FIGURE 4.
Vpu actively internalizes cell-surface BST-2. A, inhibition of endocytosis by dominant-negative dynamin rescues Vpu-induced reduction of cell-surface expression of BST-2 is shown. 293T cells transiently expressing EGFP and FLAG-tagged BST-2 with (lower panels) or without Vpu (upper panels) together with a control vector (left), dynamin-2-WT (middle; Dyn2-WT), or dominant-negative dynamin-2 (right; Dyn2-K44A) were stained for cell-surface BST-2 using the anti-FLAG monoclonal antibody and analyzed by two-color flow cytometry. B, COS7 cells transiently expressing Myc-tagged BST-2 and either HA-tagged Vpu or a control HA-tagged CD4tmVpu together with Dyn2-WT-EGFP (left half) or with Dyn2-K44A-EGFP (right half) were processed for cell-surface and intracellular immunofluorescence staining by using anti-Myc monoclonal (cell-surface) and anti-HA polyclonal (intracellular) antibodies, respectively. Squares indicate magnified regions in which the implications for the section indicated by arrows are considered under “Discussion.” Bars, 10 μm. C–E, shown is the endocytosis mutant of BST-2 remains sensitive to Vpu. C, expressions of FLAG-tagged BST-2 WT and its endocytosis mutant (Y6A/Y8A) were confirmed by immunoblotting using the anti-FLAG monoclonal antibody. D, 293T cells were transfected with Vpu-deficient HIV-1 proviral DNA and either a control or Vpu expression plasmid together with BST-2 WT or Y6A/Y8A expression plasmid. After 48 h viral supernatants were harvested and subjected to p24 ELISA. Transfection efficiencies were normalized with the luciferase activity. Data shown are the mean ± S.D. E, experiments were the same as Fig. 1B, except that a FLAG-tagged BST-2 Y6A/Y8A mutant (right) was also analyzed.