Figure 5.

Correlation of VP40 and VP40-WEA apparent brightness and frequency as well as VLP egress. (A) Frequency versus apparent brightness plot for monomeric EGFP, (B) EGFP-VP40, and (C) EGFP-VP40 WE-A from HEK293T cells. These plots show the frequency (pixels) of VP40 oligomerization in comparison to VP40 WE-A. (D) VLPs were detected with anti-EGFP antibody to compare the release of EGFP-VP40 and EGFP-VP40-WEA VLPs. Cells transfected with EGFP were used as a control for background EGFP detection. Results are plotted as a ratio of EGFP in the VLPs/EGFP in the cell lysate. Three trials were run for each condition from two separate experiments of VLP and cell lysate collection. (E) A model proposing the localization of VP40 oligomers. Imaging data from both confocal and TIRF microscopy support the notion that VP40 is predominantly monomeric in the cytoplasm as well as when associated with the plasma membrane. Plasma membrane association of VP40 then may be able to induce the oligomerization of VP40 into dimers and trimers, which are also predominantly associated with the plasma membrane. Higher order VP40 oligomers (hexamers and octamers for instance), which have been shown to exist in infectious Ebola virions are highly enriched in membrane protrusions emanating from the plasma membrane. (See the Discussion section for a more critical discussion of the factors that may regulate VP40 assembly and oligomerization).