Figure 2

Nef increases Gag proteins levels in infected primary CD4+ T cells. PHA-activated primary CD4+ T cells were exposed to VSV-G-pseudotyped WT or ∆Nef (50–150 ng of p24/ml) for 3 h. The virus was washed off and the infected cells were cultured for up to 3 days. Productive infection was followed by flow cytometry of intracellular HIV-1 Gag using the KC57 anti-HIV-1 p24 monoclonal antibody. (a) Evolution of the fraction of Gag (KC57) positive cells at the indicated days post infection. Data are Mean±SEM of cells from three independent donors. (b) MFI of intracellular Gag (KC57) staining calculated on the fraction of Gag (KC57) positive cells. Maximum, Minimum and Mean of results obtained in cells described in Figure 1a are indicated. (c) Representative dot-plot analysis of Gag (KC57) staining of primary CD4+T cells infected with WT or ∆Nef (at day 2 post-infection). Cells were exposed to a higher viral input of ∆Nef than WT, in order to obtain similar fraction of infected cells. The percentage of Gag (KC57) positive cells is indicated in the top right corner of the gated population. MFI is also indicated. (d) Analysis of infected cells from 11 independent infections (8 donors), selected for the same fraction of Gag(KC57) positive cells at day 2 post-infection (left panel). The MFI of Gag (KC57) is reduced in absence of Nef (right panel). Each infection has been symbol-coded. *p<0.05 (Mann Whitney test)