Figure 5. Defective Vpu TM mutants potently block the physical interaction between Vpu and tetherin.

293T cells were co-transfected with 1 µg VR1012 control vector or VR1012 encoding Vpu TM variants together with 2 µg HA-tetherin expression vector at a 1∶2 molar ratio to minimize the Vpu-mediated tetherin degradation. Cells were harvested 48 h later and subjected to immunoprecipitation using the anti-myc antibody and protein G agarose beads. Cell lysates or co-precipitated proteins were analyzed by immunoblotting to detect HA-tetherin and Vpu-cmyc. Equal loading was controlled by monitoring tubulin. The level of tetherin in each sample was quantified by densitometry, normalized by tubulin levels and shown beside the tetherin blot. The value obtained with the positive control Vpu WT was defined as 100%.