Activity of synthetic tat peptides in human immunodeficiency virus type 1 long terminal repeat-promoted transcription in a cell-free system.
The tat protein encoded by the human immunodeficiency virus type 1 is a potent trans-activator of gene expression from the viral long terminal repeat. The domains that are essential for trans-activation, a Pro-Xaa3-Pro triad, a cysteine-rich metal-binding sequence motif, and a cluster of basic residues, are present within the N-terminal 57 residues of tat. To determine the structural requirements for tat function and the role of metal binding at the transcription level alone, tat-(1-86) (full-length tat peptide), tat-(1-57), and tat-(1-47) were chemically synthesized. These peptides as well as the Cd2+ and Zn2+ complexes of tat-(1-86) and tat-(1-57) were evaluated for stimulation of transcription from the human immunodeficiency virus type 1 long terminal repeat by using cell-free in vitro methods. All three peptides produced a 7- to 9-fold increase over the basal level of transcription at a peptide concentration of 0.4 microM. Interestingly, at 4 microM, both tat-(1-57) and tat-(1-86) inhibited even the basal level of transcription. In contrast, tat-(1-47), which lacks the basic domain (residues 49-57), exhibited full stimulatory activity at 4 microM. Our data suggest, therefore, that the basic region may be responsible for the observed inhibitory activity of tat-(1-86) and tat-(1-57). Furthermore, binding to Zn2+ and not to Cd2+ ions only slightly augments (approximately 2-fold) the activity of the tat peptides.
Jeyapaul
J
J
Wistar Institute, Philadelphia, PA 19104.
Reddy
M R
MR
Khan
S A
SA
D000595:Amino Acid Sequence; D002104:Cadmium / Q000378:metabolism; D015696:Gene Products, tat / Q000138:chemical synthesis* / Q000235:genetics / Q000494:pharmacology; D015497:HIV-1 / Q000235:genetics*; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids; D011485:Protein Binding; D011487:Protein Conformation; D012091:Repetitive Sequences, Nucleic Acid; D015534:Trans-Activators / Q000138:chemical synthesis*; D014158:Transcription, Genetic / Q000187:drug effects; D015032:Zinc / Q000378:metabolism; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; D015534:Trans-Activators; D054322:tat Gene Products, Human Immunodeficiency Virus; D002104:Cadmium; D015032:Zinc