Reversibility of the ERAD defect requires back-transfection of enzymatically active CyPB.

<p><b>A</b> Down-regulation of CyPB and back-transfections of active or catalytically inactive (R62A) CyPB were assessed by immunoblot of total cell lysates. Tubulin is a loading control. <b>B</b> Radiolabeled BACE457Δ was immunoisolated at the end of the chase times from detergent-extracts of cells expressing normal levels of CyPB (siSCR, lanes 1–3), in cells with reduced level of CyPB (siCyPB, lanes 4–6), in cells with reduced level of CyPB back-transfected with active (siCyPB+CyPB, lanes 7–9) or catalytically inactive CyPB (siCyPB+CyPB_R62A, lanes 10–12). <b>C</b> Quantification of the labeled polypeptide bands. Error bars represent SD from at least two independent experiments.</p

Vpu induces proteasomal degradation of tetherin.

<p>(A) The MG132 proteasomal inhibitor impedes Vpu-mediated depletion of tetherin. 293T cells were transfected in duplicate with an HA-tetherin construct in the presence or absence of Vpu (with a molar ratio of 2∶1 in favor of Vpu), and were either left untreated or treated for 12 hours with the proteasome inhibitor MG132. Duplicate lysates were then pooled for western blot analysis (left panel). Actin served as a loading control. The effect of MG132 on Vpu-mediated tetherin depletion was quantified by densitometry and a plot was generated from the results of three independent experiments performed in duplicate (right panel). The values obtained in the absence of Vpu were given the arbitrary value of 100%. (B) An ubiquitin mutant that blocks the formation of the polyubiquitin chains involved in proteasomal targeting impedes Vpu-mediated depletion of tetherin. 293T cells were transfected in duplicate with a Flag-tetherin construct in the presence or absence of Vpu. In addition, cells were co-transfected with a wild type or K48R mutant version of HA-tagged ubiquitin. The molar ratio of ubiquitin to Vpu to tetherin constructs was 2.5∶1.75∶1. Duplicate extracts from these cells were pooled and analyzed by western blotting (left panel). Ezrin was used as a loading control. The effect of the different ubiquitin constructs on Vpu-mediated tetherin depletion was quantified by densitometry, and a plot was generated from the results of three independent experiments performed in duplicate (right panel). The values obtained in the absence of Vpu were given the arbitrary value of 100%. Results were statistically significant as the p value, determined by the Student test, was lower than 0.05 for indicated pairs (*). Sizes of molecular weight markers are shown in kilodaltons.</p

Vpu and β-TrCP co-immunoprecipitate with tetherin.

<p>293T cells were transfected with the indicated Vpu and β-TrCP-ΔF constructs, in the presence or absence of HA-tetherin (with a molar ratio of 2∶1 in the favor of Vpu). Equal amounts of lysates were subjected to immunoprecipitation with an anti-HA resin and analyzed by western blotting. PCNA served as a loading control. The first left lane was cut and pasted from another position from the same scan of the same blot. The figure is representative of two independent experiments. Sizes of molecular weight markers are shown in kilodaltons.</p

A β-TrCP dominant negative prevents Vpu-mediated tetherin degradation.

<p>293T were transfected with HA-tagged tetherin with or without Vpu, in the presence of either a Flag-tagged dominant negative β-TrCP-ΔF or a Flag-tagged wild type β-TrCP1. The molar ratio of β-TrCP to Vpu to tetherin constructs was 2.5∶2∶1. A GFP plasmid was included to exclude variations in transfection efficiency. The resulting duplicate lysates were pooled for gel loading, and proteins levels were determined by western blotting. Actin served as a loading control. The depicted figure is representative of four independent experiments performed in duplicate. Sizes of molecular weight markers are shown in kilodaltons.</p

Vpu requires β-TrCP2 to deplete tetherin from cells and antagonize its antiviral action.

