Extended Data Fig. 9: HRV-C and poliovirus RdRp are poorly inhibited by ddhCTP. | Nature

Extended Data Fig. 9: HRV-C and poliovirus RdRp are poorly inhibited by ddhCTP.

From: A naturally occurring antiviral ribonucleotide encoded by the human genome

Extended Data Fig. 9

a, Schematic of primer extension assay for evaluating HRV-C RdRp activity. b, HRV-C RdRp-catalysed nucleoside incorporation using CTP, 3′-dCTP or ddhCTP as nucleoside triphosphate substrates. These experiments were repeated independently at least four times with similar results. c, Increasing concentrations of ddhCTP does not efficiently inhibit HRV-C RdRp-catalysed RNA synthesis. HRV-C RdRp-catalysed nucleoside incorporation in the presence of increasing concentrations of either ddhCTP or 3′-dCTP. These experiments were repeated independently at least five times with similar results. d, Plot of the percentage inhibition as a function of either ddhCTP or 3′-dCTP concentration. Data were fit to a dose–response curve to obtain IC50 values of 900 ± 300 μM for ddhCTP and 5 ± 1 μM for 3′-dCTP. The total sample size is eight. The error reported is the standard error from the fit of the data to a dose–response curve. e, f, HRV-C (e) and poliovirus (f) RdRp-catalysed nucleoside incorporation with increasing concentrations of ddhCTP (0, 1, 10, 100 and 200 μM) at varying concentrations of CTP. Reactions were performed with the trinucleotide primer, 5′-pGGC, and 20-nt RNA template as described for dengue virus and WNV RdRp to directly compare results with HRV-C and poliovirus RdRp. At the highest concentration of ddhCTP, only approximately 2% inhibition was observed for HRV-C RdRp at 0.1 and 1 μM CTP. The IC50 values at 0.1 and 1 μM CTP are estimated to be approximately 10,000 and 20,000 μM ddhCTP, respectively. These values are three orders of magnitude higher than those obtained for dengue virus and WNV RdRp. Reactions in the presence of 3′-dCTP (200 μM) are shown as a control for inhibition. These experiments were repeated independently at least four times with similar results. g, Efficiency of incorporation and inhibition of viral RdRps. Footnotes: aCalculated for ddhCTP in direct competition with CTP (800 μM) using the linear equations obtained from the fit of the data shown in g and j. For HRV-C, the IC50 value was estimated to be two orders magnitude greater than that calculated for dengue virus and WNV RdRps as evidenced from the data shown in d. bCalculated for a ddhCTP concentration of 350 μM using the following equation: probability = [ddhCTP]/([ddhCTP] + IC50). cCalculated using the following equation: \({\rm{full\; -\; lengthgenome( \% )}}=100\times {(1-{\rm{probability}})}^{{{\rm{C}}}_{n}}\); in which Cn is the number of cytidine residues in the viral genome with values of 2,200, 2,497, 1,565 and 1,737 for dengue virus, WNV, HRV-C and poliovirus respectively.

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