Role of phosphatidic acid lipids on plasma membrane association of the Ebola virus matrix protein VP40

The Ebola virus matrix protein VP40 is responsible for the formation of the viral matrix by localizing at the inner leaflet of the human plasma membrane (PM). Various lipid types, including PI(4,5)P₂ (i.e. PIP₂) and phosphatidylserine (PS), play active roles in this process. Specifically, the negatively charged headgroups of both PIP₂ and PS interact with the basic residues of VP40 and stabilize it at the membrane surface, allowing for eventual egress. Phosphatidic acid (PA), resulting from the enzyme phospholipase D (PLD), is also known to play an active role in viral development. In this work, we performed a biophysical and computational analysis to investigate the effects of the presence of PA on the membrane localization and association of VP40. We used coarse-grained molecular dynamics simulations to quantify VP40 hexamer interactions with the inner leaflet of the PM. Analysis of the local distribution of lipids shows enhanced lipid clustering when PA is abundant in the membrane. We observed that PA lipids have a similar role to that of PS lipids in VP40 association due to the geometry and charge. Complementary experiments performed in cell culture demonstrate competition between VP40 and a canonical PA-binding protein for the PM. Also, inhibition of PA synthesis reduced the detectable budding of virus-like particles. These computational and experimental results provide new insights into the early stages of Ebola virus budding and the role that PA lipids have on the VP40-PM association.