Assessment of primer-independent de novo initiation/elongation and primer-dependent elongation polymerase activities of WT NS5 and mutant. (A) SDS-PAGE analysis of WT NS5 and NS5 mutant recombinant proteins expressed in E. coli and purified by His-tag affinity and size exclusion chromatography. Samples from left to right are (1) protein ladder, (2) WT, (3) Y838F, (4) R888A, (5) R888K, (6) D146A, (7) GAA, (8) D146A + GAA. (B) The cartoon depicts de novo initiation/elongation on the DENV 2 mini-replicon (Zhao et al. 2015b) (left) and elongation from a primed template (right). The de novo initiation/elongation and elongation polymerase activities of NS5 mutants Y838F, R888A, R888K, D146A, GAA, and D146A + GAA are compared with WT NS5 which was normalized to 100%. The data is presented as average ± SD from two to four independent repeat experiments. The statistical significance of difference between the two groups was evaluated by Student's t-test, and a P-value <0.05 was considered significant (≤0.0001, ≤0.001, ≤0.01, ≤0.05 were represented by [****], [***], [**], [*], respectively).
