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 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.

Remdesivir for the treatment of Covid-19: the value of biochemical studies

“…While the S861A mutant shows a subtle reduction in delayed chain-termination [ 26 , 27 •• ], RNA synthesis is not inhibited with the S861 G mutant [ 27 •• ]. These results guided structural studies that confirmed the original model [ 28 , 29 ].

Figure 1 How does RDV work against SARS-CoV-2?

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SARS-CoV-2 Nsp8 N-terminal domain dimerizes and harbors autonomously folded elements

“…and Nsp7 could really combine to form a hexameric ring, which would afford PCNA-like progressivity to the RTC as proposed by Zhai et al 1 (Fig 2A). By contrast, if the physiological RTC were like the recent cryo-EM structures 2,3,[8][9][10][11][12][13][14][15] (Fig. 2B), then the dimerization of the Nsp8 NTD might initially serve to self-chaperone hydrophobic patches which evolve to form interactions with Nsp12, Nsp13 and RNA.…”

Dimeric form of SARS-CoV-2 polymerase

“…We calculated nearest-neighbor (NN) distances and relative orientations of neighboring RdRp enzymes. In one of our published data sets (structure 3 11 ), we detected many particles that showed RdRps with a preferred NN distance of 80 Å and a relative orientation of 180° ( Supplemental Figure 1a, b ), indicating the existence of a structurally defined RdRp dimer.…”

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Replication and transcription of the RNA genome of the coronavirus SARS-CoV-2 rely on the viral RNA-dependent RNA polymerase (RdRp) [1][2][3][4][5] . Following the structure of the RdRp of SARS-CoV-1 6 , structures of the RdRp of SARS-CoV-2 were obtained in free form 7 and as a complex with bound RNA template-product duplex [8][9][10][11] . These structures revealed a monomeric RdRp with a subunit stoichiometry of one copy of the catalytic subunit nsp12 12 , two copies of accessory subunit nsp8 13 , and one copy of the accessory subunit nsp7 3,14 .

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