen with the somatostatin CRE (Fig. 1A, lanes 11 to 15). However, Tax was able to markedly stimulate CREB binding to this hybrid sequence (Fig. 1A, lanes 11 to 15). These results indicate that the sequences flanking the 21-bp repeat are critical for Tax stimulation of CREB binding (45). Whether these flanking sequences alter the structure of the CRE binding site to facilitate the interaction with complex composed of both CREB and Tax or whether they alter the CRE binding site to decrease the binding of CREB remains to be determined. It is important to note that when the amount of CREB protein used in these gel retardation assays was reduced fivefold, Tax was able to stimulate CREB binding to the so- FIG. 1. Tax stimulates the binding of CREB to HTLV-1 21-bp repeat. Oli- matostatin CRE about threefold. The K for CREB binding to gonucleotides corresponding to the HTLV-1 21-bp repeat III (lanes 1 to 5), the d somatostatin CRE (lanes 6 to 10), or the somatostatin CRE flanked by the the somatostatin CRE in these studies was approximately 0.5 HTLV-1 A and C domains (lanes 11 to 15) were used in gel retardation assays. nM, compared with about 5 nM for the HTLV-1 21-bp repeat. Lanes contained probe alone (lanes 1, 6, and 11) and either 100 ng (lanes 2, 3, These results indicate that the function of the flanking se- 7, 8, 12, and 13) or 500 ng (lanes 4, 5, 9, 10, 14, and 15) of recombinant CREB quences is to catalyze a process which can occur to a much protein in the presence of either 100 ng of recombinant of Tax (lanes 3, 5, 8, 10, 13, and 15) or an equivalent amount of GST protein (lanes 2, 4, 7, 9, 12, and 14). lesser degree on other promoter elements containing CRE (B) Sequences of the oligonucleotides for the 21-bp repeat, somatostatin CRE, sites. and the CRE flanked by the 21-bp repeat A and C elements. Both the basic and leucine zipper domains of CREB are critical for Tax interaction. The results of gel retardation analysis indicated that Tax could stimulate CREB binding to the HTLV-1 21-bp repeats. However, no gel-retarded complex vating properties are regulated in response to cAMP-depen- composed of Tax and CREB was detectable when a Tax-spe- dent protein kinase (30, 34, 42, 43, 63). The ability of Tax to cific antiserum was added to these complexes in gel retardation stimulate CREB binding to the 21-bp repeat has been demon- assays (data not shown). Thus, we wished to investigate strated in several studies (1, 4, 19, 45, 59, 62, 66, 67). Whether whether Tax and CREB directly interact but dissociate during this stimulation of CREB binding is due to the stable interac- the gel electrophoresis or whether other mechanisms were tion with Tax or whether Tax dissociates prior to or after responsible for Tax stimulation of CREB binding. To study CREB binding remains controversial (2, 4, 19, 62, 64, 66, 67). direct interactions between Tax and CREB, we performed Although Tax is able to markedly increase gene expression both in vivo and in vitro analyses with the mammalian two- from the HTLV-1 LTR, it does not activate gene expression hybrid system (18, 39) and gel retardation and protein inter- from most cellular and viral promoters which contain consen- action studies. For two-hybrid analysis, we made constructs in sus or palindromic CRE (42, 43) sites with the sequence which Tax was fused at its carboxy terminus to the DNA TGACGTCA, compared with the nonpalindromic sequence binding domain of the yeast transcription factor GAL4 and TGACG found in the HTLV-1 21-bp repeats (25, 45, 46, 55). CREB was fused at its carboxy terminus to the activation However, it is important to note that these other promoters domain of the herpesvirus transactivator VP16. Several previ- also do not contain sequences with homology to the A and C ous studies have used Tax fusions with GAL4 to demonstrate motifs which flank the nonconsensus CRE in the 21-bp repeats that Tax contains a weak transcriptional activation domain (11, and have been demonstrated to be critical for Tax activation of 23, 26). The 5ϫGAL-E1B TATA-CAT reporter plasmid (50) the HTLV-1 LTR (27, 41, 45, 52). In addition, these flanking was used to detect these potential protein-protein interactions sequences have been demonstrated to be important for stable following cotransfection of these constructs into the T-lympho- interaction between CREB and Tax (46). Thus, it was impor- cyte cell line Jurkat. Interaction between Tax and CREB in the tant to determine which sequences in the 21-bp repeat were two-hybrid assay would increase the CAT activity as a result of critical for Tax stimulation. We compared the binding of the effects of positioning the strong acidic activator VP16 near CREB in both the presence and absence of Tax to oligonucle- the transcription initiation complex on the E1B promoter (39). otides corresponding to the HTLV-1 21-bp repeat III, the Transfections were normalized by the addition of a ␤-galacto- somatostatin CRE (43), and hybrid DNA sequences which sidase control plasmid, and each transfection was repeated at contained the palindromic CRE site from the somatostatin least three times. promoter flanked by the A promoter and C motifs in the 21-bp The Tax-GAL construct alone gave little increase in CAT repeat III (Fig. 1). Gel retardation assays indicated that CREB activity over that of the reporter construct in the presence of bound weakly to the 21-bp repeat III and that Tax was able to VP16 (Fig. 2C, lane 1). Likewise, a construct containing the markedly stimulate CREB binding (Fig. 1A, lanes 1 to 5). In GAL DNA binding domain was not activated by CREB-VP16 contrast, using the same amount of CREB protein, we found (Fig. 2C, lane 2). In the presence of both Tax-GAL and CREB- that there was a marked increase in the ability of CREB to VP16 constructs, gene expression from the 5ϫGAL reporter 6212 YIN ET AL. J. VIROL.

