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Principal Investigator: BARRY M WILLARDSON
Organization: BRIGHAM YOUNG UNIVERSITY
Fiscal Year: 2024
Award: $447,548
Funding agency: National Institute of General Medical Sciences
PROJECT SUMMARY
The COVID-19 pandemic created the greatest infectious threat to global health in 100 years,
and monumental efforts have been made by the scientific community to combat the SARS-CoV-
2 virus. This proposal seeks to extend this effort by investigating a mechanism by which SARS-
CoV-2 hijacks the host cell chaperone system to replicate itself. We have evidence that the
SARS-CoV-2 RNA polymerase (RdRp) co-opts the cytosolic chaperonin containing TCP-1
(CCT, also called TRiC) to assemble the active polymerase complex. CCT is a large (1 MDa)
protein-folding machine that plays a major role in the cellular chaperone network responsible for
maintaining the proteome in good working condition. It uses ATP hydrolysis-driven
conformational changes to assist cytosolic proteins with multiple domains, complex folding
trajectories, or obligate binding partners to achieve their native state and assemble into
complexes. In addition to folding cellular proteins, CCT has been shown to bind several viral
proteins and contribute to viral replication of HIV, hepatitis C, influenza A, rabies, Zika and
reovirus. These observations show that CCT is a common host chaperone used by diverse
viruses to fold viral proteins, assemble viral complexes, and support viral replication. Based on
these findings, we initiated an investigation of the role of CCT in SARS-CoV-2 replication. Here,
we present robust preliminary evidence indicating that the SARS-CoV-2 non-structural protein
12 (Nsp12), the catalytic subunit of the RNA polymerase, is folded by CCT and that CCT
contributes to RdRp complex formation and SARS-CoV-2 replication. In Aim 1, we propose to
thoroughly test this hypothesis using multiple experimental approaches. In Aim 2, we propose to
determine high-resolution structures of the complex between Nsp12 and CCT. We have isolated
an Nsp12 folding intermediate bound to CCT and have determined preliminary structures of the
complex by cryogenic electron microscopy (cryo-EM). Further cryo-EM analysis will yield a high-
resolution structure of the Nsp12-CCT complex, which will be invaluable in guiding the design of
therapeutics to block Nsp12 folding by CCT, inhibit formation of the RdRp complex, and disrupt
viral replication.
Terms: <2019 novel corona virus><2019 novel coronavirus><2019-nCoV><AIDS Virus><ATP Hydrolysis><Acquired Immune Deficiency Syndrome Virus><Acquired Immunodeficiency Syndrome Virus><Anti-viral Agents><Binding><Biochemical><COVID crisis><COVID epidemic><COVID pandemic><COVID-19 crisis><COVID-19 epidemic><COVID-19 era><COVID-19 global health crisis><COVID-19 global pandemic><COVID-19 health crisis><COVID-19 pandemic><COVID-19 period><COVID-19 public health crisis><COVID-19 therapy><COVID-19 treatment><COVID-19 virus><COVID-19 years><COVID19 virus><Catalytic Core><Catalytic Domain><Catalytic Region><Catalytic Site><Catalytic Subunit><Cell Body><Cell Communication><Cell Function><Cell Interaction><Cell Physiology><Cell Process><Cell-to-Cell Interaction><Cells><Cellular Function><Cellular Physiology><Cellular Process><Chaperone><Chaperonin Family><CoV-2><CoV2><Communities><Complex><Coronaviridae><Coronavirus><Cryo-electron Microscopy><Cryoelectron Microscopy><DNA-Dependent RNA Polymerases><DNA-Directed RNA Polymerase><Diagnostic><Electron Cryomicroscopy><FK506 Binding Protein 12-Rapamycin Associated Protein 1><FKBP12 Rapamycin Complex Associated Protein 1><FRAP1><FRAP1 gene><FRAP2><Feasibility Studies><Future><G-Proteins><GTP-Binding