Journal: bioRxiv

Article Title: Dengue Virus NS1 Binds Ephrin B1 to Trigger Endothelial Dysfunction

doi: 10.1101/2025.11.19.689067

Figure Lengend Snippet: Western blot of Recombinant HA-tagged proteins using anti-HA antibodies. b-c , Sialic acid staining on the surface of HPMEC treated with the indicated recombinant proteins. Cells were treated, fixed and stained for sialic acid using wheat germ agglutinin (gold) and DAPI (blue). Representative micrographs are shown in b (scale bar, 100 μm) and fluorescence intensity quantification in c as the mean ± SEM (n=4). d , Endothelial permeability of a monolayer of treated with the indicated proteins. HPMEC were seeded in Transwells, treated and TEER measured at 0, 6 and 24 hours post treatment. Relative TEER at 6 hours is shown as mean ± SEM (n=4). e , Schematic of immunoprecipitation of HA- tagged NS1 from HPMEC lysates coupled to mass spectrometry (LC-MS/MS) bottom-up proteomics approach. f , Intensity-based absolute quantification (iBAQ) of selected host factors in each sample is illustrated as a heat map, with colors as indicated below the panel. Host proteins that were not identified (n.i.) in samples are shown in grey. g , Statistical analysis of host proteins in the DENV NS1 WT-treated sample compared to the non-phenotypic (DENV NS1 N207Q and WNV NS1) and negative ( Gaussia luciferase, Gluc) controls. Host proteins with a fold-change of log2>2 are shown in taupe and those with an enrichment of log2>2.5 are shown in ochre. EFNB1 is indicated in blue. Statistical comparisons were performed by ordinary one-way ANOVA, with ** P < 0.01, and *** P < 0.001.

Article Snippet: Recombinant DENV1-4, ZIKV, and WNV NS1 proteins, as well as the DENV2 NS1 N207Q mutant protein, were commercially acquired from Native Antigen Co. (United Kingdom).

Techniques: Western Blot, Recombinant, Staining, Fluorescence, Permeability, Immunoprecipitation, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Quantitative Proteomics, Luciferase

Journal: bioRxiv

Article Title: Dengue Virus NS1 Binds Ephrin B1 to Trigger Endothelial Dysfunction

doi: 10.1101/2025.11.19.689067

Figure Lengend Snippet: a, Western blot of EFNB1 knock-out (KO) cell lines with non-targeting guide (NTG) controls stained with mAbs against EFNB1 or β-actin. b-c , Endothelial permeability of NTG and EFNB1 KO cells treated with NS1. Relative TEER ( b ) and the area under the curve of the negative peaks ( c ) are shown as the mean ± SEM (n≥3). d , Endothelial permeability of NTG and EFNB1 KO cells treated with TNF-α and Crimean Congo hemorrhagic fever virus glycoprotein 38 (GP38). The area under the curve of the negative peaks is shown as the mean ± SEM (n≥3). e-g , Staining of EGL components of NTG control and EFNB1 treated with NS1. Cells were fixed and stained for sialic acid (gold, e-f ) or heparan sulfate (cyan, e and g ) for immunofluorescence assay (scale bar, 100 μm). Quantification of the mean fluorescence intensity is shown in f and h as the mean ± SEM (n≥3). Statistical comparisons were performed by ordinary one-way ANOVA, with * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Recombinant DENV1-4, ZIKV, and WNV NS1 proteins, as well as the DENV2 NS1 N207Q mutant protein, were commercially acquired from Native Antigen Co. (United Kingdom).

Techniques: Western Blot, Knock-Out, Staining, Permeability, Virus, Control, Immunofluorescence, Fluorescence

Journal: bioRxiv

Article Title: Dengue Virus NS1 Binds Ephrin B1 to Trigger Endothelial Dysfunction

doi: 10.1101/2025.11.19.689067

Figure Lengend Snippet: a, Schematic of the EFNB1 protein at the plasma membrane showing the extracellular RBD and the intracellular PDZ-binding motif, with phosphorylation sites indicated by “P”. b , Western blot of lysates of EFNB1 KO cells either untreated or transduced with an expression construct encoding EFNB1 WT or mutants (all Y to F, Y343/344E, Y343/344F). Proteins were detected with an anti-EFNB1 antibody and an anti-FLAG-tag antibody. c , Western blot of samples from HPMECs treated with the indicated proteins. After treatment for 30 min, FLAG-tagged EFNB1 was isolated by immunoprecipitation. Proteins were detected using a mAb binding to EFNB1 phosphorylated at Y234/329 and an EFNB1- specific mAb. d-f , Staining of EGL components of EFNB1-reconstituted cell lines treated with NS1. Cells were fixed after 6 h and stained for sialic acid (gold) and heparan sulfate (cyan). Representative images are shown in d (scale bar, 100 μm) and the quantification of sialic acid and heparan sulfate staining is shown in e and f as the mean ± SEM (n≥3). g , Endothelial permeability of EFNB1- complemented cell lines treated with NS1. TEER was measured over a time course of 24 h and the area under the curve of the negative peaks was quantified as a proxy of hyperpermeability and shown as the mean ± SEM (n≥3). Statistical comparisons were performed by ordinary one-way ANOVA, with * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Recombinant DENV1-4, ZIKV, and WNV NS1 proteins, as well as the DENV2 NS1 N207Q mutant protein, were commercially acquired from Native Antigen Co. (United Kingdom).

