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Native Antigen Inc cterminal poly histidine tag
Cterminal Poly Histidine Tag, supplied by Native Antigen Inc, used in various techniques. Bioz Stars score: 94/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 94 stars, based on 18 article reviews

cterminal poly histidine tag - by Bioz Stars, 2026-03

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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.

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

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.

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

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.

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

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.

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

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.

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

NS1 softens single endothelial cells. (A) Schematic representation of the experimental setup, illustrating the indentation of the cells by the probe. (B) Microscopic image showing a single endothelial cell and the probe. (C) Exposure to NS1 significantly lowers ( P < 0.05) the shear storage, G ′ and shear loss, G ″ of endothelial cells exposed to NS1 compared to the control. (D) P -values calculated using mixed-effects multiple comparisons with Bonferroni correction. Comparisons include mean differences of G ′ and G ″ between control and NS1-exposed condition for each applied frequency. Endothelial cells were exposed to 5 μg/mL NS1 for 4 h. Data represent the mean stiffness of 10 cells per experiment, with three independent biological replicates. Error bars indicate the standard deviation across replicates.

Journal: ACS Biomaterials Science & Engineering

Article Title: Revealing Mechanopathology Induced by Dengue NS1 Using Organ Chips and Single-Cell Force Spectroscopy

doi: 10.1021/acsbiomaterials.4c02410

Figure Lengend Snippet: NS1 softens single endothelial cells. (A) Schematic representation of the experimental setup, illustrating the indentation of the cells by the probe. (B) Microscopic image showing a single endothelial cell and the probe. (C) Exposure to NS1 significantly lowers ( P < 0.05) the shear storage, G ′ and shear loss, G ″ of endothelial cells exposed to NS1 compared to the control. (D) P -values calculated using mixed-effects multiple comparisons with Bonferroni correction. Comparisons include mean differences of G ′ and G ″ between control and NS1-exposed condition for each applied frequency. Endothelial cells were exposed to 5 μg/mL NS1 for 4 h. Data represent the mean stiffness of 10 cells per experiment, with three independent biological replicates. Error bars indicate the standard deviation across replicates.

Article Snippet: Microvascular channels were exposed to Dengue Virus Serotype 2 NS1 Protein (DENV2-NS1; The Native Antigen Company) at concentrations of 1, 5, 10, and 20 μg/mL, and incubated for 4 h. After refreshing the ECM channels with 20 μL of HBSS, the medium in the inlets and outlets of the microvascular channels was replaced with 40 μL and 30 μL of 125 μg/mL Alexa Fluor 555-conjugated albumin (A34786; Invitrogen), respectively.

Techniques: Shear, Control, Standard Deviation

NS1 softens endothelial cells in a monolayer. (A) Schematic representation of the cross-sectional layer of the AFS microfluidic chip with a transparent piezo element. Standing acoustic wave drives silica microbeads to the acoustic node, thereby stretching the cells. (B) HUVECs with silica microbeads were cultured inside the AFS microfluidic chip for 2 h at 37 °C with 5% CO 2 . (C) Exposure to 5 μg/mL of NS1 significantly softened the HUVEC cells within 4 h. No statistically significant differences in stiffness were observed after washing out the viral proteins and culturing them overnight. All experiments were performed at 37 °C, with sample sizes of n = 140, 78, and 57 for control, NS1, and recovery groups, respectively.

Journal: ACS Biomaterials Science & Engineering

Article Title: Revealing Mechanopathology Induced by Dengue NS1 Using Organ Chips and Single-Cell Force Spectroscopy

doi: 10.1021/acsbiomaterials.4c02410

Figure Lengend Snippet: NS1 softens endothelial cells in a monolayer. (A) Schematic representation of the cross-sectional layer of the AFS microfluidic chip with a transparent piezo element. Standing acoustic wave drives silica microbeads to the acoustic node, thereby stretching the cells. (B) HUVECs with silica microbeads were cultured inside the AFS microfluidic chip for 2 h at 37 °C with 5% CO 2 . (C) Exposure to 5 μg/mL of NS1 significantly softened the HUVEC cells within 4 h. No statistically significant differences in stiffness were observed after washing out the viral proteins and culturing them overnight. All experiments were performed at 37 °C, with sample sizes of n = 140, 78, and 57 for control, NS1, and recovery groups, respectively.

