a–d, HEK293T cells expressing Flag–hVIP (aqua), Flag–hVIP and hCMPK2 (purple) or cells only (dark blue). Samples were taken at 16, 24, 48 and 72 hours post infection (h.p.i.). Extraction performed with a mixture of acetonitrile, methanol and water (40:40:20 and 0.1 M formic acid). Cellular concentrations were determined using 13C915N15–CTP, 13C1015N10–ATP, 13C10N5–GTP and 13C915N2–UTP spiked into the extraction mixture at known concentrations and using equations (1) and (2) in Supplementary Information. Analysis of nucleotides ATP (a), CTP (b), GTP (c) and UTP (d) did not show statistically significant differences (ns) between Flag–hVIP, Flag–hVIP and hCMPK2 or cells only for any time point. Data are from three biologically independent samples. Statistical significance was determined using a two-way ANOVA (Supplementary Tables 12, 13, 14 and 15). e, Ratio of cellular concentrations of ddhCTP to CTP from HEK293T cells expressing Flag–hVIP (aqua) or Flag–hVIP and hCMPK2 (purple); samples were taken at 16, 24, 48 and 72 h post transfection. The overall ratio of ddhCTP to CTP remains constant when only Flag–hVIP is expressed, but the concentration of ddhCTP is boosted significantly relative to CTP when both Flag–hVIP and hCMPK2 are co-expressed (plots are derived from data shown in c and Fig. 3a).