Figure 6. Biological activity of recombinant RNPs with an ectopic NLS in PB2 protein.

(A) Quantitative estimation of the localisation of wt or mutant PB2 proteins was performed as indicated in Fig. 2C. (B) Cultures of HEK293T cells were co-transfected with plasmids expressing PB1, PA, NP and either wt or mutant PB2, with or without an additional TAg NLS fused to the C-terminus of the protein. In addition, a plasmid encoding CAT gene in negative polarity, flanked by the non-translated regions of influenza NS segment was co-transfected to provide a viral replicon. Plasmid expressing PB2 was omitted as a negative control. At 20 hours post-transfection total cell extracts were prepared and CAT protein was determined by ELISA. The averages and standard deviations of 3–9 independent experiments are shown.