Mechanism of SARS-CoV-2 polymerase stalling by remdesivir

Goran Kokic,Jana Schmitzova,Hauke S Hillen,Claudia Höbartner,Florian Seitz,Lucas Farnung,Dimitry Tegunov,Christian Dienemann,Aaron Siewert,Patrick Cramer

doi:10.1038/s41467-020-20542-0

Abstract

Remdesivir is the only FDA-approved drug for the treatment of COVID-19 patients. The active form of remdesivir acts as a nucleoside analog and inhibits the RNA-dependent RNA polymerase (RdRp) of coronaviruses including SARS-CoV-2. Remdesivir is incorporated by the RdRp into the growing RNA product and allows for addition of three more nucleotides before RNA synthesis stalls. 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 3ʹ-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 3ʹ-nucleotide of the RNA product is matched and located with the template base in the active center, and this may impair proofreading by the viral 3ʹ-exonuclease. These mechanistic insights should facilitate the quest for improved antivirals that target coronavirus replication.

Highlights

Remdesivir is the only FDA-approved drug for the treatment of COVID-19 patients
Biochemical studies showed that the RNA-dependent RNA polymerase (RdRp) can use remdesivir triphosphate (RTP) as a substrate, leading to the incorporation of remdesivir monophosphate (RMP) into the growing RNA product[10,20,22]
Consistent with recent studies[20,22], we observed that RMP is readily incorporated into RNA and that the RNA is subsequently elongated by three more nucleotides before the RdRp stalls (Fig. 1c, d)

Summary

Introduction

Remdesivir is the only FDA-approved drug for the treatment of COVID-19 patients. The active form of remdesivir acts as a nucleoside analog and inhibits the RNA-dependent RNA polymerase (RdRp) of coronaviruses including SARS-CoV-2. 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 3ʹ-nucleotide in the substrate-binding site of the RdRp and interferes with entry of the nucleoside triphosphate, thereby stalling RdRp. In the structure of the remdesivir-stalled state, the 3ʹ-nucleotide of the RNA product is matched and located with the template base in the active center, and this may impair proofreading by the viral 3ʹ-exonuclease. RMP mimics adenosine monophosphate (AMP) and forms standard Watson–Crick base pairs with uridine monophosphate (UMP) in the RNA template strand These studies explained how RMP is incorporated into RNA instead of AMP. They do not explain how remdesivir inhibits the RdRp because RdRp stalling occurs only after three more nucleotides have been added to the RNA10,20

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Methods

Results