FIG. 6.

Synthesis of cRNA and mRNA at 37 and 33°C with avian and human virus-derived polymerase complexes. COS-1 cells were cotransfected with pPolI-CAT-RT and the PB1, PB2, PA, and NP expression plasmids derived from PR8 (lanes 4 and 10) or from MAL, including in the latter case either the wild-type MAL PB2 (lanes 5 and 11) or the mutant MAL E627K-PB2 (lanes 6 and 12) expression plasmid. Controls included COS-1 cells transfected with pPolI-CAT-RT without protein expression plasmids (lanes 1 and 7), PR8 protein expression plasmids without pPolI-CAT-RT (lanes 2 and 8), and MAL protein expression plasmids without pPolI-CAT-RT (lanes 3 and 9). Following 48 h of incubation at 37°C (lanes 1 to 6) or 33°C (lanes 7 to 12), total RNAs were extracted, and 5 μg was analyzed by RNase protection assay as described in the text. The image obtained by scanning the gel with a STORM820 optical scanner is shown. The length expected for the undigested riboprobe was 190 nucleotides (nt) (lane C+). No signal was expected when 5 μg of yeast tRNA was analyzed (lane C−). The expected lengths for the riboprobe fragments protected by cRNA or mRNA were 175 and 159 nt, respectively. MW, molecular size markers.