# Mechanism of SARS-CoV-2 resistance to nucleotide analog-based antivirals

## TL;DR

This paper explains how SARS-CoV-2 resists nucleotide analog antivirals through its proofreading enzyme ExoN, offering insights for better drug design.

## Contribution

The study reveals how ExoN interacts with nucleotide analogs and identifies structural and functional determinants of resistance.

## Key findings

- NA incorporation increases RNA affinity to ExoN while weakening its interaction with RdRp.
- Cryo-EM structures reveal ExoN-NA interactions mediated by modified ribose rings of NAs.
- An allosteric regulatory loop in ExoN is displaced by NAs resilient to ExoN excision.

## Abstract

The remarkable ability of SARS-CoV-2 to resist many nucleotide analog (NA)-based antivirals represents a formidable challenge to therapeutic efforts. Here, we reveal fundamental insights into how its unique proofreading exoribonuclease (ExoN) counteracts two representative NA antivirals, bemnifosbuvir and sofosbuvir, which are designed to inhibit the viral RNA polymerase (RdRp). Our findings unveil that NA incorporation alters RNA-binding dynamics, significantly increasing the affinity of RNA to ExoN while weakening its interaction with RdRp. This shift likely facilitates RNA dissociation from RdRp, subsequent recognition by ExoN, and excision of NAs. Strikingly, we elucidate the mechanism underlying varied levels of resilience of different NAs to ExoN excision. Our cryo-EM structures of ExoN in complex with either of the two NA-incorporated RNAs reveal previously unknown ExoN-NA interactions mediated by the functional groups on the modified ribose rings of NAs, illuminating the key determinants of their recognition and excision. Furthermore, we identify an allosteric regulatory loop of ExoN that promotes the full activation of ExoN but is displaced by the binding of NAs exhibiting resilience to ExoN excision. These discoveries provide a molecular framework for understanding SARS-CoV-2 resistance to NA-based antivirals and highlight mechanisms that could be exploited to improve anti-coronavirus drug design.

The coronavirus proofreading exoribonuclease (ExoN) is a major cause of viral resistance to nucleotide analog (NA) antivirals. Here, the authors reveal how different NAs are recognized by ExoN, shedding light on improved anticoronavirus drug design.