Effects of the Tat Basic Domain on Human Immunodeficiency Virus Type 1 Transactivation, Using Chemically Synthesized

To study the structure relationship of different Tat domains, the full-length Tat protein Tat-86 ' the gene product of the first exon Tat1-72 which retains full activity of the protein, and a panel of shorter peptides mimicking different regions of the primary structure of the Tat protein.were chemically synthesized by the solid-phase method, using an efficient protocol. Synthetic Tat,-86 and Tat,-72 transactivated "-galactosidase activity in HeLa cells containing the lacZ gene under the control of the human immunodeficiency virus type 1 long terminal repeat. Analyses of the activity of Tatl86 and Tat,-72 with the sulfbydryl of cysteine residues free or protected by the acetamidomethyl group showed that only the Tat fragments with deprotected cysteine residues retain transactivation ability. In contrast, peptide Tat,-48 was inactive, with cysteine residues either free or protected. Similarly, other shorter synthetic peptides covering the different Tat domains were inactive. Interestingly, when peptides Tat,-48 and Tat38 60 were used simultaneously, a significant transactivation was obtained. This result suggests that both peptide domains are implicated in transactivation, probably by acting at two different sites. This permits us to propose a fundamentally new step in the understanding of the molecular mechanism of Tat transactivation. Tat, an 86-amino-acid transactivating protein, is directly involved in the replication of human immunodeficiency viru