Phosphatidylserine clustering by the Ebola virus matrix protein is a critical step in viral budding

Phosphatidylserine (PS) is a critical lipid factor in the assembly and spread of numerous lipid‐enveloped viruses. Here, we describe the ability of the Ebola virus (EBOV) matrix protein eVP40 to induce clustering of PS and promote viral budding in vitro , as well as the ability of an FDA‐approved drug, fendiline, to reduce PS clustering and subsequent virus budding and entry. To gain mechanistic insight into fendiline inhibition of EBOV replication, multiple in vitro assays were run including imaging, viral budding and viral entry assays. Fendiline lowers PS content in mammalian cells and PS in the plasma membrane, where the ability of VP40 to form new virus particles is greatly lower. Further, particles that form from fendiline‐treated cells have altered particle morphology and cannot significantly infect/enter cells. These complementary studies reveal the mechanism by which EBOV matrix protein clusters PS to enhance viral assembly, budding, and spread from the host cell while also laying the groundwork for fundamental drug targeting strategies. Synopsis The Ebola virus matrix protein VP40 clusters phosphatidylserine at the plasma membrane inner leaflet for efficient matrix layer formation and virus budding. The drug fendiline diminishes PS levels and clustering, reducing EBOV budding and spread. VP40 clusters phosphatidylserine (PS) in the plasma membrane of cells. VP40 PS clustering requires PS‐binding residues and VP40 oligomerization. FDA‐approved fendiline reduces PS levels and clustering. Fendiline greatly reduces VP40 oligomerization, EBOV budding and subsequent viral entry. Graphical Abstract The Ebola virus matrix protein VP40 clusters phosphatidylserine at the plasma membrane inner leaflet for efficient matrix layer formation and virus budding. The drug fendiline diminishes PS levels and clustering, reducing EBOV budding and spread.