Journal: Frontiers in Chemistry

Article Title: Identification of 6ω-cyclohexyl-2-(phenylamino carbonylmethylthio)pyrimidin-4(3 H )-ones targeting the ZIKV NS5 RNA dependent RNA polymerase

doi: 10.3389/fchem.2022.1010547

Figure Lengend Snippet: The interaction between 4w and ZIKV NS5 protein. (A) WB detection of ZIKV NS5 protein expression under different temperature gradients after 4w treatment; (B) NS5 protein grayscale analysis and ZIKV NS5 protein expression after 4w treatment observed changes in aggregation temperature; data is the mean (±SD) of three experiments. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Subsequently, the protein was transferred to a polyvinylidene fluoride (PVDF) membrane and incubated with ZIKV E (1:2,000) and NS5 antibody (1:2,000) overnight at 4°C (Sino Biological, China) then add the corresponding secondary antibody and incubate at room temperature for 2 h. A specific signal was presented with a chemiluminescent substrate.

Techniques: Expressing

Journal: Frontiers in Chemistry

Article Title: Identification of 6ω-cyclohexyl-2-(phenylamino carbonylmethylthio)pyrimidin-4(3 H )-ones targeting the ZIKV NS5 RNA dependent RNA polymerase

doi: 10.3389/fchem.2022.1010547

Figure Lengend Snippet: Compound 4w inhibited the expression of ZIKV E and NS5 protein. (A) Western blot detected the inhibitory effect of 4w on ZIKV E and NS5 protein under the concentration gradient of 4w ; (B) Grayscale analysis and statistics of the inhibition of 4w on ZIKV E and NS5 protein under the concentration gradient of WB detection; Data is the mean (±SD) of three experiments, with DMSO as a positive contro * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. (C) Detected the inhibitory effect of 4w on ZIKV E protein by immunofluorescence.

Article Snippet: Subsequently, the protein was transferred to a polyvinylidene fluoride (PVDF) membrane and incubated with ZIKV E (1:2,000) and NS5 antibody (1:2,000) overnight at 4°C (Sino Biological, China) then add the corresponding secondary antibody and incubate at room temperature for 2 h. A specific signal was presented with a chemiluminescent substrate.

Techniques: Expressing, Western Blot, Concentration Assay, Inhibition, Immunofluorescence

Journal: Emerging Microbes & Infections

Article Title: NS5-independent Ablation of STAT2 by Zika virus to antagonize interferon signalling

doi: 10.1080/22221751.2021.1964384

Figure Lengend Snippet: ZIKV infection interrupted host de novo translation. (a) Vero cells in duplicate wells were transfected with ZIKV Ubi-NS5-expressing plasmid (upper panel) or infected with ZIKV C7 (MOI = 1) for 54 h and then analysed by western blotting with the indicated antibodies. The protein abundances of each protein were quantified and plotted. A representative picture of two biological replicates is shown. Similar results were observed in multiple independent experiments. The values to the left of the blots are molecular sizes in kilodaltons. Statistical analysis was performed between the C7-infected group and the NS5-expressing group and the uninfected groups (Mock) (ns, not significant, * P < 0.05; two-tailed, unpaired t -test). (b) Schematic of the experimental design for c-n. The plasmids expressing the orthogonal tRNA (tRNA) and orthogonal aminoacyl-tRNA synthases (aaRS) and plasmid HA.ypet-TAG expressing the N-terminally HA-tagged ypet with the TAG codon replaced at amino acid 182 were cotransfected into Vero cells in triplicate wells for 24 h. Then, the transfected cells were infected or mock-infected (mock) with C7 (MOI = 5) or C7.D29 (MOI = 5) or retransfected with NS5. The cells were treated or not (control) with media containing p-azido-L-phenylalanine (0.5 mM) at 1, 2 and 3 d post infection and chased or not (control) for 4 h before harvest. For translation control, the cells were treated with 5 μg/ml puromycin (puro) for 24 h before harvesting at each time point. (c) Western blotting analysis of the 24-hour (1d)-infected cell lysates with the indicated antibodies. Representative pictures of three biological replicates are shown. The values to the left of the blots are molecular sizes in kilodaltons. (d–f) The protein abundances of protein bands in c were quantified and plotted. The mean ± SD of three biological replicates is shown ( n = 3). Statistical analysis was performed between the indicated pairs (ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001; two-tailed, unpaired t -test). (g) Western blotting analysis of the 48-hour (2d)-infected cell lysates with the indicated antibodies. Representative pictures of three biological replicates are shown. The values to the left of the blots in the panels are molecular sizes in kilodaltons. (h–j) The protein abundances of each protein in g were quantified and plotted. The mean ± SD of three biological replicates is shown ( n = 3). Statistical analysis was performed between the indicated pairs (ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001; two-tailed, unpaired t -test). (k) Western blotting analysis of the 72-hour (3d)-infected cell lysates with the indicated antibodies. Representative pictures of three biological replicates are shown. The values to the left of the blots are molecular sizes in kilodaltons. (l–n) The protein abundances of each protein in k were quantified and plotted. The mean ± SD of three biological replicates is shown ( n = 3). Statistical analysis was performed between the indicated pairs (ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001; two-tailed, unpaired t -test).

