Specific Disruption of NFAT5 Binding Impairs LTR–Reporter Gene Activity in Monocytic THP-1 Cells

<div><p>(A) Disruption of NFAT5 binding does not impair NF-κβ (p50/p65) binding. An EMSA is shown using recombinant NFAT5 and NF-κB proteins with an oligonucleotide matching the NFAT5/NF-κβ site from the HIV-1_LAI (wild-type [WT]) or an oligonucleotide with a mutation in the nt predicted to be required for NFAT5 binding (N5-Mut), but not NF-κB binding (shown at the bottom of the figure).</p><p>(B) Mutation of the NFAT5 binding site significantly reduces HIV-1_LAI LTR reporter activity in THP-1 cells. HIV-1_LAI LTR-Luc reporter constructs that contained −208 to +64 nt relative to the transcriptional initiation site of HIV-1_LAI, and that were isogenic except for the specific N5 mutation, were transfected into THP-1 cells with the <i>Renilla</i> luciferase (pRL-TK) control plasmid. Luciferase activity was determined 26 h later. The LTR mutation, which abolishes NFAT5 binding to the promoter/enhancer region, significantly (**, <i>p</i> < 0.01) suppressed LTR-dependent activity of transcription compared with WT in THP-1 cells.</p>