GS-646939 inhibits subsequent nucleotide incorporation. UTP incorporation at position “i + 1” was monitored at increasing concentrations immediately following ATP (black), GS-443902 (orange), or GS-646939 (blue) incorporation at position “i”. Concentrations of ATP, GS-443902, and GS-646939 supplemented to the reaction are shown in brackets. Viral RdRp enzymes investigated include SARS-CoV-2 (A), MERS-CoV (B), HRV-16 (C), EV-71 (D), RSV (E), HMPV (F), HPIV-3 (G), and PIV-5 (H). The product fraction was calculated as the total signal above position “i” divided by total signal in the lane. Independent 8-data point experiments were performed three times (n = 3), and error bars represent standard error associated with the fit. RdRp, RNA-dependent RNA polymerase; HRV, human rhinovirus; EV-71, enterovirus 71; RSV, respiratory syncytial virus; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; MERS-CoV, Middle East respiratory syndrome coronavirus; HMPV, human metapnuemovirus; HPIV-3, human parainfluenza virus 3; PIV-5, parainfluenza virus 5.