Fig. (2) NS5A activates NS5B during the replication reaction in an order of addition dependent manner. A. Scheme of the experimental set up. B. NS5B replication reactions showing components that were pre-incubated on ice for 30 minutes and the components that were added prior to the 30°C incubation. Lane 1, the NS5B (0.3 µM) reaction in the absence of NS5A. Lane 2, NS5A (1 pM) with no pre-incubation prior the replication reaction. Lanes 3-7, replication reactions with NS5A and various pre-incubation additions: NTPs (lane 3); the X(+) template, 0.6 µM (lane 4); NS5B (lane 5); NS5B and the X(+) template (lane 6); and NS5B with NTPs (lane 7). Arrow indicates the expected RNA product. C. Quantification of the effect of NS5A on the replication reaction (average of three independent experiments). D. Time course showing the reactions with NS5B (○) and NS5B+NS5A (■). Each data point is the average of 5 experiments.