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Principal Investigator: ELIZABETH A CAMPBELL
Organization: ROCKEFELLER UNIVERSITY
Fiscal Year: 2024
Award: $620,543
Funding agency: National Institute of Allergy and Infectious Diseases
Project Summary
COVID-19, caused by the coronavirus SARS-CoV-2, continues to devastate the world. In less than a year,
there have been more than 20 million cases with over 700,000 deaths. The viral RNA-dependent RNA
polymerase (RdRp) is the central enzyme responsible for transcription and replication of the viral RNA
genome. This enzyme is also a target for the current antiviral, remdesivir, used to ameliorate the severity and
duration of this disease. The virus also encodes several nucleic acid processing enzymes, in addition to the
RdRp, including a helicase, an endonuclease, an exonuclease, and methyltransferases. However, it is
unknown how these enzymes coordinate to transcribe and replicate the viral genome. This proposal builds
upon preliminary data of the structure of the helicase, nsp13, in complex with the RdRp and a primed substrate
RNA (nsp13-replication/transcription complex or nsp13-RTC). The aims here include completing the structural
analysis of this complex by utilizing additional data collected. The result of this aim will provide higher
resolution (better than 2.7 Å in some parts the RdRp), providing a rich basis for the development of antiviral
inhibitors. Also, having this structure in hand allows for the collaboration with expert developers of
antimicrobials, also part of the aims, including the investigation of the structural details of the pre-incorporation
state of remdesivir and antivirals produced by human microbiome.
The models resulting from the structure of nsp13-RTC serve as foundations to test how the helicase and
exonuclease function together with the RdRp. Specifically, real-time fluorescence assays, single-molecule
fluorescence resonance energy transfer (FRET), and multi-color fluorescence microscopy will be used to probe
the role of the helicase and the exonuclease in unwinding substrate RNA, backtracking, and proofreading.
Another aim applies the pipeline used to characterize the nsp13-RTC assembly, which yielded a high-
resolution structure of the complex, to other RTC assemblies. Specifically, native electrophoretic mobility
assays will be used as a starting point to probe larger assemblies of the RTC. Native mass-spectrometry will
then be used to determine the composition and stoichiometry of the complexes. Finally, cryo-EM will be
applied to solve the structures of these macromolecular machines. The resulting structures will provide a
starting point to elucidate the coordinated functions of these enzymes, provide insight into their mechanisms,
and establish novel targets for therapeutics.
In summary, this proposal aims to understand at the molecular and structural level how the SARS-CoV-2
nucleic acid processing enzymes coordinate to replicate and transcribe the viral genome, and to provide
structure-guided targets for drug discovery, with the ultimate goal of providing relief for the COVID-19
pandemic.
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2.7.7.48><Effectiveness><Electron Cryomicroscopy><Electrophoretic Mobility Shift Assay><Engineering / Architecture><Enzyme Gene><Enzymes><Exonuclease><Fluorescence><Fluorescence Light Microscopy><Fluorescence Microscopy><Foundations><GS-5734><Gel><Gene Expression><Gene Transcription><Genetic Transcription><Genome><Goals><Hand><Higher Order Chromatin Folding><Higher Order Chromatin Structure><Higher Order Structure><Holoenzymes><Human Microbiome><Hydrogen Oxide><In Vitro><Infection><Investigation><Macromolecular Structure><Manuscripts><Maps><Mass Photometry/Spectrum Analysis><Mass Spectrometry><Mass Spectroscopy><Mass Spectrum><Mass Spectrum Analyses><Mass Spectrum Analysis><Methyltransferase><Mobility Shift Assay><Modeling><Molecular><Molecular Configuration><Molecular Conformation><Molecular Interaction><Molecular Stereochemistry><Molecular Structure><N-terminal><NH2-terminal><Nucleic Acids><Nucleotide Mapping><Polymerase><Pyrophosphates><RNA Expression><RNA Polymerase Inhibitor><RNA Replicase><RNA-Dependent RNA Polymerase><RNA-Directed RNA Polymerase><Reaction><Replication Process><Replication-Associated Process><Reporting><Resolution><Rice><Role><SARS Virus><SARS corona virus><SARS corona virus 2><SARS coronavirus><SARS-Associated Coronavirus><SARS-CO-V2><SARS-COVID-2><SARS-CoV><SARS-CoV-1><SARS-CoV-2><SARS-CoV-2 epidemic><SARS-CoV-2 