Structure of the Ebola Virus Envelope Protein MPER/TM Domain and its Interaction with the Fusion Loop Explains their Fusion Activity

Abstract

Ebolavirus (EBOV), an enveloped filamentous RNA virus causing severe hemorrhagic fever, enters cells by macropinocytosis and releases its genetic material into the cytoplasm after membrane fusion in a late endosomal compartment. Membrane fusion is governed by the EBOV surface envelope glycoprotein (GP), which consists of subunits GP1 and GP2. GP1 binds to cellular receptors including Niemann-Pick C1 (NPC1) protein and GP2 is responsible for membrane fusion at low pH. GP1 undergoes multiple steps of proteolytic cleavage and binds to NPC1 at endosomal pH. GP2 is rearranged in a fashion that exposes the hydrophobic fusion loop (FL) of GP2, which is then inserted into the cellular target membrane, ultimately forming a six-helix bundle structure and resulting in the formation of the fusion pore. Although major portions of the GP2 structure that have been solved in pre- and post-fusion states and the current model places the transmembrane (TM) and FL domains of GP2 in close proximity to each other at critical steps of membrane fusion, their structures in membrane environments and especially interactions between TM and FL have not yet been characterized. Here we present the structure of the membrane proximal external region (MPER) connected to the TM domain, i.e. the missing parts of the EBOV GP2 structure. The structure, solved by solution NMR and EPR spectroscopy in membrane-mimetic environments, consists of a helix-turn-helix architecture that is independent of pH. Moreover, the MPER region, not TM region, is shown to interact in the membrane interface with the previously determined structure of the EBOV FL through several critical aromatic residues. Mutation of aromatic and neighboring residues in both binding partners decreases fusion and viral entry highlighting the functional importance of the MPER/TM - FL interaction in EBOV entry and fusion.