Structural Basis for RNA Replication by the SARS-CoV-2 Polymerase

Wang, Quan; Wu, Jiqin; Wang, Haofeng; Gao, Yan; Liu, Qiaojie; Mu, A.; Ji, Wenxin; Yan, Liming; Zhu, Yan; Zhu, Chen; Fang, Xiang; Yang, Xiaobao; Huang, Yucen; Gao, Hailong; Liu, Fengjiang; Ge, Jingpeng; Sun, Qianqian; Yang, Xiuna; Xu, Wenqing; Liu, Zhijie; Yang, Haitao; Lou, Zhiyong; Jiang, Biao; Guddat, Luke W.; Gong, Peng; Rao, Zihe

doi:10.1016/j.cell.2020.05.034

Cited by 737 publications

(616 citation statements)

References 44 publications

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“…c. nMS spectra and the corresponding deconvolved spectra for the holo-RdRp containing the RNA scaffold (a) with and without nsp13. The measured mass for the holo-RdRp:RNA complex corroborates the established stoichiometry of 1:2:1:1 for nsp7:nsp8:nsp12:RNA (Hillen et al, 2020;Kirchdoerfer and Ward, 2019;Wang et al, 2020;Yin et al, 2020), respectively (bottom). Addition of the 67.5-kDa nsp13 helicase to the RNA-bound holo-RdRp holo sample forms a transcription complex/helicase assembly with 1:1 stoichiometry (top).…”

Section: Discussionsupporting

confidence: 79%

Structural basis for helicase-polymerase coupling in the SARS-CoV-2 replication-transcription complex

Chen

Malone

Llewellyn

et al. 2020

Preprint

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SUMMARYSARS-CoV-2 is the causative agent of the 2019-2020 pandemic. The SARS-CoV-2 genome is replicated-transcribed by the RNA-dependent RNA polymerase holoenzyme (subunits nsp7/nsp82/nsp12) along with a cast of accessory factors. One of these factors is the nsp13 helicase. Both the holo-RdRp and nsp13 are essential for viral replication and are targets for treating the disease COVID-19. Here we present cryo-electron microscopic structures of the SARS-CoV-2 holo-RdRp with an RNA template-product in complex with two molecules of the nsp13 helicase. The Nidovirus-order-specific N-terminal domains of each nsp13 interact with the N-terminal extension of each copy of nsp8. One nsp13 also contacts the nsp12-thumb. The structure places the nucleic acid-binding ATPase domains of the helicase directly in front of the replicating-transcribing holo-RdRp, constraining models for nsp13 function. We also observe ADP-Mg2+ bound in the nsp12 N-terminal nidovirus RdRp-associated nucleotidyltransferase domain, detailing a new pocket for anti-viral therapeutic development.

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Section: Discussionsupporting

confidence: 79%

Structural basis for helicase-polymerase coupling in the SARS-CoV-2 replication-transcription complex

Chen

Malone

Llewellyn

et al. 2020

Preprint

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“…1 E). Overall the active site emerging from EM structures 12,14,39,40 and atomistic simulation strongly suggest that despite the reduced evolutionary refinement, SARS-CoV-2 RdRp has all the structural requirements to be an efficient RNA polymerase, both in terms of catalysis and substrate specificity. The RNA-pol reaction mechanism.…”

Section: Resultsmentioning

confidence: 99%

“…For example, mutational analysis shows that Ser861 has a crucial role in R-induced inhibition of RdRp. 40 Furthermore, large and systematic efforts to substitute the nitrile group by less problematic substituents (the nitrile group appears as a "probably undesired" consequence of the Remdesivir synthetic pathway) resulted in molecules with a lower inhibitory profile. 47 Finally, Pinner reaction has not been described for RdRp, but has been experimentally validated for many several nitrile-containing drugs designed to inhibit cysteine or serine proteases.…”

Section: Remdesivir Is Well Tolerated In An Rna Duplexmentioning

confidence: 99%

“…Ser861 is highly conserved in other CoVs RdRps (see Supplementary Fig. 14), placing a constant position in an alpha helix that adopts a highly conserved 3D arrangement (compare SARS-CoV-2 and SARS-CoV structures) 12,14,39,40,54 and this position is well preserved in those cases for which CoV RdRps structure is available. This would suggest that Remdesivir might be effective against many other CoV RdRps.…”

Section: Fig 4 Remdesivir Forms a Covalent Complex With Rdrp Of Sarsmentioning

confidence: 99%

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RNA-Dependent RNA Polymerase From SARS-CoV-2. Mechanism Of Reaction And Inhibition By Remdesivir

