Journal: The Journal of General Virology

Article Title: Mutations in the G–H loop region of ephrin-B2 can enhance Nipah virus binding and infection

doi: 10.1099/vir.0.033787-0

Figure Lengend Snippet: NiV glycoprotein pseudotyped virus infection of HeLa-USU cells expressing wild-type (wt) or mutant ephrin-B2. NiV-G and NiV-F glycoproteins were pseudotyped onto a human immunodeficiency virus (HIV)-1 based core virus using the NL4-3-Luc-E−R+ backbone (NiVpp) and used to infect HeLa-USU cells transfected with either wt or mutant ephrin-B2. Entry efficiency of NiVpp was measured by luciferase reporter-gene assay and is expressed as a percentage of wt ephrin-B2 (100 %). The enhanced infectivity of cells expressing some of the mutant ephrin-B2 molecules is highlighted by using a split y-axis. Data shown are the mean of triplicates±sd and one representative experiment of three is shown. (a) Schematic of ephrin-B2. Secondary structure elements are shown with an arrow (β-sheet) and black box (α-helix). The asterisks indicate residues that were targeted by alanine-scanning mutagenesis. (b) Mutations located within the G–H loop of ephrin-B2. (c) Mutations located outside the G–H loop of ephrin-B2.

Article Snippet: For Western blotting, incubation with different antibodies was done at 37 °C for 1 h. Expression of ephrin-B2 mutants in HeLa-USU cells was detected using a goat anti-mouse ephrin-B2 antibody as a primary antibody (R&D Systems), followed by incubation with HRP-conjugated donkey anti-goat IgG (1 : 20000) (PTGLab).

Techniques: Infection, Expressing, Mutagenesis, Transfection, Luciferase, Reporter Gene Assay

Journal: The Journal of General Virology

Article Title: Mutations in the G–H loop region of ephrin-B2 can enhance Nipah virus binding and infection

doi: 10.1099/vir.0.033787-0

Figure Lengend Snippet: Summary of ephrin-B2 substitution mutants Class I, Critical for Eph and ephrin-B2/B3 binding; class II, critical for Nipah-G and ephrin-B2 binding; class III, conservation in ephrin-B2/B3, but not in ephrin-B1 or unique sites of ephrin-B2. The expression levels of ephrin-B2 and its mutants are presented as: +++, high; ++, medium; +, low.

Article Snippet: For Western blotting, incubation with different antibodies was done at 37 °C for 1 h. Expression of ephrin-B2 mutants in HeLa-USU cells was detected using a goat anti-mouse ephrin-B2 antibody as a primary antibody (R&D Systems), followed by incubation with HRP-conjugated donkey anti-goat IgG (1 : 20000) (PTGLab).

Techniques: Binding Assay, Expressing

Journal: The Journal of General Virology

Article Title: Mutations in the G–H loop region of ephrin-B2 can enhance Nipah virus binding and infection

doi: 10.1099/vir.0.033787-0

Figure Lengend Snippet: Western blot analysis of ephrin-B2 expression. (a) Equal amounts of a plasmid encoding either the wt or a mutant ephrin-B2 gene were transfected into equivalent cultures of HeLa-USU cells. Following expression for 48 h, cells were lysed and equal amounts (volume in µl) of the various lysates were resolved by using SDS-PAGE and transferred onto a PVDF membrane. Duplicate membranes were probed with goat anti-mouse ephrin-B2 (top) or mouse anti-human β-actin (bottom), respectively. (b) Quantification of the expression of the wt and mutant ephrin-B2. The bands in (a) were quantified by densitometry using the ImageJ program (version 1.44) by using values obtained from densitometric measurements of the bands. The values of the ephrin-B2 mutations are expressed as a percentage of wt ephrin-B2 (100 %). The data were normalized with the corresponding values for β-actin.

Article Snippet: For Western blotting, incubation with different antibodies was done at 37 °C for 1 h. Expression of ephrin-B2 mutants in HeLa-USU cells was detected using a goat anti-mouse ephrin-B2 antibody as a primary antibody (R&D Systems), followed by incubation with HRP-conjugated donkey anti-goat IgG (1 : 20000) (PTGLab).

