Abstract
Ebola virus (EBOV) enters cells from late endosomes/lysosomes under mildly acidic conditions. Entry by fusion with the endosomal membrane requires the fusion loop (FL, residues 507–560) of the EBOV surface glycoprotein to undergo a pH-dependent conformational change. To find the pH trigger for this reaction we mutated multiple conserved histidines and charged and uncharged hydrophilic residues in the FL and measured their activity by liposome fusion and cell entry of virus-like particles. The FL location in the membrane was assessed by NMR using soluble and lipid-bound paramagnetic relaxation agents. While we could not identify a single residue to be alone responsible for pH triggering, we propose that a distributed pH effect over multiple residues induces the conformational change that enhances membrane insertion and triggers the fusion activity of the EBOV FL.
Highlights
Ebola virus (EBOV) is an enveloped, negative strand RNA virus that causes severe hemorrhagic fever associated with very high fatality rates [1,2]
GP is proposed to undergo a dramatic pH- and possibly Niemann-Pick protein C1 (NPC1)-triggered conformational change that results in the exposure of the hydrophobic disulfide linked fusion loop (FL) in the N-terminal region of the GP subunit 2 (GP2) [14,15,16]
Cell pellets containing EBOV FLs were lysed by sonication for 10 min on ice and purification of FLs was performed by following the previous protocol [15]
Summary
Ebola virus (EBOV) is an enveloped, negative strand RNA virus that causes severe hemorrhagic fever associated with very high fatality rates [1,2]. EBOV enters and infects cells by macropinocytosis followed by fusion of its own membrane envelope with the membrane of a late endosome [3,4,5,6]. EBOV entry and fusion are mediated by the viral surface glycoprotein (GP) [3,7,8]. GP is cleaved by cathepsins L and B in a late endosomal/lysosomal compartment, which is acidic and contains the Niemann-Pick protein C1 (NPC1) that has been shown to be an intracellular receptor for GP [9,10,11,12,13]. Low pH, and NPC1 binding are all required to produce the active form of GP that mediates membrane fusion.
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