The SARS-CoV-2 RNA polymerase is a viral RNA capping enzyme

Walker, Alexander P; Fan, H.; Keown, J.R.; Knight, Michael; Grimes, Jonathan M.; Fodor, Ervin

doi:10.1093/nar/gkab1160

Cited by 35 publications

(35 citation statements)

References 44 publications

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“…Increasing the NTP pool to 500 μM, the reaction would incorporate non-Watson–Crick base pairs and efficiently move on to the full-length product. Further, the authors in this study [ 18 ] found an IC 50 of 6.56 μM in their in vitro study, similar to our determination of 7.81 μM in the presence of 1 μM NTPs ( Figure 2 A). They also discuss several reasons why their value is much higher than the originally reported 0.77 μM.…”

Section: Discussionsupporting

confidence: 91%

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A Systematic Study on the Optimal Nucleotide Analogue Concentration and Rate Limiting Nucleotide of the SARS-CoV-2 RNA-Dependent RNA Polymerase

Vatandaslar

2022

IJMS

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The current COVID-19 pandemic has highlighted the necessity of more efficient antiviral compounds. The antiviral efficacy of adenosine-based analogs, the main repurposed drugs for SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) inhibition, is mainly assessed through in vitro or cell-free polymerization assays, under arbitrary conditions that do not reflect the physiological environment. We show that SARS-CoV-2 RdRp inhibition efficiency of remdesivir and cordycepin, two common adenosine analogs, is influenced by endogenous adenosine level, and that the current clinically approved concentrations for COVID-19 treatment are suboptimal for effective RdRp inhibition. Furthermore, we identified GTP as the rate-limiting nucleotide of SARS-CoV-2 replication. Our results demonstrate that nucleotide sensitivity of the RdRp complex and competition of nucleoside analog drugs against endogenous concentrations of nucleotides are crucial elements to be considered when designing new SARS-CoV-2 antiviral compounds.

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Section: Discussionsupporting

confidence: 91%

“…Previously, a study demonstrated that the SARS-CoV-2 RdRp complex possesses GTPase activity and also indicated that GS-443902 can inhibit this reaction [ 18 ]. While the polymerase reaction without an inhibitor was carried out at a 500 μM NTP concentration, the NTP concentration was reduced to 1 μM for reactions containing the inhibitor.…”

Section: Discussionmentioning

confidence: 99%

A Systematic Study on the Optimal Nucleotide Analogue Concentration and Rate Limiting Nucleotide of the SARS-CoV-2 RNA-Dependent RNA Polymerase

Vatandaslar

2022

IJMS

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“…In CoVs, the nsp13, nsp14 and nsp16 proteins have RTPase 25 , N 7-MTase 6 and 2′- O -MTase 7 activities, respectively. Thus, it was presupposed that the CoV capping mechanism occurs in a similar manner to the eukaryotic capping pathway, with the NiRAN domain functioning as the GTase 3 , 5 , 26 . However, evidence to support this claim is lacking.…”

