title: Ebola virus glycoprotein needs an additional trigger, beyond proteolytic priming for membrane fusion.
abstract: BACKGROUND
  Ebolavirus belongs to the family filoviridae and causes severe hemorrhagic fever in humans with 50-90% lethality. Detailed understanding of how the viruses attach to and enter new host cells is critical to development of medical interventions. The virus displays a trimeric glycoprotein (GP(1,2)) on its surface that is solely responsible for membrane attachment, virus internalization and fusion. GP(1,2) is expressed as a single peptide and is cleaved by furin in the host cells to yield two disulphide-linked fragments termed GP1 and GP2 that remain associated in a GP(1,2) trimeric, viral surface spike. After entry into host endosomes, GP(1,2) is enzymatically cleaved by endosomal cathepsins B and L, a necessary step in infection. However, the functional effects of the cleavage on the glycoprotein are unknown.
  
  
  RESULTS
  We demonstrate by antibody binding and Hydrogen-Deuterium Exchange Mass Spectrometry (DXMS) of glycoproteins from two different ebolaviruses that although enzymatic priming of GP(1,2) is required for fusion, the priming itself does not initiate the required conformational changes in the ectodomain of GP(1,2). Further, ELISA binding data of primed GP(1,2) to conformational antibody KZ52 suggests that the low pH inside the endosomes also does not trigger dissociation of GP1 from GP2 to effect membrane fusion.
  
  
  CONCLUSIONS
  The results reveal that the ebolavirus GP(1,2) ectodomain remains in the prefusion conformation upon enzymatic cleavage in low pH and removal of the glycan cap. The results also suggest that an additional endosomal trigger is necessary to induce the conformational changes in GP(1,2) and effect fusion. Identification of this trigger will provide further mechanistic insights into ebolavirus infection.
authors.affiliation: Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California, USA.
authors.forename: Shridhar
authors.initials: S
authors.lastname: Bale
authors.forename: Tong
authors.initials: T
authors.lastname: Liu
authors.forename: Sheng
authors.initials: S
authors.lastname: Li
authors.forename: Yuhao
authors.initials: Y
authors.lastname: Wang
authors.forename: Dafna
authors.initials: D
authors.lastname: Abelson
authors.forename: Marnie
authors.initials: M
authors.lastname: Fusco
authors.forename: Virgil L
authors.initials: VL
authors.lastname: Woods
authors.forename: Erica Ollmann
authors.initials: EO
authors.lastname: Saphire
mesh_terms: D057134:Antibodies, Neutralizing / Q000378:metabolism; D041961:Deuterium Exchange Measurement; D029043:Ebolavirus / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D004797:Enzyme-Linked Immunosorbent Assay; D006863:Hydrogen-Ion Concentration; D008561:Membrane Fusion / Q000502:physiology*; D008958:Models, Molecular; D009077:Mucins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation; D011134:Polysaccharides / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D013820:Thermolysin / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
chemical_list: D057134:Antibodies, Neutralizing; D009077:Mucins; D011134:Polysaccharides; D014759:Viral Envelope Proteins; C113189:envelope glycoprotein, Ebola virus; D013820:Thermolysin
references.citation: J Mol Biol. 2004 Jul 23;340(5):1185-96
references.pmid: 15236976
references.citation: Protein Sci. 1993 Apr;2(4):522-31
references.pmid: 8390883
references.citation: J Virol. 2009 Apr;83(7):2883-91
references.pmid: 19144707
references.citation: Curr Opin Struct Biol. 2009 Aug;19(4):408-17
references.pmid: 19559599
references.citation: Science. 2009 Jul 10;325(5937):204-6
references.pmid: 19590002
references.citation: Nat Struct Mol Biol. 2011 Nov 20;18(12):1424-7
references.pmid: 22101933
references.citation: J Infect Dis. 2007 Nov 15;196 Suppl 2:S251-8
references.pmid: 17940957
references.citation: J Virol. 2010 Mar;84(6):2972-82
references.pmid: 20053739
references.citation: J Cell Biochem Suppl. 2001;Suppl 37:89-98
references.pmid: 11842433
references.citation: Mol Cell. 1998 Nov;2(5):605-16
references.pmid: 9844633
references.citation: PLoS Pathog. 2008 Nov;4(11):e1000212
references.pmid: 19023410
references.citation: Crit Rev Biochem Mol Biol. 2008 May-Jun;43(3):189-219
references.pmid: 18568847
references.citation: Virol Sin. 2011 Jun;26(3):156-70
references.pmid: 21667336
references.citation: Virology. 2010 Jun 5;401(2):228-35
references.pmid: 20304456
references.citation: J Virol. 2006 Apr;80(8):4174-8
references.pmid: 16571833
references.citation: Science. 2005 Jun 10;308(5728):1643-5
references.pmid: 15831716
references.citation: Expert Rev Vaccines. 2011 Jan;10(1):63-77
references.pmid: 21162622
references.citation: Nature. 2008 Jul 10;454(7201):177-82
references.pmid: 18615077
references.citation: Adv Genet. 2011;73:121-83
references.pmid: 21310296
references.citation: Lancet. 2011 Mar 5;377(9768):849-62
references.pmid: 21084112
references.citation: Proc Natl Acad Sci U S A. 1999 Mar 16;96(6):2662-7
references.pmid: 10077567
references.citation: Nat Protoc. 2009;4(4):592-604
references.pmid: 19373230
references.citation: J Virol. 2007 Dec;81(24):13378-84
references.pmid: 17928356
references.citation: J Virol. 1998 Aug;72(8):6442-7
references.pmid: 9658086