Figure 2.

Effects of Nef Y120F/Q125H mutations on viral replication in PBMC. (A) PBMC (10⁶ cells) prepared from 2 HIV-negative donors were infected for 6 h with HIV-Nef_SF2, HIV_ΔNef or HIV-Nef_120F/125H that had been produced in HEK293T cells at 10 ng of p24 Ag, and then continuously cultured at 37 °C in fresh culture medium for an additional 15 days. Culture supernatants were collected and replaced with fresh medium every 3 days. To monitor viral replication, we quantified the concentration of p24 Ag in the culture supernatant by use of ELISA. HIV-Nef_SF2, HIV_ΔNef, and HIV-Nef_120F/125H replication kinetics are shown in each panel. P-values were calculated by using the unpaired t-test at Day 9 (HIV-Nef_SF2 vs. HIV-Nef_120F/125H). (B) A series of the same experiments was done by using PBMC prepared from 4 additional HIV-negative donors. The peak p24 Ag values were plotted and statistically analyzed by using the Wilcoxon matched-pairs signed rank test. (C) TZM-bl cells (1 × 10⁴ cells) were exposed to HIV-Nef_SF2, HIV_ΔNef or HIV-Nef_120F/125H prepared as above at 9, 6, and 3 ng of p24 Ag. Twenty-four hr later, the reporter cells were lysed and β-galactosidase activity generated as a consequence of infection was measured using a chemiluminescence substrate. Data shown are mean ± SD from 3 or 4 independent experiments. P-values were determined by ANOVA with multiple comparisons.