Figure 1: Extracellular PV-RNAs induce TLR3-mediated type I IFN production.
From: Toll-like receptor 3 recognizes incomplete stem structures in single-stranded viral RNA

(a) Mouse splenic CD11c+ DCs from WT or TLR3−/− mice (1 × 106 per ml) were stimulated with medium alone (−), poly(I:C), total RNA from PV-infected Vero cells or total RNA from uninfected Vero cells (25, 100 μg ml−1) for 24 h in FCS(−) AIM-V medium. RNAs were pre-treated with polymyxin B (5 μg ml−1) for 1 h before addition. IFN-α/β levels in culture supernatants were measured using ELISA. Representative data from three independent experiments are shown (mean±s.d.). (b) The RNA extracted from PV-infected or uninfected Vero cells was incubated in FCS(−) AIM-V medium for 30 min and then electrophoresed on a 1% agarose gel. The PV-RNAs were segmented into approximately 1,000–2,000-nt RNAs in FCS-free medium (right panel, square bracket). (c) Constructs of in vitro-transcribed PV-RNAs. Diagram of PV genome is shown (Top). Coding regions for viral capsid proteins (P1) and noncapsid proteins (P2 and P3) are indicated. Positions of the sense RNAs (PV1–10) and the complementary RNAs (cPV1–10) corresponding to the PV genome are shown as arrows. Each starting and ending position and length are described in Supplementary Table S1. (d) PV-RNA-induced TLR3-mediated IFN-β promoter activation. HEK293 cells were transfected with an empty vector (−) or expression plasmid for TLR3, together with the IFN-β promoter reporter and phRL-TK. Twenty-four hours after transfection, culture media was removed and 10 μg ml−1 poly(I:C), PV-derived dsRNAs, dsPV1–dsPV10 (left panel), sense RNAs, PV1–PV10 (middle panel) or complementary RNAs, cPV1–cPV10 (right panel), were added with FCS-free medium. Luciferase activity was measured 6 h after stimulation and shown as mean fold index induction compared with non-stimulated cells. Representative data from three independent experiments are shown (mean±s.d.). (e) Effect of FCS on the PV5-induced TLR3-mediated IFN-β promoter activation. Cells were stimulated with increasing amounts of poly(I:C) or PV5 (2.5, 10 or 25 μg ml−1) in the condition of FCS-free or -containing medium.