Davey, Robert A.Donahue, Callie Jeanne2025-03-262024https://hdl.handle.net/2144/499432024Ebola virus causes severe disease with high case-fatality rates and is a concerning public health issue. Current therapeutics are only capable of treating acute aspects of disease and require administration soon after exposure to be effective. A better understanding of virus-host dependencies will help in development of more effective treatments. For productive infection, the virus must both counteract and co-opt different host factors to successfully replicate. To identify host factors important for virus infection, we conducted full genome siRNA knockdown and CRISPR knockout screens with infectious Ebola virus as well as a protein-protein interaction screen using virus structural proteins. Like other host-pathogen interaction screens, we found little overlap in the individual gene hits identified in each screen but predicted that application of network analysis would reveal overlaps between each dataset and help in prioritizing hits for follow up and for suggesting mechanism. Prior to this work, limited tools were available to model host-pathogen interactions in the context of both the cell and virus. Hits were mapped onto existing host genetic and protein interaction networks using several algorithms including the Prize-Collecting Steiner Forest network analysis. We investigated whether network topology from the resulting network maps could predict the identification of key genes regulating viral replication. While we found no significant relationship between network topology and hit identification, topology guided network clustering and gene-set enrichment was effective at predicting the mechanistic role of genes. The orphan gene, SPNS1, was one example of a cell entry factor for Ebola virus where its interactions with other host genes within a network cluster suggested its function in entry-relevant pathways. We additionally found that protein-protein interaction data from BioID labeling of host proteins interacting with tagged virus proteins was highly productive in identification of proteins involved in replication. Annotation of this network with those virus proteins interacting with each host protein appeared to reveal instances of virus proteins interacting with host protein complexes whose functions could be deduced by corresponding gene enrichment. We then tested this outcome by studying the relationship between viral protein VP35 and members of the host mRNA decapping complex. VP35 was found to interact with the host protein EDC4, which forms a scaffold for other members of the decapping complex and controls processing body formation. We found that Ebola virus replication depended on multiple components of the complex and demonstrated that depletion of these components resulted in a reduction in viral infection. Ultimately, our work shows that during infection, Ebola virus depends on and interacts with multiple host factors in concert rather than individually, and that the functional relationships in host factors should be considered when identifying targets for therapeutic development.en-USAttribution-NonCommercial-NoDerivatives 4.0 Internationalhttp://creativecommons.org/licenses/by-nc-nd/4.0/VirologySystematic biologyCellular biologyEbola virusHigh-throughput screeningHost-pathogen interactionsmRNA decappingNetwork analysisVirus entryThesis/Dissertation2025-03-180000-0002-4284-2914