Techniques: Cell Culture, Control

NS1 disrupts vascular barrier integrity in microvessel-on-a-chip. (A) Schematic diagram of the OrganoPlate, featuring a 384-well plate interface on top and 96 integrated microfluidic chips at the bottom. (B) Diagram of a single microfluidic chip, comprising a microvessel and an ECM channel separated by a phase-guide; 1 = gel inlet, 2 = medium inlet, 3 = observation window, 4 = medium outlet. (C) Compartments of the microvessel and ECM channel. A monolayer of HUVECs forms a microvessel adjacent to the ECM channel within the microfluidic system. (D) Perfusion in the OrganoPlate is achieved through bidirectional flow generated by a tilted rocking platform with an 8 min rocking interval. (E) Dose response of microvessels to NS1-induced apparent permeability ( P app ) increases after 4 h of exposure. (F) P App of microvessels in response to 5 μg/mL NS1 at different time points (0.5, 1, 2, 4, 6 h). Data are presented as mean ± SEM; n = 6 (independent chips using HUVECs from 3 to 5 different donors). Panels A-D are reproduced or adapted from ref . Available under a CC BY license. Copyright 2021 Tang et al.

Journal: ACS Biomaterials Science & Engineering

Article Title: Revealing Mechanopathology Induced by Dengue NS1 Using Organ Chips and Single-Cell Force Spectroscopy

doi: 10.1021/acsbiomaterials.4c02410

Figure Lengend Snippet: NS1 disrupts vascular barrier integrity in microvessel-on-a-chip. (A) Schematic diagram of the OrganoPlate, featuring a 384-well plate interface on top and 96 integrated microfluidic chips at the bottom. (B) Diagram of a single microfluidic chip, comprising a microvessel and an ECM channel separated by a phase-guide; 1 = gel inlet, 2 = medium inlet, 3 = observation window, 4 = medium outlet. (C) Compartments of the microvessel and ECM channel. A monolayer of HUVECs forms a microvessel adjacent to the ECM channel within the microfluidic system. (D) Perfusion in the OrganoPlate is achieved through bidirectional flow generated by a tilted rocking platform with an 8 min rocking interval. (E) Dose response of microvessels to NS1-induced apparent permeability ( P app ) increases after 4 h of exposure. (F) P App of microvessels in response to 5 μg/mL NS1 at different time points (0.5, 1, 2, 4, 6 h). Data are presented as mean ± SEM; n = 6 (independent chips using HUVECs from 3 to 5 different donors). Panels A-D are reproduced or adapted from ref . Available under a CC BY license. Copyright 2021 Tang et al.

Techniques: Generated, Permeability

NS1-induced vascular leakage is associated with the rearrangement of VE-cadherin, formation of stress fibers, and synthesis of hyaluronan. (A) Immunostaining of endothelial cells in the microvessel-on-a-chip for VE-cadherin (green) and F-actin (red) following incubation with 5 μg/mL NS1 for 4 h. An increase in the formation of actin stress fibers and morphological rearrangement of VE-cadherin were observed (arrowheads). Scale bars: 30 μm. (B) Endothelial cells, both untreated and NS1-treated, were stained for hyaluronan (green), revealing the presence of hyaluronan-rich dot-like structures (arrowheads). Signal intensity was quantified from three independent experiments. Data are presented as mean ± SEM of 5 independent chips from 3 to 5 donors, with statistical significance indicated as *** p < 0.001. Scale bars: 30 μm.

Journal: ACS Biomaterials Science & Engineering

Article Title: Revealing Mechanopathology Induced by Dengue NS1 Using Organ Chips and Single-Cell Force Spectroscopy

doi: 10.1021/acsbiomaterials.4c02410

Figure Lengend Snippet: NS1-induced vascular leakage is associated with the rearrangement of VE-cadherin, formation of stress fibers, and synthesis of hyaluronan. (A) Immunostaining of endothelial cells in the microvessel-on-a-chip for VE-cadherin (green) and F-actin (red) following incubation with 5 μg/mL NS1 for 4 h. An increase in the formation of actin stress fibers and morphological rearrangement of VE-cadherin were observed (arrowheads). Scale bars: 30 μm. (B) Endothelial cells, both untreated and NS1-treated, were stained for hyaluronan (green), revealing the presence of hyaluronan-rich dot-like structures (arrowheads). Signal intensity was quantified from three independent experiments. Data are presented as mean ± SEM of 5 independent chips from 3 to 5 donors, with statistical significance indicated as *** p < 0.001. Scale bars: 30 μm.