<p>(A) Creation of 293T cell lines harboring stably downregulated β-TrCP1 and β-TrCP2 levels. Total RNA from 293T cell lines stably expressing the indicated shRNAmir constructs was extracted, and used to monitor β-TrCP1 and β-TrCP2 mRNA levels by real-time RT-PCR. The expression of the TBP cellular gene was used for normalization. The values of β-TrCP1 and β-TrCP2 measured in the presence of the control shRNAmir were given the arbitrary value of 100%. (B) Vpu requires β-TrCP2 to deplete tetherin from cells and antagonize its antiviral action. A Vpu-deleted HIV-1, or its Vpu-proficient counterpart, was transfected in duplicate in the indicated stable cell lines, in the presence of an HA-tagged tetherin plasmid (molar ratio of 2∶1 in favor of tetherin). The extracts of the duplicate samples were pooled for gel loading, and tetherin protein levels were monitored by western blotting (lower panel). Equal loading was controlled by monitoring PCNA, and the viral p55 Gag protein was examined to exclude variations of transfection efficiency. In parallel, titer of the viral output present in the supernatant was monitored on HeLa indicator cells (upper panel). Similar results were obtained by scoring the physical viral particle output by reverse transcription assay (data not shown). The western blot figure is assembled from the data of two gels performed in parallel, on both of which all relevant controls were present and gave identical results. The figure is representative of two independent experiments performed in duplicate. Sizes of molecular weight markers are shown in kilodaltons.</p

Vpu-induced proteasomal degradation of tetherin involves ERAD.

<p>(A) The ERAD pathway is involved in Vpu-mediated tetherin depletion. 293T cells were transfected in duplicate with 100 nM of either a non-silencing control siRNA, or of a siRNA pool targeting p97. Twenty-four hours later, these cells were transfected with Flag-tagged tetherin in the presence or absence of Vpu (with a molar ratio of 2∶1 in favor of Vpu). Duplicate cell lysates were pooled for western blot analysis (left panel). Actin served as a loading control. Note that both parts of the figure come from the same scan of the same blot. The effect of the different siRNAs on Vpu-mediated tetherin depletion was quantified by densitometry and a plot was generated from the results of two independent experiments performed in duplicate (right panel). The values obtained in the absence of Vpu were given the arbitrary value of 100%. (B) Vpu-mediated tetherin depletion does not require ubiquitination of tetherin cytosolic lysines. 293T cells were co-transfected in duplicate with or without Vpu in the presence of either HA-tagged wild type tetherin or its counterpart having both its cytosolic lysines K18 and K21 replaced with arginines (KcytoR tetherin). Duplicate extracts were pooled and analyzed by western blotting. PCNA served as a loading control. Vpu-mediated tetherin depletion was quantified by densitometry, and a plot was generated from the results of two independent experiments performed in duplicate (right panel). The values obtained in the absence of Vpu were given the arbitrary value of 100%. Sizes of molecular weight markers are shown in kilodaltons.</p

β-TrCP interaction motif is required for Vpu-induced tetherin level reduction and for its ability to rescue virion release.

<p>(A) β-TrCP interaction motif is required for Vpu-induced tetherin depletion. HIV-1 deleted for the Vpu gene was produced from 293T cells in the presence or absence of Flag-tagged tetherin. Where indicated, Vpu wild type, or mutated in one (Vpu S52A) or both serines (Vpu 2S/A) known to be required for β-TrCP interaction, was added <i>in trans</i>. The effect of these different Vpu constructs on tetherin protein level was monitored by western blotting. The extracts of duplicate samples were pooled for gel loading. Equal loading was controlled by monitoring PCNA, and the viral p55 Gag protein was examined to exclude variations of transfection efficiency. The depicted gel is representative of three independent experiments. Sizes of molecular weight markers are shown in kilodaltons. (B) β-TrCP interaction motif is required for Vpu-induced rescue of virion release. Titer of the viral output obtained during the above experiment was measured on HeLa indicator cells. The titer of the virus produced in the absence of either tetherin or Vpu was given the arbitrary score of 100%. The plot was generated from two independent experiments performed in duplicate.</p

Vpu depletes human tetherin but not murine tetherin, which parallels its ability to rescue virion release.

