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

Journal: Nucleic Acids Research

Article Title: A nanopore-based HIV-1 reference epitranscriptome

doi: 10.1093/nar/gkag220

Figure Lengend Snippet: Probing and correction of HIV-1 base modifications called by nanopore dRNA-seq. ( A ) Nanopore modification-calling results for HIV-1 viral RNA extracted from Jurkat cell cultures treated with STM2457, a drug that inhibits METTL3 m 6 A modification activity. The position of each high frequency m 6 A nucleotide in the HIV-1 genome is indicated on the x -axis. ( B ) Comparison of modifications at DRACH motif sites where m 6 A was called at high frequency. The nucleotide position of the m 6 A modification within the DRACH motif is indicated on the x -axis. Upper plot: comparison of Jurkat cell samples infected with HIV-1 without (back row) or with (front row) 30 μM STM2457 treatment. Lower graph: comparison of Jurkat cell sample infected with HIV-1 before (back row) and after (front row) baseline correction. Comparison of modification calling between NL4-3 from Jurkat cells ( C ) and two synthetic HIV-1 RNA fragments, one unmodified ( D ) and one bearing m 6 A ( E) at two DRACH motifs. The nucleotide position corresponding to the NL4-3 genome is indicated on the x -axis. All modifications called in panel (D) are considered incorrect while modifications called in panel (E), besides m 6 A at position 8975 and 8989, are considered incorrect.

Article Snippet: For replicative NL4-3, we concentrated the supernatants over a 20% sucrose cushion at 5600 × g overnight in 15 ml tubes. p24 ELISA was performed to quantify the viral preparations (R&D Systems, HIV-1 Gag p24 Quantikine ELISA Kit).

Techniques: Modification, Activity Assay, Comparison, Infection

Journal: Nucleic Acids Research

Article Title: A nanopore-based HIV-1 reference epitranscriptome

doi: 10.1093/nar/gkag220

Figure Lengend Snippet: Nanopore-based modification calling of the most reliable, baseline-subtracted HIV-1 RNA modifications on viral RNA from Jurkat cells. The HIV-1 genome architecture is illustrated above. Modifications are shown if, after baseline correction, the average modification frequency was at least 10%. ( A ) m 6 A (blue), ( B ) m 5 C (green), ( C ) pseudouridine (psi) (yellow), ( D ) inosine (purple), and ( E ) 2′- O -methylation (orange). Inset in panel (A) is a close-up of the 3′ end of the NL4-3 HIV-1 genome where m 6 A is most densely called. Results are the average of three separate biological replicates. Error bars are standard deviation.

Article Snippet: For replicative NL4-3, we concentrated the supernatants over a 20% sucrose cushion at 5600 × g overnight in 15 ml tubes. p24 ELISA was performed to quantify the viral preparations (R&D Systems, HIV-1 Gag p24 Quantikine ELISA Kit).

Techniques: Modification, Methylation, Standard Deviation

Journal: Nucleic Acids Research

Article Title: A nanopore-based HIV-1 reference epitranscriptome

doi: 10.1093/nar/gkag220

Figure Lengend Snippet: Differential modification frequencies in HIV-1 RNA splice isoforms. Comparison of modification frequency in each splice isoform as compared to the full-length (unspliced) isoform. X -axis represents individual nucleotides in the HIV-1 genome. Y -axis represents percentage of reads that contained the mutation of interest for that nucleotide, relative to the unspliced form. Bar color indicates modification type: m 6 A (blue), m 5 C (green), pseudouridine (yellow), inosine (purple), and 2′- O -methyl (orange). Only modifications with a difference of 10% from the unspliced RNA in at least one isoform are shown. Shaded background shows portion of the full transcript retained in the spliced isoform. Splice isoforms are subsets of direct-RNA sequencing sample 7C. The A6 splice site is exclusive to the HXB2 genome and therefore not indicated here.

Article Snippet: For replicative NL4-3, we concentrated the supernatants over a 20% sucrose cushion at 5600 × g overnight in 15 ml tubes. p24 ELISA was performed to quantify the viral preparations (R&D Systems, HIV-1 Gag p24 Quantikine ELISA Kit).

Techniques: Modification, Comparison, Mutagenesis, RNA Sequencing

Journal: Nucleic Acids Research

Article Title: A nanopore-based HIV-1 reference epitranscriptome

doi: 10.1093/nar/gkag220

Figure Lengend Snippet: A preliminary HIV-1 antisense epitranscriptome, the effect of cART and cell type on HIV-1 modification calling, and conservation of m 6 A in HIV-1 genomes from PLWH. (A) Nanopore modification-calling for HIV-1 antisense RNA from Jurkat cells. m 6 A (blue), m 5 C (green), pseudouridine (yellow), inosine (purple), and 2′- O -methylation (orange). Inset shows a close-up of the asp gene. (B) Nanopore modification-calling results for HIV-1 RNA taken from Jurkat cells treated without (darker color) or with (lighter color) cART treatment. Error bars represent the standard deviation from three separate biological replicates. (C) Nanopore modification-calling results for HIV-1 RNA taken from Jurkat cells, primary CD4+ T cells infected in vitro , supernatant of primary CD4+ T cells infected in vitro , and CD4+ T cells from PLWH samples. In each nucleotide position cluster, the first bar is Jurkat cell samples infected with HIV-1, the second bar is CD4+ T cells from healthy donors and infected with HIV-1, the third bar is the supernatant from the CD4+ T cells from healthy donors, and the fourth, fifth, and sixth bars are samples taken from CD4+ T cells from PLWH donors. (D) Top: comparison of m 6 A modifications called from HIV-1 RNA from Jurkat cells against preservation of the DRACH motifs for these m 6 A modifications sequenced from three PLWH samples. Bottom: analysis of conservation of known m 6 A modification sites in a larger dataset of HIV-1 mutations. Positions containing m 6 A between nucleotide positions 8000 and 9171 were identified and the average and median Shannon entropy for these positions are reported.

Article Snippet: For replicative NL4-3, we concentrated the supernatants over a 20% sucrose cushion at 5600 × g overnight in 15 ml tubes. p24 ELISA was performed to quantify the viral preparations (R&D Systems, HIV-1 Gag p24 Quantikine ELISA Kit).

Techniques: Modification, Methylation, Standard Deviation, Infection, In Vitro, Comparison, Preserving