FIG. 2. CREB basic region mutants do not interact with Tax. (A) Schematic diagram of the mammalian two-hybrid system including the 5ϫ GAL-E1B TATA-CAT reporter construct and the Tax-GAL and CREB-VP16 fusion proteins. (B) Schematic of the CREB protein showing the positions of the site- directed mutations in its basic domain. (C) Percentage of CAT conversion for the 5ϫGAL reporter construct transfected into Jurkat cells with Tax-GAL and VP16 FIG. 3. Ability of CREB leucine zipper mutants to interact with Tax. Sche- (0.3%) (lane 1), GAL and CREB-VP16 (0.7%) (lane 2), Tax-GAL and CREB- matic of CREB showing positions of mutations in the leucine zipper region. (B) VP16 (50.1%) (lane 3), GAL and CREB(284–286RKR3TTG)-VP16 (0.4%) The percentage of CAT conversion for the 5ϫGAL reporter construct trans- (lane 4), Tax-GAL and CREB(284–286RKR3TTG)-VP16 (0.2%) (lane 5), GAL fected into Jurkat cells with Tax-GAL and VP16 (1.1%) (lane 1), GAL and and CREB(301–305RRKKK3GTQQE)-VP16 (0.7%) (lane 6), Tax-GAL and wild-type (WT) CREB-VP16 (3.5%) (lane 2), Tax-GAL and CREB-VP16 CREB(301–305RRKKK3GTQQE)-VP16 (0.7%) (lane 7), GAL and CREB(282– (90.8%) (lane 3), GAL and CREB(L13V1)-VP16 (1.2%) (lane 4), Tax-GAL 282–283 283AA3PQ)-VP16 (0.6%) (lane 8), Tax-GAL and CREB( AA3PQ)- and CREB(L13V1)-VP16 (25.6%) (lane 5), GAL and CREB(L23V2)-VP16 VP16 (44) (lane 9), GAL and CREB(282–284AAR3PQL)-VP16 (0.4%) (lane (2.0%) (lane 6), Tax-GAL and CREB(L23V2)-VP16 (87.3%) (lane 7), GAL 10), Tax-GAL and CREB(282–284AAR3PQL)-VP16 (46) (lane 11), GAL and and CREB(L33V3)-VP16 (1.9%) (lane 8), Tax-GAL and CREB(L33V3)- CREB(304K3E)-VP16 (lane 12), and Tax-GAL and CREB(304K3E)-VP16 VP16 (89.2%) (lane 9), GAL and CREB(L43V4)-VP16 (1.3%) (lane 10), and (0.9%) (lane 13). Tax-GAL and CREB(L43V4)-VP16 (89.8%) (lane 11). (C) Percentage of CAT conversion for the 5ϫGAL reporter construct following transfection into Jurkat cells with Tax-GAL and VP16 (1.3%) (lane 1), GAL and CREB-VP16 (4.1%) (lane 2), Tax-GAL and CREB-VP16 (84.3%) (lane 3), GAL and CREB(L1L23 construct was stimulated approximately 50-fold, indicating the V1V2)-VP16 (4.2%) (lane 4), Tax-GAL and CREB(L1L23V1V2)-VP16 (3.2%) direct and stable interaction between CREB and Tax proteins (lane 5), GAL and CREB(L2L33V2V3)-VP16 (5.1%) (lane 6), Tax-GAL and CREB(L2L33V2V3)-VP16 (16.8%) (lane 7), GAL and CREB(⌬L2L3L4)- (Fig. 2C, lane 3). Next, we further defined domains in CREB VP16 (2.4%) (lane 8), Tax-GAL and CREB(⌬L2L3L4)-VP16 (2.1%) (lane 9), which were required for interaction with Tax. CREB contains and GAL-VP16 (86.3%) (lane 10). in its carboxy terminus a classical leucine zipper region with four leucine residues spaced seven residues apart which is preceded by a basic amino acid domain that is critical for its DNA-binding properties (14, 31, 34). Previously, we demon- Thus, our results indicate the requirement for specific amino strated that the bZIP domain of CREB and the amino termi- acid residues in the CREB basic domain for interaction be- nus of Tax were critical for interactions in the two-hybrid tween CREB and Tax in the two-hybrid system. system (64). To further map the regions within the CREB To determine the role of the leucine zipper of CREB on bZIP domain which were required for Tax interaction, a vari- interactions with Tax, we constructed both point mutations and ety of site-directed mutations were constructed in either the truncations in this region (14). Mutation of each leucine resi- CREB basic domain or the leucine zipper (14) (Fig. 2B). All due to valine (Fig. 3A) did not disrupt the ability of CREB to mutations in the CREB basic domain, including CREB(281–284 interact with Tax-GAL (Fig. 3B, lanes 5, 7, 9, and 11), although RKR3TTG)-VP16 (Fig. 2C, lane 5), CREB(301–305RRKKK3 a mutant of the first leucine residue in the CREB leucine GTQQE)-VP16 (Fig. 2C, lane 7), and CREB(304K3E)-VP16 zipper (Fig. 3B, lane 5) exhibited decreased interaction with (Fig. 2C, lane 13), were unable to interact with Tax-GAL. A Tax compared with the wild type. We also examined the ability previous study using gel retardation analysis indicated that of Tax to interact with CREB proteins containing mutations in CREB interaction with Tax was mediated entirely through the more than one leucine residue or truncations of the leucine Ala-Ala-Arg domain, situated at amino acids 282 to 284 of zipper region (Fig. 3A). CREB(L1L23V1V2)-VP16 (Fig. 3C, CREB, which flank its basic domain (2). We tested this and a lane 5) and CREB(L2L33V2V3)-VP16 (Fig. 3C, lane 7) and similar mutant in two-hybrid system and found that both the a truncation of the leucine zipper, CREB(⌬L2L3L4)-VP16 CREB(282–283AA3PQ)-VP16 and CREB(282–284AAR3PQL)- (Fig. 3C, lane 9), were each defective compared with wild-type VP16 mutants were able to interact with Tax similarly to the CREB-VP16 (Fig. 3C, lane 3) for interaction with Tax-GAL. wild-type CREB-VP16 construct (Fig. 2C, lanes 9 and 11). As demonstrated below, the mutations in the basic and leucine VOL. 69, 1995 HTLV-1 Tax AND CREB PROTEIN INTERACTIONS 6213

L 1 L 2 L 3 L 4

FIG. 4. Domain swap between CREB and Jun deﬁne domains required for interaction with Tax. (A) Schematic of CREB (CR)-Jun chimeric proteins which were fused to the activation domain of VP16. (B) Percentage of CAT conversion of the 5ϫGAL reporter following transfection into Jurkat cells with Tax-GAL and VP16 (0.2%) (lane 1), GAL and wild-type (WT) CREB (CR)-VP16 (0.2%) (lane 2), Tax-GAL and CREB-VP16 (48.8%) (lane 3), GAL and Jun-VP16 (0.2%) (lane 4), Tax-GAL and Jun-VP16 (0.4%) (lane 5), GAL and CREB-JunbZIP1-VP16 (0.1%) (lane 6), Tax-GAL and CREB-JunbZIP1-VP16 (0.8%) (lane 7) GAL and CREB-JunbZIP2-VP16 (0.2%) (lane 8), Tax-GAL and CREB-JunbZIP2-VP16 (0.3%) (lane 9), GAL and CREB-JunbZIP-VP16 (0.2%) (lane 10), Tax-GAL and CREB-JunbZIP-VP16 (0.3%) (lane 11), and GAL-VP16 (68.1%) (lane 12). (C) Percentage CAT conversion for the 5ϫGAL reporter construct following transfection of Jurkat cells with Tax-GAL and VP16 (3.1%) (lane 1), GAL and CREB-VP16 (3.7%) (lane 2), Tax-GAL and CREB-VP16 (93.4%) (lane 3), GAL and CREBL1-JunL1-VP16 (0.2%) (lane 4), Tax-GAL and CREBL1-Jun-VP16 (1.4%) (lane 5), GAL and CREB-JunL2-VP16 (3.1%) (lane 6), Tax-GAL and CREB- JunL2-VP16 (88.6%) (lane 7), GAL and CREB-JunL3-VP16 (2.1%) (lane 8), Tax-GAL and CREBL3-Jun-VP16 (91.2%) (lane 9), GAL and CREBL4-Jun-VP16 (0.8%) (lane 10), and Tax-GAL and CREBL4-Jun-VP16 (90.4%) (lane 11).

