Mariam DAO, Ottawa University

Mariam Dao, Athish Marutharaj, Agnitha Xavier, Marceline Coté

Discovering & Evolving

Filoviruses, including Ebola virus (EBOV) and Marburg virus (MARV), rely on the matrix protein VP40 as the primary driver of viral assembly, budding, and egress. In EBOV, co-expression of the nucleoprotein (NP) and glycoprotein (GP) enhances VP40-mediated particle formation, whereas their contribution in MARV remains incompletely defined. Furthermore, host factors that facilitate VP40-dependent processes are poorly characterized. This study aims to systematically define the host protein interactome of VP40 and determine how NP and GP co-expression remodels these interactions for each virus. To capture proximal and transient host-viral interactions during egress, we used proximity-dependent biotinylation with UltraID, a compact engineered biotin ligase fused to the N-terminus of VP40. Upon biotin supplementation, neighboring proteins were biotinylated in living cells. Fusion of UltraID to EBOV and MARV VP40 did not impair filovirus-like particle production and enabled robust biotinylation of proteins in cells and viral particles. Importantly, co-expression of EBOV NP and GP increased particle production and markedly altered the intensity and profile of biotinylated proteins. In contrast, these pronounced changes were not observed for MARV, suggesting distinct VP40 interactomes between the two viruses. Biotinylated proteins will be affinity purified using beads, digested with trypsin, and analyzed by mass spectrometry to generate high-confidence VP40 interactomes. Comparative analyses across conditions will define virus-specific and NP/GP-dependent remodeling events. These findings establish a molecular framework for understanding host-pathogen interactions governing filovirus egress and budding and identify candidate host pathways that may be targeted for development of host-directed antiviral strategies.