Ebolavirus, one of two members of the family of filoviruses, causes a severe hemorrhagic fever with 50–90% human mortality. That no vaccines or treatments are yet available combined with the frequent re-emergence of the virus, its high prevalence among wildlife, and ease of importation of the virus make it a significant public health concern. A team of researchers from the Scripps Research Institute, using diffraction data collected at ALS Beamline 5.0.2, has recently determined the crystal structure of an oligomeric glycoprotein from the viral surface in complex with a rare antibody derived from a human survivor. This work explains how the glycoprotein, termed GP, mediates recognition of the host cell, drives fusion of the viral and host membranes (necessary for viral entry into the host), and masks itself from immune surveillance. The structure also explains why antibodies that neutralize the virus are so rare, identifies the very few sites to which a neutralizing antibody might bind, and thus, provides templates for vaccines and antibodies against the virus.

The crystal structure of Ebolavirus GP reveals a three-lobed chalice-like structure. The three GP1 subunits (colored blue and green), mediate attachment to new host cells and are tethered together by the three GP2 subunits (white). GP2 forms the protein machinery that drives fusion of the viral membrane with the host cell. The human antibody KZ52 (yellow) binds the GP at the base of the chalice, where it bridges GP1 to GP2, before fusion of the membranes.

Glycoproteins are proteins that contain carbohydrate chains (glycans) covalently attached to their polypeptide side chains, a process known as glycosylation. The glycoprotein GP is the sole resident of the Ebolavirus surface and is responsible for attaching to and entering new host cells, shielding the viral surface from immune surveillance, and maintaining viral stability when outside host cells (often for long periods of time). However, structures of viral glycoproteins in their native, viral surface forms can be difficult to achieve as the proteins are oligomeric, metastable, and heavily glycosylated. To find one crystal that would diffract to 3.4 Å and permit structure determination, the Scripps researchers had to grow ~50,000 crystals and screen the 800 largest crystals. Their crystallized trimeric, pre-fusion form of GP in complex with a neutralizing antibody derived from a human survivor of the 1995 outbreak in Kikwit, Zaire, retained all the regions required for attachment, fusion, and entry.

GP-antibody (KZ52) complex. Click on the figure to see how the GP2 subunits (white) are wound around GP1 (blue) like thread around a spool. GP1 forms a hydrophobic clamp on GP2, holding it in this metastable, pre-fusion conformation on the viral surface.

In its biologically active form, Ebolavirus GP contains two subunits with separate structural and functional roles. GP1 is responsible for receptor engagement, while GP2 mediates fusion of viral and host membranes. The crystal structure showed that the 450-kDa GP is a trimer shaped like a three-lobed chalice with the bowl of the chalice formed by three GP1 subunits and the stem of the chalice fashioned from three GP2 subunits that cradle and encircle the GP1 trimer. Here, portions of the GP2 (the internal fusion loop and heptad repeat region) together wrap around GP1, and in turn, hydrophobic residues of GP1 clamp the heptad repeat of GP2 into its metastable, pre-fusion conformation. This clamp is released upon entry into the host cell through an as-yet unidentified process, allowing GP2 to spring into its more stable, six-helix bundle conformation and trigger fusion of virus and host membranes.

This structure, the first nearly complete structure of any filovirus glycoprotein, identified a putative receptor-binding site on GP that is sequestered in the bowl of the GP trimer and further masked by a novel glycan cap domain and an unstructured mucin-like domain (mucins are heavily glycosylated proteins). GP cleavage was known to be an essential step in entry, but the precise site or role of cleavage was unknown. The structure identifies the probable cleavage site and illustrates how cleavage at this site uncaps the receptor-binding regions, freeing them for interaction with host-cell receptor(s). The crystal structure also reveals that most of GP is shielded by a thick cloak of carbohydrate and identifies the very few sites left exposed and available for antibody binding, making this structure suitable as a template for vaccines and antibodies to target these newly revealed slits in Ebolavirus’s cloak.

Research conducted by J.E. Lee, M.L. Fusco, W.B. Oswald, A.J. Hessell, D.R. Burton, and E.O. Saphire (The Scripps Research Institute).

Research Funding: U.S. National Institutes of Health, the Burroughs Wellcome Fund, and the Canadian Institutes of Health Research. Operation of the ALS is supported by the U.S. Department of Energy, Office of Basic Energy Sciences.

Publication about this research: J.E. Lee, M.L. Fusco, W.B. Oswald, A.J. Hessell, D.R. Burton, and E.O. Saphire, "Structure of the Ebola virus glycoprotein bound to an antibody from a human survivor," Nature 454, 177 (2008).