Characterization of IFNβ promoter activation by Nsp2(C-E). Overview of the IFNβ promoter (A). Impact of Nsp2 expression on the IFNβ promoter (B), the NF-κB responsive elements, positive regulatory domain II (PRDII) (C) and IRF3-responsive elements, the positive regulatory domains I and III (PRDI-III) (D) of those promoters. Reporter essays were performed as described above with 300 ng, 200 ng, and 100 ng of vector encoding for Nsp2. All experiments were performed twice in triplicate, and one representative experiment is shown. Results are expressed as fold activation relative to the pcDNA mock-infected control (mean ± SD, n = 3 replicates/condition). Effect of Nsp2 expression on the IFNβ1 production (E). Twenty-four hours post-seeding, A549-hACE2 SB-Nsp2 or SB-GFP cells were induced with 0.5 µg/mL of Doxycycline. Thirty-two hours post-induction, cells were stimulated with poly(I:C) for sixteen hours and IFNβ1 was measured in the supernatant by ELISA. All experiments were performed twice in triplicate and the compilation of the data is shown. Results are expressed as activation percentage relative to the poly I:C mock-infected control (mean ± SD (n = 6 replicates/condition)). Statistical analyses were performed by comparing corresponding condition with mock-transfected control with a nonparametric one-way ANOVA with Dunn’s correction. ns: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.