Aranda

Orozco

2020

Preprint

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We combine sequence analysis, molecular dynamics and hybrid quantum mechanics/molecular mechanics simulations to obtain the first description of the mechanism of reaction of SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) and of the inhibition of the enzyme by Remdesivir. Despite its evolutionary youth, the enzyme is highly optimized to have good fidelity in nucleotide incorporation and a good catalytic efficiency. Our simulations strongly suggest that Remdesivir triphosphate (the active form of drug) is incorporated into the nascent RNA replacing ATP, leading to a duplex RNA which is structurally very similar to an unmodified one. We did not detect any reason to explain the inhibitory activity of Remdesivir at the active site. Displacement of the nascent Remdesivir-containing RNA duplex along the exit channel of the enzyme can occur without evident steric clashes which would justify delayed inhibition. However, after the incorporation of three more nucleotides we found a hydrated Serine which is placed in a perfect arrangement to react through a Pinner's reaction with the nitrile group of Remdesivir. Kinetic barriers for crosslinking and polymerization are similar suggesting a competition between polymerization and inhibition. Analysis of SARS-CoV-2 mutational landscape and structural analysis of polymerases across different species support the proposed mechanism and suggest that virus has not explored yet resistance to Remdesivir inhibition.

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“…NSP8 is a 198 amino acid long protein and rich in alpha helical content. Notably, R51 plays a major role in interaction with the double stranded RNA at the minor groove side and M129 occurs at the interface of NSP8-NSP12 (Figure 4, PDB ID: 6YYT) 28 indicating that these mutations may have an influence on the viral replication mechanism. The other mutations that prevail either in the first (A16V, A21V, T89I, S177L and T187I) or in the second phase (Q19H, V34F, S41F, T123I, T145I and I156V) have a low significant rate (less than 0.1%).…”

Section: L37f Is the Dominant Mutation In Nsp6mentioning

confidence: 99%

Global variation in the SARS-CoV-2 proteome reveals the mutational hotspots in the drug and vaccine candidates

Patro

Sathyaseelan

Uttamrao

et al. 2020

Preprint

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To accelerate the drug and vaccine development against the severe acute respiratory syndrome virus 2 (SARS-CoV-2), a comparative analysis of SARS-CoV-2 proteome has been performed in two phases by considering manually curated 31389 whole genome sequences from 84 countries. Among the 9 mutations that occur at a high significance (T85I-NPS2, L37F-NSP6, P323L-NSP12, D614G-spike, Q57H-ORF3a, G251V-ORF3a, L84S-ORF8, R203K-nucleocapsid and G204R-nucleocapsid), R203K-nucleocapsid and G204R-nucleocapsid are co-occurring mutations and P323L-NSP12 and D614G-spike often appear simultaneously. Other notable variations that appear with a moderate to low significance are, M85-NSP1 deletion, D268-NSP2 deletion, 112 amino acids deletion in ORF8, a phenylalanine insertion amidst F34-F36 (NSP6) and several co-existing substitution/deletion (I559V & P585S in NSP2, P504L & Y541C in NSP13, G82 & H83 deletions in NSP1 and K141, S142 & F143 deletions in NSP2) mutations. P323L-NSP12, D614G-spike, L37F-NSP6, L84S-ORF8 and the sequences deficient of the high significant mutations has led to 4 major SARS-CoV-2 clades. The top 5 countries bearing all the high significant and majority of the moderate significant mutations are: USA, England, Wales, Australia and Scotland. Further, the majority of the significant mutations has evolved in the first phase and has already transmitted around the globe indicating the positive selection pressure. Among the 26 SARS-CoV-2 proteins, nucleocapsid protein, ORF3a, ORF8, RNA dependent RNA polymerase and spike exhibit a higher heterogeneity compared with the rest of the proteins. However, NSP9, NSP10, NSP8, the envelope protein and NSP4 are highly resistant to mutations and can be exploited for drug/vaccine development.

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Structural Basis for RNA Replication by the SARS-CoV-2 Polymerase

Cited by 737 publications

References 44 publications

Structural basis for helicase-polymerase coupling in the SARS-CoV-2 replication-transcription complex

Structural basis for helicase-polymerase coupling in the SARS-CoV-2 replication-transcription complex

RNA-Dependent RNA Polymerase From SARS-CoV-2. Mechanism Of Reaction And Inhibition By Remdesivir

Global variation in the SARS-CoV-2 proteome reveals the mutational hotspots in the drug and vaccine candidates

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