Influenza Polymerase Fidelity Figure 1. NTP concentration dependent ApG-primed RNA polymerase activity of IAV Pol complex. ApG primer (0.3 mM) was extended by IAV Pol using RNA templates encoding the first 14 (A), 50 (B) and 137 (C) nucleotides (nt) 39 end sequences of the IAV PA gene. RNA polymerization was initiated by adding 1 mM ATP, 1 mM UTP, 1 mM CTP, 0.08 mM a-32P-GTP, and varying concentrations of nonradioactive GTP (lanes 1-5; 0.1, 1.0, 10, 100 and 1000 mM). M: Size markers C: Negative control without polymerase. F: Fully extended products. The fully extended products observed in the reaction with 50-nt (D) and 137-nt (E) were determined by densitometric analysis (observed activity) and these values were plotted against the actual GTP concentrations used in each reaction. The activity at 0.1 mM GTP was set arbitrarily to 100%. doi:10.1371/journal.pone.0010372.g001 extended products (14-nt, 50-nt and 137-nt), implying that the polymerase activity of the Pol complex was actually elevated when the total GTP concentration increased. This observation with the different sized templates indicates that the effect of GTP concentration on the activity of the enzyme is consistent, regardless of the sequence and size of the template. Next, we plotted the actual extent of the full length products in the 50-nt (Fig. 1D) and 137-nt (Fig. 1E) template reactions, which corresponds to the total IAV Pol activity in each reaction. The observed activity is then compared with the expected activity i.e., the predicted product extent which should decrease ten times with every 10-fold increase of nonradioactive GTP concentration. As seen in the plots of the data from the 50-nt (Fig. 1D) and 137-nt products (Fig. 1E), the total IAV Pol activity measured from the gel analysis was significantly higher than the expected activity. Strikingly, fully extended products were able to be detected in the reaction with an equal 1000 mM of all four NTPs (Fig. 1, lanes 5) where only ,0.01% of the total GTP was radioactive. Thus, this data suggests that the polymerase activity of H3N2 IAV Pol must have been elevated at least 200–500 times when we used a 1000 times higher concentration of the final GTP than the concentration that has typically been used in various recent studies. This observation also implies that the IAV Pol activity has been underestimated in the IAV Pol assay that employed limited GTP because the reaction rate at this limited GTP pool is likely below Vmax. PLoS ONE | www.plosone.org 4 April 2010 | Volume 5 | Issue 4 | e10372