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Principal Investigator: Gaya K. Amarasinghe
Organization: WASHINGTON UNIVERSITY
Fiscal Year: 2019
Award: $2,563,154
Funding agency: National Institute of Allergy and Infectious Diseases
Abstract: Project Overview for Structural and Functional Studies of Ebola Virus RNA synthesis
Ebolaviruses (EBOVs) are non-segmented negative-strand RNA viruses (NNSVs) and Category A Priority
biodefense pathogens that cause frequent lethal hemorrhagic fever, including the devastating and ongoing
outbreak in West Africa. Approved anti-EBOV therapeutics are lacking. EBOV replicates with high efficiency in
vivo while effectively evading host innate antiviral immunity. The unrestrained replication and associated
inflammation leads to death. A complete understanding of EBOV pathogenesis therefore requires
understanding how the viral RNA dependent RNA polymerase (RDRP) complex interacts with host factors.
The overarching goal of this program project proposal is to understand the mechanisms of RDRP function by
defining the interactions among its components and with the host proteome and by defining signals that
regulate its function. Toward this goal Project 1 will identify cis- and trans-acting factors that impact EBOV
RDRP activity. Project 2 will define a structural basis for the EBOV RDRP complex, identify regulatory
mechanisms, and determine species and strain specificities, and Project 3 will use genome-wide RNAi and
proteomic strategies to identify host factors that modulate RDRP function. Project 1 provides functional context
into which a subset of Project 3 “hits” can be interpreted, while Project 3 will likely identify host factors relevant
to the findings of Project 1. Project 2 will provide a structural framework that will be used to integrate findings
from both Projects 1 and 3. Together, these studies will provide mechanistic insights into how protein-protein
and protein-vRNA interactions control EBOV RDRP function. Research projects are supported by an
Administrative Core, a Protein Production and Protein Interaction Core, and critically, a Biosafety Level 4
(BSL4) Core. Core B will generate unique reagents, including monoclonal and synthetic antibodies. Core C
(BSL4) will provide unique capabilities to obtain materials from and perform tests using wild type and mutant
EBOVs as well as pathogenesis evaluation. The work will be performed by highly productive and collaborative
investigators with expertise in every aspect of the proposed studies, including biochemistry, EBOV
pathogenesis, high throughput screening and data analysis, immunology, proteomics, structural biology, and
virology. These studies will provide unprecedented insights into the components of the EBOV RDRP and in its
interaction with the host as well as species and strain differences that affect the RDRP complex. We also
expect to identify specific viral and host factor interactions as novel therapeutic targets.
Terms: <Address><Affect><Africa><Assay><Attenuated><BSL-4 facility><BSL4 facility><Bioassay><Biochemistry><Biologic Assays><Biological Assay><Biological Chemistry><Biological Testing><Biology><Case Study><Categories><Cell Communication and Signaling><Cell Signaling><Cessation of life><Clinical Treatment Moab><Collaborations><Complex><Cryo-electron Microscopy><Cryoelectron Microscopy><DNA-Dependent RNA Polymerases><DNA-Directed RNA Polymerase><Data Analyses><Data Analysis><Death><Development><Disease><Disease Outbreaks><Disorder><EBOV><EC 2.7.7.48><Ebola><Ebola virus><Electron Cryomicroscopy><Elements><Ensure><Epidemic><Europe><Evaluation><Event><Filoviridae><Filovirus><General Viruses><Goals><Health><High Throughput Assay><History><Host Factor><Host Factor Protein><Human><Human Resources><Immunology><In Vitro><Inflammation><Infrastructure><Integration Host Factors><Intracellular Communication and Signaling><Investigators><Knowledge><Light><Link><Manpower><Manuscripts><Maps><Modern Man><Molecular><Monoclonal Antibodies><Non-Polyadenylated RNA><Nucleoproteins><Outbreaks><P01 Mechanism><P01 Program><Pathogenesis><Photoradiation><Post-Transcriptional Gene Silencing><Posttranscriptional Gene Silencing><Production><Program Project Grant><Program Research Project Grants><Proteins><Proteome><Proteomics><Quelling><R-Series Research Projects><R01 Mechanism><R01 Program><RNA><RNA Editing><RNA Gene Products><RNA Interference><RNA Polymerases><RNA Replicase><RNA Silencing><RNA Viruses><RNA analysis><RNA chemical synthesis><RNA synthesis><RNA, Messenger, Editing><RNA-Dependent RNA Polymerase><RNA-Directed RNA Polymerase><RNAi><Reagent><Recording of previous events><Regulation><Reporting><Research><Research Grants><Research Personnel><Research Program Projects><Research Project Grants><Research Projects><Research Resources><Researchers><Resolution><Resources><Ribonucleic Acid><Sequence-Specific Posttranscriptional Gene Silencing><Signal Transduction><Signal Transduction Systems><Signaling><Single Crystal Diffraction><Specificity><Structure><Structure-Activity Relationship><Technology><Testing><Therapeutic><Time><Training><Trans-Acting Factors><Trans-Activators><Transactivators><Treatment Efficacy><Variant><Variation><Viral><Viral Gene Products><Viral Gene Proteins><Viral Genome><Viral Hemorrhagic Fevers><Viral Proteins><Virus><Virus Replication><Work><X Ray Crystallographies><X-Ray Crystallography><X-Ray Diffraction Crystallography><X-Ray/Neutron Crystallography><Xray Crystallography><anti-viral immunity><antiviral immunity><biodefense><biological signal transduction><biophysical analysis><biophysical studies><biosafety level 4 facility><case report><chemical structure function><cryo-EM><cryoEM><data interpretation><developmental><ebolavirus><genome scale><genome-wide><genomewide><global health><health economics><hemorrhagic fever><high throughput screening><in vivo><in vivo Model><innovate><innovation><innovative><insight><intervention efficacy><mAbs><multidisciplinary><mutant><new drug target><new druggable target><new pharmacotherapy target><new therapeutic target><new therapy target><novel><novel drug target><novel druggable target><novel pharmacotherapy target><novel therapeutic target><novel therapy target><pathogen><pathogenic virus><personnel><programs><side effect><structural biology><structure function relationship><synthetic antibodies><therapeutic agent development><therapeutic development><therapeutic efficacy><therapeutically effective><therapy efficacy><viral RNA><viral multiplication><viral pathogen><viral replication><virology><virus RNA><virus genome><virus multiplication><virus protein>