Proteins><GTP-Regulatory Proteins><Gene Transcription><Genetic Transcription><Guanine Nucleotide Coupling Protein><Guanine Nucleotide Regulatory Proteins><HCV infection><HIV><Hepatitis C><Hepatitis C virus infection><Hepatitis, Viral, Non-A, Non-B, Parenterally-Transmitted><Hepatitus C><Human Immunodeficiency Viruses><Influenza A><Influenza A virus><Influenza Viruses Type A><Influenzavirus A><Investigation><Knowledge><LAV-HTLV-III><Lymphadenopathy-Associated Virus><Mechanistic Target of Rapamycin><Mediating><Molecular><Molecular Chaperones><Molecular Configuration><Molecular Conformation><Molecular Interaction><Molecular Stereochemistry><Non-Polyadenylated RNA><Non-structural Protein><Nonstructural Protein><Nucleotides><Orthomyxovirus Type A><Play><Polymerase><Position><Positioning Attribute><Process><Process Measure><Proteins><Proteome><RAFT1><RNA><RNA Expression><RNA Gene Products><RNA Polymerases><Rabies><Reovirus><Resolution><Respiratory Enteric Orphan Viruses><Ribonucleic Acid><Role><SARS corona virus 2><SARS-CO-V2><SARS-COVID-2><SARS-CoV-2><SARS-CoV-2 epidemic><SARS-CoV-2 global health crisis><SARS-CoV-2 global pandemic><SARS-CoV-2 pandemic><SARS-CoV-2 therapy><SARS-CoV-2 treatment><SARS-CoV2><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><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-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 pandemic><Severe acute respiratory syndrome related corona virus 2><Structure><Subcellular Process><System><Techniques><Testing><Therapeutic><Transcription><Type A Influenza><Variant><Variation><Viral><Viral Diseases><Viral Gene Products><Viral Gene Proteins><Viral Proteins><Virus><Virus Assembly><Virus Diseases><Virus Replication><Virus-HIV><Work><Wuhan coronavirus><ZIKA><anti-viral compound><anti-viral drugs><anti-viral medication><anti-viral therapeutic><anti-virals><c-cpn gene product><chaperonin><chaperonin CCT><combat><conformation><conformational><conformational state><conformationally><conformations><corona virus><coronavirus disease 2019 crisis><coronavirus disease 2019 epidemic><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 therapy><coronavirus disease 2019 treatment><coronavirus disease 2019 virus><coronavirus disease crisis><coronavirus disease epidemic><coronavirus disease pandemic><coronavirus disease-19 global pandemic><coronavirus disease-19 pandemic><coronavirus disease-19 virus><cryo-EM><cryoEM><cryogenic electron microscopy><cytosolic chaperonin><design><designing><experience><experiment><experimental research><experimental study><experiments><global health><hCoV19><hep C><hepatitis non A non B><infection by hepatitis c virus><inhibitor><lyssa><mTOR><mammalian target of rapamycin><nCoV2><new drug treatments><new drugs><new pharmacological therapeutic><new therapeutics><new therapy><next generation therapeutics><non A, non B hepatitis><non-A, non-B hepatitis><novel drug treatments><novel drugs><novel pharmaco-therapeutic><novel pharmacological therapeutic><novel therapeutics><novel therapy><protein complex><protein folding><resolutions><scaffold><scaffolding><severe acute respiratory syndrome coronavirus 2 global health crisis><severe acute respiratory syndrome coronavirus 2 global pandemic><severe acute respiratory syndrome coronavirus 2 therapy><severe acute respiratory syndrome coronavirus 2 treatment><small molecular inhibitor><small molecule><small molecule inhibitor><social role><treat COVID-19><treat SARS-CoV-2><treat coronavirus disease 2019><treat severe acute respiratory syndrome coronavirus 2><viral RNA><viral assembly><viral infection><viral multiplication><viral replication><virus RNA><virus infection><virus multiplication><virus protein><virus-induced disease>