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Principal Investigator: Jamie Jon Arnold
Organization: UNIV OF NORTH CAROLINA CHAPEL HILL
Fiscal Year: 2024
Award: $404,270
Funding agency: National Institute of Allergy and Infectious Diseases
Abstract
The RNA-dependent RNA polymerase (RdRp) drives viral evolution by mediating both genetic drift (mutation)
and genetic/antigenic shift (recombination) of RNA viruses. RdRp speed and fidelity contribute to the rate of
mutation and formation of the mutant swarm, a well-established determinant of viral fitness and pathogenesis.
For more than two decades, we have used the RdRp from poliovirus (PV) as a model system to elucidate
fundamental biochemical and biophysical principles governing speed and fidelity of nucleotide addition. A major
conclusion of this work is that both speed and fidelity are controlled by the dynamics of a conserved array of
amino acid residues in the active site. Importantly, these dynamics can be manipulated genetically or exploited
pharmacologically, thus contributing to the creation of attenuated strains and antiviral agents.
RNA recombination in PV occurs by a template-switching mechanism, a process in which the RdRp initiates
elongation on one template (donor) but completes elongation on a different template (acceptor). PV RdRp is
sufficient to catalyze template switching in vitro. However, the trigger(s) and mechanism of template switching
remain largely unknown. Using a novel cell-based assay for PV RNA recombination, the Evans laboratory
observed a direct correlation between RdRp infidelity and the frequency of RNA recombination. RdRp
misincorporation frequency as a biochemical property governing template switching was not expected but
motivated our foray into the study of RNA recombination. Over the past five years, we have established an
experimental paradigm to elucidate the mechanism of RdRp-catalyzed RNA recombination, discover
biochemical and biophysical properties and structural determinants of the RdRp governing RNA recombination,
and reveal the biological consequences of perturbations to the mechanism and/or efficiency of RNA
recombination.
Here, we will utilize our experimental paradigm to achieve the following: (1) Link structural determinants of
the RdRp to elementary steps and mechanisms of RNA recombination; (2) Investigate the impact of RNA
modifications on elongation and recombination by the RdRp; and (3) Reveal the mechanism and biological
function of forced-copy-choice RNA recombination.
Terms: <Achievement><Achievement Attainment><Active Sites><Amino Acids><Anti-viral Agents><Assay><Attenuated><Base Pairing><Bioassay><Biochemical><Biologic Models><Biological><Biological Assay><Biological Function><Biological Models><Biological Process><Biology><Biophysics><Bypass><Cell Body><Cells><Complex><DNA><DNA Recombination><Defect><Deoxyribonucleic Acid><Development><EBOV><EC 2.7.7.48><EV-68><EV-71><EV-A71><EV-D68><Ebola virus><Ebola-like Viruses><Enterovirus><Enterovirus 68><Enterovirus 71><Enterovirus A71><Enterovirus D68><Evolution><Frequencies><Genetic><Genetic Alteration><Genetic Change><Genetic Differentiation><Genetic Divergence><Genetic Drift><Genetic Recombination><Genetic defect><Genome><Goals><Guanine><Human poliovirus><In Vitro><Individual><Knowledge><Laboratories><Link><Mediating><Model System><Modeling><Modification><Mutation><Non-Polyadenylated RNA><Nucleotides><Polio Virus><Poliovirus><Polymerase><Position><Positioning Attribute><Process><Property><Public Health><RNA><RNA Gene Products><RNA Replicase><RNA Virus Infections><RNA Viruses><RNA viral infection><RNA-Dependent RNA Polymerase><RNA-Directed RNA Polymerase><Recombination><Ribonucleic Acid><SARS Virus><SARS corona virus><SARS coronavirus><SARS-Associated Coronavirus><SARS-CoV><SARS-CoV-1><SARS-Related Coronavirus><Severe Acute Respiratory Coronavirus><Severe Acute Respiratory Syndrome Virus><Severe Acute Respiratory Syndrome corona virus><Severe Acute Respiratory Syndrome coronavirus><Speed><Testing><Viral><Viral Gene Products><Viral Gene Proteins><Viral Pathogenesis><Viral Proteins><Virion><Virus Particle><Work><aminoacid><anti-viral compound><anti-viral drugs><anti-viral medication><anti-viral therapeutic><anti-virals><attenuate><attenuates><base><bases><biologic><biophysical analysis><biophysical characteristics><biophysical characterization><biophysical foundation><biophysical measurement><biophysical parameters><biophysical principles><biophysical properties><biophysical sciences><biophysical studies><develop a vaccine><develop vaccines><development of a vaccine><developmental><epitranscriptome><gene manipulation><genetic manipulation><genetically manipulate><genetically perturb><genome mutation><in vitro Assay><in vivo><inhibitor><insight><mutant><novel><pharmacologic><poliomyelitis virus><posttranscriptional><prevent><preventing><reconstitute><reconstitution><severe acute respiratory syndrome-CoV><structural determinants><structural factors><sugar><vaccine development><viral RNA><viral fitness><virus RNA><virus pathogenesis><virus protein>