Induction of Cell-Cell Fusion by Ebola Virus Glycoprotein: Low pH Is Not a Trigger

PLoS Pathog. 2016 Jan 5;12(1):e1005373. doi: 10.1371/journal.ppat.1005373. eCollection 2016 Jan.

Abstract

Ebola virus (EBOV) is a highly pathogenic filovirus that causes hemorrhagic fever in humans and animals. Currently, how EBOV fuses its envelope membrane within an endosomal membrane to cause infection is poorly understood. We successfully measure cell-cell fusion mediated by the EBOV fusion protein, GP, assayed by the transfer of both cytoplasmic and membrane dyes. A small molecule fusion inhibitor, a neutralizing antibody, as well as mutations in EBOV GP known to reduce viral infection, all greatly reduce fusion. By monitoring redistribution of small aqueous dyes between cells and by electrical capacitance measurements, we discovered that EBOV GP-mediated fusion pores do not readily enlarge-a marked difference from the behavior of other viral fusion proteins. EBOV GP must be cleaved by late endosome-resident cathepsins B or L in order to become fusion-competent. Cleavage of cell surface-expressed GP appears to occur in endosomes, as evidenced by the fusion block imposed by cathepsin inhibitors, agents that raise endosomal pH, or an inhibitor of anterograde trafficking. Treating effector cells with a recombinant soluble cathepsin B or thermolysin, which cleaves GP into an active form, increases the extent of fusion, suggesting that a fraction of surface-expressed GP is not cleaved. Whereas the rate of fusion is increased by a brief exposure to acidic pH, fusion does occur at neutral pH. Importantly, the extent of fusion is independent of external pH in experiments in which cathepsin activity is blocked and EBOV GP is cleaved by thermolysin. These results imply that low pH promotes fusion through the well-known pH-dependent activity of cathepsins; fusion induced by cleaved EBOV GP is a process that is fundamentally independent of pH. The cell-cell fusion system has revealed some previously unappreciated features of EBOV entry, which could not be readily elucidated in the context of endosomal entry.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Blotting, Western
  • COS Cells
  • Cathepsins / metabolism
  • Chlorocebus aethiops
  • Ebolavirus / physiology*
  • Flow Cytometry
  • Fluorescent Antibody Technique
  • HEK293 Cells
  • Hemorrhagic Fever, Ebola / virology*
  • Humans
  • Hydrogen-Ion Concentration
  • Immunohistochemistry
  • Patch-Clamp Techniques
  • Viral Fusion Proteins / metabolism*
  • Virus Internalization*

Substances

  • Viral Fusion Proteins
  • Cathepsins