Remdesivir is one nucleotide analogue prodrug capable to terminate RNA synthesis in SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) by two distinct mechanisms. Although the "delayed chain termination" mechanism has been extensively investigated, the "template-dependent" inhibitory mechanism remains elusive. In this study, we have demonstrated that remdesivir embedded in the template strand seldom directly disrupted the complementary NTP incorporation at the active site. Instead, the translocation of remdesivir from the +2 to the +1 site was hindered due to the steric clash with V557. Moreover, we have elucidated the molecular mechanism characterizing the drug resistance upon V557L mutation. Overall, our studies have provided valuable insight into the "template-dependent" inhibitory mechanism exerted by remdesivir on SARS-CoV-2 RdRp and paved venues for an alternative antiviral strategy for the COVID-19 pandemic. As the "template-dependent" inhibition occurs across diverse viral RdRps, our findings may also shed light on a common acting mechanism of inhibitors.
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