mouse monoclonal antibody recognizing zikv ns1 protein (gtx634158)  (GeneTex)

Journal: bioRxiv

Article Title: A genus-wide interaction atlas across NS4B orthologues identifies a conserved role for UFMylation in orthoflavivirus replication

doi: 10.1101/2025.05.15.653649

Figure Lengend Snippet: a . Systematic comparison of interactomes and host proteome changes (Effectome) of the orthoflavivirus NS4Bs and four control proteins (ZIKV capsid, Gaussia luciferase, HCV-NS4B and naïve JEG-3 cells), using eight orthologues of pathogenic orthoflaviviruses (DENV, YFV, ZIKV, JEV, WNV, USUV, TBEV, POWV). The numbers of unique and shared (significantly-enriched in at least 6 viral baits) host interactions across the orthoflavivirus NS4B proteins (“NS4Bome”, Interactome) and significantly modulated proteins in NS4B-expressing cells (Effectome) is shown. b. Combined virus–host protein–protein interaction network of orthoflavivirus NS4Bs measured by AP–MS. Shared interacting proteins amongst two species are denoted by the orange shadows. Selected biological functions and processes are denoted in grey shades. Only high-confidence interactors are shown (Log2(Fold-change) ≥ 5; p-value ≤ 0.01). Interactions between viral and host proteins are indicated by grey lines. Colored circles and nodes represent a manually-curated selection of gene-ontology annotations and organellar distribution, respectively. ERGIC, ER-Golgi intermediate compartment, SLC, solute carrier; BSG, basigin; TMEM, transmembrane proteins; tRNA, transfer RNA.

Article Snippet: The mouse monoclonal antibody recognizing ZIKV NS1 protein (GTX634158), and polyclonal rabbit anti-ZIKV NS2B (GTX133318), NS4A (GTX133704), and NS4B (GTX133311) antibodies, were purchased from GeneTex.

Techniques: Comparison, Control, Luciferase, Expressing, Virus, Protein-Protein interactions, Selection

Journal: bioRxiv

Article Title: A genus-wide interaction atlas across NS4B orthologues identifies a conserved role for UFMylation in orthoflavivirus replication

doi: 10.1101/2025.05.15.653649

Figure Lengend Snippet: a. Specificity of shortlisted NS4B interacting host proteins across NS4B orthologues selected for functional validation and experimental setup of the phenotypic screen to assess their functional relevance. b . Intensity-based absolute quantification (iBAQ) of protein abundance of orthoflavivirus NS4B-interacting host proteins across baits. The profile of 58 host proteins selected for RNA interference (RNAi) screening are shown. (N.I. = not identified). c. RNAi screening of NS4B-binding proteins to identify host factors involved in ZIKV replication. JEG-3 cells were transduced with lentiviruses encoding shRNAs targeting each of the 58 NS4B - interacting host proteins (2–3 shRNAs/gene), and infected with a full-length ZIKV reporter strain expressing Renilla luciferase at 72 hpt. The extent of virus replication was determined by luciferase activity at 48 hpi. The results of each biological replicate for one shRNA per gene are shown as a heatmap (relative to shNT; complete dataset in Supplementary Figure 3). Newly identified host-restriction and -dependency factors are highlighted in red and blue, respectively (cut-off criteria: 50% difference in viral replication with two out of three shRNAs or >75% difference with one out of three shRNAs). Mean cell viability for each shRNA is also shown as a heatmap (cut-off criteria: cell viability ≥75% of shNTs; n=3). B4GALT7 shRNA is slightly toxic (cell viability 74%, highlighted in red. d. Volcano plots comparing interactors of ZIKV-NS4B and HCV-NS4B (log2(fold change) ≥ 2.5, FDR-corrected Welch’s t-test p ≤ 0.01). n = 4 independent experiments. A selected group of previously reported interactors of ZIKV-NS4B is shown in black. Newly identified host-interactors functionally- and orthogonally-validated in this study are shown in red. Viral baits are shown in blue. e , Co-immunoprecipitation of ZIKV-NS4B–HA with endogenous host proteins. Cell lysates of JEG-3 cells infected with wild-type ZIKV (NT) or NS4B-HA-tagged ZIKV (ZIKV-NS4B HA ) proteins were used for anti-HA immunoaffinity purification and probed with the indicated antibodies against newly-identified NS4B-interacting host proteins. Representative blots are shown (n = 3 independent experiments).

Article Snippet: The mouse monoclonal antibody recognizing ZIKV NS1 protein (GTX634158), and polyclonal rabbit anti-ZIKV NS2B (GTX133318), NS4A (GTX133704), and NS4B (GTX133311) antibodies, were purchased from GeneTex.

Techniques: Functional Assay, Biomarker Discovery, Quantitative Proteomics, Binding Assay, Transduction, Infection, Expressing, Luciferase, Virus, Activity Assay, shRNA, Immunoprecipitation, Immunoaffinity Purification

Journal: bioRxiv

Article Title: A genus-wide interaction atlas across NS4B orthologues identifies a conserved role for UFMylation in orthoflavivirus replication

doi: 10.1101/2025.05.15.653649

Figure Lengend Snippet: a-b. JEG-3 cells were transduced with lentiviruses expressing shRNAs targeting each of the 58 NS4B host-interacting proteins (2-3 shRNAs/gene), and infected with a full-length ZIKV reporter strain expressing Renilla luciferase at 72 hpt. The extent of virus replication was determined by luciferase activity 48 hpi. The results (Fold-of-shNT) are shown as a heatmap ( a ). Newly identified host-restriction and -dependency factors are highlighted in red and blue, respectively (cut-off criteria: 50% difference in viral replication with 2/3 shRNAs or >75% difference with 1/3 shRNA). Mean cell viability for each shRNA is also shown as a heatmap ( b ) (cut-off criteria: cell viability ≥75% of shNTs; n=3). Crossed squares indicate individual shRNAs missing. c. Co-immunoprecipitation of HA-tagged NS4B protein of Orthoflaviviruses with endogenous host proteins. Cell lysates of JEG-3 cells stably expressing HA-tagged NS4B protein of 8 Orthoflaviviruses (DENV2, JEV, POWV, TBEV, USUV, WNV, YFV, and ZIKV) were used for HA-immunoaffinity purification and probed with the indicated antibodies. Lysates from uninfected JEG-3 cells, cells expressing HA tagged Gaussia Luciferase (GLuc), and cells stably expressing HA-tagged NS4B protein of HCV were used as control. Panel c shows representative immunoblots.

Article Snippet: The mouse monoclonal antibody recognizing ZIKV NS1 protein (GTX634158), and polyclonal rabbit anti-ZIKV NS2B (GTX133318), NS4A (GTX133704), and NS4B (GTX133311) antibodies, were purchased from GeneTex.

Techniques: Transduction, Expressing, Infection, Luciferase, Virus, Activity Assay, shRNA, Immunoprecipitation, Stable Transfection, Immunoaffinity Purification, Control, Western Blot

Journal: bioRxiv

Article Title: A genus-wide interaction atlas across NS4B orthologues identifies a conserved role for UFMylation in orthoflavivirus replication

doi: 10.1101/2025.05.15.653649

Figure Lengend Snippet: a. Schematic representation of the NS4B protein membrane topology depicting the position of the internal HA tag. b. Following infection of JEG-3 cells with either ZIKV H/PF/2013 wild-type strain (ZIKV, black) or ZIKV-NS4B HA (blue) at an MOI 0.01, supernatants were collected at the indicated time points to determine the kinetics of infectious virus release by TCID50 (Tissue Culture Infectious Dose) assay. c. Western blot analysis confirmed the expression of HA tagged NS4B protein during ZIKV infection (top and middle panels, third lane). GAPDH was used as a loading control (bottom panel). The cell lysates were harvest in RIPA buffer 48 hpi. d. Representative images of JEG-3 cells infected with replication-competent ZIKV H/PF/2013 molecular clone carrying an HA-tag within the NS4B ORF (ZIKV-NS4B HA ) at an MOI of 5. The cells were fixed 24 hpi, stained with anti-NS4B (green) and anti-HA (red) antibodies, and visualized by confocal microscopy. Scale bar 20 µm. Panel b, n=3 biological replicates are shown, each circle represents the mean and error bars represent the standard deviation of the mean. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, or ns, not significant as determined by unpaired two-tailed t-test on log₁₀-transformed PFU/mL values with Holm–Šidák correction for multiple comparisons. Panel c shows representative immunoblot.

Article Snippet: The mouse monoclonal antibody recognizing ZIKV NS1 protein (GTX634158), and polyclonal rabbit anti-ZIKV NS2B (GTX133318), NS4A (GTX133704), and NS4B (GTX133311) antibodies, were purchased from GeneTex.

Techniques: Membrane, Infection, Virus, Western Blot, Expressing, Control, Staining, Confocal Microscopy, Standard Deviation, Two Tailed Test, Transformation Assay

Journal: bioRxiv

Article Title: A genus-wide interaction atlas across NS4B orthologues identifies a conserved role for UFMylation in orthoflavivirus replication

doi: 10.1101/2025.05.15.653649

Figure Lengend Snippet: UBA5 knock-down JEG-3 cells (shUBA5) or control cells (shNT) were infected with ZIKV H/PF/2013 wild-type strain (a-c) or ZIKV H/PF/2013 reporter strain expressing Renilla luciferase (d) at an MOI of 0.1. At 24 hpi, intracellular viral protein levels were determined by western blotting ( a ), viral RNA levels were determined by RT-qPCR ( b ), and infectious particle production was determined by plaque assay on cell culture supernatants ( c ), while viral replication was determined by measuring luciferase activity ( d ). e. Schematic representation of the UFMylation pathway showing the substrate UFM1 being conjugated onto the lysine residues of the target protein through an enzymatic pathway involving the E1 activase, UBA5, the E2 conjugase, UFC1, and the E3 ligase, UFL1. f. Western blot analyzing the reconstitution of the UFMylation pathway after the complementation of UBA5 knock-out (KO) JEG-3 cells with either wild-type UBA5 or UFMylation-dead UBA5 mutants. g. UBA5 KO JEG-3 cells were infected with ZIKV H/PF/2013 wild-type strain (MOI 0.01) and supernatants were collected at different times post-infection. The kinetics of infectious virus release were determined by plaque assay. h. UBA5 KO JEG-3 cells, complemented with either wild-type UBA5 or UFMylation-dead UBA5 mutants (Mut1 and Mut2), were infected with ZIKV H/PF/2013 wild-type strain (MOI 0.01). UBA5_Mut1 fails to activate UFM1 (UBA5 C250R) and Uba5_Mut2 fails to bind to UFC1 (UBA5 L397R, M401R). Virus titers measured in the supernatants at 48 hpi by plaque assay showed that only wild-type UBA5 could rescue ZIKV replication. Panels b,c,d, and h, n=3 biological replicates; bars represent the mean and error bars represent the standard deviation of the mean. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, or ns, not significant as determined by two-way ANOVA with Tukey’s multiple comparisons test (b,c and d), unpaired two-tailed t-test on log₁₀-transformed PFU/mL values with Holm–Šidák correction for multiple comparisons (g) or one-way ANOVA with Dunnett’s multiple comparisons test (h). a and f, representative immunoblots from n=3 biological replicates are shown.

Article Snippet: The mouse monoclonal antibody recognizing ZIKV NS1 protein (GTX634158), and polyclonal rabbit anti-ZIKV NS2B (GTX133318), NS4A (GTX133704), and NS4B (GTX133311) antibodies, were purchased from GeneTex.

Techniques: Knockdown, Control, Infection, Expressing, Luciferase, Western Blot, Quantitative RT-PCR, Plaque Assay, Cell Culture, Activity Assay, Knock-Out, Virus, Standard Deviation, Two Tailed Test, Transformation Assay

Journal: bioRxiv

Article Title: A genus-wide interaction atlas across NS4B orthologues identifies a conserved role for UFMylation in orthoflavivirus replication

doi: 10.1101/2025.05.15.653649

Figure Lengend Snippet: a. Cell lysates of JEG-3 cells that were mock-infected or infected with wild-type ZIKV H/PF/2013 (MOI=0.1) were harvested at 24 and 48 hpi and subjected to non-reducing SDS-PAGE. The top two panels stained with anti-UFM1 antibodies show the UFM1 conjugates and UFM1, respectively. NS4A was stained as an infection marker (third panel), and β-actin was used as a loading control (bottom panel). b. Immunoblot analysis of anti-FLAG immunoprecipitated extracts (right panels) and inputs (left panels) from both mock and ZIKV H/PF/2013 wild-type strain-infected (MOI 0.01) JEG-3 cells stably expressing FLAG UFM1ΔSC. Eluates were stained with antibodies specific to UFMylation pathway members as indicated on the left. c. Subcellular distribution of UFMylation pathway members (UFSP2 and UFL1). JEG-3 cells infected with a ZIKV infectious molecular clone carrying an HA tag internal to NS4B (ZIKV-NS4B HA ) were stained with anti-UFSP2 (top two rows) or anti-UFL1 (bottom two rows), anti-HA and anti-NS3 antibodies (MOI 5; 24 hpi). Scale bar = 10 μm. A representative experiment of n=3 is shown. d. Immunoblot analysis of anti-FLAG immunoprecipitated extracts (right panels) and inputs (left panels) from both mock and ZIKV H/PF/2013 wild-type strain (MOI 0.01) infected JEG-3 cells stably expressing FLAG UFM1ΔSC. Eluates were stained with antibodies specific to viral proteins as indicated on the left. e. Heat map of all detected UFMylation pathway members across the extended NS4B PPI networks. Intensity-based absolute quantification (iBAQ) of protein abundance of UFMylation-related host proteins across baits. N.I.; not identified. F. Replication of multiple orthoflaviviruses is inhibited in UBA5 KO cells. Control (gNT) or UBA5 KO (gUBA5) JEG-3 cells were infected with the orthoflaviviruses ZIKV, DENV2, JEV, WNV and YFV. At 48 hpi, infectious titers in the supernatant were determined by plaque assay. Herpes simplex virus 1 (HSV-1) was used as control. Panel f, n=3 biological replicates; bars represent the mean and error bars represent the standard deviation of the mean. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, or ns, not significant as determined by two-way ANOVA with Sidak’s multiple comparisons test.

Article Snippet: The mouse monoclonal antibody recognizing ZIKV NS1 protein (GTX634158), and polyclonal rabbit anti-ZIKV NS2B (GTX133318), NS4A (GTX133704), and NS4B (GTX133311) antibodies, were purchased from GeneTex.

