10.1371/journal.ppat.1002620.g003 Figure 3  Specific disruption of NFAT5 or NF-κB binding sites in the LTR of HIV-1 subtype B.
(A) Mutations introduced into a HIV-1Lai/Bal-env infectious molecular clone. NF-κB and NFAT5 binding site mutations were introduced into the LTR of the HIV-1Lai/Bal-env-wild type (WT) infectious molecular clone. The NF-κB binding site mutants (HIV-1Lai/Bal-env-κB I-Mut, HIV-1Lai/Bal-env-κB II-Mut, HIV-1Lai/Bal-env-κB I+II-Mut), and NFAT5 binding site mutant (HIV-1Lai/Bal-env-N5-Mut) are shown along with the HIV-1Lai/Bal-env-WT sequence. Mutations were introduced into the 3′ LTR of the HIV-1Lai/Bal-env–WT proviral sequence. (B) Specific mutation of the NFAT5 binding site abolishes NFAT5 binding to the viral LTR but does not affect NF-κB p50/p65 binding to the overlapping NF-κB binding site. Quantitative DNaseI footprinting analysis is shown using HIV-1 LTR fragments (-262 to +4 nt relative to the transcription start site) from HIV-1Lai/Bal-env-WT and HIV-1Lai/Bal-env-N5-Mut and increasing concentrations of recombinant NF-κB (p50/p65) (25 ng, 100 ng, and 500 ng), or NFAT5 (10 ng, 50 ng, and 250 ng). The regions that are protected from DNase I cleavage by the binding of NF-κB and NFAT5 are indicated with a bars. (C) Specific disruption of the HIV-1 subtype B NF-κB binding sites effectively abrogates recombinant p50/p65 binding. Quantitative DNaseI footprinting analysis is shown of nucleotides −262 to +4 from HIV-1Lai/Bal-env-WT, HIV-1Lai/Bal-env-κB I-Mut, HIV-1Lai/Bal-env-κB II-Mut, and HIV-1Lai/Bal-env-κB I+II-Mut and increasing concentrations of recombinant NF-κB (p50/p65) (25 ng, 100 ng, and 500 ng). The regions that are protected from DNase I cleavage by the binding of recombinant NF-κB are indicated with a bars. (D) Specific disruption of the HIV-1 subtype B NF-κB binding site does not inhibit but enhances NFAT5 binding to this region. Quantitative DNaseI footprinting analysis is shown of nucleotides −262 to +4 from HIV-1Lai/Bal-env-WT, HIV-1Lai/Bal-env-κB I-Mut, HIV-1Lai/Bal-env-κB II-Mut, and HIV-1Lai/Bal-env-κB I+II-Mut and increasing concentrations of recombinant NFAT5 (10 ng, 50 ng, and 250 ng). The regions that are protected from DNase I cleavage by the binding of recombinant NFAT5 are indicated with a bars.