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Principal Investigator: Deborah F Kelly
Organization: WAKE FOREST UNIVERSITY
Fiscal Year: 2024
Award: $458,225
Funding agency: National Institute of Allergy and Infectious Diseases
PROJECT SUMMARY/ABSTRACT (DESCRIPTION):
RNA viruses can be devastating human pathogens that impart large medical and economic burdens to society
(e.g., SARS-CoV-2, influenza A virus, Ebola virus, rotavirus, etc.). While these viruses can differ quite
dramatically in their pathogenesis, they share a common replication feature—they must synthesize new RNA
molecules from RNA templates. Because host cell enzymes lack this activity, RNA viruses encode a specialized
RNA-dependent RNA polymerase (RdRp). Viral RdRps are structurally- and functionally-conserved among
diverse RNA viral families, and they directly catalyze all stages of viral transcription and genome replication.
However, these enzymes rarely function alone in the context of infected cells. Instead, the viral RdRps are tightly
regulated by other proteins in multi-subunit transcriptase/replicase complexes so as to maximize the type and
timing of viral RNA synthesis. The overall objective of this application is to gain mechanistic insight into RdRp
regulation for rotavirus—an 11-segmented, double-stranded (ds) RNA virus that causes life-threatening diarrhea
in young children. The rotavirus VP1 RdRp functions only when bound beneath the icosahedral VP2 core shell
layer of intact, or partially intact, particles. Engagement of VP1 by VP2 during early particle assembly activates
the RdRp so that it functions as a replicase, converting packaged, single-stranded positive sense (+) RNA
templates into dsRNA genome segments. Early assembly intermediates then morph into double-layered
particles, wherein the VP2-bound, VP1 RdRp switches to a transcriptase activity, synthesizing +RNAs using
dsRNA templates. Still, major gaps in knowledge remain about the structure of the VP1 RdRp as a replicase
during dsRNA synthesis and its regulation by the VP2 core shell protein. Here, well-established in vitro
biochemical and genetic techniques are combined with state-of-the-art structural approaches to close these gaps
in knowledge and inform a deep understanding of rotavirus RdRp regulation. AIM 1 will elucidate VP2 core shell
determinants critical for VP1 replicase activity, and AIM 2 will determine the first-ever in situ 3D atomic structures
of VP1 as a replicase in both ice (using cryo-EM) and liquid (using a microfluidics system). The work outlined in
this application is significant, as it is expected to reveal detailed structure-function information about the rotavirus
RdRp that could be applied to rational antiviral drug design.
Terms: <0-11 years old><2019 novel corona virus><2019 novel coronavirus><2019-nCoV><3-D><3-D structure><3-Dimensional><3-dimensional structure><3D><3D structure><ARM Domain><Anti-viral Agents><Armadillo Repeat><Armadillo/Beta-Catenin-Like Repeat><Assay><Beta-Catenin Binding Repeat><Beta-Catenin-Binding Domain><Beta-Catenin-Like Repeat><Binding><Bioassay><Biochemical><Biologic Models><Biological Assay><Biological Models><COVID-19 virus><COVID19 virus><Cell Body><Cells><Child><Child Youth><Children (0-21)><CoV-2><CoV2><Complex><Cryo-electron Microscopy><Cryoelectron Microscopy><Data><Defect><Diarrhea><Disease><Disorder><Double Stranded RNA Virus><Double-Stranded RNA><Drug Design><EBOV><EC 2.7.7.48><Ebola virus><Ebola-like Viruses><Economic Burden><Electron Cryomicroscopy><Engineering><Enzyme Gene><Enzymes><Family><Foundations><Gene Transcription><Genetic Technics><Genetic Techniques><Genetic Transcription><Genome><Genomic Segment><Ice><Image><In Situ><In Vitro><Infection><Influenza A><Influenza A virus><Influenza Viruses Type A><Influenzavirus A><Knowledge><Lesion><Life><Liquid substance><Mammalian Cell><Maps><Medical><Microfluidics><Mission><Model System><Molecular Configuration><Molecular Conformation><Molecular Interaction><Molecular Stereochemistry><N-terminal><NH2-terminal><NIH><National Institutes of Health><Non-Polyadenylated RNA><Orthomyxovirus Type A><Pathogenesis><Performance><Plasmids><Proteins><Publishing><RNA><RNA Expression><RNA Gene Products><RNA Replicase><RNA Viruses><RNA chemical synthesis><RNA synthesis><RNA-Dependent RNA Polymerase><RNA-Directed RNA Polymerase><Recombinants><Regulation><Reoviridae><Research><Resolution><Ribonucleic Acid><Rotavirus><SARS corona virus 2><SARS-CO-V2><SARS-COVID-2><SARS-CoV-2><SARS-CoV2><SARS-associated corona virus 2><SARS-associated coronavirus 2><SARS-coronavirus-2><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-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 related corona virus 2><Site><Societies><Structure><System><Techniques><Testing><Transcriptase><Transcription><Type A Influenza><United States National Institutes of Health><Viral><Virion><Virus><Virus Particle><Virus Replication><Virus-like particle><Work><Wuhan coronavirus><anti-viral compound><anti-viral drugs><anti-viral medication><anti-viral therapeutic><anti-virals><conformation><conformational><conformational state><conformationally><conformations><coronavirus disease 2019 virus><coronavirus disease-19 virus><cryo-EM><cryoEM><cryogenic electron microscopy><dsRNA><dsRNA Virus><flexibility><flexible><fluid><genome segment><genomic region><hCoV19><human disease><human pathogen><imaging><in vitro activity><insight><kids><liquid><mutant><nCoV2><particle><prevent><preventing><public health relevance><rational design><reconstruction><replicase><resolutions><reverse genetics><scaffold><scaffolding><structural biology><three dimensional><three dimensional structure><viral RNA><viral multiplication><viral replication><virus RNA><virus multiplication><virus-like nanoparticles><viruslike particle><youngster><µfluidic>