Structural basis for inhibition of the RNA-dependent RNA polymerase from SARS-CoV-2 by remdesivir

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 of ribosomal frameshifting during translation of the SARS-CoV-2 RNA genome

Science ◽

10.1126/science.abf3546 ◽

2021 ◽

pp. eabf3546

Author(s):

Pramod R. Bhatt ◽

Alain Scaiola ◽

Gary Loughran ◽

Marc Leibundgut ◽

Annika Kratzel ◽

...

Keyword(s):

Electron Microscopy ◽

Rna Polymerase ◽

Viral Rna ◽

Structural Basis ◽

Rna Dependent Rna Polymerase ◽

Ribosomal Frameshifting ◽

Cryo Electron Microscopy ◽

Ribosomal Tunnel ◽

Rna Genome ◽

Viral Polyprotein

Programmed ribosomal frameshifting is a key event during translation of the SARS-CoV-2 RNA genome allowing synthesis of the viral RNA-dependent RNA polymerase and downstream proteins. Here we present the cryo-electron microscopy structure of a translating mammalian ribosome primed for frameshifting on the viral RNA. The viral RNA adopts a pseudoknot structure that lodges at the entry to the ribosomal mRNA channel to generate tension in the mRNA and promote frameshifting, whereas the nascent viral polyprotein forms distinct interactions with the ribosomal tunnel. Biochemical experiments validate the structural observations and reveal mechanistic and regulatory features that influence frameshifting efficiency. Finally, we compare compounds previously shown to reduce frameshifting with respect to their ability to inhibit SARS-CoV-2 replication, establishing coronavirus frameshifting as a target for antiviral intervention.

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Structural basis of ribosomal frameshifting during translation of the SARS-CoV-2 RNA genome

10.1101/2020.10.26.355099 ◽

2020 ◽

Author(s):

Pramod R. Bhatt ◽

Alain Scaiola ◽

Gary Loughran ◽

Marc Leibundgut ◽

Annika Kratzel ◽

...

Keyword(s):

Rna Polymerase ◽

Viral Proteins ◽

Viral Rna ◽

Structural Basis ◽

Rna Dependent Rna Polymerase ◽

Ribosomal Frameshifting ◽

Infected Cells ◽

Rna Genome ◽

Exit Tunnel ◽

Viral Polyprotein

AbstractProgrammed ribosomal frameshifting is the key event during translation of the SARS-CoV-2 RNA genome allowing synthesis of the viral RNA-dependent RNA polymerase and downstream viral proteins. Here we present the cryo-EM structure of the mammalian ribosome in the process of translating viral RNA paused in a conformation primed for frameshifting. We observe that the viral RNA adopts a pseudoknot structure lodged at the mRNA entry channel of the ribosome to generate tension in the mRNA that leads to frameshifting. The nascent viral polyprotein that is being synthesized by the ribosome paused at the frameshifting site forms distinct interactions with the ribosomal polypeptide exit tunnel. We use biochemical experiments to validate our structural observations and to reveal mechanistic and regulatory features that influence the frameshifting efficiency. Finally, a compound previously shown to reduce frameshifting is able to inhibit SARS-CoV-2 replication in infected cells, establishing coronavirus frameshifting as target for antiviral intervention.

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Structure of the RNA-dependent RNA polymerase from COVID-19 virus

Science ◽

10.1126/science.abb7498 ◽

2020 ◽

Vol 368 (6492) ◽

pp. 779-782 ◽

Cited By ~ 312

Author(s):

Yan Gao ◽

Liming Yan ◽

Yucen Huang ◽

Fengjiang Liu ◽

Yao Zhao ◽

...

Keyword(s):

Rna Polymerase ◽

Antiviral Drug ◽

Central Component ◽

Analysis Model ◽

Primary Target ◽

Rna Dependent Rna Polymerase ◽

Viral Rdrp ◽

Cryo Electron Microscopy ◽

N Terminus ◽

Novel Coronavirus

A novel coronavirus [severe acute respiratory syndrome–coronavirus 2 (SARS-CoV-2)] outbreak has caused a global coronavirus disease 2019 (COVID-19) pandemic, resulting in tens of thousands of infections and thousands of deaths worldwide. The RNA-dependent RNA polymerase [(RdRp), also named nsp12] is the central component of coronaviral replication and transcription machinery, and it appears to be a primary target for the antiviral drug remdesivir. We report the cryo–electron microscopy structure of COVID-19 virus full-length nsp12 in complex with cofactors nsp7 and nsp8 at 2.9-angstrom resolution. In addition to the conserved architecture of the polymerase core of the viral polymerase family, nsp12 possesses a newly identified β-hairpin domain at its N terminus. A comparative analysis model shows how remdesivir binds to this polymerase. The structure provides a basis for the design of new antiviral therapeutics that target viral RdRp.

