FIG. 6.

Cap-snatching activity in vitro of purified RNPs containing wild-type (WT) or mutant PB2 subunits. Recombinant RNPs, prepared and purified as indicated in the legend to Fig. 5, and virion-derived RNPs were tested for cap-snatching activity with a labeled probe capped and O-methylated in vitro. No cap snatching was observed in the absence of either capping or O-methylation of the probe. (A) The reaction products were separated in a 18% polyacrylamide-urea gel. The positions of the labeled probe and capped oligonucleotide product are indicated. (B) Western blot of parallel aliquots of the same RNPs used for cap snatching, with anti-PA monoclonal antibodies. MOCK, material obtained after purification of RNPs reconstituted in the absence of RNA template. The values are the percentage of capped oligonucleotide bands (A) or PA bands (B), with the wild-type value set at 100%.