anti-ns1-zikv protein clone ea88 mouse monoclonal antibody  (Thermo Fisher)

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: 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: Infectious Diseases of Poverty

Article Title: Armigeres subalbatus is a potential vector for Zika virus but not dengue virus

doi: 10.1186/s40249-022-00990-0

Figure Lengend Snippet: The blood-fed infection of ZIKV or DENV-2 in Ar. subalbatus . A Schematic diagram of oral infection experiment. B Infection rates and ( C ) RNA copies of ZIKV in various tissues at different days post inoculation (dpi). The results are present as the means ± SD . Error bars indicate SD s. The experiment was repeated three times. SD : Standard deviation.

Article Snippet: F Virus particles were detected with ZIKV NS1 protein antibody (Thermo Fisher) by IHC and are displayed as obvious brownish-red marked by red arrow The detection result of fourth instar larvae (41 pools) showed that the larvae of Ar. subalbatus could not be infected by DENV-2 in the continuous addition experiment. (Additional file : Table S3).

Techniques: Infection, Standard Deviation

Journal: Infectious Diseases of Poverty

Article Title: Armigeres subalbatus is a potential vector for Zika virus but not dengue virus

doi: 10.1186/s40249-022-00990-0

Figure Lengend Snippet: The artificial urine infection of ZIKV or DENV-2 in the larvae of Ar. subalbatus . A Schematic diagram of the artificial virus urine infection experiment. B Susceptibility of different instar larvae reared in artificial urine contained ZIKV, and virus was detected in the fourth instar larvae. C RNA copies of ZIKV in infected midguts, ovaries, and salivary glands of adults. The results are expressed as the means ± standard errors (SEs). D The third instar larvae were reared in artificial urine with ZIKV, and the infection of larvae was detected at 1, 2, 3, 4 days post inoculation (dpi) with RT-PCR and RT-qPCR. E ZIKV was detected in midgut, anal papillae and carcass of fourth instar larvae at 4dpi with RT-PCR and RT-qPCR. F Virus particles were detected with ZIKV NS1 protein antibody (Thermo Fisher) by IHC and are displayed as obvious brownish-red marked by red arrow

Article Snippet: F Virus particles were detected with ZIKV NS1 protein antibody (Thermo Fisher) by IHC and are displayed as obvious brownish-red marked by red arrow The detection result of fourth instar larvae (41 pools) showed that the larvae of Ar. subalbatus could not be infected by DENV-2 in the continuous addition experiment. (Additional file : Table S3).

Techniques: Infection, Virus, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Infectious Diseases of Poverty

Article Title: Armigeres subalbatus is a potential vector for Zika virus but not dengue virus

doi: 10.1186/s40249-022-00990-0

Figure Lengend Snippet: Infection rate of larvae for ZIKV in artificial urine

Article Snippet: F Virus particles were detected with ZIKV NS1 protein antibody (Thermo Fisher) by IHC and are displayed as obvious brownish-red marked by red arrow The detection result of fourth instar larvae (41 pools) showed that the larvae of Ar. subalbatus could not be infected by DENV-2 in the continuous addition experiment. (Additional file : Table S3).

Techniques: Infection