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