Extended Data Fig. 8: ddhCTP is used as a substrate by dengue virus and WNV RdRp and chain terminates RNA synthesis.

a, Schematic of primer-extension assay for evaluating dengue virus and WNV RdRp activity. b, Dengue virus RdRp-catalysed nucleoside incorporation using CTP, 3′-dCTP or ddhCTP as nucleoside triphosphate substrates. Some full-length product was observed in the presence of ddhCTP (more than 99% pure), which is due to residual contaminating CTP that could not be removed. c, Reaction products resolved by denaturing PAGE containing 40% formamide showing the trace amount of CTP contaminate in the ddhCTP preparation. These experiments were repeated independently at least four times with similar results. d, Longer incubation times and more dengue virus RdRp enzyme does not increase the yield of extended product. e, Dengue virus RdRp-catalysed nucleoside incorporation with increasing concentrations of ddhCTP (0, 1, 10, 100, 200 and 750 μM) at varying concentrations of CTP. This experiment was repeated independently three times with similar results. f, Plot of the percentage inhibition as a function of ddhCTP concentration at varying concentrations of CTP. Data were fit to a dose–response curve to obtain half-maximum inhibitory concentration (IC₅₀) values of ddhCTP of 60 ± 10, 120 ± 20, 520 ± 90 and 3,900 ± 700 μM at 0.1, 1, 10 and 100 μM CTP, respectively. This experiment was repeated at least three times with similar results. The total sample size is 24. The error reported is the standard error from the fit of the data to a dose–response curve. g, Plot of IC₅₀ values as a function of CTP concentration. The data were fit to a line with a slope of 38 ± 1 and an intercept of 91 ± 25. The error reported is the standard error from the fit of the data to a line. h, WNV RdRp-catalysed nucleoside incorporation with increasing concentrations of ddhCTP (0, 1, 10, 100, 200 and 750 μM) at varying concentrations of CTP. This experiment was repeated at least three times with similar results. i, Plot of the percentage inhibition as a function of ddhCTP concentration at varying concentrations of CTP. Data were fit to a dose–response curve to obtain IC₅₀ values of ddhCTP of 20 ± 10, 70 ± 10, 300 ± 40 and 2,700 ± 300 μM at 0.1, 1, 10 and 100 μM CTP, respectively. The total sample size is 24. The error reported is the standard error from the fit of the data to a dose–response curve. j, Plot of IC₅₀ values as a function of CTP concentration. The data were fit to a line with a slope of 27 ± 1 and an intercept of 31 ± 8. Both of these results demonstrate that once ddhCMP is incorporated, it effectively terminates synthesis and that the small amount of extended product is from a trace amount of CTP contamination. The error reported is the standard error from the fit of the data to a line.