Abstract
Nucleoside analogs are successful in treating viral infections. dNTP analogs are primarily DNA chain terminators, while NTP analog remdesivir can inhibit RNA synthesis by delayed chain termination or when embedded in the template strand. Here, enzymatic assays, mass spectrometry, and cryo-EM structures 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 1'-cyano-imposed conformational restriction of the remdesivir:UMP base-pair is resistant to translocation, reducing successive RTP incorporations. The S759A mutant confers RTP resistance. We show that the mutation switches the RdRp preference to ATP; RTP is incorporated only at 10-fold excess to ATP. 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.