Investigation of Ebola VP40 assembly and oligomerization in live cells using number and brightness analysis

Ebola virus assembles and buds from the inner leaflet of the plasma membrane of mammalian cells, which is primarily attributed to its major matrix protein VP40. Oligomerization of VP40 has been shown to be essential to the life cycle of the virus including formation of virions from infected cells. To date, VP40 oligomerization has mainly been assessed by chemical cross-linking following cell fractionation studies with VP40 transfected cells. This has made it difficult to discern the spatial and temporal dynamics of VP40 oligomerization. To gain a better understanding of the VP40 assembly and oligomerization process in live cells, we have employed real-time imaging of enhanced green fluorescent protein tagged VP40. Here, we use both confocal and total internal reflection microscopy coupled with number and brightness analysis to show that VP40 oligomers are localized on the plasma membrane and are highly enriched at sites of membrane protrusion, consistent with sites of viral budding. These filamentous plasma membrane protrusion sites harbor VP40 hexamers, octamers, and higher order oligomers. Consistent with previous reports, abrogation of VP40 oligomerization through mutagenesis greatly diminished VP40 egress and also abolished membrane protrusion sites enriched with VP40. In sum, real-time single-molecule imaging of fluorescently labeled Ebola VP40 is able to resolve the spatial and temporal dynamics of VP40 oligomerization.