Techniques: Infection, SDS Page, Staining, Marker, Control, Western Blot, Immunoprecipitation, Stable Transfection, Expressing, Quantitative Proteomics, Plaque Assay, Virus, Standard Deviation

Journal: bioRxiv

Article Title: A genus-wide interaction atlas across NS4B orthologues identifies a conserved role for UFMylation in orthoflavivirus replication

doi: 10.1101/2025.05.15.653649

Figure Lengend Snippet: a. Schematic representation of the UFMylation pathway inhibition by the UBA5 inhibitor DKM 2-93. DKM 2-93 competitively binds to the catalytic cysteine of UBA5, and prevents the activation of UFM1. b. Dose-response curve of DKM 2-93 for inhibition of ZIKV infection in JEG-3 cells. JEG-3 cells were treated with increasing concentrations of the inhibitor for 24 h, then infected with the ZIKV H/PF/2013 reporter strain expressing Renilla luciferase (MOI 0.1), and treated with the inhibitor for another 24 h. Cell viability and virus replication were determined at 24 hpi by resazurin and luciferase assays, respectively. c. Western blot analysis confirmed the impairment of virus replication. NS1 was stained as an infection marker (top panel), and GAPDH was used as a loading control (bottom panel). d. A reduction in ZIKV titers was observed in the presence of non-cytotoxic concentrations of the inhibitor in JEG-3 cells. JEG-3 cells were infected with ZIKV H/PF/2013 wild-type strain (MOI 0.01), and treated with either DMSO or two non-cytotoxic concentrations of DKM 2-93. Supernatants were harvested at 48 hpi and the virus titers were measured by plaque assay. e. Time-of-addition analysis of the antiviral activity of DKM 2-93. JEG-3 cells were either pre-treated (grey bar) with the inhibitor (32 µM) for 3 h prior to infection with ZIKV H/PF/2013 wild-type strain (MOI 0.01), or co-treated (blue bar) by adding the inhibitor (32 µM) to the viral inoculum during 1 h of virus adsorption, or post-treated (orange bar) 3 h after the removal of the viral inoculum. In each case, the supernatant was collected at 48 hpi, and the virus titers were determined by plaque assay. f-g. Huh7 cells were electroporated with wild-type subgenomic replicon (sgZIKV) reporter virus RNA expressing Renilla luciferase, and treated with DKM 2-93 (32 µM) immediately thereafter. Luciferase activity was measured 4 hours post electroporation to assess effects on viral RNA translation (f, sgZIKV-R2A), and up to 96 hours post electroporation to assess effects of viral RNA replication (g, sgZIKV-R2A). Panels d, e, and f, n=3 biological replicates; bars represent the mean and error bars represent the standard deviation of the mean. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, or ns, not significant as determined by one-way ANOVA with Dunnett’s multiple comparisons test (d) or two-way ANOVA with Sidak’s multiple comparisons test (e and f) or unpaired two-tailed t-test on log₁₀-transformed PFU/mL values with Holm–Šidák correction for multiple comparisons (g). Panel c, representative immunoblots from n=3 biological replicates are shown.

Article Snippet: The mouse monoclonal antibody recognizing ZIKV NS1 protein (GTX634158), and polyclonal rabbit anti-ZIKV NS2B (GTX133318), NS4A (GTX133704), and NS4B (GTX133311) antibodies, were purchased from GeneTex.

Techniques: Inhibition, Activation Assay, Infection, Expressing, Luciferase, Virus, Western Blot, Staining, Marker, Control, Plaque Assay, Activity Assay, Adsorption, Electroporation, Standard Deviation, Two Tailed Test, Transformation Assay

Journal: bioRxiv

Article Title: A genus-wide interaction atlas across NS4B orthologues identifies a conserved role for UFMylation in orthoflavivirus replication

doi: 10.1101/2025.05.15.653649

Figure Lengend Snippet: RNA-seq analysis on mock- or ZIKV-infected cells upon UBA5 silencing. Control (shNT) or UBA5 KD cells (shUBA5) were mock-infected or infected with ZIKV H/PF/2013 and cellular RNA was extracted at 48 hpi for global transcriptomic analysis. a. Volcano plot showing differentially expressed genes shUBA5 vs. shNT mock-infected JEG-3 cells. Significantly-regulated genes are displayed in red (p-adj < 0.1 and |log2-fold-change| > 0.5). DE genes list was extracted after correcting for within group variation using “contrast = c(“condition”,“shUBA5”,“shNT”)”. b. Gene ontology enrichment plot showing significantly enriched GO terms in mock-infected shUBA5 compared to controls (shNT) JEG-3 cells. c. Volcano plot showing differentially expressed genes shUBA5 vs. shNT JEG-3 upon ZIKV infection. Significantly regulated genes are displayed in red (p-adj < 0.1 and |log2-fold-change| > 0.5). DE genes list was extracted to show effect of shUBA5-treated cells upon ZIKV infection using “contrast = list(c(“condition_shUBA5_vs_shNT”,“groupZIKV.conditionshUBA5”)”. d. Gene ontology enrichment plot showing significantly enriched GO terms in ZIKV-infected shUBA5 compared to controls (shNT) JEG-3 cells. In b and c Gene ontology enrichment plot displays significantly enriched GO terms ranked based on lowest p-adjusted value and highest proportion of DE genes in corresponding GO term compared to background genes expressed in the experiment. No fold-change cutoff was used for GO enrichment analysis. e . Heatmap showing significant/non-significant log2-fold-changes of interferon-related gene expression in ZIKV-infected vs Mock-infected in independent comparisons for shNT- and shUBA5-silenced condition.

Article Snippet: The mouse monoclonal antibody recognizing ZIKV NS1 protein (GTX634158), and polyclonal rabbit anti-ZIKV NS2B (GTX133318), NS4A (GTX133704), and NS4B (GTX133311) antibodies, were purchased from GeneTex.

Techniques: RNA Sequencing, Infection, Control, Gene Expression

Journal: bioRxiv

Article Title: A genus-wide interaction atlas across NS4B orthologues identifies a conserved role for UFMylation in orthoflavivirus replication

doi: 10.1101/2025.05.15.653649

Figure Lengend Snippet: a. Control (gNT) and UBA5 KO (gUBA5) JEG-3 cells cultured for two days were imaged by confocal microscopy. Representative images of mitochondria labeled with anti-COXIV (green) and the nuclear stain DAPI (blue). Scale bar, 20 µm. Quantitative analysis of mitochondrial morphology using the Mitochondrial Analyzer plugin in ImageJ/Fiji. Panels b–g, represent the mean area which calculates the average size of individual mitochondria per cell ( b ), the mean perimeter ( c ), the mean form factor (perimeter² / 4π × area) which reflects the mitochondrial shape, with higher values indicating more elongated structures and values closer to 1 indicating rounder mitochondria ( d ), the aspect ratio, the ratio of the major axis to the minor axis with higher AR >1.5 - 2.0 indicates elongated mitochondria associated with fusion ( e ), the mean branch length, the average length of individual mitochondrial branches within the network ( f ), and the branches per mitochondria ( g ) in UBA5 KO JEG-3 cells. h-k. Knock-out of UBA5 impairs mitochondrial respiration. The Oxygen Consumption Rate (OCR) of UBA5 knock-out JEG-3 cells was measured at the indicated time points using the Seahorse technology. OCR values were first normalized to total protein content (µg per condition) and then to the mean basal OCR of control cells in each independent experiment (h). The basal respiration (i), ATP production (j), and maximal respiration (k) were quantified from the mitochondrial respiration profile. l-o. ZIKV infection modulates mitochondrial respiration. Data from n=3 biological replicates are shown in all panels; Panels b–g, the middle line of the floating bars corresponds to the mean; Panels h and l, each circle represents the mean and error bars represent the standard deviation of the mean; and panels i–k, and m-o, the bars represent the mean and error bars represent the standard error of the mean. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, or ns, not significant as determined by Mann-Whitney test (panels b–g), paired t-test (panels h and l) or unpaired t-test (panels i–k, m–o).

Article Snippet: The mouse monoclonal antibody recognizing ZIKV NS1 protein (GTX634158), and polyclonal rabbit anti-ZIKV NS2B (GTX133318), NS4A (GTX133704), and NS4B (GTX133311) antibodies, were purchased from GeneTex.

Techniques: Control, Cell Culture, Confocal Microscopy, Labeling, Staining, Knock-Out, Infection, Standard Deviation, MANN-WHITNEY

Journal: bioRxiv

Article Title: A genus-wide interaction atlas across NS4B orthologues identifies a conserved role for UFMylation in orthoflavivirus replication

doi: 10.1101/2025.05.15.653649

Figure Lengend Snippet: a. Experimental scheme of ZIKV infection in zebrafish. b . Representative images of zebrafish larvae at 2 days post-fertilization (dpf). c. ZIKV RNA levels in zebrafish larvae at 2 dpf determined by ddPCR (N=5). Bars represent the mean and error bars represent the standard deviation of the mean. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, or ns, not significant as determined by one-way ANOVA followed by Tukey’s post hoc test. d . Phenotype proportions of zebrafish larvae at 2 dpf were examined across conditions as previously described (N=3): Mock-infected (n=74), ZIKV-infected (n=74), and ZIKV-infected treated with 20 µM DKM 2-93 (n=77). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, or ns, not significant as determined by pairwise chi-squared test with p-value adjustment by Holm method.

Article Snippet: The mouse monoclonal antibody recognizing ZIKV NS1 protein (GTX634158), and polyclonal rabbit anti-ZIKV NS2B (GTX133318), NS4A (GTX133704), and NS4B (GTX133311) antibodies, were purchased from GeneTex.

Techniques: Infection, Standard Deviation

Journal: Frontiers in Pharmacology

Article Title: Antiviral activity of eicosapentaenoic acid against zika virus and other enveloped viruses

doi: 10.3389/fphar.2025.1564504

Figure Lengend Snippet: Inhibition of ZIKV activity by EPA in vitro . The mRNA levels of ZIKV E (A) and NS1 (B) was evaluated in ZIKV-infected Vero cells treated with EPA via qRT−PCR. (C) The protein expression of ZIKV E was evaluated in ZIKV-infected Vero cells treated with EPA via Western blotting. (D) ZIKV activity was evaluated in ZIKV-infected Vero cells treated with EPA via cell-based immunodetection assay. (E) ZIKV activity was evaluated in ZIKV-infected Vero cells treated with EPA via indirect immunofluorescence analysis. All experiments were repeated three times. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; n. s = not significant.

Article Snippet: The cells were incubated with a primary antibody against ZIKV E or NS1 (1:1000) (BioFront Technologies, United States) at 37°C for 1 h, followed by incubation with a horseradish peroxidase (HRP)-labeled secondary antibody (1:2000) (Cell Signalling Technology, United States).

Techniques: Inhibition, Activity Assay, In Vitro, Infection, Quantitative RT-PCR, Expressing, Western Blot, Immunodetection, Immunofluorescence

Journal: Frontiers in Pharmacology

Article Title: Antiviral activity of eicosapentaenoic acid against zika virus and other enveloped viruses

doi: 10.3389/fphar.2025.1564504

Figure Lengend Snippet: EPA rapidly inhibited ZIKV replication and targeted the virus. Determination of mRNA levels of ZIKV E (A) and NS1 (B) in cells infected with EPA–ZIKV mixture pre-incubated for 0, 5, 10, 30, and 60 min. Determination of the mRNA levels of ZIKV E (C) and NS1 (D) in cells, which were divided into four groups according to the treatment of EPA and ZIKV. Cell-treatment group: EPA was incubated with Vero cells for 1 h before ZIKV infection. Virus-treatment group: ZIKV was pre-incubated with EPA for 30 min and subsequently inoculated into cells. Co-treatment group: EPA and ZIKV were simultaneously inoculated into cells. Post-treatment group: Cells were incubated with ZIKV for 1 h and washed before being treated with EPA. All experiments were repeated three times. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; n. s = not significant.

Article Snippet: The cells were incubated with a primary antibody against ZIKV E or NS1 (1:1000) (BioFront Technologies, United States) at 37°C for 1 h, followed by incubation with a horseradish peroxidase (HRP)-labeled secondary antibody (1:2000) (Cell Signalling Technology, United States).

Techniques: Virus, Infection, Incubation

Journal: Frontiers in Pharmacology

Article Title: Antiviral activity of eicosapentaenoic acid against zika virus and other enveloped viruses

doi: 10.3389/fphar.2025.1564504

Figure Lengend Snippet: EPA disrupted the early stages of the ZIKV replication cycle. (A, B) Results of time-of-addition assay. The mRNA levels of ZIKV E (A) and NS1 (B) were evaluated via qRT-PCR. EPA was added at 0, 5, 10, 20, and 40 min after ZIKV infection or at 0, 0.5, 1, 2, and 4 h after 1 h of ZIKV infection. The effects of EPA on the binding of ZIKV to host cells (C, D) , its adsorption on host cells (E–G) , and its entry into host cells (H) are shown. The effects of EPA on the mRNA (C–F) and protein (G, H) expression of ZIKV E are shown. All experiments were repeated three times. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; n. s = not significant.

Article Snippet: The cells were incubated with a primary antibody against ZIKV E or NS1 (1:1000) (BioFront Technologies, United States) at 37°C for 1 h, followed by incubation with a horseradish peroxidase (HRP)-labeled secondary antibody (1:2000) (Cell Signalling Technology, United States).

Techniques: Quantitative RT-PCR, Infection, Binding Assay, Adsorption, Expressing

Journal: Advanced Science

Article Title: Ubiquitination of NS1 Confers Differential Adaptation of Zika Virus in Mammalian Hosts and Mosquito Vectors

doi: 10.1002/advs.202408024

Figure Lengend Snippet: Post‐translational modification of ZIKV viral proteins. A) SH‐sy5y cells were infected with ZIKV (MR766) at a multiplicity of infection (MOI) of 1, and cell samples were collected 48 h post‐infection. Mass spectrometry was employed to analyze the acetylation, methylation, phosphorylation, and ubiquitination patterns of viral proteins. B and C) 293T cells were transfected with plasmids encoding various ZIKV proteins tagged with Flag (NS1‐Flag, NS3‐Flag, NS5‐Flag, PrM/E‐Flag, NS2A‐Flag, NS2B‐Flag, NS4A‐Flag, NS4B‐Flag, or C‐Flag). B) In another set of experiments, these proteins were co‐transfected with a plasmid expressing ubiquitin tagged with HA (Ub‐HA). C) After 24 h, cells were treated with MG132 (5 µM) for 4 h. Cell lysates were collected, and immunoprecipitation with Flag antibody‐coupled magnetic beads was performed. The ubiquitination levels of viral proteins were then analyzed by Western Blot. The presented data are representative of three independent experiments.

Article Snippet: The following antibodies were used in this study: GAPDH Mouse Antibody (Proteintech, Cat # 60004‐I‐Ig), DYKDDDDK Tag Mouse Antibody (ABclonal, Cat # AE005), anti‐rabbit IgG HRP‐linked antibody (CST, Cat # 7074), HRP goat anti‐mouse IgG (BioLegend, Cat # 405 306), HA‐tag Rabbit Polyclonal Antibody (CST, Cat # 3724), Ubiquitin Antibody, (Santa Cruz, Cat # sc‐8017), K63‐linkage Specific Polyubiquitin (D7A11) Rabbit mAb (CST, Cat # 12 930), K48‐linkage Specific Polyubiquitin (D9D5) Rabbit mAb (CST, Cat # 12 805), CACYBP Polyclonal Antibody (Abclonal, Cat # A8757), TRIM4 Polyclonal Antibody (Abclonal, Cat # A15922), WWP2 Polyclonal Antibody (Abclonal, Cat # 12197‐1‐AP), Myc‐tag Rabbit Polyclonal Antibody (Proteintech, Cat # I6286‐I‐AP), Goat Anti‐Mouse IgG Antibody (H+L), DyLight 488 (SeraCare, Cat # 5230‐0391), Goat Anti‐Rabbit IgG H&L (Alexa Fluor 647) (abcam, Cat # ab150083), GST Tag Antibody (ABGENT), ZIKV virus NS1 protein antibody (GeneTex, Cat # GTX133307, GTX634158).

