Figure 2

Vpx treatment of quiescent CD4⁺T-cells results in increased susceptibility to HIV-1 infection. a. Impact of Vpx treatment on quiescent CD4⁺ T-cells susceptibility to HIV-CMV-EGFP infection. Upper right panel: expression vectors used to produce HIV-CMV-EGFP virions. Left panel: PBMCs were treated 12 h with VLP-Mock or VLP-Vpx then infected with HIV-CMV-EGFP or uninfected (control). Proportions of EGFP positive quiescent CD4⁺ T-cells (CD69- HLA-DR-) were assessed 4 days post infection. Lower right panel: Results are expressed as percentage of EGFP positive cells. Graphic shows mean and standard deviation for 6 healthy donors. b. PBMCs were cultured as in a. after staining with the eFluor670 proliferation dye. Cell division and EGFP expression were assessed in CD4⁺ T-cells (a representative experiment is shown). c. Impact of Vpx on quiescent CD4⁺ T-cell subsets susceptibility to HIV-CMV-EGFP infection. Left panel: gating strategy to define CD4⁺ T-cell subsets: Naïve (Tn; CD45RA⁺, CCR7⁺), Central Memory (Tcm; CD45RA^-, CCR7⁺) and Effector Memory (Tem; CD45RA^-, CCR7^-). Middle panel: Permissiveness of Tn, Tcm and Tem to HIV-CMV-EGFP performed as described in a (a representative experiment; n = 3). d. Kinetics of full length HIV-CMV-EGFP DNA accumulation quantified by quantitative PCR in purified non-stimulated CD4⁺ T-cells from 2 healthy donors #5 and #6. Proportions of EGFP positive quiescent CD4⁺ T-cells (CD69- HLA-DR-) assessed by flow cytometry 4 days post infection by HIV-CMV-EGFP are indicated on the right. e. Effect of Vpx on the permissiveness of quiescent CD4⁺ T-cells to infection by HIV-EGFP. Upper panel: expression vectors used to produce HIV-EGFP virions. Lower left panel: PBMCs were cultured as in a. except cells were infected with HIV-EGFP. Proportions of EGFP positive quiescent CD4⁺ T-cells (CD69- HLA-DR-) and CD14⁺ monocytes were assessed 4 days post infection for 3 donors. Lower right panel: Full length HIV-EGFP DNA accumulation was performed as in d. for 1 donor.