Highly effective viral RdRP translocation hindrance by the remdesivir nucleotide

Nucleoside/nucleotide analogs (NAs) typically interfere with substrate selection and catalysis steps of polymerase nucleotide addition cycle (NAC), thereby affecting polymerase catalytic efficiency. In contrast, their interference with post-chemistry translocation is rarely considered in rational design, partly due to a lack of direct and quantitative evidence for such interference. Remdesivir (RDV) is an adenosine analog with broad-spectrum antiviral activity against diverse RNA virus families and has been approved for COVID-19 treatment. RDV is capable of inducing a characteristic "delayed" intervention, wherein the interference occurs several NACs after its incorporation. By combining X-ray crystallography, quench-flow kinetics, and molecular dynamics simulations, we show that the composite rate of RDV translocation from product strand position -3 to -4 and subsequent catalysis is at least 100-fold slower than that of adenosine in an enterovirus RdRP. However, mutating the corresponding roadblock residue serine417 to glycine largely abolishes this difference. Nonetheless, the incorporated RDV nucleotide can still alter the free-energy landscape even in the S417G mutant complex, enabling the capture of an over-translocated state. Our work demonstrates translocation-related intervention as an effective and broadly applicable polymerase intervention strategy in NA rational design.