Abstract
Nucleoside analogs are successful in treating viral infections. dNTP analogs are primarily DNA chain terminators, while NTP analog remdesivir can inhibit RNA synthesis by delayed chain termination or when embedded in the template strand. Here, enzymatic assays, mass spectrometry, and cryo-EM structures 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 1′-cyano-imposed conformational restriction of the remdesivir:UMP base-pair is resistant to translocation, reducing successive RTP incorporations. The S759A mutant confers RTP resistance. We show that the mutation switches the RdRp preference to ATP; RTP is incorporated only at 10-fold excess to ATP. 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.
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Data availability
The data that support this study are available from the corresponding author upon reasonable request. The coordinates and cryo-EM density maps for the structures WT-AMP, WT-RMP, S759A-AMP, and S759A-RMP are deposited with PDB accession codes/EMDB codes 9SAR /EMD-54695, 9SAP/EMD-54693, 9SAQ/EMD-54694, and 9SAO/EMD-54692, respectively. All MS data can be obtained from the Open Science Framework (OSF) online repository at https://doi.org/10.17605/OSF.IO/YDRT3.
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Acknowledgements
This study was supported by Canada Excellence Research Chair (CERC) funding and KU Leuven research grant C24E/22/035 to K.D. K.D. and M.G. also acknowledge support from The Alberta Ministry of Technology and Innovation, through SPP-ARC (Striving for Pandemic Preparedness—The Alberta Research Consortium). C.J.G. acknowledges support from CIHR funding reference number 181545. Q.G. acknowledges financial support from the Research Foundation - Flanders (FWO-Vlaanderen) through doctoral fellowship grant number 1162823N and 1162825N. The authors acknowledge Dr. Abhimanyu Singh for advice on protein expression, Dr. Randy Whittal and Dr. Joseph Utomo at the Mass Spectrometry facility at the University of Alberta for the MS analysis.
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Conceptualization: C.J.G., M.G., K.D.; Investigation and Methodology: C.J.G. and H.W.L.; Biochemistry: C.J.G.; MS: C.J.G.; Protein purification: M.F.O.; Cryo-EM and structural studies: M.F.O., Q.G., and B.D-W.; Data analysis: C.J.G., H.W.L., M.G.; Structural biology: M.F.O., Q.G., and K.D.; Funding acquisition: K.D. and M.G.; Project administration and supervision: K.D. Original draft written by: C.J.G., M.F.O., K.D.; Reviewed and edited by all authors.
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Gordon, C.J., Oliva, M.F., Lee, H.W. et al. Preferential remdesivir triphosphate incorporation by SARS-CoV-2 polymerase is altered to ATP by the S759A mutation. Commun Biol (2026). https://doi.org/10.1038/s42003-026-09844-z
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DOI: https://doi.org/10.1038/s42003-026-09844-z
