FIG. 2.

Purification of influenza virus mini-RNPs reconstituted in vivo. The viral RNPs reconstituted in vivo as indicated in Fig. 1 were purified by two successive glycerol gradients as indicated in Materials and Methods. The analyses corresponding to the fractionation of the second gradient are presented. Aliquots of each fraction were processed for Western blotting by using anti-PB1 (A) or anti-NP antibodies (B) or were analyzed by SDS-PAGE and Coomassie blue staining (C). The activity of each fraction was determined by in vitro transcription and TCA precipitation, filtration on a dot blot apparatus, and autoradiography (D, COMP). As a control, the activity of a parallel gradient with a sample in which no template was transfected (−RT) was determined. Pol and NP indicate the positions of the polymerase proteins and the NP in the gel, respectively.