Effect of HIV-1 Vpu and HIV-2 Env on tetherin. (A) HeLa cells were transfected with 2 μg of either a Vpu expression plasmid (pcDNA-Vphu) or a ROD10 HIV-2 Env expression plasmid and analyzed by confocal microscopy. Cell surface tetherin was detected by addition of an anti-tetherin antibody prior to fixation and permeabilization, while incubation with anti-Vpu or anti-Env antibodies was performed after permeabilization. The cell surface rim of tetherin was reduced in cells co-expressing Vpu or ROD10 Env (arrowed cells). Scale bars represent 10 μM. (B) HeLa cells were co-transfected with 10 μg of pHIV-1-pack, together with 2 μg of expression plasmids for HIV-2 Env ROD10, ROD10Y707A or ROD14. Proteins in cell lysates were analyzed by Western blotting using an anti-HIV-2 Env antibody. (C) FACS analyses of HeLa-CD4 P4.R5 cells transfected with a plasmid expressing GFP, together with either an empty vector control (Ctrl.), Vpu (pcDNA-Vphu), or Env-expression vectors from HIV-2 ROD10, ROD10Y707A or ROD14. Staining for tetherin with HM1.24 monoclonal antibody and gating on the GFP-expressing population allowed for enrichment of cells that had been transfected. The mean fluorescence intensity of tetherin staining is shown for the GFP-expressing population. (D) HeLa cells were co-transfected with 10 μg of pHIV-1-pack, together with 2 μg of expression plasmids for Vpu (pcDNA-Vphu) or the ROD10 Env. Proteins in cell lysates or VLPs were analyzed by Western blotting as indicated. Lysates were deglycosylated prior to analysis of tetherin. (E) Mean relative levels of tetherin in lysates of HeLa cells expressing Vpu or ROD10 Env. Error bars represent SEM. ** indicates statistical significance, p < 0.01 compared to control, non-transfected cells, n = 9. (F) Mean relative level of VLP release from HeLa cells expressing Vpu or ROD10 Env, calculated as the ratio of p24 signal in VLPs:lysates, made relative to the pHIV-1-pack control (Ctrl.). Error bars represent SEM, n = 7.