DISTINCT REGIONS IN HUMAN T-CELL LYMPHOTROPIC VIRUS TYPE-I TAX MEDIATE INTERACTIONS WITH ACTIVATOR PROTEIN CREB AND BASAL TRANSCRIPTION FACTORS

Human T-cell lymphotropic virus type I (HTLV-I) transactivator Tax aug ments transcription from three (cyclic AMP response element (CRE)-cont aining 21-bp repeats in the viral long terminal repeat and several oth er cis regulatory elements, including the NF-kappa B binding sites and the serum response element. Tax does not bind DNA directly; rather, i t acts via cellular sequence-specific DNA binding proteins to stimulat e transcription. We have shown recently that Tax forms multiprotein co mplexes with the heterodimeric and homodimeric forms of a ubiquitous c ellular transcription factor, CREB (CRE binding protein). In vitro sel ection for preferred Tax-CREB binding sites indicates that the Tax-CRE B complex exhibits greatly increased DNA recognition specificity and a ssembles preferentially on CRE motifs, TGACGT/C, flanked by long runs of G (5') and/or C (3') residues, as found is the HTLV-I 21-bp repeats . The indirect tethering of Tax to the 21-bp repeats via CREB is cruci al for Tax transactivation. We now report the domain organization of T ax by characterizing its mutants. Tax mutants with alterations in the NH2 terminus, including three deletion mutants, Tax(6-353), Tax(21-353 ), and Tax(89-353), and two amino acid substitution mutants, M1 (H3S) and M7 (C29A, P30S), all failed to interact with CREB in vitro. In con trast, a short COOH-terminal deletion, Tax(1-319), and a Tax mutant wi th amino acid substitutions near the COOH end, M47 (L319R, L320S), wer e able to interact with CREB and the 21-bp repeats to assemble ternary Tax-CREB-DNA complexes. As demonstrated earlier, M1, M7, and M47 ail failed to transactivate the HTLV-I long terminal repeat, Our data indi cate that the defects in MI and M7 result from an inability to interac t with CREB. In contrast, the COOH-terminal mutations in M47 most like ly inactivated the transactivation domain of Tax. As anticipated, a Ta x mutant, M22 (G137A, L138S) which activated transcription from the 21 -bp repeats with reduced capacity and was defective in trans activatin g the NF-KB binding sites, continued to interact with CREB in vitro, a lbeit with a lower level of efficiency. Finally, a glutathione S-trans ferase (GST)-Tax fusion protein with the GST moiety fused to the NH2 t erminus of Tax failed to interact with CREB. Removal of the GST domain from GST-Tax by thrombin restores Tax's ability to assemble a ternary Tax-CREB-21-bp-repeat complex These data support the notion that the NH2-terminal region of Tax is important for interaction with CREB whil e the COOH end of Tax most likely is involved in communication with th e basal transcriptional machinery.