HeLa cells were infected with the indicated VSV-G pseudotyped HxBH10-derived virus. Forty-eight hours post-infection, cells were immunostained for cell-surface Tetherin, fixed, permeabilized and co-stained with anti-Tetherin (red), anti-Vpu (green) and anti-TGN46 (blue) specific Abs. Nuclei were counterstained with DAPI (cyan). Cells were observed by confocal microscopy. The white bar represents a distance of 10 µm.

In absence of Vpu, Tetherin traffics along the secretory pathway and reaches the plasma membrane. The protein is endocytosed, transported to the TGN and is then recycled back to the cell-surface. In presence of Vpu, Tetherin is forming complexes with the viral protein in the membrane of the ER. Recruitment of SCF^β-TrCP by Vpu leads to ubiquitination and proteosomal degradation of a fraction of Tetherin. Tetherin escaping degradation exits the ER. In the membrane of the TGN, Vpu binds endocytosed Tetherin and/or Tetherin arriving from the ER and causes the sequestration of the restriction factor. Subsequently, Vpu induces Tetherin ubiquitination through the recruitment of SCF^β-TrCP, thus targeting the ubiquitinated proteins to lysosomal degradation.

HeLa cells were co-transfected with the indicated HxBH10 constructs and a GFP-expressing plasmid. Forty-eight hours post-transfection, cell-surface Tetherin was labeled with Tetherin specific Abs at 4°C before incubating cells at 37°C for the indicated time intervals to allow endocytosis. Cells were then incubated at 4°C in presence of the appropriate secondary Abs. The graph depicts the relative levels of Tetherin at the surface of GFP-expressing cells (time 0 = 100%) and represents the loss of Tetherin-specific signal resulting from endocytosis. Full lines: HxBH10-vpu+; dashed lines: HxBH10-vpu-. Error bars indicate the standard deviation of the mean from two independent experiments.

(A) HeLa cells were infected with VSV-G pseudotyped HxBH10-vpu-, HxBH10-vpu+ or HxBH10-vpu S52D,S56D virus. Forty-eight hours post-infection, cells were immunostained for cell-surface Tetherin, fixed, permeabilized and co-stained with anti-Tetherin (red), anti-Vpu (green) and anti-TGN46 (blue) specific Abs. Nuclei were counterstained with DAPI (cyan). Cells were observed by confocal microscopy. The white scale bar represents a distance of 10 µm. (B) Quantitation of the absolute Tetherin signal in the TGN was determined by measuring the specific Tetherin signal in the region delineated by the TGN46 marker on digital picture produced using similar acquisition time. Error bars indicate the standard deviation of the mean from the quantitative analysis of at least 25 distinct cells per conditions.

(A) Schematic representation of WT Vpu, Vpu KSL and Vpu RD. The TM domain is represented by the unfilled box while the cytosolic domain with the two predicted α-helices (H-1 and H-2) is indicated by the filled box. Phosphorylation sites are represented by the small circles. The amino-acid sequence of the TM anchor region is represented below. Hyphens represent identical amino-acids. (B-E) Co-immunoprecipitation experiments of Tetherin and Vpu. (B) HEK 293T cells were transfected with a HA-human Tetherin-expressing plasmid and the indicated HxBH10-derived proviral constructs. Forty-eight hours post-transfection, cells were lysed and proteins were co-immunoprecipitated using anti-Tetherin Abs. Co-immunoprecipitated proteins were analyzed by western blot using anti-Tetherin and anti-Vpu Abs. (C-E) HEK 293T cells were transfected with the indicated HA-Tetherin-expressing plasmids and the specified HxBH10 proviral constructs. Forty-eight hours post-transfection, cells were lysed and proteins were co-immunoprecipitated and analyzed as described above. In (D), HA-Tetherin proteins were revealed by western blot using anti-HA Abs. Amounts of protein in the lysates prior to immunoprecipitation (input) are shown in the left panels. Non-specific bands, depicted by the asterisks, were used as loading controls.

HEK 293T cells were co-transfected with a fixed amount of the HA-Tetherin-expressing plasmid (235 ng) and the indicated amounts of HxBH10 proviral constructs. Forty-eight hours post-transfection, cells and virus-containing supernatants were harvested and processed for western blot analysis. (A) Proteins in the viral and cell lysates were detected using specific Abs. (B) Quantification of HIV-1 release efficiency. Bands corresponding to Gag products in cells and viral particles in panel (A), as detected using anti-p24 Abs, were scanned by laser densitometry. The virus particle release efficiency was determined as described in the Materials and Methods and calculated as a percentage of the release of HxBH10-vpu+ (100%) in absence of HA-Tetherin. The values within the graph represent the fold increase of virus particle release upon Vpu expression.

(A-C) Turnover of exogenously-expressed Tetherin. (A) HEK 293T cells were co-transfected with the indicated HxBH10 proviral constructs and the pcDNA-Tetherin plasmid. Forty-eight hours post-transfection, cells were pulse-labeled and chased for the indicated time intervals. Tetherin and Vpu were immunoprecipitated with specific Abs, and analyzed by autoradiography. The asterisk indicates the presence of a non-specific signal. (B) Quantitation of (A). The graph represents the percentage of Tetherin recovered relative to time 0. The signal intensity of all Tetherin-specific bands was determined for each time point by densitometric scanning. The percentage of Tetherin recovered was calculated as the ratio of the Tetherin bands signal at a given time relative to the signal at time 0. Error bars indicate the standard deviation of the mean from 2 independent experiments. (C) Virus-containing supernatants and a fraction of the cells from (A) were collected prior to labeling. Gag proteins from the cell and viral lysates were analyzed by western blot using anti-p24 Abs. (D-E) Turnover of endogenous Tetherin. (D) HeLa cells were infected with the indicated VSV-G-pseudotyped HxBH10 virus. Forty-eight hours post-infection, cells were pulse-labeled and chased for the indicated time intervals. Tetherin was immunoprecipitated with specific Abs, and analyzed by autoradiography. Vpu and actin were detected by western blot from cell lysates harvested prior to labeling. (E) Quantitation of (D) as described in (B). Full black line: HxBH10-vpu+; Dashed line: HxBH10-vpu-; Full grey line: HxBH10-vpu S52D,S56D.

