RP03 Project Summary/Abstract Filoviruses critically depend on cellular proteins to facilitate replication and are susceptible to inhibition by cellular antiviral systems. Research Project 3 (RP03) tests the roles of host protein complexes in virus replication and establishes an approach to evaluate large datasets for biological importance in virus infection. The work builds on our genome-wide siRNA, CRISPR-Cas9, BioID, yeast two-hybrid (Y2H), and co-affinity purification plus mass spectrometry screening data that has identified 100s of host factors that play roles in EBOV and MARV replication. Traditionally, follow up on such large hit sets has been slow, hampered by assays of sufficient throughput and informative read-out to provide needed prioritization. Additionally, hits tend to addressed in isolation, disregarding the relatedness of each hit by function or cellular association. Here, we apply an advanced computer algorithm, the Prize Collecting Steiner Forest (PCSF) algorithm to associate hit proteins by known, high confidence, published, protein-protein interaction (PPI) networks. Overlaying virus protein interactions detected in Y2H and proteomics work revealed clusters of host proteins that interact with a common virus protein. Furthermore, overlaying highest probability protein function using Gene Ontology (GO) terms from different databases, revealed clusters of host proteins related by likely cellular function, with actin regulation and RNA processing being the most over-represented but also pathways related to protein modifications through ubiquitinylation, sumoylation or phosphorylation being evident (as seen in RP01 and RP02). Based on these novel findings, we propose the hypothesis that the host factors residing in subnetworks related by common virus proteins, function or both play the same role in a specific virus replication step. Here, we test this hypothesis by applying a novel, statistically high powered optical pooled screening platform that phenotypically evaluates infection outcome by measuring both virus protein and virus RNA expression levels together with subcellular staining patterns to associate viral functional relatedness to relatedness by known host PPI. This approach allows efficient prioritization of groups of factors for evaluation by mechanistic assays that identify affected steps in virus infection and then defining regions of the host and virus proteins responsible for the infection outcome. The work starts with high priority validated leads representing the actin and RNA processing networks, then evaluates our existing high stringency network, which is then expanded and enriched through new data fed from RP01 and RP02. Our team consists of experts with strong track records in performing and analyzing large host factor genetic and proteomic screens, and performing virus mechanistic analysis. Through extensive interaction between each group, we expect to gain mechanistic insight into roles for identified host ...