Consecutive catalytic steps of viral RNA polymerase and exonuclease suggest a way to overcome intrinsic nucleotide analogue resistance
Abstract
Nucleotide analogues (NAs) have been successfully used for the treatment of various RNA virus infections by selectively targeting the viral RNA-dependent RNA polymerase (RdRp) for incorporation into the viral genome. However two major families of human-infecting RNA viruses, Coronaviridae (CoV) and Arenaviridae, encode exonuclease domains that may recognize and remove incorporated NAs, thus providing natural resistance against some of these drugs. Both polymerization and excision reactions are mechanistically centered on the nucleotide α-phosphate, enabling the potential for sequential inhibition of both RNA synthesis and repair. Here, we provide structural evidence of inversion of configuration at the phosphorus center during polymerization, demonstrating that the SARS-CoV-2 RdRp proceeds through an S N 2 mechanism. A 2.39 Å resolution cryo-EM structure of a ternary replication complex bound to RNA and an α-thio–modified NTP shows that incorporation of the preferred S P isomer at the 3′ end of the RNA yields a phosphorothioate linkage in the R P configuration. This R P -phosphorothioate RNA product shows reduced cleavage by both the SARS-CoV-2 and three arenavirus RNA exonucleases, revealing a stereochemical preference opposite to that of structurally related DNA exonucleases. This observation contradicts the prevailing assumption that sulfur substitution at the metal-coordinating oxygen universally blocks catalysis. Instead, RNA exonuclease stereoselectivity appears to be shaped not only by metal–sulfur interactions but also by the geometry of nucleophile activation. These findings provide mechanistic insights into phosphoryl transfer in viral polymerases and exonucleases and highlight opportunities to counteract intrinsic nuclease-mediated resistance against antiviral nucleotide analogues.
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BibTeX
@article{shannon2026consecutive,
title = {{Consecutive catalytic steps of viral RNA polymerase and exonuclease suggest a way to overcome intrinsic nucleotide analogue resistance}},
author = {Ashleigh Shannon and Véronique Fattorini and Candice Sartre and Aurélie Chazot and Adel Moussa and Jean-Pierre Sommadossi and Yingxiao Zhu and Manfu Wang and Hui Shi and François Ferron and Karine Alvarez and Bruno Canard},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
year = {2026},
month = jun,
doi = {10.1073/pnas.2605725123},
url = {https://hal.science/hal-05783752v1},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Consecutive catalytic steps of viral RNA polymerase and exonuclease suggest a way to overcome intrinsic nucleotide analogue resistance
AU - Ashleigh Shannon
AU - Véronique Fattorini
AU - Candice Sartre
AU - Aurélie Chazot
AU - Adel Moussa
AU - Jean-Pierre Sommadossi
AU - Yingxiao Zhu
AU - Manfu Wang
AU - Hui Shi
AU - François Ferron
AU - Karine Alvarez
AU - Bruno Canard
PY - 2026
DA - 2026/06/09
JO - Proceedings of the National Academy of Sciences of the United States of America
DO - 10.1073/pnas.2605725123
UR - https://hal.science/hal-05783752v1
AB - Nucleotide analogues (NAs) have been successfully used for the treatment of various RNA virus infections by selectively targeting the viral RNA-dependent RNA polymerase (RdRp) for incorporation into the viral genome. However two major families of human-infecting RNA viruses, Coronaviridae (CoV) and Arenaviridae, encode exonuclease domains that may recognize and remove incorporated NAs, thus providing natural resistance against some of these drugs. Both polymerization and excision reactions are mechanistically centered on the nucleotide α-phosphate, enabling the potential for sequential inhibition of both RNA synthesis and repair. Here, we provide structural evidence of inversion of configuration at the phosphorus center during polymerization, demonstrating that the SARS-CoV-2 RdRp proceeds through an S N 2 mechanism. A 2.39 Å resolution cryo-EM structure of a ternary replication complex bound to RNA and an α-thio–modified NTP shows that incorporation of the preferred S P isomer at the 3′ end of the RNA yields a phosphorothioate linkage in the R P configuration. This R P -phosphorothioate RNA product shows reduced cleavage by both the SARS-CoV-2 and three arenavirus RNA exonucleases, revealing a stereochemical preference opposite to that of structurally related DNA exonucleases. This observation contradicts the prevailing assumption that sulfur substitution at the metal-coordinating oxygen universally blocks catalysis. Instead, RNA exonuclease stereoselectivity appears to be shaped not only by metal–sulfur interactions but also by the geometry of nucleophile activation. These findings provide mechanistic insights into phosphoryl transfer in viral polymerases and exonucleases and highlight opportunities to counteract intrinsic nuclease-mediated resistance against antiviral nucleotide analogues.
SN - 0027-8424
SN - 1091-6490
ER -