Nucleobase Methylation Enhances SARS-CoV-2 Chain Terminator Evasion of Exonuclease Proofreading

Abstract

Nucleoside analogues (NuAs) targeting the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) represent a key antiviral strategy. However, their efficacy is fundamentally limited by the viral proofreading exoribonuclease nsp14, which excises misincorporated NuAs from nascent viral RNA. To identify NuAs capable of achieving chain termination while resisting nsp14-nsp10-mediated excision, we sequentially assessed a panel of nucleotide analogues encompassing ribose 2', 3', and nucleobase modifications for RdRp chain termination followed by nsp14-nsp10 ExoN resistance. Among the candidates, 5-methyl-3'-dUTP emerges as a standout inhibitor. It functions as an efficient immediate chain terminator, and the 5-methyl modification confers substantial resistance to nsp14-nsp10-mediated proofreading excision, notably outperforming its unmodified counterpart 3'-dUTP. RdRp also exhibited improved incorporation efficiency for this analogue. RNA chains terminated with 5-methyl-3'-dUTP exhibit barely detectable primer extension even following nsp14-nsp10-mediated exonucleolytic cleavage. The superior chain termination and ExoN resistance of 5-methyl-3'-dUTP were independently corroborated using an smFRET assay. Mechanistically, molecular dynamics simulations reveal that this enhanced ExoN resistance arises from destabilization of F146 loop stacking in the nsp14-nsp10 active site. Collectively, these findings establish 5-methyl-3'-dUTP as a promising antiviral lead compound and provide a structural framework for designing next-generation nucleoside analogues capable of evading coronavirus proofreading surveillance.