Replication of nef point mutant viruses in primary human T cells. Activated CD4⁺ T cells were inoculated with wild-type HIV-1 and nef mutant HIV-1 (1 ng of p24 antigen), and supernatants from cell cultures were sampled every 2 days following inoculation and assayed for virus production by p24 enzyme-linked immunosorbent assay. The data shown are averages of duplicate parallel cultures and are representative of three independent experiments. Wild-type (WT) and nef-defective (ΔNef) virus replication kinetics are shown in each panel for comparison. dpi, days postinfection.

Infectivity of nef point mutant viruses does not correlate with replication efficiency in primary T cells. (A) Viruses containing nef point mutations were produced by transfection of 293T cells and assayed for infection of P4 reporter target cells. Infected cells were quantified following 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal) staining, and infectivity was calculated as the number of infected cells per nanogram of p24 in the inoculum. The values shown are averages of triplicate infections with three independently generated virus stocks. (B) Graphic representation of infectivity as a function of virus replication in activated CD4⁺ T cells. WT, wild type; DPI, days postinfection.

Nef-dependent CD4 downregulation correlates with HIV-1 replication efficiency. (A) FACS analysis of CD4 downregulation during HIV-1 infection of primary CD4⁺ T cells. Three days following infection with the indicated HSA-encoding viruses, cell surface CD4 levels were quantified by two-color flow cytometry following staining with HSA- and CD4-specific monoclonal antibodies. Percentages of HSA⁺ CD4⁺ and HSA⁺ CD4⁻ populations are shown to the right of each FACS plot. (B) CD4 downregulation by mutant Nef proteins was calculated as a percentage of the difference in CD4 levels on cells infected with wild-type HIV-1 and those infected with nef-defective HIV-1. Shown are mean values and standard deviations of the six values obtained (two concentrations of input virus and three time points each) from a representative experiment. (C) Graphic representation of relative CD4 downregulation activity versus HIV-1 replication efficiency. Replication efficiency was determined by interpolation of the day following virus inoculation when p24 production exceeded 250 ng/ml; therefore, higher values represent slower replication kinetics. WT, wild type; DPI, days postinfection.