BCA2/Rabring7 Promotes Tetherin-Dependent HIV-1 Restriction
Figure 5
The targeted depletion of BCA2 blocks the intracellular accumulation of viral particles.
(A) Single-round virus release analysis of HeLa cells treated with control siRNA or two different BCA2-targeted siRNA vectors for 24 hours, prior to transfection with pNL4–3 or pNL4–3ΔVpu. At 48 hours following transfection, cell supernatants were analyzed by p24 ELISA. Immunoblotting analysis with a BCA2 antibody to detect endogenous BCA2 expression is shown in the bottom panel. (B) Confocal microscopic analysis of HeLa cells treated with control siRNA (upper panels) or BCA2-targeted siRNA (lower panels), prior to transfection with pNL4–3ΔVpu (scale bar, 10 µm). After 48 hours following transfection, cells were fixed and immunostained with anti-p24 (green) and anti-CD63 (red) antibodies followed by confocal microscopy. (C) In the cultures described in (B), over 100 cells were analyzed for the subcellular localization of p24, as described in Fig. 4B. (D) HeLa cells were treated with control siRNA or BCA2-targeted siRNA for 24 hours, prior to transfection with pNL4–3 or pNL4–3ΔVpu as in (A). At 48 hours following transfection, cell supernatants were harvested (first supernatants) and cells were treated with either PBS or buffer containing the protease subtilisin (1 mg/ml) for 15 min, prior to re-harvesting of the cell supernatants (second supernatants). Both the first and second supernatants were then mixed and analyzed by p24 ELISA.