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. Author manuscript; available in PMC: 2009 Nov 25.
Published in final edited form as: Virology. 2008 Oct 1;381(2):161–167. doi: 10.1016/j.virol.2008.08.033

Figure 4.

Figure 4

FOXP3 inhibits HIV-1 infection by interfering with NFAT and/or NFκB function on the HIV-1 LTR. (A) FOXP3 inhibits LTR activity in 293T cells. 293T cells were co-transfected with a control or wild-type FOXP3 expression vector, an NFAT2 expression vector, and an HIV-1 LTR luciferase reporter plasmid as described in the Materials and Methods. Data show that FOXP3 inhibits NFAT2 induced LTR activity in 293T cells. One representative of 3 experiments is presented. (B) FOXP3 decreases the binding of NFAT2 to the LTR. ChIP with anti-NFAT2 and anti-FOXP3 antibodies was performed using control (without FOXP3) and FOXP3 transfected 293 cells, which were co-transfected with an HIV-1 LTR reporter plasmid. NFAT2 and FOXP3 engagement in vivo of the HIV-1 LTR reporter sequence was analyzed using PCR amplification primers specific to the proximal HIV-1 LTR sequence as described in the Materials and Methods. ChIP analysis data are presented as fold differences (mean ± SEM) between specific antibody and isotype control from 3 independent experiments (*p=0.053, compared to NFAT2 without FOXP3). (C) FOXP3 does not substantially inhibit productive infection by NL4-3 viruses mutated at either the dual NFAT or NFκB binding sites. FOXP3 or control lentivirus transduced T-cells were infected with NL4-3 wild type, NFAT-mutant, or NFκB-mutant HIV-1 viruses and were monitored for p24-gag expression. The wild type and NFAT and NFκB mutant sequences are depicted above the bar graphs with the mutant sequences underlined and bolded. Data in the bar graph to the left are represented as % inhibition of infection in FOXP3 transduced cells compared to control cells for the wild type and each binding mutant virus. Data are the mean ± SEM of 3 experiments (**p=0.005; *p=0.024). Data shown on the right is a representative example of 1 of 3 experiments. Data are presented as %GFP+, p24+ cells for each virus and for both the control and FOXP3 transduced cells. The binding mutant viruses have reduced but not absent HIV-1 production in response to T cell activation.