The Tax Protein-DNA Interaction is Essential for HTLV-I Transactivation in Vitro Brian A. Lenzmeier 1 , Eldon E. Baird 2 , Peter B. Dervan 2 and Jennifer K. Nyborg 1 * 1 Department of Biochemistry and Molecular Biology Colorado State University, Fort Collins, CO, 80523-1870, USA 2 Division of Chemistry and Chemical Engineering, 164-30 California Institute of Technology, Pasadena, CA 91125, USA The human T-cell leukemia virus type-1 (HTLV-I)-encoded Tax protein enhances viral gene transcription through interaction with three repeated DNA elements located in the viral promoter. These elements, called viral CREs, are composed of an off-consensus eight base-pair cyclic AMP response element (CRE), immediately ¯anked by sequences that are rich in guanine and cytosine residues. Recent biochemical experiments have demonstrated that in the presence of the cellular protein CREB, Tax directly binds the viral CRE GC-rich sequences via interaction with the minor groove. To determine the functional signi®cance of the Tax-DNA interaction, we synthesized minor groove-binding pyrrole-imidazole polyamides which bind speci®cally to the GC-rich sequences in the viral CREs. At concentrations where the polyamides speci®cally protect the GC-rich sequences from MPE:Fe cleavage, the polyamides block the Tax-DNA interaction. At precisely these same concentrations, the polya- mides speci®cally inhibit Tax transactivation in vitro, without altering CREB-activated transcription or basal transcription from the same promo- ter. Together, these data provide strong evidence that Tax-viral CRE interaction is essential for Tax function in vitro, and suggest that targeted disruption of the Tax-DNA minor groove interaction with polyamides may provide a novel approach for inhibiting viral replication in vivo. # 1999 Academic Press Keywords: polyamides; transcription; oncoprotein; CBP; CREB *Corresponding author Introduction The human T-cell leukemia virus type-1 (HTLV- 1) is a retrovirus etiologically linked to several human diseases, including an in¯ammatory neuro- degenerative disorder called tropical spastic para- paresis (TSP), and an aggressive malignancy called adult T-cell leukemia (ATL) (Watanabe, 1997). Although the molecular basis of HTLV-I pathogen- esis is not well understood, there is strong emer- ging evidence that expression of the virally encoded Tax protein is responsible for the clinical manifestations associated with the virus. Ef®cient HTLV-I virion production is dependent upon Tax expression, as Tax is required for high level tran- scription of the viral genome (Cann et al., 1985; Chen et al., 1985; Felber et al., 1985; Sodroski et al., 1985). Tax stimulates HTLV-1 gene expression through interaction with three 21 bp repeat enhan- cer elements, called viral CREs (cAMP response elements), located in the transcriptional control region of the virus (Rosen et al., 1985; Paskalis et al., 1986; Shimotohno et al., 1986; Brady et al., 1987; Fujisawa et al., 1986; Jeang et al., 1988). The viral CREs are composed of a central eight base-pair off- consensus CRE core, and short run of highly con- served guanine and cytosine (GC) base-pairs immediately adjacent to the CRE core. Both the CRE core and G  C-rich ¯anking sequences are critical for Tax-mediated activation of HTLV-I tran- scription (Lenzmeier & Nyborg, 1999). For Tax stimulation of HTLV-I transcriptional initiation, Tax makes a series of protein-protein and protein-DNA contacts that lead to the for- mation of an elaborate nucleoprotein structure on the HTLV-I promoter. The assembly of the Tax- containing complex requires the binding of the cel- Present address: B. A. Lenzmeier, Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA. Abbreviations used: HTLV-1, human T-cell leukemia virus type-1; CRE, cAMP response element; bZIP, basic- leucine zipper; EMSA, electrophoretic mobility shift assay; GST, glutathione-S-transferase. E-mail address of the corresponding author: jnyborg@vines.colostate.edu Article No. jmbi.1999.2969 available online at http://www.idealibrary.com on J. Mol. Biol. (1999) 291, 731±744 0022-2836/99/340731±14 $30.00/0 # 1999 Academic Press