Fig. 3

UPR-induced antiviral signaling following infection is independent of interferon signaling. a Tunicamycin treatment inhibited TBEV yields independently of IFNAR. Primary embryonic MEF Ifnar^-/- cells were either infected with TBEV (blue bars) or treated with TM immediately after infection (black bars). At the indicated time points virus yields were measured (PFU/ml). b Tunicamycin treatment of TBEV infected cells led to early IFNβ induction. Total RNA from MEF Ifnar^-/- infected as above in a was extracted at the indicated time points and used as template for real-time qPCR using primers specific for IFNβ. c Tunicamycin treatment led to early IRF3 phosphorylation. U2OS cells were infected with TBEV and treated with TM immediately after infection. At the indicated time points the total protein content was extracted and subjected to immunoblotting with the indicated antibodies. d Tunicamycin treatment led to early IRF3 nuclear translocation. U2OS cells were infected with TBEV and treated with TM immediately after infection. At the indicated time points, cells were fixed and stained for IRF3 (AlexaFluor 488, green) and dsRNA (AlexaFluor 594, red). Scale bar = 10 μm. e Quantification of IRF3 translocation following TBEV infection and Tunicamycin treatment. Cells treated as in d were manually quantified for IRF3 nuclear translocation at each time point. The graph shows the results from two independent experiments, ∼ 200 cells for each time point. f, g Tunicamycin treatment of TBEV infected cells led to early ISG induction. Total RNA from U2OS cells infected with TBEV moi = 1 was extracted at the indicated time points and used as template for real-time qPCR using primers specific for IFIT1 or Viperin. Values of ISG mRNA from mock-infected cells treated with Tunicamycin were also indicated (magenta bars). h Tunicamycin treatment of TBEV infected cells led an early ISG signature. Transcriptome analysis of cells infected with TBEV compared with infected and treated with TM was performed at 8 h.p.i. in U2OS cells. The cluster of 39 genes differentially regulated in the two conditions is shown in the heatmap (upregulated, red, downregulated, blue, following the indicated gradient). Statistics as already described in the legend to Fig. 1. Source data are provided as a Source Data file