Techniques: Modification, Infection, Mass Spectrometry, Methylation, Phospho-proteomics, Ubiquitin Proteomics, Transfection, Plasmid Preparation, Expressing, Immunoprecipitation, Magnetic Beads, Western Blot

Journal: Advanced Science

Article Title: Ubiquitination of NS1 Confers Differential Adaptation of Zika Virus in Mammalian Hosts and Mosquito Vectors

doi: 10.1002/advs.202408024

Figure Lengend Snippet: E3 ubiquitin ligase WWP2 interacts with NS1. A) NS1‐Flag was transfected into 293T cells, followed by ZIKV infection (MOI = 0.5) 24 h later. Immunoprecipitation of NS1‐Flag was performed with Flag antibody‐coupled magnetic beads 24 h post‐infection to analyze the ubiquitin‐associated enzymes interacting with NS1‐Flag protein using mass spectrometry. B–E) WWP2‐Myc was co‐transfected with NS1‐Flag expression plasmid in 293T cells. After 24 h, immunoprecipitation of NS1‐Flag was performed, and WWP2‐Myc protein was detected by Western Blot. B) In another set of experiments, immunoprecipitation of WWP2‐Myc was carried out, and NS1‐Flag protein was detected by Western Blot. C) In addition, 293T cells were infected with ZIKV (MOI = 1), and after 48 h, endogenous WWP2 was immunoprecipitated. ZIKV NS1 protein was then detected by Western Blot D), and endogenous WWP2 protein was detected by immunoprecipitation of ZIKV NS1 and Western Blot E). F) 293T cells were infected with ZIKV(MOI = 0.5) for 48 h. The intracellular localization of the endogenous WWP2 and NS1 proteins was observed using laser confocal imaging. G and H) Each of the WWP2 truncates was co‐transfected with NS1‐Flag in 293T cells, and the cells were collected after 24 h. Immunoprecipitation with NS1‐Flag was performed, and Western Blot detected the expression of Myc‐tagged truncated proteins. I) Molecular docking prediction results for WWP2‐WW and ZIKV NS1 proteins. The data presented are representative of three independent experiments.

Article Snippet: The following antibodies were used in this study: GAPDH Mouse Antibody (Proteintech, Cat # 60004‐I‐Ig), DYKDDDDK Tag Mouse Antibody (ABclonal, Cat # AE005), anti‐rabbit IgG HRP‐linked antibody (CST, Cat # 7074), HRP goat anti‐mouse IgG (BioLegend, Cat # 405 306), HA‐tag Rabbit Polyclonal Antibody (CST, Cat # 3724), Ubiquitin Antibody, (Santa Cruz, Cat # sc‐8017), K63‐linkage Specific Polyubiquitin (D7A11) Rabbit mAb (CST, Cat # 12 930), K48‐linkage Specific Polyubiquitin (D9D5) Rabbit mAb (CST, Cat # 12 805), CACYBP Polyclonal Antibody (Abclonal, Cat # A8757), TRIM4 Polyclonal Antibody (Abclonal, Cat # A15922), WWP2 Polyclonal Antibody (Abclonal, Cat # 12197‐1‐AP), Myc‐tag Rabbit Polyclonal Antibody (Proteintech, Cat # I6286‐I‐AP), Goat Anti‐Mouse IgG Antibody (H+L), DyLight 488 (SeraCare, Cat # 5230‐0391), Goat Anti‐Rabbit IgG H&L (Alexa Fluor 647) (abcam, Cat # ab150083), GST Tag Antibody (ABGENT), ZIKV virus NS1 protein antibody (GeneTex, Cat # GTX133307, GTX634158).

Techniques: Ubiquitin Proteomics, Transfection, Infection, Immunoprecipitation, Magnetic Beads, Mass Spectrometry, Expressing, Plasmid Preparation, Western Blot, Imaging

Journal: Advanced Science

Article Title: Ubiquitination of NS1 Confers Differential Adaptation of Zika Virus in Mammalian Hosts and Mosquito Vectors

doi: 10.1002/advs.202408024

Figure Lengend Snippet: WWP2 ubiquitinates NS1 and leads to NS1 degradation. A and B) NS1‐Flag and WWP2‐Myc/shWWP2 (1 µg) were co‐transfected in 293T cells, and the protein levels of NS1‐Flag were detected by Western Blot 48 h later. C and D) NS1‐Flag and WWP2‐Myc/shWWP2 (1 µg) were co‐transfected in 293T cells, and the mRNA levels of NS1 were detected by qRT‐PCR 48 h later. E) NS1‐Flag and WWP2‐Myc were co‐transfected in 293T cells, which were then treated with MG132 (5 µM, 4 h) 24 h later. The protein levels of NS1‐Flag were detected by the Western Blot method. F) NS1‐Flag and WWP2‐Myc were co‐transfected in 293T cells. 24 h later, cells were treated with Chloroquine (10 µM, 6 h). The protein levels of NS1‐Flag were detected by the Western Blot. G and H) NS1‐Flag and shWWP2/WWP2‐Myc (1 µg) were co‐transfected in 293T cells and treated with MG132 (5 µM) for 4 h after 48 h. The ubiquitination level of NS1‐Flag protein was detected by the Western Blot method after immunoprecipitation of NS1‐Flag. I and J) NS1‐Flag and WWP2‐Myc (WT or C838A) plasmids were co‐transfected in 293T cells. After 24 h, immunoprecipitation of NS1‐Flag was performed, and the ubiquitination levels of NS1‐Flag protein were detected by Western Blot (F). The protein levels of NS1‐Flag were detected by Western Blot (G). K) NS1‐Flag, purified WWP2 (or WWP2‐C838A), E1 (Hdm2), and E2 (UbcH5a) were incubated for 1 h in the presence of ATP. The in vitro ubiquitination level of NS1 was analyzed by Western Blot. The data presented are representative of three independent experiments. ns, non‐significant (Student's t‐test).

Article Snippet: The following antibodies were used in this study: GAPDH Mouse Antibody (Proteintech, Cat # 60004‐I‐Ig), DYKDDDDK Tag Mouse Antibody (ABclonal, Cat # AE005), anti‐rabbit IgG HRP‐linked antibody (CST, Cat # 7074), HRP goat anti‐mouse IgG (BioLegend, Cat # 405 306), HA‐tag Rabbit Polyclonal Antibody (CST, Cat # 3724), Ubiquitin Antibody, (Santa Cruz, Cat # sc‐8017), K63‐linkage Specific Polyubiquitin (D7A11) Rabbit mAb (CST, Cat # 12 930), K48‐linkage Specific Polyubiquitin (D9D5) Rabbit mAb (CST, Cat # 12 805), CACYBP Polyclonal Antibody (Abclonal, Cat # A8757), TRIM4 Polyclonal Antibody (Abclonal, Cat # A15922), WWP2 Polyclonal Antibody (Abclonal, Cat # 12197‐1‐AP), Myc‐tag Rabbit Polyclonal Antibody (Proteintech, Cat # I6286‐I‐AP), Goat Anti‐Mouse IgG Antibody (H+L), DyLight 488 (SeraCare, Cat # 5230‐0391), Goat Anti‐Rabbit IgG H&L (Alexa Fluor 647) (abcam, Cat # ab150083), GST Tag Antibody (ABGENT), ZIKV virus NS1 protein antibody (GeneTex, Cat # GTX133307, GTX634158).

Techniques: Transfection, Western Blot, Quantitative RT-PCR, Ubiquitin Proteomics, Immunoprecipitation, Purification, Incubation, In Vitro

Journal: Advanced Science

Article Title: Ubiquitination of NS1 Confers Differential Adaptation of Zika Virus in Mammalian Hosts and Mosquito Vectors

doi: 10.1002/advs.202408024

Figure Lengend Snippet: WWP2 expression was upregulated during ZIKV infection. A) SH‐sy5y or 293T cells were infected with ZIKV (MR766) (MOI = 0.5). WWP2 levels was analyzed by qRT‐PCR and Western Blot. B) THP‐1 or 293T cells were treated with IFN‐α (500 U ml −1 ) for 6 h, and WWP2 levels were determined using qRT‐PCR and Western Blot. C) WWP2 expression was up‐regulated during ZIKV infection based on GEO databases. Data are representative of 3 independent experiments and presented as mean ± SD. ** P < 0.01, and *** P < 0.001, **** P < 0.0001 (Student's t‐test).

Article Snippet: The following antibodies were used in this study: GAPDH Mouse Antibody (Proteintech, Cat # 60004‐I‐Ig), DYKDDDDK Tag Mouse Antibody (ABclonal, Cat # AE005), anti‐rabbit IgG HRP‐linked antibody (CST, Cat # 7074), HRP goat anti‐mouse IgG (BioLegend, Cat # 405 306), HA‐tag Rabbit Polyclonal Antibody (CST, Cat # 3724), Ubiquitin Antibody, (Santa Cruz, Cat # sc‐8017), K63‐linkage Specific Polyubiquitin (D7A11) Rabbit mAb (CST, Cat # 12 930), K48‐linkage Specific Polyubiquitin (D9D5) Rabbit mAb (CST, Cat # 12 805), CACYBP Polyclonal Antibody (Abclonal, Cat # A8757), TRIM4 Polyclonal Antibody (Abclonal, Cat # A15922), WWP2 Polyclonal Antibody (Abclonal, Cat # 12197‐1‐AP), Myc‐tag Rabbit Polyclonal Antibody (Proteintech, Cat # I6286‐I‐AP), Goat Anti‐Mouse IgG Antibody (H+L), DyLight 488 (SeraCare, Cat # 5230‐0391), Goat Anti‐Rabbit IgG H&L (Alexa Fluor 647) (abcam, Cat # ab150083), GST Tag Antibody (ABGENT), ZIKV virus NS1 protein antibody (GeneTex, Cat # GTX133307, GTX634158).

Techniques: Expressing, Infection, Quantitative RT-PCR, Western Blot

Journal: Advanced Science

Article Title: Ubiquitination of NS1 Confers Differential Adaptation of Zika Virus in Mammalian Hosts and Mosquito Vectors

doi: 10.1002/advs.202408024

Figure Lengend Snippet: WWP2 restricts ZIKV infection. A–C) SH‐sy5y (A)/U3A (B) cells were infected with lentivirus overexpressing or knocking down WWP2 (MOI = 10). Subsequently, cells were infected with ZIKV (MOI = 0.5) 48 h later. Viral mRNA levels in the cells were detected 24 h later using qRT‐PCR. Viral load in the supernatant was visualized by TCID50, and the infectious viral load in U3A supernatants was determined by plaque assay (C). D) WWP2‐Myc (WT or C838A) plasmid was transfected into 293T cells and infected with ZIKV (MOI = 0.5) after 24 h. Cellular RNA was extracted at 24 and 48 h, and the viral RNA levels were analyzed by qRT‐PCR. The viral supernatant titer after 48 h was determined by TCID50. E‐H) Ifnar1 −/− mice (6 weeks old, 12 mice per group) were injected with 5 × 10 7 PFU shmWWP2 lentivirus via the tail‐vein route; 7 days later, mice were injected intraperitoneally with 10 7 PFU ZIKV. Hemocytes and serum were collected on days 3 and 5. Blood cell RNA was extracted, and qRT‐PCR was used to detect the RNA content of ZIKV and shmWWP2 in the cells E). Viral titers in the serum of mice on day 5 were detected by TCID50 F). Infectious virus in the serum of mice on day 5 was detected by the plaque assay G). The status and survival of mice were recorded by daily observation (* P < 0.05, Log‐rank test) (H). Data are representative of 3 independent experiments and presented as mean ± SD. ns, non‐significant, * P < 0.05, ** P < 0.01, and *** P < 0.001 (Student's t‐test).

Article Snippet: The following antibodies were used in this study: GAPDH Mouse Antibody (Proteintech, Cat # 60004‐I‐Ig), DYKDDDDK Tag Mouse Antibody (ABclonal, Cat # AE005), anti‐rabbit IgG HRP‐linked antibody (CST, Cat # 7074), HRP goat anti‐mouse IgG (BioLegend, Cat # 405 306), HA‐tag Rabbit Polyclonal Antibody (CST, Cat # 3724), Ubiquitin Antibody, (Santa Cruz, Cat # sc‐8017), K63‐linkage Specific Polyubiquitin (D7A11) Rabbit mAb (CST, Cat # 12 930), K48‐linkage Specific Polyubiquitin (D9D5) Rabbit mAb (CST, Cat # 12 805), CACYBP Polyclonal Antibody (Abclonal, Cat # A8757), TRIM4 Polyclonal Antibody (Abclonal, Cat # A15922), WWP2 Polyclonal Antibody (Abclonal, Cat # 12197‐1‐AP), Myc‐tag Rabbit Polyclonal Antibody (Proteintech, Cat # I6286‐I‐AP), Goat Anti‐Mouse IgG Antibody (H+L), DyLight 488 (SeraCare, Cat # 5230‐0391), Goat Anti‐Rabbit IgG H&L (Alexa Fluor 647) (abcam, Cat # ab150083), GST Tag Antibody (ABGENT), ZIKV virus NS1 protein antibody (GeneTex, Cat # GTX133307, GTX634158).

Techniques: Infection, Quantitative RT-PCR, Plaque Assay, Plasmid Preparation, Transfection, Injection, Virus

Journal: Advanced Science

Article Title: Ubiquitination of NS1 Confers Differential Adaptation of Zika Virus in Mammalian Hosts and Mosquito Vectors

doi: 10.1002/advs.202408024

Figure Lengend Snippet: WWP2 ubiquitinates amino acids K265 and K284 of ZIKV NS1. A and B) WWP2‐Myc was co‐transfected with NS1‐WT or its mutants in 293T cells for 24 h. After 24 h, the cells were treated with MG132 (5 µM) for 4 h. NS1‐Flag was immunoprecipitated, and the ubiquitylation level of NS1 proteins was detected by Western Blot A). The protein level of NS1 was detected by Western Blot B). C) NS1‐WT or its mutants were transfected into 293T cells and treated with actinomycin ketone CHX (50 µM) for 0, 2, 4, and 8 h after 24 h. NS1 protein levels were detected by Western Blot. D) Secondary mass spectrometry analysis of ubiquitinations at positions K265 and K284 of NS1. E) WWP2‐Myc, NS1‐Flag, and ubiquitin molecule mutant plasmids (K6, K11, K27, K29, K33, K48, and K63) were co‐transfected into 293T cells. After 24 h, the cells were treated with MG132 (5 µM, 4 h), and NS1‐Flag was immunoprecipitated. NS1 proteins were detected by Western Blot method, and the ubiquitination level was assessed. F and G) WWP2‐Myc or shWWP2 (1 µg), NS1‐Flag, were co‐transfected into 293T cells. After 24 h, the cells were treated with MG132 (5 µM, 4 h), and NS1‐Flag was immunoprecipitated. NS1 proteins were detected by Western Blot, and the ubiquitination types of NS1 were detected using K48 and K63 antibodies. H and I) WWP2‐Myc, NS1‐Flag individual point mutants, and ubiquitin molecule mutant K48/K63‐HA were co‐transfected into 293T cells. After 24 h, the cells were treated with MG132 (5 µM, 4 h), and immunoprecipitated with NS1‐Flag. The level of ubiquitination of NS1 proteins was detected by Western Blot. Data are representative of 3 independent experiments.

Article Snippet: The following antibodies were used in this study: GAPDH Mouse Antibody (Proteintech, Cat # 60004‐I‐Ig), DYKDDDDK Tag Mouse Antibody (ABclonal, Cat # AE005), anti‐rabbit IgG HRP‐linked antibody (CST, Cat # 7074), HRP goat anti‐mouse IgG (BioLegend, Cat # 405 306), HA‐tag Rabbit Polyclonal Antibody (CST, Cat # 3724), Ubiquitin Antibody, (Santa Cruz, Cat # sc‐8017), K63‐linkage Specific Polyubiquitin (D7A11) Rabbit mAb (CST, Cat # 12 930), K48‐linkage Specific Polyubiquitin (D9D5) Rabbit mAb (CST, Cat # 12 805), CACYBP Polyclonal Antibody (Abclonal, Cat # A8757), TRIM4 Polyclonal Antibody (Abclonal, Cat # A15922), WWP2 Polyclonal Antibody (Abclonal, Cat # 12197‐1‐AP), Myc‐tag Rabbit Polyclonal Antibody (Proteintech, Cat # I6286‐I‐AP), Goat Anti‐Mouse IgG Antibody (H+L), DyLight 488 (SeraCare, Cat # 5230‐0391), Goat Anti‐Rabbit IgG H&L (Alexa Fluor 647) (abcam, Cat # ab150083), GST Tag Antibody (ABGENT), ZIKV virus NS1 protein antibody (GeneTex, Cat # GTX133307, GTX634158).

