Figure 4.

Comparison of the PV RdRp kinetic fidelity variants. (A) In most cases, nucleotide incorporation fidelity was defined as the second-order rate constant for correct nucleotide incorporation divided by the second-order rate constant for incorrect nucleotide incorporation (i.e. k_pol/K_d,app (ATP) /k_pol/K_d,app (GTP)). The low catalytic activities for K359R and R174K precluded accurate rate measurements at lower nucleotide concentrations (especially for incorrect nucleotide), and so nucleotide incorporation fidelities for these variants were based on estimates of k_pol using a higher nucleotide concentration^{38, 52}. In the K359R and R174K variants, fidelity is defined as the k_pol for correct nucleotide incorporation divided by the estimated k_pol for incorrect nucleotide incorporation (i.e. k_pol (ATP) / k_pol (GTP)). Viruses encoding the H273R¹⁵, T362I⁴⁶, G64S^{20, 21} and K359R²⁶ variants all have reduced virulence in a PV mouse model. Asterisks indicate that the kinetic parameters for the variant were statistically different from WT values, according to p value (0.01<*≤0.05; 0.001<**≤0.01; ***≤0.001; n=3) (B) Structure of PV RdRp⁵³ (PDB 1RA6), showing the locations of amino acid substitutions used to generate lower and higher fidelity variants. Conserved structural motifs are also colored: A, red; B, green; C, yellow; D, blue; E, purple; F, pink.