Figure 3: Both N- and C-terminal dsRNA-binding sites of TLR3 are required for PV-RNA-induced TLR3 activation.
From: Toll-like receptor 3 recognizes incomplete stem structures in single-stranded viral RNA

(a) Secondary structure of PV-RNAs (cPV1, PV5 and PV6) predicted by the mfold software. Thick lines indicate dsRNA regions (1–11 bp). The starting and ending positions of ds regions are shown with the nt number. A total number of base pair and the number of nts involved in base pairing in cPV1, PV5 and PV6 are described under the secondary-structure model. (b) Binding affinity of PV-RNAs to TLR3 ECD under different pH conditions. 32P-labelled PV-RNAs (cPV1 and PV5) were mixed with varying concentrations of hTLR3 ECD protein and passed through a nitrocellulose filter. After washing, bound radioactivity was measured, and binding activities were calculated as a percentage of input RNA before filtration. The apparent dissociation constant (Kd) values calculated for cPV1 and PV5 were 39±16 and 10±3 nM (pH 5.0), and 31±13 and 7±3 nM (pH 6.0), respectively. (c) HEK293 cells were transfected with an empty vector or expression plasmid for WT TLR3 or each TLR3 mutant (H39A, H39E, H60A, H60E and H539A), together with the IFN-β promoter reporter plasmid and phRL-TK. Twenty-four hours post transfection, cells were stimulated with 10 μg ml−1 poly(I:C) or PV5 in FCS-free medium. Luciferase activity was measured 6 h after stimulation. Representative data from three independent experiments are shown.