Journal: Viruses

Article Title: Extracellular Vesicles Are Conveyors of the NS1 Toxin during Dengue Virus and Zika Virus Infection

doi: 10.3390/v15020364

Figure Lengend Snippet: ZIKV and DENV2-infected A549 cells produce exosome-like particles associated with NS1 protein. ( A ) The NS1 protein of both ZIKV and DENV2 is found associated with the exosomal fraction obtained from the cell culture supernatants (CCS) of A549 infected cells. The tetraspanin CD63 detected on the dot blot of exosomal fractions provides insight into the quantity of exosomes extracted. ( B ) Western blot analysis of the exosome fraction, heated (HT) (+) or not (−), produced by DENV2 infected cells, show the presence of NS1 in dimeric form. A unique band with an apparent mass of ~48 kDa is observed after heating (+). ( C ) DLS analysis of exosomes produced by A549 cells during ZIKV (green) or DENV2 (blue) infection. On the y axis, the intensity gives the proportions of the different vesicle populations, depending on their size on the x axis. Both DENV2 and ZIKV infections appear to slightly reshape the distribution of vesicles.

Article Snippet: Recombinant ZIKV-NS1 HIS-tag (strain Zika SPH2015) and DENV2-NS1 HIS-tag (Dengue type 2, strain New Guinea C) were from Sino Biological.

Techniques: Infection, Cell Culture, Dot Blot, Western Blot, Produced

Journal: Viruses

Article Title: Extracellular Vesicles Are Conveyors of the NS1 Toxin during Dengue Virus and Zika Virus Infection

doi: 10.3390/v15020364

Figure Lengend Snippet: The overexpressed ZIKV-NS1 FLAG-tag and DENV2-NS1 V5-tag co-elute with exosome-like particles. ( A ) The recombinant ZIKV-NS1 FLAG-tag and DENV2-NS1 V5-tag , overexpressed in stable HEK cell lines, are present in the CCS, mainly as dimers. ( B ) The tetraspanins CD81 and CD63 are immunodetected in the exosome fractions of CCS from cells expressing the different secreted recombinant proteins. ( C ) When detecting the recombinant proteins in the CCS and exosomal fractions, only the NS1 proteins were detected in the exosomal fractions, and a similar signal was obtained whether or not the vesicles had been lysed. ( D ) SEAP activity was estimated by quantiblue assay. CCS and the flowthrough remaining from the exosome extraction, but not purified exosomes, exhibited SEAP activity. Ordinary one-way ANOVA test was performed using GraphPad Prism **** p < 0.0001, ns (not significant) ( E ) Western blot analysis of exosome protein extracts shows that ZIKV-NS1 FLAG-tag and DENV2-NS1 V5-tag are found associated with exosomes in their dimeric form.

Article Snippet: Recombinant ZIKV-NS1 HIS-tag (strain Zika SPH2015) and DENV2-NS1 HIS-tag (Dengue type 2, strain New Guinea C) were from Sino Biological.

Techniques: FLAG-tag, Recombinant, Expressing, Activity Assay, Purification, Western Blot

Journal: Viruses

Article Title: Extracellular Vesicles Are Conveyors of the NS1 Toxin during Dengue Virus and Zika Virus Infection

doi: 10.3390/v15020364

Figure Lengend Snippet: ZIKV-NS1 FLAG-tag is present in the exosome fraction. ( A ) Western blot analysis of HEK293-NS1 supernatant following ultrafiltration. Mouse anti-FLAG antibody was used to detect ZIKV-NS1 FLAG-tag , while the detection of albumin served as protein loading control. ( B ) Dot blot analysis of putative exosome fractions. After the ultrafiltration step, size exclusion chromatography was carried out on the >100 kDa fraction. Detection of ZIKV-NS1 FLAG-tag was achieved using a mouse anti-FLAG antibody, whereas CD81 was chosen as a marker for exosomes. ( C ) CCS containing CD63-positive EVs and secreted ZIKV-NS1 FLAG-tag (S1) were treated with PEG and centrifuged to obtain a pellet enriched in EVs. CD63 was found in the first pellet (P PEG) and after the second ultracentrifugation step (P2), validating the enrichment of exosomes which were also found associated with ZIKV-NS1 FLAG-tag . ( D ) ELISA capture of the exosomes in the supernatant of HEK293-ZIKV-NS1 FLAG-tag versus control. Exosomes were captured by an anti-CD81 antibody coated on Maxisorp ® plate. NS1 FLAG-tag protein was detected using a mouse anti-FLAG antibody. Ordinary one-way ANOVA test was performed using GraphPad Prism, ** p < 0.001. ( E ) DLS analysis of the exosomal fractions with the proportion of vesicles by size graph and the corresponding z-average-table .

