Ebola virus requires phosphatidylinositol (3,5) bisphosphate production for efficient viral entry

Highlights: • The PIKfyve-ArPIKfyve-Sac3 complex is required for efficient EBOV entry. • PIKfyve inhibitors block entry by all pathogenic filoviruses and EBOV replication. • PIKfyve kinase activity is critical for EBOV delivery to its viral receptor NPC1. • EBOV increases PtdIns(3,5)P2 positive vesicles in cells during viral entry. For entry, Ebola virus (EBOV) requires the interaction of its viral glycoprotein with the cellular protein Niemann-Pick C1 (NPC1) which resides in late endosomes and lysosomes. How EBOV is trafficked and delivered to NPC1 and whether this is positively regulated during entry remain unclear. Here, we show that the PIKfyve-ArPIKfyve-Sac3 cellular complex, which is involved in the metabolism of phosphatidylinositol (3,5) bisphosphate (PtdIns(3,5)P₂), is critical for EBOV infection. Although the expression of all subunits of the complex was required for efficient entry, PIKfyve kinase activity was specifically critical for entry by all pathogenic filoviruses. Inhibition of PIKfyve prevented colocalization of EBOV with NPC1 and led to virus accumulation in intracellular vesicles with characteristics of early endosomes. Importantly, genetically-encoded phosphoinositide probes revealed an increase in PtdIns(3,5)P₂-positive vesicles in cells during EBOV entry. Taken together, our studies suggest that EBOV requires PtdIns(3,5)P₂ production in cells to promote efficient delivery to NPC1.