Figure 1

Nef increases the amount of Gag proteins in HeLa cells. HeLa cells were infected with VSV-G-pseudotyped wild type (WT) or nef-deleted (∆Nef) viruses. At day 2 post-infection cells were stained with the anti HIV-1 p24 KC57 antibody and the percentage of KC57 positive cells and their relative mean fluorescence intensity (MFI) were analyzed by flow cytometry. (a) Representative dot plots of infected HeLa cells (left panels). Percentage of KC57 positive cells is indicated in the top right corner of the gated population. MFI for the gated population is also indicated. A compilation of 10 independent experiments (Mean+SEM) of the percentage of KC57 positive cells and the Gag (KC57) MFI are shown (right panels). (b). Levels of HIV-1 p24 (in ng/ml) in the supernatants and cell lysates derived from cells described in (a) and measured by ELISA using the anti HIV-1 p24 183-H12-5C antibody. (c) The efficiency of viral release was calculated as the ratio between the levels of HIV-1 p24 in the supernatants and the total antigen HIV-1 p24 (supernatant + cell associated). Mean+SEM is shown. (d). HeLa cells were infected with VSV-G-pseudotyped WT- or ∆Nef viruses in which the green fluorescent protein (GFP) was inserted in frame with Gag-p17 (Gag-GFP). Cells were harvested at day 2 post-infection and analyzed by flow cytometry. Of note GFP signal does not distinguish between the mature and immature forms of Gag-GFP. The Gag-GFP MFI was measured on gated Gag-GFP positive HeLa cells. Representative dot plot analysis (left panel) in which the percentage of Gag-GFP positive cells is indicated in the top right corner of the gated population together with the MFI. Mean+SEM of 3 independent experiments of Gag-GFP MFI (right panel). *p<0.05; **p<0.01 (Mann Whitney test).