Article Snippet: Recombinant ZIKV-NS1 HIS-tag (strain Zika SPH2015) and DENV2-NS1 HIS-tag (Dengue type 2, strain New Guinea C) were from Sino Biological.

Techniques: FLAG-tag, Western Blot, Dot Blot, Size-exclusion Chromatography, Marker, Enzyme-linked Immunosorbent Assay

Journal: Viruses

Article Title: Extracellular Vesicles Are Conveyors of the NS1 Toxin during Dengue Virus and Zika Virus Infection

doi: 10.3390/v15020364

Figure Lengend Snippet: Overexpression of ZIKV and DENV2 NS1 lead to an increase in exosomes size.

Article Snippet: Recombinant ZIKV-NS1 HIS-tag (strain Zika SPH2015) and DENV2-NS1 HIS-tag (Dengue type 2, strain New Guinea C) were from Sino Biological.

Techniques: Over Expression, Standard Deviation

Journal: Viruses

Article Title: Extracellular Vesicles Are Conveyors of the NS1 Toxin during Dengue Virus and Zika Virus Infection

doi: 10.3390/v15020364

Figure Lengend Snippet: ZIKV and DV2 NS1 recombinant proteins bind to extracellular vesicles. ( A ) HEK 293 cells expressing ZIKV-NS1 Flag--tag protein were treated or not with GW4869 at 10 µM for 16 h in order to inhibit exosome’s biogenesis. The cell culture supernatant (CCS) was collected for exosomes extraction using the Exoeasy kit. A dot blot was performed using anti-Flag and then quantified using imageJ. The flow through (FT) is the remaining part of the first exosome extraction step, and the wash is the second flow through, considered as a negative control. The quantity of NS1 associated with exosomes was reduced when cells were treated with GW4869, thus eliminating the co-elution bias of NS1 protein with exosomes. Unpaired t-test was performed using GraphPad Prism, * p < 0.05. ( B ) CCS of HEK was collected 48 h post-platting and incubated with ZIKV-NS1 HIS-tag or DENV2-NS1 HIS-tag for 30 min and 4 h. CCS was then proceeded to PEG exosome precipitation. The negative control (CTL−) corresponds to panserin. The positive control (CTL+) corresponds to 0.2 ng of NS1 solubilized in panserin. Dot blots were performed using anti-His and anti-NS1 antibodies to detect ZIKV and DV2 NS1 proteins, respectively. NS1 of ZIKV and DV2 were found associated with EVs as early as 30 min post-incubation.

Article Snippet: Recombinant ZIKV-NS1 HIS-tag (strain Zika SPH2015) and DENV2-NS1 HIS-tag (Dengue type 2, strain New Guinea C) were from Sino Biological.

Techniques: Recombinant, Expressing, FLAG-tag, Cell Culture, Dot Blot, Negative Control, Co-Elution Assay, Incubation, Positive Control

Journal: Viruses

Article Title: Extracellular Vesicles Are Conveyors of the NS1 Toxin during Dengue Virus and Zika Virus Infection

doi: 10.3390/v15020364

Figure Lengend Snippet: Graphical overview of the association of ZIKV and DENV NS1 proteins with exosomes.

Article Snippet: Recombinant ZIKV-NS1 HIS-tag (strain Zika SPH2015) and DENV2-NS1 HIS-tag (Dengue type 2, strain New Guinea C) were from Sino Biological.

Techniques:

Journal: Frontiers in Immunology

Article Title: Active Human Complement Reduces the Zika Virus Load via Formation of the Membrane-Attack Complex

doi: 10.3389/fimmu.2018.02177

Figure Lengend Snippet: Effect of human serum on ZIKV infectivity. ZIKV [1 x 10 6 PFU/mL] was incubated with active normal human serum (NHS) or 50% heat-inactivated NHS (hiNHS) for 1 h at 37°C. Thereafter, 10-fold dilutions of pre-incubated virus-serum mixtures were titrated on Vero cells. Plaques were visualized 4 days post infection using crystal violet staining. Data were analyzed with one-way-ANOVA with Bonferroni post-hoc comparison (*** p < 0.001; ns indicates not significant). Mean data of at least 3 independent experiments are shown. The error bars represent the standard deviation.

