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

Na/K pump subunits distribution upon subcellular fractionation of CsA-treated or CypB-silenced HK-2 cells.

<p>Sh Luc, Sh Luc treated with CsA, and Sh CypB cells show different patterns of sedimentation on sucrose gradients of α and β Na/K ATPase and CypB. Cells treated or untreated with CsA were harvested and loaded on sucrose gradients (10 to 50% w/v) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013930#s4" target="_blank">Methods</a>. (A) Western Blot of fractions eluted from sucrose gradient of Sh Luc cells probed against protein markers of specific cell compartments: Syntaxin-6 (Golgi), Calnexin (ER), E-cadherin (Plasmatic Membrane) and S6 (Ribosomes). (B) Western Blot of fractions eluted from sucrose gradient of Sh Luc, Sh Luc + CsA, and Sh CypB cells probed against α and β Na/K ATPase and CypB. Figures are representative of at least three independent experiments.</p

Na/K-ATPase activity decreases in CypB silenced human kidney cells (HK-2).

<p>A: HK-2 cells were silenced for CypB using microRNA-based shRNA lentiviral vectors (mRNAi-CypB). Interference of the luciferase gene was used as control (ctl-luciferase). Western blot assays show a complete lack of CypB in CypB silenced cells, even at saturating protein concentrations from cell lysates (20 µg). B: Na/K-ATPase activity decreases in silenced CypB cells in a similar manner than 10 µM CsA treatment for 24 h does in HK-2 cells. Luciferase silenced HK-2 cells were used as a control. CypA silencing in HK-2 cells does not have any effect on activity (not shown). Figures are representative of at least three independent experiments.</p

Interaction between CypB and Na/K-ATPase beta and alfa subunits.

<p>A: Yeast two hybrid assay. CDNA for CypB was cloned in frame into the yeast expression vector (pGBKT7) that harbours the GAL4 activation domain. The recombinant plasmids plus Human Kidney Matchmaker cDNA Library were co-transformed into AH109 yeast strain. The co-transformed cells were selected on Ade-/Leu-/His-/Trp-, 10 µM Aminotriazol dropout plates to monitor for growth. The positive control represents co-transformation of p53 and T-antigen in two-hybrid expression vectors, pGBKT7-P53 and pGADT7 (BD Biosciences, Clontech), respectively. B: GST-pull-down assays. CypB was fused in frame with the GST gene. GST and GST-HCypB products were immobilized on Sepharose 4B and incubated with COS-7 cells lysates expressing Na/K-β1 (pCMV-HA-Na/K). The bound proteins were analyzed by immunoblotting with anti-Na/K-β1 rabbit polyclonal antibody. C–E: Co-Immunoprecipitation assays. HK-2 lysates were immunoprecipitated with mouse monoclonal antibodies against Na/K-β1 or Na/K-α1 and rabbit polyclonal antibody against CypB. The immunoprecipitates were subjected to Western blotting analyses, as indicated in the figure. As control, ChromePure mouse IgG and ChromePure rabbit IgG were used. Figures 1B to 1E are representative of at least three independent experiments.</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

The decrease in T cell transduction efficiency by SIV_MAC is not explained by differences in reporter gene expression.

<p>(A) CRFK cells (left panel) and Jurkat T cells (right panel) were transduced with VSV G-pseudotyped, single-cycle, two-part HIV-1_NL4-3GFP or SIV_MAC239-GFP vectors, as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005050#ppat.1005050.g001" target="_blank">Fig 1</a>_. Virus stocks were normalized by reverse transcriptase activity prior to transduction. 48 hrs after transduction, cells were visualized by phase contrast and fluorescence microscopy. Shown are representative fields for each condition at 100x magnification. Fluorescence intensity of individual T cells transduced with SIV_MAC239-GFP is at least as strong as that in cells transduced with HIV-1_NL4-3GFP. (B) VSV G-pseudotyped, HIV-1_NL4-3 (black squares) and SIV_MAC239 (white circles) three-part vectors were generated by plasmid transfection of 293T cells. In each case, the viral genomic RNA was designed to transduce an identical SFFV-GFP reporter gene. Vector stocks were normalized by titer on CRFK cells, and then used to challenge Jurkat T cells. 48 hrs post vector challenge, the percentage GFP-expressing cells was determined by FACS. Data is plotted as percent GFP⁺ (infected) cells (Y axis) versus CRFK infectious units (IU) x 1,000 (X axis).</p

Evidence for a dominant-acting, capsid-specific, restriction activity in Jurkat T cells.

