Journal: bioRxiv

Article Title: Dengue virus NS1 undergoes partial nuclear translocation to modulate host transcription and support viral replication

doi: 10.64898/2026.04.13.718202

Figure Lengend Snippet: A) BHK-21 cells were infected with DENV-2, fixed at the indicated times post-infection, and processed for confocal microscopy. NS1 (green) and nuclei (DAPI, blue). B) Quantification by two different bioinformatics software, ICY © (upper panel and ImageJ (lower panel), of the amount of NS1 present inside the cell nuclei. Results are expressed as a percentage, taking as 100% the total pool of NS1 present inside the cell (right panels). **/*** p=0.005. There are no significant differences between the two methods

Article Snippet: BHK-21 cells were incubated with commercial recombinant DENV2 NS1 protein (R&D Systems, 9439-DG) diluted in EMEM supplemented with 5% fetal bovine serum to a final concentration of 2 μg/mL.

Techniques: Infection, Confocal Microscopy, Software

Journal: bioRxiv

Article Title: Dengue virus NS1 undergoes partial nuclear translocation to modulate host transcription and support viral replication

doi: 10.64898/2026.04.13.718202

Figure Lengend Snippet: BHK-21 cells were infected with DENV-2, fixed at 24 hpi, and examined using confocal microscopy. A) C olocalization of NS1 with cell structures of interest. B) Labelling of different cell proteins for fraction quality control. Sequential removal of different cell parts was achieved throughout the process based on buffer compositions. DIC, differential interference contrast. Results from one of two independent experiments are shown.

Article Snippet: BHK-21 cells were incubated with commercial recombinant DENV2 NS1 protein (R&D Systems, 9439-DG) diluted in EMEM supplemented with 5% fetal bovine serum to a final concentration of 2 μg/mL.

Techniques: Infection, Confocal Microscopy, Control

Journal: bioRxiv

Article Title: Dengue virus NS1 undergoes partial nuclear translocation to modulate host transcription and support viral replication

doi: 10.64898/2026.04.13.718202

Figure Lengend Snippet: BHK-21 cells were infected with DENV2 or mock - infected, harvested at 24 hpi, and subjected to subcellular fractionation. (A) Localization of DENV2 NS1 by western blot after fully denaturing SDS-PAGE electrophoresis, or (B) after non-denaturing blue native PAGE electrophoresis. (C) Western blot for quality control of each fraction using appropriate protein markers. Results from one of two independent experiments are shown.

Article Snippet: BHK-21 cells were incubated with commercial recombinant DENV2 NS1 protein (R&D Systems, 9439-DG) diluted in EMEM supplemented with 5% fetal bovine serum to a final concentration of 2 μg/mL.

Techniques: Infection, Fractionation, Western Blot, SDS Page, Electrophoresis, Blue Native PAGE, Control

Journal: bioRxiv

Article Title: Dengue virus NS1 undergoes partial nuclear translocation to modulate host transcription and support viral replication

doi: 10.64898/2026.04.13.718202

Figure Lengend Snippet: (A) BHK-21 cells were infected with DENV2 and processed at 24 hpi. Colocalization was analyzed by confocal microscopy, detailing the spatial interaction between NS1 (green) and the nuclear lamina (red). Signal overlap was quantified by Pearson’s (0.162) and Manders (0.324) correlation coefficients. (B) Co-immunoprecipitation assay from BHK-21 cells transfected with a plasmid expressing DENV2 NS1. Cells were collected at 24 hpt and precipitation was performed using NS1 as a bait. Results from one of two independent experiments are shown.

Article Snippet: BHK-21 cells were incubated with commercial recombinant DENV2 NS1 protein (R&D Systems, 9439-DG) diluted in EMEM supplemented with 5% fetal bovine serum to a final concentration of 2 μg/mL.

Techniques: Infection, Confocal Microscopy, Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation, Expressing

Journal: bioRxiv

Article Title: Dengue virus NS1 undergoes partial nuclear translocation to modulate host transcription and support viral replication

doi: 10.64898/2026.04.13.718202

Figure Lengend Snippet: A) Bipartite NLS sequence predicted in DENV-2 NS1 by NLS mapper (score of 5.9, with a total of 1.0 cytoplasmic location and 10.0 nuclear location). B) Sequence alignment of the predicted bipartite NLS across DENV serotypes 1 to 4 and Zika virus. C) Structural models of dimeric (left) and hexameric (right) DENV-2 NS1 generated by Alphafold 3.0 showing the exposed nature of the predicted NLS (highlighted in red and N-glycosylation site (N207) in blue).

Article Snippet: BHK-21 cells were incubated with commercial recombinant DENV2 NS1 protein (R&D Systems, 9439-DG) diluted in EMEM supplemented with 5% fetal bovine serum to a final concentration of 2 μg/mL.

