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1.
EuropePMC; 2022.
Preprint in English | EuropePMC | ID: ppcovidwho-337015

ABSTRACT

The SARS-CoV-2 RNA-dependent RNA polymerase coordinates viral RNA synthesis as part of an assembly known as the replication-transcription complex (RTC) 1 . Accordingly, the RTC is a target for clinically approved antiviral nucleoside analogs, including remdesivir 2 . Faithful synthesis of viral RNAs by the RTC requires recognition of the correct nucleotide triphosphate (NTP) for incorporation into the nascent RNA. To be effective inhibitors, antiviral nucleoside analogs must compete with the natural NTPs for incorporation. How the SARS-CoV-2 RTC discriminates between the natural NTPs, and how antiviral nucleoside analogs compete, has not been discerned in detail. Here, we use cryo-electron microscopy to visualize the RTC bound to each of the natural NTPs in states poised for incorporation. Furthermore, we investigate the RTC with the active metabolite of remdesivir, remdesivir triphosphate (RDV-TP), highlighting the structural basis for the selective incorporation of RDV-TP over its natural counterpart ATP 3,4 . Our results elucidate the suite of interactions required for NTP recognition, informing the rational design of antivirals. Our analysis also yields insights into nucleotide recognition by the nsp12 NiRAN, an enigmatic catalytic domain essential for viral propagation 5 . The NiRAN selectively binds GTP, strengthening proposals for the role of this domain in the formation of the 5’ RNA cap 6 .

2.
Nat Struct Mol Biol ; 29(3): 250-260, 2022 03.
Article in English | MEDLINE | ID: covidwho-1735263

ABSTRACT

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 backward on the RNA template (backtracking), prompting questions as to how the RdRp can efficiently synthesize RNA in the presence of nsp13. Here we use cryogenic-electron microscopy and molecular dynamics simulations to analyze the nsp132-RTC, revealing four distinct conformational states of the helicases. The results indicate 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.


Subject(s)
COVID-19 , SARS-CoV-2 , Cryoelectron Microscopy , Humans , RNA Helicases/chemistry , Viral Nonstructural Proteins/chemistry , Virus Replication
3.
EuropePMC; 2020.
Preprint in English | EuropePMC | ID: ppcovidwho-318315

ABSTRACT

Recent advances in single particle cryogenic electron microscopy (cryo-EM) have enabled the structural determination of numerous protein assemblies at high resolution, yielding unprecedented insights into their function. However, despite its extraordinary capabilities, cryo-EM remains time-consuming and resource-intensive. It is therefore beneficial to have a means for rapidly assessing and optimizing the quality of samples prior to lengthy cryo-EM analyses. To do this, we have developed a native mass spectrometry (nMS) platform that provides rapid feedback on sample quality and highly streamlined biochemical screening. Because nMS enables accurate mass analysis of protein complexes, it is well-suited for routine evaluation of the composition, integrity, and homogeneity of samples prior to their plunge-freezing on EM grids. We demonstrate the utility of our nMS-based platform for facilitating cryo-EM studies using structural characterizations of exemplar bacterial transcription complexes as well as the replication-transcription assembly from the SARS-CoV-2 virus that is responsible for the COVID-19 pandemic.Funding: This work is supported by the Pels Foundation to The Rockefeller University, NIH grants P41 GM109824 and P41 GM103314 to BTC, R35 GM118130 to SAD, and R01 GM114450 to EAC. Access to the cryo-EM microscopes and support was through The Rockefeller University Evelyn Gruss Lipper Cryo-EM Resource Center and at The Simons Electron Microscopy Center (SEMC), National Resource for Automated Molecular Microscopy (NRAMM), and National Center for CryoEM Access and Training (NCCAT) at the NYSBC, supported by NIH NIGMS (P41 GM103310), NYSTAR, the Simons Foundation (SF349247), the NIH Common Fund Transformative High Resolution CryoElectron Microscopy program (U24 GM129539) and NY State Assembly Majority. Conflict of Interest: The authors declare there are no competing interests.

4.
Nat Rev Mol Cell Biol ; 23(1): 21-39, 2022 01.
Article in English | MEDLINE | ID: covidwho-1537322

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has killed millions of people and continues to cause massive global upheaval. Coronaviruses are positive-strand RNA viruses with an unusually large genome of ~30 kb. They express an RNA-dependent RNA polymerase and a cohort of other replication enzymes and supporting factors to transcribe and replicate their genomes. The proteins performing these essential processes are prime antiviral drug targets, but drug discovery is hindered by our incomplete understanding of coronavirus RNA synthesis and processing. In infected cells, the RNA-dependent RNA polymerase must coordinate with other viral and host factors to produce both viral mRNAs and new genomes. Recent research aiming to decipher and contextualize the structures, functions and interplay of the subunits of the SARS-CoV-2 replication and transcription complex proteins has burgeoned. In this Review, we discuss recent advancements in our understanding of the molecular basis and complexity of the coronavirus RNA-synthesizing machinery. Specifically, we outline the mechanisms and regulation of RNA translation, replication and transcription. We also discuss the composition of the replication and transcription complexes and their suitability as targets for antiviral therapy.


