# Structural basis for selective remdesivir incorporation by SARS-CoV-2 polymerase, and S759A resistance

## TL;DR

This study reveals how the SARS-CoV-2 virus incorporates the drug remdesivir and how a mutation can make the virus resistant to it.

## Contribution

The study provides structural insights into remdesivir incorporation and resistance mechanisms in SARS-CoV-2 RNA polymerase.

## Key findings

- SARS-CoV-2 RNA polymerase preferentially incorporates remdesivir triphosphate over ATP.
- The remdesivir:UMP base pair is structurally stable, limiting successive incorporations.
- The S759A mutation in RdRp causes resistance by altering the ribose conformation and primer positioning.

## Abstract

Nucleoside analogs are successfully used to treat viral infections. dNTP analogs are primarily DNA chain terminators, while NTP analog remdesivir can inhibit RNA synthesis by delayed chain termination or when in the template strand. Here, enzymatic assays, mass spectrometry, and cryo-EM demonstrate that SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) preferentially incorporates remdesivir triphosphate (RTP), outcompeting 10-fold excess ATP; however, successive RTP incorporations are disfavored when ATP is present. The RdRp structures demonstrate that the remdesivir:UMP base pair is resilient to translocation, reducing successive RTP incorporations. Consequently, the relative concentration of ATP-to-RTP and structural rigidity of remdesivir:UMP, would limit remdesivir occupancy to < 16% of available positions in a copied genomic RNA strand. The S759A mutant confers RTP resistance. The structures of S759A RdRp reveal that the primer 3′-end nucleotide repositioning and its altered ribose-ring conformation contribute to RTP resistance. These findings have implications for designing non-obligate nucleoside analogs with different inhibition mechanisms.