Journal: PLoS Pathogens

Article Title: HIV-1 Nef Targets MHC-I and CD4 for Degradation Via a Final Common β-COP–Dependent Pathway in T Cells

doi: 10.1371/journal.ppat.1000131

Figure Lengend Snippet: (A) Reduction of surface expression of HLA-A2, CD4 and A2/CD4 as measured by flow cytometry. CEM HA-HLA-A2 and CEM HA-A2/CD4 cells were transduced with a control adenovirus ( nef − ) or adeno-Nef ( nef + ) and stained for surface HLA-A2 and CD4. The histograms shaded gray represent cells treated with control adenovirus, and the solid black line indicates cells treated with adeno-Nef. (B-C) Measurements of surface stability. CEM HA-HLA-A2 and HA-A2/CD4 cells were treated with adenovirus as in part A and the internalization of endogenous CD4 (B) or A2/CD4 (C) was compared to the internalization of HLA-A2. The filled squares represent control ( nef− ) cells, and the open squares represent adeno-Nef ( nef + ) cells. Quantitation of part B was compiled from four independent experiments performed in duplicate. Part C is representative of two experiments performed in triplicate. (D–E) HLA-A2 is inefficiently transported to the cell surface in T cells expressing Nef. CEM HA-HLA-A2 and CEM HA-A2/CD4 cells were transduced as in part A. Metabolic labeling with continuous surface biotinylation was performed in the presence of a cell-impermeable biotinylation reagent [NHS-biotin, (Pierce)] to label cell surface proteins. The cells were lysed and immunoprecipitated first with an antibody against HLA-A2 or CD4 (part D) or anti-HA (part E), then 2/3 was re-precipitated with avidin beads to selectively precipitate the HLA-A2 on the cell surface. Normalized surface MHC-I was calculated as follows: ((surface MHC-I /total MHC-I×2)×100). Quantitation for parts D and E represents the mean±standard deviation for four and three independent experiments respectively. For part D, quantitation is derived from data for the lower of the two Nef levels shown.

Article Snippet: CEM T cells stably expressing HA-tagged HLA-A2 (CEM HA-HLA-A2) have already been described .

Techniques: Expressing, Flow Cytometry, Transduction, Staining, Quantitation Assay, Labeling, Immunoprecipitation, Avidin-Biotin Assay, Standard Deviation, Derivative Assay

Journal: PLoS Pathogens

Article Title: HIV-1 Nef Targets MHC-I and CD4 for Degradation Via a Final Common β-COP–Dependent Pathway in T Cells

doi: 10.1371/journal.ppat.1000131

Figure Lengend Snippet: (A) Three-way co-localization of HLA-A2, CD4 and Rab7. CEM HLA-A2 cells stably expressing YFP-Rab7 were transduced with adeno-Nef. HLA-A2 (red), CD4 (green), and YFP-Rab7 (blue) were simultaneously detected using three-color confocal microscopy. The top row shows the x – y projection of the cell, while the bottom row displays the x – z projection. Ten sequential optical sections were compiled to generate a projection of each cell about the x – z plane. Scale bar = 5 microns. (B) Immunogold labeling of HLA-A2 and CD4. Representative electron micrographs of CEM HA-HLA-A2 cells treated with control adeno (column 1) or adeno-Nef (columns 2 and 3). Thawed cryosections of cells were labeled with anti-HA (HLA-A2) and anti-CD4 antibodies followed by 15 and 10 nm protein A-gold respectively. (C) Immunogold labeling of HA-HLA-A2 and γ-adaptin in Nef-expressing CEM T cells. Thawed cryosections of cells were labeled with anti-HA (HLA-A2) and anti-γ-adaptin antibodies followed by 15 and 10 nm protein A-gold, respectively.

Article Snippet: CEM T cells stably expressing HA-tagged HLA-A2 (CEM HA-HLA-A2) have already been described .

