FIG. 1.

GM1 and Env expression in uninfected and infected T cells. (A) Jurkat CE6.1 T cells (top panels), naïve CD4⁺ T cells negatively enriched from peripheral blood mononuclear cells (PBMC) (middle panels), or 3-day PHA (5 μg/ml)- and interleukin-2 (10 IU)-activated primary CD4⁺ T cells (bottom panels) were washed and resuspended in RPMI-1% fetal calf serum (WB), and 5 × 10⁵ cells were allowed to settle onto poly-l-lysine-coated coverslips, fixed in ice-cold 4% paraformaldehyde in phosphate-buffered saline-1% bovine serum albumin, and stained for GM1-containing lipid rafts (green) with 10 μg/ml biotinylated B-Ctx (Sigma, United Kingdom) at 4°C followed by streptavidin-FITC (Jackson Immunoresearch). All staining with B-Ctx was performed on fixed cells to prevent Ctx-induced cross-linking and GM1 capping. (B) HIV-1_LAI-infected (Jurkat_LAI) effector cells were washed, allowed to settle onto poly-l-lysine-coated coverslips, and stained for surface Env (red) and GM1 (green) with 10 μg/ml of the human Env-specific antibody 50-69 (CFAR, United Kingdom) and 10 μg/ml B-Ctx followed by anti-human-TRITC and streptavidin-FITC, respectively. (C) HIV-1_LAI-infected 3-day PHA- and interleukin-2-activated primary CD4⁺ T-cells negatively enriched from PBMC by magnetic selection (Miltenyi Biotech, United Kingdom) were stained for Env (red) and GM1 (green) as described above. All images represent single x-y sections through the center of a cell and were acquired using a Bio-Rad MicroRadiance laser scanning confocal microscope and subsequently processed using Metamorph v6 (Universal Imaging Corporation) and Photoshop 7.0 (Adobe Inc.). Fluorescence images are shown next to the corresponding Nomarski images, and the colocalization of red and green gives yellow staining.