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Investigation into the proteolytic processing and localization of the replicase polyprotein of Grapevine rupestris stem pitting-associated virus

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Title: Investigation into the proteolytic processing and localization of the replicase polyprotein of Grapevine rupestris stem pitting-associated virus
Author: Udaskin, Molly Lizabeth
Department: Department of Molecular and Cellular Biology
Program: Molecular and Cellular Biology
Advisor: Meng, Baozhong
Abstract: The replicase polyprotein of Grapevine rupestris stem pitting-associated virus (genus Foveavirus, family Betaflexiviridae), which infects grapevines, contains domains required for RNA replication. To determine the cleavage pattern of the polyprotein, we designed a unique strategy to detect and compare cleavage products wherein a full-length construct and two N-terminal truncations, beginning directly before the alkylationB or the tandem Ovarian tumor-like/Papain-like protease (PPro) domains, were constructed. Constructs were designed such that their translation products were N- and C-terminally tagged with FLAG and HA epitopes, respectively. After agroinfiltration into Nicotiana benthamiana, protein expression was examined via immunoprecipitation and Western blot. Results suggest that there are at least two cleavages occurring to the replicase polyprotein (localized after the methyltransferase domain and between the PPro and helicase domains). Further work is still needed to determine the exact cleavage location. It was determined through confocal microscopy that the replicase does not co-localize with the mitochondrion.
URI: http://hdl.handle.net/10214/9462
Date: 2016-01
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