Subject(s)
Antiviral Agents/pharmacology , Drug Design , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Transcription, Genetic , Virus Replication/physiology , Animals , Humans , RNA, Viral/metabolism , Transcription, Genetic/drug effects , Virus Replication/drug effects
5.
Non-conventional in English | [Unspecified Source], Grey literature | ID: grc-750506

ABSTRACT

SARS-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.

7.
Enzymes ; 49: 1-37, 2021.
Article in English | MEDLINE | ID: covidwho-1370416

ABSTRACT

The ongoing Covid-19 pandemic has spurred research in the biology of the nidovirus severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Much focus has been on the viral RNA synthesis machinery due to its fundamental role in viral propagation. The central and essential enzyme of the RNA synthesis process, the RNA-dependent RNA polymerase (RdRp), functions in conjunction with a coterie of viral-encoded enzymes that mediate crucial nucleic acid transactions. Some of these enzymes share common features with other RNA viruses, while others play roles unique to nidoviruses or CoVs. The RdRps are proven targets for viral pathogens, and many of the other nucleic acid processing enzymes are promising targets. The purpose of this review is to summarize recent advances in our understanding of the mechanisms of RNA synthesis in CoVs. By reflecting on these studies, we hope to emphasize the remaining gaps in our knowledge. The recent onslaught of structural information related to SARS-CoV-2 RNA synthesis, in combination with previous structural, genetic and biochemical studies, have vastly improved our understanding of how CoVs replicate and process their genomic RNA. Structural biology not only provides a blueprint for understanding the function of the enzymes and cofactors in molecular detail, but also provides a basis for drug design and optimization. The concerted efforts of researchers around the world, in combination with the renewed urgency toward understanding this deadly family of viruses, may eventually yield new and improved antivirals that provide relief to the current global devastation.


Subject(s)
RNA, Viral , SARS-CoV-2/genetics , RNA, Viral/biosynthesis , RNA, Viral/genetics , RNA-Dependent RNA Polymerase/genetics
8.
Proc Natl Acad Sci U S A ; 118(19)2021 05 11.
Article in English | MEDLINE | ID: covidwho-1254144

ABSTRACT

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.


Subject(s)
COVID-19/virology , SARS-CoV-2/physiology , Virus Replication/genetics , Adenosine Monophosphate/pharmacology , Antiviral Agents/pharmacology , COVID-19/genetics , COVID-19/metabolism , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Cryoelectron Microscopy/methods , DNA Helicases/metabolism , Genome, Viral , Humans , Molecular Dynamics Simulation , RNA Helicases/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , RNA-Dependent RNA Polymerase/metabolism , RNA-Dependent RNA Polymerase/physiology , SARS-CoV-2/drug effects , SARS-CoV-2/genetics , Viral Nonstructural Proteins/genetics
9.
Proc Natl Acad Sci U S A ; 118(19)2021 05 11.
Article in English | MEDLINE | ID: covidwho-1196910

ABSTRACT

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.


Subject(s)
COVID-19/virology , SARS-CoV-2/physiology , Virus Replication/genetics , Adenosine Monophosphate/pharmacology , Antiviral Agents/pharmacology , COVID-19/genetics , COVID-19/metabolism , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Cryoelectron Microscopy/methods , DNA Helicases/metabolism , Genome, Viral , Humans , Molecular Dynamics Simulation , RNA Helicases/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , RNA-Dependent RNA Polymerase/metabolism , RNA-Dependent RNA Polymerase/physiology , SARS-CoV-2/drug effects , SARS-CoV-2/genetics , Viral Nonstructural Proteins/genetics
10.
Sci Rep ; 10(1): 22375, 2020 12 23.
Article in English | MEDLINE | ID: covidwho-997939