Article Snippet: To investigate the interaction of the complement component C1q with ZIKV-derived proteins, 5 μg/mL of recombinant ZIKV envelope or NS1 protein [MyBioSource, San Diego, USA] or 100 μL of ZIKV supernatant derived from C6/36 cells [containing 5 x 10 6 PFU/well] were coated in carbonate buffer (pH 9.6) on a 96-well flat-bottom plate (Maxisorp, Nunc, Roskilde, Denmark) overnight at 4°C.

Techniques: Infection, Incubation, Virus, Staining, Comparison, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Active Human Complement Reduces the Zika Virus Load via Formation of the Membrane-Attack Complex

doi: 10.3389/fimmu.2018.02177

Figure Lengend Snippet: Time as a limiting factor on ZIKV infectivity. ZIKV [1 x 10 6 PFU/mL] was mixed with 50% active or heat-inactivated NHS and incubated for different time points (ranging from 1 min to 1 h) at 37°C. Virus-containing samples were then serially diluted and titrated on Vero cells. After 1 h incubation at 37°C, the cells were overlaid with agarose. Viral concentration was determined 4 days post infection using crystal violet staining. Mean data of 3 independent experiments are shown. The error bars represent the standard deviation.

Article Snippet: To investigate the interaction of the complement component C1q with ZIKV-derived proteins, 5 μg/mL of recombinant ZIKV envelope or NS1 protein [MyBioSource, San Diego, USA] or 100 μL of ZIKV supernatant derived from C6/36 cells [containing 5 x 10 6 PFU/well] were coated in carbonate buffer (pH 9.6) on a 96-well flat-bottom plate (Maxisorp, Nunc, Roskilde, Denmark) overnight at 4°C.

Techniques: Infection, Incubation, Virus, Concentration Assay, Staining, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Active Human Complement Reduces the Zika Virus Load via Formation of the Membrane-Attack Complex

doi: 10.3389/fimmu.2018.02177

Figure Lengend Snippet: (A) EDTA inhibition confirms complement contribution to viral lysis. ZIKV [1 x 10 6 PFU/mL] was incubated with active or heat-inactivated human serum in the presence of increasing amounts of EDTA or Mg 2+ -EGTA. DMEM (not shown) and hiNHS were used as controls. (B) To prevent activation of the classical and lectin complement pathways, active or heat-inactivated human serum was pre-incubated with increasing amounts of C1 esterase inhibitor as indicated. (C) In the absence of C1q, most of the virus remained infectious whereas addition of purified C1q (70 μg/mL) restored the lytic effect on the virus. HiNHS was set to 100%. (D) Blocking of the lectin pathway by synthetic peptides (SFMI-1/2) did not rescue the virus. Combination of C1q-depleted serum and SFMI-1/2 served as additional control showing that the peptides had no effect on the infectivity of the virus. In all experiments, the viral titer was determined by plaque assays using Vero cells. Data were analyzed with one-way-ANOVA with Bonferroni post-hoc comparison (** p < 0.005). All virus lysis experiments were conducted in duplicates and repeated three times. The data represent mean values, and the error bars show standard deviations.

Article Snippet: To investigate the interaction of the complement component C1q with ZIKV-derived proteins, 5 μg/mL of recombinant ZIKV envelope or NS1 protein [MyBioSource, San Diego, USA] or 100 μL of ZIKV supernatant derived from C6/36 cells [containing 5 x 10 6 PFU/well] were coated in carbonate buffer (pH 9.6) on a 96-well flat-bottom plate (Maxisorp, Nunc, Roskilde, Denmark) overnight at 4°C.

Techniques: Inhibition, Lysis, Incubation, Activation Assay, Virus, Purification, Blocking Assay, Control, Infection, Comparison

Journal: Frontiers in Immunology

Article Title: Active Human Complement Reduces the Zika Virus Load via Formation of the Membrane-Attack Complex

doi: 10.3389/fimmu.2018.02177

Figure Lengend Snippet: Natural IgM blocking results in ZIKV rescue. Anti-human IgM blocking antibodies were incubated with 50% NHS or hiNHS for 30 min on ice before ZIKV was added. After incubation of 1 h at 37°C, the virus-serum mixture was serially diluted and titrated on Vero cells. After 1 h r incubation at 37°C, the cells were overlaid with agarose. Viral concentration was determined4 days post infection using crystal violet staining. All virus lysis experiments were conducted in triplicate, and the error bars show standard deviations.

