Rapid incorporation of Favipiravir by the fast and permissive viral RNA polymerase complex results in SARS-CoV-2 lethal mutagenesis

Abstract The ongoing Corona Virus Disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has emphasized the urgent need for antiviral therapeutics. The viral RNA-dependent-RNA-polymerase (RdRp) is a promising target with polymerase inhibitors successfully used for the treatment of several viral diseases. We demonstrate here that Favipiravir predominantly exerts an antiviral effect through lethal mutagenesis. The SARS-CoV RdRp complex is at least 10-fold more active than any other viral RdRp known. It possesses both unusually high nucleotide incorporation rates and high-error rates allowing facile insertion of Favipiravir into viral RNA, provoking C-to-U and G-to-A transitions in the already low cytosine content SARS-CoV-2 genome. The coronavirus RdRp complex represents an Achilles heel for SARS-CoV, supporting nucleoside analogues as promising candidates for the treatment of COVID-19.

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Favipiravir strikes the SARS-CoV-2 at its Achilles heel, the RNA polymerase

10.1101/2020.05.15.098731 ◽

2020 ◽

Cited By ~ 20

Author(s):

A. Shannon ◽

B. Selisko ◽

NTT Le ◽

J. Huchting ◽

F. Touret ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Synthesis ◽

Virus Disease ◽

Antiviral Effect ◽

Error Rates ◽

Viral Rna ◽

Lethal Mutagenesis ◽

Nucleotide Analogue ◽

Nucleotide Incorporation ◽

Promising Target

AbstractThe ongoing Corona Virus Disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has emphasized the urgent need for antiviral therapeutics. The viral RNA-dependent-RNA-polymerase (RdRp) is a promising target with polymerase inhibitors successfully used for the treatment of several viral diseases. Here we show that Favipiravir exerts an antiviral effect as a nucleotide analogue through a combination of chain termination, slowed RNA synthesis and lethal mutagenesis. The SARS-CoV RdRp complex is at least 10-fold more active than any other viral RdRp known. It possesses both unusually high nucleotide incorporation rates and high-error rates allowing facile insertion of Favipiravir into viral RNA, provoking C-to-U and G-to-A transitions in the already low cytosine content SARS-CoV-2 genome. The coronavirus RdRp complex represents an Achilles heel for SARS-CoV, supporting nucleoside analogues as promising candidates for the treatment of COVID-19.

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Mechanism of Inhibition of Ebola Virus RNA-Dependent RNA Polymerase by Remdesivir

Viruses ◽

10.3390/v11040326 ◽

2019 ◽

Vol 11 (4) ◽

pp. 326 ◽

Cited By ~ 179

Author(s):

Egor Tchesnokov ◽

Joy Feng ◽

Danielle Porter ◽

Matthias Götte

Keyword(s):

Rna Polymerase ◽

Rna Synthesis ◽

Ebola Virus ◽

Virus Disease ◽

Chain Termination ◽

Mitochondrial Rna ◽

Long Distance ◽

Rna Dependent Rna Polymerase ◽

Nucleotide Analogue ◽

Nucleotide Incorporation

Remdesivir (GS-5734) is a 1′-cyano-substituted adenosine nucleotide analogue prodrug that shows broad-spectrum antiviral activity against several RNA viruses. This compound is currently under clinical development for the treatment of Ebola virus disease (EVD). While antiviral effects have been demonstrated in cell culture and in non-human primates, the mechanism of action of Ebola virus (EBOV) inhibition for remdesivir remains to be fully elucidated. The EBOV RNA-dependent RNA polymerase (RdRp) complex was recently expressed and purified, enabling biochemical studies with the relevant triphosphate (TP) form of remdesivir and its presumptive target. In this study, we confirmed that remdesivir-TP is able to compete for incorporation with adenosine triphosphate (ATP). Enzyme kinetics revealed that EBOV RdRp and respiratory syncytial virus (RSV) RdRp incorporate ATP and remdesivir-TP with similar efficiencies. The selectivity of ATP against remdesivir-TP is ~4 for EBOV RdRp and ~3 for RSV RdRp. In contrast, purified human mitochondrial RNA polymerase (h-mtRNAP) effectively discriminates against remdesivir-TP with a selectivity value of ~500-fold. For EBOV RdRp, the incorporated inhibitor at position i does not affect the ensuing nucleotide incorporation event at position i+1. For RSV RdRp, we measured a ~6-fold inhibition at position i+1 although RNA synthesis was not terminated. Chain termination was in both cases delayed and was seen predominantly at position i+5. This pattern is specific to remdesivir-TP and its 1′-cyano modification. Compounds with modifications at the 2′-position show different patterns of inhibition. While 2′-C-methyl-ATP is not incorporated, ara-ATP acts as a non-obligate chain terminator and prevents nucleotide incorporation at position i+1. Taken together, our biochemical data indicate that the major contribution to EBOV RNA synthesis inhibition by remdesivir can be ascribed to delayed chain termination. The long distance of five residues between the incorporated nucleotide analogue and its inhibitory effect warrant further investigation.

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The mechanism of action of ribavirin: lethal mutagenesis of RNA virus genomes mediated by the viral RNA-dependent RNA polymerase

Current Opinion in Infectious Diseases ◽

10.1097/00001432-200112000-00015 ◽

2001 ◽

Vol 14 (6) ◽

pp. 757-764 ◽

Cited By ~ 70

Author(s):

Craig E. Cameron ◽

Christian Castro

Keyword(s):

Rna Polymerase ◽

Mechanism Of Action ◽

Rna Virus ◽

Viral Rna ◽

Rna Dependent Rna Polymerase ◽

Lethal Mutagenesis ◽

Virus Genomes

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Structural Basis for the Inhibition of the RNA-Dependent RNA Polymerase from SARS-CoV-2 by Remdesivir

10.1101/2020.04.08.032763 ◽

2020 ◽

Cited By ~ 17

Author(s):

Wanchao Yin ◽

Chunyou Mao ◽

Xiaodong Luan ◽

Dan-Dan Shen ◽

Qingya Shen ◽

...

