Integrity of membrane fluidity and fatty acid environment are necessary for Ebola virus VP40 assembly and release of viral particles

Plasma membrane (PM) domains and order phases have been shown to play a key role in the assembly, release, and entry of several lipid-enveloped viruses. In the present study, we provide a mechanistic understanding of the Ebola virus (EBOV) matrix protein VP40 interaction with PM lipids and their effect on VP40 oligomerization, a crucial step for viral assembly and budding. VP40 matrix formation is sufficient to induce changes in the PM fluidity. We demonstrate that the distance between the lipid headgroups, the fatty acid tail saturation and the order between the two leaflets are important factors for the stability of VP40 binding and oligomerization at the PM. Use of FDA-approved drugs (dibucaine, propranolol and trifluoperazine) to fluidize the plasma membrane, destabilizes the viral matrix assembly leading to a reduction in budding efficiency. Lastly, we show that VP40 can tether and cluster lipid vesicles upon protein enrichment at the membrane. This is a new characteristic of the protein, and it opens the door to new avenues of exploration to deepen our understanding of VP40 host interactions and EBOV assembly. Indeed, our findings support a complex assembly mechanism of the EBOV viral matrix that reaches beyond lipid headgroup specificity using ordered PM lipid regions independent of cholesterol. ### Competing Interest Statement The authors have declared no competing interest.