Fig. 1. RNA from reovirus and L-A virus requires 5′-phosphates to induce a RIG-I-dependent response.

(a-d) RNA samples were tested in an IFN-β promoter reporter assay in HEK293 cells: (a) RNA from reoT3D- or IAV-infected cells +/−CIP, (b) reoT3D vRNA +/−CIP, (c) reoT1L genome segments, and (d) reoT3D segments +/− CIP. For (b) and (d), RNA integrity was verified by gel electrophoresis. (e) IFN-α levels from transfected DCs. (f-h) IFN-α levels (f) or relative expression (RE) of ifit1 (g-h) from control (MDA5^+/−), RIG-I^−/−, MDA5^−/− or RIG-I/MDA5^−/− MEFs transfected with reo vRNA (f,h) or isolated reoT1L L segments (g) +/− CIP. Water and ppp-IVT-RNA^99nts are controls. For (e-h), cells were treated with ribavirin to block virus replication. (i) Total L-A RNA (genome and transcript), L-A genomes and L-A transcripts were analysed as in (a). (j) Total L-A RNA was analysed as in (e). (k-l) Total L-A RNA +/− shrimp alkaline phosphatase (SAP) was analysed as in (a) or transfected into MDA5^−/− DCs and analysed as in (g). Water, ppp-IVT-RNA^99nts, poly(dA:dT) and cyclic-di-GMP were included as controls. All experiments were performed at least twice. For PCR and IFN-α data, the mean (±s.d.) of triplicate technical replicates is shown (* = not detected).