Direct internalization of cell-surface BST-2/tetherin by the HIV-1 accessory protein Vpu

Abstract

The host transmembrane protein BST-2/tetherin is a powerful antiviral factor that blocks the production of enveloped viruses. The HIV-1 accessory protein Vpu inhibits the antiviral activity of BST-2; however, the degradation pathway by which Vpu downregulates BST-2 from the cell surface and the actual subcellular location where Vpu targets BST-2 for downregulation remain controversial. Whereas one study showed that Vpu acts on constitutively endocytosed BST-2, we recently reported that Vpu can internalize BST-2 from the cell surface. Because the evidence for this conclusion was derived from indirect results, we present direct evidence in this study using an antibody internalization assay with an endocytosis-defective mutant of BST-2. The internalization of the BST-2 protein into cells coexpressing wild-type Vpu was observed when the cells were preincubated with antibodies against BST-2 at 37˚C, but not at 4˚C, for 10 min. These results strongly support our previous finding that continuously expressed de novo BST-2 at the cell surface is internalized by functional Vpu protein.

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Acknowledgements

This work was supported by a grant from the Ministry of Health, Labor and Welfare of Japan.

Figures and Tables

Figure 1 Antibody internalization assay (with preincubation at 4°C for 10 min). COS7 cells were co-transfected as described previously,10 with pCA-Vpu-EGFP-RRE (WT or CD4tm mutant), pCA-Rev, and Myc-tagged BST-2 (pCA-BST-2-exMyc-Y6A/Y8A) and cultured for 24 h. To detect Myc-tagged BST-2 internalized at the plasma membrane, transfected cells were cultured in complete medium in the presence of anti-Myc mouse monoclonal antibodies (1:50 dilution) at 4°C for 10 min. After an additional 80 min in complete medium in the presence of lysosomal protease inhibitors (40 μM of leupeptin and pepstatin A), the cells were washed in PBS at 4°C and then fixed with 4% paraformaldehyde. The fixed cells were permeabilized with 0.05% saponin and immunostained with rabbit anti-cathepsin D (1:200). Cy3-conjugated goat anti-mouse and Cy5-conjugated goat anti-rabbit secondary antibodies were used at 5 μg/ml. DN A staining with Hoechst was performed at 0.5 μg/ml. All immunofluorescence images were captured as described previously.10 Bars, 10 μm.

Figure 2 Antibody internalization assay (with preincubation at 37°C for 10 min). Same as in Figure 1, but the cells transfected with the WT (A) or mutant (B) Vpu expression plasmid were cultured in complete medium with anti-Myc mouse monoclonal antibodies at 37°C instead of 4°C for 10 min, and then fixed with 4% paraformaldehyde either immediately (0 min) or after an additional incubation (20, 50 or 80 min). Squares outline magnified regions where the colocalization of BST-2, Vpu, and a lysosome marker cathepsin D is indicated by arrows. Bars, 10 µm.

Figure 3 Schematic diagram of the direct internalization of cell-surface BST-2 by HIV-1 Vpu followed by lysosomal degradation.

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