Techniques: Transfection, Immunoprecipitation, Western Blot, Mass Spectrometry, Ubiquitin Proteomics, Mutagenesis

Journal: Advanced Science

Article Title: Ubiquitination of NS1 Confers Differential Adaptation of Zika Virus in Mammalian Hosts and Mosquito Vectors

doi: 10.1002/advs.202408024

Figure Lengend Snippet: Amino acid mutation at position NS1 K265, K284 alters ZIKV virulence. A and B) Schematic diagram of the ZIKV packaging process (A): The full‐length plasmid of the 2016 GZ01 strain was used as a template. The full‐length plasmid of the NS1 point‐mutated K265R, K284R, and K265/284R viral genomes was obtained by targeted mutagenesis. The full‐length plasmid was transfected with RNA into BHK21 cells after in vitro transcription, and the viral supernatant was collected after culture to obtain the WT viruses and mutant viruses (B). C–E) WT, K265R, K284R, and K265/284R viruses were packaged with the same mass of RNA, and the titer of the viral particles was detected by TCID50 (C). 293T cells were infected with the same titer of the mutant viruses (MOI = 1), and the intracellular viral load was detected by qRT‐PCR 48 h later (D). The same titer of mutant viruses was used to infect 293T cells (MOI = 1), and the level of ZIKV NS1 in the supernatant was detected by ELISA after 72 h (E). F and G) 293T (F) and SH‐sy5y (G) cells were infected with lentiviruses knocking down the expression of WWP2, and then infected with WT and mutant viruses after 48 h. Cells were collected after 48 h to extract the RNA, and the viral load in the cells was detected by qRT‐PCR. H) Ifnar1 −/− mice were infected with 10 7 PFU viruses (WT, K265R, K284R, and K265/284R), and viral loads were detected by qRT‐PCR on day 5 after infection. I) A search of the Virus Sequence Library ( https://nextstrain.org ) revealed the existence of a naturally occurring strain of ZIKV NS1 mutated at amino acid positions 265/284. J) Model for regulation of ZIKV NS1 by WWP2. Data are representative of 3 independent experiments and presented as mean ± SD. ns, non‐significant, * P < 0.05, ** P < 0.01, and *** P < 0.001, **** P < 0.0001 (Student's t‐test).

Article Snippet: The following antibodies were used in this study: GAPDH Mouse Antibody (Proteintech, Cat # 60004‐I‐Ig), DYKDDDDK Tag Mouse Antibody (ABclonal, Cat # AE005), anti‐rabbit IgG HRP‐linked antibody (CST, Cat # 7074), HRP goat anti‐mouse IgG (BioLegend, Cat # 405 306), HA‐tag Rabbit Polyclonal Antibody (CST, Cat # 3724), Ubiquitin Antibody, (Santa Cruz, Cat # sc‐8017), K63‐linkage Specific Polyubiquitin (D7A11) Rabbit mAb (CST, Cat # 12 930), K48‐linkage Specific Polyubiquitin (D9D5) Rabbit mAb (CST, Cat # 12 805), CACYBP Polyclonal Antibody (Abclonal, Cat # A8757), TRIM4 Polyclonal Antibody (Abclonal, Cat # A15922), WWP2 Polyclonal Antibody (Abclonal, Cat # 12197‐1‐AP), Myc‐tag Rabbit Polyclonal Antibody (Proteintech, Cat # I6286‐I‐AP), Goat Anti‐Mouse IgG Antibody (H+L), DyLight 488 (SeraCare, Cat # 5230‐0391), Goat Anti‐Rabbit IgG H&L (Alexa Fluor 647) (abcam, Cat # ab150083), GST Tag Antibody (ABGENT), ZIKV virus NS1 protein antibody (GeneTex, Cat # GTX133307, GTX634158).

Techniques: Mutagenesis, Plasmid Preparation, Transfection, In Vitro, Infection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing, Virus, Sequencing

Journal: Advanced Science

Article Title: Ubiquitination of NS1 Confers Differential Adaptation of Zika Virus in Mammalian Hosts and Mosquito Vectors

doi: 10.1002/advs.202408024

Figure Lengend Snippet: WWP2 is a broad‐spectrum arthropod‐borne flavivirus suppressor. A) Conservation of site 284 of the arthropod‐borne flavivirus NS1 protein. B–D) After overexpression or knockdown of WWP2 in 293T (B) and U3A (C) cells, the cells were infected with JEV (MOI = 0.5), and cellular RNA was extracted after 24 and 48 h. The viral content of the cells was detected by qRT‐PCR; the amount of infectious viruses in the supernatant of U3A was detected by plaque assay (D). E–G) Using WT and Wwp 2 −/− mice, 10 7 PFU JEV (SA14) was injected intraperitoneally, and hemocytes and serum were collected by orbital blood sampling on days 3 and 5, respectively. Blood cell RNA was extracted, and the amount of JEV in the cells was detected using qRT‐PCR (E); the viral titer in the serum of mice on day 5 was detected by TCID50 (F); the survival of mice was observed and recorded daily (* P<0.05, Log‐rank test) (G). H) JEV NS1‐Flag and WWP2‐Myc/shWWP2 (1 µg) plasmids were co‐transfected in 293T cells and treated with MG132 (5 µM) for 4 h after 48 h. NS1‐Flag was immunoprecipitated, and ubiquitination of JEV NS1‐Flag protein was detected by Western Blot. I) JEV NS1‐Flag and WWP2‐Myc/shWWP2 (1 µg) plasmids were co‐transfected in 293T cells, and the cells were collected after 48 h. NS1 protein levels were detected by Western Blot. J) JEV NS1‐Flag and WWP2‐Myc were co‐transfected in 293T cells, which were treated with MG132 (5 µM) for 4 h after 24 h. JEV NS1 protein levels were detected by Western Blot. K‐N) Cells were infected with LGTV after overexpression or knockdown of WWP2 in 293T (K) and U3A cells (M) (MOI = 1), and cellular RNA was extracted after 48 h. The viral RNA load in the cells was detected by using qRT‐PCR; etch‐a‐sketch assay was performed to detect the amount of infectious virus in the supernatants of U3A cells (N). Data are representative of 3 independent experiments and presented as mean ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001 (Student's t‐test).

Article Snippet: The following antibodies were used in this study: GAPDH Mouse Antibody (Proteintech, Cat # 60004‐I‐Ig), DYKDDDDK Tag Mouse Antibody (ABclonal, Cat # AE005), anti‐rabbit IgG HRP‐linked antibody (CST, Cat # 7074), HRP goat anti‐mouse IgG (BioLegend, Cat # 405 306), HA‐tag Rabbit Polyclonal Antibody (CST, Cat # 3724), Ubiquitin Antibody, (Santa Cruz, Cat # sc‐8017), K63‐linkage Specific Polyubiquitin (D7A11) Rabbit mAb (CST, Cat # 12 930), K48‐linkage Specific Polyubiquitin (D9D5) Rabbit mAb (CST, Cat # 12 805), CACYBP Polyclonal Antibody (Abclonal, Cat # A8757), TRIM4 Polyclonal Antibody (Abclonal, Cat # A15922), WWP2 Polyclonal Antibody (Abclonal, Cat # 12197‐1‐AP), Myc‐tag Rabbit Polyclonal Antibody (Proteintech, Cat # I6286‐I‐AP), Goat Anti‐Mouse IgG Antibody (H+L), DyLight 488 (SeraCare, Cat # 5230‐0391), Goat Anti‐Rabbit IgG H&L (Alexa Fluor 647) (abcam, Cat # ab150083), GST Tag Antibody (ABGENT), ZIKV virus NS1 protein antibody (GeneTex, Cat # GTX133307, GTX634158).

Techniques: Over Expression, Knockdown, Infection, Quantitative RT-PCR, Plaque Assay, Injection, Sampling, Transfection, Immunoprecipitation, Ubiquitin Proteomics, Western Blot, Virus

Journal: Advanced Science

Article Title: Ubiquitination of NS1 Confers Differential Adaptation of Zika Virus in Mammalian Hosts and Mosquito Vectors

doi: 10.1002/advs.202408024

Figure Lengend Snippet: Ubiquitination of NS1 by WWP2 homologs in mosquitoes promotes ZIKV infection of mosquitoes. A) ZIKV NS1‐Flag was transfected in C6/36 cells and treated with MG132 (5 µM) for 4 h after 24 h. Cells were collected and immunoprecipitated with Flag antibody‐coupled magnetic beads, and the ubiquitination level of viral proteins was detected by Western Blot. B) The E3 ligase Su(dx), which is highly homologous to human WWP2, is present in Aedes albopictus. (WWP2 GenBank: U96114.2; Su(dx) GenBank: XM_01 969 6185.2) C) NS1‐Flag was co‐transfected with Su(dx)‐His expression plasmid in C6/36 cells and infected with ZIKV (MR766) (MOI = 0.5) 24 h later for 24 h. NS1‐Flag was immunoprecipitated, and Su(dx)‐His protein was detected by Western Blot. D) NS1‐Flag and different doses of Su(dx) were co‐transfected in C6/36 cells, and NS1‐Flag protein levels were detected by Western Blot 24 h later. E) Co‐transfected siSu(dx) (50 nM) with NS1‐Flag (1 µg) in C6/36 cells, treated with MG132 (5 µM) for 4 h after 24 h. Immunoprecipitation of NS1‐Flag was performed, and the ubiquitination level of NS1 protein was detected by Western Blot method. F) Su(dx)‐Flag (1 µg) was transfected into C6/36 cells. After 24 h, the cells were infected with WT, K265R, K284R and K265, 284R viruses (MOI = 0.5), respectively. 48 h later, the cells were treated with MG132 (5 µM, 4 h) and NS1 was immunoprecipitated. NS1 protein was detected by Western Blot and its ubiquitination level was determined. G) Transfection of siSu(dx) (50 nM) in C6/36 cells was followed by infection with ZIKV (MOI = 1) after 48 h. Viral mRNA levels in the cells, as well as Su(dx) knockdown efficiency, were detected after 24 h using qRT‐PCR. H) Su(dx)‐His was transfected in C6/36 cells, infected with ZIKV (MOI = 1) 24 h later, and the viral mRNA level as well as the efficiency of Su(dx) overexpression was detected in the cells 48 h later using qRT‐PCR. I) NSC2805 (10 µM, 4 h) treated C6/36 cells were infected with ZIKV and viral RNA levels were detected by qRT‐PCR at 24 h J) Aedes aegypti mosquitoes were divided into two groups, the experimental group was injected with Su(dx) dsRNA, and the control group was injected with Luc dsRNA. 100 PFU of MR766 strain virus was injected into each mosquito. The viral mRNA level and Su(dx) knockdown efficiency in mosquitoes were detected by qRT‐PCR on day 7 after infection. K) Recombinant viruses (WT, K265R, K284R, and K265/284R) of the same titer were injected into the thoracic cavity of Aedes aegypti mosquitoes (50 PFU of virus per mosquito), and viral loads in the mosquitoes were detected by qRT‐PCR on day 7 after infection. Data are representative of 3 independent experiments and presented as mean ± SD. ns, non‐significant, * P < 0.05, ** P < 0.01, and *** P < 0.001, **** P < 0.0001 (Student's t‐test).

Article Snippet: The following antibodies were used in this study: GAPDH Mouse Antibody (Proteintech, Cat # 60004‐I‐Ig), DYKDDDDK Tag Mouse Antibody (ABclonal, Cat # AE005), anti‐rabbit IgG HRP‐linked antibody (CST, Cat # 7074), HRP goat anti‐mouse IgG (BioLegend, Cat # 405 306), HA‐tag Rabbit Polyclonal Antibody (CST, Cat # 3724), Ubiquitin Antibody, (Santa Cruz, Cat # sc‐8017), K63‐linkage Specific Polyubiquitin (D7A11) Rabbit mAb (CST, Cat # 12 930), K48‐linkage Specific Polyubiquitin (D9D5) Rabbit mAb (CST, Cat # 12 805), CACYBP Polyclonal Antibody (Abclonal, Cat # A8757), TRIM4 Polyclonal Antibody (Abclonal, Cat # A15922), WWP2 Polyclonal Antibody (Abclonal, Cat # 12197‐1‐AP), Myc‐tag Rabbit Polyclonal Antibody (Proteintech, Cat # I6286‐I‐AP), Goat Anti‐Mouse IgG Antibody (H+L), DyLight 488 (SeraCare, Cat # 5230‐0391), Goat Anti‐Rabbit IgG H&L (Alexa Fluor 647) (abcam, Cat # ab150083), GST Tag Antibody (ABGENT), ZIKV virus NS1 protein antibody (GeneTex, Cat # GTX133307, GTX634158).

Techniques: Ubiquitin Proteomics, Infection, Transfection, Immunoprecipitation, Magnetic Beads, Western Blot, Expressing, Plasmid Preparation, Knockdown, Quantitative RT-PCR, Over Expression, Injection, Control, Virus, Recombinant

Journal: Journal of Virology

Article Title: The Raf kinase inhibitors Dabrafenib and Regorafenib impair Zika virus replication via distinct mechanisms

doi: 10.1128/jvi.00618-24

Figure Lengend Snippet: Dabrafenib and Regorafenib inhibit ZIKV but not DENV2 replication. ( A and B ) A549 cells were mock-infected or infected with ZIKV (MOI = 0.01 PFU/cell) and subsequently treated with the indicated SMKIs or DMSO (vehicle control). ( A ) Viral titers in the supernatant were determined at 72 hpi by endpoint dilution assay. ( B ) ZIKV infection was visualized at 72 hpi by immunofluorescence staining for ZIKV E protein (green). Nuclei were stained with Hoechst 33342 (blue). Scale bar, 200 µm. Representative images of two independent experiments are shown. ( C ) A549 cells were infected with ZIKV (MOI = 0.01 PFU/cell) and subsequently treated with DMSO, bosutinib (5 µM), Dabrafenib (10 µM), or Regorafenib (2.5 µM). Viral titers in the supernatant were determined at 2, 24, 48, and 72 hpi by endpoint dilution assay. ( D ) A549 cells were infected with ZIKV (MOI = 1 PFU/cell) and subsequently treated with DMSO, bosutinib (5 µM), Dabrafenib (10 µM), or Regorafenib (2.5 µM). Viral titers in the supernatant were determined at 48 hpi by endpoint dilution assay. ( E ) A549 cells were infected with ZIKV (MOI = 0.01 PFU/cell) and subsequently treated with the indicated SMKIs or DMSO. Viral titers in the supernatant were determined at 72 hpi by endpoint dilution assay. ( F ) Vero cells were infected with ZIKV (MOI = 0.01 PFU/cell) and subsequently treated with DMSO, Dabrafenib (2.5 µM), or Regorafenib (1 µM). Viral titers in the supernatant were determined at 48 hpi by endpoint dilution assay. ( G ) A549 cells were mock-infected or infected with DENV2 (MOI = 0.01 PFU/cell) and subsequently treated with DMSO, Dabrafenib (10 µM), or Regorafenib (2.5 µM). Viral titers in the supernatant were determined at 72 hpi by endpoint dilution assay. Data are expressed as mean ± standard error of the mean (SEM) from two independent experiments with three biological replicates per experiment. *, P < 0.05; **, P < 0.01 (Mann–Whitney U test vs “DMSO”).

