scholarly journals Mechanism of SARS-CoV-2 polymerase stalling by remdesivir

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Goran Kokic ◽  
Hauke S. Hillen ◽  
Dimitry Tegunov ◽  
Christian Dienemann ◽  
Florian Seitz ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Rna Polymerase ◽  
Binding Site ◽  
Molecular Mechanism ◽  
Active Center ◽  
Rna Synthesis ◽  
Active Form ◽  
Nucleoside Triphosphate ◽  
Rna Dependent Rna Polymerase ◽  
Cryo Electron Microscopy

AbstractRemdesivir 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.

scholarly journals Mechanism of SARS-CoV-2 polymerase inhibition by remdesivir

2020 ◽  
Author(s):  
Goran Kokic ◽  
Hauke S. Hillen ◽  
Dimitry Tegunov ◽  
Christian Dienemann ◽  
Florian Seitz ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Rna Polymerase ◽  
Binding Site ◽  
Molecular Mechanism ◽  
Nucleoside Analogue ◽  
Rna Synthesis ◽  
Active Form ◽  
Nucleoside Triphosphate ◽  
Rna Dependent Rna Polymerase ◽  
Cryo Electron Microscopy

Remdesivir is the only FDA-approved drug for the treatment of COVID-19 patients1–4. The active form of remdesivir acts as a nucleoside analogue and inhibits the RNA-dependent RNA polymerase (RdRp) of coronaviruses including SARS-CoV-25–7. Remdesivir is incorporated by the RdRp into the growing RNA product and allows for addition of three more nucleotides before RNA synthesis stalls6,8. 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 3’-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 3’-nucleotide of the RNA product is matched with the template base, and this may prevent proofreading by the viral 3’-exonuclease that recognizes mismatches9,10. These mechanistic insights should facilitate the quest for improved antivirals that target coronavirus replication.

scholarly journals Initial Location of the RNA-dependent RNA Polymerase in the Bacteriophage Φ6 Procapsid Determined by Cryo-electron Microscopy

2008 ◽  
Vol 283 (18) ◽  
pp. 12227-12231 ◽  
Author(s):  
Anindito Sen ◽  
J. Bernard Heymann ◽  
Naiqian Cheng ◽  
Jian Qiao ◽  
Leonard Mindich ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Rna Polymerase ◽  
Rna Dependent Rna Polymerase ◽  
Cryo Electron Microscopy ◽  
Initial Location

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

2021 ◽  
Vol 118 (19) ◽  
pp. e2102516118
Author(s):  
Brandon Malone ◽  
James Chen ◽  
Qi Wang ◽  
Eliza Llewellyn ◽  
Young Joo Choi ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Molecular Dynamics ◽  
Structural Basis ◽  
Nucleoside Triphosphate ◽  
Cross Linking ◽  
Rna Dependent Rna Polymerase ◽  
Present Evidence ◽  
Cryo Electron Microscopy ◽  
Dynamics Simulations ◽  
Transcription And Replication

Backtracking, 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 severe acute respiratory syndrome coronavirus 2 (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 cross-linking, 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 nucleoside triphosphate (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.

scholarly journals A Library of Nucleotide Analogues Terminate RNA Synthesis Catalyzed by Polymerases of Coronaviruses Causing SARS and COVID-19

2020 ◽  
Author(s):  
Steffen Jockusch ◽  
Chuanjuan Tao ◽  
Xiaoxu Li ◽  
Thomas K. Anderson ◽  
Minchen Chien ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Safety Profile ◽  
Rna Synthesis ◽  
Viral Infections ◽  
Nucleoside Triphosphate ◽  
Exonuclease Activity ◽  
Base Pairs ◽  
Rna Dependent Rna Polymerase ◽  
Nucleotide Analogues ◽  
Tenofovir Alafenamide