Techniques: Stable Transfection, Expressing, Transduction, Confocal Microscopy, Labeling

Journal: PLoS Pathogens

Article Title: HIV-1 Nef Targets MHC-I and CD4 for Degradation Via a Final Common β-COP–Dependent Pathway in T Cells

doi: 10.1371/journal.ppat.1000131

Figure Lengend Snippet: (A) Analysis of protein expression in β-COP and μ1 knockdown cells. CEM HA-HLA-A2 cells were transduced with a lentivirus expressing both GFP and a control shRNA (shNC) or an shRNA targeting either β-COP (shβ-COP) or μ1 (shμ1). At 72 hours later, they were transduced with adeno-Nef or control adenovirus. Three days later they were harvested, and western blot analysis was used to assess protein levels of β-COP, μ1 and Nef. (B,C) Quantification of μ1 and β-COP expression in shRNA treated cells. The amount of either μ1 (B) or β-COP (C) was quantified using Adobe Photoshop software. The average percent remaining±standard deviation for four experiments (B) and three experiments (C) is shown. To adjust for protein loading in part B, the nonspecific background band directly below μ1 (shown in part A) was used to normalize protein loading. (D) Knockdown of β-COP does not affect HLA-A2 transport to the cell surface. CEM HA-HLA-A2 cells were transduced with lentivirus expressing either shNC or shβ-COP as in part A. Cell surface transport was assessed using a metabolic labeling assay with biotinylation as described in . (E) Knockdown of β-COP does not disrupt the Golgi apparatus. CEM HA-HLA-A2 cells were transduced with lentivirus expressing the indicated shRNA and GFP as in part A and treated with brefeldin A (BFA) at 50 µM or DMSO for 30 minutes. The integrity of the Golgi apparatus was assessed by immunofluorescence staining for giantin and analyzed by confocal microscopy. Images were taken using a Zeiss confocal microscope and analyzed with LSM Image Browser and Adobe Photoshop software. Single Z-sections are shown. The results shown for parts D and E are representative of three independent experiments.

Article Snippet: CEM T cells stably expressing HA-tagged HLA-A2 (CEM HA-HLA-A2) have already been described .

Techniques: Expressing, Transduction, shRNA, Western Blot, Software, Standard Deviation, Labeling, Immunofluorescence, Staining, Confocal Microscopy, Microscopy

Journal: PLoS Pathogens

Article Title: HIV-1 Nef Targets MHC-I and CD4 for Degradation Via a Final Common β-COP–Dependent Pathway in T Cells

doi: 10.1371/journal.ppat.1000131

Figure Lengend Snippet: (A) β-COP and μ1 are required for Nef to reduce cell surface expression of HLA-A2. CEM HA-HLA-A2 cells were transduced with a lentivirus expressing GFP and a control (shNC), β-COP (shβ-COP) or μ1 (shμ1) shRNAs and with control adenovirus ( nef − ) or adeno-Nef ( nef + ). Cell surface expression of HLA-A2 or CD4 in the GFP-positive cells was assessed by flow cytometry. The gray shaded histogram represents control adenovirus ( nef − ) treated cells and the solid black line represents adeno-Nef ( nef + ) treated cells. (B) Quantitation of HLA-A2 and CD4 downmodulation in Nef expressing cells transduced with shRNA. The median fold downmodulation (median fluorescence of control/median fluorescence of Nef-expressing cells)±standard deviation derived from five (HLA-A2) and four (CD4) independent experiments. A p value was calculated using a two tailed t-test and significant differences were indicated with asterisks (* p <0.02, *** p <10 −3 , **** p <10 −4 ). (C) Knockdown of β-COP stabilizes intracellular levels of HLA-A2 and A2/CD4 in Nef expressing cells. CEM HA-HLA-A2 and CEM HA-A2/CD4 were treated as in part A. Lysates from these cells were generated and treated with endoglycosidase H (endo H). Protein levels of HLA-A2 and A2/CD4 were assessed by western blot using an anti-HA antibody. Endo H–resistant bands are marked with an R and endo H–sensitive bands are marked with an S. The results shown are representative of three independent experiments for HLA-A2 and two independent experiments for A2/CD4. (D) Quantification of endo H–resistant protein. Adobe Photoshop software was used to quantify each band for the Nef-expressing samples. The percentage of endo H–resistant protein in each condition was calculated as follows: [resistant band/(resistant band+sensitive band)]×100. The fold stabilization was then calculated as: (% endo H–resistant in experimental sample)/[% endo H–resistant in control (shNC)]. The data shown is the mean of two experiments±standard deviation.