Techniques: Clinical Proteomics, Membrane, Binding Assay, Phospho-proteomics, Western Blot, Transduction, Expressing, Construct, FLAG-tag, Isolation, Immunoprecipitation, Staining, Permeability

Journal: bioRxiv

Article Title: Dengue Virus NS1 Binds Ephrin B1 to Trigger Endothelial Dysfunction

doi: 10.1101/2025.11.19.689067

Figure Lengend Snippet: a-j, Relative binding of EFNB1 Fc over Gluc Fc to flavivirus NS1 ( a-b ), DENV1-4 NS1 ( c ), DENV NS1 WT and N207Q ( d-e ), individual domains of DENV2 NS1 ( f-g ), or chimeric DENV-WNV NS1 ( i-j ). Luminex beads were coupled to the different antigens, and a dilution series of six 4-fold dilutions of the EFNB1 RBD Fc or the Gluc Fc fusion proteins was added. Binding was detected using fluorescently conjugated anti-mouse Fc antibodies, and the ratio of EFNB1 Fc MFI over the Gluc Fc MFI was plotted either as a curve with the mean ± SEM ( a, d, f, i ) or as a single point dilution at the peak of the curve with the mean as a bar ( b, c, e, g, j ) (n=3 technical replicates). Statistical comparisons were performed by t-test or ordinary one-way ANOVA, with * P < 0.05, ** P < 0.01 and *** P < 0.001. h , Schematic of the design of the chimeric NS1 proteins based on a WNV NS1 backbone (tan) with domain swaps of DENV2 NS1 (blue). k-l , Boltz2 structure prediction of the EFNB1 RBD (blue, UniProt: P98172) binding to DENV2 NS1 (tan, UniProt: P29990) as a cartoon representation. k , Dimeric NS1 shown from the bottom with an inset of the wing domain interacting with EFNB1. The major interacting residues and their distances are annotated. l , Dimeric NS1 shown from the top with an inset of the β-ladder domain interacting with the GH loop on EFNB1. The major interacting residues and their distances are annotated.

Article Snippet: Recombinant DENV1-4, ZIKV, and WNV NS1 proteins, as well as the DENV2 NS1 N207Q mutant protein, were commercially acquired from Native Antigen Co. (United Kingdom).

Techniques: Binding Assay, Luminex

Journal: bioRxiv

Article Title: Dengue Virus NS1 Binds Ephrin B1 to Trigger Endothelial Dysfunction

doi: 10.1101/2025.11.19.689067

Figure Lengend Snippet: a, Schematic of the tested approaches to target EFNB1. b , Endothelial permeability of HPMEC treated with NS1 in presence of decreasing concentrations of the PKC inhibitor Go 6983. The area under the curve of the negative peaks was calculated and plotted against the inhibitor concentration in nM as the mean ± SEM (n≥2). c , Endothelial permeability of HPMEC treated with NS1 or with a combination of NS1 and Fc fusion proteins. NS1 was complexed with EFNB1 RBD Fc fusion proteins or as a negative control with the Gluc Fc fusion protein and TEER was measured. The area under the curve of the negative peaks was quantified as the mean ± SEM (n≥3). d-e , Vascular leak in the mouse dermis. NS1 was complexed with the EFNB1 RBD Fc fusion proteins and the complex or the individual proteins were injected intradermally into the shaved skin of C57BL/6 mice. PBS was used as a negative control. The tracer dye Dextran-680 was simultaneously injected intravenously, and dye extravasation was visualized using a LiCor Scanner after extraction of the skin. A representative scan is shown in d and the quantification of the mean fluorescent intensity (MFI) of the extravasated tracer dye is shown in e as the mean ± SEM (n=5). Statistical comparisons were performed by ordinary one-way ANOVA, with * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Recombinant DENV1-4, ZIKV, and WNV NS1 proteins, as well as the DENV2 NS1 N207Q mutant protein, were commercially acquired from Native Antigen Co. (United Kingdom).

Techniques: Permeability, Concentration Assay, Negative Control, Injection, Extraction