Structural basis for Marburg virus neutralization by a cross-reactive human antibody

Abstract

The filoviruses, including Marburg and Ebola, express a single glycoprotein on their surface, termed GP, which is responsible for attachment and entry of target cells. Filovirus GPs differ by up to 70% in protein sequence, and no antibodies are yet described that cross-react among them. Here, we present the 3.6 Å crystal structure of Marburg virus GP in complex with a cross-reactive antibody from a human survivor, and a lower resolution structure of the antibody bound to Ebola virus GP. The antibody, MR78, recognizes a GP1 epitope conserved across the filovirus family, which likely represents the binding site of their NPC1 receptor. Indeed, MR78 blocks binding of the essential NPC1 domain C. These structures and additional small-angle X-ray scattering of mucin-containing MARV and EBOV GPs suggest why such antibodies were not previously elicited in studies of Ebola virus, and provide critical templates for development of immunotherapeutics and inhibitors of entry.

Figure 1. Structure of Marburg virus GP

(A) Crystal structure of MARV GPcl (GP1; purple and GP2; dark gray) superimposed with the equivalent structure of EBOV (PDB ID; 3CSY, GP1; blue and GP2; light gray). The glycan cap of EBOV GP is deleted for clarity. The yellow box outlines the MR78 epitope and putative receptor-binding site. The black box outlines the interaction site of the MARV-specific helix α2 of GP1 (purple) with the fusion loop of GP2 (dark grey). The visible N-linked sugars on MARV and EBOV GPcl crystal structures are shown as dot models. MARV GPcl bears glycans at positions N94 and N171, which are not glycosylated in EBOV. See also Figure S1. (B) Top view of GP. (C) MARV GP lacks the intra-GP1 disulfide bond of EBOV. C147 of EBOV (blue) is replaced by H131 in MARV (purple), and the corresponding polypeptide traces outward from the trimer center. The orange box outlines the glycan attachment sites at the base of each GP. (D) Residues 172-180 of MARV form an α helix (α2) that packs against both N- and C-terminal arms of the fusion loop. In ebolaviruses, the equivalent residues are predict to form a loop rather than a helix and are disordered in crystal structures. (E) At the base of GP, MARV bears a glycan attached to N171 while EBOV bears a glycan attached to N40 (drawn as an oval as it was not included in the EBOV crystal structure).

Figure 2. MR78 binds both MARV and EBOV GPcl at the apex of GP1

(A) 3.6 Å crystal structure of MARV GPcl in complex with Fab MR78. Each GP1 is colored a different shade of purple, GP2 is gray, and the MR78 Fab is in yellow. (B) 8 Å structure of EBOV GPcl in complex with Fab MR78, determined by molecular replacement and rigid body refinement. Each EBOV GP1 is colored a different shade of blue and GP2 is gray. See also Figure S2. Fab MR78 (yellow) binds the apex of GP1 of both viruses.

Figure 3. MR78 recognizes a conserved epitope at the apex of cleaved GP1

(A) Conservation of the MR78 epitope among filovirus GPs, mapped onto one monomer of MARV GPcl. Sequence alignment was performed in ebolavirus (Ebola, Sudan, Reston, Taï Forest, Bundibugyo), marburgvirus (Musoke, Angola, Popp, Ci67, DRC1999, Ravn), and cuevavirus (Lloviu) genuses. Residues identical across the filoviruses are colored red; residues that possess strong similarity, magenta; weak similarity, pink; no similarity, gray. (B) The apex of cleaved MARV GP1, where Fab MR78 binds, forms a wave crest-and-trough morphology (magenta). The hydrophilic crest and the hydrophobic trough each contain residues previously shown to be critical for virus entry (Dube et al., 2009; Manicassamy et al., 2005; Manicassamy et al., 2007; Mpanju et al., 2006). The diagonal black line indicates the base of the trough. See also Figure S4. (C) Surface representation of the interface between one monomer of MARV GPcl (bottom) and Fab MR78 (top). CDR H1 is colored red; CDR H2, orange; CDR H3, purple; CDR L1, blue; CDR L2, green; CDR L3, forest green. The footprint on MARV GPcl is colored according to the CDR that mediates the contact. GP residues contacted by MR78 are indicated, and colored according to the CDR that mediates the contact (CDR names in parentheses).

Figure 4. Similarity in recognition of the putative receptor-binding site by MR78 and the Ebola virus glycan cap

(A) The CDR H3 of MR78 (yellow) reaches into the hydrophobic trough of GP1 (purple). F111.2 and Y112.2 of CDR H3 interact with P63, S67, W70, F72, I95 and I125 of MARV GP. (B) Similar residues of the EBOV glycan cap (white blue) bind into this trough on the surface of EBOV GP (blue), prior to enzymatic cleavage. Here, F225 and Y232 of the glycan cap interact with P80, T83, W86, F88, L111 and V141 in the trough (PDB ID; 3CSY).

Figure 5. MARV and EBOV present different surfaces for antibody recognition

(A and B) Molecular envelopes of mucin-containing MARV and EBOV GP ectodomains determined by SAXS. Rendered Gaussian distributions of molecular envelopes are illustrated in light gray, with ribbon models of the crystallized MARV GPcl and EBOV GPΔmuc trimers to scale and overlaid for comparison. The trimers are illustrated as ribbons. Note that the glycan cap was removed from MARV GP used in crystallization in order to improve diffraction, but was contained in the complete MARV GP used for SAXS. The glycan cap did not inhibit diffraction of EBOV GP and is included in the EBOV GP crystal structure. MARV GPcl is colored in purple (GP1) and gray (GP2). EBOV GPΔmuc is colored blue (GP1), white blue (GP1 glycan cap) and gray (GP2). MARV GP is drawn in two possible orientations because definitive placement of polypeptide is challenging at this resolution. In either orientation however, the mucin-like domains of MARV project sideways, equatorially or downwards from the core of GP. In MARV, the mucin-like domain is attached to both GP1 and GP2. By contrast, in EBOV, the mucin-like domain is attached solely to GP1, there is no anchor at the base. Both these SAXS experiments and previous electron tomography (Tran et al., 2014) agree on the upward projection of the mucin-like domains in EBOV. See also Figure S5. (C) Differing positions of the mucin-like domains between MARV and EBOV may lead to elicitation of different types of antibodies. The lower position and GP2 anchor of the mucin-like domain of MARV may better mask the base of GP, but expose its upper surfaces, allowing antibodies like mAb MR78 to be elicited. The upwards projection of the EBOV mucin-like domain and absence of any GP2 anchor, appear to better mask upper surfaces, but expose the base, allowing antibodies such as KZ52 (Lee et al., 2008), 2G4, 4G7 (Murin et al., 2014) and 16F6 (directed against Sudan ebolavirus (Dias et al., 2011; Bale et al., 2012)) to be elicited.