Article Snippet: The following primary antibodies were used: mouse monoclonal anti-ZIKV NS1 antibody (1:1,000; clone GT5212, GeneTex), rabbit polyclonal anti-ZIKV E protein antibody (1:1,000; Biorbyt), rabbit polyclonal anti-phospho-eIF2α (Ser51) antibody (1:2,000; Proteintech), rabbit polyclonal anti-eIF2α antibody (1:2,000; Proteintech), rabbit monoclonal anti-GAPDH antibody (1:2,000; clone D16H11, Cell Signaling Technology), and rabbit monoclonal anti-vinculin antibody (1:5,000; clone 3M13, Sigma-Aldrich).

Techniques: Infection, Control, Endpoint Dilution Assay, Immunofluorescence, Staining, MANN-WHITNEY

Journal: Journal of Virology

Article Title: The Raf kinase inhibitors Dabrafenib and Regorafenib impair Zika virus replication via distinct mechanisms

doi: 10.1128/jvi.00618-24

Figure Lengend Snippet: Dabrafenib and Regorafenib target ZIKV infection at the post-entry stage. ( A ) Schematic representation of the time-of-addition assays. ( B ) A549 cells were pre-treated with DMSO (vehicle control), Dabrafenib (10 µM), or Regorafenib (2.5 µM) for 2 h prior to infection with ZIKV (MOI = 3 PFU/cell) as well as during the adsorption period. ZIKV infection was visualized at 18 hpi by immunofluorescence staining for ZIKV E protein (green). Nuclei were stained with Hoechst 33342 (blue). Scale bar, 200 µm. Images are representative of three independent experiments. Bar graph shows the proportions of infected cells determined from immunofluorescence images (one per experiment) using ImageJ. ( C and D ) A549 cells were infected with ZIKV (MOI = 3 PFU/cell) and subsequently treated with DMSO, Dabrafenib (10 µM), or Regorafenib (2.5 µM). ( C ) ZIKV infection was visualized at 24, 48, and 72 hpi by immunofluorescence staining for ZIKV E protein (green). Nuclei were stained with Hoechst 33342 (blue). Scale bar, 200 µm. Images are representative of three independent experiments. ( D ) Viral titers in the supernatant were determined at 24, 48, and 72 hpi by endpoint dilution assay. Data are expressed as mean ± SEM from three independent experiments with three biological replicates per experiment. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Mann–Whitney U test vs “DMSO”).

Article Snippet: The following primary antibodies were used: mouse monoclonal anti-ZIKV NS1 antibody (1:1,000; clone GT5212, GeneTex), rabbit polyclonal anti-ZIKV E protein antibody (1:1,000; Biorbyt), rabbit polyclonal anti-phospho-eIF2α (Ser51) antibody (1:2,000; Proteintech), rabbit polyclonal anti-eIF2α antibody (1:2,000; Proteintech), rabbit monoclonal anti-GAPDH antibody (1:2,000; clone D16H11, Cell Signaling Technology), and rabbit monoclonal anti-vinculin antibody (1:5,000; clone 3M13, Sigma-Aldrich).

Techniques: Infection, Control, Adsorption, Immunofluorescence, Staining, Endpoint Dilution Assay, MANN-WHITNEY

Journal: Journal of Virology

Article Title: The Raf kinase inhibitors Dabrafenib and Regorafenib impair Zika virus replication via distinct mechanisms

doi: 10.1128/jvi.00618-24

Figure Lengend Snippet: Dabrafenib, but not Regorafenib, reduces viral dsRNA levels. A549 cells were infected with ZIKV (MOI = 3 PFU/cell) and subsequently treated with DMSO (vehicle control), Dabrafenib (10 µM), or Regorafenib (2.5 µM). ZIKV infection was visualized at 24, 48, and 72 hpi by immunofluorescence staining for ZIKV E protein (red) and dsRNA (green). Nuclei were stained with Hoechst 33342 (blue). Scale bars, 100 µm. Dot plot shows image-based quantification of the integrated density of dsRNA in individual cells. Data are expressed as mean ± SEM. Data from one of three independent experiments with similar results are shown. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (unpaired Student’s t test vs “DMSO”).

Article Snippet: The following primary antibodies were used: mouse monoclonal anti-ZIKV NS1 antibody (1:1,000; clone GT5212, GeneTex), rabbit polyclonal anti-ZIKV E protein antibody (1:1,000; Biorbyt), rabbit polyclonal anti-phospho-eIF2α (Ser51) antibody (1:2,000; Proteintech), rabbit polyclonal anti-eIF2α antibody (1:2,000; Proteintech), rabbit monoclonal anti-GAPDH antibody (1:2,000; clone D16H11, Cell Signaling Technology), and rabbit monoclonal anti-vinculin antibody (1:5,000; clone 3M13, Sigma-Aldrich).

Techniques: Infection, Control, Immunofluorescence, Staining

Journal: Journal of Virology

Article Title: The Raf kinase inhibitors Dabrafenib and Regorafenib impair Zika virus replication via distinct mechanisms

doi: 10.1128/jvi.00618-24

Figure Lengend Snippet: Dabrafenib, but not Regorafenib, attenuates viral RNA synthesis. ( A ) A549 cells were infected with ZIKV (MOI = 3 PFU/cell) and subsequently treated with DMSO (vehicle control), Dabrafenib (10 µM), or Regorafenib (2.5 µM). Total cell-associated RNA was isolated at 24, 48, and 72 hpi, and analyzed by positive strand-specific RT-qPCR (upper left panel) and negative strand-specific RT-qPCR (upper right panel). Lower left panel: Results shown in the upper panels plotted as the ratio of positive-strand RNA [(+)RNA] to negative-strand RNA [(−)RNA]. Data are expressed as mean ± SEM from two independent experiments with two biological replicates per experiment. *, P < 0.05; **, P < 0.01 (two-way ANOVA vs “DMSO”). ( B ) A549 cells were mock-infected or infected with ZIKV (MOI = 3 PFU/cell) and were left untreated (Untr) or were treated with DMSO (vehicle control), Dabrafenib (Db; 10 µM), or Regorafenib (Rg; 2.5 µM) for the indicated durations. Whole-cell lysates were analyzed by Western blotting with antibodies against ZIKV NS5 and GAPDH (loading control). Black dotted lines indicate removal of portions of the blots for clarity.

Article Snippet: The following primary antibodies were used: mouse monoclonal anti-ZIKV NS1 antibody (1:1,000; clone GT5212, GeneTex), rabbit polyclonal anti-ZIKV E protein antibody (1:1,000; Biorbyt), rabbit polyclonal anti-phospho-eIF2α (Ser51) antibody (1:2,000; Proteintech), rabbit polyclonal anti-eIF2α antibody (1:2,000; Proteintech), rabbit monoclonal anti-GAPDH antibody (1:2,000; clone D16H11, Cell Signaling Technology), and rabbit monoclonal anti-vinculin antibody (1:5,000; clone 3M13, Sigma-Aldrich).

Techniques: Infection, Control, Isolation, Quantitative RT-PCR, Western Blot

Journal: Journal of Virology

Article Title: The Raf kinase inhibitors Dabrafenib and Regorafenib impair Zika virus replication via distinct mechanisms

doi: 10.1128/jvi.00618-24

Figure Lengend Snippet: Regorafenib reduces ZIKV E protein, but not NS1, levels and does not activate the integrated stress response. ( A ) A549 cells were either left untreated (Untr) or were treated with DMSO (vehicle control), Axitinib (Ax; 10 µM), Dabrafenib (Db; 10 µM), or Regorafenib (Rg; 2.5 µM) for the indicated durations. As a positive control, cells were treated with DTT (2 mM) for 30 min prior to harvesting. Whole-cell lysates were analyzed by Western blotting with antibodies against phospho-eIF2α (Ser51), total eIF2α, and vinculin (loading control). ( B ) A549 cells were mock-infected or infected with ZIKV (MOI = 3 PFU/cell) and were left untreated (Untr) or were treated with DMSO (vehicle control), Axitinib (Ax; 10 µM), Dabrafenib (Db; 10 µM), or Regorafenib (Rg; 2.5 µM) for the indicated durations. As a positive control, cells were treated with DTT (2 mM) for 30 min prior to harvesting. Whole-cell lysates were analyzed by Western blotting with antibodies against phospho-eIF2α (Ser51), total eIF2α, ZIKV E protein, and vinculin (loading control). Ratios of phospho-eIF2α to total eIF2α (P/T) and the relative levels of ZIKV E protein and ZIKV NS1 were determined by densitometric analysis. The latter are shown as bar graphs and are expressed as mean ± standard deviation (SD). **, P < 0.01; ****, P < 0.0001 (multiple t test with Holm–Šidák correction vs “DMSO”). Blots in ( A ) and ( B ) are representative of two independent experiments. ( C ) A549 cells were treated with DTT (2 mM) for 30 min prior to fixation and SGs were visualized by immunofluorescence staining for G3BP1 (red). Nuclei were stained with Hoechst 33342 (blue). Scale bar, 50 µm. ( D ) A549 cells were mock-infected or infected with ZIKV (MOI = 3 PFU/cell) and were left untreated (Untr) or treated with DMSO (vehicle control) or Regorafenib (2.5 µM) for 24 h. ZIKV infection and SGs were visualized by immunofluorescence staining for ZIKV E protein (green) and G3BP1 (red), respectively. Scale bars, 50 µm. Images are representative of two independent experiments.

Article Snippet: The following primary antibodies were used: mouse monoclonal anti-ZIKV NS1 antibody (1:1,000; clone GT5212, GeneTex), rabbit polyclonal anti-ZIKV E protein antibody (1:1,000; Biorbyt), rabbit polyclonal anti-phospho-eIF2α (Ser51) antibody (1:2,000; Proteintech), rabbit polyclonal anti-eIF2α antibody (1:2,000; Proteintech), rabbit monoclonal anti-GAPDH antibody (1:2,000; clone D16H11, Cell Signaling Technology), and rabbit monoclonal anti-vinculin antibody (1:5,000; clone 3M13, Sigma-Aldrich).

Techniques: Control, Positive Control, Western Blot, Infection, Standard Deviation, Immunofluorescence, Staining

Journal: Journal of Virology

Article Title: The Raf kinase inhibitors Dabrafenib and Regorafenib impair Zika virus replication via distinct mechanisms

doi: 10.1128/jvi.00618-24

Figure Lengend Snippet: Regorafenib treatment results in inefficient ZIKV NS1 secretion. ( A ) Schematic representation of the cycloheximide (CHX)–chase experiment. ( B ) A549 cells were mock-infected or infected with ZIKV (MOI = 3 PFU/cell) and subsequently treated with DMSO (vehicle control), Axitinib (Ax; 10 µM), Dabrafenib (Db; 10 µM), or Regorafenib (Rg; 2.5 µM). At 48 hpi, cells were exposed to cycloheximide (10 µg/mL) and chased for 0, 12, and 24 h. Whole-cell lysates were analyzed by Western blotting with antibodies against ZIKV E protein, ZIKV NS1, and GAPDH (loading control). Blots are representative of two independent experiments. ( C ) A549 cells were mock-infected or infected with ZIKV (MOI = 3 PFU/cell) and subsequently treated with DMSO (vehicle control), Axitinib (Ax; 10 µM), Dabrafenib (Db; 10 µM), or Regorafenib (Rg; 2.5 µM). At 56 hpi, cells were exposed to MG132 (10 µM) or left untreated. Whole-cell lysates were collected at 72 hpi and analyzed by Western blotting with antibodies against ZIKV E protein, ZIKV NS1, and vinculin (loading control). ( D ) A549 cells were infected with ZIKV (MOI = 3) and subsequently treated with DMSO (vehicle control), Axitinib (Ax; 10 µM), Dabrafenib (Db; 2.5 µM), or Regorafenib (Rg; 2.5 µM). Culture supernatants were collected at 72 hpi, precipitated with TCA and analyzed by Western blotting with an antibody against ZIKV NS1. Ponceau S staining served as a loading control. Whole-cell lysates were processed in parallel and analyzed by Western blotting with antibodies against ZIKV NS1 and GAPDH (loading control). Black dotted lines indicate removal of portions of the blots for clarity.

Article Snippet: The following primary antibodies were used: mouse monoclonal anti-ZIKV NS1 antibody (1:1,000; clone GT5212, GeneTex), rabbit polyclonal anti-ZIKV E protein antibody (1:1,000; Biorbyt), rabbit polyclonal anti-phospho-eIF2α (Ser51) antibody (1:2,000; Proteintech), rabbit polyclonal anti-eIF2α antibody (1:2,000; Proteintech), rabbit monoclonal anti-GAPDH antibody (1:2,000; clone D16H11, Cell Signaling Technology), and rabbit monoclonal anti-vinculin antibody (1:5,000; clone 3M13, Sigma-Aldrich).

Techniques: Infection, Control, Western Blot, Staining

Journal: Journal of Virology

Article Title: The Raf kinase inhibitors Dabrafenib and Regorafenib impair Zika virus replication via distinct mechanisms

doi: 10.1128/jvi.00618-24

Figure Lengend Snippet: Regorafenib alters ER morphology in ZIKV-infected cells and limits viral egress. ( A and B ) A549 cells were mock-infected or infected with ZIKV (MOI = 3 PFU/cell) and subsequently treated with DMSO (vehicle control) or Regorafenib (2.5 µM). At 24 hpi ( A ) and 48 hpi ( B ), ZIKV infection and ER were visualized by immunofluorescence staining for ZIKV E protein (green) and calnexin (red), respectively. Nuclei were stained with Hoechst 33342 (blue). Scale bars, 25 µm. Images are representative of two independent experiments. ( C ) A549 cells were infected with ZIKV (MOI = 3 PFU/cell) and subsequently treated with DMSO (vehicle control), Dabrafenib (10 µM), or Regorafenib (2.5 µM). At 24, 48, and 72 hpi, total cell-associated RNA and extracellular viral RNA were isolated, and quantified by RT-qPCR. Data were normalized to DMSO control, and are expressed as mean ± SEM from two independent experiments with two biological replicates per experiment. *, P < 0.05; **, P < 0.01 (unpaired Student’s t test, “cell associated” vs “extracellular”).

Article Snippet: The following primary antibodies were used: mouse monoclonal anti-ZIKV NS1 antibody (1:1,000; clone GT5212, GeneTex), rabbit polyclonal anti-ZIKV E protein antibody (1:1,000; Biorbyt), rabbit polyclonal anti-phospho-eIF2α (Ser51) antibody (1:2,000; Proteintech), rabbit polyclonal anti-eIF2α antibody (1:2,000; Proteintech), rabbit monoclonal anti-GAPDH antibody (1:2,000; clone D16H11, Cell Signaling Technology), and rabbit monoclonal anti-vinculin antibody (1:5,000; clone 3M13, Sigma-Aldrich).