(A) Association of Vpu with endogenous Tetherin. HeLa cells were transfected with the indicated HxBH10 proviral constructs. Forty-eight hours post-transfection, cells were lysed and proteins were co-immunoprecipitated using anti-Tetherin Abs. Co-immunoprecipitated proteins were analyzed by western blot using anti-Tetherin, anti-Vpu and anti-Env Abs. The amount of proteins in the lysate prior to immunoprecipitation (input) is shown in the left panel. Non-specific bands, depicted by the asterisks, were used as loading controls. (B) Cell-surface expression of Tetherin. HeLa cells were transfected with the indicated HxBH10 proviral constructs and a GFP-expressing plasmid. Cell-surface Tetherin expression was analyzed on GFP-positive cells by flow cytometry, 48h post-transfection. MFI values are shown beside the histogram. Filled histogram: pre-immune control; dashed line: HxBH10-vpu-; full line: HxBH10-vpu+; dotted line: HxBH10-vpu KSL. (C) Effect of Vpu on HIV-1 particle release. Cells and virus-containing supernatants were collected from the experiment described in (A), lysed and analyzed for the detection of Gag-related products by western blot using specific Abs. (D) Quantitation of virus particle release. Bands corresponding to Gag products in cells and virus particles were scanned by laser densitometry. The virus particle release efficiency was determined as described in the Materials and Methods and calculated as a percentage of the HxBH10-vpu+ virus release (100%). The error bars represent the standard deviation from the mean of three independent experiments.

(A-B) HeLa cells were transfected with the indicated HxBH10 proviral constructs and a GFP-expressing plasmid. Forty-eight hours post-transfection, cells were harvested and treated with pronase (0.05%). After quenching of the proteolytic reaction, cells were cultured at 37°C for different time intervals and then immunostained for cell-surface Tetherin. (A) Dot plots representing cell-surface Tetherin levels relative to GFP expression. MFI values are presented for each gate. Treatment with BFA (10 µM) served as a positive control for intracellular retention. BFA was added to the media immediately after treatment with pronase and kept throughout the chase period. (B) Quantitative representation of panel (A). Kinetics of Tetherin re-expression were determined by calculating the percentage of Tetherin expression (MFI) at the surface of pronase-treated cells relative to the corresponding untreated GFP-negative control at different time intervals. Equations pertaining to each linear equation are shown above the graph. Error bars represent the standard deviation of at least two separate experiments. Black dashed line: HxBH10-vpu+/GFP-; black full line: HxBH10-vpu-/GFP+; red full line: HxBH10-vpu+/GFP+; blue full line: HxBH10-vpu S52D,S56D/GFP+. Similar results were obtained when the proportion of cells expressing Tetherin at the cell-surface was used as a read-out instead of Tetherin MFI. For these latter analyses, the cut-off was arbitrarily set on the pronase-treated HxBH10-vpu+/GFP+ time 0 sample (illustrated by the dotted line in Figure 5A) so that the background level of Tetherin-positive cells corresponded to 1%.

(A) Schematic representation of HA-Tetherin wt and HA-Tetherin Y6Y8. The overlapping Tyr-based motifs are presented in the grey box. X and Φ correspond to variable and hydrophobic amino-acid residues, respectively. Hyphens represent unchanged amino-acids. The site that is cleaved prior to addition of the GPI lipid anchor is represented by the dashed line. Glycosylation sites are represented at position 65 and 92. (B) Kinetics of HA-Tetherin Y6Y8 internalization. HEK 293T cells were co-transfected with the indicated HxBH10 constructs and a GFP-expressing plasmid. Forty-eight hours post-transfection, cell-surface Tetherin was labeled with Tetherin specific Abs at 4°C before incubating cells at 37°C for the indicated time intervals to allow endocytosis. Cells were then incubated at 4°C in presence of appropriate secondary Abs. The graph depicts the relative levels of Tetherin at the surface of GFP-expressing cells (time 0 = 100%) and represents the loss of Tetherin-specific signal following endocytosis. (C) Cell-surface expression of HA-Tetherin Y6Y8. HEK 293T cells were co-transfected with the indicated HA-Tetherin plasmid and HxBH10 proviral constructs in presence of a GFP-expressing plasmid. Cell-surface Tetherin expression was analyzed on GFP-positive cells by flow cytometry 48 h post-transfection. Geo mean values (depicted as MFI) are shown. Full lines: HxBH10-vpu+; dashed lines: HxBH10-vpu-; filled histogram: pre-immune control. The results are representative of two independent experiments. (D) Effect of HA-Tetherin Y6Y8 on HIV-1 particle release. HEK 293T cells were co-transfected with the indicated HxBH10 proviral constructs and HA-Tetherin-expressing plasmids. Forty-eight hours post-transfection, transfected cells and virus-containing supernatants were harvested. Proteins from cell and viral lysates were analyzed by western blot using specific Abs. (E) Quantitation of HIV-1 particle release. Bands corresponding to Gag products in cells and viral particles were scanned by laser densitometry. The virus particle release efficiency was determined as described in the Materials and Methods and calculated as a percentage of the release of HxBH10-vpu+ (100%) in absence of HA-Tetherin. Error bars indicate the standard deviation of the mean from two independent experiments.