Capacite de liaison de la proteine NFAT1 a la region 'enhancer' du VIH-1 et son role potentiel dans la modulation transcriptionnelle

Members of the NFAT family seem to play an important role in transcription modulation through their binding to the HIV-1 enhancer region located in the LTR (Long Terminal Repeat). In the present study, we wanted to determine the degree of implication of this factor in the modulation of LTR activity. The ultimate goals of this study were to evaluate the possible role of NFAT1 in transcriptional regulation of the HIV-1 clade LTR and the essential nucleotides permitting an effective binding on the kappaB motifs. EMSA competition assays were performed using competitor oligonucleotides designed from the HIV-1 clade enhancer regions or oligonucleotides differently mutated in the kappaB region. Nuclear extracts from Jurkat cells stimulated with TNFalpha, from 293T cells transiently expressing NFAT1 or recombinant NFAT1 purified from E. coli were also used. Our analysis indicated that clade E showed a lesser binding capacity to NFATI when compared to other clades. We have also demonstrated that nucleotides 5' - GGAAA - 3' of the NFAT1 consensus sequence, located on the antisense strand, remain essential for the formation of the NFAT1/DNAv complex. Evidence also suggested that the first nucleotide of the AP-2-like region (C; antisense strand) could play a role in this binding. We also demonstrated that mutation on the NFAT recognition site in the enhancer region does not completely inhibit the binding of NF-kappaB factor. These results clarify the role played by NFAT1 in HIV-1 transcriptional modulation and specify its binding requirement to the kappaB repeat of the LTR region.