Figure 7: Mapping of TLR3-activating RNA structure in PV5.

(a) Secondary structure of PV5-derived RNAs (PV5-D1–PV5-D5) predicted by the mfold software. (b) RNAs were incubated in FCS-free or -containing medium at 37 °C. Non-treated RNA or RNA incubated for 30 min were loaded onto a 1% agarose gel. (c) TLR3-mediated IFN-β promoter activation induced by PV5 or PV5-derived RNAs in HEK293 cells transiently expressing TLR3. Cells were stimulated with indicated RNAs (10 μg ml⁻¹) (upper panel) or RNAs complexed with DOTAP (1 μg ml⁻¹) (lower panel). After 6 h, luciferase reporter activity was measured and expressed as the fold induction relative to the activity of unstimulated cells. Data are shown as the mean±s.d. Representative data from three independent experiments are shown. (d) Splenic CD11c⁺ DCs (upper panels) or bone marrow-derived macrophages (lower panels) (2 × 10⁶ per ml) isolated from TLR3^−/− or WT mice were stimulated with 100 pmol ml⁻¹ PV5 or PV5-derived RNAs in FCS-free AIM medium. Twenty-four hours after stimulation, culture supernatants were collected, and IFN-β in the supernatants was quantified using ELISA. TNF-α and IL-6 levels were measured using CBA. Representative data from three independent experiments are shown (mean±s.d.).