Mechanistic insights into the activity of SARS-CoV-2 RNA polymerase inhibitors using single-molecule FRET

Abstract The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has resulted in significant global mortality, with over 7 million cases reported. Despite extensive research and high vaccination rates, highly mutated forms of the virus continue to circulate. It is therefore important to understand the viral lifecycle and the precise molecular mechanisms underlying SARS-CoV-2 replication. To address this, we developed a single-molecule Förster resonance energy transfer (smFRET) assay to directly visualize and analyse in vitro RNA synthesis by the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). We purified the minimal replication complex, comprising nsp12, nsp7, and nsp8, and combined it with fluorescently labelled RNA substrates, enabling real-time monitoring of RNA primer elongation at the single-molecule level. This platform allowed us to investigate the mechanisms of action of key inhibitors of SARS-CoV-2 replication. In particular, our data provides evidence for remdesivir’s mechanism of action, which involves polymerase stalling and subsequent chain termination dependent on the concentration of competing nucleotide triphosphates. Our study demonstrates the power of smFRET to provide dynamic insights into SARS-CoV-2 replication, offering a valuable tool for antiviral screening and mechanistic studies of viral RdRp activity.