Vpu induces the degradation of human tetherin in 293T cells but stabilizes its expression in COS cells.

<p>293T (A) or COS (B) cells were transfected with vectors expressing HA-tagged human, mouse, rhesus, and agm tetherins without or with Vpu expression vector at a tetherin:Vpu DNA ratio of 1∶5. Total transfected DNA was held constant with empty vector. Truncated human tetherins that lack the cytoplasmic tail (delCT) or putative GPI anchor (delGPI) were also used to identify the regions of tetherin required for Vpu-mediated degradation. One day posttransfection, cells were lysed and subjected to western blot analysis with the indicated antibodies. Vpu decreased tetherin expression by 3.1 fold in 293T cells whereas it increased tetherin expression by ∼5-fold in COS cells; levels of the delCT mutant were increased ∼3-fold in COS cells and 1.6 fold in 293T cells by Vpu. (C) COS cells were transfected with HA-tagged human tetherin expression vector and cell lysates were subjected to western blot analysis with anti-HA or anti-tubulin antibodies. Vpu was also co-expressed to compare the pattern of tetherin expression. Molecular mass markers are shown on the right (in kDa). In this experiment, proteasomal inhibitors (MG132 and ALLN) increased the expression of the ∼23 kDa tetherin species by 3.2 fold, whereas lysosomal inhibitors (bafilomycin and concanamycin) rescued the expression of the ∼26 kDa tetherin species by 2.6 fold and the ∼23 kDa tetherin species by 1.7 fold compared to the no inhibitor control. Co-expression of Vpu increased the expression of the ∼26 kDa tetherin species by 5.2 fold and the ∼23 kDa tetherin species by 1.7 fold. (D) 293T, COS, and Vero cells were transfected with HA-tagged human tetherin expression vector in the absence and presence of Vpu expression vector, and one-day posttransfection cells were lysed and subjected to Western blot analysis as above. Vpu reduced the expression of tetherin by 2.4 fold in 293T cells, whereas the levels were increased by ∼20-fold in COS cells and ∼4-fold in Vero cells. Similar results were obtained in an independent experiment.