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 Tat1-86, 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 Tat1-86 and Tat1-72 transactivated beta-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 Tat1-86 and Tat1-72 with the sulfhydryl 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 Tat1-48 was inactive, with cysteine residues either free or protected. Similarly, other shorter synthetic peptides covering the different Tat domains were inactive. Interestingly, when peptides Tat1-48 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.
Vives
E
E
Laboratoire de Biochimie, Ingénierie des protéines, CNRS, URA1455, Faculté de Médecine Secteur Nord, Marseille, France.
Charneau
P
P
van Rietschoten
J
J
Rochat
H
H
Bahraoui
E
E
D000595:Amino Acid Sequence; D001483:Base Sequence; D003545:Cysteine / Q000494:pharmacology; D015696:Gene Products, tat / Q000138:chemical synthesis / Q000494:pharmacology*; D016325:HIV Long Terminal Repeat / Q000235:genetics; D015497:HIV-1 / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000494:pharmacology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D015533:Transcriptional Activation / Q000187:drug effects*; D001616:beta-Galactosidase / Q000096:biosynthesis; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D013438:Sulfhydryl Compounds; D054322:tat Gene Products, Human Immunodeficiency Virus; D001616:beta-Galactosidase; D003545:Cysteine