FIG. 6.

HIV-1 endocytosis in CEM_GFP target cells can occur independently of the internalization of cell-free viral particles. Shown are FACS analyses for the detection of both GFP and HIV-1 Gag products in CEM_GFP cells cocultured for 6 and 16 h with U937_HIV-1 cells after incubation for 3 h at 4°C in the presence of 0.5 μg/ml of Leu3A anti-CD4 MAbs. CEM_GFP cells (5 × 10⁴) were pelleted with 10⁵ U937_HIV-1 in a U-bottom 96-well plate and suspended in 20 μl of complete medium in the presence of both 1 μg/ml of T-20 and the anti-CD4 MAbs. After 3 h at 4°C, 80 μl of complete medium complemented with the anti-CD4 MAbs was added, and the plate was immediately incubated at 37°C. As controls, parallel cocultures were run in the absence of anti-CD4 MAbs or upon pretreatment of CEM_GFP cells for 30 min at 4°C with the anti-CD4 MAbs, which were afterwards maintained over time. The cells were then harvested at the indicated times and analyzed by FACS. The quadrants are set on the basis of uninfected CEM_GFP cells. The percentages of Gag⁺ cells are indicated. The results are representative of two independent experiments.