zipper domains of CREB did not alter the overall levels of taining bZIP domains to their cognate DNA binding sequences these proteins, as determined by Western blot analysis of cel- (4, 19, 62). We wished to determine whether Tax was able to lular extracts transfected with these constructs. These results specifically interact with the other bZIP proteins through their indicate that Tax interaction with CREB requires either spe- basic and leucine zipper regions. To investigate this question, cific amino acid sequences within the leucine zipper or the a series of CREB-Jun chimeric proteins was constructed as ability of CREB to dimerize through the maintenance of this shown in Fig. 4A. Like the CREB protein, Jun contains a basic region. domain flanked by a leucine zipper with four equally spaced Chimeric proteins composed of CREB and Jun define do- leucine residues (3). These fusion proteins were tested for the mains required for specific interaction with Tax. In addition to ability to interact with Tax by using the two-hybrid system (Fig. the stimulation of CREB binding, Tax has also been reported 4), in vitro protein-protein binding assays (see Fig. 6), and gel to stimulate the binding of a variety of different proteins con- retardation assays (see Fig. 7). 6214 YIN ET AL. J. VIROL.

As shown in Fig. 4B, the CREB-VP16 but not the Jun-VP16 protein interacted with Tax-GAL, indicating that Tax could specifically interact with CREB but not another bZIP protein such as Jun (Fig. 4B, lanes 3 and 5). Western blot analysis indicated equal expression of the CREB-VP16 and Jun-VP16 proteins (data not shown) and that the Jun-VP16 protein could strongly interact with Fos-GAL (see below). Next, we tested whether chimeric proteins between CREB and Jun could be used to map domains in these proteins required for interaction with Tax. CREB-Jun fusion proteins which had domain swaps within or immediately after the basic domain of Jun did not interact with Tax-GAL (Fig. 4B, lane 7, 9, and 11). These results suggested that amino acids located carboxyl to the basic domain were also critical for interactions with Tax. To better define the domains which were important for CREB interaction with Tax, other domain swaps between CREB and Jun were tested. The CREBL1-Jun-VP16 construct, which contained the N terminus of CREB extending to the first leucine residue in its zipper region linked to the remainder of the zipper region of the Jun protein, also did not interact with Tax (Fig. 4C, lane 5). However, another con- FIG. 5. Chimeric proteins composed of CREB and Jun define domains re- struct, CREBL2-Jun-VP16, which contained the CREB coding quired for interaction with Fos. Percentage of CAT conversion for the 5ϫGAL sequence extending to the second leucine residue in its zipper reporter construct following transfection of Jurkat cells with GAL-Fos and VP16 (1.0%) (lane 1), GAL and Jun-VP16 (1.5%) (lane 2), GAL-Fos and Jun-VP16 region linked to the remainder of the Jun zipper region, was (90.7%) (lane 3), GAL-Fos and CREB-JunbZIP1-VP16 (92.4%) (lane 4), GAL- able to interact with Tax (Fig. 4C, lane 7). Two constructs, Fos and CREB-JunbZIP2-VP16 (93.7%) (lane 5), GAL-Fos and CREB-Jun- CREBL3-Jun-VP16 and CREBL4-Jun-VP16, which had do- bZIP-VP16 (95.2%) (lane 6), GAL-Fos and CREBL1-Jun-VP16 (91.9%) (lane main swaps after either the third or the fourth leucine residues 7), GAL-Fos and CREBL2-Jun-VP16 (59.6%) (lane 8), GAL-Fos and CREBL3- Jun-VP16 (13.8%) (lane 9), GAL-Fos and CREBL4-Jun-VP16 (5.5%) (lane 10), in the CREB protein joined to either the third or the fourth and GAL-VP16 (94.8%) (lane 11). (B) Western blot analysis to detect the leucine residues of Jun, interacted with Tax similarly to the protein levels for each CREB mutant and CREB-Jun fusion protein, using 20 ␮l wild-type CREB-VP16 construct (Fig. 4C, lanes 9 and 11). of cell lysates prepared from cells harvested 48 h posttransfection and monoclo- 304 These results indicate that the residues in CREB required for nal antibody 12CA5. Shown are results for CREB (lane 1), CREB( K3E) (lane 2), CREB(L13V1) (lane 3), CREB(L1L23V1V2) (lane 4), CREBL1-Jun interaction with Tax extend to the second leucine residue in (lane 5), CREBL2-Jun (lane 6), CREBL3-Jun (lane 7), CREBL4-Jun (lane 8), the zipper region. and mock-infected cells (lane 9). Positions of size markers are shown in kilodal- As a further test of the ability of the CREB-Jun fusion tons. proteins to heterodimerize, we tested the interaction of these constructs with the Jun dimerization partner Fos (49). We cotransfected the 5ϫGAL reporter construct together with (17). All of these constructs expressed similar quantities of Fos-GAL and each of the CREB-Jun-VP16 constructs used for protein as the wild-type CREB construct (Fig. 5B). These two-hybrid analysis. The CREB-Jun fusion proteins, including results indicate that the CREB mutants and CREB-Jun fusion those with domain swaps within the basic domain of CREB constructs which were defective in two-hybrid analysis with and extending to the first leucine residue of CREB, which did GAL-Tax did not produce lower levels of protein than the not interact with Tax, were able to associate with Fos-GAL wild-type construct. similarly to the wild type Jun-VP16 construct (Fig. 5A, lanes 3 Dimerization is required but not sufficient for CREB inter- to 7). Proteins with domain swaps which contained larger por- action with Tax. Since the leucine zipper region is required for tions of the CREB protein and extended to either the second, CREB dimerization, we performed gluteraldehyde cross-link- third, or fourth leucine residue in the CREB zipper were ing on each of the in vitro-translated CREB mutants and defective for association with Fos-GAL compared with wild- CREB-Jun fusions to determine if these proteins had altered type Jun-VP16 (Fig. 5A, lanes 8 to 10), although they were able dimerization properties (14). The dimerization reaction was to interact with Tax. performed by using the 35S-labeled in vitro translation prod- Finally, it was critical to determine whether the inability of a ucts from each of these constructs synthesized in rabbit reticu- number of the CREB-VP16 mutants and the CREB-Jun fusion locyte lysate, incubating these proteins with 0.02% gluteralde- constructs to interact with Tax was due to the low or unstable hyde for 30 min at 30ЊC, and performing SDS-PAGE and protein expression following transfection of these constructs. A autoradiography. Both monomer and dimer forms were de- variety of the CREB-VP16 and CREB-Jun fusion constructs, tected for wild-type CREB (Fig. 6, lane 1) and CREB proteins including the basic domain mutant CREB(304K3E)-VP16 containing mutations of single leucine residues in the CREB (Fig. 5B, lane 2), the double leucine residue mutant CREB zipper region (Fig. 6, lanes 2 to 5 and 15). However, a protein (L1L23V1V2)-VP16 (Fig. 5B, lane 3), the first leucine residue