Techniques: Immunostaining, Incubation, Staining

Phylogenetic tree of Taiwan DENV2 strains from 1995 to 2015 based on the DENV2 NS1 full-length sequence. The maximum likelihood phylogenetic tree was constructed using the IQ-TREE program with 1000 bootstrap replications. The sequences of DENV2 Taiwan strains from 1995 to 2014 were collected from GenBank while the 2015 DENV2 outbreak strains were previously sequenced with the Illumina Miseq platform . The 2015 Taiwan outbreak strains were marked in red, the Taiwan DENV2 strains from 1995 to 2014 were marked in orange, and the global reference strains were marked in black. Virus names were shown as country, accession number, and reported year of each sequence. Numbers on nodes were bootstrap support value exceeding 75%

Journal: Journal of Biomedical Science

Article Title: A non-structural protein 1 substitution of dengue virus enhances viral replication by interfering with the antiviral signaling pathway

doi: 10.1186/s12929-024-01116-4

Figure Lengend Snippet: Phylogenetic tree of Taiwan DENV2 strains from 1995 to 2015 based on the DENV2 NS1 full-length sequence. The maximum likelihood phylogenetic tree was constructed using the IQ-TREE program with 1000 bootstrap replications. The sequences of DENV2 Taiwan strains from 1995 to 2014 were collected from GenBank while the 2015 DENV2 outbreak strains were previously sequenced with the Illumina Miseq platform . The 2015 Taiwan outbreak strains were marked in red, the Taiwan DENV2 strains from 1995 to 2014 were marked in orange, and the global reference strains were marked in black. Virus names were shown as country, accession number, and reported year of each sequence. Numbers on nodes were bootstrap support value exceeding 75%

Article Snippet: The standard used in this experiment was purchased from the Native Antigen Company, which is DENV2 NS1 hexamer produced in mammalian HEK293 cells with purity higher than 95% determined by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE).

Techniques: Sequencing, Construct, Virus

Phylogenetic tree of DENV2 strains of Asian countries from 1995 to 2019 based on the DENV2 NS1 full-length sequence. The maximum likelihood phylogenetic tree was constructed using the IQ-TREE program with 1000 bootstrap replications. The sequences of DENV2 of Asian countries from 1995 to 2019 were collected from GenBank while the 2015 DENV2 outbreak strains were previously sequenced with the Illumina Miseq platform . The 2015 Taiwan outbreak strains were marked in red, the global reference strains were marked in black, and the DENV2 strains of Asian countries from the other countries were marked in different colors. Virus names were shown as country, accession number, and reported year of each sequence. Numbers on nodes were bootstrap support value exceeding 75%. TW: Taiwan; CN: China; IN: India; ID: Indonesia; SG: Singapore; MY: Malaysia; TH: Thailand; VN: Vietnam; PH: Philippines; KH: Cambodia; LA: Laos

Journal: Journal of Biomedical Science

Article Title: A non-structural protein 1 substitution of dengue virus enhances viral replication by interfering with the antiviral signaling pathway

doi: 10.1186/s12929-024-01116-4

Figure Lengend Snippet: Phylogenetic tree of DENV2 strains of Asian countries from 1995 to 2019 based on the DENV2 NS1 full-length sequence. The maximum likelihood phylogenetic tree was constructed using the IQ-TREE program with 1000 bootstrap replications. The sequences of DENV2 of Asian countries from 1995 to 2019 were collected from GenBank while the 2015 DENV2 outbreak strains were previously sequenced with the Illumina Miseq platform . The 2015 Taiwan outbreak strains were marked in red, the global reference strains were marked in black, and the DENV2 strains of Asian countries from the other countries were marked in different colors. Virus names were shown as country, accession number, and reported year of each sequence. Numbers on nodes were bootstrap support value exceeding 75%. TW: Taiwan; CN: China; IN: India; ID: Indonesia; SG: Singapore; MY: Malaysia; TH: Thailand; VN: Vietnam; PH: Philippines; KH: Cambodia; LA: Laos

