Cellular determinants of reovirus entry and infection
2012-10
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Cellular determinants of reovirus entry and infection
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2012-10
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Abstract
Efficient entry is a critical determinant of reovirus oncolysis. While a model
for the entry of reovirus virions has been described, the method by which partially
uncoated intermediate subviral particles (ISVPs) enter host cells remains
unknown. Biochemical studies have suggested that ISVPs can directly penetrate
the plasma membrane since these particles can cause membrane damage and
hemolysis at high multiplicities of infection. We used a combination of techniques
to show that these particles can use endocytosis to enter and productively infect
host cells. We found that uptake of reovirus virions and ISVPs was inhibited in
cells treated with a small molecule inhibitor of dynasore. Analysis of reovirus
replication in cells treated with genistein and in cells expressing dominantnegative
caveolin-1 revealed that ISVPs use dynamin-dependent caveolar
endocytosis to enter host cells. These studies also showed that reovirus virions
were able to take advantage of caveolar endocytosis to infect cells. Because
many viruses have recently been found to enter cells through dynamindependent and –independent endocytic pathways, we also assessed the role of
cholesterol in reovirus infection. The growth of reovirus virions, but not ISVPs,
was inhibited in cells treated with methyl-β-cyclodextrin, which extracts
membrane cholesterol. We also wanted to learn whether reovirus entry had cell
type-specific differences. Analysis of reovirus infection in a rat embryo fibroblast
(CREF) and Ras-transformed CREF cell line suggested that reovirus particles
lose the ability to use clathrin-mediated endocytosis in CREF cells following Ras transformation. Reovirus can also infect cells that lack caveolae. To assess
whether reovirus used different endocytic pathways to infect cells devoid of
caveolae, we analyzed reovirus entry and infection in polarized Caco-2 cells.
While reovirus infection of these cells was dynamin-independent, particle
internalization occurred through both dynamin-dependent and –independent
pathways. Visualization of particle uptake showed that particles internalized
through dynamin-dependent endocytosis were transcytosed to the basolateral surface. These findings have demonstrated that reovirus can use a diverse set of
endocytic pathways to enter and infect cells. The data presented in this thesis
show that reovirus entry is a complex process that can vary in different cell types.
In addition, it has revealed that particles internalized into various endocytic
pathways are trafficked to different destinations in polarized epithelial cells.
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University of Minnesota Ph.D. dissertation. October 2012. Major: Microbiology, Immunology and Cancer Biology. Advisor: Leslie A. Schiff, Ph.D. 1 computer file (PDF); ix, 154 pages.
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Schulz, Wade Loren. (2012). Cellular determinants of reovirus entry and infection. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/143969.
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