Identification of three NFAT binding motifs in the 5′-upstream region of the human CD3γ gene that differentially bind NFATc1, NFATc2, and NF-κB p50

Human immunodeficiency virus, type 1 (HIV-1) infection of CD4(+) T cells progressively abrogates T cell receptor (TCR)-CD3 function and surface expression by specifically interfering with CD3gamma gene transcription. Our data show that the loss of CD3gamma transcripts begins very early after infection and accumulates to a >90% deficiency before a significant effect on surface receptor density is apparent. Blocking TCR-CD3-directed NFAT activation with cyclosporin A provokes a partial re-expression of CD3gamma gene transcripts and surface complexes in a time- and dose-dependent manner. We have identified three NFAT consensus sequences (5'-GGAAA-3') in the 5'-upstream region of the human CD3gamma gene at: -124 to -120 (NFAT(gamma1)), -384 to -380 (NFAT(gamma2)), and +450 to +454 (NFAT(gamma3)) from the first transcription initiation site. Using electrophoretic mobility shift and supershift assays, we show that NFATc2 alone binds to the NFAT(gamma2) motif, however, complexes containing either NFATc2 or NFATc1 plus NF-kappaB p50 bind to the NFAT(gamma1) and NFAT(gamma3) sites. We further demonstrate that NFATc1 and NF-kappaB p50 bind in the same protein-DNA complex and that a fourth Ala added to the core sequence (5'-GGAAA (A) under bar -3') in NFAT(gamma1), and NFAT(gamma3) is critical for their binding. Finally, we have shown that an increase in the binding of nuclear NFATc2, NFATc1, and NF-kappaB p50 to these three motifs is correlated with a progressive loss of CD3gamma transcripts after HIV-1 infection.