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.
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Data availability
Atomic coordinates of the four structures determined in this study have been deposited in the Protein Data Bank with accession codes 9YRK (SARS-CoV-2 ExoN•T20P14-B complex, dimeric form), 9YRL (SARS-CoV-2 ExoN•T20P14-B complex, protomer A), 9YRN (SARS-CoV-2 ExoN•T20P14-S complex, tetrameric form), and 9YRO (SARS-CoV-2 ExoN•T20P14-S complex, monomeric form). The cryo-EM maps have been deposited in the Electron Microscopy Data Bank with accession numbers EMD-73369 (SARS-CoV-2 ExoN•T20P14-B complex, dimeric form), EMD-73370 (SARS-CoV-2 ExoN•T20P14-B complex, protomer A focus-refined map), EMD-73371 (SARS-CoV-2 ExoN•T20P14-S complex, tetrameric form), and EMD-73372 (SARS-CoV-2 ExoN•T20P14-S complex, monomeric form). Source data are provided with this paper.
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Acknowledgements
We thank P. Juneja for support with cryo-EM sample screening at the Iowa State University cryo-EM facility, cryo-EM facility staff at the Hormel Institute and G. Nye at the Stanford-SLAC Cryo-EM Center (S2C2) for support during the collection of cryo-EM datasets. The S2C2 is supported by a National Institutes of Health grant R24GM154186. This work was supported by a National Institutes of Health grant DP2AI177906 to C.L., an award from the Searle Scholars Program SSP-2024-106 to C.L., a grant from the Hormel Institute, University of Minnesota, to B.L., and a National Institutes of Health grant R35GM150607 to Y.Y.
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Y.Y. and C.L. conceived and designed the experiments. C.L., Y.Y., Y.L., X.C., and R.J.G. performed protein and RNA purifications. C.L., Y.L., and Y.Y. conducted biochemical characterizations. Y.Y. and C.L. prepared the cryo-EM samples. B.L. and C.L. collected cryo-EM data. C.L. and Y.Y. processed the cryo-EM data and performed model building and structural analyses. Y.Y. and C.L. wrote the manuscript with input from B.L.
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Liu, C., Li, Y., Cao, X. et al. Mechanism of SARS-CoV-2 resistance to nucleotide analog-based antivirals. Nat Commun (2026). https://doi.org/10.1038/s41467-026-68304-8
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DOI: https://doi.org/10.1038/s41467-026-68304-8
