FIG. 2.

Expression of Nef during HIV-1 production: impact on viral infectivity. GFP reporter viruses were produced in 293T cells in the presence of increasing amounts of a Nef-coding plasmid (0.125 to 2 μg/T75 flasks). (A) Producer cells were solubilized (left) and viruses were pelleted from the cell culture supernatant by ultracentrifugation (right). Samples were resolved by SDS-PAGE and visualized by Western blotting (WB) followed by immunodetection with p24 (top)- and HA (bottom)-specific antibodies. (B) Molecular species derived from the processing of Nef in mature viruses. The lane showing the incorporation of Nef into viral particles (panel A, lane 10) is shown next to a schematic representation of Nef processed by HIV-1 protease. The position of the processing sites (green arrows) was determined according to published data and the measurement of the mobility of the anti-HA reactive proteins in the gel. (C) Viral infectivity was assayed with HeLa CD4 cells. HeLa CD4 cells were incubated with increasing amounts of viruses (50 × 10³ to 400 × 10³ RT cpm/ml) produced in the presence of the indicated amounts of Nef-coding plasmid, and the percentages of GFP-positive cells were measured by fluorescence-activated cell sorting 60 h postinfection.