Techniques: Western Blot, Expressing, Plasmid Preparation, Mutagenesis, Transfection, SDS Page

Journal: The Journal of General Virology

Article Title: Mutations in the G–H loop region of ephrin-B2 can enhance Nipah virus binding and infection

doi: 10.1099/vir.0.033787-0

Figure Lengend Snippet: Immunofluorescence detection and flow cytometry of ephrin-B2 expression on cell surface. HeLa-USU cells were transfected with the indicated expression plasmids. Following expression for 24 h, cells were incubated with goat anti-ephrin-B2 antibody, followed by probing with FITC-conjugated donkey anti-goat IgG. The surface expression of wt or mutant ephrin-B2 was detected by confocal immunofluorescence microscopy (a) and quantified by flow cytometry (b). (a) The expression plasmids are shown at the left side of the panels. The columns (from left to right) represent staining of expressed ephrin-B2 (FITC, in green), cell nuclei (Hoechst 33258, in blue) and the superimposed images of the two. (b) Surface expression of wt or mutant ephrin-B2 was quantified by flow cytometry. The data are shown as the mean fluorescence intensity of the transfected cells indicated.

Article Snippet: For Western blotting, incubation with different antibodies was done at 37 °C for 1 h. Expression of ephrin-B2 mutants in HeLa-USU cells was detected using a goat anti-mouse ephrin-B2 antibody as a primary antibody (R&D Systems), followed by incubation with HRP-conjugated donkey anti-goat IgG (1 : 20000) (PTGLab).

Techniques: Immunofluorescence, Flow Cytometry, Expressing, Transfection, Incubation, Mutagenesis, Microscopy, Staining, Fluorescence

Journal: The Journal of General Virology

Article Title: Mutations in the G–H loop region of ephrin-B2 can enhance Nipah virus binding and infection

doi: 10.1099/vir.0.033787-0

Figure Lengend Snippet: Kinetic analysis of NiV-glycoprotein pseudovirus entry into HeLa-USU cells expressing wt or mutant L124A ephrin-B2. (a) Various pseudovirus preparations were adsorbed to ephrin-B2-expressing cells at 4, 25 or 37 °C for 1 h. The inoculum was removed and cells were washed with PBS and cultured with fresh media at 37 °C. Entry efficiency of the NiVpp at the different incubation temperatures was measured by luciferase reporter-gene assay at 48 h post-infection (p.i.) and is expressed as the percentage of wt ephrin-B2 at the corresponding temperature (100 %). (b) Pseudotyped viruses were adsorbed to ephrin-B2-expressing cells at 37 °C for different time intervals, and entry efficiency of the NiVpp was measured by luciferase reporter-gene assay at 48 h p.i. and is expressed as the percentage of wt ephrin-B2 at the corresponding time point (100 %). Error bars indicate sd.

Article Snippet: For Western blotting, incubation with different antibodies was done at 37 °C for 1 h. Expression of ephrin-B2 mutants in HeLa-USU cells was detected using a goat anti-mouse ephrin-B2 antibody as a primary antibody (R&D Systems), followed by incubation with HRP-conjugated donkey anti-goat IgG (1 : 20000) (PTGLab).

Techniques: Expressing, Mutagenesis, Cell Culture, Incubation, Luciferase, Reporter Gene Assay, Infection

Journal: The Journal of General Virology

Article Title: Mutations in the G–H loop region of ephrin-B2 can enhance Nipah virus binding and infection

doi: 10.1099/vir.0.033787-0

Figure Lengend Snippet: Binding kinetic analysis of NiV-G to wt or mutant L124A ephrin-B2 by bio-layer interferometry. (a) Real-time binding analysis of NiV-G to the wt or mutant L124A ephrin-B2. Bio-layer interferometry was used to measure the binding kinetics of soluble NiV-G to both ephrin-B2 (wt) and its mutant (L124A) in response units (nm). Biotinylated soluble NiV glycoprotein G (sNiV-G) was immobilized to streptavidin-coated biosensors, and the binding of wt and mutant ephrin-B2 was assessed at the indicated concentrations. (b) Steady-state analysis of real-time binding data obtained with the Octet Red system. The plots in each panel show response versus protein concentration curves derived from the raw binding data. One representative experiment of two is shown.

Article Snippet: For Western blotting, incubation with different antibodies was done at 37 °C for 1 h. Expression of ephrin-B2 mutants in HeLa-USU cells was detected using a goat anti-mouse ephrin-B2 antibody as a primary antibody (R&D Systems), followed by incubation with HRP-conjugated donkey anti-goat IgG (1 : 20000) (PTGLab).

