Journal: eLife

Article Title: HIV-1 envelope glycoprotein modulates CXCR4 clustering and dynamics on the T cell membrane

doi: 10.7554/eLife.110354

Figure Lengend Snippet: ( A ) Representative transmission electron micrographs of gp120-VLP particles. Original scale bar 100 nm. ( B ) Culture media of transfected HEK-293T cells (cm) with different constructs, as indicated, and the corresponding clarified samples (cs) containing the VLPs or the LVPs generated, were analyzed by western blot with anti-p24, and -gp120 mAbs. The anti-p24 mAb also recognizes Pr55Gag, a precursor of p24 presents in immature particles. Molecular weight markers are indicated (kDa). ( C ) Flow cytometry analysis of VLPs (Env(-) and expressing x4-gp120) bound to latex beads in the presence of soluble human CD4, using an anti-Histidine mAb. A representative experiment is shown of 3 performed. ( D ) Representative transduction experiments using the indicated LVPs, where the reporter expression was captured using the Tecan SparkCyto reader. Upper panels show bright-field images of target HEK-293 CD4 cells. Lower panels show fluorescence signal from GFP expression in transduced cells. Images were captured with a 4× objective, using an exposure of 200ms in all cases and 80ms for control VSVG-VLPs (positive control). Env(-) LVPs were used as negative control (n=3). ( E ) Quantification of the Mean Fluorescence Intensity (MFI) of images obtained in transduction experiments. Figure 2—figure supplement 1—source data 1. Original files for western blot analysis for . Figure 2—figure supplement 1—source data 2. PDF file containing original western blots for . Original membranes corresponding to , panel B. Culture media of transfected HEK-293T cells (cm) with different constructs, as indicated, and the corresponding clarified samples (cs) containing the VLPs or the LVPs generated, were analyzed by western blot with anti-p24, and -gp120 mAbs. The anti-p24 mAb also recognizes Pr55Gag, a precursor of p24 presents in immature particles. Molecular weight markers are indicated (kDa). , panel B shows the last six lanes of these membranes reorganized to separate LVPs from VLPs. Original files for western blot analysis displayed in .

Article Snippet: The following antibodies were used: anti-human CXCR4 monoclonal antibody (mAb; clone 44717) and phycoerythrin-conjugated anti-human CXCR4 mAb (clone 12G5; both from R&D Systems, Minneapolis, MN); goat F(ab’)2 anti-mouse IgG-PE (Southern Biotech, Birmingham, AL); anti-human CD4 mAb (clone OKT4; Biolegend, San Diego, CA); anti-histidine mAb (clone AD1.1.10; R&D Systems); rabbit anti-gp120 IIIb Ab ( ); rabbit anti-Gag p24 HIV-1 mAb (R&D Systems); and anti-phospho-AKT mAb (S473; #4060), anti-phospho-ERK1,2 mAb (T202/Y204; #9191), and anti-phospho-Lck mAb (Y505; #2751; all from Cell Signaling Technology, Danvers, MA); anti-tubulin mAb conjugated with rhodamine (Bio-Rad, Hercules, CA); phalloidin-TRITC (#P1951, Sigma-Merck, St Louis, MO); anti-ICAM 3 mAb (clone HP2/19) kindly donated by Dr. Francisco Sánchez Madrid (Instituto Sanitario Hospital Universitario La Princesa); goat anti-mouse-AF488 Ab (Thermo Fisher Scientific); anti-human gp120 mAb Fab fragments (clone 2G12; Polymun Scientific, Vienna, Austria); anti-human IgG Fab fragments (Jackson ImmunoResearch, West Grove, PA) conjugated to Abberior STAR RED (Abberior GmbH, Gottingen, Germany), kindly donated by Dr. Jakub Chojnacki (Germans Trias i Pujol Research Institute (IGTP)); anti-p24 HIV-1 (clone 37G12; Polymun Scientific) conjugated with Abberior STAR ORANGE.

Techniques: Transmission Assay, Transfection, Construct, Generated, Western Blot, Molecular Weight, Flow Cytometry, Expressing, Transduction, Fluorescence, Control, Positive Control, Negative Control

Journal: eLife

Article Title: HIV-1 envelope glycoprotein modulates CXCR4 clustering and dynamics on the T cell membrane

doi: 10.7554/eLife.110354

Figure Lengend Snippet: ( A ) Representative images of clarified VLPs visualized by STED microscopy. Upper panels show images of the indicated VLPs stained for Gag p24 (blue) and gp120 (red). Lower panels show ×10 magnification of equivalent images. White arrows indicate mature VLPs (p24 condensation). ( B ) Percentage of mature VLPs, analyzed from the images in ( A ) using TrackAnalyzer in ImageJ, based on p24 intensity and aggregation level (mean ± SD; n=2; ****p≤0.0001; the significance indicated on immature VLPs bar shows the difference with all other conditions). ( C ) Percentage of VLPs expressing gp120 on their surface, as analyzed in ImageJ (mean ± SD; n=2; ***p≤0.001). ( D ) Distribution of gp120 mean fluorescence intensity. Each spot corresponds to the mean fluorescence intensity for each analyzed VLP in a.u. The black line represents the mean of all values (****p≤0.0001). ( E ) Frequency of gp120 intensity/particle. Statistical significance was determined by one-way-ANOVA followed by Tukey’s multiple comparisons test in panels B and C and by Mann-Whitney analysis for panel D.

