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Fidelity Variants of RNA Dependent RNA Polymerases Uncover an Indirect, Mutagenic Activity of Amiloride Compounds

Figure 2

In vitro biochemical assays confirm the higher (A372V) and lower (S299T) incorporation fidelities of CVB3 variants.

(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.

Figure 2