Figure 1

Characterization of CCR5-tropic HIV-1-WT and HIV-1-∆Vpu proviral DNA and viruses. HeLa cells were transfected with WT or Vpu-deficient pNL4.3-Ada-GFP proviral DNA. Transfected cells and virus-containing supernatants were analyzed by western blot using the indicated antibodies (A). Relative virus particle release efficiency. The particle release efficiency of HIV-1-WT was set at 100% (average of 3 independent experiments) (B). An aliquot of transfected cells was used for analysis of BST2 levels at the surface of GFP-positive (HIV-expressing cells; dashed line) and -negative (non-transfected cells; continuous line) cells by flow cytometry (representative of n = 3). Gray filled histogram represents preimmune staining with normal rabbit serum (C). Primary CD4⁺ T cells were isolated from healthy donors and activated with PHA and IL-2. Activated T cells were infected with HIV-1-WT or HIV-1-∆Vpu at an MOI of 1. At different time points post infection, an aliquot of culture was collected for flow cytometry analysis of CD4 and BST2 expression at the surface of GFP-positive (infected; dashed line) or -negative (non infected; continuous line) cells. Data is shown for cells collected 3 days post infection (dpi). Relative BST2 and CD4 levels at 3-dpi on p24^- (open bar) and p24⁺ (filled bar) cells are shown (MFI on p24-negative = 100%; n = 2, * p ≤ 0.05, N.S.: not significant). (D). Infectious virus production in supernatant was determined by assessing the levels of luciferase activity (in duplicate) following infection of HeLaTZM-bl reporter cells (E). Data are representative of 3 independent experiments. Error bars represent standard deviations (SD).