containing a mutation of the first leucine residue, which only mutant CREB(L13V1)-VP16 (Fig. 5B, lane 4), and a variety weakly interacted with Tax in the two-hybrid system, was of CREB-Jun fusion constructs (Fig. 5B, lanes 5 to 8), were defective in dimerization and required 10-fold more lysate cloned downstream of the T7 polymerase promoter in the to detect the dimer form (14) (Fig. 6, lanes 2 and 15). Pro- pTM1 expression vector and transfected into HeLa cells. The teins containing double mutants in the leucine zipper region cells were then infected with a recombinant vaccinia virus of CREB, CREB(L1L23V1V2)-VP16 and CREB(L2L33 which expressed T7 polymerase as described elsewhere (15, V2V3)-VP16, which did not interact with Tax in the two-hybrid 52). Cellular extracts were prepared, and Western blot analysis system, were unable to dimerize (Fig. 6, lanes 6 and 7) even was performed with monoclonal antibody 12CA5, which was when 10-fold more lysate was used. The CREB basic domain directed against the influenza virus hemagglutinin epitope mutants which did not interact with Tax were able to dimerize VOL. 69, 1995 HTLV-1 Tax AND CREB PROTEIN INTERACTIONS 6215

Finally, we wanted to determine whether the ability of wild- type and mutant CREB proteins to interact with Tax correlated with the ability of Tax to stimulate CREB binding to the HTLV-1 21-bp repeat. Gel retardation was performed with purified CREB or CREB-Jun fusion proteins which contained amino acids 271 to 341 of CREB or amino acids 271 to 362 of the CREB-Jun fusions and were of greater than 90% purity. These truncated wild-type CREB proteins interacted as well as wild-type CREB with GAL-Tax in two-hybrid analysis (64). Tax was able to strongly induce wild-type CREB binding to the 21-bp repeat oligonucleotides (Fig. 8, lanes 1 and 2), while CREB proteins containing mutations in either the basic domain (Fig. 8, lane 3 and 4) or leucine residues in the CREB zipper region (Fig. 8, lanes 5 and 6) were unable to bind alone, and there was no stimulation of binding by Tax. The CREBL1- Jun fusion protein, which was defective for interaction with Tax but had wild-type dimerization properties, was able to bind to the 21-bp repeat but was not stimulated by the addition of Tax (Fig. 8, lanes 7 and 8). However, fusion proteins composed FIG. 6. Analysis of in vitro dimerization of mutant CREB proteins and of CREB with portions of Jun beginning at leucine zipper CREB-Jun chimeras. Following in vitro translation in rabbit reticulocyte lysate in 35 residues 2, 3, or 4 were each stimulated by the addition of Tax the presence of [ S]methionine, 0.02% gluteraldehyde was added, and the lysate (Fig. 8, lanes 9 to 14). Thus, the in vitro protein interaction was subjected to SDS-PAGE and autoradiography. Assays were performed with 2 ␮l of wild-type (WT) CREB (CR)-VP16 (lane 1), CREB(L13V1)-VP16 (lane studies and gel retardation analysis correlated with the in vivo 2), CREB(L23V2)-VP16 (lane 3), CREB(L33V3)-VP16 (lane 4), CREB two-hybrid analysis with CREB. (L43V4)-VP16 (lane 5), CREB(L1L23V1V2)-VP16 (lane 6), CREB(L2L33 V2V3)-VP16 (lane 7), CREB(284–286RKR3TTG)-VP16 (lane 8), CREB(304K3 E)-VP16 (lane 9), CREB-JunbZIP-VP16 (lane 10), CREBL1-Jun-VP16 (lane DISCUSSION 11), CREBL2-Jun-VP16 (lane 12), CREBL3-Jun-VP16 (lane 13), and CREBL4- Jun-VP16 (lane 14) and 20 ␮l of CREB(L13V1)-VP16 (lane 15). Positions of Previous mutagenesis studies have demonstrated that three size markers are indicated in kilodaltons. imperfectly conserved 21-bp repeats in the HTLV-1 LTR are required for Tax activation (8, 25, 46, 55). Within these 21-bp repeats, three domains designated A, B, and C have been (Fig. 6, lanes 8 and 9) as expected, indicating that the mainte- shown to be important for Tax activation (27, 29, 41, 51, 60). nance of CREB dimerization alone is not sufficient for inter- The B domain contains sequences with strong homology to action with Tax. All of the CREB-Jun fusion proteins were sequences found in the CRE binding sites of various viral and able to dimerize, which again demonstrated the functional cellular promoters (42, 43). Mutagenesis of this B motif elim- integrity of these proteins (Fig. 6, lanes 10 and 11). These data inates Tax activation (27, 29, 41, 51, 60), suggesting the poten- in conjunction with the two-hybrid analysis indicate that the tial importance of different ATF/CREB (45, 59, 62, 66, 67) dimer structure of CREB is important but not sufficient for factors that have been demonstrated to bind to this element. interaction with Tax. Tax stimulates the binding of CREB to the B motif in the 21-bp In vitro assays of Tax interaction with CREB correlate with two-hybrid analysis. Next, we performed in vitro analysis to further define the requirements for interactions between CREB and Tax and correlate these results with those obtained from two-hybrid analysis. A number of the previously described CREB mutants and CREB-Jun fusions were expressed as GST fusion proteins in bacteria and then purified by using glutathione beads. Equivalent amounts of each of these proteins were then incubated with 32P-labeled Tax, and after strin- gent washing, the amount of labeled Tax that remained bound to the GST-fusion proteins was analyzed. N-terminal Tax mutants (1, 54, 64) did not bind to wild-type or mutant CREB constructs, nor did wild-type Tax bind to GST alone (data not shown). The wild-type CREB protein (Fig. 7, lane 1) and CREB proteins containing mutations of each of the leucine residues in the CREB leucine zipper were each able to interact with radiolabeled Tax (Fig. 7, lanes 2 to 5). However, the GST-CREB(304K3E) basic domain mutant (Fig. 7, lane 7), the leucine zipper mutant GST-CREB(L1L23V1V2) (Fig. 7, FIG. 7. The CREB bZIP domain is required for in vitro interaction with Tax. lane 8), and the CREB-Jun fusion protein beginning at the first 32 leucine zipper residue, GST-CREBL1-Jun, did not retain the Purified P-labeled Tax-E6K protein (lane 6) was incubated with the indicated of GST fusion protein; following extensive washing, the labeled protein which labeled Tax protein (Fig. 7, lane 9), while the CREB-Jun pro- was retained on glutathione beads was subjected to SDS-PAGE. The GST fusion tein which began at the second leucine residue, GST- proteins incubated with the labeled Tax proteins were GST-wild-type (WT) CREBL2-Jun, bound radiolabeled Tax (Fig. 7, lane 10). These CREB (CR) (lane 1), GST-CREB(L13V1) (lane 2), GST-CREB(L23V2) results indicate that the in vitro binding data correlated very (lane 3), GST-CREB(L33V3) (lane 4), GST-CREB(L43V4) (lane 5), labeled Tax probe alone (lane 6), GST-CREB(304K3E) (lane 7), GST-CREB(L1L23 well with the results of two-hybrid interaction studies to define V1V2) (lane 8), GST-CREBL1-Jun (lane 9), and GST-CREBL2-Jun (lane 10). domains required for Tax and CREB interaction. Positions of size markers are indicated in kilodaltons. 6216 YIN ET AL. J. VIROL.