Article Snippet: Anti-YFV NS5 antibody (GTX134141; Genetex) were used in Western blots at 1:1000 dilution in immunostaining.

Techniques: Infection, Transfection, Expressing, Plasmid Preparation, Western Blot, Two Tailed Test, Control

Journal: Emerging Microbes & Infections

Article Title: NS5-independent Ablation of STAT2 by Zika virus to antagonize interferon signalling

doi: 10.1080/22221751.2021.1964384

Figure Lengend Snippet: Murine STAT2, which was refractory to ZIKV-induced ablation, elicited robust antiviral signalling upon IFN treatment. (a) Murine STAT2 was refractory to ZIKV-induced reduction. Huh7.5 cells stably expressing GFP, hSTAT2-HA and murine STAT2-HA (mSTAT2-HA) were infected with ZIKV (MOI = 5) for 2 d and then harvested for western blotting analysis with the indicated antibodies. Representative pictures of three biological replicates are shown. The values to the left of the blots are molecular sizes in kilodaltons. Statistical analysis was performed between the C7-infected group and NS5-expressing group and the uninfected groups (Mock) (ns, not significant, ** P < 0.01; two-tailed, unpaired t-test). (b) Murine STAT2 was refractory to ZIKV NS5 induced reduction. The plasmids expressing the ZIKV Ubi-NS5 or Vector were co-transfected with hSTAT2-HA or murine STAT2-HA (mSTAT2-HA)-expressing plasmid and GFP-expressing plasmid (6:3:1) in to Vero cells for 48 h, and then harvested for western blotting analysis with the indicated antibodies. Representative pictures of three biological replicates are shown. The values to the left of the blots are molecular sizes in kilodaltons. Statistical analysis was performed between the indicated groups (ns, not significant, ** P < 0.01; two-tailed, unpaired t-test). (c) Experimental design for C. Huh7.5-GFP, Huh7.5-hSTAT2-HA and Huh7.5-mSTAT2-HA cells were infected with C7-Gluc (MOI = 0.4) for 8 h and then treated with IFN-α (2000 U/ml). At the indicated time points after IFN-α treatment, cells were harvested. (d) Gluc activities in the cell lysates of Huh7.5-GFP, Huh7.5-hSTAT2-HA and Huh7.5-mSTAT2-HA cells were determined. The mean ± SD of three biological replicates is shown ( n = 3). Statistical analysis was performed between the indicated groups at each time point (** P < 0.01, *** P < 0.001; two-tailed, unpaired t-test). (e) Type I interferon (IFN) binds to interferon receptors, activating the Janus kinases Jak1 and Tyk2 to engage STAT1 and STAT1 and phosphorylate STAT1 and STAT2. Phosphorylated STAT1 and STAT2 form a heterodimer and recruit IFN regulatory factor 9 (IRF-9) to assemble interferon-stimulating gene factor 3 (ISGF3). ISGF3 enters the nucleus to elicit the expression of interferon-stimulating genes. ZIKV infection interrupts host de novo protein synthesis, accelerating the degradation of the pool of ubiquitinated short-lived proteins and STAT2. As reported, NS5 may contribute to this process by facilitating NS5 ubiquitination. See details in the text.

Article Snippet: Anti-YFV NS5 antibody (GTX134141; Genetex) were used in Western blots at 1:1000 dilution in immunostaining.

Techniques: Stable Transfection, Expressing, Infection, Western Blot, Two Tailed Test, Plasmid Preparation, Transfection, Ubiquitin Proteomics

Journal: Viruses

Article Title: TIM-1 Promotes Japanese Encephalitis Virus Entry and Infection

doi: 10.3390/v10110630

Figure Lengend Snippet: JEV infection and TIM-1 expression in cell lines. BHK, A549, and 293T cells were infected with JEV NJ2008 (MOI of 0.5) for 40 h. ( A ) Supernatants were collected and the production of progeny virions were determined by a plaque assay titrated on BHK-21 cells. Data are presented as mean ± standard deviation (SD) from three independent experiments; ( B ) Cell lysates were harvested to detect the level of JEV NS5 and TIM-1 protein by Western blot analysis using specific antibodies. One representative experiment out of three is shown. Endogenous TIM-1 expression in A549 and 293T cells was detected by ( C ) Western blot analysis and ( D ) confocal microscopy using an anti-TIM-1 monoclonal antibody. Scale bars, 10 µm.

Article Snippet: Specific monoclonal antibodies for JEV E and NS1 were produced in our lab, and an NS5 monoclonal antibody was purchased from Gene Tex (Irvine, CA, USA).

Techniques: Infection, Expressing, Plaque Assay, Standard Deviation, Western Blot, Confocal Microscopy