FIG. 5.
Transfected BST-2 in 293T cells is destabilized by VphU but not Vpu. (A) 293T cells were transfected with 1 μg of pCDNA-BST-2 with 4 μg of either wt NL4-3 virus (closed circles) or NL4-3/Udel virus (open circles). (B) 293T cells were transfected with 1 μg of pcDNA-BST2 with (closed circles) or without (open circles) 2 μg pcDNA-VphU. Total DNA was adjusted to 5 μg with empty vector DNA. (C) 293T cells were transfected as described above (B), with the addition of brefeldin A during the pulse and chase at 5 μg/ml. In all experiments, 24 h after transfection cells were pulse-labeled for 1 h at 37°C by the addition of 35S-labeled amino acids and chased for the indicated time intervals in complete medium. BST-2 was immunoprecipitated with specific antibodies. Representative results are shown. The position of molecular weight standards is shown on the left. (D to F) The percentage of immature BST-2 remaining at each time point was calculated relative to the signal at time zero. Plotted data are derived from three independent experiments. Error bars represent standard errors of the means. (G) Cell lysates from 293T cells transfected with 1 μg of BST-2 as well as HeLa and CEMx174 cell extracts were separated by SDS-PAGE and subjected to immunoblot analysis with antibodies to BST-2 and tubulin (tub). The position of molecular weight standards is shown on the left. (H) 293T cells were transfected with 1 μg of pcDNA-BST2 in the absence of Vpu (lane 1) or together with 2 μg of pcDNA-VphU (lane 2), 4 μg of pNL4-3/Udel (lane 3), or 4 μg of pNL4-3 DNA (lane 4). Total DNA was adjusted to 5 μg with empty vector DNA. Lysates were separated by SDS-PAGE and subjected to immunoblot analysis with antibodies to Vpu, or tubulin (tub).