Mechanism of SARS-CoV-2 polymerase stalling by remdesivir

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 3'-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 3'-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 3'-exonuclease. These mechanistic insights should facilitate the quest for improved antivirals that target coronavirus replication.

Fig. 1. Remdesivir impairs RNA elongation by RdRp.

a Chemical structure of remdesivir triphosphate (RTP) showing the ribose 1ʹ cyano group. b RNA template-product duplex. The direction of RNA elongation is indicated. c Remdesivir-induced RdRp stalling. Replacing ATP with RTP leads to an elongation barrier after addition of three more nucleotides. The barrier can be overcome at higher NTP concentrations. The RNA 5ʹ-end contains a fluorescent label. Asterisk indicates 3ʹ-dGTP. Source data are provided as a Source Data file. d Quantification of the experiment in panel c after triplicate measurements. Standard deviations are shown. Source data are provided as a Source Data file.

Fig. 2. Preparation of remdesivir-containing RNA.

a Scheme of the synthesis of 5ʹ-O-DMT-2ʹ-O-TBDMS-protected 3ʹ-cyanoethyl diisopropyl phosphoramidite (Rem-PA), which was used to synthesize RNA oligos with remdesivir monophosphate (RMP) at defined positions. b Analysis of RMP-containing RNA by denaturing HPLC confirms the presence of RMP. c Analysis of the RMP-containing RNA by LC-MS after digestion into mononucleosides confirms the presence of RMP. d Minimal RNA template-product scaffold with RMP (R) or AMP (A) in a synthesized RNA oligonucleotide product strand. e The presence of RMP in a synthesized RNA oligonucleotide inhibits RNA extension by RdRp on the minimal RNA scaffold (d). Source data are provided as a Source Data file.

Fig. 3. Structural analysis of remdesivir-induced RdRp stalling.

a Position of RNA scaffolds 1–3 as observed in RdRp-RNA complex structures 1–3. Template and product strands are on the top and bottom, respectively. b Cryo-EM density of RNA in the active center of structures 1–3. The active site metal ion was modeled and is shown as a magenta sphere. c The C1ʹ-cyano group of the RMP ribose moiety (violet) is accommodated at position –3 (left), but would clash with the side chain of nsp12 residue serine-861 (red) at position –4 (right). Spheres indicate atomic van der Waals surfaces.