A Yeast Two-Hybrid Protein Domain Screening Approach for Ebola Virus-Human Protein Interactions Identifies PABPC1 as a Host Factor Required for Replication

Abstract Ebola virus (EBOV) causes severe hemorrhagic fever and remains a global health threat despite advances in vaccines and antibody-based therapeutics. Viral replication and immune evasion depend on interactions between EBOV proteins and host factors, yet the full spectrum of these interactions is unknown. To systematically identify EBOV-human protein-protein interactions, we performed more than 200 yeast two-hybrid (Y2H) screens using full-length EBOV proteins and domain fragments as baits and cDNA libraries derived from human liver tissue, macrophages, and interferon-stimulated macrophages. This approach revealed 521 unique interactions supported by confidence scoring based on retest assays and library screen data. The interactome includes proteins involved in RNA metabolism, transcriptional regulation, and post-translational modification, with enrichment for RNA-binding proteins among partners of NP, VP30, and L. By employing multiple constructs for each EBOV gene, interaction interfaces could be localized to specific viral domains. This approach identified a likely MYND domain binding site in the intrinsically ordered region of NP. Similarly, overlapping cDNA fragments from Y2H screens revealed potential binding sites within human proteins, including putative VP30 binding sites on NUFIP2 and PABPC1 that lacked canonical PPPPxY motifs. PABPC1 interacted with VP30 in uninfected human cells and accumulated in inclusion bodies in infected cells that resembled respiratory syncytial virus inclusion body associated granules (IBAGs). Inhibiting PABPC1 expression reduced EBOV replication early in infection. These findings expand the EBOV-host interaction network, identify candidate regulators of viral RNA synthesis, and provide a resource for mechanistic studies and antiviral target discovery. Importance Ebola virus (EBOV) remains a major global health threat due to its high mortality rate and limited treatment options. Viral replication and immune evasion depend on interactions between EBOV proteins and host factors, yet these interactions are incompletely understood. This study uses a large-scale yeast two-hybrid approach to systematically map EBOV-human protein-protein interactions, revealing hundreds of previously unreported partners and identifying cellular pathways that may be exploited by the virus. By integrating domain mapping, confidence scoring, and functional validation, we provide a resource that advances understanding of EBOV biology and may be used to discover candidates for antiviral development.