FIGURE 3.
Vpu-induced BST-2 degradation is partially βTrCP-dependent. A, Vpu connects BST-2 with either βTrCP-1 or -2. Precleared cell extracts from 293T cells transiently expressing CD4, Vpu (WT or 2/6 mutant), and either FLAG-tagged βTrCP-1 or -2 were immunoprecipitated (IP) with an anti-CD4 antibody (left half). Similarly, precleared cell extracts from 293T cells transiently expressing BST-2, Vpu (WT or 2/6 mutant), and either FLAG-tagged βTrCP-1 or -2 were immunoprecipitated with the anti-BST-2 antibody (right half). The resulting complexes were analyzed by immunoblotting with antibodies to Vpu and FLAG (upper two panels). Aliquots of cell lysates were also analyzed by immunoblotting in parallel for Vpu and FLAG (lower two panels). B, βTrCP interaction-defective mutant of Vpu retains some activity to enhance virion release. HeLa cells were infected with vesicular stomatitis virus glycoprotein-pseudotyped WT, Vpu2/6, and ΔVpu viruses. After 40 h, viral supernatants were harvested and subjected to p24 ELISA. Data are presented as a percentage of the amount of WT release and shown as the mean ± S.D. Vpu expressions from the proviral DNAs were confirmed by immunoblotting. C, shown is partial inhibitory effect of βTrCP knockdown on Vpu activity. HeLa cells were transduced with a control lentiviral vector or with vectors producing βTrCP-1 and/or -2-specific shRNA. After 48 h, transduced cells were infected as described in B. Data are presented as a percentage of the amount of WT release in the presence of control shRNA and are representative of three independent experiments.