Properties of IFN-inducing RNA generated by SFV replicase.
(A) COP-5 cells were transfected with either pRep-RDR, pRep-RDR/GAA or poly(I:C) or were mock-transfected. After 48 hr, the cells were lysed and the total RNA was size-fractionated on a silica column. The two resulting fractions, containing either large RNAs (>200 nt) or small RNAs (<200 nt), were transfected into MEFs, and IFN-β levels were measured by ELISA after 24 hr. Mock, transfection without DNA or RNA; ND, not detectable; 10×RNA <200, 10-fold molar excess of small RNAs as compared to large RNAs. (B) COP-5 cells transfected with pRep-RDR were lysed at 48 hr post transfection. The total RNA was extracted and fractionated into polyadenylated and non-polyadenylated RNA fractions using oligo(dT)-affinity chromatography. Increasing amounts of the obtained RNA fractions were transfected into COP-5 cells, while the total RNA amount was kept constant with “stuffer” RNA (naïve COP-5 total RNA). At 24 hr after transfection, the amount of IFN-β was determined in the cell culture medium by ELISA. polyA+, polyadenylated; polyA−, non-polyadenylated. (C) Upper panel: dsRNA probes were treated with various amounts of the indicated RNase, separated on agarose gel, and then visualized by ethidium bromide staining. Lower panel: Total RNA was extracted from pRep-RDR-transfected cells and digested with various amounts of the indicated RNase or treated with DNase I or alkaline phosphatase (AP) and analyzed as described in (C, upper panel). Mock, no enzyme added. (D) RNAs (C, lower panel) were transfected into COP-5 cells, and ELISA was used to measure the amount of secreted IFN-β after 24 hr. Error bars (A, B, and D) represent the standard deviation of two experiments.