Activation of transcription factor NF-kappaB by the Tat protein of human immunodeficiency virus type 1.
A recombinant Tat protein was used to investigate the molecular mechanisms of transcriptional activation of the human immunodeficiency virus type 1 long terminal repeat (LTR). Liposome-mediated delivery of this protein to responsive cells results in dose-dependent LTR activation. As evaluated by mRNA quantitation with competitive PCR, the activation response is rapid and transient, peaking at 5 h after the beginning of Tat treatment. In vivo footprinting experiments at the LTR showed that transcriptional activation is concomitant with a modification of the protein-DNA interaction pattern at the downstream kappaB site of the enhancer and at the adjacent Sp1 boxes. The effects of Tat on the enhancer are mediated by Tat-induced nuclear translocation of NF-kappaB, which parallels the kinetics of transcriptional activation. This induction results from degradation of the inhibitor IkappaB-alpha, is blocked under antioxidant conditions and by a protease inhibitor, and occurs as a rapid response in different cell types. The functional response to Tat is impaired upon cell treatment with a kappaB site decoy or with sodium salicylate, an inhibitor of NF-kappaB activation. These results show that NF-kappaB activation by Tat is important for LTR transcriptional activation. Furthermore, they suggest that some of the pleiotropic effects of Tat on cellular functions can be mediated by induction of NF-kappaB.
Demarchi
F
F
International Centre for Genetic Engineering and Biotechnology, Trieste, Italy.
d'Adda di Fagagna
F
F
Falaschi
A
A
Giacca
M
M
D000975:Antioxidants / Q000494:pharmacology; D001483:Base Sequence; D002461:Cell Line, Transformed; D004247:DNA / Q000378:metabolism; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D015870:Gene Expression; D015696:Gene Products, tat / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D020672:I-kappa B Proteins; D007700:Kinetics; D008969:Molecular Sequence Data; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D012980:Sodium Salicylate / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects; D014407:Tumor Cells, Cultured; D054322:tat Gene Products, Human Immunodeficiency Virus
D000975:Antioxidants; D017931:DNA Primers; D004268:DNA-Binding Proteins; D015696:Gene Products, tat; D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D011480:Protease Inhibitors; D011993:Recombinant Fusion Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; D000072000:NF-KappaB Inhibitor alpha; D004247:DNA; D004229:Dithiothreitol; D012980:Sodium Salicylate