Techniques: Sequencing, Virus, Generated, Glycoproteomics

Journal: bioRxiv

Article Title: Dengue virus NS1 undergoes partial nuclear translocation to modulate host transcription and support viral replication

doi: 10.64898/2026.04.13.718202

Figure Lengend Snippet: A) Mutations introduced in part 1 (Mut1), part 2 (Mut2) or both parts (Mut3) of the bipartite NLS (underlined in red). Residue changes and positions are indicated. B) Evaluation of the amount of NS1 present in the cell nuclei after transfection with the different mutant constructions. Left panels, plasmid expressing GFP with 4 NLS (4-GFP NLS) used as a positive control. Right panels, wild type (pNS1) and the 3 different mutant constructions. Ivermectin treatment of 4-GFP NSL and pNS1-transfected cells was included as an additional positive control. Confluent monolayers of BHK-21 cells were processed for confocal microscopy 24 hpt. Cell nuclei were stained with DAPI (blue), and NS1 was visualized with Alexa488 (green). C) Quantification of the amount of NS1 present inside the cell nuclei, expressed as a percentage of the total signal. For each construction, 30 cells were analyzed. *p<0.05

Article Snippet: BHK-21 cells were incubated with commercial recombinant DENV2 NS1 protein (R&D Systems, 9439-DG) diluted in EMEM supplemented with 5% fetal bovine serum to a final concentration of 2 μg/mL.

Techniques: Residue, Transfection, Mutagenesis, Plasmid Preparation, Expressing, Positive Control, Confocal Microscopy, Staining

Journal: bioRxiv

Article Title: Dengue virus NS1 undergoes partial nuclear translocation to modulate host transcription and support viral replication

doi: 10.64898/2026.04.13.718202

Figure Lengend Snippet: A) Monolayers of BHK-21 were simultaneously transfected with in vitro transcribed RNA from DENV2-mut-mCherry, together with plasmids that express wild-type NS1 (p wt-NS1) or NS1 mutated in both parts of the NSL (p Mut3-NS1), and 6 days after, cells were fixed and processed for confocal microscopy. Controls include non-transfected cells (N/T), cells transfected with a plasmid expressing wt-NS1 wild type alone (p wt-NS1), and RNA derived from either infectious clone alone (DENV2-wt-mCherry and DENV2-mut-mCherry). B) Cell supernatants collected from the trans complemented cells after 6 days were tested for infectivity in monolayers of BHK-21 cells. Cells were fixed and processed for confocal microscopy 7 days after infection. Cell nuclei were stained with DAPI (blue), and DENV NS1 was visualized using an anti-NS1 Mab as primary antibody and an Alexa 488 conjugated secondary antibody (green). Scale bar: 50uM. Results from one of two independent experiments are shown.

Article Snippet: BHK-21 cells were incubated with commercial recombinant DENV2 NS1 protein (R&D Systems, 9439-DG) diluted in EMEM supplemented with 5% fetal bovine serum to a final concentration of 2 μg/mL.

Techniques: Transfection, In Vitro, Confocal Microscopy, Plasmid Preparation, Expressing, Derivative Assay, Infection, Staining

Journal: bioRxiv

Article Title: Dengue virus NS1 undergoes partial nuclear translocation to modulate host transcription and support viral replication

doi: 10.64898/2026.04.13.718202

Figure Lengend Snippet: A) Volcano plot showing differential gene expression in BHK-21 cells expressing the NS1-M3 mutant relative to wild-type NS1-Wt. The x-axis represents the fold change (log₂), and the y-axis indicates statistical significance (-log₁₀ crude P-value). Orange dots denote significantly upregulated genes in NS1-M3 compared to NS1-Wt, while blue dots represent downregulated genes (log₂ FC ≥ 1, P < 0.05). The extensive distribution of differentially expressed genes highlights the distinct biological impact of the M3 mutation compared to the native protein. B) Bidirectional hierarchical clustering heatmap of 1,755 genes with significant differential expression (times change > 2 and cutoff P-value). Rows represent individual genes and columns represent experimental conditions. Expression levels are presented as Z-scores of log2 normalized values, ranging from low expression (blue) to high expression (yellow). The upper dendrogram reveals a distinct clustering of the NS1-M3 mutant, separate from the NS1-Wt and control groups. (Right)

Article Snippet: BHK-21 cells were incubated with commercial recombinant DENV2 NS1 protein (R&D Systems, 9439-DG) diluted in EMEM supplemented with 5% fetal bovine serum to a final concentration of 2 μg/mL.

Techniques: Gene Expression, Expressing, Mutagenesis, Quantitative Proteomics, Control

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