Sphingosine Kinases Promote Ebola Virus Infection and Can Be Targeted to Inhibit Filoviruses, Coronaviruses, and Arenaviruses Using Late Endocytic Trafficking to Enter Cells

[Image: see text] Entry of enveloped viruses in host cells requires the fusion of viral and host cell membranes, a process that is facilitated by viral fusion proteins protruding from the viral envelope. These viral fusion proteins need to be triggered by host factors, and for some viruses, this event occurs inside endosomes and/or lysosomes. Consequently, these ‘late-penetrating viruses’ must be internalized and delivered to entry-conducive intracellular vesicles. Because endocytosis and vesicular trafficking are tightly regulated cellular processes, late-penetrating viruses also depend on specific host proteins for efficient delivery to the site of fusion, suggesting that these could be targeted for antiviral therapy. In this study, we investigated a role for sphingosine kinases (SKs) in viral entry and found that chemical inhibition of sphingosine kinase 1 (SK1) and/or SK2 and knockdown of SK1/2 inhibited entry of Ebola virus (EBOV) into host cells. Mechanistically, inhibition of SK1/2 prevented EBOV from reaching late-endosomes and lysosomes that contain the EBOV receptor, Niemann Pick C1 (NPC1). Furthermore, we present evidence that suggests that the trafficking defect caused by SK1/2 inhibition occurs independently of sphingosine-1-phosphate (S1P) signaling through cell-surface S1P receptors. Lastly, we found that chemical inhibition of SK1/2 prevents entry of other late-penetrating viruses, including arenaviruses and coronaviruses, and inhibits infection by replication-competent EBOV and SARS-CoV-2 in Huh7.5 cells. In sum, our results highlight an important role played by SK1/2 in endocytic trafficking, which can be targeted to inhibit entry of late-penetrating viruses and could serve as a starting point for the development of broad-spectrum antiviral therapeutics.