Spatial Localization of the Ebola Virus Glycoprotein Mucin-Like Domain Determined by Cryo-Electron Tomography

Abstract

bThe Ebola virus glycoprotein mucin-like domain (MLD) is implicated in Ebola virus cell entry and immune evasion. Using cryo-electron tomography of Ebola virus-like particles, we determined a three-dimensional structure for the full-length glycoprotein in a near-native state and compared it to that of a glycoprotein lacking the MLD. Our results, which show that the MLD is located at the apex and the sides of each glycoprotein monomer, provide a structural template for analysis of MLD function. The filoviruses Ebolavirus (EBOV) and Marburgvirus cause fatal hemorrhagic fevers in humans, with mortality rates approaching 90% (1, 2). Consequently, considerable effort is focused on developing vaccines and therapeutics to prevent and treat filoviral infections. All filoviruses display multiple copies of a single membrane-anchored glycoprotein (GP) projecting from their envelopes (reviewed in reference 3). Like most class I viral fusion proteins, GP is a trimer in which each monomer is a disulfide-bonded complex of a receptor binding subunit (GP1) and a fusion subunit (GP2) (reviewed in references 3 and 4). The structure of the trimeric ectodomain was described as a "chalice" consisting of a glycan cap, a head, and a base (5). The Ebola virus GP is the target of multiple neutralizing antibodies (Ab), several of which are effective in preventing the onset of disease in nonhuman primates when administered as part of a monoclonal Ab (MAb) cocktail 1 or 2 days after viral exposure (6, 7).The mucin-like domain (MLD) is a highly glycosylated region spanning EBOV GP1 residues 313 to 501 (Fig. 1A). Although it is dispensable for EBOV infections in vitro (8, 9) and is not highly conserved (5), many functions have been attributed to the MLD. These include influencing GP structure (10), enhancing viral attachment to target cell surfaces (11, 12), protecting conserved regions of GP, such the receptor binding site, from antibody recognition (10, 13), and masking immune regulatory molecules, such as major histocompatibility complex 1 (MHC1), on infected cell surfaces (14, 15). In addition, many neutralizing antibodies, including two that comprise part of a promising therapeutic cocktail (7), are directed against the MLD (13,16,17). The goal of the present study was to provide structural information on the MLD of GP from EBOV as an aid to understanding the multiple functions of filoviral MLDs.The Ebola virus GP crystal structure was obtained using a truncated protein that lacks both the MLD and the transmembrane domain (5) (Fig. 1A) and gives only a partial representation of the GP structure. To localize the MLD on Ebola virus GP in as near a native state as possible, we produced entry-competent virus-like particles (VLPs), as described previously (18), and performed cryo-electron tomography and subtomogram averaging of GP spikes using previously described methods (19).We imaged Ebola virus VLPs that express GPs in either the full-length (FL-GP) (Fig. 1B and C) or MLD-deleted (⌬Muc-GP) state ( Fig. 1D and E), with the goal of identify...

Structures of protective antibodies reveal sites of vulnerability on Ebola virus

“…These data also support the current model of the mucin-like domain as a physically separate portion of GP1, likely extending outward from the top of GP (39,41), because no mucin-like domain antibody in this study inhibits binding of any antibody against the remaining core of GP.…”

“…S2A). The mucin-like domains comprise nearly half the mass of GP and are thought to be heavily glycosylated and disordered (39,(41)(42)(43)(44). Notably, these domains are not visible in our class averages (Fig.…”

Both Epistasis and Diversifying Selection Drive the Structural Evolution of the Ebola Virus Glycoprotein Mucin-Like Domain

“…This clustering of epistatic sites could be the product of a relatively high mutation rate in this region, reflecting its dispensability for cell entry (10), but this possibility has, to date, not been fully investigated. Third, and most critically, this small cluster is right in the mucin-like domain (MLD), a region that spans residues 308 to 501 (7,8) and that has been hypothesized to shield the glycoprotein from antibodies (9,39). The human antibody Fab KZ52 was removed to facilitate visualization.…”

“…They discovered a strikingly high ratio of nonsynonymous-to-synonymous mutations concentrated in the heavily glycosylated mucin-like domain (MLD) of GP, a region that spans residues 308 to 501 (7,8). While it has been proposed that the MLD plays a key role in immune evasion (9), the flexible structure and dispensability of this domain (10) might explain its propensity to maintain high substitution rates throughout the outbreak.…”

Composite Model of Full GP Structure of Ebola Virus Envelope Glycoprotein

“…This figure combines the published X-ray crystallographic (core) [1] and cryoelectron tomography (global) [2] structures, together now with in silico modeled sections of the mucinlike domain and separately the GP stalk [3]. The latter are missing from earlier published crystal structure determinations.…”