<p>(A) Jurkat and HeLa cells stably expressing the ALV-A receptor (TvA) or TagRFP-657, as indicated, were fused by treatment with PEG and transduced with ALV-A Env-pseudotyped HIV-1_NL4-3-GFP, or with isogenic vector bearing the SIV_MAC239 CA^1-202. Shown are flow cytometry dot plots obtained 48 hrs post-transduction. HeLa-TagRFP-657 cells are only permissive to infection with ALV-A Env-pseudotyped vectors after fusion with Jurkat-TvA. Infected heterokaryons were visualized as GFP and TagRFP-657 double-positive cells. As a positive transduction control, TagRFP-657 and TvA were also co-expressed in HeLa cells, as indicated. The percentage of transduced cells are indicated. (B) Bar graph showing the infectivity of the SIV_MAC239 CA^1-202-bearing vector relative to the isogenic vector bearing HIV-1 CA, for the HeLa, Jurkat and heterokaryons. Data from the flow cytometry data shown in A, and two repeat experiments, is shown with the standard deviation.</p

The capsid of SIV_MAC, HIV-2, or SIV_SM is sufficient to decrease HIV-1 transduction efficiency in a T cell-specific manner.

<p>(A) Chimeric vectors were generated in which the coding sequence for HIV-1 CA amino acid residues 1 to 202 of the two-part HIV-1_NL4-3GFP vector (white squares) was replaced with sequence encoding the corresponding amino acid residues from HIV-2_ROD (grey triangles) or SIV_MAC239 (black circles). VSV G-pseudotyped vector was generated for each by transfection of 293T cells. Stocks were normalized by RT and used to challenge CRFK cells (B) or HeLa cells (C). Stocks were then normalized for CRFK transduction activity and used to challenge Jurkat T cells (D). 48 hrs post-challenge, the percentage of GFP-expressing cells was determined by FACS. Data is plotted as percent GFP⁺ (infected) cells (Y axis) versus RT activity (B and C), or versus CRFK infectious units (IU) x 1000 (X axis). (E) Chimeric vectors were generated in which the coding sequence for HIV-1 CA amino acid residues 1 to 202 of the HIV-1 <i>gag-pol</i> expression vector (white) was replaced with sequence encoding the corresponding amino acid residues from various HIV-2 isolates (grey) or SIV_SME041 (black). Three-part, VSV G-pseudotyped, SFFV-GFP bearing vectors were generated for each CA chimera by transfection of 293T cells. Stocks were then normalized for CRFK transduction activity and used to challenge Jurkat T cells. 48 hrs post-challenge, the percentage of GFP-expressing cells was determined by FACS. Data is plotted as CRFK normalized transduction of Jurkat cells, relative to the parental HIV-1 vector (F). Accession numbers for the different CA coding sequences are as follows: HIV-2(AB), 731744; HIV-2(A), GH123; HIV-2(D), L33083; HIV-2(E), L33087; HIV-2(F), U75441; HIV-2(H), AY5308; SIV_SME041, HM059825.</p

The block to SIV_MAC infection of Jurkat T cells occurs after formation of 2-LTR circles.

<p>CRFK and Jurkat (A), or Hela and Jurkat (B), or PBMCs (C) were infected with VSV G-pseudotyped HIV-1_NL4-3-GFP, or with isogenic vector bearing the SIV_MAC239 CA residues 1 to 202. 24 hrs post-infection, DNA was collected from the cells and subjected to qPCR using primers specific for full-length linear viral cDNA, 2-LTR circles, or proviral DNA, as indicated. Shown is the abundance of signal from vector bearing the SIV_MAC239 CA^1-202, relative to the amount of signal from HIV-1_NL4-3-GFP. In each case, infection was performed in the presence of an RT inhibitor to control for background levels of signal.</p

The transduction defect associated with SIV_MAC CA is independent of the virus entry pathway.

<p>A two-part, <i>env</i>-minus HIV-1 vector with GFP in place of nef (black squares), or an isogenic vector in which CA^1-202 coding sequences were replaced with those from SIV_MAC239 (white circles), were produced by 293T transfection. Each vector was pseudotyped with Env glycoprotein from either HIV-1_HXB2(A), HIV-2_MCN (B), ecotropic MLV (C), or ALV-A (D) and transduction efficiency was measured on HeLa cells bearing human CD4 (A and B), the mCAT1 ecotropic receptor (C), or the avian TVA receptor (D), and then used to challenge Jurkat cells bearing the same receptors. 48 hrs post-challenge, the percentage of GFP-expressing cells was determined by FACS.</p