Techniques: Sequencing, Construct, Virus

K272R mutant virus replicated faster than WT virus in type I IFN producing cells. A Schematic diagram of NS1 amino acid mutations of Taiwan 2015 DENV2 outbreak strains. The red region represents the NS1 protein of DENV2. The stars indicate the amino acid substitutions isolated from the 2015 Taiwan dengue outbreak. B Production of DENV2-EGFP rg viruses containing each amino acid substitution in BHK-21 cells. The fluorescent images were captured under 10 × magnification of fluorescent microscope. C , D Growth kinetics of each rg virus in C A549 cells and D Vero cells. E Viral competition assay of each rg virus in Vero cells. Vero cells were infected with WT virus, mutant viruses or 1:1 mixture of WT and each mutant, then each group of viruses were passaged up to P5. Sanger sequencing was performed to determine the dominant variant at P1 and P5. All data are representative data from at least two independent experiments with comparable results and plotted as mean ± SEM with *p < 0.05 and ns, p > 0.05 by two-way ANOVA. WT: wildtype

Journal: Journal of Biomedical Science

Article Title: A non-structural protein 1 substitution of dengue virus enhances viral replication by interfering with the antiviral signaling pathway

doi: 10.1186/s12929-024-01116-4

Figure Lengend Snippet: K272R mutant virus replicated faster than WT virus in type I IFN producing cells. A Schematic diagram of NS1 amino acid mutations of Taiwan 2015 DENV2 outbreak strains. The red region represents the NS1 protein of DENV2. The stars indicate the amino acid substitutions isolated from the 2015 Taiwan dengue outbreak. B Production of DENV2-EGFP rg viruses containing each amino acid substitution in BHK-21 cells. The fluorescent images were captured under 10 × magnification of fluorescent microscope. C , D Growth kinetics of each rg virus in C A549 cells and D Vero cells. E Viral competition assay of each rg virus in Vero cells. Vero cells were infected with WT virus, mutant viruses or 1:1 mixture of WT and each mutant, then each group of viruses were passaged up to P5. Sanger sequencing was performed to determine the dominant variant at P1 and P5. All data are representative data from at least two independent experiments with comparable results and plotted as mean ± SEM with *p < 0.05 and ns, p > 0.05 by two-way ANOVA. WT: wildtype

Techniques: Mutagenesis, Virus, Isolation, Microscopy, Competitive Binding Assay, Infection, Sequencing, Variant Assay

K272R mutant promoted higher secretion of soluble NS1. A , B A549 cells infected with K272R mutant or WT virus at or MOI 0.01 or MOI 0.1 for the indicated time points. C 293 T cells were transfected with 1 µg or 2 µg of plasmid overexpressing NS1-K272R or NS1-WT. A – C NS1 concentration in culture supernatants were measured by ELISA. All data are representative data from at least two independent experiments with ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05 and ns, p > 0.05 by two-way ANOVA

Journal: Journal of Biomedical Science

Article Title: A non-structural protein 1 substitution of dengue virus enhances viral replication by interfering with the antiviral signaling pathway

doi: 10.1186/s12929-024-01116-4

Figure Lengend Snippet: K272R mutant promoted higher secretion of soluble NS1. A , B A549 cells infected with K272R mutant or WT virus at or MOI 0.01 or MOI 0.1 for the indicated time points. C 293 T cells were transfected with 1 µg or 2 µg of plasmid overexpressing NS1-K272R or NS1-WT. A – C NS1 concentration in culture supernatants were measured by ELISA. All data are representative data from at least two independent experiments with ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05 and ns, p > 0.05 by two-way ANOVA

Techniques: Mutagenesis, Infection, Virus, Transfection, Plasmid Preparation, Concentration Assay, Enzyme-linked Immunosorbent Assay