Article Snippet: The following antibodies were used: anti-human CXCR4 monoclonal antibody (mAb; clone 44717) and phycoerythrin-conjugated anti-human CXCR4 mAb (clone 12G5; both from R&D Systems, Minneapolis, MN); goat F(ab’)2 anti-mouse IgG-PE (Southern Biotech, Birmingham, AL); anti-human CD4 mAb (clone OKT4; Biolegend, San Diego, CA); anti-histidine mAb (clone AD1.1.10; R&D Systems); rabbit anti-gp120 IIIb Ab ( ); rabbit anti-Gag p24 HIV-1 mAb (R&D Systems); and anti-phospho-AKT mAb (S473; #4060), anti-phospho-ERK1,2 mAb (T202/Y204; #9191), and anti-phospho-Lck mAb (Y505; #2751; all from Cell Signaling Technology, Danvers, MA); anti-tubulin mAb conjugated with rhodamine (Bio-Rad, Hercules, CA); phalloidin-TRITC (#P1951, Sigma-Merck, St Louis, MO); anti-ICAM 3 mAb (clone HP2/19) kindly donated by Dr. Francisco Sánchez Madrid (Instituto Sanitario Hospital Universitario La Princesa); goat anti-mouse-AF488 Ab (Thermo Fisher Scientific); anti-human gp120 mAb Fab fragments (clone 2G12; Polymun Scientific, Vienna, Austria); anti-human IgG Fab fragments (Jackson ImmunoResearch, West Grove, PA) conjugated to Abberior STAR RED (Abberior GmbH, Gottingen, Germany), kindly donated by Dr. Jakub Chojnacki (Germans Trias i Pujol Research Institute (IGTP)); anti-p24 HIV-1 (clone 37G12; Polymun Scientific) conjugated with Abberior STAR ORANGE.

Techniques: Microscopy, Staining, Expressing, Fluorescence, MANN-WHITNEY

Journal: eLife

Article Title: HIV-1 envelope glycoprotein modulates CXCR4 clustering and dynamics on the T cell membrane

doi: 10.7554/eLife.110354

Figure Lengend Snippet: The presence of CXCR4 R334X on JKCD4 + cells does not alter gp120 binding and increases fusion events with target cells expressing HIV pHXB2 envelope. ( A ) Binding of X4-gp120 to target cells expressing CD4 and CXCR4 or CD4 and CXCR4 R334X analyzed by flow cytometry. Cells were incubated with 0.3 mg/mL of X4-gp120 at 37 °C for 30 min. Data show MFI (arbitrary units, a.u.) mean ± SD; (n=2). Statistical significance was determined using Student’s t-test (n.s.=not significant). ( B ) Cell-cell fusion between JKHXBc2-expressing HIV-1 envelope and different target cells (JKCD4 + CXCR4 + , JKCD4 + CXCR4 - , and JKCD4 + CXCR4 R334X ). Prior to co-culture, each cell type was loaded with the corresponding cell-tracker. Data show the percentage of fusion events ± SD (n=6). We used as reference the fusions events detected in JKCD4 + CXCR4 + cells (100%). Statistical significance was determined by one-way-ANOVA (*p<0.05, ****p≤0.0001). ( C ) Representative biparametric histograms from cells in B showing CMAC versus orange fluorophores. ( D ) Human PBMCs isolated from a WHIM patient (WHIM) and three healthy donors (HD1-3) in two independent experiments were infected with X4-pseudotyped HIV-1 NL4-3 (MOI: 0.001). At 2 hr post infection (p.i.), supernatant samples were obtained at different time points (days post-infection) and p24 levels (pg/mL) in each sample were determined using a commercial ELISA. Results show mean ± SD (n=2).

Article Snippet: The following antibodies were used: anti-human CXCR4 monoclonal antibody (mAb; clone 44717) and phycoerythrin-conjugated anti-human CXCR4 mAb (clone 12G5; both from R&D Systems, Minneapolis, MN); goat F(ab’)2 anti-mouse IgG-PE (Southern Biotech, Birmingham, AL); anti-human CD4 mAb (clone OKT4; Biolegend, San Diego, CA); anti-histidine mAb (clone AD1.1.10; R&D Systems); rabbit anti-gp120 IIIb Ab ( ); rabbit anti-Gag p24 HIV-1 mAb (R&D Systems); and anti-phospho-AKT mAb (S473; #4060), anti-phospho-ERK1,2 mAb (T202/Y204; #9191), and anti-phospho-Lck mAb (Y505; #2751; all from Cell Signaling Technology, Danvers, MA); anti-tubulin mAb conjugated with rhodamine (Bio-Rad, Hercules, CA); phalloidin-TRITC (#P1951, Sigma-Merck, St Louis, MO); anti-ICAM 3 mAb (clone HP2/19) kindly donated by Dr. Francisco Sánchez Madrid (Instituto Sanitario Hospital Universitario La Princesa); goat anti-mouse-AF488 Ab (Thermo Fisher Scientific); anti-human gp120 mAb Fab fragments (clone 2G12; Polymun Scientific, Vienna, Austria); anti-human IgG Fab fragments (Jackson ImmunoResearch, West Grove, PA) conjugated to Abberior STAR RED (Abberior GmbH, Gottingen, Germany), kindly donated by Dr. Jakub Chojnacki (Germans Trias i Pujol Research Institute (IGTP)); anti-p24 HIV-1 (clone 37G12; Polymun Scientific) conjugated with Abberior STAR ORANGE.

Techniques: Binding Assay, Expressing, Flow Cytometry, Incubation, Co-Culture Assay, Isolation, Infection, Enzyme-linked Immunosorbent Assay