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Vaccinia Virus Gene Acquisition Through Non-Homologous Recombination
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- Author / Creator
- Vallee, Gregory Thomas
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Poxviruses encode many genes that are orthologs of cellular genes. These orthologs serve many functions, but those that are of most interest are ones that have evolved further and now serve an immune-evasion function. Such genes were likely first acquired by poxviruses through some form of non-homologous recombination (NHR). Using an infection-transfection assay, I investigated vaccinia virus's (VAC) ability to recombine with (“capture”) a non-homologous (NH) substrate that encoded a fluorescent and drug selectable protein. This study showed that VAC can capture duplex DNA and cDNA·RNA hybrid molecules, but the recombinant frequencies were much lower in the absence of VAC homology. VAC recombined most efficiently with DNA duplexes encoding homology to the N2L locus. The recombinant frequency (RF) that was measured in these control reactions was 670×10^(-6). Forty-fold fewer recombinants were recovered from VAC-infected cells transfected with homologous cDNA·RNA hybrid molecules (RF=21 ×10^(-6)). However, when these experiments were repeated using substrates lacking any VAC homology, the linear duplex DNA yielded even fewer recombinants (RF=1.6×10^(-6)) and cDNA·RNA hybrid substrates yielded hardly any recombinants at all (RF=3×10^(-8)).
NHR generated a variety of viruses with genome rearrangements ranging from insertions with flanking duplications to large-scale indels. Most of the viruses that captured the NH substrate generated defective genomes. These viruses suffered large deletions of essential sequence and their replication and packaging were dependent on a wild-type co-infecting helper virus. In many cases, we also observed partial duplications of the sequences encoding the selectable substrate and these would rapidly rearrange through homologous recombination. A review of the sequences surrounding the insert junctions suggests that VAC NHR is likely catalyzed by a microhomology-independent mechanism. The junctions exhibited little to no pre-existing microhomologies, and we did not observe any disproportionate increase in sequence identity at the junctions or surrounding sequences (~30 nt on either side). In two cases we also found examples of the VAC topoisomerase I recognition sequence [(C/T)CCTT] overlapping the site of the VAC genome that had been targeted by recombination. These studies show that VAC can catalyze NHR through a process that may reflect a form of aberrant replication fork repair supplemented by topoisomerase I action. -
- Subjects / Keywords
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- Graduation date
- Fall 2021
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- Type of Item
- Thesis
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- Degree
- Master of Science
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- License
- This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.