Ebola Virus Triggers Receptor Tyrosine Kinase-Dependent Signaling To Promote The Delivery Of Viral Particles To Entry-Conducive Intracellular Compartments

Filoviruses, such as the Ebola virus (EBOV) and Marburg virus (MARV), are causative agents of sporadic outbreaks of hemorrhagic fevers in humans. To infect cells, filoviruses are internalized via macropinocytosis and traffic through the endosomal pathway where host cathepsin-dependent cleavage of the viral glycoproteins occurs. Subsequently, the cleaved viral glycoprotein interacts with the late endosome/lysosome resident host protein, Niemann-Pick C1 (NPC1). This interaction is hypothesized to trigger viral and host membrane fusion, which results in the delivery of the viral genome into the cytoplasm and subsequent initiation of replication. Some studies suggest that EBOV viral particles activate signaling cascades and host-trafficking factors to promote their localization with host factors that are essential for entry. However, the mechanism through which these activating signals are initiated remains unknown. By screening a kinase inhibitor library, we found that receptor tyrosine kinase inhibitors potently block EBOV and MARV GP-dependent viral entry. Inhibitors of epidermal growth factor receptor (EGFR), tyrosine protein kinase Met (c-Met), and the insulin receptor (InsR)/insulin like growth factor 1 receptor (IGF1R) blocked filoviral GP-mediated entry and prevented growth of replicative EBOV in Vero cells. Furthermore, inhibitors of c-Met and InsR/IGF1R also blocked viral entry in macrophages, the primary targets of EBOV infection. Interestingly, while the c-Met and InsR/IGF1R inhibitors interfered with EBOV trafficking to NPC1, virus delivery to the receptor was not impaired in the presence of the EGFR inhibitor. Instead, we observed that the NPC1 positive compartments were phenotypically altered and rendered incompetent to permit viral entry. Despite their different mechanisms of action, all three RTK inhibitors tested inhibited virus-induced Akt activation, providing a possible explanation for how EBOV may activate signaling pathways during entry. In sum, these studies strongly suggest that receptor tyrosine kinases initiate signaling cascades essential for efficient post-internalization entry steps.Author summaryEbola virus (EBOV) and Marburg virus (MARV) are zoonotic pathogens that can cause severe hemorrhagic fevers in humans and non-human primates. They are members of the growing Filoviridae family that also includes three other species of Ebolaviruses known to be highly pathogenic in humans. While vaccines for EBOV are being deployed and showed high efficacy, pan-filoviral treatment is still lacking. To infect cells, EBOV requires the endosomal/lysosomal resident protein Niemann-Pick C1 (NPC1). Accordingly, viral particles require extensive trafficking within endosomal pathways for entry and delivery of the viral genome into the host cell cytoplasm. Here, we used chemical biology to reveal that EBOV triggers receptor tyrosine kinase (RTK)-dependent signaling to traffic to intracellular vesicles that contain the receptor and are conducive to entry. The characterization of host trafficking factors and signaling pathways that are potentially triggered by the virus are important as these could be targeted for antiviral therapies. In our study, we identified several RTK inhibitors, some of which are FDA-approved drugs, that potently block EBOV infection. Since all filoviruses known to date, even Mengla virus that was recently discovered in bats in China, use NPC1 as their entry receptor, these inhibitors have the potential to be effective pan-filovirus antivirals.