K272R amino acid substitution contributed to immune evasion from antiviral response. A – C A549 cells were infected with K272R mutant virus or WT virus at MOI 0.1 for 72 h followed by IFN-α treatment (1000 IU/mL) for 6 h. Quantitative RT-PCR analysis of ISGs (A) IFIT1 , (B) ISG15 , and C MxA were performed. D Immunoassay of extracts of 293 T cells transfected with 1 µg or 2 µg of plasmid overexpressing NS1-K272R or NS1-WT. (E–H) 293 T cells transfected with E , F 1 µg or G , H 2 µg of plasmids overexpressing NS1-K272R and NS1-WT, followed by IFN-α treatment (1000 IU/mL) for 6 h. Quantitative RT-PCR analysis of ISGs E , G IFIT1 and F , H ISG15 were performed. I , J STAT1 phosphorylation status was analyzed by western blot analysis. I A549 cells extracts with K272R mutant virus or WT virus infection at MOI 0.5 for 72 h and J 293 T cells were transfected with 2 µg of vector control, overexpressing NS1-WT and K272R mutant plasmids followed by IFN-α treatment (2000 IU/mL) for 1 h. The phosphorylated STAT1 protein expression was detected K A549 cells were infected with K272R mutant virus or WT virus at MOI 0.01 for 72 h and quantitative RT-PCR analysis of SOCS3 was performed. All data are representative data from at least two independent experiments with ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05 by one-way ANOVA. UT: untreated; VC: vector control

Journal: Journal of Biomedical Science

Article Title: A non-structural protein 1 substitution of dengue virus enhances viral replication by interfering with the antiviral signaling pathway

doi: 10.1186/s12929-024-01116-4

Figure Lengend Snippet: K272R amino acid substitution contributed to immune evasion from antiviral response. A – C A549 cells were infected with K272R mutant virus or WT virus at MOI 0.1 for 72 h followed by IFN-α treatment (1000 IU/mL) for 6 h. Quantitative RT-PCR analysis of ISGs (A) IFIT1 , (B) ISG15 , and C MxA were performed. D Immunoassay of extracts of 293 T cells transfected with 1 µg or 2 µg of plasmid overexpressing NS1-K272R or NS1-WT. (E–H) 293 T cells transfected with E , F 1 µg or G , H 2 µg of plasmids overexpressing NS1-K272R and NS1-WT, followed by IFN-α treatment (1000 IU/mL) for 6 h. Quantitative RT-PCR analysis of ISGs E , G IFIT1 and F , H ISG15 were performed. I , J STAT1 phosphorylation status was analyzed by western blot analysis. I A549 cells extracts with K272R mutant virus or WT virus infection at MOI 0.5 for 72 h and J 293 T cells were transfected with 2 µg of vector control, overexpressing NS1-WT and K272R mutant plasmids followed by IFN-α treatment (2000 IU/mL) for 1 h. The phosphorylated STAT1 protein expression was detected K A549 cells were infected with K272R mutant virus or WT virus at MOI 0.01 for 72 h and quantitative RT-PCR analysis of SOCS3 was performed. All data are representative data from at least two independent experiments with ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05 by one-way ANOVA. UT: untreated; VC: vector control

Techniques: Infection, Mutagenesis, Virus, Quantitative RT-PCR, Transfection, Plasmid Preparation, Western Blot, Control, Expressing

Schematic diagram of effects of K272R amino acid substitution on the NS1 properties upon viral infection. During K272R mutant virus infection, the NS1-K272R inhibited the phosphorylation of STAT1 protein and downstream expression of ISGs to enhance the K272R mutant replication. Besides, the infection of K272R mutant virus induced more intense phosphorylation of p65 and higher secretion of pro-inflammatory cytokines which may be due to the higher production of sNS1

Journal: Journal of Biomedical Science

Article Title: A non-structural protein 1 substitution of dengue virus enhances viral replication by interfering with the antiviral signaling pathway

doi: 10.1186/s12929-024-01116-4

Figure Lengend Snippet: Schematic diagram of effects of K272R amino acid substitution on the NS1 properties upon viral infection. During K272R mutant virus infection, the NS1-K272R inhibited the phosphorylation of STAT1 protein and downstream expression of ISGs to enhance the K272R mutant replication. Besides, the infection of K272R mutant virus induced more intense phosphorylation of p65 and higher secretion of pro-inflammatory cytokines which may be due to the higher production of sNS1

Techniques: Infection, Mutagenesis, Virus, Expressing

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