FIG 7.

Variation in type I IFN response activation by DI-RNAs in the presence of the MV N and P proteins. (A) Western blot analysis of T7 polymerase, MV-N, MV-P, and β-actin in HEK293-T7 and HEK293-T7-NP cells. A total of 1 × 10⁶ cells were plated, and lysis was performed 12 h later. (B) Absolute quantification of 1,212-, 450-, and 1,260-nt DI-RNAs produced by HEK293-T7 and HEK293-T7-NP cells transfected with the p2RZ-DI1212, p2RZ-DI450, and p2RZ-DI1260 vectors. Total RNA (400 ng) purified from cells at 24 h posttransfection was treated with RQ1 RNase-free DNase, and 100 ng was analyzed by RT-qPCR. Absolute quantification was performed using serial dilutions of in vitro-transcribed MV DI-RNAs and normalized with actin. Results are expressed in copy numbers of RNA molecules. Experiments were performed two times, and samples were tested in triplicate. (C and D) HEK293-T7 and HEK293-T7-NP cells were transfected with p2RZ vectors encompassing DI-RNAs or an actin sequence (C) or with in vitro-transcribed RNAs (actin, 5′3P, or 3 different IVT DI-RNAs) (D). Relative IFN-β mRNA expression levels were measured at 24 h posttransfection, normalized to GAPDH, and compared to those of mock-transfected cells. Experiments were performed two times, and the upper and lower 95% confidence intervals are shown as error bars for technical triplicates of the most representative experiment.