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Subcellular Investigation of Poxvirus Coinfections and the Impact on the Evolution of Cidofovir Resistance
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- Author / Creator
- Lee, Stephen Z
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The Orthopoxviruses remain important re-emerging pathogens, yet there are only two monotherapy antiviral drugs for treating infections. One of the two approved drugs is brincidofovir, which is a prodrug of the acyclic nucleoside phosphonate cidofovir (CDV). CDV inhibits the vaccinia virus DNA polymerase E9, and substitutions in the polymerase confer resistance to the drug. The molecular mechanisms explaining how rare, CDV-resistant (CDVR) mutants emerge in a population are poorly understood. It is unclear how a rare mutant would ever gain a foothold in a predominantly wildtype population. We were particularly interested in understanding how coinfection between CDVR and CDV-sensitive (CDVS) viruses would impact the selection for this rare trait. To this end, we assembled recombinant CDVR or CDVS vaccinia viruses expressing EGFP or mKate2 fluorescent proteins fused to the λ bacteriophage cro DNA binding peptide. The cro-EGFP and cro-mKate2 proteins were useful markers to distinguish virus plaques in cell cultures and fluorescent labels for tracking the expansion of virus factories, cytoplasmic sites of replication for poxviruses.
Surprisingly, we showed that in coinfection experiments with CDV treatment, CDVR viruses appeared to lose their selective advantage over CDVS viruses. When the viruses replicated in separate cells, there was a strong selection for the CDVR viruses, which showed that the presence of CDVS viruses somehow interfered with the selection for CDVR viruses during coinfection. This prompted us to look at the subcellular behavior of the two viruses using fluorescence microscopy. In single infections with CDV treatment, the CDVR virus displayed a significantly higher virus factory growth rate than the CDVS virus. However, when the two viruses were present in the same cell, there was no difference in the growth of the CDVR and CDVS viruses despite the presence of CDV. Additionally, increasing the number of CDVS viruses in the cell significantly slowed factory growth across both CDVR and CDVS viruses. Thus, in a coinfected cell, the CDVS virus parasitized the growth advantage of the CDVR virus. This allowed the CDVS virus to survive the strong selective pressure from CDV treatment. However, when the two viruses replicated in isolated cells, the CDVS virus could not gain this advantage. Thus, the purifying selection from CDV treatment can only act when viruses remain in separate cells and cannot interact. These data suggest that mechanisms that maintain single infections could play an important role in maintaining fitness in a population of poxviruses. -
- Subjects / Keywords
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- Graduation date
- Fall 2024
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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 Library 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.