Subcellular Characterization of the Molecular Determinants of Ebola VP40 Trafficking and Assembly

Ebola virus is a single-stranded negative-sense RNA virus that can cause severe hemorrhagic fevers in humans. Ebola virus, along with other members of the filoviridae family, produce virions with a characteristic filamentous morphology. VP40, the filovirus matrix protein, is responsible for curving the host plasma membrane (PM). Expression of VP40 and the assembly of the matrix layer results in the budding of filamentous particles. VP40 forms cytosolic homodimers via interactions in its N-terminal domain, while interactions in its C-terminal domain drive oligomerization into the 2D-crystalline matrix layer. While VP40 is expressed throughout the host cytosol and assembles on the inner leaflet of the PM, VP40 does not appear to directly bind the PM but instead requires interactions with components of the host secretory machinery. Here, we characterize a series of VP40 mutants targeted to the molecular determinants of Ebola VP40 assembly and trafficking using confocal microscopy and genetically-encoded fluorescent tags. Using this approach, we characterize the subcellular distribution of these mutants, showing novel cellular phenotypes. Several mutants previously characterized as trafficking deficient show aggregation dependent on membrane binding, suggesting a possible route of VP40 trafficking. We then co-expressed these mutants with organelle markers, which provide insights into the affected parts of the host trafficking pathways. Together, our results provide new insights into molecular interactions that drive the assembly of the Ebola matrix layer.