Figure 4. Vpu-mediated down-regulation of BST-2 in the context of the complete HIV-1 genome requires the Vpu transmembrane domain and conserved serines in the cytoplasmic domain but is not prevented by inhibition of the proteasome.

A) HeLa cells were transfected to express complete HIV-1 genomes, either wild-type (“WT”), vpu-negative (“ΔVpu”), a mutant encoding a Vpu protein whose transmembrane domain is scrambled (“VpuRD”), or a mutant in which serines 52 and 56 in the Vpu cytoplasmic domain are replaced by alanines (“Vpu2/6”), in each case along with GFP encoded on a separate plasmid. The cells were stained the next day for surface BST-2 and analyzed by two-color flow cytometry. B) HeLa cells were transfected with the indicated proviral genomes, and the fraction of the total p24 capsid antigen produced that was secreted into the media was measured one day later. C) HeLa cells were transfected to express transiently either no viral protein (“mock”) or a codon-optimized version of HIV-1_NL4-3 Vpu along with GFP encoded on a separate plasmid. The next day, cells were either treated for five hours with 25 μM MG-132 (an inhibitor of the proteasome) or left untreated, then stained for surface BST-2 and analyzed by two-color flow cytometry. In panels A and C, horizontal and vertical lines indicate gates set using either GFP-negative cells or a primary antibody isotype control for BST-2; colors are arbitrary and distinguish GFP-negative, -low positive, and -high positive cells.