Article Snippet: CEM T cells stably expressing HA-tagged HLA-A2 (CEM HA-HLA-A2) have already been described .

Techniques: Expressing, Transduction, Flow Cytometry, Quantitation Assay, shRNA, Fluorescence, Standard Deviation, Derivative Assay, Two Tailed Test, Generated, Western Blot, Software

Journal: PLoS Pathogens

Article Title: HIV-1 Nef Targets MHC-I and CD4 for Degradation Via a Final Common β-COP–Dependent Pathway in T Cells

doi: 10.1371/journal.ppat.1000131

Figure Lengend Snippet: (A) Knockdown of β-COP stabilizes CD4 + vesicles in Nef expressing cells. CEM HA-HLA-A2 cells transduced with a lentivirus expressing GFP and either control shRNA (shNC) or shRNA targeting β-COP (shβ-COP) were transduced with control adenovirus ( nef − ) or adeno-Nef ( nef + ). The cells were incubated with CD4 antibody on ice and then shifted to 37°C for internalization for the indicated times. Images were taken with a Zeiss confocal microscope and processed using LSM Image Browser and Adobe Photoshop software. Single Z-sections are shown. (B) Quantitation of CD4 + vesicles is shown for 15 GFP + , nef + cells treated with shNC and 17 GFP + , nef + cells treated with shβ-COP. The mean±standard deviation is shown. (C) Quantitation is shown for 5 GFP + , nef + cells treated with shNC and 5 GFP + , nef + cells treated with shβ–COP. The mean±standard deviation is shown. (D) CEM HA-HLA-A2 cells were transduced with a lentivirus expressing either GFP and control (shNC) or β-COP (shβ–COP) shRNA, infected with HIV, treated with bafilomycin or DMSO and stained for HLA-A2 and LAMP-1 as previously described . Images were taken with a Zeiss confocal microscope and processed as in part A. Single Z-sections are shown. (E) Relative co-localization of HLA-A2 with LAMP-1 in 10 GFP + , adeno-Nef-expressing T cells treated with shNC and 15 GFP + , adeno-Nef-expressing T cells treated with shβ-COP. (F) Relative co-localization of HLA-A2 with LAMP-1 in 6 GFP + , HIV- nef + infected T cells treated with shNC and, 6 GFP + , HIV- nef + –infected T cells treated with shβ-COP. Quantitation of microscopy data was performed independently by two blinded investigators who scored maximal observable co-localization among all cells at an arbitrary value of 5. Each cell was then scored relative to that. The mean±standard deviation is shown.

Article Snippet: CEM T cells stably expressing HA-tagged HLA-A2 (CEM HA-HLA-A2) have already been described .

Techniques: Expressing, Transduction, shRNA, Incubation, Microscopy, Software, Quantitation Assay, Standard Deviation, Infection, Staining

Journal: PLoS Pathogens

Article Title: HIV-1 Nef Targets MHC-I and CD4 for Degradation Via a Final Common β-COP–Dependent Pathway in T Cells