Article Snippet: To investigate the interaction of the complement component C1q with ZIKV-derived proteins, 5 μg/mL of recombinant ZIKV envelope or NS1 protein [MyBioSource, San Diego, USA] or 100 μL of ZIKV supernatant derived from C6/36 cells [containing 5 x 10 6 PFU/well] were coated in carbonate buffer (pH 9.6) on a 96-well flat-bottom plate (Maxisorp, Nunc, Roskilde, Denmark) overnight at 4°C.

Techniques: Blocking Assay, Incubation, Virus, Concentration Assay, Infection, Staining, Lysis

Journal: Frontiers in Immunology

Article Title: Active Human Complement Reduces the Zika Virus Load via Formation of the Membrane-Attack Complex

doi: 10.3389/fimmu.2018.02177

Figure Lengend Snippet: Binding of C1q to recombinant ZIKV envelope (E) and NS1 proteins. ZIKV proteins or viral particles were coated to ELISA plates and incubated with decreasing amounts of C1q as indicated. Bound C1q was detected using a polyclonal anti-C1q antibody followed by a HRP-labeled goat anti-rabbit IgG and visualized by TMB. The absorbance was measured at 450 nm, using a Bio-Rad plate reader. Data show the mean of two experiments performed in duplicate.

Article Snippet: To investigate the interaction of the complement component C1q with ZIKV-derived proteins, 5 μg/mL of recombinant ZIKV envelope or NS1 protein [MyBioSource, San Diego, USA] or 100 μL of ZIKV supernatant derived from C6/36 cells [containing 5 x 10 6 PFU/well] were coated in carbonate buffer (pH 9.6) on a 96-well flat-bottom plate (Maxisorp, Nunc, Roskilde, Denmark) overnight at 4°C.

Techniques: Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Incubation, Labeling

Journal: Frontiers in Immunology

Article Title: Active Human Complement Reduces the Zika Virus Load via Formation of the Membrane-Attack Complex

doi: 10.3389/fimmu.2018.02177

Figure Lengend Snippet: Dissecting the role of the lectin and classical pathways on ZIKV derived from the human cell line A549. NHS (50%) reduced the viral titer for about one order of magnitude. Inhibition of the lectin pathway by a peptide mix of SFMI-1 and 2 had no effect. By contrast, C1q depletion rescued the virus and most of the virus remained infectious. Viral titer was determined by plaque assays using Vero cells. Data were analyzed with one-way-ANOVA with Bonferroni post-hoc comparison (** p < 0.005). All virus lysis experiments were conducted in duplicates and repeated two times. The data represent mean values, and the error bars show standard deviations.

Article Snippet: To investigate the interaction of the complement component C1q with ZIKV-derived proteins, 5 μg/mL of recombinant ZIKV envelope or NS1 protein [MyBioSource, San Diego, USA] or 100 μL of ZIKV supernatant derived from C6/36 cells [containing 5 x 10 6 PFU/well] were coated in carbonate buffer (pH 9.6) on a 96-well flat-bottom plate (Maxisorp, Nunc, Roskilde, Denmark) overnight at 4°C.

Techniques: Derivative Assay, Inhibition, Virus, Comparison, Lysis

Journal: Frontiers in Immunology

Article Title: Active Human Complement Reduces the Zika Virus Load via Formation of the Membrane-Attack Complex

doi: 10.3389/fimmu.2018.02177

Figure Lengend Snippet: Reduction of viral infectivity is linked to decreased RNA levels. After incubating ZIKV with active or heat-inactivated human serum, the viral RNA was digested by addition of RNases. Three hours after incubation at 37°C, the remaining genomic material was extracted and quantified by RT-PCR. The RNA copy number was calculated from the amount of RNA obtained by incubation of the virions with 50% hiNHS, which was set to 100%. Results are given as % of RNA loss. Data were analyzed with one-way-ANOVA with Bonferroni post-hoc comparison (**** p < 0.0001). All virus lysis experiments were conducted in triplicate. The data represent mean values, and the error bars show standard deviations.

Article Snippet: To investigate the interaction of the complement component C1q with ZIKV-derived proteins, 5 μg/mL of recombinant ZIKV envelope or NS1 protein [MyBioSource, San Diego, USA] or 100 μL of ZIKV supernatant derived from C6/36 cells [containing 5 x 10 6 PFU/well] were coated in carbonate buffer (pH 9.6) on a 96-well flat-bottom plate (Maxisorp, Nunc, Roskilde, Denmark) overnight at 4°C.