Techniques: Binding Assay, Mutagenesis, Protein Concentration, Derivative Assay

Journal: The Journal of General Virology

Article Title: Mutations in the G–H loop region of ephrin-B2 can enhance Nipah virus binding and infection

doi: 10.1099/vir.0.033787-0

Figure Lengend Snippet: NiV-glycoprotein-mediated cell-to-cell fusion in HeLa-USU cells transfected with wt or L124A mutant ephrin-B2. (a) HeLa-USU cells were co-transfected with plasmids encoding NiV-F, NiV-G and the indicated ephrin-B2 genes. Cells were fixed 24h post-transfection and nuclei were visualized by Hoechst 33258 staining. Twenty randomly selected syncytia were photographed. (b) Syncytium formation in HeLa-USU cells expressing wt or mutant ephrin-B2 was measured by counting the mean number of nuclei per syncytium in 20 randomly selected syncytia. Data shown are the means of 20 syncytia±sem. Statistical analysis was performed by using Student’s t-tests with spss (version 13.0). Results where P<0.05 were considered to be significant.

Article Snippet: For Western blotting, incubation with different antibodies was done at 37 °C for 1 h. Expression of ephrin-B2 mutants in HeLa-USU cells was detected using a goat anti-mouse ephrin-B2 antibody as a primary antibody (R&D Systems), followed by incubation with HRP-conjugated donkey anti-goat IgG (1 : 20000) (PTGLab).

Techniques: Transfection, Mutagenesis, Staining, Expressing

Journal: The Journal of General Virology

Article Title: Mutations in the G–H loop region of ephrin-B2 can enhance Nipah virus binding and infection

doi: 10.1099/vir.0.033787-0

Figure Lengend Snippet: Infection by NiV or HeV in HeLa-USU cells stably expressing wt (USU-hB2) or L124A mutant (USU-hB2 m) ephrin-B2. Cells were seeded at 20 000 cells per well 1 day before infection. Cells were infected with 500 TCID50 of NiV (a) or HeV (b) in 50 µl. Virus and cells were incubated for the time indicated on the x-axis and then virus was washed off by washing five times with PBS. DMEM+10 % FCS was added to wells and the cells were incubated for 24 h. Cells were then fixed and infected cells visualized by immunofluorescence by using polyclonal rabbit anti-HeV P antisera. Infected cell foci were counted microscopically (n = 4). Statistical analysis was performed by using Student’s t-tests with spss.

Article Snippet: For Western blotting, incubation with different antibodies was done at 37 °C for 1 h. Expression of ephrin-B2 mutants in HeLa-USU cells was detected using a goat anti-mouse ephrin-B2 antibody as a primary antibody (R&D Systems), followed by incubation with HRP-conjugated donkey anti-goat IgG (1 : 20000) (PTGLab).

Techniques: Infection, Stable Transfection, Expressing, Mutagenesis, Incubation, Immunofluorescence

Journal:

Article Title: The Notch target genes Hey1 and Hey2 are required for embryonic vascular development

doi: 10.1101/gad.291004

Figure Lengend Snippet: Lack of vascular ephrin-B2, CD44, and neuropilin1 expression in Notch1 and Hey1/2 KO embryos. Expression of the arterial endothelial markers ephrin-B2 (A–C), CD44 (D–F), and neuropilin1 (G–I) in wild-type (A,D,G), Notch1–/– (B,E,H), and Hey1/2 DKO (C,F,I) embryos at E9.5. Immunohistochemistry of transverse sections reveals staining of both aortae (arrows) in control embryos, whereas the cardinal veins (arrowheads) are negative. Similar sections of the mutant embryos still exhibit cardiac CD44 and neuropilin1 expression as well as ephrin-B2 staining in the neural tube, but the aortae are clearly not stained. (h) Heart; (nt) neural tube. Enlarged views of an aortic vessel for each case are shown in insets.

Article Snippet: Ephrin-B2 antibody (R&D Systems) staining was done according to Batlle et al. ( 2002 ).

