Co-staining of tetherin with calnexin and endocytosed transferrin. HeLa cells transfected with either 2 μg of Vpu (Vphu-HcRed) or ROD10 Env plasmids were analyzed for co-localization with the ER marker, calnexin (A), or with endocytosed transferrin (B). Triple color merged images are shown. Scale bars represent 10 μM.

Vpu and ROD10 Env have no effect on TfR distribution. HeLa cells were either mock treated or transfected with 2 μg Vphu-HcRed or 2 μg ROD10 Env expression plasmids, permeabilized and stained with specific antibodies against human transferrin receptor (TfR) or HIV-2 Env, or visualized by HcRed fluorescence, and analyzed by confocal microscopy. TfR was found at the cell surface and in a perinuclear concentration, and its distribution was unaltered by expression of either viral protein. Scale bars represent 10 μm.

Tetherin redistribution by HIV-1 and HIV-2 proviral clones. HeLa cells were either mock treated or transfected with 8 μg of HIV-1_NL4-3 or HIV-2_ROD10 proviral clones. Cells were fixed, permeabilized, and stained for endogenous tetherin (green), the TGN46 marker (blue), HIV-1 Vpu (red) or HIV-2 Env (red). Triple color merged images are shown. NL4-3 transfected cells showed tetherin co-localized with Vpu and the TGN. ROD10 transfected cells had two distinct appearances. ~25% of cells showed tetherin localized with a compact TGN marker (upper panels), while the majority of the cells had tetherin in a more diffuse perinuclear location that co-localized with more distorted TGN staining (lower panels). Scale bars represent 10 μM.

Redistribution of tetherin to an intracellular compartment by HIV anti-tetherin factors. (A) The percentage of HeLa cells displaying tetherin concentrated in a perinuclear compartment (PNC) was calculated for 100 cells, from either control (Ctrl.) cells or cells transfected with 2 μg of Vpu or ROD10 Env expression plasmids. Mean +/- SEM is shown for n = 2 independent experiments. (B) HeLa cells transfected with either Vpu (Vphu-HcRed) or ROD10 Env, showed increased concentration of tetherin in a perinuclear compartment (arrowed), that co-stained with the TGN marker, TGN46. The triple color merged image is shown. Scale bars represent 10 μM.

Redistribution of EGFP-tetherin by functional anti-tetherin factors. (A) HeLa cells were co-transfected with 100 ng of EGFP-tetherin and the indicated HIV proteins. EGFP-tetherin was detected using an anti-GFP antibody, and was found to be removed from the cell surface and concentrated internally by expression of both Vpu (Vphu-HcRed) and ROD10 Env. The non-functional Env proteins from ROD14 or ROD10(Y707A) had no effect on cell surface EGFP-tetherin levels, although we frequently observed that the EGFP-tetherin signal was brighter with more visible intracellular puncta in the co-transfected cells. Scale bars represent 10 μM. (B) The degree of co-localization of EGFP-tetherin with markers for the TGN or endocytosed transferrin, in the presence of Vpu or ROD10 Env, was calculated using Pearson's coefficients. Statistical significance was calculated using unpaired t-tests, ** indicates p < 0.01 compared to control, non-transfected cells.

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, ROD10_Y707A 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, ROD10_Y707A 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.

Cellular distribution of tetherin. (A) Confocal analysis of HeLa cells showing the distribution of endogenous tetherin, detected with a specific antiserum. Cells that were fixed but not permeablized (left panel) allowed visualization of tetherin at the cell surface, while permeabilized cells revealed tetherin concentrated in a perinuclear compartment that was visible in ~50% of cells. This intracellular pool co-localized with a marker for the TGN (TGN-46), as shown by the PDM analysis in the upper right corner of the merged image, where positive co-localization is pseudocolored in orange. Scale bars represent 10 μM. (B) 293A cells were co-transfected with 10 μg HIV-1-pack and 100 ng of expression plasmids for either untagged tetherin or EGFP-tagged tetherin. Cell lysates were analyzed by Western blotting, using antibodies against GFP and tetherin. (C) Cell lysates and pelleted supernatant fractions (VLPs) from same experiment as (B) were probed for HIV-1 p24 expression. Both tetherin constructs inhibited VLP release. (D) HeLa and 293A cells were transfected with either 100 ng or 300 ng of the EGFP-tetherin plasmid. With 300 ng, a punctate pattern of EGFP fluorescence was observed throughout the cells; with 100 ng, the protein could only be detected using an anti-GFP antibody, that revealed an intense surface rim and a fainter PNC in both types of cells. Cells were fixed and permeabilized before staining. Scale bars represent 10 μM. (E) The intracellular concentration of EGFP-tetherin in transiently transfected HeLa cells (100 ng plasmid) was analyzed by confocal microscopy using anti-GFP antibody and specific markers for the TGN (TGN46) and recycling endosomes (endocytosed transferrin). The degree of co-localization was calculated using Pearson's coefficients. Mean +/- SEM is shown for 20 individual cells analyzed.

Analysis of perinuclear concentration of tetherin by Vpu and ROD10 Env. (A) HeLa cells were incubated with anti-tetherin antibody at 4°C for 1 hour, followed by incubation at 37°C for either 15 or 45 minutes to allow endocytosis of antibody-tetherin complexes. The antibody was found in a perinuclear region by 15 minutes (arrowed), but became more diffuse by 45 minutes, suggesting that tetherin quickly exits this compartment and recycles back to the plasma membrane. Scale bars represent 10 μM. (B) HIV-1 VLPs produced in 293A cells in the presence of 100 ng of wild-type (WT) or YY-AA tetherin, with and without co-transfection of 2 μg Vpu (pCMV-Vphu). Expression of proteins was confirmed by Western blotting with specific antibodies; tetherin levels were visualized for both untreated and PNGase F treated cell lysates. Mean +/- SEM fold-enhancement of VLP release by Vpu is shown in presence of tetherin or tetherin (YY-AA), n = 2. (C) HeLa cells expressing EGFP-tetherin or EGFP-tetherin(YY-AA) were incubated for 1.5 hours, with or without 20 μg/ml cycloheximide, and subsequently fixed, permeabilized, and stained with anti-EGFP antibody. In the absence of drug, both WT and YY-AA EGFP-tetherin proteins were found at the cell surface and in a perinuclear region (arrowed). After cycloheximide treatment, only EGFP-tetherin produced this population, with EGFP-tetherin(YY-AA) forming a more punctuate pattern, dispersed throughout the cytoplasm. Two different cells are shown for the drug treatment. Scale bars represent 10 μM. (D) HeLa cells were co-transfected with 60 ng EGFP-tetherin(YY-AA) and 2 μg of expression plasmids for either Vphu-HcRed or ROD10 Env. Cells were incubated with or without cycloheximide, as above, and subsequently fixed, permeabilized, and stained with anti-EGFP (green), anti-TGN46 (blue) and anti-HIV-2 Env (red) antibodies. Vpu was detected by HcRed expression (red). EGFP-tetherin(YY-AA) was concentrated in a perinuclear compartment by Vpu or ROD10 Env, overlapping with the TGN marker, irrespective of cycloheximide treatment. Scale bars represent 10 μM.