Long and short poly I:C preferentially activate ATPase activities of MDA5 and RIG-I. ATPase activity of RIG-I or MDA5 protein was measured in the presence of the indicated RNAs. The x axis shows the concentration of RNAs. (A) Long and short poly I:C. (B) 5′-tri-phosphate ssRNA. Several quantities (1, 0.2, 0.04, and 0.008 μg) of poly I:C were used. Error bars show SDs between triplicates.

RIG-I and MDA5 selectively recognize dsRNA in a length-dependent manner. (A and B) Indicated genotype of MEFs were treated with 1 μg/ml of the indicated RNAs (10 μg/ml in the case of high dose) for 24 h. s, sense ssRNA; as, antisense ssRNA; s+as, dsRNA generated by annealing s with as. s and as are chemically synthesized 70-bp ssRNAs having a 5′ hydroxyl end in A. s and as are in vitro transcribed capped-ssRNAs in (B). The production of IFN-β in the supernatant was measured by ELISA. (C) WT, Rig-I^−/−, Mda5^−/−, and Rig-I^−/−Mda5^−/− MEFs were treated with 1 μg/ml of in vitro–transcribed capped dsRNAs for 16 h. The production of IFN-β in the supernatant was measured by ELISA. Error bars show SDs between triplicates.

dsRNA generated during VSV replication induces IFNs in a RIG-I–dependent manner. (A) RNA samples harvested from uninfected (mock), EMCV-, VSV-, or influenza virus (flu)–infected cells were transfected into WT, Rig-I^−/−, and Mda5^−/− MEFs. The production of IFN-β in the culture supernatant 10 h after transfection was measured by ELISA. (B) RNA harvested from noninfected (mock) or EMCV-, VSV-, or influenza virus–infected cells with CIAP-, RNase III-, both CIAP-, and RNase III-treatments or nontreatment (enzyme-) was transfected into WT MEFs. The production of IFN-β in the supernatant 10 h after transfection was measured by ELISA. (C) dsRNA in uninfected (mock), EMCV-, VSV-, or influenza virus-infected cells was measured by ELISA. (D) Immunostaining for dsRNA in MEFs infected with EMCV, VSV, and influenza virus for 8 h. Red, dsRNA; blue, nucleus. Error bars show SDs between triplicates. (E) RNA harvested from noninfected (mock) or VSV-infected cells (indicated periods) was electrophoresed in 1.5% agarose gel, transferred to a nylon membrane, and blotted by anti-dsRNA antibody. Reovirus genome RNAs were indicated as the size control. The arrow shows VSV dsRNA. (F) dsRNA blotting of RNA harvested from EMCV- or VSV-infected cells. RNAs were electrophoresed in nondenaturing 10% polyacrylamide gel. Reovirus genome RNAs were indicated (left). Arrows (right) show EMCV and VSV dsRNA.

Preferential recognition of long and short poly I:C by MDA5 and RIG-I. (A) The indicated RNAs are shown on the ethidium bromide–stained agarose gel. M, DNA marker. (B) WT, Rig-I^−/−, Mda5^−/−, and Rig-I^−/−Mda5^−/− MEFs were treated with 1 μg/ml of untreated or RNase III–treated poly I:C for 16 h. The production of IFN-β in the supernatant was measured by ELISA. mock, no RNA. (C) Relative induction of IFN-β after stimulation with poly I:C treated with RNase III for the indicated periods. (D) The production level of IFN-β in the stimulation with RNase A–treated, Bal31-treated, or alkaline-hydrolyzed poly I:C. Error bars show the SDs between triplicates.

RIG-I and MDA5 specifically bind to short and long poly I:C. (A) Complex of indicated poly I:C and protein (MDA5 or RIG-I) was observed by AFM. Height is on a scale from 0 to 4 nm, with a low area depicted in dark brown and a higher area depicted in brighter color. Scale area, 500 nm. Bars, 100 nm. Those are representative images from several pictures. (B and C) Statistical height analyses of molecules corresponding to pictures in A.

Reovirus genome dsRNA includes both RIG-I and MDA5 ligands. (A) WT, Rig-I^−/−, and Mda5^−/− GM-CSF-DCs were infected with the indicated multiplicity of infection of reovirus. The production of IFN-β in the supernatant was measured by ELISA. (B and C) The indicated genotypes of MEFs were treated with 1 μg/ml of reovirus genome RNA (B) or 0.1 μg/ml of dsRNA segments (C) for 16 h. The production of IFN-β in the supernatant was measured by ELISA. The reoviral genome is shown on the ethidium bromide-stained gel (C, right), and the S (1.2–1.4 kbp), M (2.2–2.3 kbp), and L (3.9 kbp) segments are indicated. Error bars show SDs between triplicates.