Remdesivir is the only FDA-approved drug for the treatment of COVID-19 patients 1–4 . The active form of remdesivir acts as a nucleoside analogue and inhibits the RNA-dependent RNA polymerase (RdRp) of coronaviruses including SARS-CoV-2 5–7 . Remdesivir is incorporated by the RdRp into the growing RNA product and allows for addition of three more nucleotides before RNA synthesis stalls 6,8 . Here we use synthetic RNA chemistry, biochemistry and cryo-electron microscopy to establish the molecular mechanism of remdesivir-induced RdRp stalling. We show that addition of the fourth nucleotide following remdesivir incorporation into the RNA product is impaired by a barrier to further RNA translocation. This translocation barrier causes retention of the RNA 3’-nucleotide in the substrate-binding site of the RdRp and interferes with entry of the next nucleoside triphosphate, thereby stalling RdRp. In the structure of the remdesivir-stalled state, the 3’-nucleotide of the RNA product is matched with the template base, and this may prevent proofreading by the viral 3’-exonuclease that recognizes mismatches 9,10 . These mechanistic insights should facilitate the quest for improved antivirals that target coronavirus replication.

Mechanism of SARS-CoV-2 polymerase inhibition by remdesivir