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. 2023 May 1;11(3):e04138-22. doi: 10.1128/spectrum.04138-22

FIG 1.

FIG 1

EV-D68 VP3 suppresses virus-induced IFN-β activation. (A) The UV-inactivated EV-D68 virus dose-dependently inhibited the IFN-β promoter induced by SeV infection. RD cells were treated with an increasing dose of UV-EV-D68 (MOI = 0.5, 1, 2, 3) for 24 h. The positive control group was cells infected with active EV-D68 (MOI = 0.5). HEK293T cells were transfected with 200 ng of the reporter plasmid IFN-β-Luc and 2 ng of pRLSV40 for 24 h. After transfection, the cells were treated with SeV (20 HA/mL) or UV-EV D68 (MOI = 0.5, 1, 2, 3) for 24 h, and the cell lysates were subjected to a luciferase assay. (B) Detection of anti-IFN-I responses by viral structural proteins. HEK293T cells were transfected with 200 ng of the reporter plasmid IFN-β-Luc, 2 ng of pRL-SV40, and 600 ng of VP1, VP2/4, or VP3. Twenty-four hours later, the cells were treated with medium or SeV (20 HA/mL) for 16 h, the cell lysates were subjected to a luciferase assay, and the expression of plasmid was confirmed by Western blotting. (C) The treatment of EV-D68 VP3 inhibits IFN-β mRNA synthesis. HEK293T cells were transfected with 600 ng VP3-HA or empty vector. Twenty-four hours after transfection, cells were infected with SeV (20 HA/mL) for 16 h. Total RNA was extracted, and the expression of IFN-β mRNA was determined by quantitative PCR (qPCR) assay. Results are expressed as IFN-β mRNA levels relative to GAPDH RNA levels. Cell lysates were subjected to Western blotting with antibodies against anti-HA and GAPDH. (D) VP3 dose-dependently inhibits SeV-induced IFN-β promoter activity. HEK293T cells were transfected with an increasing dose of VP3 (200 ng, 400 g, 600 ng, and 800 ng). Twenty-four hours later, the cells were treated with medium or SeV (20 HA/mL) for 16 h, the cell lysates were subjected to a luciferase assay, and the expression of plasmid was confirmed by Western blotting. A statistically significant difference was determined using SPSS with a two-tailed Student’s unpaired t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., not significant).