Document text
Principal Investigator: Robert N Kirchdoerfer
Organization: UNIVERSITY OF WISCONSIN-MADISON
Fiscal Year: 2021
Award: $92,225
Funding agency: National Institute of Allergy and Infectious Diseases
SUMMARY
Coronaviruses are sporadically emerging viruses responsible for SARS and MERS disease
outbreaks. There are currently no direct treatments for these viruses, nor is there a vaccine which
induces broad protection from infection. However, several stages in the virus life cycle are promising
targets for therapeutic intervention. Cell entry is mediated by the large glycoprotein spike, which binds
to host receptors and mediates fusion of the viral and host membranes. The ability of coronaviruses to
adapt to new species or escape from the immune system is attributed to the viral spike protein. Once
inside the cell, the viral RNA synthesis complex is assembled from 16 non-structural proteins (NSP)
which transcribe, edit and modify viral RNAs and remodel ER membranes to create RNA replication
factories. Expression of the viral structural proteins involves the RNA synthesis complex carrying out
discontinuous strand synthesis to produce a nested set of viral mRNAs with truncations of the 5' open
reading frames. Discontinuous strand synthesis is essential for the production of new virions and
understanding its mechanisms will shed light on related viral processes such as viral recombination to
generate spike variants with altered serotypes or host tropisms.
During the K99 phase, I will obtain training in cryo-electron microscopy to complement my
expertise in X-ray crystallography. I will use cryo-electron microscopy to examine the distinct
conformations of the coronavirus spike protein as it binds host receptors and is primed for the fusion
process by host proteases as I transition to the R00 phase of the award. These studies build on the
recent structure determination of the HKU1-CoV spike protein from Dr. Andrew Ward's laboratory to
which I contributed. Not only does this spike structure demonstrate the feasibility of the proposed
experiments, but also provides a basis for new hypotheses of spike protein function.
Also during the K99 phase, I will utilize the expertise of Dr. Erica Saphire's laboratory to
develop RNA helicase assays to assess the function of the viral NSP13 helicase. I will use these
assays to provide mechanistic, biochemical evidence to identify RNA templates upon which the
NSP13 helicase stalls and may lead to induction of the RNA synthesis complex to carryout
discontinuous strand synthesis. During the R00 phase, I will complement these studies with
biochemistry, X-ray crystallography and cryo-electron microscopy to identify molecular mechanisms
by which NSP13 recognizes RNA substrates and communicates with the RNA synthesis complex.
These proposed studies will illuminate novel targets for antiviral therapy.
Terms: <Address><Antiviral Agents><Antiviral Drugs><Antiviral Therapy><Antivirals><Assay><Aves><Avian><Award><Binding><Binding Proteins><Bioassay><Biochemical><Biochemistry><Biologic Assays><Biological Assay><Biological Chemistry><Birds><Cell Body><Cells><CoV S protein><CoV emergence><CoV glycoprotein S><CoV spike glycoprotein><CoV spike protein><Complement><Complement Proteins><Complex><Coronaviridae><Coronavirus><Coronavirus glycoprotein S><Coronavirus spike protein><Cryo-electron Microscopy><Cryoelectron Microscopy><Crystallographies><Crystallography><DNA Helicases><DNA Recombination><DNA Unwinding Proteins><DNA unwinding enzyme><Data><Development><Disease Outbreaks><Dissociation><Electron Cryomicroscopy><Elements><Epidemic><Esteroproteases><Event><Gene Transcription><Genetic Recombination><Genetic Transcription><Genome><Glycoproteins><Goals><Health><Homologous Sequences><Human><Immune