Importance of NFAT in HIV-1 LTR regulation by bpV[pic].

(A) Jurkat cells were transiently transfected by the DEAE-Dextran protocol with 15 μg pLTR-LUC plus 15 μg of either the control vector pRc/β-actin (■) or the pRc/β-actin VIVIT-GFP vector (▪). Following a 24-hour incubation period, cells were either left untreated or stimulated with PHA (3 μg/mL), PMA (20 ng/mL), or bpV[pic] (10 μM) and lysed after 8 hours of stimulation. Luciferase activity was evaluated as the mean of 3 different measured samples and is represented as fold increase over untreated samples. This is representative of 3 independent experiments. (B) Nuclear extracts from bpV-treated Jurkat cells were incubated with the HIV-1 enhancer probe and competed with either 100-fold cold excess of NFAT oligonucleotide (lane 2), NF-κB oligonucleotide (lane 3), wild-type HIV-1 enhancer oligonucleotide (lane 4), or HIV-1 enhancer oligonucleotide mutated at the NF-κB–binding sites (lane 5). Samples were then migrated on a native polyacrylamide gel, dried, and exposed on Kodak X-OMAT film. Free probe and specific complexes are indicated on the left side of the panel. (C) 293T cells were transiently transfected by the calcium phosphate protocol with 5 μg pLTR-LUC plus 5 μg of either pREP4, pREP4-NFAT1, or pREP4-NFAT2. Cells were washed 16 hours after transfection and incubated for another 24 hours in fresh medium before lysis. Luciferase activity was evaluated as the mean of 3 different measured samples. This is representative of 3 independent experiments.