genome><SARS-CoV-2 global health crisis><SARS-CoV-2 global pandemic><SARS-CoV-2 inhibitor><SARS-CoV-2 pandemic><SARS-CoV2><SARS-CoV2 genome><SARS-Related Coronavirus><SARS-associated corona virus 2><SARS-associated coronavirus 2><SARS-coronavirus-2><SARS-coronavirus-2 epidemic><SARS-coronavirus-2 pandemic><SARS-related corona virus 2><SARS-related coronavirus 2><SARSCoV2><Scientist><Severe Acute Respiratory Coronavirus><Severe Acute Respiratory Coronavirus 2><Severe Acute Respiratory Distress Syndrome CoV 2><Severe Acute Respiratory Distress Syndrome Corona Virus 2><Severe Acute Respiratory Distress Syndrome Coronavirus 2><Severe Acute Respiratory Syndrome CoV 2><Severe Acute Respiratory Syndrome CoV 2 epidemic><Severe Acute Respiratory Syndrome CoV 2 pandemic><Severe Acute Respiratory Syndrome Virus><Severe Acute Respiratory Syndrome corona virus><Severe Acute Respiratory Syndrome coronavirus><Severe Acute Respiratory Syndrome-associated coronavirus 2><Severe Acute Respiratory Syndrome-related coronavirus 2><Severe acute respiratory syndrome associated corona virus 2><Severe acute respiratory syndrome coronavirus 2><Severe acute respiratory syndrome coronavirus 2 epidemic><Severe acute respiratory syndrome coronavirus 2 inhibitor><Severe acute respiratory syndrome coronavirus 2 pandemic><Severe acute respiratory syndrome related corona virus 2><Severities><Site><Structure><Systematics><Testing><Time><Transcription><Transcription Process><Veklury><Viral><Viral Activity><Viral Function><Viral Genome><Viral Physiology><Virus><Virus Inhibitors><Virus Replication><Water><Wuhan coronavirus><anti-microbial><anti-viral compound><anti-viral development><anti-viral drug development><anti-viral drugs><anti-viral medication><anti-viral therapeutic><anti-viral therapeutic development><anti-viral therapy development><anti-virals><antimicrobial><antiviral development><antiviral drug development><antiviral therapeutic development><antiviral therapy development><block SARS-CoV-2><block severe acute respiratory syndrome coronavirus 2><cofactor><conformation><conformational><conformational state><conformationally><conformations><coronavirus disease 2019><coronavirus disease 2019 crisis><coronavirus disease 2019 epidemic><coronavirus disease 2019 genome><coronavirus disease 2019 global health crisis><coronavirus disease 2019 global pandemic><coronavirus disease 2019 health crisis><coronavirus disease 2019 pandemic><coronavirus disease 2019 public health crisis><coronavirus disease 2019 virus><coronavirus disease 2019 virus genome><coronavirus disease crisis><coronavirus disease epidemic><coronavirus disease pandemic><coronavirus disease-19><coronavirus disease-19 global pandemic><coronavirus disease-19 pandemic><coronavirus disease-19 virus><coronavirus infectious disease-19><cryo-EM><cryoEM><cryogenic electron microscopy><developing anti-viral agent><developing anti-viral drug><developing anti-viral therapeutic><developing anti-viral therapy><developing antiviral agent><developing antiviral drug><developing antiviral therapeutic><developing antiviral therapy><developmental><disease duration><disease length><drug discovery><endonuclease><experiment><experimental research><experimental study><experiments><gel shift assay><hCoV19><hands><helicase><human-associated microbiome><illness length><improved><in vivo><inhibit SARS-CoV-2><inhibit severe acute respiratory syndrome coronavirus 2><inhibitor><insight><large data sets><large datasets><methylase><nCoV2><new drug target><new drug treatments><new druggable target><new drugs><new pharmacological therapeutic><new pharmacotherapy target><new therapeutic target><new therapeutics><new therapy><new therapy target><next generation therapeutics><novel><novel drug target><novel drug treatments><novel druggable target><novel drugs><novel pharmaco-therapeutic><novel pharmacological therapeutic><novel pharmacotherapy target><novel therapeutic target><novel therapeutics><novel therapy><novel therapy target><nucleotide analog><pathogen><protein protein interaction><remdesivir><resolutions><severe acute respiratory syndrome coronavirus 2 genome><severe acute respiratory syndrome coronavirus 2 global health crisis><severe acute respiratory syndrome coronavirus 2 global pandemic><severe acute respiratory syndrome-CoV><single molecule><single-molecule FRET><single-molecule fluorescence resonance energy transfer><smFRET><social role><stoichiometry><transmethylase><viral inhibitor><viral multiplication><viral replication><virus genome><virus multiplication>