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Permanent link (DOI): https://doi.org/10.7939/R3RR1PV61

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Investigations into Poxvirus-Host Interactions Open Access

Descriptions

Other title
Subject/Keyword
RNAi screen
cell cycle
myxoma virus
poxvirus
chromatin
vaccinia virus
glycolysis
Type of item
Thesis
Degree grantor
University of Alberta
Author or creator
Teferi, Wondimagegnehu Mulatu
Supervisor and department
Evans, David H. (Department of Medical Microbiology and Immunology)
Examining committee member and department
Gotte, Matthias (Department of Medical Microbiology and Immunology)
Hazes, Bart (Department of Medical Microbiology and Immunology)
Mahoney, Douglas J. (Department of Microbiology, Immunology and Infectious Disease, University of Calgary)
Foley, Edan (Department of Medical Microbiology and Immunology)
Evans, David H. (Department of Medical Microbiology and Immunology)
Michalak, Marek (Department of Biochemistry)
Department
Department of Medical Microbiology and Immunology
Specialization
Virology
Date accepted
2016-01-13T15:13:45Z
Graduation date
2016-06
Degree
Doctor of Philosophy
Degree level
Doctoral
Abstract
Poxviruses, such as vaccinia virus (VACV) and myxoma virus (MYXV), actively modulate various cellular structures and functions to ensure effective replication and transmission. In the contrary, cells use several restriction mechanisms to mitigate these viruses. This evolutionary relationship is the basis for poxvirus-host interactions. Although some of these interactions have been described, given the large number of proteins encoded by poxviruses, a significant number of them are yet to be discovered. To further our understanding of poxvirus-host interactions, we performed large-scale small interfering RNA (siRNA) screens in MDA-MB-231 cells and identified human host factors that modulate MYXV replication. Using human whole-genome (21585 genes) and sub-genomic (kinases and phosphatases, 986 genes) siRNA libraries, we identified 711 antiviral (siRNA pools that enhanced MYXV replication) and 333 proviral genes (siRNA pools that inhibited MYXV replication). Cluster analysis of these genes identified a number of enriched pathways and processes including inflammatory and mitogen-activated protein kinase pathways, the cell cycle and glycolysis. We further studied some of these pathways. Decreasing the glycolytic activity of cells through siRNA silencing of key glycolytic enzymes, such as phosphofructokinase (PFK)-1, or treatment with 2-deoxy-D-glucose reduced the replication of MYXV. In contrast, enhancing glycolytic activity through over-expression of glycolytic enzymes, such as 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), increased virus replication confirming that infection is favoured by aerobic glycolytic metabolism. Similarly, according to our siRNA screen results, siRNAs that trap cells in G1-phase of the cell cycle by inhibiting the G1/S-checkpoint stimulated MYXV growth. This observation was reproduced by arresting cells at G1 with a chemical inhibitor of cyclin-dependent kinases 4/6. Moreover, the inhibitor also enhanced the oncolytic potentials of the virus. We also investigated the interaction between poxviruses and the repressive chromatin. VACV infection enhanced markers of the repressive chromatin through SUV39H1/2 dependent manner while MYXV did not change them. Our preliminary studies suggested that VACV might use the repressive chromatin to produce a widespread reduction in cellular gene expression. Overall these studies demonstrate that poxviruses interact with various cellular processes. A detailed understanding of these processes could help to identify anti-poxvirus drug targets and enhance the oncolytic potentials of these viruses.
Language
English
DOI
doi:10.7939/R3RR1PV61
Rights
This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for the purpose of private, scholarly or scientific research. 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.
Citation for previous publication
Teferi, W. M., Dodd, K., Maranchuk, R., Favis, N., and Evans, D. (2013). A whole-genome RNA interference screen for human cell factors affecting myxoma virus replication. Journal of Virology. 87(8): 4623-41.

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