Vpu Antagonizes BST-2–Mediated Restriction of HIV-1 Release via β-TrCP and Endo-Lysosomal Trafficking
Figure 1
β-TrCP is required for the optimal down-regulation of cell-surface BST-2: inhibition of Vpu activity by ΔF-box β-TrCP and shRNA targeting β-TrCP-1 and -2.
(A) ΔF-box β-TrCP inhibits Vpu-mediated down-regulation of cell-surface BST-2. Cells (HeLa) were transfected with either an empty plasmid or a plasmid expressing Vpu, along with a plasmid expressing GFP as a transfection marker. The cells were also transfected with either an empty plasmid (“mock”), a plasmid expressing β-TrCP, or a plasmid expressing a β-TrCP protein lacking the F-box (ΔFbox β-TrCP). The next day, the cells were stained for surface BST-2 and analyzed by two-color flow cytometry. Histograms represent the relative cell number vs. BST-2 fluorescence intensity for the GFP-positive cells. The percentage of GFP-positive cells varied between 30 and 33% for the six transfections shown. Gray-shaded histograms represent cells not transfected to express Vpu; unshaded histograms represent cells transfected to express Vpu. ΔF-box β-TrCP inhibited the Vpu-mediated down-regulation of cell surface BST-2 in each of four experiments; statistical analysis is described in the text. (B) Cells (HeLa) were transfected with a plasmid expressing β-TrCP (WT β-TrCP), or a plasmid expressing a β-TrCP protein lacking the F-box (ΔFbox β-TrCP), or not transfected; cell lysates were analyzed by immunoblot to detect the HA-tagged β-TrCP proteins. (C) shRNA targeting β-TrCP inhibits Vpu-mediated down-regulation of cell-surface BST-2. Cells (HeLa) were transfected with plasmids expressing shRNAs targeting either Renilla GFP (rGFP) as an irrelevant control, β-TrCP-1, β-TrCP-2, or both β-TrCP-1 and -2; in two cases these plasmids also expressed jellyfish GFP (the plasmids targeting β-TrCP-1 and both β-TrCP-1 and -2); for the others a separate plasmid expressing GFP was co-transfected. Two days later, the cells were re-transfected with an empty plasmid or a plasmid expressing Vpu, along with a plasmid expressing Tac antigen (IL-2 receptor α; CD25) as a transfection marker. The next day, the cells were stained for surface BST-2 and Tac, and then analyzed by three-color flow cytometry. Two-color dot plots are the BST-2 vs.Tac intensity of the individual GFP-positive cells. The results shown are representative of two independent experiments. (D) HeLa cells were transfected with the indicated plasmids expressing shRNAs used in (C) along with the plasmid expressing β-TrCP-1-HA; cell lysates were analyzed by immunoblot to detect the HA-tagged β-TrCP.