FIG 8.

Influence of remdesivir-TP incorporation on incorporation of the next nucleotide. (A) P/T complex used in the experiment whose result is shown in panel B. The bold “U” in the template indicates the position base-pairing with ATP or remdesivir-TP. (B) Influence of single remdesivir-TP incorporation on incorporation of the next nucleotide. nsp12 (50 nM) and nsp8-7 (2 μM) were incubated with 5 nM P/T (A), 0.1 μM CTP (the first nucleotide to be incorporated), and 0.1 μM ATP or remdesivir-TP (the second nucleotide to be incorporated) in reaction buffer for 30 min at 37°C and then rapidly mixed with different concentrations (indicated above the gel, in micromolar units) of UTP. The reactions were continued at 22°C for 20 s before the addition of stopping solution, and the products were resolved by denaturing PAGE. The identity of the tested nucleotide (ATP or remdesivir-TP) is indicated at the bottom of the gel. The locations of the 30-mer primer and the 31-mer (CTP incorporation), 32-mer (single ATP or remdesivir-TP incorporation), and 42-mer (full-length extension products) are indicated on the left of the gel. The influence of double (C and D), triple (E and F), and quadruple (G and H) incorporations of remdesivir-TP on incorporation of the subsequent nucleotide was analyzed using a similar method.