EFFECTS OF THE TAT BASIC DOMAIN ON HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 TRANSACTIVATION, USING CHEMICALLY SYNTHESIZED TAT PROTEIN AND TAT PEPTIDES

To study the structure relationship of different Tat domains, the full -length Tat protein Tat(1-86), the gene product of the first exon Tat( 1-72) which retains full activity of the protein, and a panel of short er peptides mimicking different regions of the primary structure of th e Tat protein were chemically synthesized by the solid-phase method, u sing an efficient protocol. Synthetic Tat(1-86) and Tat(1-72) transact ivated beta-galactosidase activity in HeLa cells containing the lacZ g ene under the control of the human immunodeficiency virus type 1 long terminal repeat. Analyses of the activity of Tat(1-86) and Tat(1-72) w ith the sulfhydryl of cysteine residues free or protected by tile acet amidomethyl group showed that only the Tat fragments with deprotected cysteine residues retain transactivation ability. In contrast, peptide Tat(1-48) was inactive, with cysteine residues either free or protect ed. Similarly, other shorter synthetic peptides covering the different Tat domains were inactive. Interestingly, when peptides Tat(1-48) and Tat(38-60) were used simultaneously, a significant transactivation wa s obtained. This result suggests that both peptide domains are implica ted in transactivation, probably by acting at two different sites. Thi s permits us to propose a fundamentally new step in the understanding of the molecular mechanism of Tat transactivation.