The remarkable ability of SARS-CoV-2 to resist nucleotide analog (NA)-based antivirals, such as remdesivir, represents a formidable challenge to therapeutic efforts. Here, we reveal fundamental insights into how its unique proofreading exoribonuclease (ExoN) counteracts multiple representative NA antivirals, which are designed to disrupt RNA synthesis by the viral RNA polymerase (RdRp). Our findings unveil 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, even when they are embedded within an RNA substrate. Strikingly, we elucidate, for the first time, the mechanism underlying varied levels of the resilience of different NAs to ExoN excision. Our cryo-EM structures of ExoN in complex with each of the three 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.
Yang et al. (Sun,) studied this question.