PPi-dependent excision of a chain terminator and rescue of RNA synthesis. (A) The schematic depicts a chain-terminated product after addition of only GTP, ATP, and 2′-C-methyl-CTP. Following this reaction, the excision of the chain terminator and the rescue of RNA synthesis are initiated with the addition of PPi, UTP, and CTP. (B) Excision and rescue occur in a dose-dependent manner with the addition of up to 320 μM PPi. The reaction is shown for wild-type NS5B and the mutant enzymes. Lanes C, the excision reaction alone, which produces a product at position +15. (C) The amount of excised and rescued product was quantified as the amount of product at position +20 as a percentage of the total amount of product in the lane. Data points are the means ± standard deviations for three independent experiments.