(A) 293T cells are co-transfected with plasmids encoding PB2, PB1, and PA from Nanchang/1995, an NP variant, and a reporter plasmid expressing a negative-sense influenza viral-RNA encoding GFP. The four influenza proteins (PB2, PB1, PA, and NP) collaborate to transcribe the reporter GFP vRNA into mRNA, which is translated into protein, causing the cells to fluoresce. Activity is quantified by flow cytometry as the mean fluorescence intensity (MFI) above the background of control cells that receive no NP, relative to the activity of three wild-type Aichi/1968 NP replicates. (B) Example flow cytometry plots. (C) Total transcriptional activity as a function of the amount of transfected NP plasmid. The PB2, PB1, PA, and GFP reporter plasmids are transfected at 200 ng each. The NP plasmid was varied from 0 to 300 ng, and the fluorescence was quantified. At low levels of NP plasmid, the signal increases with increasing plasmid concentration, but at high levels the signal is saturated. We performed our assays with 50 ng of NP plasmid, which is near the midpoint of the curve—with this amount of plasmid, the signal changes in a sensitive manner with amount of NP.