Tunneling nanotubes as an alternate route of Ebola virus dissemination

Ebola virus (EBOV) is an emerging, dangerous virus that causes increasingly more frequent outbreaks of a systemic, hemorrhagic disease in human populations. Approved countermeasures to prevent or treat EBOV disease are currently limited. Macrophages are the initial cells targeted by EBOV, and due to their migratory properties are believed to rapidly disseminate the virus to distant tissues and organs despite the lack of experimental evidence. In current models, EBOV propagates infection through the cell-free form, where virus particles enter the cell, replicate the genome, and then assemble/egress to challenge neighboring cells. We have preliminary data suggesting that EBOV may exploit an alternative mode to spread infection (in parallel with the established model): viral nucleocapsids via tunneling nanotubes (TNTs), an actin-based intercellular communication system that allows direct exchange of cytoplasmic material between connecting cells. EBOV infection induces formation of intercellular connections containing virus nucleocapsid protein in primary human endothelial cell and macrophage populations. These connections support cell-to-cell transfer of the nucleocapsid protein in the absence of the virus. The data also show that EBOV can efficiently replicate in endothelial cells devoid of factors critical for virus entry, after initial retardation, and that the replication is compromised in cells depleted of host M-Sec, a central factor for TNT formation. This proposal aims to interrogate the interactions between EBOV and TNTs through two Specific Aims. In Aim 1, we will determine if TNTs are the intercellular connections induced by EBOV to spread infection in human endothelial cells and macrophages. In Aim 2, we will determine if EBOV spreads infection through intercellular transfer of nucleocapsids. Our discoveries will establish an alternate model of EBOV dissemination within the host, laying the groundwork for further investigations into pathogenesis of filoviruses. Importantly, these findings may lead to development of novel strategies to target EBOV and related viruses.