AbstractSARS-CoV-2, a member of the coronavirus family, is responsible for the current COVID-19 worldwide pandemic. We previously demonstrated that five nucleotide analogues inhibit the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp), including the active triphosphate forms of Sofosbuvir, Alovudine, Zidovudine, Tenofovir alafenamide and Emtricitabine. We report here the evaluation of a library of additional nucleoside triphosphate analogues with a variety of structural and chemical features as inhibitors of the RdRps of SARS-CoV and SARS-CoV-2. These features include modifications on the sugar (2’ or 3’ modifications, carbocyclic, acyclic, or dideoxynucleotides) or on the base. The goal is to identify nucleotide analogues that not only terminate RNA synthesis catalyzed by these coronavirus RdRps, but also have the potential to resist the viruses’ exonuclease activity. We examined these nucleotide analogues with regard to their ability to be incorporated by the RdRps in the polymerase reaction and then prevent further incorporation. While all 11 molecules tested displayed incorporation, 6 exhibited immediate termination of the polymerase reaction (Carbovir triphosphate, Ganciclovir triphosphate, Stavudine triphosphate, Entecavir triphosphate, 3’-O-methyl UTP and Biotin-16-dUTP), 2 showed delayed termination (Cidofovir diphosphate and 2’-O-methyl UTP), and 3 did not terminate the polymerase reaction (2’-fluoro-dUTP, 2’-amino-dUTP and Desthiobiotin-16-UTP). The coronavirus genomes encode an exonuclease that apparently requires a 2’ -OH group to excise mismatched bases at the 3’-terminus. In this study, all of the nucleoside triphosphate analogues we evaluated form Watson-Cricklike base pairs. All the nucleotide analogues which demonstrated termination either lack a 2’-OH, have a blocked 2’-OH, or show delayed termination. These nucleotides may thus have the potential to resist exonuclease activity, a property that we will investigate in the future. Furthermore, prodrugs of five of these nucleotide analogues (Brincidofovir/Cidofovir, Abacavir, Valganciclovir/Ganciclovir, Stavudine and Entecavir) are FDA approved for other viral infections, and their safety profile is well known. Thus, they can be evaluated rapidly as potential therapies for COVID-19.

scholarly journals Coronavirus-Replikation: Mechanismus und Inhibition durch Remdesivir

BIOspektrum ◽  
2021 ◽  
Vol 27 (1) ◽  
pp. 49-53
Author(s):  
Patrick Cramer ◽  
Goran Kokic ◽  
Christian Dienemann ◽  
Claudia Höbartner ◽  
Hauke S. Hillen
Keyword(s):  
Electron Microscopy ◽  
Rna Polymerase ◽  
Structure Function ◽  
Rna Dependent Rna Polymerase ◽  
Large Genome ◽  
Cryo Electron Microscopy ◽  
Rna Genome ◽  
Short Time ◽  
Viral Enzyme

AbstractCoronaviruses use an RNA-dependent RNA polymerase to replicate and transcribe their RNA genome. The structure of the SARS-CoV-2 polymerase was determined by cryo-electron microscopy within a short time in spring 2020. The structure explains how the viral enzyme synthesizes RNA and how it replicates the exceptionally large genome in a processive manner. The most recent structure-function studies further reveal the mechanism of polymerase inhibition by remdesivir, an approved drug for the treatment of COVID-19.

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

Science ◽  
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.

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

2021 ◽  
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.

scholarly journals Ensemble cryo-electron microscopy reveals conformational states of the nsp13 helicase in the SARS-CoV-2 helicase replication-transcription complex

2021 ◽  
Author(s):  
James Chen ◽  
Qi Wang ◽  
Brandon Malone ◽  
Eliza Llewellyn ◽  
Yakov Pechersky ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Active Site ◽  
Rna Synthesis ◽  
Genome Replication ◽  
Nonstructural Proteins ◽  
Rna Dependent Rna Polymerase ◽  
Conformational States ◽  
Cryo Electron Microscopy ◽  
Allosteric Mechanism ◽  
Dynamics Simulations

