Nef rescues the release of Gag VLPs from the L domain-deleted and L domain-mutated Gag polyproteins.A)Efficient production of Gag VLPs from a mutant hybrid GagΔ p6.Nef chimera. Two days after the transfection, supernatants from 293T cells expressing wild-type Gag as well as mutant GagΔ p6 proteins and the mutant hybrid GagΔ p6.Nef chimera were collected and submitted to ultracentrifugation for the purification of Gag VLPs. Purified Gag VLPs and cell lysates were processed as in Fig. 1. Lane 1: Wild type Gag protein; Lane 2: Mutant GagΔ p6 protein; Lane 3: Mutant hybrid GagΔ p6.Nef chimera. (B)Hybrid Vpr.Nef protein increases the release of Gag VLPs from a mutated p6 and Pol-deleted virus. The mutant GagLTAL provirus was co-expressed with Vpr or with the Vpr.Nef chimera in 293T cells. Two days after the transfection, supernatants and cells were collected. Purified Gag VLPs and cell lysates were processed as in Fig. 1. Equivalent amounts of the mutant GagLTAL protein were loaded in the lysate to facilitate comparisons between GagVLPs in the supernatant. Gag VLPs were detected with α p24 antibodies. Ratios between the mutant GagLTAL proteins in supernatants and lysates are presented in the bar graph below the western blots. Lane 1: Mutant GagLTAL protein with Vpr; Lanes 2 and 3; Mutant GagLTAL protein and increasing concentrations of the hybrid Vpr.Nef protein.