Techniques: Infection, Control, Immunofluorescence, Staining, Isolation, Quantitative RT-PCR

Journal: EMBO Molecular Medicine

Article Title: In vitro and in vivo inhibition of the host TRPC4 channel attenuates Zika virus infection

doi: 10.1038/s44321-024-00103-4

Figure Lengend Snippet: ( A ) Representative immunofluorescence images of ZIKV-infected wild-type BHK cells taken at 72 hpi. The scale bar represents 50 μm. ( B ) Pearson correlation analysis. The correlation plot shows strong correlations between TRPC4 protein expression and ZIKV E-protein production ( r = 0.83). The fluorescence intensity ( F ) was quantified using ImageJ software. ( C ) Representative immunoblot images show relatively elevated TRPC4 protein amounts in U87 cells infected with higher MOI of ZIKV at 72 hpi. ( D ) Representative western blot images show TRPC4, NS1, and tubulin protein expression in ZIKV-infected or Mock BHK cells (left). Right, a comparison of the relative gray intensity for TRPC4 in western blots depicted on the left ( n = 6 biological replicates), with tubulin staining being used as a loading control. ( E ) Western blot analysis of the ZIKV viral NS1 protein expression in U87 cells overexpressing variable amounts of the TRPC4 cDNA and subjected to a 72-h challenge with the ZIKV virus. ( F ) The shown plots compare the relative RNA levels of TRPC4 (left) and ZIKV (right, n = 4 biological replicates) in BHK cells infected with ZIKV at 48 hpi and 72 hpi. qRT-PCRs were performed to amplify the TRPC4 and ZIKV (the linker region of membrane protein and envelope protein) RNA. ( G ) The correlation analysis. There is a strong correlation between mRNA levels of TRPC4 and ZIKV (NS5) in the ZIKV-infected mouse brains ( r = 0.86). ( H ) Representative immunoblot images are shown. Mock- or ZIKV-infected mouse brains were collected to perform the western blot analysis. Increased TRPC4 protein amounts were detected in ZIKV-infected mouse brains compared to Mock (control)-infected brains. The unpaired T test (two-tailed) was used to determine if there was a significant difference between two groups, and the correlation coefficient was obtained through linear regression analysis. Data information: In ( D , F ), data are presented as mean ± SEM, ** P ≤ 0.01. .

Article Snippet: Anti-ZIKV-NS1 monoclonal antibody , GeneTex , Cat # BF-1225-06.

Techniques: Immunofluorescence, Infection, Expressing, Fluorescence, Software, Western Blot, Comparison, Staining, Control, Virus, Membrane, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: In vitro and in vivo inhibition of the host TRPC4 channel attenuates Zika virus infection

doi: 10.1038/s44321-024-00103-4

Figure Lengend Snippet: ( A ) ZIKV infection results in brain inflammation in A129 mice. Representative images of brain section immunolabeled for GFAP (red) and DAPI (blue). The scale bar represents 500 μm. ( B ) Representative images of the hippocampus and cortex from mock or ZIKV-infected A129 adult mice at 12 dpi immunolabeled for TRPC4 (green) and viral E protein (red). The scale bars represent 1000 μm and 100 μm, respectively. The right insert in ( B ) presents the Pearson correlation analysis, revealing an association between TRPC4 protein levels and viral E-protein levels ( r = 0.91). ( C ) ZIKV infection also leads to an increase of the TRPC4 protein in neonatal mouse brain. The scale bar represents 100 μm. ( D ) the knockdown of TRPC4 by siRNA resulted in a reduction in NS1 production in HT22 cells ( n = 3 biological replicates). ( E ) the mock-infected cell viability was assessed in the presence of either AC1903, NITD008, HC-070, or Pyr3 ( n = 3 biological replicates). The unpaired T test (two-tailed) was employed to determine if there was a significant difference between two groups. Data information: In ( D , E ), data are presented as mean ± SEM, ** P ≤ 0.001.

Article Snippet: Anti-ZIKV-NS1 monoclonal antibody , GeneTex , Cat # BF-1225-06.

Techniques: Infection, Immunolabeling, Knockdown, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: In vitro and in vivo inhibition of the host TRPC4 channel attenuates Zika virus infection

doi: 10.1038/s44321-024-00103-4

Figure Lengend Snippet: ( A ) Representative immunoblot images are shown. The treatments are indicated above the immunoblots. ( B , C ) The shown plots demonstrate that ZIKV-infected shRNA-TRPC4-BHK cells exhibited decreased levels of TRPC4 and ZIKV-NS1 proteins compared to ZIKV-infected scRNA-BHK cells. TRPC4 protein expression was also decreased in mock-infected shRNA-TRPC4-BHK cells compared to mock-infected scRNA-BHK cells. Western blots were used to compare TRPC4 ( n = 4 biological replicates) and NS1 ( n = 5 biological replicates) protein expression in the indicated groups. ( D ) The percentage of cell survival was increased to 68% in the TRPC4-shRNA group ( n = 9 biological replicates) compared to the scRNA control group (9.6%). The unpaired T test (two-tailed) and one-way ANOVA followed by the Tukey test as the post hoc were employed to determine if there was a significant difference between two groups or among multiple groups, respectively. GAPDH staining was used as a loading control. Data information: In ( B – D ), data are presented as mean ± SEM, ** P ≤ 0.01, *** P ≤ 0.0001. .

Article Snippet: Anti-ZIKV-NS1 monoclonal antibody , GeneTex , Cat # BF-1225-06.

Techniques: Western Blot, Infection, shRNA, Expressing, Control, Two Tailed Test, Staining

Journal: EMBO Molecular Medicine

Article Title: In vitro and in vivo inhibition of the host TRPC4 channel attenuates Zika virus infection

doi: 10.1038/s44321-024-00103-4

Figure Lengend Snippet: ( A ) Cell viability following ZIKV infection in the presence of specific channel modulators ( n = 3 biological replicates). ( B ) The plot shows the results of qRT-PCR quantification of relative ZIKV RNA copies in the mRNA isolated from various cell lines infected with ZIKV in the presence of HC-070 ( n = 3–9 biological replicates). ( C , D ) Representative immunoblots and the quantification results. BHK cells were seeded on six-well plates and pretreated with HC-070 (10 μM), AC1903 (10 μM, a specific TRPC5 inhibitor), or the vehicle control for 1 h and then infected with ZIKV (SMGC-1, MOI = 0.001). Cell lysates were collected at 72 hpi. The western blot analysis was used to quantify the ZIKV-NS1 ( n = 4 biological replicates) and TRPC4 ( n = 3 biological replicates) protein levels. Bands’ intensities were quantified using ImageJ software. Tubulin staining was used as a loading control. The plot in the lower panel of C shows that HC-070 but not AC1903 decreased the ZIKV-NS1 protein level in ZIKV-infected cells. The plot in the lower panel of D shows that HC-070 decreased the TRPC4 protein level in ZIKV-infected cells. ( E , F ) Cells were inoculated in 96-well plates treated as above. Seventy-two hours later, cells were stained with the anti-ZIKV-E antibody. Representative images of BHK immunostaining for the ZIKV E protein (red) and summary data of mean F intensity ( n = 4–7 biological replicates). The scale bar represents 50 μm. ( G , H ) The production of ZIKV RNA ( n = 3 biological replicates) and ZIKV-NS1 proteins was attenuated in BHK cells treated with a series of concentrations of HC-070. The Dunnett’s test as the post hoc following one-way ANOVA on ranks ( B ) or The Student–Newman–Keuls Method as the post hoc following one-way ANOVA ( C – G ) was employed to determine if there was a significant difference among multiple groups. Data information: In ( A – D , F , G ), data are presented as mean ± SEM, * P ≤ 0.05, ** P ≤ 0.0001, **** P ≤ 0.0001. .

Article Snippet: Anti-ZIKV-NS1 monoclonal antibody , GeneTex , Cat # BF-1225-06.

Techniques: Infection, Quantitative RT-PCR, Isolation, Western Blot, Control, Software, Staining, Immunostaining

Journal: EMBO Molecular Medicine

Article Title: In vitro and in vivo inhibition of the host TRPC4 channel attenuates Zika virus infection

doi: 10.1038/s44321-024-00103-4

Figure Lengend Snippet: Comparison of anti-flavivirus activity of various TRP channel modulators that are commonly used.

Article Snippet: Anti-ZIKV-NS1 monoclonal antibody , GeneTex , Cat # BF-1225-06.

Techniques: Comparison, Activity Assay, Positive Control

Journal: EMBO Molecular Medicine

Article Title: In vitro and in vivo inhibition of the host TRPC4 channel attenuates Zika virus infection

doi: 10.1038/s44321-024-00103-4

Figure Lengend Snippet: ( A , B ) BHK cells infected with ZIKV were co-stained with anti-TRPC4 (green) and anti-ZIKV-E-protein (red) antibodies. The scale bar represents 10 μm. The regions of interest (ROI), where correlation analyses are conducted, are depicted in the white boxes. Pearson’s Coefficient ( r = 0.46) was determined to assess the co-localization of the TRPC4 protein and the viral E protein. ( C ) the Co-IP of TRPCs protein and ZIKV-E protein in BHK cells. The anti-flag antibody was used to immunoprecipitate TRPC proteins tagged with flag from cell lysates. The immunoprecipitated complexes were subsequently analyzed using Western blots and probed using the anti-E-protein antibody. The data indicates that there was no association between TRPC and ZIKV-E proteins under our experimental conditions. ( D ) BHK cells were transfected with R-GECO Ca 2+ sensor plasmid to monitor the intracellular Ca 2+ levels in mock or ZIKV-infected cells ( n = 4–5 biological replicates). ΔRFU represents the change in fluorescence intensity relative to the baseline. ( E , F ) A representative immunoblot and the results of densitometry analyses comparing relative TRPC4 protein levels in control, ZIKV-NS3, or ZIKV-NS3 + TRPC4 expressing BHK cells. GAPDH was used as a loading control. Co-expression of ZIKV-NS3 and TRPC4 cDNAs in BHK cells augmented the expression rate of the TRPC4 protein. ( G ) Normalized fluorescence intensity changes (F/F o ) in R-GECO cells co-expressing NS3 and/or TRPC4 in the presence or absence of 10 μM HC-070 ( n = 9–12; 3–4 wells per each experiment; 3 biological replicates). The MANOVA test with Bonferroni correction was employed to determine if there was a significant difference among multiple groups. Data information: In ( D , F , G ), data are presented as mean ± SEM.

Article Snippet: Anti-ZIKV-NS1 monoclonal antibody , GeneTex , Cat # BF-1225-06.

Techniques: Infection, Staining, Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot, Transfection, Plasmid Preparation, Fluorescence, Control, Expressing

Journal: EMBO Molecular Medicine

Article Title: In vitro and in vivo inhibition of the host TRPC4 channel attenuates Zika virus infection

doi: 10.1038/s44321-024-00103-4

Figure Lengend Snippet: ( A ) Survival rates of ZIKV-infected BHK cells were increased in the HC-070 (10 μM), EGTA (1 mM, the extracellular calcium chelating agent), and KN-93 (10 μM, CaMKII inhibitor) treatment groups ( n = 5–7 biological replicates). ( B ) Representative images (left) and summary data (right) of ZIKV- or mock-infected BHK cells immunoassayed for the ZIKV E protein (red). The cells were pretreated with DMSO, HC-070 (10 μM), EGTA (1 mM), or KN-93 (10 μM). The fluorescence intensity (F) was quantified using ImageJ software, and the obtained data were compared in the right panel ( n = 4–6 biological replicates). The scale bar represents 50 μm. ( C ) Western blots were performed to detect and quantify the production of ZIKV-NS1 proteins (NS1) in KN-93 (10 μM) or HC-070 (10 μM) pretreated BHK cells infected with ZIKV ( n = 4 biological replicates). ( D ) Viral replication was quantified by counting infectious viral particles isolated from the supernatants of BHK cells infected with ZIKV and treated with either DMSO (vehicle control), HC-070 (10 μM), or KN-93 (10 μM). Infectious viral particles were detected using the viral plaque-forming unit assay ( n = 4 biological replicates). ( E ) Representative immunoblot images showing a decrease of TRPC4 protein levels in KN-93-treated BHK cells without ZIKV infection. ( F ) Co-immunoprecipitation (Co-IP) assays are shown. Whole-cell extracts from ZIKV-NS3 overexpressed BHK cells were subjected to immunoprecipitation (IP) using either anti-NS3 or anti-CaMKII antibody. The co-immunoprecipitated proteins were subsequently detected by western blotting using specific antibodies against CaMKII or NS3. ( G ) The graph shows that ZIKV-infected neonatal mouse brains exhibit an enhanced immunofluorescence staining of pCREB ( n = 4 mice) compared to mock-infected mice. ( H ) Western blot analysis was performed to detect (left) and quantify (right) the phosphorylation level of CREB (pCREB) relative to total CREB in KN-93 (10 μM) or HC-070 (10 μM) pretreated BHK cells infected with ZIKV ( n = 5 biological replicates). ( I – L ) Representative immunoblot images show the efficacy of siRNA-mediated knockdown of the ZIKV-NS1 protein production in ZIKV-infected BHK cells ( n = 4 biological replicates). Prior to ZIKV challenge (MOI 0.01), cells were transfected with scRNAs or siRNAs targeting CaMKII ( I , J ) or CREB ( K , L ) for a duration of 48 h ( n = 4 biological replicates). ( M ) Shown are the changes in normalized R-GECO fluorescence (F/F o ) induced by Gd 3+ (100 μM) in TRPC4 (black line), NS3 (green line), or TRPC4 + NS3 (red line)-expressing cells. Intracellular Ca 2+ levels were monitored using the R-GECO biosensor. Cells were transfected with the indicated cDNAs. The horizontal bar shows the times when Gd 3+ was added to the wells with cells. The right panel displays a comparison of normalized R-GECO fluorescence increases in each tested group ( n = 9–12, 3–4 wells were for each experiment, with three biological replicates). The unpaired T test (two-tailed) and one-way ANOVA followed by the Dunnett’s test as the post hoc were employed to determine if there was a significant difference between two groups or among multiple groups, respectively. Data information: In ( A – D , G , H , J , L , M ), data are presented as mean ± SEM, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. .

Article Snippet: Anti-ZIKV-NS1 monoclonal antibody , GeneTex , Cat # BF-1225-06.

Techniques: Infection, Fluorescence, Software, Western Blot, Isolation, Control, Immunoprecipitation, Co-Immunoprecipitation Assay, Immunofluorescence, Staining, Phospho-proteomics, Knockdown, Transfection, Expressing, Comparison, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: In vitro and in vivo inhibition of the host TRPC4 channel attenuates Zika virus infection

doi: 10.1038/s44321-024-00103-4

Figure Lengend Snippet: ( A ) ZIKV infection of the neonatal mouse brain elevates the protein level of pCREB. 1-day-old ICR mice were infected with ZIKV, and then the brains were collected at 12 dpi. Tissues were fixed and stained with anti-CREB (green), and anti-pCREB (red) antibodies. The scale bar represents 100 μm. ( B , C ) Representative Western blot images demonstrate the effectiveness of siRNA-induced reduction of ZIKV-NS1 protein production in HT22 cells ( n = 4 biological replicates). Prior to exposing the cells to ZIKV (MOI 0.01), a 48-h transfection was performed using scRNAs or siRNAs that specifically target CaMKII ( n = 4 biological replicates) or CREB ( n = 3 biological replicates). ( D ) cells were transfected with R-GECO Ca 2+ sensor plasmid to monitor the intracellular Ca 2+ levels. No difference was observed between the Gd 3+ (100 μM)-induced fluorescence increases in the presence of the vehicle (DMSO) or 10 μM KN-93 in R-GECO + TRPC4 expressing cells ( n = 6 biological replicates). Gd 3+ was applied at the times indicated with the horizontal bar. ( E ) The averaged normalized fluorescence increases induced by histamine (10 μM) in H1R (histamine receptor, green line), H1R + NS3 (orange line), TRPC4 + H1R (black line), or NS3 + TRPC4 + H1R (red line) expressing cells are shown (n = 6 biological replicates). Histamine was added at the times indicated by the horizontal bar. ( E ) Right panel, a comparison of averaged peak values for data shown in the left panel. The unpaired T test (two-tailed) was employed to determine if there was a significant difference between two groups. Data information: In ( B – E ), data are presented as mean ± SEM, ** P ≤ 0.001.

Article Snippet: Anti-ZIKV-NS1 monoclonal antibody , GeneTex , Cat # BF-1225-06.