Techniques: Expressing, Immunohistochemistry, Staining, Mutagenesis

Journal: PLoS Pathogens

Article Title: The Mouse IAPE Endogenous Retrovirus Can Infect Cells through Any of the Five GPI-Anchored EphrinA Proteins

doi: 10.1371/journal.ppat.1002309

Figure Lengend Snippet: A. Scheme of the screening strategy to identify the protein used as a receptor by the IAPE Env. More technical details are provided in the main text and in the section. LV: lentiviral vector. B. Scheme representing the cellular localisation of the proteins encoded by the two candidate receptor genes. TMEM9 is an endosomal protein, whereas Ephrin A4 (encoded by the EFNA4 gene) is expressed at the cell membrane where it can interact with extracellular molecules, including the EphA proteins. C. Quantification of the mRNA level of the two genes identified as potential receptors for the IAPE elements. The amount of the corresponding mRNAs was measured by qRT-PCR performed on total RNA extracted from the parental Vero cells as a control, or the five clones found to be the most sensitive to infection by IAPE pseudotypes (re-numbered from 1 to 5 in this panel). Clones 1,4 and 5 contained an EFNA4 cDNA, and clones number 2 and 3 a TMEM9 cDNA. The mRNA levels were normalised using the RPLO gene as a reference, and the parental Vero cell line level was set at 1 for both genes.

Article Snippet: Staining of specific Ephrin A proteins was done using either a goat anti mouse Ephrin A2 antibody (R&D systems) or a rabbit anti Ephrin A5 antibody (Novus Biologicals), following the recommendations provided.

Techniques: Plasmid Preparation, Membrane, Quantitative RT-PCR, Control, Clone Assay, Infection

Journal: PLoS Pathogens

Article Title: The Mouse IAPE Endogenous Retrovirus Can Infect Cells through Any of the Five GPI-Anchored EphrinA Proteins

doi: 10.1371/journal.ppat.1002309

Figure Lengend Snippet: (A) WOP cells transduced with the EFNA4 gene or a control gene were stained with the soluble His-tagged IAPE envelope SU subunit (or that of syncytin1 (syn1) as a control) followed by an Alexa488 anti-His antibody and subjected to FACS analysis. Only the cells transduced with EFNA4 bind the IAPE SU protein, and none of the cells were stained with the control syncytin1-SU. The expression level of Ephrin A proteins in the two populations was checked using the EphA2-Fc soluble protein (that can bind all Ephrin A proteins) and are shown in the small panels on the right. The data presented correspond to one representative experiment out of three. (B) 293T cells were transiently transfected with an expression vector for IAPE Env, or Ampho MLV Env as a control. At day 2 post transfection, cells were stained with a soluble Ephrin A4-Fc fusion protein, or a control Fc protein, followed by an Alexa 488 fluorescent anti-Fc antibody before being subjected to FACS analysis. Only the cells expressing the IAPE Env bind to the recombinant Ephrin A4 protein, The data presented correspond to one representative experiment out of three. Expression of the two envelope proteins in the transfected cells was checked by Western blot using specific antibodies, as shown on the right. (C) The soluble recombinant IAPE or syn1 His-tagged SU proteins were tested for interaction with Ephrin A4-Fc (or Fc-only and EphA2 controls) in a pull down assay as schematised on the left. Pellets were analysed by Western blot using an antibody directed against the His tag (upper part). The only interaction detected is between Ephrin A4-Fc and IAPE SU. We ensured that the 2 His-tagged SU proteins were produced in similar amount, as shown on the right (Before IP panel) and that all 3 Fc-tagged proteins were efficiently pulled down by the protein A-agarose beads and recovered in similar amount in the pellet, as shown on the lower panel (Western blot performed with an anti mouse IgG antibody).

Article Snippet: Staining of specific Ephrin A proteins was done using either a goat anti mouse Ephrin A2 antibody (R&D systems) or a rabbit anti Ephrin A5 antibody (Novus Biologicals), following the recommendations provided.

Techniques: Transduction, Control, Staining, Expressing, Transfection, Plasmid Preparation, Recombinant, Western Blot, Pull Down Assay, Produced

Journal: PLoS Pathogens

Article Title: The Mouse IAPE Endogenous Retrovirus Can Infect Cells through Any of the Five GPI-Anchored EphrinA Proteins

doi: 10.1371/journal.ppat.1002309

Figure Lengend Snippet: (A) Comparison of the efficacy of human (Hs), mouse (Mm) and rat (Rn) Ephrin A4 proteins as receptors for IAPE Env pseudotypes. WOP cells were transduced with a lentiviral vector encoding one of the 3 versions of EFNA4, or a control gene. Three days later, they were challenged with GFP-marked lentiviral particles pseudotyped with the IAPE Env, the Friend ecotropic Env or no Env (none). Viral titres were measured by FACS three days post infection. The results (mean titre ± standard deviation) are from 5 independent experiments. (B) Test of the mouse Ephrin A and Ephrin B proteins for their activity as receptors for the IAPE Env pseudotypes. WOP cells were transduced with lentiviral vectors containing the different EFNA/B cDNA or a control gene, and tested for their ability to be infected with IAPE Env pseudotypes as described in (A). Results (mean ± standard deviation calculated from 3–4 independent experiments) are expressed as the percentage of the titre measured for each gene as compared to that obtained with the human EFNA4 (Hs_EFNA4, shown on the right). Asteriks indicate values significantly different (p<0.05) from that obtained with the untreated cells (unpaired Student test).

