Figure 1

RNAi-mediated silencing of endogenous EloB expression impairs Vif functions. (A) 293 T cells were seeded in 6-well plates at 0.8 × 10⁶ cells per well just prior to transfection with siRNAs at a final concentration of 20 nM. The cells were harvested at 72 h post-transfection, and EloB protein expression was analyzed by Western blot. Mock, cells treated with Hiperfect transfection reagent only; siNC, cells transfected with AllStars Negative Control siRNA; siEB1-3, cells transfected with EloB-siRNA1-3. Band intensities were quantified using Bandscan software, normalized to tubulin and expressed as percentages of the mock value. (B) 293 T cells transfected with siRNAs were harvested at 72 h post-transfection and analyzed by qRT-PCR for EloB mRNA levels normalized to β-actin mRNA. Fold changes in EloB gene expression relative to that of the mock control are shown. (C) At 24 h after siRNA treatment, 293 T cells (siRNA-treated cells were grown in 25 cm² flasks to about 80% confluency) were co-transfected with 450 ng of VR-A3G-HA, 3.5 μg of NL4-3ΔVif, and 1.2 μg of VR-Vif or VR1012. After 48 h of transfection, cells were examined for A3G expression (lanes 1–3, control siRNA treated cells; lanes 4–6, EloB siRNA treated cells), and corresponding supernatants were used in (D) and (E). (D) Pelleted virions obtained from (C) were examined for A3G–HA virion packaging. (E) Relative infectivity of viruses obtained from (C) was assessed by the MAGI assay, with the infectivity of viruses produced from cells co-transfected with NL4-3ΔVif and VR-Vif in the presence of control siRNA was set to 100%. Virus input was normalized according to the p24 level. Error bars in (B) represent standard deviations from three independent experiments and in (E) from triplicate wells.