(A) Activated primary rhesus macaque lymphocytes were infected with wild-type SIV, SIV Δnef, SIV ΔnefP and SIV ΔnefP Y721A. Five days post-infection, the cells were treated with IFNα, and stained 24 hours later for the surface expression of CD4 and tetherin (BST-2) and the intracellular expression of SIV p27. The MFI of BST-2 staining for lymphocytes infected with each virus is shown after gating on p27+ CD4+ cells. (B) Virus-infected lymphocytes were examined by confocal microscopy for the co-localization of Env and BST-2. Twenty-four hours after the addition of IFNα, the cells were fixed, permeabilized and stained with monoclonal antibodies to BST-2 (green), Env (red), and with a nuclear dye (blue). The scale bar corresponds to 1 µm. (C) The extent of Env and tetherin co-localization was estimated by calculating the Pearson’s correlation coefficients for images of 20 randomly selected cells infected with each virus (see also Figure S4), and the differences in these values were compared for wild-type SIV- versus SIV Δnef-infected cells and for SIV Δnef- versus SIV ΔnefP-infected cells using an unpaired Student’s t-test.