proteins to their cognate binding sites (4, 19, 62), our results suggest that Tax stimulation of binding is relatively restricted and is strongly dependent on the primary sequence of the 21-bp repeat. Tax was able to stably interact with CREB, and mutagenesis demonstrated that the requirements for this binding were within the bZIP region of CREB. A variety of proteins containing mutations in the CREB basic domain prevented interactions with Tax, suggesting that these amino acids may be important for direct interaction with Tax. However, in contrast to a previous study which used gel retardation analysis with CREB as an assay for Tax interaction, we did not find that mutation of amino acids 284 to 286 flanking the CREB basic domain prevented interaction with Tax (2). These mutations decreased CREB binding to the 21-bp repeats (14), and so the role of Tax on the stimulation of binding of these mutants was difficult to interpret. Though CREB constructs containing point mutations in each of the leucine residues in its zipper region were still able to interact with Tax, proteins containing mutations of multiple leucine residues in the zipper which interrupt CREB dimerization were unable to interact with Tax. FIG. 8. Domains in CREB required for stimulation of binding by Tax. The These results suggest that the ability of CREB to dimerize was HTLV-1 21-bp repeat III oligonucleotide was end labeled with [␥-32P]ATP by polynucleotide kinase and tested in a gel retardation assay with 100 ng of essential for interaction with Tax. bacterially expressed wild-type (WT) CREB (CR) (lanes 1 and 2), CREB(304 However, it appears that there was added specificity within K3E) (lanes 3 and 4), CREB(L1L23V1V2) (lanes 5 and 6), CREBL1-Jun the leucine zipper for interactions between CREB and Tax. (lanes 7 and 8), CREBL2-Jun (lanes 9 and 10), CREBL3-Jun (lanes 11 and 12), Domain swaps between CREB and Jun demonstrated that the CREBL4-Jun (lanes 13 and 14), and probe alone (lane 15). Lanes 1, 3, 5, 7, 9, 11, and 13 contained 100 ng of GST; lanes 2, 4, 6, 8, 10, 12 and 14 contained 100 ng Jun basic domain could not substitute for that of CREB to of bacterially expressed Tax. allow interaction with Tax. Similar experiments indicated that the entire leucine zipper of Jun could not substitute for the CREB zipper even though there was equivalent dimerization of these proteins in in vitro gluteraldehyde cross-linking assays. repeats, though whether a stable association between the Tax Thus, an intact bZIP domain in the context of the CREB basic and CREB proteins is maintained in gel retardation analysis is domain was not sufficient for interaction with Tax. By including unclear (4, 19, 62, 66, 67). Tax has also been shown to stimulate both the CREB basic domain and the first seven amino acids of the binding of a variety of other bZIP proteins to their cognate its leucine zipper region fused to the remainder of the leucine binding sites (4, 19, 62), though this broad range of Tax func- zipper region of Jun, we found that this chimeric protein was tion has been somewhat difficult to reconcile with the restricted able to interact with Tax. Since a CREB truncation which left ability of Tax to increase gene expression from viral and cel- the first seven amino acids of the leucine zipper region intact lular promoters. but removed the remainder of the leucine zipper was unable to To further explore the specificity of Tax interaction with interact with Tax, we would suggest that the minimal require- CREB, we used several complementary techniques to deter- ments for CREB interaction with Tax include the CREB basic mine the requirements for interactions between Tax and domain and a portion of its leucine zipper in the context of the CREB in both in vitro and in vivo assays. First, we used the ability to undergo dimerization. mammalian two-hybrid system so that interactions between Finally, we wished to correlate the in vivo interactions of Tax CREB and Tax could be studied in the absence of CREB DNA and CREB with the ability of Tax to stimulate CREB binding binding, since mutations in the CREB basic and leucine zipper to the HTLV-1 21-bp repeat. The ability of Tax to stimulate domains affect both binding and interaction. Furthermore, do- CREB binding to the 21-bp repeat correlated very well with its main swaps could be performed between two different bZIP ability to interact with CREB in both in vitro binding assays proteins, CREB and Jun, each of whose binding has previously and two-hybrid analysis. This was most clearly demonstrated been reported to be stimulated by Tax (4, 62). Thus, the im- with the CREB-Jun chimeric proteins. For example, although portance of the basic and leucine zipper domains of these the CREB-Jun chimera which began at the first leucine residue proteins on interaction with Tax could be better ascertained. in the zipper region was able to bind to the 21-bp repeat, its These studies were extended by using in vitro protein inter- binding was not stimulated by Tax and it did not interact with action and gel retardation studies so that we could correlate Tax either in vitro or in vivo. In contrast, the CREB-Jun chi- the in vivo interactions between CREB and Tax with the ability mera which began at the second leucine residue in the CREB of Tax to stimulate CREB binding to the HTLV-1 21-bp re- leucine zipper region exhibited binding to the 21-bp repeat and peats. In agreement with previous studies (2, 4, 