IRF1 impacts VACV replication in glucose/IFN-γ primed but not galactose conditions.

(A) Western blot analysis of IRF1 KO A549 cell lines generated with pLentiCRISPR V2 vectors. Two sgRNAs were used per target gene; sgCtrl–non targeting control. (B) Glucose: VSV-Luc (luciferase) replication assays in IRF1 KO cells primed with IFN-γ (N = 4). (C) Galactose: VSV-Luc (luciferase) replication assays in IRF1 KO cells primed with IFN-γ (N = 4). (D) ISRE reporter assay of 293T cells transfected with human FLAG-IRF1 plasmids. EV: empty vector, FL-hIRF1: WT human FLAG-IRF1, FL-hIRF (YLP): human FL-IRF1 Y109A/L112A/P113A mutant [75]. (E) Glucose: no IFN-priming vaccinia (luciferase) replication assays ‐ 24 hours post infection ‐ in IRF1 KO cells (sgIRF1_1) transiently transfected with human FLAG-IRF1 plasmids for 24 hours or 48 hours prior to infection (N = 4); VACV-Luc-GFP (MOI = 0.01). EV: empty vector, FL-hIRF1: WT human FLAG-IRF1, FL-hIRF1 (YLP): human FL-IRF1 Y109A/L112A/P113A mutant [75]. (F) Western blot analysis of glucose: no IFN-priming VACV-infected IRF1 KO cells transiently transfected with human FLAG-IRF1 plasmids. Infection was performed with VACV-Luc-GFP (MOI = 0.01) at 48 hrs post-transfection, and protein was harvested at 24 hours post-infection. (G) Glucose: VACV-Luc-GFP (luciferase) replication assays in IRF1 KO cells primed with IFN-γ (N = 4). (H) Glucose: western blot of vaccinia virus infected IRF1 KO A549 cells with or without IFN-γ priming. (I) Galactose: VACV-Luc-GFP (luciferase) replication assays in IRF1 KO cells primed with IFN-γ (N = 4). (J) Galactose: western blot of vaccinia virus infected IRF1 KO A549 cells with or without IFN-γ priming. IRF1 blot in galactose is long exposure. Statistical analysis was performed using an unpaired t-test in GraphPad Prism 9.5.1: n.s. not significant, * P ≤ 0.05, ** P< 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.