Ebola virus (EBOV) and other members of the Filoviridae family are enveloped RNA viruses that are the causative agents of sporadic outbreaks of highly lethal disease in humans and non-human primates. EBOV entry into host cells requires attachment, internalization, and subsequent trafficking to the late endosomal/lysosomal compartment in order to reach the filovirus entry receptor, Niemann-Pick C1 (NPC1) and other triggering factors required for EBOV glycoprotein (GP)-mediated fusion between the viral and host membranes. The highly regulated nature of endosomal trafficking coupled with the dependence of EBOV on accurate endolysosomal trafficking for entry led us to hypothesize that the virus depends on—and potentially actively regulates—a consortium of specific host trafficking factors. In this thesis, we investigated the role of two trafficking complexes involved in endosomal maturation and trafficking, the Homotypic Fusion and Vacuole Protein Sorting (HOPS) complex and the PIKfyve-ArPIKfyve-Sac3 (PAS) complex, in EBOV entry. Furthermore, in order to further dissect how the PAS complex is regulated and performs its effector functions, we performed a protein-protein interaction screen using BioID in order to define the PAS cellular interactome. Using an inducible CRISPR/Cas9 system, we found that depletion of each HOPS subunit, as well as depletion of a positive regulator of the HOPS complex, UVRAG, impaired EBOV entry. Furthermore, we mapped a region of UVRAG spanning residues 269-442 to be key for binding to the HOPS complex and mediating EBOV entry, indicating that expression of and coordination between the HOPS complex and UVRAG are required for EBOV entry. Similarly, knockout of each subunit of the PAS complex was found to impair EBOV entry. Further molecular dissection using small molecule inhibitors and enzymatic mutants of PIKfyve and Sac3 demonstrated that PIKfyve kinase activity is required for EBOV entry, while Sac3 phosphatase activity is dispensable. Using a fluorescent probe for phosphatidylinositol(3,5)bisphosphate, the lipid product generated by PIKfyve, we also found evidence that stimulation of cells by EBOV virus-like-particles enhances PIKfyve activity, suggesting that the virus can promote its entry by activating the PAS complex. Finally, using BioID to screen for interacting proteins of the PAS complex, we identified candidate interactors involved in endosomal trafficking as well as other cell processes including mitochondrial function and cell cycle regulation. Further characterization of one candidate interactor, the coatomer complex I (COPI), using proximity ligation assays validated the interaction between ArPIKfyve and COPI subunit COPB1, and provides further evidence for a role of COPI in endosomal trafficking. Taken together, these results highlight the importance of cellular trafficking factors involved in diverse facets of endosomal dynamics, from lipid metabolism to membrane tethering, for the entry of EBOV and other filoviruses, and further shed light on how EBOV can actively modulate host trafficking networks to promote successful viral entry and infection. Further molecular dissection of how the virus hijacks cell trafficking will facilitate the development of antiviral therapeutics as well as elucidate how these fundamental cellular processes are regulated.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/42269 |
| Date | 08 June 2021 |
| Creators | Qiu, Shirley |
| Contributors | Côté, Marceline |
| Publisher | Université d'Ottawa / University of Ottawa |
| Source Sets | Université d’Ottawa |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
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