Figure S3.

Picornavirus ssRNAs Do Not Exert IFN-β-Stimulatory Activity, Related to Figure 4

(A) ssRNA fractions (ssPools) of mock- and CVB3-infected cells were subjected to rRNA removal. The input (2%) and resulting materials were analyzed by agarose gel electrophoresis. The dsRNA fractions were loaded in parallel as a comparison.

(B) In vitro transcribed ssRNA was prepared from CVB3 cDNA clone (CVB3 ivt) and treated with either calf intestinal alkaline phosphatase (CIP), which removes all three phosphate groups from the 5′ end of the RNA, or 5′ polyphosphatase (PyP), which removes the beta- and gamma- phosphate groups. Remaining 5′ groups of these RNAs are indicated in brackets. RIG-I^−/− MEFs were transfected with indicated amounts of these CVB3 IVT RNAs in the presence of CHX (10 μg/ml), and IFN-β induction was measured at 8 hr.p.t. by real-time qPCR. Data presented as mean ± SD.

(C) Mock- and CIP-treated CVB3 IVT RNAs as on the left were transfected into WT MEFs in the presence of CHX (10 μg/ml), and IFN-β induction at 8 hr.p.t. was measured by real-time qPCR. The 5′ppp-containing RNA, but not the 5′OH-containing RNA, induced high levels of IFN-β, confirming the quality of the in vitro transcribed RNAs produced and the efficiency of the CIP treatment.