Keyword(s):

Rna Polymerase ◽

Virus Disease ◽

Complex Structure ◽

Antiviral Drug ◽

Viral Rna ◽

Chain Elongation ◽

Structural Basis ◽

Rna Dependent Rna Polymerase ◽

Working Mechanism ◽

Primer Rna

The pandemic of Corona Virus Disease 2019 (COVID-19) caused by SARS-CoV-2 has become a global crisis. The replication of SARS-CoV-2 requires the viral RNA-dependent RNA polymerase (RdRp), a direct target of the antiviral drug, Remdesivir. Here we report the structure of the SARS-CoV-2 RdRp either in the apo form or in complex with a 50-base template-primer RNA and Remdesivir at a resolution range of 2.5-2.8 Å. The complex structure reveals that the partial double-stranded RNA template is inserted into the central channel of the RdRp where Remdesivir is incorporated into the first replicated base pair and terminates the chain elongation. Our structures provide critical insights into the working mechanism of viral RNA replication and a rational template for drug design to combat the viral infection.

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Structural basis for repurpose and design of nucleoside drugs for treating COVID-19

10.1101/2020.11.01.363812 ◽

2020 ◽

Author(s):

Wanchao Yin ◽

Xiaodong Luan ◽

Zhihai Li ◽

Yuanchao Xie ◽

Ziwei Zhou ◽

...

Keyword(s):

Rna Polymerase ◽

Effective Treatment ◽

Viral Rna ◽

Structural Basis ◽

Template Strand ◽

Viral Rdrp ◽

Nucleotide Analog ◽

Global Pandemic ◽

Nucleoside Drugs ◽

Covalently Linked

SARS-CoV-2 has caused a global pandemic of COVID-19 that urgently needs an effective treatment. Nucleoside analog drugs including favipiravir have been repurposed for COVID-19 despite of unclear mechanism of their inhibition of the viral RNA polymerase (RdRp). Here we report the cryo-EM structures of the viral RdRp in complex with favipiravir and two other nucleoside inhibitor drugs ribavirin and penciclovir. Ribavirin and the ribosylated form of favipiravir share a similar ribose scaffold that is distinct from penciclovir. However, the structures reveal that all three inhibitors are covalently linked to the primer strand in a monophosphate form despite the different chemical scaffolds between favipiravir and penciclovir. Surprisingly, the base moieties of these inhibitors can form mismatched pairs with the template strand. Moreover, in view of the clinical disadvantages of remdesivir mainly associated with its prodrug form, we designed several orally-available remdesivir parent nucleoside derivatives, including VV16 that showed 5-fold more potent than remdesivir in inhibition of viral replication. Together, these results demonstrate an unexpected promiscuity of the viral RNA polymerase and provide a basis for repurpose and design of nucleotide analog drugs for COVID-19.One Sentence SummaryCryo-EM structures of the RNA polymerase of SARS-CoV-2 reveals the basis for repurposing of old nucleotide drugs to treat COVID-19.

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Releasing the Genomic RNA Sequestered in the Mumps Virus Nucleocapsid

Journal of Virology ◽

10.1128/jvi.01422-16 ◽

2016 ◽

Vol 90 (22) ◽

pp. 10113-10119 ◽

Cited By ~ 7

Author(s):

Chelsea Severin ◽

James R. Terrell ◽

James R. Zengel ◽

Robert Cox ◽

Richard K. Plemper ◽

...

Keyword(s):

Rna Polymerase ◽

Nucleocapsid Protein ◽

Rna Synthesis ◽

Rna Virus ◽

Mumps Virus ◽

Viral Rna ◽

Genomic Rna ◽

Rna Dependent Rna Polymerase ◽

Negative Strand ◽

Viral Rdrp

ABSTRACT In a negative-strand RNA virus, the genomic RNA is sequestered inside the nucleocapsid when the viral RNA-dependent RNA polymerase uses it as the template for viral RNA synthesis. It must require a conformational change in the nucleocapsid protein (N) to make the RNA accessible to the viral polymerase during this process. The structure of an empty mumps virus (MuV) nucleocapsid-like particle was determined to 10.4-Å resolution by cryo-electron microscopy (cryo-EM) image reconstruction. By modeling the crystal structure of parainfluenza virus 5 into the density, it was shown that the α-helix close to the RNA became flexible when RNA was removed. Point mutations in this helix resulted in loss of polymerase activities. Since the core of N is rigid in the nucleocapsid, we suggest that interactions between this region of the mumps virus N and its polymerase, instead of large N domain rotations, lead to exposure of the sequestered genomic RNA. IMPORTANCE Mumps virus (MuV) infection may cause serious diseases, including hearing loss, orchitis, oophoritis, mastitis, and pancreatitis. MuV is a negative-strand RNA virus, similar to rabies virus or Ebola virus, that has a unique mechanism of viral RNA synthesis. They all make their own RNA-dependent RNA polymerase (RdRp). The viral RdRp uses the genomic RNA inside the viral nucleocapsid as the template to synthesize viral RNAs. Since the template RNA is always sequestered in the nucleocapsid, the viral RdRp must find a way to open it up in order to gain access to the covered template. Our work reported here shows that a helix structural element in the MuV nucleocapsid protein becomes open when the sequestered RNA is released. The amino acids related to this helix are required for RdRp to synthesize viral RNA. We propose that the viral RdRp pulls this helix open to release the genomic RNA.

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