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

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causative agent of the COVID-19 pandemic, is a highly pathogenic and transmissible β-coronavirus characterized by frequent variant emergence. The rapid evolution of SARS-CoV-2 highlights its RNA-dependent RNA polymerase (RdRP), the conserved central machinery for viral replication and transcription, remains a valuable drug target. Nucleoside analogue (NuA) is a class of drugs exerting anti-viral effect by interfering SARS-CoV-2 genome synthesis through RNA chain incorporation by RdRP. Nevertheless, all coronaviruses encode a proofreading exoribonuclease in nonstructural protein nsp14 to excise misincoporated nucleotides that impedes the efficacy of NuAs. We investigated a range of chemically modified NuAs, including those with 2’-fluoro, 2’-O-methyl, and 3’-deoxy ribose substitutions as well as nucleobase modifications, to ascertain their RdRP chain termination effect and resistance to nsp14/nsp10 exonuclease proofreading. Through a combination of biochemical experiments and MD simulations, we elucidated the influence of these modifications on anti-viral efficiency. Our findings demonstrated that 3’-deoxy substitution offers superior chain termination and proofreading evasion abilities which can be further enhanced by nucleobase methylation. The study’s comprehensive analysis not only furthers our understanding of SARS-CoV-2’s molecular biology but also contributes to the strategic development of antiviral agents with the potential to combat the current pandemic and prepare for future coronavirus outbreaks.