Alternative role of motif B in template dependent polymerase inhibition

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) relies on the central molecular machine RNA-dependent RNA polymerase (RdRp) for the viral replication and transcription. Remdesivir at the template strand has been shown to effectively inhibit the RNA synthesis in SARS-CoV-2 RdRp by deactivating not only the complementary UTP incorporation but also the next nucleotide addition. How-ever, the underlying molecular mechanism of the second inhibitory point remains unclear. In this work, we have performed molecular dynamics simulations and demonstrated that such inhibition has not directly acted on the nucleotide addition at the active site. Instead, the translocation of Remdesivir from + 1 to − 1 site is hindered thermodynamically as the post-translocation state is less stable than the pre-translocation state due to the motif B residue G683. Moreover, another conserved residue S682 on motif B further hinders the dynamic translocation of Remdesivir due to the steric clash with the 1′-cyano substitution. Overall, our study has unveiled an alternative role of motif B in mediating the translocation when Remdesivir is present in the template strand and complemented our understanding about the inhibitory mechanisms exerted by Remdesivir on the RNA synthesis in SARS-CoV-2 RdRp.