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dc.contributor.supervisorMcKenna, Sean (Chemistry)en_US
dc.contributor.authorSoumya R., Deo
dc.date.accessioned2015-09-09T12:31:55Z
dc.date.available2015-09-09T12:31:55Z
dc.date.issued2014-03-20en_US
dc.date.issued2015-04-11en_US
dc.identifier.citationDOI: 10.1371/journal.pone.0092545en_US
dc.identifier.citationdoi: 10.1016/j.jsb.2015.04.005en_US
dc.identifier.urihttp://hdl.handle.net/1993/30733
dc.description.abstract2'-5'-oligoadenylate synthetases (OAS) are interferon-stimulated proteins that act in the innate immune response to viral infection. Upon binding to viral double-stranded RNAs, OAS enzymes produce 2'-5'-linked oligoadenylates that stimulate RNase L and ultimately slow viral propagation. Studies have linked mutations in the OAS1 gene to increased susceptibility to West Nile virus (WNV) infection, highlighting the importance of the OAS1 enzyme. Here I report that the 5'-terminal region (5'-TR) of the WNV genome, comprising both the 5'-untranslated region (5'-UTR) and initial coding region, is capable of OAS1 activation in vitro. This region contains three RNA stem loops (SLI, SLII, and SLIII), whose relative contribution to OAS1 binding affinity and activation were investigated using electrophoretic mobility shift assays and enzyme kinetics experiments. Stem loop I (SLI) is dispensable for maximum OAS1 activation, as a construct containing only SLII and SLIII was capable of enzymatic activation. Mutations to the RNA binding site of OAS1 confirmed the specificity of the interaction. Solution conformations of both the 5'-TR RNA of WNV and OAS1 were then elucidated using small-angle x-ray scattering. I also report that the 3' terminal region (3'-TR) is able to mediate specific interaction with and activation of OAS1. Binding and kinetic experiments identified a specific stem loop within the 3'-TR that is sufficient for activation of the enzyme. The solution confirmation of the 3'-terminal region was determined by small angle X-ray scattering, and computational models suggest a conformationally restrained structure comprised of a helix and short stem loop. Structural investigation of the 3'-TR in complex with OAS1 is also presented. Finally, we show that genome cyclization by base pairing between the 5'- and 3'-TRs, a required step for replication, is not sufficient to protect WNV from OAS1 recognition. The purity, monodispersity and homogeneity of all samples subjected to SAXS analysis were evaluated using dynamic light scattering and/or analytical ultra-centrifuge. These data provide a framework for understanding recognition of the highly structured terminal regions of a flaviviral genome by an innate immune enzyme.en_US
dc.language.isoengen_US
dc.publisherPlos.orgen_US
dc.publisherElsevieren_US
dc.rightsinfo:eu-repo/semantics/openAccess
dc.subjectviral RNAen_US
dc.subjectinnate immunityen_US
dc.subjectOAS1en_US
dc.subjectbiophysical analysisen_US
dc.subjectsmall angle X-ray scatteringen_US
dc.titleCharacterization of the terminal region RNAs of the West Nile virus genome and their interaction with the small isoform of 2' 5'-oligoadenylate synthetases (OAS)en_US
dc.typeinfo:eu-repo/semantics/doctoralThesis
dc.typedoctoral thesisen_US
dc.degree.disciplineChemistryen_US
dc.contributor.examiningcommitteeKhajehpour, Mazdak (Chemistry) Bieringer, Mario (Chemistry) Mark, Brian (Microbiology) Jan, Eric (University of British Columbia)en_US
dc.degree.levelDoctor of Philosophy (Ph.D.)en_US
dc.description.noteOctober 2015en_US


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