FIG 2.

HIV-1 group M Nef antagonism of human tetherin. The indicated HIV-1 group M Nef proteins were tested for their ability to rescue virus release for HIV-1 NL4-3 Δvpu Δnef in HEK293T cells expressing human tetherin, as described in the legend of Fig. 1. (A and B) The accumulation of HIV-1 p24 in the cell culture supernatant was measured by an antigen capture ELISA (A), and the amounts of p24 in the culture supernatants versus Nef and p55 Gag in cell lysates were compared by Western blot analysis (B). (C) Tetherin downmodulation was assessed by transfecting a HEK293T cell line that stably expresses human tetherin (293T-hBST-2 cells) with bicistronic pCGCG constructs that coexpress Nef and eGFP. Histograms show tetherin (BST-2) staining on the surface of viable, eGFP-positive cells relative to cells transfected with the empty pCGCG vector (dark blue) or stained with an isotype control antibody (shaded). (D) Differences in the surface expression levels of tetherin are plotted as a percentage of tetherin staining on 293T-hBST-2 cells transfected with an empty vector. The error bars represent the standard deviations of the means for replicate assays, and differences in mean virus release (A) and tetherin downmodulation (D) compared to empty vector controls were estimated by ordinary one-way ANOVA (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001). The data shown in panels A to D are representative of results from three independent experiments.