UBC Theses and Dissertations
Study of the minute virus of mice cell entry Garcin, Pierre
The minute virus of mice prototype (MVMp) is a non-enveloped single stranded DNA virus of the family Parvoviridae. MVMp is one of the smallest viruses and shows intriguing abilities to preferentially infect and kill cancer cells (oncotropism/oncolytism), suggesting a potential for MVMp as an anti-cancer agent. Unfortunately, there is a lack of knowledge of the early events of MVMp infection cycle, such as binding to the cell surface and subsequent endocytosis. In an attempt to identify cellular partners of MVMp infection, our lab performed a mass spectrometry analysis of MVMp potential binding partners. Following this analysis, the galactose-binding lectin (galectin) 3 (Gal-3) was identified as binding partner for MVMp. Given the involvement of this extra-cellular matrix protein in the clustering and endocytosis of cell surface receptors, and its up-regulation in various aggressive tumor cells, I hypothesized that Gal-3 could play a role in MVMp cell entry, and potentially in its oncotropism. Using siRNA knockdown of Gal-3 in different cells followed by immunofluorescence microscopy analysis, I found that Gal-3 is necessary for an efficient MVMp cell entry and infection in different cells. Moreover, I discovered that the Golgi enzyme β1,6-acetylglucosaminyltransferase 5 (Mgat5), whose role is the addition of complex N-glycosylation to various cell surface receptors for Gal-3 binding, is required for MVMp infection. I also found that cancer cells with higher Gal-3 expression are more susceptible to MVMp infection than cells with lower Gal-3 levels. Next I used a combination of flow cytometry, immuno-fluorescence and transmission electron microscopy to characterize the early events of MVMp infection in various tissue-culture cell lines. My results show that many crucial parameters of the mesenchymal cell migration process regulate MVMp cellular entry and infection. I found that MVMp relies on cell protrusions to cluster at the leading edge of migrating cells rapidly after binding to the plasma membrane, from where it is subsequently endocytosed. Moreover, transmission electron microscopy analysis revealed that MVMp uses various endocytic pathways, which was confirmed using drug inhibitors of endocytosis. Finally, I found that epithelial-mesenchymal transition, an inducer of cancer cell migration, triggers MVMp infection in highly dividing non-permissive cancer cells.
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