Figure 1

Expression of ferroportin inhibits HIV-1 transcription. A and B, Expression of ferroportin in 293T cells. 293T cells were transfected with vectors expressing wild type ferroportin or CD4. At 24 hours posttransfection, the cells were stained with APC-linked antibodies against c-myc or CD4 and analyzed by FACS (A) or the cells were lysed and expression of ferroportin and CD4 was verified by SDS-PAGE and immunoblotting (B). In panel A, solid line - the cells stained with antibodies, shadow line - cells stained with non-specific IgG linked to APC. C, Inhibition of Tat-induced transcription. 293T cells were transfected with vectors expressing CD4 or wild type ferroportin. At 24 hours posttransfection, the cells were re-transfected with HIV-1 LTR-LacZ (lane 1) or and HIV-1 LTR-LacZ and HIV-1 Tat expression vectors (lane 2) combined with EGFP expression vector. After 24 hours of culturing, the cells were lyzed and β-galactosidase activity was determined using ONPG-based assay. D, Efficiency of transfection verified by co-expression of EGFP. 293T cells were transiently transfected with HIV-1 LTR-Luciferase reporters in combination with CMV-EGFP. The cells were cultured for 24 hrs posttransfection, then lysed and EGFP fluorescence was measured on Luminescence spectrometer. The results are averages of 4 independent transfections. Lane 1, WT HIV-1 LTR (-105 to +77). Lane 2, HIV-1 LTR (-81 to+77) with NF-kB deleted sites. Lane 3, HIV-1 LTR (-105 to +77) with Sp1 inactivated sites. E, Inhibition of basal HIV-1 transcription. 293T cells were transiently transfected with indicated HIV-1 LTR-Luciferase reporters in combination with control CMV-EGFP, ferroportin-EGFP or cdNIPP1-EGFP expression vectors. Lane 1, WT HIV-1 LTR (-105 to +77). Lane 2, HIV-1 LTR (-81 to+77) with NF-κB deleted sites. Lane 3, HIV-1 LTR (-105 to +77) with Sp1 inactivated sites. The cells were cultured for 24 hrs posttransfection, then lysed and the lysates were used to measure GFP fluorescence and luciferase activity.