HIV TAT GENE AND TRANSCRIPTIONAL ACTIVATION

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The Tat gene of human immunodeficiency virus (HIV) plays a central role in the activation and life cycle of HIV. Tat exerts its effects at the level of transcriptional initiation and elongation. We have previously reported the direct physical interaction between the HIV-1 Tat protein and the basal transcription factor, TBP/TFIID. Affinity chromatography demonstrated that wild type Tat, but not a transactivation mutant of Tat, was capable of depleting TBP/TFIID from cell extracts. These experiments represented the first demonstration of a basal transcription factor that binds, in an activation-dependent manner, to Tat. We now report that the Tat-TBP interaction can be detected in HIV-1-infected cells. The domain of TBP interacting with Tat has been mapped from amino acids 163 to 196 using deletion and site-specific mutants of TBP. This domain of TBP, which includes the H1 and S2 domain, is distinct from the H2 binding site for other activator proteins such as E1A. The interaction of Tat with TFIID regulates the binding of accessory proteins to TFIID. Tat stabilizes the interaction of TFIID with TFIIA in a gel shift assay. In addition, Tat competes for Dr1 interaction with TBP. Our results suggest that the basal transcription factor, TBP/TFIID, represents an important regulatory molecule in HIV transcription. The human immunodeficiency virus type 1 (HIV-1) transactivator Tat protein is essential for efficient viral gene expression and virus replication. Tat interacts with the basal transcription factor TFIID. This interaction is mediated through the amino acid 36-50 core domain of Tat. We now demonstrate that soluble peptide analogs of the Tat core domain are able to effectively block LTR transactivation. In cotransfection experiments, Tat peptide analogs inhibited Tat transactivation of an HIV-1 LTR-CAT reporter construct up to 80-fold. Inhibition of control promoters was approximately two-fold. Tat peptide analogs inhibited HIV virus replication by 85 percent in latently infected U1 cells induced with Tat. While both short and long peptide analogs (amino acids 36-50 vs 36-72) inhibited Tat transactivation in transient assays, the short peptides were more effective inhibitors of virus replication in U1 cells. Furthermore, U1 cells treated with the Tat peptide analog showed markedly delayed virus transmission when co-cultivated with parental U937 cells. The Tat peptide analog did not decrease expression of cellular genes including beta-actin, GAPDH and histone H2Bb.