Techniques: Infection, Incubation, Reverse Transcription Polymerase Chain Reaction, Comparison, Virus, Lysis

Journal: Frontiers in Immunology

Article Title: Active Human Complement Reduces the Zika Virus Load via Formation of the Membrane-Attack Complex

doi: 10.3389/fimmu.2018.02177

Figure Lengend Snippet: Blocking the assembly of MAC leads to virus rescue. ZIKV [1 x 10 6 PFU/mL] was exposed to either 50% C9-depleted (C9dep) human serum or 50% heat-inactivated C9-depleted (hiC9dep) serum for 1 h at 37°C. As a control, the depleted serum was reconstituted with purified C9 protein, adjusted to its natural concentration in serum [60 μg/mL]. During incubation, the RNA of lysed ZIKV was digested by external RNase addition. Subsequently, the amount of complement lysis-resistant virions was determined by RT-PCR. The RNA copy number was calculated by incubation of the virions with 50% hiNHS, which was set to 100%. Results are given as % of RNA loss. All assays were performed in triplicate. The data represent mean values, and the error bars show standard deviations.

Article Snippet: To investigate the interaction of the complement component C1q with ZIKV-derived proteins, 5 μg/mL of recombinant ZIKV envelope or NS1 protein [MyBioSource, San Diego, USA] or 100 μL of ZIKV supernatant derived from C6/36 cells [containing 5 x 10 6 PFU/well] were coated in carbonate buffer (pH 9.6) on a 96-well flat-bottom plate (Maxisorp, Nunc, Roskilde, Denmark) overnight at 4°C.

Techniques: Blocking Assay, Virus, Control, Purification, Concentration Assay, Incubation, Lysis, Reverse Transcription Polymerase Chain Reaction

Journal: Sensors (Basel, Switzerland)

Article Title: Label-Free Electrochemical Biosensors for the Determination of Flaviviruses : Dengue, Zika, and Japanese Encephalitis

doi: 10.3390/s20164600

Figure Lengend Snippet: Survey of electrochemical label-free biosensors for Zika diagnostic.

Article Snippet: Microtiter plate precoated with ZIKV NS1/ Euroimmun anti-ZIKV IgM , Indirect ELISA , Antibody-HRP conjugates , DENV Ig M antibodies , 56% , West Nile virus , d, f , a, c , [ , ] .

Techniques: Diagnostic Assay, Virus, Polymer, Membrane, Indirect ELISA

Journal: Scientific Reports

Article Title: Immune outcomes of Zika virus infection in nonhuman primates

doi: 10.1038/s41598-020-69978-w

Figure Lengend Snippet: Adaptive immune responses. ( a , b ) viSNE analyses of T cell activation in rhesus ( a ) and cynomolgus ( b ) macaques, as measured by CD69 expression. Dot plots are concatenated for CD8-depleted and nondepleted animals within each species. The viSNE clustering profiles of CD4 and CD8 T cell subsets ( left ), including naïve, central memory (CM), and effector memory (EM) T cells correspond to cell populations in the CD69 heatmaps in nondepleted and CD8-depleted animals at 1 and 10 dpi ( right ). Black ovals indicate EM CD4 populations in CD8-depleted animals at 10 dpi, which showed higher levels of CD69 expression compared to nondepleted animals at the same timepoint. ( c ) Proliferation of EM CD4 T cells by Ki67 expression. Area-under-the-curve analysis revealed a significant difference in EM CD4 T cell proliferation among CD8-depleted (n = 4) and nondepleted (n = 5) animals by a Mann–Whitney test ( p = 0.0159). viSNE plots were generated using viSNE software (Cytobank version 7.0, https://www.cytobank.org/platform.html ). ( d ) CD4 T cell responses in rhesus macaques, assessed by intracellular cytokine staining (ICS) of PBMCs stimulated with viral peptides derived from the indicated ZIKV proteins (C = capsid; M = membrane; E = envelope; NS1 = nonstructural protein 1, consistent throughout). CD4 T cell responses were identified by co-positivity for IL-2 and IFNγ. ( Inset ): representative antigen-specific cytometry plots for R64357 (CD8-depleted) at 30 dpi. Dot plots were generated using FlowJo (version X.10.4.2, https://www.flowjo.com/ ). ( e ) CD8 T cell responses, determined by ICS for perforin and IFNγ co-positivity. ( f ) Serum neutralizing antibody titers in rhesus macaques, represented as PRNT90.

Article Snippet: PBMCs from the indicated timepoints were thawed and rested overnight prior to stimulation with peptide pools comprising ZIKV capsid (C), membrane (M), envelope (E), and nonstructural protein 1 (NS1) (BEI Resources).

Techniques: Activation Assay, Expressing, MANN-WHITNEY, Generated, Software, Staining, Derivative Assay, Cytometry

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