Techniques: Infection, Staining, Western Blot, Transfection, Plasmid Preparation, Fluorescence, Expressing, Comparison, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: In vitro and in vivo inhibition of the host TRPC4 channel attenuates Zika virus infection

doi: 10.1038/s44321-024-00103-4

Figure Lengend Snippet: ( A ) Timeline of time-of-drug-addition assay. BHK cells were inoculated with the virus during the times indicated with a horizontal green bar (2 h). The orange horizontal bars represent the times of HC-070 (10 μM) or NITD008 (5 μM) treatment. At 24 hpi, total mRNA was extracted for qRT-PCR. BHK cells were infected with ZIKV at a multiplicity of infection of 0.05. The control virus group was treated with DMSO. ( B ) During the time-of-drug-addition assay, the quantification of viral RNA copies in the total mRNA extracted from cell lysates was performed using qRT-PCR ( n = 4 biological replicates). ( C ) The ZIKV (2 × 10 6 PFU) samples were incubated with the indicated compounds or an equal volume of vehicle at 4 °C, 25 °C, and 37 °C for a duration of 1 h. Subsequently, the infectious viral particles in each sample were quantified using the PFU assay ( n = 3 biological replicates). EGCG was employed as the positive control. Neither HC-070 nor KN-93 exhibited any impact on the infectivity of ZIKV particles in vivo. ( D ) Representative immunofluorescence images of BHK cells stained with DDX3X (green) and ZIKV E-protein (red) antibodies are shown. The right panel displays graphs with the quantification of data shown in the left panel. The fluorescence intensity of the ZIKV E protein and the nuclear/cytoplasmic DDX3X fluorescence intensity ratio in ZIKV-infected cells exhibited a reduction in both HC-070 and EGTA treatment groups (three independent biological replicates). ( E ) Representative immunoblot images showing the protein levels of DDX3X and Lamin A/C in the nuclei and TRPC4 and GAPDH in the cytoplasm of ZIKV-infected cells overexpressing TRPC4 versus control ZIKV-infected cells ( n = 3 biological replicates). The graph in the right panel shows that overexpression of the TRPC4 protein increased the relative level of the DDX3X protein in the nucleus of ZIKV-infected cells. The unpaired T test (one-tailed) and one-way ANOVA followed by the Dunnett’s test as the post hoc were employed to determine if there was a significant difference between two groups or among multiple groups, respectively. GAPDH and Lamin A/C staining were used as loading control. Data information: In ( B – E ), data are presented as mean ± SEM, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. .

Article Snippet: Anti-ZIKV-NS1 monoclonal antibody , GeneTex , Cat # BF-1225-06.

Techniques: Virus, Quantitative RT-PCR, Infection, Control, Incubation, Positive Control, In Vivo, Immunofluorescence, Staining, Fluorescence, Western Blot, Over Expression, One-tailed Test

Journal: EMBO Molecular Medicine

Article Title: In vitro and in vivo inhibition of the host TRPC4 channel attenuates Zika virus infection

doi: 10.1038/s44321-024-00103-4

Figure Lengend Snippet: ( A ) Design of experiments involving human brain organoids. Human brain organoids were infected with 1.2 × 10 6 PFU ZIKV or mock medium at 30 days. At 2 dpi, the supernatant was collected for the luciferase assay and viral content assay, and organoids were used to extract viral RNA. ( B ) Luciferase Assay. The luminescence was measured as described in the Method section in the presence or absence of 10 μM HC-070 or 10 μM KN-93 ( n = 6 brain organoids). ( C , D ) Viral RNA analyses by qRT-PCR. The viral RNA copy number was determined in the supernatant and organoids of each tested group ( n = 6 brain organoids) and it was decreased in the presence of HC-070 and KN-93. The one-way ANOVA test followed by the Dunnett’s post hoc test was employed to determine if there was a significant difference among multiple groups. Data information: In ( B – D ), data are presented as mean ± SEM, * P ≤ 0.05. .

Article Snippet: Anti-ZIKV-NS1 monoclonal antibody , GeneTex , Cat # BF-1225-06.

Techniques: Infection, Luciferase, Quantitative RT-PCR

Journal: EMBO Molecular Medicine

Article Title: In vitro and in vivo inhibition of the host TRPC4 channel attenuates Zika virus infection

doi: 10.1038/s44321-024-00103-4

Figure Lengend Snippet: Mice were injected intraperitoneally with 2 × 10 4 PFU ZIKV at post-natal day 1, and HC-070 (0.3 mg/kg and 1 mg/kg), KN-93 (3 mg/kg), or vehicle was IP injected starting at 2 dpi and then for 10 consecutive days. ( A , B ) The graphs show body weight changes and animal survival curves ( n = 10–20 mice). HC-070 and KN-93 treatments markedly improved the survival of ZIKV-infected mice. ( C ) The viral loads in the brain were quantified using qRT-PCR ( n = 5–7 biological replicates). HC-070 and KN-93 treatments decreased ZIKV RNA copies. ( D – F ) The grasping reflex ( D ) was assessed in each group of mice at 7 dpi ( n = 10–20 mice). Subsequently, the latency to fall in the hindlimb suspension test was measured at 9 dpi ( E ), n = 10–20 mice). In addition, Racine scores were recorded to evaluate seizures in ZIKV-infected animals ( F ). The one-way ANOVA test followed by the Dunnett’s post hoc test was employed to determine if there was a significant difference among multiple groups. Data information: In ( A , C – F ), data are presented as mean ± SEM, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. .

Article Snippet: Anti-ZIKV-NS1 monoclonal antibody , GeneTex , Cat # BF-1225-06.

Techniques: Injection, Infection, Quantitative RT-PCR, Suspension

Journal: EMBO Molecular Medicine

Article Title: In vitro and in vivo inhibition of the host TRPC4 channel attenuates Zika virus infection

doi: 10.1038/s44321-024-00103-4

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Anti-ZIKV-NS1 monoclonal antibody , GeneTex , Cat # BF-1225-06.

Techniques: Mouse Assay, Recombinant, Plasmid Preparation, Virus, Sequencing, Staining, Software, Modification, Luciferase, Cell Viability Assay, Lysis

Journal: Journal of Biomedical Science

Article Title: The glycosylation deficiency of flavivirus NS1 attenuates virus replication through interfering with the formation of viral replication compartments

doi: 10.1186/s12929-024-01048-z

Figure Lengend Snippet: N-linked glycosylation of ZIKV NS1 is important for ER remodeling. A The glycosylation levels of WT and mutated ZIKV NS1. Intracellular and secreted ZIKV NS1 expressed in HeLa cells were analyzed by Western blotting. < < , glycosylated NS1; < , non-glycosylated NS1(N130AN207A). B Quantification of secreted NS1. The percentage was calculated as secreted NS1 divided by the total NS1 (the sum of intracellular and secreted NS1). C NS1 induced ER morphology changes. HeLa cells expressing Myc-tagged WT or mutated ZIKV NS1 were stained with Myc and climp63 antibodies, with climp63 serving as the ER marker. NS1-positive cells were categorized into two groups based on ER morphology. “Aggregated ER” denotes ER aggregations above 5 μm 2 , as indicated by arrows; while “normal ER” indicates the ER maintaining sheet and tubule structures with ER aggregations below 5 μm 2 , as indicated by arrowheads. Scale bar, 20 μm. D Quantification of the cell population with different ER morphologies involved analyzing over 200 cells in each group. The percentage was calculated as the number of cells with aggregated ER divided by the total number of NS1-positive cells. E Ultrastructure of the ER in HeLa cells overexpressing WT or mutated ZIKV NS1 was captured by TEM. Ctrl, untransfected cells. Scale bar, 1 μm (upper), 500 nm (lower). F Quantification of TEM images in ( E ). The percentage of cells displaying specific ER morphologies among 50 cells was determined. All statistical data represent mean ± SEM ( n = 3; *, p < 0.05; ***, p < 0.001; ****, p < 0.0001; n.s., non-significant; two-tailed t test)

Article Snippet: After being blocked with 3% Bovine Serum Albumin (BSA), cells were incubated with the primary antibodies, Myc (MBL, 1:500) and climp63 (Proteintech, 1:300) or ZIKV/DENV NS1 (BioFront, 1:200) for 2 h at 37 °C.

Techniques: Glycoproteomics, Western Blot, Expressing, Staining, Marker, Two Tailed Test

Journal: Journal of Biomedical Science

Article Title: The glycosylation deficiency of flavivirus NS1 attenuates virus replication through interfering with the formation of viral replication compartments

doi: 10.1186/s12929-024-01048-z

Figure Lengend Snippet: Tunicamycin blocks ZIKV NS1 glycosylation and interferes with its ER remodeling. A The glycosylation levels of ZIKV NS1 WT under tunicamycin (Tu) treatment. Intracellular and secreted NS1 proteins expressed in HeLa cells were immunoblotted with the Myc antibody. < < , glycosylated NS1; < , non-glycosylated NS1. DMSO as a control. B Quantification of glycosylated NS1. The percentage was calculated as glycosylated NS1 divided by the total intracellular NS1. C Quantification of secreted NS1. The percentage was calculated as secreted NS1 divided by the total NS1 (the sum of intracellular and secreted NS1). D ZIKV NS1 induced ER morphology changes with Tu treatment. HeLa cells were treated with 0.5 μg/mL Tu or transfected with Myc-tagged WT NS1 plasmid, followed by treatment with 0.5 μg/mL Tu. Cells were fixed and stained with Myc and climp63 antibodies. Arrows indicate cells with aggregated ER, arrowheads indicate cells with normal ER. Scale bar, 20 μm. E Quantification of the cell population with different ER morphologies. The percentage was calculated as the number of cells with aggregated ER divided by the total number of NS1-positive cells. F TEM images showed the ER ultrastructure in HeLa cells treated with 0.25 μg/mL Tu or transfected with WT ZIKV NS1 plasmid and treated with 0.25 μg/mL Tu. Scale bar, 1 μm (upper), 500 nm (lower). G Quantification of TEM images in ( F ). The percentage of cells displaying specific ER morphologies upon transfection with the ZIKV NS1 plasmid among 50 cells was determined and listed in the table. H Tu induced ZIKV NS1 aggregation. HeLa cells were transfected with WT ZIKV NS1 plasmid and treated with 0.5 μg/mL Tu. Then the soluble supernatants and detergent-resistant pellets were analyzed by Western blotting. I Quantification of functional NS1 (detergent-soluble supernatant) in ( H ). The percentage was calculated as soluble NS1 in the supernatant divided by the total NS1 (the sum of soluble and insoluble NS1). J Quantification of glycosylated or non-glycosylated insoluble NS1 upon Tu treatment in ( H ). The percentage of insoluble NS1 was calculated by dividing glycosylated or non-glycosylated insoluble NS1 by the total glycosylated or non-glycosylated NS1, respectively. All statistical data represent mean ± SEM ( n = 3; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; n.s., non-significant; two-tailed t test)

Article Snippet: After being blocked with 3% Bovine Serum Albumin (BSA), cells were incubated with the primary antibodies, Myc (MBL, 1:500) and climp63 (Proteintech, 1:300) or ZIKV/DENV NS1 (BioFront, 1:200) for 2 h at 37 °C.

Techniques: Glycoproteomics, Control, Transfection, Plasmid Preparation, Staining, Western Blot, Functional Assay, Two Tailed Test

Journal: Journal of Biomedical Science

Article Title: The glycosylation deficiency of flavivirus NS1 attenuates virus replication through interfering with the formation of viral replication compartments

doi: 10.1186/s12929-024-01048-z

Figure Lengend Snippet: Glycosylation deficiency of ZIKV NS1 leads to protein aggregation. A The NS1 proteins with Twin-Strep-Tag, purified from HEK293T cells, were analyzed by Coomassie blue staining. B Schematic shows the liposome co-floating assay. NS1 proteins were incubated with liposomes, and then the mixture was applied to density gradient centrifugation. The proteins that floated up with liposomes were harvested from the top layer. C Co-floating assay of WT and mutated NS1. Input (2 times diluted with protein loading buffer) and float-up samples were analyzed by Western blotting. D Quantification of the co-floating NS1. Percentage was calculated as float-up NS1 divided by the input. E Glycosylation deficiency led to NS1 aggregation. HeLa cells were transfected with plasmids encoding for WT or mutated ZIKV NS1 proteins, and the soluble lysates and detergent-resistant pellets were analyzed by Western blotting. F Quantification of insoluble NS1 (detergent-resistant pellet) in ( E ). Percentage was calculated as insoluble NS1 divided by the total NS1 (the sum of soluble and insoluble NS1). All statistical data represent mean ± SEM ( n = 3; **, p < 0.01; ****, p < 0.0001; n.s., non-significant; two-tailed t test)

Article Snippet: After being blocked with 3% Bovine Serum Albumin (BSA), cells were incubated with the primary antibodies, Myc (MBL, 1:500) and climp63 (Proteintech, 1:300) or ZIKV/DENV NS1 (BioFront, 1:200) for 2 h at 37 °C.

Techniques: Glycoproteomics, Strep-tag, Purification, Staining, Incubation, Liposomes, Gradient Centrifugation, Western Blot, Transfection, Two Tailed Test

Journal: Journal of Biomedical Science

Article Title: The glycosylation deficiency of flavivirus NS1 attenuates virus replication through interfering with the formation of viral replication compartments

doi: 10.1186/s12929-024-01048-z

Figure Lengend Snippet: Glycosylation deficiency affects ER lumenal ZIKV NS1 solubility and ER association. A ZIKV NS1 and membrane-anchored CNX accumulated in pellet. HeLa cells expressing ZIKV NS1 proteins were subjected to ultrasonication and sequential centrifugations. The sup and pellet samples were analyzed by Western blotting. Sup, supernatant. CNX, calnexin. B The distributions of ZIKV NS1 and CNX in the pellets from ( A ) were separated by sucrose density gradient centrifugation. C The mutated NS1 was enriched in the high-density fractions. Distributions of ZIKV NS1 in fractions 1–7 and 8–10 were calculated as the amount of NS1 divided by that of CNX. D ZIKV NS1 N130AN207A mutant was accumulated in detergent-resistant pellet. Fractions 1–7 and 8–10 from ( B ) were harvested and treated with 1% NP-40, and the solubility was determined by Western blotting. E Quantification of insoluble NS1 (detergent-resistant pellet) in ( D ). The percentage was calculated as insoluble NS1 divided by the total NS1 (the sum of soluble and insoluble NS1). F N130AN207A mutant of ZIKV NS1 induced ER stress. Total RNA was extracted from HeLa cells and the mRNA levels of ER stress sensors were quantified by qPCR. Ctrl means untransfected cells. All statistical data represent mean ± SEM ( n = 3; *, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s., non-significant; two-tailed t test)

Article Snippet: After being blocked with 3% Bovine Serum Albumin (BSA), cells were incubated with the primary antibodies, Myc (MBL, 1:500) and climp63 (Proteintech, 1:300) or ZIKV/DENV NS1 (BioFront, 1:200) for 2 h at 37 °C.

Techniques: Glycoproteomics, Solubility, Membrane, Expressing, Western Blot, Gradient Centrifugation, Mutagenesis, Two Tailed Test

Journal: Journal of Biomedical Science

Article Title: The glycosylation deficiency of flavivirus NS1 attenuates virus replication through interfering with the formation of viral replication compartments

doi: 10.1186/s12929-024-01048-z

Figure Lengend Snippet: N-linked glycosylation of ZIKV NS1 ensures effective virus replication. A Renilla luciferase assay of ZIKV replicons. The luciferase activity was measured at 10 h and 48 h after transfection of BHK-21 cells with WT or mutated ZIKV replicon RNA. B Immunofluorescence assay detecting the replication efficiency. BHK-21 cells transfected with ZIKV replicons were stained with a dsRNA antibody (Green: dsRNA, Blue: DAPI). Scale bar, 20 μm. C Quantification of dsRNA-positive cells involved analyzing 20 views for each group. WT ZIKV replicon is set as 100%. D Plaque assays detecting infectious ZIKV production were performed in Vero cells on day 5 after infection. E The number of plaques per mL was counted and analyzed statistically. F Ultrastructure of the ER in BHK-21 cells infected with WT or mutated ZIKV was captured by TEM. Scale bar, 1 μm (upper), 500 nm (lower). All statistical data represent mean ± SEM ( n = 3; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; n.s., non-significant; two-tailed t test)

Article Snippet: After being blocked with 3% Bovine Serum Albumin (BSA), cells were incubated with the primary antibodies, Myc (MBL, 1:500) and climp63 (Proteintech, 1:300) or ZIKV/DENV NS1 (BioFront, 1:200) for 2 h at 37 °C.