<p>(A) Vpu counteracts human but not murine tetherin antiviral activity. 293T cells were transfected with an HIV-1 provirus either proficient or deficient for the Vpu gene, together with the indicated tetherin constructs. Viral output was then scored by titration of the resulting supernatant on HeLa indicator cells. (B) Vpu depletes human, but not murine, tetherin. Duplicate cell extracts from the above experiment were monitored for tetherin level (as detected with an anti-HA antibody). The viral p55 Gag protein was monitored to exclude variations of transfection efficiency. The depicted tetherin bands correspond to the heterogeneously glycosylated monomer of tetherin of around 30 kDa, but equivalent depletion could be observed for the 60 kDa dimeric form (data not shown). PCNA was used as a loading control. Note that all parts come from the same blot, but the detection of murine tetherin required longer exposure due to its lower expression. (C) Vpu depletes human tetherin in a dose dependent manner, in the absence of other viral components. Increasing doses of a Vpu-expressing plasmid was co-transfected with a Flag-tagged human tetherin in 293T cells (molar ratios of 1∶1 and 2∶1, indicated by + and ++ respectively). Steady state levels of tetherin were monitored by western blot analysis of duplicate cell extracts using an anti-Flag antibody. Transfection levels were assessed with a GFP plasmid, and actin was used as a loading control. All three sections of this figure are representative of five independent experiments performed in duplicate. Sizes of molecular weight markers are shown in kilodaltons.</p

The depletion of tetherin by Vpu correlates with its ability to block tetherin antiviral activity.

<p>(A) The depletion of tetherin by Vpu correlates in a dose-dependent manner with its ability to block tetherin antiviral activity. 293T cells were transfected with an HIV-1 provirus deleted for Vpu, together with a fixed dose of human HA-tagged tetherin, and either without or with increasing doses of Vpu added <i>in trans</i> (molar ratios are indicated). Viral output was scored by titration of the supernatant on HeLa indicator cells. In parallel, the level of tetherin was monitored by western blotting and subsequently quantified by densitometry. Both the cellular content of tetherin and its antiviral activity were then plotted. The values obtained in the absence of Vpu were given the arbitrary score of 100%. The plot was generated from two independent experiments performed in duplicate. The extracts of duplicate samples were pooled for gel loading. Equal loading was controlled by monitoring PCNA, and the viral p24 protein was examined to exclude variations of transfection efficiency. (B) The depletion of tetherin by Vpu correlates across different time points with its ability to block tetherin antiviral activity. 293T cells were transfected with an HIV-1 provirus deleted for the Vpu gene, with or without a given dose of HA-tagged human tetherin, in the presence or the absence of Vpu added <i>in trans</i>. At indicated time points, the titer of the viral output was scored on HeLa indicator cells. In parallel, the level of tetherin was monitored by western blotting and subsequently quantified by densitometry. For each condition, both the cellular content of tetherin and its antiviral activity were plotted as a percent of the values obtained in parallel in the absence of Vpu, which were given the arbitrary score of 100%. The plot was generated from two independent experiments performed in duplicate. The extracts of duplicate samples were pooled for gel loading. Equal loading was controlled by monitoring actin, and the viral p55 Gag protein was examined to exclude variations of transfection efficiency. For both figures, Pearson coefficients of correlation and Student p values were computed for tetherin expression versus antiviral activity. Sizes of molecular weight markers are shown in kilodaltons.</p

Localization of Na/K-ATPase beta and CypB in silenced or CsA-treated HK-2 cells.

<p>Immunocitochemistry assays using anti-CypB rabbit polyclonal antibody and mouse monoclonal anti-Na/K-β1 antibody were performed on luciferase silenced HK-2 control cells (ctl-luciferase) (Panel A), 10 µM CsA treated ctl-luciferase cells, for 24 h (Panel B) and Cyp B silenced cells (Panel C). White arrows point to plasma membrane and yellow arrows to endoplasmic reticulum localization. Fluorescence labelling was visualized in a Leica DM IRBE confocal microscope. Figures are representative of at least three independent experiments.</p