RNA elongation by respiratory syncytial virus polymerase is calibrated by conserved region V
Fig 7
BI-D inhibited transcription by preventing release of small le RNA and caused accumulation of abortive RNAs.
(A) Schematic diagram of the 3' end of the transcription-competent minigenome. The RNA products are shown in blue and the regions contained within the primers and probes are indicated in red. (B and C) Primer extension analysis of RNAs produced from the minigenome in the presence of varying concentrations of BI-D, using Sensiscript reverse transcriptase and primers 56–75 (B) and 15–39 (C). (D, E) Primer extension analysis of RNAs with Thermoscript reverse transcriptase and either the 56–75 (D) or 91–113 primer (E). In panels B, D, and E, lane 1 shows markers of oligonucleotides representing the primer extension products from the respective initiation sites; in panel C, lanes 1 and 2 show markers. Note that in panels C-E, the markers were migrated on the same gels as the adjacent samples, but a different exposure was required to visualize them. (F, G) Quantification of replicates of the experiments shown in panels B and C. Panel F shows levels of RNA initiated at +1, +3, and gs detected by primer 56–75. Panel G shows levels of RNA initiated at +1 and +3, detected by primer extension with primer 15–39. The +1 and gs products in panel F and the data in panel G were normalized to the—BI-D control in each experiment, which was set to 100%. In panel F, because the +3 products analyzed with primer 56–75 were at the level of background in the absence of BI-D, the +3 products were normalized to the value obtained with 800 nM BI-D, which was set to 100%. The bars show the mean and standard deviation of three independent experiments. (H, I) Northern blot analysis of small RNAs produced from the transcription-competent minigenomes by L protein in the absence or presence of BI-D. (J) Comparison of the sizes of abortive RNAs produced by a capping-deficient L variant (R1339) or by wt L protein in the presence of 800 nM BI-D. In H-J, RNA was migrated on 6% urea-acrylamide gels and detected with the indicated probe. The ladders are described in materials and methods. The 26 nt marker was based on the migration of the bromophenol blue (BB) dye on a 6% gel.