19, 45, 59, 62, Tax stimulation and interacted with Tax in both in vitro and in 64, 66, 67), our data indicate that Tax can markedly increase vivo assays. These results suggest that the ability of Tax to CREB binding to the HTLV-1 21-bp repeat but that this bind- stimulate CREB binding to the 21-bp repeat correlates well ing is strongly dependent on a CRE motif in the context of with its ability to directly interact with Tax. 21-bp flanking sequences. These results indicate that the se- However, our studies and those of several other groups (4, quences flanking the CRE in the 21-bp repeat are critical for 19, 62, 64) have not demonstrated a stable interaction between Tax stimulation of CREB binding (45) and thus may help Tax and CREB in gel retardation assays, though in vitro pro- explain the restricted ability of Tax to activate gene expression tein-protein interaction studies clearly demonstrate such an from upstream promoter elements. Though Tax has been dem- interaction. In contrast, other studies have demonstrated such onstrated to stimulate the binding of a variety of different bZIP an interaction in gel retardation assays (2, 59, 66, 67). The VOL. 69, 1995 HTLV-1 Tax AND CREB PROTEIN INTERACTIONS 6217 reasons for these differences are not clear, though dissociation 13. Cullen, B. R. 1992. Mechanism of action of regulatory proteins encoded by of Tax from the CREB protein during electrophoresis may be complex retroviruses. Microbiol. Rev. 56:375–394. 14. Dwarki, V. J., M. Montminy, and I. M. Verma. 1990. Both the basic region one explanation. Our results indicate that Tax is able to inter- and the leucine zipper domain of the cyclic AMP response element binding act with the basic domain of CREB in addition to approxi- (CREB) protein are essential for transcriptional activation. EMBO J. 9:225– mately seven amino acids of its leucine zipper domain. In 232. addition, the ability of CREB to dimerize is also critical for Tax 15. Elroy-Stein, O., T. R. Fuerst, and B. Moss. 1989. Cap-independent translation of mRNA conferred by encephalomyocarditis virus 5Ј sequence im- interaction. Thus, the most likely models by which Tax inter- proves the performance of the vaccinia virus/bacteriophage T7 hybrid ex- action with CREB stimulates CREB binding would be for Tax pression system. Proc. Natl. Acad. Sci. USA 86:6126–6130. to stimulate CREB dimerization or to associate with CREB 16. Felber, B. K., H. Paskalis, D. Kleinman-Ewing, F. Wong-Staal, and G. N. after dimerization via direct interaction with its basic and a Pavlakis. 1985. The pX protein of HTLV-I is a transcriptional activator of its long terminal repeats. Science 229:54–58. portion of its leucine zipper region, leading to an increase in 17. Field, J., J. Nikawa, D. Broek, B. MacDonald, L. Rodgers, I. A. Wilson, R. A. the on rate of CREB binding to the 21-bp repeat. In both of Lerner, and M. Wigler. 1988. Purification of a RAS-responsive adenylyl these models, Tax may dissociate before or after CREB bind- cyclase complex from Saccharomyces cerevisiae by use of an epitope addition ing. We did not find that Tax was able to stimulate the rate of method. Mol. Cell. Biol. 8:2159–2165. 18. Fields, S., and O. Song. 1989. A novel genetic system to detect protein- CREB dimerization as previously suggested (62, 63a). We protein interactions. Nature (London) 340:245–246. would favor a model in which Tax associates with CREB 19. Franklin, A. A., M. F. Kubik, M. N. Uittenbogaard, A. Brauweiler, P. Utais- dimers which bind to the 21-bp repeat, leading to an increase incharoen, M.-A. H. Matthews, W. S. Dynan, J. P. Hoeffler, and J. K. Nyborg. in the on rate of CREB binding (62). Whether the sequences 1993. Transactivation by the human T-cell leukemia virus tax protein is mediated through enhanced binding of activating transcription factor-2 flanking the B domain in the 21-bp repeats directly interact (ATF-2) ATF-2 response and cAMP element-binding protein (CREB). J. with a complex composed of both Tax and CREB followed by Biol. Chem. 268:21225–21231. Tax dissociation during gel electrophoresis remains to be de- 20. Fujii, M., T. Niki, T. Mori, T. Matsuda, M. Matsui, N. Nomura, and M. termined. Further analysis of the kinetics and the DNA struc- Seiki. 1991. HTLV-1 Tax induces expression of various immediate early serum responsive genes. Oncogene 6:2349–2352. ture of the HTLV-1 21-bp repeats will be necessary to further 21. Fujii, M., P. Sassone-Corsi, and I. M. Verma. 1988. c-fos promoter trans- elucidate the mechanism by which Tax regulates the DNA- activation by the tax1 protein of human T-cell leukemia virus type I. Proc. binding properties of CREB and activates HTLV-1 gene ex- Natl. Acad. Sci. USA 85:8526–8530. pression. 22. Fujii, M., H. Tsuchiya, T. Chuhjo, T. Akizawa, and M. Seiki. 1992. Interac- tion of HTLV-I Tax1 with p67SRF causes the aberrant induction of cellular immediate early genes through CArG boxes. Genes Dev. 6:2066–2076. ACKNOWLEDGMENTS 23. Fujii, M., H. Tsuchiya, and M. Seiki. 1991. HTLV-1 Tax has distinct but overlapping domains for transcriptional activation and for enhancer speci- We thank Ty Lawrence and Elizabeth Folta for manuscript prepa- ficity. Oncogene 6:2349–2352. ration. 24. Fujisawa, J., M. Seiki, T. Kiyokawa, and M. Yoshida. 1985. Functional This work was supported by grants from the NIH and the Council activation of the long terminal repeat of human T-cell leukemia virus type I for Tobacco Research. by a trans-acting factor. Proc. Natl. Acad. Sci. USA 82:2277–2281. 