Section: Mainmentioning

confidence: 99%

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confidence: 99%

The mechanism of RNA capping by SARS-CoV-2

Park

Osinski

Hernandez

et al. 2022

Nature

120

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The SARS-CoV-2 RNA genome contains a 5′-cap that facilitates translation of viral proteins, protection from exonucleases and evasion of the host immune response [1][2][3][4] . How this cap is made is not completely understood. Here, we reconstitute the SARS-CoV-2 7Me GpppA2′-O-Me-RNA cap using virally encoded non-structural proteins (nsps). We show that the kinase-like NiRAN domain 5 of nsp12 transfers RNA to the amino terminus of nsp9, forming a covalent RNA-protein intermediate (a process termed RNAylation). Subsequently, the NiRAN domain transfers RNA to GDP, forming the cap core structure GpppA-RNA. The nsp14 6 and nsp16 7 methyltransferases then add methyl groups to form functional cap structures. Structural analyses of the replicationtranscription complex bound to nsp9 identified key interactions that mediate the capping reaction. Furthermore, we demonstrate in a reverse genetics system 8 that the N-terminus of nsp9 and the kinase-like active site residues in the NiRAN domain are required for successful SARS-CoV-2 replication. Collectively, our results reveal an unconventional mechanism by which SARS-CoV-2 caps its RNA genome, thus exposing a new target in the development of antivirals to treat COVID-19. 3 Main TextCoronaviruses (CoVs) are a family of positive-sense, single-stranded RNA viruses that cause disease in humans, ranging from mild common colds to more severe respiratory infections 9 . The most topical of these is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of the ongoing COVID-19 pandemic, which to date has resulted in over 5.7million deaths and almost 400-million cases globally 10 .The SARS-CoV-2 RNA genome contains two open reading frames (ORF1a and ORF1ab), which are translated by host ribosomes to form two large polyproteins 2 . These polyproteins are subsequently cleaved by viral proteases to form 16 non-structural proteins (nsp1-16), some of which make up the Replication-Transcription Complex (RTC) 2 . At the core of the RTC is the nsp12 RNA-dependent RNA polymerase (RdRp), which is the target of several promising antivirals used to treat COVID-19 including remdesivir 11 and molnupiravir 12 . In addition to the RdRp domain, nsp12 contains an N-terminal Nidovirus RdRp-Associated Nucleotidyltransferase (NiRAN) domain (Fig. 1a) 5 . The NiRAN domain shares sequence and structural similarity with the pseudokinase selenoprotein-O (SelO), which transfers AMP from ATP to protein substrates (a process termed AMPylation) [13][14][15] . Notably, the active site kinase-like residues of the NiRAN domain are highly conserved in Nidovirales (Extended Data Fig. 1) and are required for equine arteritis virus (EAV) and SARS-CoV-1 replication in cell culture 5 . Several hypotheses for the function of the NiRAN domain have been proposed, including roles in protein-primed RNA synthesis, RNA ligation, and mRNA capping 5,16 .The CoV RNA genome, like eukaryotic mRNAs, contains a methylated guanosine linked to the first nucleotide of the RNA via a reverse 5′ to 5′ triphosphat...

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“…More than 95% identical to SARS-CoV counterpart, nsp12 of SARS-CoV-2 exhibits a similar sensitivity to the inhibitory effect of remdesivir, and the decreased enzymatic activity and thermostability ( Peng et al, 2020 ). Besides RdRP activity, SARS-CoV-2 nsp12 is responsible for viral RNA capping as a GTPase, adding a GTP nucleotide to the 5’ end of viral RNA via a 5’–5’ triphosphate linkage ( Walker et al, 2021 ).…”

Section: Introductionmentioning

confidence: 99%

Genome-Wide Analysis of the Indispensable Role of Non-structural Proteins in the Replication of SARS-CoV-2

Jin

Ouyang

et al. 2022

Front. Microbiol.

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Understanding the process of replication and transcription of SARS-CoV-2 is essential for antiviral strategy development. The replicase polyprotein is indispensable for viral replication. However, whether all nsps derived from the replicase polyprotein of SARS-CoV-2 are indispensable is not fully understood. In this study, we utilized the SARS-CoV-2 replicon as the system to investigate the role of each nsp in viral replication. We found that except for nsp16, all the nsp deletions drastically impair the replication of the replicon, and nsp14 could recover the replication deficiency caused by its deletion in the viral replicon. Due to the unsuccessful expressions of nsp1, nsp3, and nsp16, we could not draw a conclusion about their in trans-rescue functions. Our study provided a new angle to understand the role of each nsp in viral replication and transcription, helping the evaluation of nsps as the target for antiviral drug development.

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The SARS-CoV-2 RNA polymerase is a viral RNA capping enzyme

Cited by 35 publications

References 44 publications

A Systematic Study on the Optimal Nucleotide Analogue Concentration and Rate Limiting Nucleotide of the SARS-CoV-2 RNA-Dependent RNA Polymerase

A Systematic Study on the Optimal Nucleotide Analogue Concentration and Rate Limiting Nucleotide of the SARS-CoV-2 RNA-Dependent RNA Polymerase

The mechanism of RNA capping by SARS-CoV-2

Genome-Wide Analysis of the Indispensable Role of Non-structural Proteins in the Replication of SARS-CoV-2

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