FIG 3.
Nef and Vpu affect the cell surface levels of tetraspanins in the context of infection. CD4-positive T cells were negatively isolated from fresh buffy coats, infected with wt HIV-1NL4-3-IRES-GFP (wt) or a Nef- and Vpu-defective counterpart (ΔΔ), and stained for surface expression of selected TSPANs 72 h later. Cells were fixed and analyzed by flow cytometry. (A) Representative flow cytometry dot plots of gated living cells. The MFI (YGeoMean) of uninfected (gate R2) and infected (GFP-positive) (gate R3) cells is shown in red for the indicated surface receptors. (B) Histogram of the relative surface expression of the depicted TSPANs and controls (CD4, HLA-ABC, and CD317) after CD4+ T cell infection with wt HIV-1NL4-3-IRES-GFP, ΔΔ, and the single Nef-defective (ΔNef) or Vpu-defective (ΔVpu) counterparts. For each marker, the MFI ratio of infected (R3) to uninfected (R2) cells was determined. Subsequently, the value for ΔΔ-infected cultures was set to 100%. Values are the arithmetic means of data from two independent experiments (two donors) and standard deviations.