FIG. 2.
HIV-1 Env colocalizes with the raft markers GM1 and CD59 but not with the nonraft marker TfR. JurkatLAI cells were incubated for 1 h at 12°C with B-Ctx, the human Env-specific antibody 50-69, and either a mouse CD59-specific monoclonal antibody (Serotec) or a mouse anti-human TfR-specific antibody (from T. Harder, University of Oxford). Cells were washed at 4°C and incubated for a further 1 h at 12°C with anti-human-TRITC, streptavidin-Cy5, and anti-mouse-FITC to promote antibody-induced patching of CD59 and TfR as described previously (14). Cells were then washed, fixed in paraformaldehyde, and analyzed by confocal microscopy. (A) CD59 (green) colocalizes with HIV-1 Env (red) and GM1 (blue). (B) TfR (green) does not colocalize with HIV-1 Env (red) and GM1 (blue). (C) Incorporation of GM1 into a budding HIV-1 virion. JurkatLAI cells were fixed sequentially in 4% and 8% paraformaldehyde, stained for Env with the human MAb 2G12 (20 μg/ml) and for GM1 with B-Ctx (20 μg/ml), washed, and labeled with anti-human immunoglobulin G and anti-biotin conjugated with 5-nm and 10-nm gold colloids, respectively (Agar, United Kingdom). Cells were then fixed in a mixture of glutaraldehyde and paraformaldehyde, washed, and postfixed in 1% osmium tetroxide in cacodylate buffer. After extensive washing, the cells were incubated in 0.5% magnesium uranyl acetate, dehydrated in ethanol and propylene oxide, and embedded in Epon resin. Ultrathin sections were examined using a Phillips FEI Technai 12 transmission electron microscope, and digital images were captured using Soft imaging software and processed using Photoshop. Bar, 100 nm.