doi: 10.1371/journal.ppat.1000131

Figure Lengend Snippet: (A) The HLA-A2 cytoplasmic tail is necessary for co-precipitation of AP-1. Parental HLA-A2-negative CEM T cells (CEM) or CEM T cell lines expressing HA-HLA-A2 or HA-A2/CD4 were transduced with adeno-Nef or a control adenovirus. Lysates were immunoprecipitated with an antibody directed against HLA-A2 (BB7.2) and the presence of Nef or AP-1 was detected by western blot analysis. Results are representative of three independent experiments. (B) The cytoplasmic tail is necessary for the HLA-A2/Nef fusion protein to co-precipitate AP-1 in Nef expressing T cells. CEM T cells were transduced with a murine retroviral vector expressing no protein (vector), A2/Nef or A2/CD4/Nef fusion proteins. These cells were immunoprecipitated with an anti-HLA-A2 antibody (BB7.2) and western blot analysis was performed to detect co-precipitation of AP-1. Spaces between lanes indicate where intervening lanes were cropped out to remove irrelevant data. Results are representative of two independent experiments.

Article Snippet: CEM T cells stably expressing HA-tagged HLA-A2 (CEM HA-HLA-A2) have already been described .

Techniques: Expressing, Transduction, Immunoprecipitation, Western Blot, Plasmid Preparation

Journal: PLoS Pathogens

Article Title: HIV-1 Nef Targets MHC-I and CD4 for Degradation Via a Final Common β-COP–Dependent Pathway in T Cells

doi: 10.1371/journal.ppat.1000131

Figure Lengend Snippet: (A) Flow cytometric analysis of Nef mutants defective at MHC-I downmodulation. CEM T cells treated with control adenovirus ( nef − ), adeno-Nef ( nef + ) or the indicated mutant were stained either with an anti-HLA-A2 antibody (BB7.2) or an antibody directed at CD4. Cells were analyzed by flow cytometry as described in . (B) Quantitation of MHC-I and CD4 downmodulation by Nef and Nef mutants. Fold downmodulation was determined by dividing the mean fluorescence intensity (MFI) of control virus treated cells by the MFI of Nef-expressing cells. The average value from three (wild-type) or two (mutant Nef) experiments was plotted±the standard deviation. (C) Nef D 123 G and V 10 EΔ17–26 mutants are defective at β-COP binding. CEM T cells were treated with control adenovirus ( nef − ), adeno-Nef ( nef + ), or the indicated mutant and immunoprecipitated with a control antibody (BB7.2) or an antibody directed against β-COP (M3A5). The presence of Nef was detected by western blot analysis. Arrows indicate the positions of wild type Nef and Nef V 10 EΔ17–26. Results are representative of at least two independent experiments. (D) V 10 EΔ17–26 Nef is defective at MHC-I, but not CD4, degradation. CEM cells expressing HA-HLA-A2 and HA-A2/CD4 were transduced with adeno-viral vectors encoding wild-type Nef (Nef + ), V 10 EΔ17–26 Nef, or a control adenoviral vector (Nef − ). Two days later, the media on half of the cells was replaced with media containing 20 mM ammonium chloride to inhibit lysosomal degradation. The next day, the cells were harvested, lysed, and normalized. Each sample was split equally and one set was treated with endo H. Protein levels of HA-HLA-A2 and HA-A2/CD4 were assessed by western blot analysis using an anti-HA antibody. Endo H–resistant bands are marked with an R and endo H–sensitive bands are marked with an S.

Article Snippet: CEM T cells stably expressing HA-tagged HLA-A2 (CEM HA-HLA-A2) have already been described .

Techniques: Mutagenesis, Staining, Flow Cytometry, Quantitation Assay, Fluorescence, Expressing, Standard Deviation, Binding Assay, Immunoprecipitation, Western Blot, Transduction, Plasmid Preparation

Journal: PLoS Pathogens

Article Title: HIV-1 Nef Targets MHC-I and CD4 for Degradation Via a Final Common β-COP–Dependent Pathway in T Cells

