Figure 3.

Picornavirus RF Is a Potent MDA5 Agonist

(A) dsRNA fractions from mock- or CVB3-infected HeLa cells were transfected-into MEFs of indicated genotypes, and IFN-β mRNA level at 2 hr.p.t. was measured by real-time qPCR. Data presented as mean ± SD.

(B) dsRNA fractions (dsPool) of mock- and CVB3-infected cells were treated with 10 ng/μl RNase A, 10 mU RNase III, or 10 mU DNase I at 37°C for 15 min, and analyzed on agarose gel.

(C) DNA and RF bands observed in CVB3 dsRNA fraction were gel purified and analyzed on agarose gel. CVB3 dsRNA fraction (10 ng per well in 24-well format), and the gel-purified RNAs (10, 2, or 0.4 ng/well) were transfected into WT, RIG-I^−/−, MDA5^−/−, or MAVS^−/− MEFs in the presence of CHX (10 μg/ml). IFN-β mRNA induction was determined by real-time qPCR at 8 hr.p.t.

(D) dsRNA fractions of mock-, CVB3-, or mengovirus-infected cells were analyzed on agarose gel.

(E) DNA and RF bands from mengovirus dsRNA fraction were gel purified and transfected into RIG-I^−/− MEFs in the presence of CHX (10 μg/ml). IFN-β response at 8 hr.p.t. was measured by real-time qPCR.

(F) Gel-purified RF and DNA bands from CVB3 dsRNA fraction, as well as a in vitro transcribed dsRNA of CVB3 sequence (ivt dsRNA) (0.3 μg/ml), were incubated with recombinant MDA5 (0.3 μM) at 37°C in the presence of 2mM ATP, and free Pi was measured using Green Reagent at 0, 30, and 60 min after reaction was started. M, dsDNA marker with indicated size in kbp. Bands number 1 and 2 on gel corresponds to the samples used in RNA transfection. Data presented as mean ± SD.