system><In Vitro><Infection><Intervention><Intervention Strategies><Investigators><Laboratories><Lead><Learning><Life Cycle><Life Cycle Stages><Ligand Binding Protein><Ligand Binding Protein Gene><Light><MERS><MERS corona virus><MERS coronavirus><MERS coronavirus disease><MERS virus><MERS-CoV><MERS-CoV disease><Mediating><Membrane><Messenger RNA><Middle East Respiratory Syndrome><Middle East Respiratory Syndrome CoV disease><Middle East Respiratory Syndrome Corona Virus><Middle East Respiratory Syndrome Coronavirus><Middle East Respiratory Syndrome Virus><Middle East Respiratory Syndrome coronavirus disease><Middle East Respiratory Syndrome-CoV><Middle Eastern Respiratory Syndrome><Middle Eastern Respiratory Syndrome CoV disease><Middle Eastern Respiratory Syndrome Corona virus><Middle Eastern Respiratory Syndrome Coronavirus><Middle Eastern Respiratory Syndrome Virus><Middle Eastern Respiratory Syndrome coronavirus disease><Middle Eastern Respiratory Syndrome-CoV><Modeling><Modern Man><Molecular><Molecular Configuration><Molecular Conformation><Molecular Interaction><Molecular Stereochemistry><Mutate><Nature><Non-Polyadenylated RNA><Non-structural Protein><Nonstructural Protein><ORFs><Open Reading Frames><Outbreaks><Pb element><Peptidases><Peptide Hydrolases><Phase><Photoradiation><Polymerase><Position><Positioning Attribute><Process><Production><Protease Gene><Proteases><Protein Binding><Protein Coding Region><Proteinases><Proteins><Proteolytic Enzymes><RNA><RNA Expression><RNA Gene Products><RNA Helicase><RNA Synthesis Induction><RNA chemical synthesis><RNA replication><RNA synthesis><Receptor Protein><Recombination><Replication Error><Research Personnel><Researchers><Ribonucleic Acid><SARS><SARS Virus><SARS corona virus><SARS coronavirus><SARS coronavirus disease><SARS-Associated Coronavirus><SARS-CoV><SARS-CoV disease><SARS-Related Coronavirus><Sequence Homologs><Serotyping><Severe Acute Respiratory Syndrome><Severe Acute Respiratory Syndrome CoV disease><Severe Acute Respiratory Syndrome Virus><Severe Acute Respiratory Syndrome corona virus><Severe Acute Respiratory Syndrome coronavirus><Severe Acute Respiratory Syndrome coronavirus disease><Single Crystal Diffraction><Site><Specificity><Structure><Surface><TRS><Therapeutic Intervention><Time><Training><Transcription><Transcription Process><Traumeel S><Tropism><Vaccine Design><Vaccines><Variant><Variation><Viral><Viral Activity><Viral Function><Viral Physiology><Viral Structural Proteins><Virion><Virus><Virus Particle><X Ray Crystallographies><X-Ray Crystallography><X-Ray Diffraction Crystallography><X-Ray/Neutron Crystallography><Xray Crystallography><allergic/immunologic body system><allergic/immunologic organ system><anti-viral agents><anti-viral drugs><anti-viral therapy><anti-virals><bound protein><conformation><conformational state><corona virus><corona virus emergence><coronavirus S protein><coronavirus emergence><coronavirus spike glycoprotein><coronavirus vaccine><cryo-EM><cryoEM><developmental><emergent CoV><emergent corona virus><emergent coronavirus><emerging CoV><emerging corona virus><emerging coronavirus><experiment><experimental research><experimental study><heavy metal Pb><heavy metal lead><helicase><intervention therapy><interventional strategy><life course><mRNA><membrane structure><nCoV><new CoV><new corona virus><new coronavirus><novel><novel CoV><novel corona virus><novel coronavirus><programs><protein function><receptor><receptor binding><receptor bound><severe acute respiratory syndrome-CoV><skills><structural biology><targeted drug therapy><targeted drug treatments><targeted therapeutic><targeted therapeutic agents><targeted therapy><targeted treatment><vaccine against coronavirus><viral RNA><viral infectious disease treatment><virus RNA><ward>