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An exploration of viral RNA-mediated strategies to stall and repress the cellular exoribonuclease XRN1

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dc.contributor.authorCharley, Phillida A., author
dc.contributor.authorWilusz, Jeffrey, advisor
dc.contributor.authorZabel, Mark, committee member
dc.contributor.authorPerera, Rushika, committee member
dc.contributor.authorReddy, Anireddy, committee member
dc.date.accessioned2019-01-07T17:19:17Z
dc.date.available2021-01-03T17:19:23Z
dc.date.issued2018
dc.description.abstractThe regulation of mRNA decay plays a vital role in determining both the level and quality control of cellular gene expression in eukaryotes. Since they are likely recognized as foreign/unwanted transcripts, viral RNAs must also successfully navigate around the cellular host RNA decay machinery to establish a productive infection. This bypass of the cellular RNA decay machinery can be accomplished in many ways, including the sequestering of regulatory proteins or inactivating enzymatic components. One attractive way for RNA viruses to undermine the cellular RNA decay machinery is to target the cellular exoribonuclease XRN1 since this enzyme plays a major role in mRNA decay, appears to coordinate transcription rates with RNA decay rates, and is localized to the cytoplasm and thus readily accessible to cytoplasmic RNA viruses. We have previously shown that many members of Flaviviridae (e.g. Dengue, West Nile, Hepatitis C and Bovine Viral Diarrhea viruses) use RNA structures in their 5' or 3' untranslated regions (UTRs) to stall and repress XRN1. This results in the stabilization of viral RNAs while also causing significant dysregulation of cellular RNA stability (and thus dysregulation of overall cellular gene expression). In this dissertation we first extend this observation to another member of the Flaviviridae, Zika virus, by demonstrating that structures in the 3' UTR of the viral genomic RNA can stall and repress XRN1. Significantly, we also demonstrate that the 3' UTR of the N mRNA of the ambisense segment of Rift Valley Fever virus, as well as two other phleboviruses of the Phenuiviridae, also can effectively stall and repress XRN1. This observation establishes XRN1 stalling in an additional family of RNA viruses, in this case in the order Bunyavirales. We have mapped the region responsible for XRN1 stalling to a G-rich core of ~50 nucleotides and provide evidence that the formation of a G-quadruplex is contributing to stalling of XRN1. In addition to phleboviruses, we also detected RNA regions that stall XRN1 in the non-coding regions of two other virus families. The 3' UTRs of all four ambisense transcripts of Junin virus, an arenavirus, stall and repress XRN1. This observation was extended to two additional arenaviruses, suggesting that XRN1 stalling may be a conserved property of the 3' UTRs in the Arenaviridae. Finally, we demonstrate that the non-coding RNA from beet necrotic yellow vein virus RNA segment 3 is produced by XRN1 stalling and requires a conserved sequence called the coremin motif. Collectively, these observations establish XRN1 stalling and repression as a major strategy used by many virus families to effectively interface with the cellular RNA decay machinery during infection. We performed two proof of principle studies to extend the significance of the observation of XRN1 stalling during RNA virus infections. First, since XRN1 stalling may be associated with successful viral gene expression as well as cytopathology, we explored whether we could identify a small molecule compound that could interfere with the knot-like three helix RNA junction structure that stalls XRN1 in the 3' UTR of flaviviruses. We tested several triptycene-based molecules, compounds that have been previously shown to intercalate into three helix junctions and identified four triptycene derivatives that interfere with XRN1 stalling. Lastly, we explored whether there might be a cellular exoribonuclease that could navigate through the well-characterized flavivirus structure that effectively stalls XRN1. Our efforts focused on the mammalian Dom3z/DXO enzyme which contains both 5' decapping and 5'-3' exoribonuclease activity. Interestingly, recombinant Dom3z/DXO enzyme did not stall on RNAs containing the 3' UTR of either Dengue virus or the Rift Valley Fever Virus N mRNA. This may suggest that there is a molecular arms race of sorts between the cell and the virus for supremacy of regulating the 5'-3' decay of RNA during infection.
dc.format.mediumborn digital
dc.format.mediumdoctoral dissertations
dc.identifierCharley_colostate_0053A_15156.pdf
dc.identifier.urihttps://hdl.handle.net/10217/193131
dc.languageEnglish
dc.language.isoeng
dc.publisherColorado State University. Libraries
dc.relation.ispartof2000-2019
dc.rightsCopyright and other restrictions may apply. User is responsible for compliance with all applicable laws. For information about copyright law, please see https://libguides.colostate.edu/copyright.
dc.subjectJUNV
dc.subjectRVFV
dc.subjectXRN1
dc.subjectMERCoV
dc.subjectBNYVV
dc.subjecttriptycenes
dc.titleAn exploration of viral RNA-mediated strategies to stall and repress the cellular exoribonuclease XRN1
dc.typeText
dcterms.embargo.expires2021-01-03
dcterms.embargo.terms2021-01-03
dcterms.rights.dplaThis Item is protected by copyright and/or related rights (https://rightsstatements.org/vocab/InC/1.0/). You are free to use this Item in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s).
thesis.degree.disciplineMicrobiology, Immunology, and Pathology
thesis.degree.grantorColorado State University
thesis.degree.levelDoctoral
thesis.degree.nameDoctor of Philosophy (Ph.D.)

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