Neutralizing ebolavirus: structural insights into the envelope glycoprotein and antibodies targeted against it

Abstract

The ebolavirus (EBOV) envelope glycoprotein (GP) is solely responsible for viral attachment to, fusion with, and entry of new host cells, and consequently is a major target of vaccine design efforts. Recently determined crystal structures of key antibodies in complex with their EBOV epitopes have provided insights into the molecular architecture of GP and defined likely hotspots for viral neutralization. In this review, we discuss the structural basis for antibody-mediated neutralization of ebolavirus and its implications for novel therapeutic or vaccine strategies.

Figure 1. Overall structure of EBOV GP

(a) Molecular surface of the GP trimer viewed on its side and down its three-fold axis. Monomer A is colored according to its subdomains: GP1 base- green; GP1 head- blue; GP1 glycan cap- cyan; GP2 N-terminus- red; GP2 internal fusion loop- orange; and GP2 HR1- yellow. (b) Molecular surface of the EBOV GP chalice and cradle. Three lobes of GP1, shown in shades of gray, form the GP chalice, and three subunits of GP2 (orange) wrap around the base of the chalice to form the cradle. Adapted from [22].

Figure 2. Ebolavirus GP-mediated entry

Ebolavirus is thought to enter cells through receptor-mediated endocytosis (a) Initially, the metastable, prefusion EBOV GP may bind low affinity lectins or another unidentified receptor at the cell surface for viral attachment. (b) Subsequently, Ebolavirus is internalized and trafficked to the endosome, where host cathepsins cleave GP to remove the glycan cap and mucin-like domain. (c) The newly exposed surface may allow either tighter binding to the host surface receptor or binding to a second cellular factor or receptor in the endosome that could then trigger conformational changes in the GP2 fusion subunit. (d) Structural rearrangements in GP2 allow HR1 to form a single 44-residue helix and position the internal fusion loop for insertion into the host endosomal membrane. Upon insertion in the host membrane, the internal fusion loop adopts a 3₁₀ helix. (e) Based on studies in the influenza virus, more than one trimer of GP2 may be required during the membrane fusion process. (f) The formation of the low energy 6-helix bundle (6HB) requires HR2 and MPER to swing from the viral membrane towards the host membrane and pack against the trimeric bundle of HR1. These rearrangements juxtapose the EBOV GP’s internal fusion loop and transmembrane domain, thus facilitating the fusion of the host and viral membranes. This figure is adapted from [22].

Figure 3. Locations of neutralizing epitopes on EBOV GP

The locations of the Zaire ebolavirus neutralizing antibodies are mapped onto the molecular surface of the prefusion EBOV GP structure. In general, there are at least three regions on EBOV GP which have elicited neutralizing or protective epitopes. The KZ52 neutralizing epitope, which likely overlaps with mAb 133/3.16 (green), is located at a non-neutralizing site at the base of the EBOV GP chalice (colored in orange). This epitope is primarily composed of GP2 residues 505–514 and 549–556. A second neutralizing epitope termed 226/8.1 (colored red) is centered in the vicinity of the cathepsin cleavage site around residues 134, 194 and 195. The loop between residues 189–213 is disordered in the crystal structure and is shown as green dots. The mucin-like domain is the site of at least three linear neutralizing epitopes (modeled as white lines). These three linear neutralizing epitopes (residues 401–417, 389–405 and 477–493) map to unstructured and non-glycosylated regions on the mucin-like domain.

Figure 4. EBOV GP-neutralizing antibody interactions

(a) EBOV GP-KZ52 interactions. KZ52 recognizes a discontinuous epitope at the base of the EBOV GP chalice, and bridges the N terminus and internal fusion loop of GP2 to the N terminus of GP1. One EBOV GP monomer is colored and labeled according to Figure 1a and the Fab heavy and light chains are colored in black and gray, respectively. Selected side-chain interactions at the GP-KZ52 interface are magnified in the inset box. Note that in the wild-type Zaire ebolavirus sequence, position 42 contains a threonine, rather than the valine mutant used here for crystallization. (b) 2-D schematic of the interactions between EBOV GP and KZ52. Van der Waals interactions are illustrated by blue semi-circles and hydrogen bonds by dashed lines. (c) EBOV GP-13F6-1-2 interactions. 13F6-1-2 utilizes a rare Vλ_x light chain and in contrast to other antibody-peptide interactions, the EBOV GP peptide epitope of 13F6-1-2 binds in a diagonal fashion, recognizing an unstructured, non-glycosylated linear epitope corresponding to residues 404–412 in the mucin-like domain. The EBOV GP peptide is colored in yellow and 13F6-1-2 heavy and light chains are colored in green and blue, respectively. (d) Comparison of peptide binding orientations in Vθ/Vκ and Vλ_x-containing antibodies. Left panel, the peptide binding to the Vλ_x-containing 13F6-1-2 antibody (PDB code: 2QHR) is shown in yellow. Right panel, the light and heavy chains of Vλ/Vκ antibodies were superimposed, but for clarity only the peptides are shown (PDB code: 1CU4, red; 1TJG, brown; 1F58, blue; 1ACY, green; 1NAK, yellow; 1SM3, magenta; 1GGI, cyan; 1CFN, orange; and 1CE1, black). This figure is adapted from [22,54].