FIG. 7.

Effect of cholesterol-binding drugs on HIV-1 Gag trafficking in HEK 293T cells. HEK 293T cells were transfected with the HxBc2 provirus. Two days after transfection, cells were pretreated with filipin (4 μg/ml) or MβCD (8 mM) for 30 min before incubation for another 30 min at 0°C with (A) Tfr- or (B) ChTxβ-Alexa-488 conjugates. Tfr or ChTxβ internalization was allowed for 10 min at 37°C. Endocytosis inhibitors were maintained throughout the experiment. Samples were subsequently analyzed by immunofluorescence microscopy (bar, 10 μm) as described in Materials and Methods. Data shown are representative of two independent experiments. In parallel, cells were metabolically labeled with [³⁵S]Met-Cys for 10 min and chased for 2 h in the presence of (C) filipin or (D) MβCD prior to cell lysis and subcellular fractionation by Optiprep gradient centrifugation. Gag-related products in Optiprep fractions were detected by immunoprecipitation using a monoclonal anti-p24 Ab for each collected fraction. (E) Quantification of the relative amounts of Pr55^gag detected in PM- and LE/MVB-associated fractions (1 to 6 and 13 and 14, respectively) at zero h of chase. (F) Quantification of the relative amounts of mature Gag products (p25/p24) detected in PM- or MVB-associated fractions at 2 h of chase. Gag-related signals were quantified as described in the legend to Fig. 6. Data shown are means ± standard deviations of the results of five control, three filipin, and two MβCD independent experiments.