The human T-cell lymphotropic virus type I (HTLV-I) transactivator, Tax, the ubiquitous transcriptional factor cyclic AMP (cAMP) response element-binding protein (CREB protein), and the 21-bp repeats in the HTLV-I transcriptional enhancer form a ternary nucleoprotein complex (L. J. Zhao and C. Z. Giam, Proc. Natl. Acad. Sci. USA 89:7070-7074, 1992). Using an antibody directed against the COOH-terminal region of Tax along with purified Tax and CREB proteins, we selected DNA elements bound specifically by the Tax-CREB complex in vitro. Two distinct but related groups of sequences containing the cAMP response element (CRE) flanked by long runs of G and C residues in the 5' and 3' regions, respectively, were preferentially recognized by Tax-CREB.In contrast, CREB alone binds only to CRE motifs (GNTGACG[T/C]) without neighboring G-or C-rich sequences. The Tax-CREB-selected sequences bear a striking resemblance to the 5' or 3' two-thirds of the HTLV-I 21-bp repeats and are highly inducible by Tax. Gel electrophoretic mobility shift assays, DNA transfection, and DNase I footprinting analyses indicated that the G-and C-rich sequences flanking the CRE motif are crucial for Tax-CREB-DNA ternary complex assembly and Tax transactivation but are not in direct contact with the Tax-CREB complex. These data show that Tax recruits CREB to form a multiprotein complex that specifically recognizes the viral 21-bp repeats. The expanded DNA binding specificity of Tax-CREB and the obligatory role the ternary Tax-CREB-DNA complex plays in transactivation reveal a novel mechanism for regulating the transcriptional activity of leucine zipper proteins like CREB.Human T-cell lymphotropic virus type I (HTLV-I) is the prototype of a group of retroviruses (including HTLV-I, HTLV-II, simian T-cell leukemia virus, and bovine leukemia virus [BLV]) that produce regulatory proteins to modulate viral mRNA synthesis and utilization (6). The 40-kDa nuclear protein, Tax, encoded by the 3' region of the HTLV-I genome is a transcriptional activator (6). Tax does not bind DNA directly (13,21). Like many other viral transactivators such as herpes simplex virus VP16 and adenovirus Ela (for a review, see reference 20), Tax forms multiprotein complexes with host cell transcription factors to gain control of the cellular mRNA synthetic machinery for viral replication (1,18,(27)(28)(29). The ability of Tax to alter gene expression in HTLV-I-infected cells appears to be causally linked to HTLV-I pathogenesis, which is manifested clinically as adult T-cell leukemia (5, 16, 24) and tropical spastic paraparesis/HTLV-I-associated myelopathy (2,12,17,22).Tax activates viral transcription from three 21-bp repeats in the U3 region of the HTLV-I long terminal repeat (3,23,25,26). Mutational analyses indicated that two copies of the repeat and the CRE (cyclic AMP [cAMP]-responsive-element)-like sequences, TGACG, in the repeats are crucial for transactivation by Tax (3,13,23,25,26 Tax action (10,19). This is best shown by the inability of HTLV-I Tax to transactivate thre...

The human T-cell lymphotropic virus type I (HTLV-I) transactivator, Tax, the ubiquitous transcriptional factor cyclic AMP (cAMP) response element-binding protein (CREB protein), and the 21-bp repeats in the HTLV-I transcriptional enhancer form a ternary nucleoprotein complex (L. J. Zhao and C. Z. Giam, Proc. Natl. Acad. Sci. USA 89:7070-7074, 1992). Using an antibody directed against the COOH-terminal region of Tax along with purified Tax and CREB proteins, we selected DNA elements bound specifically by the Tax-CREB complex in vitro. Two distinct but related groups of sequences containing the cAMP response element (CRE) flanked by long runs of G and C residues in the 5' and 3' regions, respectively, were preferentially recognized by Tax-CREB. In contrast, CREB alone binds only to CRE motifs (GNTGACG[T/C]) without neighboring G- or C-rich sequences. The Tax-CREB-selected sequences bear a striking resemblance to the 5' or 3' two-thirds of the HTLV-I 21-bp repeats and are highly inducible by Tax. Gel electrophoretic mobility shift assays, DNA transfection, and DNase I footprinting analyses indicated that the G- and C-rich sequences flanking the CRE motif are crucial for Tax-CREB-DNA ternary complex assembly and Tax transactivation but are not in direct contact with the Tax-CREB complex. These data show that Tax recruits CREB to form a multiprotein complex that specifically recognizes the viral 21-bp repeats. The expanded DNA binding specificity of Tax-CREB and the obligatory role the ternary Tax-CREB-DNA complex plays in transactivation reveal a novel mechanism for regulating the transcriptional activity of leucine zipper proteins like CREB.