Article Snippet: Staining of specific Ephrin A proteins was done using either a goat anti mouse Ephrin A2 antibody (R&D systems) or a rabbit anti Ephrin A5 antibody (Novus Biologicals), following the recommendations provided.

Techniques: Comparison, Transduction, Plasmid Preparation, Control, Infection, Standard Deviation, Activity Assay

Journal: PLoS Pathogens

Article Title: The Mouse IAPE Endogenous Retrovirus Can Infect Cells through Any of the Five GPI-Anchored EphrinA Proteins

doi: 10.1371/journal.ppat.1002309

Figure Lengend Snippet: Ephrin A proteins in these serial cryosections were labelled using a commercial soluble EphA7-Fc tagged protein (a soluble form of EphA7 that can bind all-Ephrin A proteins) (B and D), or a Fc-only control protein (A and C). A and B show high magnifications of a Graäfian follicle with the oocyte (filled arrowheads) surrounded by follicle cells (open arrowheads). C and D show cross-sections of seminiferous tubules, with spermatozoa in the central lumen. Filled arrowheads point to stained germinal cells located apart from the periphery of the tubule (inset: higher magnification of stained cells).

Article Snippet: Staining of specific Ephrin A proteins was done using either a goat anti mouse Ephrin A2 antibody (R&D systems) or a rabbit anti Ephrin A5 antibody (Novus Biologicals), following the recommendations provided.

Techniques: Control, Staining

Journal: PLoS ONE

Article Title: Pericardial Patch Angioplasty Heals via an Ephrin-B2 and CD34 Positive Cell Mediated Mechanism

doi: 10.1371/journal.pone.0038844

Figure Lengend Snippet: Scale bar, 10 microns, except 40 microns for subcutaneous histology. n = 3; representative pictures are shown. Immunofluorescence shows CD34 and Ephrin-B2-positive cells colocalize in the middle of the bovine arterial patch, day 7. Photomicrographs of immunofluorescence: blue color, DAPI; green color, CD34; red color, Ephrin-B2; merge, green + red. n = 3; representative pictures are shown. Scale bar, 10 microns. Similarly, merge of CD34 and VEGFR2 is shown. Bar graphs show increased expression of Ephrin-B2 and CD34 mRNA and protein in arterial, but not subcutaneous, patches. n = 4−5. Panels A, B, D, and E, mRNA; Panels C and F, protein.

Article Snippet: Tissue sections were de-paraffinized and stained using primary antibodies against CD31 (BD Pharmingen, 550300), CD34 (R&D, AF4117) Ephrin-B2 (Novus, NBP1-48610), or ED-1 (Abcam).

Techniques: Immunofluorescence, Expressing

Journal: PLoS ONE

Article Title: Pericardial Patch Angioplasty Heals via an Ephrin-B2 and CD34 Positive Cell Mediated Mechanism

doi: 10.1371/journal.pone.0038844

Figure Lengend Snippet: 30. A ) Representative photomicrographs of bovine and porcine patches, low (10x) and high (40x) magnification, scale bar 80 and 30 (porcine, 40) microns. Arrow shows the neointima; arrowhead shows the patch; L, lumen. B ) Bar graph of mean neointimal thickness, bovine and porcine patches, day 30. C ) Photomicrographs of immunofluorescence: blue color, DAPI; green color, CD34; red color, Ephrin-B2 or VEGFR2; merge, green + red. n = 2; representative pictures are shown. Scale bar, 10 microns. D ) Western blot of Ephrin-B2 or CD34, in bovine or porcine pericardial patch, day 0 (preimplantation) or day 30; n = 2.

Article Snippet: Tissue sections were de-paraffinized and stained using primary antibodies against CD31 (BD Pharmingen, 550300), CD34 (R&D, AF4117) Ephrin-B2 (Novus, NBP1-48610), or ED-1 (Abcam).

Techniques: Immunofluorescence, Western Blot