Terminal loop adenines are necessary for high IFN expression. (A) Predicted RNA structures of strong immunostimulatory and weak immunostimulatory stem–loops derived from cbVGs. (B–F) Indicated mutations were generated for cbVG sequences attached to the X RNA. The loop sequences of (B) SeV 70–114, (C) RSV 1–48, (D) NiV 85–136, and (E) NiV 324–361 were mutated. (F) RSV 1–48 stem nucleotides were mutated to alter RNA structure. RNAs were in vitro transcribed, and 1 pmol of IVT RNA was transfected into A549 cells. Expression of IFNL1 was measured by qPCR at 6 hpt. One-way ANOVAs with Tukey's HSD post hoc test were performed for statistical analysis. (ns) P > 0.05, (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, (****) P < 0.0001. Data are represented as fold change over the wild-type stem–loops. Data reported are biological triplicates.