doi: 10.1371/journal.ppat.1000131

Figure Lengend Snippet: (A) Flow cytometric analysis of HLA-A2 and CD4 expression in cells expressing Nef mutants. CEM T cells stably expressing HLA-A2 were spin-transduced with murine retroviral supernatants that express the indicated Nef construct and a GFP cassette from an internal ribosomal entry site. The cells were gated for GFP expression and results of HLA-A2 (top panel) or endogenous CD4 (bottom panel) staining are shown. R/E stands for R 17,19 A/E 154,155 A double mutant. Open light gray curve, parental cell line; shaded dark gray curve, empty vector; black shaded curve, wild type Nef; and open dark gray curve, Nef mutant. (B) Quantification of down-modulation. The mean±SD for greater than or equal to six experiments (actual number varies depending on the mutant) is shown. (C,D) R 17/19 is needed for optimal HLA-A2 degradation, whereas the E 154/155 is dispensable. CEM T cells expressing HLA-A2 and Nef were generated as described in part A. The cells were pulse labeled with 35 S-labeled amino acids, chased for 0 or 12 hours in complete medium and lysed. HLA-A2 was immunoprecipitated with the anti-HLA-A2 antibody BB7.2, separated by SDS-PAGE and quantified using a phosphorimager. “Ig Control” indicates results from HLA-A2-negative parental CEM cells immunoprecipitated with BB7.2 antibody. (D) Quantification of degradation. Nef activity was calculated as follows: (the fraction of HLA-A2 remaining in control cells /the fraction of HLA-A2 remaining in Nef expressing cells). The value obtained for each mutant was divided by that for wild type Nef and multiplied by 100 to calculate % wild type activity. The mean±SD for two experiments is shown. (E) R 17/19 and E 154/155 are required for the β-COP/Nef interaction. CEM T cells expressing HA-A2 were transduced with a retroviral vector expressing either wild-type Nef or the indicated Nef mutant. The cells were immunoprecipitated with an anti–β-COP antibody and the presence of Nef was assessed by western blot as described in . The Ig control is HLA-A2–negative parental CEM cells expressing wild-type Nef immunoprecipitated with a control antibody (BB7.2) and the vector only control is CEM cells expressing HLA-A2 transduced with the empty retroviral vector.

Article Snippet: CEM T cells stably expressing HA-tagged HLA-A2 (CEM HA-HLA-A2) have already been described .

Techniques: Expressing, Stable Transfection, Transduction, Construct, Staining, Mutagenesis, Plasmid Preparation, Generated, Labeling, Immunoprecipitation, SDS Page, Activity Assay, Western Blot

Journal: PLoS Pathogens

Article Title: HIV-1 Nef Targets MHC-I and CD4 for Degradation Via a Final Common β-COP–Dependent Pathway in T Cells

doi: 10.1371/journal.ppat.1000131

Figure Lengend Snippet: (A) Cells expressing HA-HLA-A2/CD4 were treated as in , lysed and treated with endo H as indicated. The samples were separated by SDS-PAGE and western blotted for the HA tag on HA-A2/CD4. (B) Quantification of degradation. Western blots were quantified using Adobe Photoshop software. Nef activity was calculated as follows (fraction of total protein that was endo H–resistant for wild type Nef/fraction endo H–resistant for each mutant)×100. The mean±SD for four experiments is shown. (C) Model for the mechanism by which Nef affects CD4 and MHC-I trafficking. HIV Nef binds the CD4 cytoplasmic tail at the cell surface, and recruits AP-2 and/or the vacuolar-ATPase to facilitate internalization. CD4 is internalized and is transported to an endosomal compartment associated with Rab7 and β-COP. In contrast, Nef binds the MHC-I cytoplasmic tail early in the secretory pathway, AP-1 is recruited and facilitates transport to an intermediate endosomal compartment marked with Rab7. If AP-1 falls off the Nef-MHC-I complex after arrival in the endosome, Nef binds β-COP and targets MHC-I (and CD4) to lysosomes for degradation. If AP-1 remains bound, it promotes recycling of the Nef-MHC-I complex to the TGN. LY = lysosome, LE/MVB = late endosome/multi-vesicular body.

Article Snippet: CEM T cells stably expressing HA-tagged HLA-A2 (CEM HA-HLA-A2) have already been described .

Techniques: Expressing, SDS Page, Western Blot, Software, Activity Assay, Mutagenesis