Structure and Function of the Fusion Loop from Ebolavirus GP2

Abstract

Ebolavirus is an enveloped virus belonging to the Filoviridae family that causes hemorrhagic fever in humans. The mechanism of virus-host membrane fusion is not well characterized, but is mediated by the single viral glycoprotein, GP. Ebolavirus membrane fusion occurs from an endosomal compartment after proteolytic activation of GP by endosomal cathepsins and is governed by a patch of hydrophobic residues located within the internal fusion loop of the GP fusion subunit, GP2. Here we present liposome fusion data and NMR structures for the disulfide bound internal fusion loop (Ebov FL) at neutral and mildly acidic pH conditions. Membrane fusion activity of Ebov FL was measured in a liposome fusion assay after acidification to pH 5.5. Liposome fusion studies with varying lipid compositions showed that anionic lipids do not greatly contribute to lipid mixing, but the dependence on head group structure played a greater role. Replacement of POPG with POPS showed a decrease in lipid mixing whereas replacement with bis(monoacylglycero)phosphate (BMP) showed comparable lipid mixing. CD experiments indicated that moderate secondary structure was formed upon the addition of DPC at pH 7.0 reaching 14% α-helical structure, which further increased upon acidification to 23%. The NMR structures in DPC micelles at neutral and acidic conditions revealed a conformational change forming a bend in the fusion loop at the lower pH. The bend guides the reorganization of residues located in the hydrophobic patch. This rearrangement is thought to promote lipid disruption resulting in membrane fusion.