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Characterization of Particle-Associated Rotavirus Polymerase Activities

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title
Characterization of Particle-Associated Rotavirus Polymerase Activities
author
Anderson, Mackenzie
abstract
Viruses are obligate intracellular parasites that impart devastating medical, agricultural, and economic impacts. The successful replication of viruses with RNA genomes is dependent on the synthesis of new RNA molecules. RNA synthesis by rotaviruses and other segmented, double-stranded RNA (dsRNA) viruses in the Reovirales order occurs within the confines of proteinaceous particles. Specifically, the rotavirus polymerase (VP1) requires the core shell protein (VP2) for both the transcription of positive-sense, single-stranded RNAs (+ssRNAs) as well as replication of the viral dsRNA genome. However, a mechanistic understanding into how VP2 regulates VP1 activities and contributes to formation of infectious rotavirus particles remains limited. A major goal of this dissertation research was to identify VP2 residues critical for supporting VP1-mediated dsRNA synthesis. First, recombinant VP2 proteins were engineered to contain alanine amino acid changes at structurally determined polymerase contact sites. The proteins were then assayed for the capacity to support VP1-mediated dsRNA synthesis using a robust in vitro assay. Several VP2 mutants supported significantly increased or decreased VP1 activity in vitro, suggesting they are regulatory residues of the core shell. During these experiments, it was discovered that VP1 possessed a particle-associated terminal transferase activity, which is seen in some other viral polymerases. VP1 was shown to transfer a uridine monophosphate (UMP) onto viral +ssRNA templates in reactions that contain VP2 virus-like particles. Furthermore, this dissertation also characterized a mutant rotavirus with a temperature sensitive lesion in VP2 (tsVP2). While this virus has a >2-log reduction in viral titers at 39ºC versus 31ºC, in vitro assays with tsVP2 demonstrated no differences in either transcription (+ssRNA synthesis) or genome replication (dsRNA synthesis) at either temperature. This observation suggests tsVP2 may have an early assembly defect at 39ºC, which only indirectly impacts viral RNA synthesis. Overall, results from this dissertation research further the mechanistic understanding of rotavirus intra-particle RNA synthesis. Such knowledge may inform rational drug design to prevent rotavirus disease.
contributor
Esstman, Sarah M (advisor)
Dos Santos, Patricia C (committee member)
Ornelles, David A (committee member)
Johnson, Erik C (committee member)
Muday, Gloria K (committee member)
date
2023-09-08T08:35:20Z (accessioned)
2023-09-08T08:35:20Z (available)
2023 (issued)
degree
Biology (discipline)
identifier
http://hdl.handle.net/10339/102600 (uri)
language
en (iso)
publisher
Wake Forest University
type
Dissertation

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