Ebola Virus Spike Glycoprotein Recruits Cholesterol for Efficient Fusion

Abstract

Cholesterol serves critical roles in membrane fusion of enveloped viruses by modulating physical properties such as thickness, fluidity, and curvature of biological membranes. The spike glycoprotein GP of Ebola virus (EBOV) is a class 1 viral fusion protein sharing similar structural features with Influenza hemagglutinin and HIV gp120/gp41. Fusion of EBOV in the late endosome requires Niemann-Pick C1 protein as a receptor, which is an endosomal cholesterol transporter. Thus, cholesterol might be important in EBOV membrane fusion and cell entry. Here we present evidence that the EBOV GP membrane proximal external region/transmembrane (MPER/TM) domains recruit cholesterol to a local environment that enhances fusion. Virus-like particle (VLP) cell entry and lipid mixing fusion assays show that entry and fusion depend on the cholesterol concentration in the viral membrane. Cholesterol titration experiments using NMR revealed that several glycines, most notably G660, on the GP2 TM domain are critical for its interaction with cholesterol. The interaction in the G660L mutant is diminished and shows a more open angle between the MPER and TM domains in the mutant compared to WT, as measured by pulsed EPR experiments. Single particle fusion experiments (by TIRF microscopy) show that cholesterol in the viral membrane enhances the probability and kinetics of fusion. The G660L mutant shows a higher probability of hemifusion, which correlates with a 4-fold lower cell entry efficiency of mutant compared to WT VLPs. Taken together, it appears that a quite strong interaction of cholesterol with the TM domain of GP is important to facilitate cell entry of EBOV by membrane fusion.