The SARS-CoV-2 nonstructural proteins coordinate genome replication and gene expression. Structural analyses revealed the basis for coupling of the essential nsp13 helicase with the RNA dependent RNA polymerase (RdRp) where the holo-RdRp and RNA substrate (the replication-transcription complex, or RTC) associated with two copies of nsp13 (nsp132-RTC). One copy of nsp13 interacts with the template RNA in an opposing polarity to the RdRp and is envisaged to drive the RdRp backwards on the RNA template (backtracking), prompting questions as to how the RdRp can efficiently synthesize RNA in the presence of nsp13. Here, we use cryo-electron microscopy and molecular dynamics simulations to analyze the nsp132-RTC, revealing four distinct conformational states of the helicases. The results suggest a mechanism for the nsp132-RTC to turn backtracking on and off, using an allosteric mechanism to switch between RNA synthesis or backtracking in response to stimuli at the RdRp active site.

scholarly journals Structural basis of transcription-translation coupling

Science ◽  
2020 ◽  
Vol 369 (6509) ◽  
pp. 1359-1365 ◽  
Author(s):  
Chengyuan Wang ◽  
Vadim Molodtsov ◽  
Emre Firlar ◽  
Jason T. Kaelber ◽  
Gregor Blaha ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Escherichia Coli ◽  
Transcription Factors ◽  
Rna Polymerase ◽  
Active Center ◽  
Messenger Rna ◽  
Coupled Processes ◽  
Structural Basis ◽  
Cryo Electron Microscopy ◽  
Translation Coupling

In bacteria, transcription and translation are coupled processes in which the movement of RNA polymerase (RNAP)–synthesizing messenger RNA (mRNA) is coordinated with the movement of the first ribosome-translating mRNA. Coupling is modulated by the transcription factors NusG (which is thought to bridge RNAP and the ribosome) and NusA. Here, we report cryo–electron microscopy structures of Escherichia coli transcription-translation complexes (TTCs) containing different-length mRNA spacers between RNAP and the ribosome active-center P site. Structures of TTCs containing short spacers show a state incompatible with NusG bridging and NusA binding (TTC-A, previously termed “expressome”). Structures of TTCs containing longer spacers reveal a new state compatible with NusG bridging and NusA binding (TTC-B) and reveal how NusG bridges and NusA binds. We propose that TTC-B mediates NusG- and NusA-dependent transcription-translation coupling.

scholarly journals Fe-S cofactors in the SARS-CoV-2 RNA-dependent RNA polymerase are potential antiviral targets

Science ◽  
2021 ◽  
pp. eabi5224
Author(s):  
Nunziata Maio ◽  
Bernard A. P. Lafont ◽  
Debangsu Sil ◽  
Yan Li ◽  
J. Martin Bollinger ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Metal Binding ◽  
Rna Dependent Rna Polymerase ◽  
Iron Sulfur Clusters ◽  
Metal Binding Sites ◽  
Iron Sulfur ◽  
Cryo Electron Microscopy ◽  
Metal Cofactors ◽  
Antiviral Targets ◽  
Viral Helicase

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of coronavirus disease 2019 (COVID-19), uses an RNA-dependent RNA polymerase (RdRp) for the replication of its genome and the transcription of its genes. We found that the catalytic subunit of the RdRp, nsp12, ligates two iron-sulfur metal cofactors in sites that were modeled as zinc centers in the available cryo-electron microscopy structures of the RdRp complex. These metal binding sites are essential for replication and for interaction with the viral helicase. Oxidation of the clusters by the stable nitroxide TEMPOL caused their disassembly, potently inhibited the RdRp, and blocked SARS-CoV-2 replication in cell culture. These iron-sulfur clusters thus serve as cofactors for the SARS-CoV-2 RdRp and are targets for therapy of COVID-19.

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