Techniques: Glycoproteomics, Virus, Luciferase, Activity Assay, Transfection, Immunofluorescence, Staining, Infection, Two Tailed Test

Journal: Journal of Biomedical Science

Article Title: The glycosylation deficiency of flavivirus NS1 attenuates virus replication through interfering with the formation of viral replication compartments

doi: 10.1186/s12929-024-01048-z

Figure Lengend Snippet: Tunicamycin inhibits ZIKV replication. A Renilla luciferase assay of ZIKV replicons. The luciferase activity was measured at 10 h and 36 h after the transfection of BHK-21 cells with WT ZIKV replicon RNA. Cells were treated with Tu (0.5 μg/mL) at 10 h post-transfection. B Immunofluorescence assay detecting replication efficiency. BHK-21 cells transfected with WT ZIKV replicon were treated with Tu (0.5 μg/mL) and stained with a dsRNA antibody (Green: dsRNA, Blue: DAPI). Scale bar, 20 μm. C Tu inhibited ZIKV replication. ZIKV-infected BHK-21 cells were treated with Tu (0.25 μg/mL) at 8 h post-infection (hpi) and stained with NS1 antibody at 24 hpi. Scale bar, 100 μm. D IC50 curve of Tu for ZIKV replication. ZIKV replication levels in BHK-21 cells under Tu treatment were quantified by qPCR assay at 24 hpi. The IC50 value is 68.59 nM. The statistical data represent mean ± SEM. E Tu inhibited ZIKV RCs formation. TEM images showed the ER ultrastructure in BHK-21 cells treated with 0.25 μg/mL Tu or infected with ZIKV and treated with 0.25 μg/mL Tu. Scale bar, 1 μm (upper), 500 nm (lower). F Quantification of TEM images in ( E ). The percentage of cells containing RCs upon infection with ZIKV among 50 cells was determined. G The glycosylation status of ZIKV NS1 in ZIKV-infected BHK-21 cells treated with Tu was detected by Western blotting with NS1 antibody at 24 hpi

Article Snippet: After being blocked with 3% Bovine Serum Albumin (BSA), cells were incubated with the primary antibodies, Myc (MBL, 1:500) and climp63 (Proteintech, 1:300) or ZIKV/DENV NS1 (BioFront, 1:200) for 2 h at 37 °C.

Techniques: Luciferase, Activity Assay, Transfection, Immunofluorescence, Staining, Infection, Glycoproteomics, Western Blot

Journal: Journal of Biomedical Science

Article Title: The glycosylation deficiency of flavivirus NS1 attenuates virus replication through interfering with the formation of viral replication compartments

doi: 10.1186/s12929-024-01048-z

Figure Lengend Snippet: N-linked glycosylation of ZIKV NS1 is important for ER remodeling. A The glycosylation levels of WT and mutated ZIKV NS1. Intracellular and secreted ZIKV NS1 expressed in HeLa cells were analyzed by Western blotting. < < , glycosylated NS1; < , non-glycosylated NS1(N130AN207A). B Quantification of secreted NS1. The percentage was calculated as secreted NS1 divided by the total NS1 (the sum of intracellular and secreted NS1). C NS1 induced ER morphology changes. HeLa cells expressing Myc-tagged WT or mutated ZIKV NS1 were stained with Myc and climp63 antibodies, with climp63 serving as the ER marker. NS1-positive cells were categorized into two groups based on ER morphology. “Aggregated ER” denotes ER aggregations above 5 μm 2 , as indicated by arrows; while “normal ER” indicates the ER maintaining sheet and tubule structures with ER aggregations below 5 μm 2 , as indicated by arrowheads. Scale bar, 20 μm. D Quantification of the cell population with different ER morphologies involved analyzing over 200 cells in each group. The percentage was calculated as the number of cells with aggregated ER divided by the total number of NS1-positive cells. E Ultrastructure of the ER in HeLa cells overexpressing WT or mutated ZIKV NS1 was captured by TEM. Ctrl, untransfected cells. Scale bar, 1 μm (upper), 500 nm (lower). F Quantification of TEM images in ( E ). The percentage of cells displaying specific ER morphologies among 50 cells was determined. All statistical data represent mean ± SEM ( n = 3; *, p < 0.05; ***, p < 0.001; ****, p < 0.0001; n.s., non-significant; two-tailed t test)

Article Snippet: Antibodies used for Western blotting include Myc (MBL, Cat. #M192-3), ZIKV/DENV NS1 (BioFront, Cat. # B-1191–46), Calnexin (MBL, Cat. #M178-3).

Techniques: Glycoproteomics, Western Blot, Expressing, Staining, Marker, Two Tailed Test

Journal: Journal of Biomedical Science

Article Title: The glycosylation deficiency of flavivirus NS1 attenuates virus replication through interfering with the formation of viral replication compartments

doi: 10.1186/s12929-024-01048-z

Figure Lengend Snippet: Tunicamycin blocks ZIKV NS1 glycosylation and interferes with its ER remodeling. A The glycosylation levels of ZIKV NS1 WT under tunicamycin (Tu) treatment. Intracellular and secreted NS1 proteins expressed in HeLa cells were immunoblotted with the Myc antibody. < < , glycosylated NS1; < , non-glycosylated NS1. DMSO as a control. B Quantification of glycosylated NS1. The percentage was calculated as glycosylated NS1 divided by the total intracellular NS1. C Quantification of secreted NS1. The percentage was calculated as secreted NS1 divided by the total NS1 (the sum of intracellular and secreted NS1). D ZIKV NS1 induced ER morphology changes with Tu treatment. HeLa cells were treated with 0.5 μg/mL Tu or transfected with Myc-tagged WT NS1 plasmid, followed by treatment with 0.5 μg/mL Tu. Cells were fixed and stained with Myc and climp63 antibodies. Arrows indicate cells with aggregated ER, arrowheads indicate cells with normal ER. Scale bar, 20 μm. E Quantification of the cell population with different ER morphologies. The percentage was calculated as the number of cells with aggregated ER divided by the total number of NS1-positive cells. F TEM images showed the ER ultrastructure in HeLa cells treated with 0.25 μg/mL Tu or transfected with WT ZIKV NS1 plasmid and treated with 0.25 μg/mL Tu. Scale bar, 1 μm (upper), 500 nm (lower). G Quantification of TEM images in ( F ). The percentage of cells displaying specific ER morphologies upon transfection with the ZIKV NS1 plasmid among 50 cells was determined and listed in the table. H Tu induced ZIKV NS1 aggregation. HeLa cells were transfected with WT ZIKV NS1 plasmid and treated with 0.5 μg/mL Tu. Then the soluble supernatants and detergent-resistant pellets were analyzed by Western blotting. I Quantification of functional NS1 (detergent-soluble supernatant) in ( H ). The percentage was calculated as soluble NS1 in the supernatant divided by the total NS1 (the sum of soluble and insoluble NS1). J Quantification of glycosylated or non-glycosylated insoluble NS1 upon Tu treatment in ( H ). The percentage of insoluble NS1 was calculated by dividing glycosylated or non-glycosylated insoluble NS1 by the total glycosylated or non-glycosylated NS1, respectively. All statistical data represent mean ± SEM ( n = 3; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; n.s., non-significant; two-tailed t test)

Article Snippet: Antibodies used for Western blotting include Myc (MBL, Cat. #M192-3), ZIKV/DENV NS1 (BioFront, Cat. # B-1191–46), Calnexin (MBL, Cat. #M178-3).

Techniques: Glycoproteomics, Control, Transfection, Plasmid Preparation, Staining, Western Blot, Functional Assay, Two Tailed Test

Journal: Journal of Biomedical Science

Article Title: The glycosylation deficiency of flavivirus NS1 attenuates virus replication through interfering with the formation of viral replication compartments

doi: 10.1186/s12929-024-01048-z

Figure Lengend Snippet: Glycosylation deficiency of ZIKV NS1 leads to protein aggregation. A The NS1 proteins with Twin-Strep-Tag, purified from HEK293T cells, were analyzed by Coomassie blue staining. B Schematic shows the liposome co-floating assay. NS1 proteins were incubated with liposomes, and then the mixture was applied to density gradient centrifugation. The proteins that floated up with liposomes were harvested from the top layer. C Co-floating assay of WT and mutated NS1. Input (2 times diluted with protein loading buffer) and float-up samples were analyzed by Western blotting. D Quantification of the co-floating NS1. Percentage was calculated as float-up NS1 divided by the input. E Glycosylation deficiency led to NS1 aggregation. HeLa cells were transfected with plasmids encoding for WT or mutated ZIKV NS1 proteins, and the soluble lysates and detergent-resistant pellets were analyzed by Western blotting. F Quantification of insoluble NS1 (detergent-resistant pellet) in ( E ). Percentage was calculated as insoluble NS1 divided by the total NS1 (the sum of soluble and insoluble NS1). All statistical data represent mean ± SEM ( n = 3; **, p < 0.01; ****, p < 0.0001; n.s., non-significant; two-tailed t test)

Article Snippet: Antibodies used for Western blotting include Myc (MBL, Cat. #M192-3), ZIKV/DENV NS1 (BioFront, Cat. # B-1191–46), Calnexin (MBL, Cat. #M178-3).

Techniques: Glycoproteomics, Strep-tag, Purification, Staining, Incubation, Liposomes, Gradient Centrifugation, Western Blot, Transfection, Two Tailed Test

Journal: Journal of Biomedical Science

Article Title: The glycosylation deficiency of flavivirus NS1 attenuates virus replication through interfering with the formation of viral replication compartments

doi: 10.1186/s12929-024-01048-z

Figure Lengend Snippet: Glycosylation deficiency affects ER lumenal ZIKV NS1 solubility and ER association. A ZIKV NS1 and membrane-anchored CNX accumulated in pellet. HeLa cells expressing ZIKV NS1 proteins were subjected to ultrasonication and sequential centrifugations. The sup and pellet samples were analyzed by Western blotting. Sup, supernatant. CNX, calnexin. B The distributions of ZIKV NS1 and CNX in the pellets from ( A ) were separated by sucrose density gradient centrifugation. C The mutated NS1 was enriched in the high-density fractions. Distributions of ZIKV NS1 in fractions 1–7 and 8–10 were calculated as the amount of NS1 divided by that of CNX. D ZIKV NS1 N130AN207A mutant was accumulated in detergent-resistant pellet. Fractions 1–7 and 8–10 from ( B ) were harvested and treated with 1% NP-40, and the solubility was determined by Western blotting. E Quantification of insoluble NS1 (detergent-resistant pellet) in ( D ). The percentage was calculated as insoluble NS1 divided by the total NS1 (the sum of soluble and insoluble NS1). F N130AN207A mutant of ZIKV NS1 induced ER stress. Total RNA was extracted from HeLa cells and the mRNA levels of ER stress sensors were quantified by qPCR. Ctrl means untransfected cells. All statistical data represent mean ± SEM ( n = 3; *, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s., non-significant; two-tailed t test)

Article Snippet: Antibodies used for Western blotting include Myc (MBL, Cat. #M192-3), ZIKV/DENV NS1 (BioFront, Cat. # B-1191–46), Calnexin (MBL, Cat. #M178-3).

Techniques: Glycoproteomics, Solubility, Membrane, Expressing, Western Blot, Gradient Centrifugation, Mutagenesis, Two Tailed Test

Journal: Journal of Biomedical Science

Article Title: The glycosylation deficiency of flavivirus NS1 attenuates virus replication through interfering with the formation of viral replication compartments

doi: 10.1186/s12929-024-01048-z

Figure Lengend Snippet: N-linked glycosylation of ZIKV NS1 ensures effective virus replication. A Renilla luciferase assay of ZIKV replicons. The luciferase activity was measured at 10 h and 48 h after transfection of BHK-21 cells with WT or mutated ZIKV replicon RNA. B Immunofluorescence assay detecting the replication efficiency. BHK-21 cells transfected with ZIKV replicons were stained with a dsRNA antibody (Green: dsRNA, Blue: DAPI). Scale bar, 20 μm. C Quantification of dsRNA-positive cells involved analyzing 20 views for each group. WT ZIKV replicon is set as 100%. D Plaque assays detecting infectious ZIKV production were performed in Vero cells on day 5 after infection. E The number of plaques per mL was counted and analyzed statistically. F Ultrastructure of the ER in BHK-21 cells infected with WT or mutated ZIKV was captured by TEM. Scale bar, 1 μm (upper), 500 nm (lower). All statistical data represent mean ± SEM ( n = 3; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; n.s., non-significant; two-tailed t test)

Article Snippet: Antibodies used for Western blotting include Myc (MBL, Cat. #M192-3), ZIKV/DENV NS1 (BioFront, Cat. # B-1191–46), Calnexin (MBL, Cat. #M178-3).

Techniques: Glycoproteomics, Virus, Luciferase, Activity Assay, Transfection, Immunofluorescence, Staining, Infection, Two Tailed Test

Journal: Journal of Biomedical Science

Article Title: The glycosylation deficiency of flavivirus NS1 attenuates virus replication through interfering with the formation of viral replication compartments

doi: 10.1186/s12929-024-01048-z

Figure Lengend Snippet: Tunicamycin inhibits ZIKV replication. A Renilla luciferase assay of ZIKV replicons. The luciferase activity was measured at 10 h and 36 h after the transfection of BHK-21 cells with WT ZIKV replicon RNA. Cells were treated with Tu (0.5 μg/mL) at 10 h post-transfection. B Immunofluorescence assay detecting replication efficiency. BHK-21 cells transfected with WT ZIKV replicon were treated with Tu (0.5 μg/mL) and stained with a dsRNA antibody (Green: dsRNA, Blue: DAPI). Scale bar, 20 μm. C Tu inhibited ZIKV replication. ZIKV-infected BHK-21 cells were treated with Tu (0.25 μg/mL) at 8 h post-infection (hpi) and stained with NS1 antibody at 24 hpi. Scale bar, 100 μm. D IC50 curve of Tu for ZIKV replication. ZIKV replication levels in BHK-21 cells under Tu treatment were quantified by qPCR assay at 24 hpi. The IC50 value is 68.59 nM. The statistical data represent mean ± SEM. E Tu inhibited ZIKV RCs formation. TEM images showed the ER ultrastructure in BHK-21 cells treated with 0.25 μg/mL Tu or infected with ZIKV and treated with 0.25 μg/mL Tu. Scale bar, 1 μm (upper), 500 nm (lower). F Quantification of TEM images in ( E ). The percentage of cells containing RCs upon infection with ZIKV among 50 cells was determined. G The glycosylation status of ZIKV NS1 in ZIKV-infected BHK-21 cells treated with Tu was detected by Western blotting with NS1 antibody at 24 hpi

Article Snippet: Antibodies used for Western blotting include Myc (MBL, Cat. #M192-3), ZIKV/DENV NS1 (BioFront, Cat. # B-1191–46), Calnexin (MBL, Cat. #M178-3).

Techniques: Luciferase, Activity Assay, Transfection, Immunofluorescence, Staining, Infection, Glycoproteomics, Western Blot