25. Fujisawa, J., M. Seiki, M. Sato, and M. Yoshida. 1986. A transcriptional REFERENCES enhancer sequence of HTLV-I is responsible for trans-activation mediated 1. Adya, N., and C. Z. Giam. 1995. Distinct regions in human T-cell lympho- by p40x of HTLV-I. EMBO J. 5:713–718. tropic virus type 1 Tax mediate interactions with activator protein CREB and 26. Fujisawa, J., M. Toita, T. Yoshimura, and M. Yoshida. 1991. The indirect basal transcription factors. J. Virol. 69:1834–1841. association of human T-cell leukemia virus Tax proteins with DNA results in 2. Adya, N., L.-J. Zhao, W. Huang, I. Boros, and C.-Z. Giam. 1994. Expansion transcriptional activation. J. Virol. 65:4525–4528. of CREB’s DNA recognition specificity by TAX results from interaction with 27. Fujisawa, J.-I., M. Toita, and M. Yoshida. 1989. A unique enhancer element tax Ala-Ala-Arg at positions 282-284 near the conserved DNA-binding domain for the trans activator (p40 ) of human T-cell leukemia virus type I that is of CREB. Proc. Natl. Acad. Sci. USA 91:5642–5646. distinct from cyclic AMP and 12-O-tetradecanoylphorbol-13-acetate-respon- 3. Angel, P., E. A. Allegretto, S. T. Okino, K. Hattori, W. J. Boyle, T. Hunter, sive elements. J. Virol. 63:3234–3239. and M. Karin. 1988. Oncogene jun encodes a sequence-specific trans-acti- 28. Gessain, A., F. Barin, J. C. Vernant, O. Gout, L. Maurs, A. Calender, and G. vator similar to AP1. Nature (London) 322:166–170. De The. 1985. Antibodies to human T-lymphotropic virus type I in patients 4. Armstrong, A. P., A. A. Franklin, M. N. Henbogaard, H. A. Giebler, and J. K. with tropical spastic paraparesis. Lancet ii:407–410. Nyborg. 1993. Pleiotropic effect of the human T-cell leukemia virus TAX 29. Giam, C. Z., and Y. L. Xu. 1989. HTLV-I tax gene product activates tran- protein on the DNA binding activity of eukaryotic transcription factors. Proc. scription via pre-existing cellular factors and cAMP responsive element. J. Natl. Acad. Sci. USA 90:7303–7307. Biol. Chem. 264:15236–15241. 5. Beimling, P., and K. Moelling. 1992. Direct interaction of CREB protein 30. Gonzalez, G. A., and M. R. Montiminy. 1989. Cyclic AMP stimulates soma- with 21 bp Tax-response elements of HTLV-I LTR. Oncogene 7:257–262. tostatin gene transcription by phosphorylation of CREB at serine 133. Cell 6. Be´raud, C., G. Lombard-Platet, Y. Michal, and P. Jalinot. 1990. Binding of 59:675–680. the HTLV-1 Tax1 transactivator to the inducible 21bp enhancer is mediated 31. Gonzalez, G. A., K. K. Yamamoto, W. H. Fischer, D. Karr, P. Menzel, W. by the cellular factor HEB1. EMBO J. 10:3795–3803. Biggs III, W. W. Vale, and M. R. Montminy. 1989. A cluster of phosphory- 7. Be´raud, C., S.-C. Sun, P. Ganchi, D. W. Ballard, and W. C. Greene. 1994. lation sites on the cyclic AMP-regulated nuclear factor CREB predicted by Human T-cell leukemia virus type I Tax associates with and is negatively its sequence. Nature (London) 337:749–752. regulated by the NF-␬B2 p100 gene product: implications for viral latency. 32. Grassmann, R., C. Dengler, I. Muller-Fleckenstein, B. Fleckenstein, K. Mol. Cell. Biol. 14:1374–1382. McGuire, M. Dokhelar, J. Sodroski, and W. Haseltine. 1989. Transformation 8. Brady, J., K. T. Jeang, J. Duvall, and G. Khoury. 1987. Identification of to continuous growth of primary human T lymphocytes by human T-cell p40x-responsive regulatory sequences within the human T-cell leukemia vi- leukemia virus type I X-region genes transduced by a Herpesvirus saimiri rus type I long terminal repeat. J. Virol. 61:2175–2181. vector. Proc. Natl. Acad. Sci. USA 86:3351–3355. 9. Cann, A. J., J. D. Rosenblatt, W. Wachsman, N. P. Shah, and I. S. Y. Chen. 33. Hinuma, Y., H. Komoda, T. Chosa, T. Kondo, M. Kohakura, T. Takenaka, 1985. Identification of the gene responsible for human T-cell leukemia virus M. Kikuchi, M. Ichimaru, K. Yunoki, I. Sato, R. Matsuo, Y. Takiuchi, H. transcriptional regulation. Nature (London) 318:571–574. Uchino, and M. Hanaoka. 1982. Antibodies to adult T-cell leukemia-virus- 10. Caron, C., R. Rousset, C. Beraud, V. Moncollin, J. M. Egly, and P. Jalinot. associated antigen (ATLA) in sera from patients with ATL and controls 1993. Functional and biochemical interaction of the HTLV-I Tax1 transac- in Japan: a nation-wide sero epidemiologic study. Int. J. Cancer 29:631– tivator with TBP. EMBO J. 12:4269–4278. 635. 11. Connor, L. M., M. N. Oxman, J. N. Brady, and S. J. Marriott. 1993. Twenty- 34. Hoeffler, J. P., T. E. Meyer, Y. Yun, J. L. Jameson, and J. F. Habener. 1988. one base pair repeat elements influence the ability of a GAL4-Tax fusion Cyclic AMP-responsive DNA-binding protein: structure based on a cloned protein to transactivate the HTLV-I long terminal repeat. Virology 195:569– placental CDNA. Science 242:1430–1433. 577. 35. Inoue, J., M. Seiki, T. Taniguchi, S. Tsuru, and M. Yoshida. 1986. Induction 12. Cross, S. L., M. B. Feinberg, J. B. Wolf, N. J. Holbrook, F. Wong-Staal, and of interleukin 2 receptor gene expression by p40x encoded by human T-cell W. J. Leonard. 1987. Regulation of the human interleukin-2 receptor alpha leukemia virus type 1. EMBO J. 5:2883–2888. chain promoter: activation of a nonfunctional promoter by the transactivator 36. Jeang, K. T., I. Boros, J. Brady, M. Radonovich, and G. Khoury. 1988. gene of HTLV-I. Cell 49:47. Characterization of cellular factors that interact with the human T-cell leu- 6218 YIN ET AL. J. VIROL.

