Figure 3

90K-myc reduces cell surface levels of HIV-1 Env and is not a global inhibitor of furin-mediated proteolytic activity. (A) 293T cells were cotransfected with pBR.HIV-1 IRES GFP and vector or pcDNA6.90K-myc. Two days post transfection, levels of surface HIV-1 gp120 were assessed by flow cytometry following staining with a human anti-HIV-1 Env antibody. Shown are representative dot plots from one experiment out of three. Red numbers indicate the MFI in gate P2 (HIV-negative cells) and P3 (HIV-positive cells). (B) Quantitative analysis of relative HIV-1 Env surface levels in the absence or presence of 90K-myc. Env cell surface levels were calculated by comparing, within the same sample, Env levels on non-GFP-expressing cells (gate P2) with Env levels on cells with medium-high expression levels (gate P3). The ratio obtained for vector-transfected cells was set to 100%. Shown is the arithmetic mean ± S.E.M. of three independent experiments. * : p < 0.05 (Student’s T-Test). (C-F) 293T cells were cotransfected with an expression vector for the indicated furin-dependent glycoproteins (1.3 μg) and vector or pcDNA6.90K-myc (1.3 μg). Two days post transfection, cells were lysed and proteins were subjected to Western Blotting using the indicated antibodies. Depicted are the efficiencies of precursor processing, measured by Infrared imaging-based quantification of the respective precursor and the cleavage product.