FIG 7.
Both 5′-di/triphosphates and the stem region in MV DI RNA are important for IFN induction. (A) DI RNA samples used in this experiment. (Left) Diagrams of MV DI RNA (DI), DI RNA treated with calf intestinal phosphatase (DI-CIP), and the DI RNA mutant containing the loop only (DI-lp). (Right) In vitro-transcribed RNA samples were loaded into a formaldehyde agarose gel and visualized under UV. (B to E) Requirement for 5′-di/triphosphates and the stem region. Poly(I·C) (IC), DI RNA, DI-CIP, and DI-lp were transfected into A549 cells. Quantitative real-time RT-PCR analysis was performed after 6 h to measure the levels of the IFN-β (B), CCL5 (C), IFN-α1 (D), IFN-γ (E), and GAPDH mRNA transcripts. The levels of target gene mRNA expression relative to the level of GAPDH mRNA expression were calculated by the comparative CT method. All expression values were normalized to those for the mock-transfected control sample. Results are the means from three independent experiments, and error bars indicate SDs. The differences in the levels of IFN-β mRNA between DI RNA and DI-CIP as well as between DI RNA and DI-lp were statistically significant, as judged by a two-tailed Student's t test with P values equal to 3.2 × 10−5 (highlighted by ***) and 0.00028 (highlighted by ***), respectively (B), and the differences in the levels of CCL5 mRNA between DI RNA and DI-CIP as well as between DI and DI-lp were statistically significant, as judged by a two-tailed Student's t test with P values equal to 0.00074 (highlighted by ***) and 0.00055 (highlighted by ***), respectively (C).