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Intermolecular Interaction Among Remdesivir, RNA and RNA-Dependent RNA Polymerase of SARS-CoV-2 Analyzed by Fragment Molecular Orbital Calculation

10.26434/chemrxiv.12362222 ◽

2020 ◽

Author(s):

Koichiro Kato ◽

Teruki Honma ◽

Kaori Fukuzawa

Keyword(s):

Rna Polymerase ◽

Molecular Orbital ◽

Antiviral Drug ◽

Molecular Orbital Calculation ◽

Data Bank ◽

Rna Dependent Rna Polymerase ◽

Fragment Molecular Orbital ◽

Global Pandemic ◽

The World ◽

Primer Rna

<p>COVID-19, a disease caused by a new strain of coronavirus (SARS-CoV-2) originating from Wuhan, China, has now spread around the world, triggering a global pandemic, leaving the public eagerly awaiting the development of a specific medicine and vaccine. In response, aggressive efforts are underway around the world to overcome COVID-19. In this study, referencing the data published on the Protein Data Bank (PDB ID: 7BV2) on April 22, we conducted a detailed analysis of the interaction between the complex structures of the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 and Remdesivir, an antiviral drug, from the quantum chemical perspective based on the fragment molecular orbital (FMO) method. In addition to the hydrogen bonding and intra-strand stacking between complementary strands as seen in normal base pairs, Remdesivir bound to the terminus of an primer-RNA strand was further stabilized by diagonal π-π stacking with the -1A base of the complementary strand and an additional hydrogen bond with an intra-strand base, due to the effect of chemically modified functional group. Moreover, stable OH/π interaction is also formed with Thr687 of the RdRp. We quantitatively revealed the exhaustive interaction within the complex among Remdesivir, template-primer-RNA, RdRp and co-factors, and published the results in the FMODB database.</p>

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Intermolecular Interaction Among Remdesivir, RNA and RNA-Dependent RNA Polymerase of SARS-CoV-2 Analyzed by Fragment Molecular Orbital Calculation

10.26434/chemrxiv.12362222.v1 ◽

2020 ◽

Author(s):

Koichiro Kato ◽

Teruki Honma ◽

Kaori Fukuzawa

Keyword(s):

Rna Polymerase ◽

Molecular Orbital ◽

Antiviral Drug ◽

Molecular Orbital Calculation ◽

Data Bank ◽

Rna Dependent Rna Polymerase ◽

Fragment Molecular Orbital ◽

Global Pandemic ◽

The World ◽

Primer Rna

<p>COVID-19, a disease caused by a new strain of coronavirus (SARS-CoV-2) originating from Wuhan, China, has now spread around the world, triggering a global pandemic, leaving the public eagerly awaiting the development of a specific medicine and vaccine. In response, aggressive efforts are underway around the world to overcome COVID-19. In this study, referencing the data published on the Protein Data Bank (PDB ID: 7BV2) on April 22, we conducted a detailed analysis of the interaction between the complex structures of the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 and Remdesivir, an antiviral drug, from the quantum chemical perspective based on the fragment molecular orbital (FMO) method. In addition to the hydrogen bonding and intra-strand stacking between complementary strands as seen in normal base pairs, Remdesivir bound to the terminus of an primer-RNA strand was further stabilized by diagonal π-π stacking with the -1A base of the complementary strand and an additional hydrogen bond with an intra-strand base, due to the effect of chemically modified functional group. Moreover, stable OH/π interaction is also formed with Thr687 of the RdRp. We quantitatively revealed the exhaustive interaction within the complex among Remdesivir, template-primer-RNA, RdRp and co-factors, and published the results in the FMODB database.</p>

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Structural basis of viral RNA-dependent RNA polymerase nucleotide addition cycle in picornaviruses

10.1016/bs.enz.2021.06.002 ◽

2021 ◽

pp. 215-233

Author(s):

Peng Gong

Keyword(s):

Rna Polymerase ◽

Viral Rna ◽

Structural Basis ◽

Rna Dependent Rna Polymerase ◽

Nucleotide Addition

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Structural basis for backtracking by the SARS-CoV-2 replication-transcription complex

10.1101/2021.03.13.435256 ◽

2021 ◽

Cited By ~ 2

Author(s):

Brandon Malone ◽

James Chen ◽

Qi Wang ◽

Eliza Llewellyn ◽

Young Joo Choi ◽

...

Keyword(s):

Electron Microscopy ◽

Molecular Dynamics ◽

Rna Polymerase ◽

Critical Role ◽

Structural Basis ◽

Rna Dependent Rna Polymerase ◽

Present Evidence ◽

Cryo Electron Microscopy ◽

Dynamics Simulations ◽

Transcription And Replication

AbstractBacktracking, the reverse motion of the transcriptase enzyme on the nucleic acid template, is a universal regulatory feature of transcription in cellular organisms but its role in viruses is not established. Here we present evidence that backtracking extends into the viral realm, where backtracking by the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) may aid viral transcription and replication. Structures of SARS-CoV-2 RdRp bound to the essential nsp13 helicase and RNA suggested the helicase facilitates backtracking. We use cryo-electron microscopy, RNA-protein crosslinking, and unbiased molecular dynamics simulations to characterize SARS-CoV-2 RdRp backtracking. The results establish that the single-stranded 3’-segment of the product-RNA generated by backtracking extrudes through the RdRp NTP-entry tunnel, that a mismatched nucleotide at the product-RNA 3’-end frays and enters the NTP-entry tunnel to initiate backtracking, and that nsp13 stimulates RdRp backtracking. Backtracking may aid proofreading, a crucial process for SARS-CoV-2 resistance against antivirals.Significance StatementThe COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The SARS-CoV-2 genome is replicated and transcribed by its RNA-dependent RNA polymerase (RdRp), which is the target for antivirals such as remdesivir. We use a combination of approaches to show that backtracking (backwards motion of the RdRp on the template RNA) is a feature of SARS-CoV-2 replication/transcription. Backtracking may play a critical role in proofreading, a crucial process for SARS-CoV-2 resistance against many antivirals.

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