Fidelity Variants of RNA Dependent RNA Polymerases Uncover an Indirect, Mutagenic Activity of Amiloride Compounds
(A) A radiolabeled primer/template is used to measure the incorporation of bases in vitro using purified RdRp enzymes, conditions detailed in Methods. The templating U (in bold) permits the quantification of incorporation of the correct (ATP) or incorrect (GTP) nucleotide over time. (B) Visualization of GTP mis-incorporation by wild type, A372V and S299T RdRp. The incorporation of GTP is monitored over time (m) as the accumulation of elongation product (n+1) minus blank control. (C) Quantification of relative incorporation product at 2 and 5 minutes reaction time.