JOURNAL OF VIROLOGY, Oct. 1995, p. 6209–6218 Vol. 69, No. 10 0022-538X/95/$04.0010 Copyright q 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 a,c-fos, and those of human im- Tax activation of the interleukin-2 receptor a (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 b gene (37). Thus, Tax mediated by NF-kB 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 associa- tion 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 Ge´ne´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 48C. 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 48C. 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 2808C. For 63His-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 48C. 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 2808C. 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-63His and the CREB-63His mutants in either the CREB binding to the HTLV-1 21-bp repeats. presence or absence of bacterially produced Tax-63His proteins was incubated with 1 mg 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-ml final volume. Synthetic double-stranded oligonucleotides contained the Plasmid constructs. The 53GAL-E1B TATA-CAT reporter plasmid contain- somatostatin CRE (59-GGTTCCTCCTTGGCTGACGTCAGAGAGAGA-39), ing five GAL4 DNA binding sites and the E1B TATA box linked to the chlor- HTLV-1 LTR 21-bp repeat III (59-TCGACGTCCTCAGGCGTTGACGACAA amphenicol acetyltransferase (CAT) gene has previously been described (50).