Abstract
Nucleoside analogs are successfully used to treat viral infections. dNTP analogs are primarily DNA chain terminators, while NTP analog remdesivir can inhibit RNA synthesis by delayed chain termination or when in the template strand. Here, enzymatic assays, mass spectrometry, and cryo-EM demonstrate that SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) preferentially incorporates remdesivir triphosphate (RTP), outcompeting 10-fold excess ATP; however, successive RTP incorporations are disfavored when ATP is present. The RdRp structures demonstrate that the remdesivir:UMP base pair is resilient to translocation, reducing successive RTP incorporations. Consequently, the relative concentration of ATP-to-RTP and structural rigidity of remdesivir:UMP, would limit remdesivir occupancy to < 16% of available positions in a copied genomic RNA strand. The S759A mutant confers RTP resistance. The structures of S759A RdRp reveal that the primer 3'-end nucleotide repositioning and its altered ribose-ring conformation contribute to RTP resistance. These findings have implications for designing non-obligate nucleoside analogs with different inhibition mechanisms.