ABSTRACT

The global population is at present suffering from a pandemic of Coronavirus disease 2019 (COVID-19), caused by the novel coronavirus Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The goal of this study was to use artificial intelligence (AI) to predict blueprints for designing universal vaccines against SARS-CoV-2, that contain a sufficiently broad repertoire of T-cell epitopes capable of providing coverage and protection across the global population. To help achieve these aims, we profiled the entire SARS-CoV-2 proteome across the most frequent 100 HLA-A, HLA-B and HLA-DR alleles in the human population, using host-infected cell surface antigen presentation and immunogenicity predictors from the NEC Immune Profiler suite of tools, and generated comprehensive epitope maps. We then used these epitope maps as input for a Monte Carlo simulation designed to identify statistically significant "epitope hotspot" regions in the virus that are most likely to be immunogenic across a broad spectrum of HLA types. We then removed epitope hotspots that shared significant homology with proteins in the human proteome to reduce the chance of inducing off-target autoimmune responses. We also analyzed the antigen presentation and immunogenic landscape of all the nonsynonymous mutations across 3,400 different sequences of the virus, to identify a trend whereby SARS-COV-2 mutations are predicted to have reduced potential to be presented by host-infected cells, and consequently detected by the host immune system. A sequence conservation analysis then removed epitope hotspots that occurred in less-conserved regions of the viral proteome. Finally, we used a database of the HLA haplotypes of approximately 22,000 individuals to develop a "digital twin" type simulation to model how effective different combinations of hotspots would work in a diverse human population; the approach identified an optimal constellation of epitope hotspots that could provide maximum coverage in the global population. By combining the antigen presentation to the infected-host cell surface and immunogenicity predictions of the NEC Immune Profiler with a robust Monte Carlo and digital twin simulation, we have profiled the entire SARS-CoV-2 proteome and identified a subset of epitope hotspots that could be harnessed in a vaccine formulation to provide a broad coverage across the global population.


Subject(s)
COVID-19 Vaccines/immunology , COVID-19/prevention & control , Machine Learning , Pandemics/prevention & control , Proteome , SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/immunology , Algorithms , Alleles , Amino Acid Sequence , COVID-19/virology , Drug Evaluation, Preclinical/methods , Epitopes, T-Lymphocyte/immunology , HLA Antigens/genetics , Haplotypes , Humans , Immunogenicity, Vaccine , Mutation , Proteomics/methods , SARS-CoV-2/genetics , Software
11.
Structure ; 29(2): 186-195.e6, 2021 02 04.
Article in English | MEDLINE | ID: covidwho-939287

ABSTRACT

Recent advances in single-particle cryogenic electron microscopy (cryo-EM) have enabled the structural determination of numerous protein assemblies at high resolution, yielding unprecedented insights into their function. However, despite its extraordinary capabilities, cryo-EM remains time-consuming and resource-intensive. It is therefore beneficial to have a means for rapidly assessing and optimizing the quality of samples prior to lengthy cryo-EM analyses. To do this, we have developed a native mass spectrometry (nMS) platform that provides rapid feedback on sample quality and highly streamlined biochemical screening. Because nMS enables accurate mass analysis of protein complexes, it is well suited to routine evaluation of the composition, integrity, and homogeneity of samples prior to their plunge-freezing on EM grids. We demonstrate the utility of our nMS-based platform for facilitating cryo-EM studies using structural characterizations of exemplar bacterial transcription complexes as well as the replication-transcription assembly from the SARS-CoV-2 virus that is responsible for the COVID-19 pandemic.


Subject(s)
Cryoelectron Microscopy/methods , Mass Spectrometry/methods , Single Molecule Imaging/methods , Escherichia coli , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Methyltransferases/chemistry , Methyltransferases/metabolism , RNA Helicases/chemistry , RNA Helicases/metabolism , SARS-CoV-2/enzymology , SARS-CoV-2/ultrastructure , Transcription Factors/chemistry , Transcription Factors/metabolism , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/metabolism
12.
Cell ; 182(6): 1560-1573.e13, 2020 09 17.
Article in English | MEDLINE | ID: covidwho-710427

ABSTRACT

SARS-CoV-2 is the causative agent of the 2019-2020 pandemic. The SARS-CoV-2 genome is replicated and 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 cryoelectron microscopic structures of the SARS-CoV-2 holo-RdRp with an RNA template product in complex with two molecules of the nsp13 helicase. The Nidovirales 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 therapy development.


Subject(s)
Methyltransferases/chemistry , RNA Helicases/chemistry , RNA-Dependent RNA Polymerase/chemistry , Viral Nonstructural Proteins/chemistry , Virus Replication , Adenosine Diphosphate/chemistry , Adenosine Diphosphate/metabolism , Betacoronavirus/genetics , Betacoronavirus/metabolism , Betacoronavirus/ultrastructure , Binding Sites , Coronavirus RNA-Dependent RNA Polymerase , Cryoelectron Microscopy , Holoenzymes/chemistry , Holoenzymes/metabolism , Magnesium/metabolism , Methyltransferases/metabolism , Protein Binding , RNA Helicases/metabolism , RNA, Viral/chemistry , RNA-Dependent RNA Polymerase/metabolism , SARS-CoV-2 , Viral Nonstructural Proteins/metabolism
13.
bioRxiv ; 2020 Jul 13.
Article in English | MEDLINE | ID: covidwho-663149

ABSTRACT

SARS-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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