FIG 6.

Tetherin antagonism by Nef protects HIV-1-infected cells from ADCC. (A) CEM.NKR-_CCR5-sLTR-Luc cells were infected with wild-type HIV-1 NL4-3 (Vpu⁺), HIV-1 NL4-3 Δvpu, or HIV-1.Δvpu.IeG-Nef recombinants expressing the indicated Nef alleles and incubated with a CD16⁺ NK cell line at an effector-to-target cell ratio of 10:1 in the presence of serial dilutions of purified IgG from HIV-1-positive donors (HIVIG). ADCC was calculated from the luciferase activity (RLU) after an 8-h incubation. Error bars indicate the standard deviations of the means for triplicate wells at each antibody concentration, and the dotted line indicates 50% killing of HIV-1-infected cells. (B) Nef and p55 Gag expression in virus-infected cells was confirmed by Western blot analysis of cell lysates with staining for β-actin as a control for sample loading. (C and D) Histograms showing the fluorescence intensity of tetherin (BST-2) (C) and Env (D) staining on the surface of viable, HIV-1-infected (p55⁺ CD4^low) cells as described above for panel A. Tetherin and Env staining on cells infected with virus expressing NL4-3 Nef is shown as a reference (red histograms). The shaded histograms indicate nonspecific staining with either an isotype control for the BST-2-specific antibody (C) or IgG from HIV-negative donors (D). (E and F) Susceptibility to ADCC correlates with the fluorescence intensity of tetherin (E) and Env (F) staining on the surface of virus-infected cells (Pearson correlation test). The data are representative of results from three independent experiments.