kemia virus type I p40x-responsive 21-base-pair sequence. J. Virol. 62:4499– expression. J. Virol. 68:1002–1009. 4509. 53. Semmes, O. J., and K.-T. Jeang. 1992. Mutational analysis of human T-cell 37. Jeang, K. T., S. G. Widen, O. J. Semmes, and S. H. Wilson. 1990. HTLV-I leukemia virus type I Tax: regions necessary for function determined with 47 trans-activator protein, tax, is a trans-repressor of the human beta-poly- mutant proteins. J. Virol. 66:7183–7192. merase gene. Science 247:1082–1084. 54. Semmes, O. J., and K. T. Jeang. 1995. Definition of a minimal activation 38. Leung, K., and G. J. Nabel. 1988. HTLV-1 transactivator induces interleu- domain in human T-cell leukemia virus type 1 Tax. J. Virol. 69:1827–1833. kin-2 receptor expression through an NF-kappa B-like factor. Nature 333: 55. Shimotohno, K., M. Takano, T. Teruchi, and M. Miwa. 1986. Requirement 776–778. of multiple copies of a 21-nucleotide sequence in the U3 region of human 39. Liu, F., and M. R. Green. 1990. A specific member of the ATF transcription T-cell leukemia virus type I and type II long terminal repeats for trans-acting factor family can mediate transcription activation by adenovirus E1A pro- activation of transcription. Proc. Natl. Acad. Sci. USA 83:8112–8116. tein. Cell 61:1217–1224. 56. Siekevitz, M., S. F. Josephs, M. Dukovich, N. Peffer, F. Wong-Staal, and 40. Marriott, S. J., I. Boros, J. F. Duvall, and J. N. Brady. 1989. Indirect binding W. C. Greene. 1987. Activation of the HIV-1 LTR by T cell mitogens and the of human T-cell leukemia virus type I tax1 to a responsive element in the trans-activator protein of HTLV-I. Science 238:1575–1578. viral long terminal repeat. Mol. Cell. Biol. 9:4152–4160. 57. Smith, D. B., and K. S. Johnson. 1988. Single-step purification of polypep- 41. Montagne, J., C. Beraud, I. Crenon, L. Platet, A. Gazzolo, A. Sergeant, and tides expressed in Escherichia coli as fusions with glutathione S-transferase. P. Jalinot. 1990. Tax1 induction of the HTLV-I 21 bp enhancer requires Gene 67:31–40. cooperation between two cellular DNA-binding protein. EMBO J. 9:957– 58. Sodroski, J., C. Rosen, W. C. Goh, and W. Haseltine. 1985. A transcriptional 964. activator protein encoded by the x-lor region of the human T-cell leukemia 42. Montminy, M. R., and L. M. Bilezikjian. 1987. Binding of a nuclear protein virus. Science 228:1430–1434. to the cyclic-AMP response element of the somatostatin gene. Nature (Lon- 59. Suzuki, T., J. I. Fujisawa, M. Toita, and M. Yoshida. 1993. The trans- don) 328:175–178. activator tax of human T-cell leukemia virus type 1 (HTLV-I) interacts with 43. Montminy, M. R., K. A. Sevarino, J. A. Wagner, G. Mandel, and R. H. cAMP-responsive element (CRE) binding and CRE modulator proteins that Goodman. 1986. Identification of a cyclic-AMP-responsive element within bind to the 21-base-pair enhancer of HTLV-1. Proc. Natl. Acad. Sci. USA the rat somatostatin gene. Proc. Natl. Acad. Sci. USA 83:6682–6686. 90:610–614. 44. Osame, M., K. Usuku, S. Izumo, N. Ijichi, H. Amitani, A. Igata, M. Mat- 60. Tan, T. H., M. Horikoshi, and R. G. Roeder. 1989. Purification and charac- sumoto, and M. Tara. 1986. HTLV-I associated myelopathy, a new clinical terization of multiple nuclear factors that bind to the TAX-inducible en- entity. Lancet i:1031–1032. hancer within the human T-cell leukemia virus type 1. Mol. Cell. Biol. 45. Paca-Uccaralertkun, S., L. J. Zhao, N. Adya, J. V. Cross, B. R. Cullen, and 9:1733–1745. I. M. Boros. 1994. In vitro selection of DNA elements highly responsive to 61. Tanaka, A., C. Takahashi, S. Yamaoka, T. Nosaka, M. Maki, and M. Ha- the human T-cell lymphotropic virus type I transcriptional activator, Tax. tanaka. 1990. Oncogenic transformation by the tax gene of human T-cell Mol. Cell Biol. 14:456–462. leukemia virus type I in vitro. Proc. Natl. Acad. Sci. USA 87:1071–1075. 46. Paskalis, H., K., B. K. Felber, and G. N. Pavlakis. 1986. Cis-acting sequences 62. Wagner, S., and M. R. Green. 1993. HTLV-I tax protein stimulation of DNA responsible for the transcriptional activation of a human T-cell leukemia binding of bZIP proteins by enhancing dimerization. Science 262:395–399. virus type I constitute a conditional enhancer. Proc. Natl. Acad. Sci. USA 63. Yamamoto, K. K., G. A. Gonzalez, W. H. Biggs, and M. R. Montminy. 1988. 83:6558–6562. Phosphorylation-induced binding and transcriptional efficacy of nuclear fac- 47. Poiesz, B. J., R. W. Ruscetti, A. F. Gazdar, P. A. Bunn, J. D. Minna, and R. C. tor CREB. Nature (London) 334:494–498. Gallo. 1980. Detection and isolation of type C retrovirus particles from fresh 63a.Yin, M.-J., and R. Gaynor. Unpublished observations. and cultured lymphocytes of a patient with cutaneous T-cell lymphoma. 64. Yin, M.-J., E. J. Paulssen, J.-S. Seeler, and R. B. Gaynor. 1995. Protein Proc. Natl. Acad. Sci. USA 77:7415–7419. domains involved in both in vivo and in vitro interactions between human 48. Poteat, H. T., P. Kadison, K. McGuire, L. Park, R. E. Park, J. G. Sodroski, T-cell leukemia virus type I Tax and CREB. J. Virol. 69:3420–3432. and W. A. Haseltine. 1989. Response of the human T-cell leukemia virus type 65. Yoshimura, T., J. Fujisawa, and M. Yoshida. 1990. Multiple cDNA clones 1 long terminal repeat to cyclic AMP. J. Virol. 63:1604–1611. encoding nuclear proteins that bind to the tax-dependent enhancer of 49. Ransone, L. J., J. Visvader, P. Sassone-Corsi, and I. M. Verma. 1989. Fos- HTLV-1: all contain a leucine zipper structure and basic amino acid domain. jun interaction: mutational analysis of the leucine zipper domain of both EMBO J. 9:2537–2542. proteins. Genes Dev. 3:770–781. 66. Zhao, L. J., and C. Z. Giam. 1991. Interaction of the human T-cell lympho- 50. Sadowski, I., and M. Ptashne. 1989. A vector for expressing GAL4 (1-147) tropic virus type I (HTLV-I) transcriptional activator Tax with cellular fac- fusions in mammalian cells. Nucleic Acids Res. 17:7539. tors that bind specifically to the 21-base-pair repeats in the HTLV-I en- 51. Seeler, J. S., C. Muchardt, M. Podar, and R. B. Gaynor. 1993. Regulatory hancer. Proc. Natl. Acad. Sci. USA 88:11445–11449. elements involved in tax-mediated transactivation of the HTLV-I LTR. 67. Zhao, L. J., and C. Z. Giam. 1992. Human T-cell lymphotropic virus type I Virology 196:442–450. (HTLV-I) transcriptional activator, Tax, enhances CREB binding to 52. Seeler, J.-S., C. Muchardt, A. Suessle, and R. B. Gaynor. 1994. Transcription HTLV-I 21-base-pair repeats by protein-protein interaction. Proc. Natl. factor PRDII-BF1 activates human immunodeficiency virus type 1 gene Acad. Sci. USA 89:7070–7074.