Journal: iScience

Article Title: Virus-host interactome reveals host cellular pathways perturbed by tick-borne encephalitis virus infection

doi: 10.1016/j.isci.2026.115821

Figure Lengend Snippet: TBEV NS5 interacts with P300 to modulate G0/G1 cell cycle progression (A) Cells were transfected with the plasmids of Flag-tagged EV and ten TBEV viral proteins, the cell distribution was analyzed by flow cytometry. (B) The expression plasmids of Flag-EV, 0.5 and 1.0 μg Flag-NS5 were transfected into A549 cells, the cell distribution was analyzed by flow cytometry. The experiment was repeated for three times, and the percentages of cells were shown in column graph. (C) Cells were transfected with Flag-EV or Flag NS5 together with HA-P300, the interaction between NS5 and P300 was analyzed by co-immunoprecipitation analysis. (D) The colocalization of P300 (green) and NS5 (red) were examined by immunofluorescence analysis. Scale bars, 10 μm. (E–G) Cells transfected with NS5 were collected after 48 h, the expression of CDK4, CDK6, and P16 was analyzed by immunoblot (E). (F) Quantification of protein levels from (E). Data are normalized to actin and presented as fold change relative to control (mean ± SD, n = 3). (G) qPCR analysis of mRNA expression of CDK4, CDK6, and P16 upon NS5 overexpression. Data are normalized to GAPDH using the 2ˆ(-ΔΔCt) method and shown as fold change relative to control (mean ± SD, n = 3). (H) A549 cells transfected with HA-EV, HA P300 and P300 Hm were further transfected with NS5, the distribution of cells was analyzed by flow cytometry. The experiments were repeated for three times, and the proportions of cells were shown in column graph, the expression of indicated proteins was assessed by immunoblot assay. (I) Cells transfected with P300 siRNA and NC siRNA (siNC) were further transfected with NS5, the distribution of cells was analyzed by flow cytometry, and the proportions of cells and the relative expression of P300 mRNA were shown in column graph. (J) Cells transfected with HA-EV, HA P300, and P300 Hm were mock-infected or infected TBEV, the distribution of cells was analyzed by flow cytometry. (K) Cells transfected with P300 siRNA and NC siRNA were mock-infected or infected TBEV, the distribution of cells was analyzed by flow cytometry. (L) Outline of G0/G1 cell-cycle arrest regulated by TBEV NS5. Data in column graphs are represented as mean ± SEM of three independent experiments. The p values are calculated and reported using one-way ANOVA.

Article Snippet: For small interfering RNA (siRNA) targeting P300, 5′-GGACUACCCUAUCAAGUAATT-3′, was synthesized at Sangon Biotech (Shanghai, China).

Techniques: Transfection, Flow Cytometry, Expressing, Immunoprecipitation, Immunofluorescence, Western Blot, Control, Over Expression, Infection

Journal: iScience

Article Title: Virus-host interactome reveals host cellular pathways perturbed by tick-borne encephalitis virus infection

doi: 10.1016/j.isci.2026.115821

Figure Lengend Snippet: P300 inhibitors and CDK4 agonist restrict viral gene expression by interfering with TBEV-induced cell-cycle arrest (A and B) A549 cells were either mock-infected or infected with TBEV, the cells were then treated with indicated concentration of C646 (A) and CPI-637 (B), the supernatants were collected at 48 hpi, and the mRNA level of TBEV E gene was analyzed by probe qPCR. (C–F) Cells mock-infected or infected with TBEV at the MOI of ten were treated with 10 μM C646 and CPI-637, the expression of TBEV NS1 protein was detected by immunoblot (C and D) and immunofluorescence analysis (E), and the cell cycle distribution was analyzed by flow cytometry (F). Scale bars, 50 μm. (G) Cells transfected with HA-EV, HA P300, and P300Hm were infected TBEV, the mRNA level of TBEV E gene in the supernatants was analyzed by probe qPCR. (H) Cells transfected with P300 siRNA and NC siRNA were infected TBEV, the mRNA level of TBEV E gene in the supernatants was analyzed by probe qPCR. (I) A549 cells were treated with the indicated concentrations of chrysin for 2 h, the cells were then infected with TBEV, the supernatants were collected at 48 hpi, and the mRNA level of TBEV E gene was analyzed by probe qPCR. (J–L) Cells were treated with 10 μM chrysin for 2 h, the cells were then mock-infected or infected with TBEV at the MOI of ten, the expression of TBEV NS1 protein was detected by immunoblot (J), and the cell distribution was analyzed by flow cytometry (K). The percentage of cells (L) upon chrysin treatment was shown in column graphs. Scale bars, 50 μm. Data in column graphs are represented as mean ± SEM of three independent experiments. The p values are calculated and reported using one-way ANOVA.

Article Snippet: For small interfering RNA (siRNA) targeting P300, 5′-GGACUACCCUAUCAAGUAATT-3′, was synthesized at Sangon Biotech (Shanghai, China).

Techniques: Gene Expression, Infection, Concentration Assay, Expressing, Western Blot, Immunofluorescence, Flow Cytometry, Transfection