Reconstitution strategies for exploring interactions of Ebola virus matrix protein with host membrane mimetics

Ebola virus (EBOV), an enveloped virus of the Filoviridae family, is an excellent biophysical model for studying enveloped virus assembly, despite its status as a BSL-4 pathogen. The expression of the EBOV matrix protein VP40 (viral protein 40 kDa) is necessary and sufficient for virus assembly/budding in human cells. VP40, in its dimeric form, interacts with the host plasma membrane (PM) inner leaflet and forms higher-order hexamers critical for EBOV assembly. The dependence of viral maturation on VP40 dynamics provides the opportunity to quantify biophysical requirements for the EBOV life cycle using a reductionist approach to measure VP40-membrane interactions. There are significant gaps in our understanding of the regulation underlying VP40 recruitment at the host cell PM, and how it culminates in the assembly/budding of mature, infectious virions. Here, we describe (i) reconstitution strategies for mimicking the VP40-host cell membrane interface in a controlled, biologically relevant manner and (ii) total internal reflection fluorescence microscopy-based detection of endogenous VP40 association with membranes under regulation of anionic lipid content. These methods are applicable for analyzing peripheral proteins and their interactions with lipid membranes.