Crystal Structure of the Marburg Virus GP2 Core Domain in Its Postfusion Conformation

Marburg virus (MARV) and Ebola virus (EBOV) are members of the family <i>Filoviridae</i> (“filoviruses”) and cause severe hemorrhagic fever with human case fatality rates of up to 90%. Filovirus infection requires fusion of the host cell and virus membranes, a process that is mediated by the envelope glycoprotein (GP). GP contains two subunits, the surface subunit (GP1), which is responsible for cell attachment, and the transmembrane subunit (GP2), which catalyzes membrane fusion. The GP2 ectodomain contains two heptad repeat regions, N-terminal and C-terminal (NHR and CHR, respectively), that adopt a six-helix bundle during the fusion process. The refolding of this six-helix bundle provides the thermodynamic driving force to overcome barriers associated with membrane fusion. Here we report the crystal structure of the MARV GP2 core domain in its postfusion (six-helix bundle) conformation at 1.9 Å resolution. The MARV GP2 core domain backbone conformation is virtually identical to that of EBOV GP2 (reported previously), and consists of a central NHR core trimeric coiled coil packed against peripheral CHR α-helices and an intervening loop and helix–turn–helix segments. We previously reported that the stability of the MARV GP2 postfusion structure is highly pH-dependent, with increasing stability at lower pH [Harrison, J. S., Koellhoffer, J. K., Chandran, K., and Lai, J. R. (2012) <i>Biochemistry</i> <i>51</i>, 2515–2525]. We hypothesized that this pH-dependent stability provides a mechanism for conformational control such that the postfusion six-helix bundle is promoted in the environments of appropriately mature endosomes. In this report, a structural rationale for this pH-dependent stability is described and involves a high-density array of core and surface acidic side chains at the midsection of the structure, termed the “anion stripe”. In addition, many surface-exposed salt bridges likely contribute to the stabilization of the postfusion structure at low pH. These results provide structural insights into the mechanism of MARV GP2-mediated membrane fusion

Conformational Properties of Peptides Corresponding to the Ebolavirus GP2 Membrane-Proximal External Region in the Presence of Micelle-Forming Surfactants and Lipids

Ebola virus and Sudan virus are members of the family <i>Filoviridae</i> of nonsegmented negative-strand RNA viruses (“filoviruses”) that cause severe hemorrhagic fever with fatality rates as high as 90%. Infection by filoviruses requires membrane fusion between the host and the virus; this process is facilitated by the two subunits of the envelope glycoprotein, GP1 (the surface subunit) and GP2 (the transmembrane subunit). The membrane-proximal external region (MPER) is a Trp-rich segment that immediately precedes the transmembrane domain of GP2. In the analogous glycoprotein for HIV-1 (gp41), the MPER is critical for membrane fusion and is the target of several neutralizing antibodies. However, the role of the MPER in filovirus GP2 and its importance in membrane fusion have not been established. Here, we characterize the conformational properties of peptides representing the GP MPER segments of Ebola virus and Sudan virus in the presence of micelle-forming surfactants and lipids, at pH 7 and 4.6. Circular dichroism spectroscopy and tryptophan fluorescence indicate that the GP2 MPER peptides bind to micelles of sodium dodecyl sulfate and dodecylphosphocholine (DPC). Nuclear magnetic resonance spectroscopy of the Sudan virus MPER peptide revealed that residues 644–651 interact directly with DPC, and that this interaction enhances the helical conformation of the peptide. The Sudan virus MPER peptide was found to moderately inhibit cell entry by a GP-pseudotyped vesicular stomatitis virus but did not induce leakage of a fluorescent molecule from a large unilammellar vesicle comprised of 1-palmitoyl-2-oleoylphosphatidylcholine or cause hemolysis. Taken together, this analysis suggests the filovirus GP2 MPER binds and inserts shallowly into lipid membranes

Synthetic Antibodies with a Human Framework That Protect Mice from Lethal Sudan Ebolavirus Challenge

The ebolaviruses cause severe and rapidly progressing hemorrhagic fever. There are five ebolavirus species; although much is known about Zaire ebolavirus (EBOV) and its neutralization by antibodies, little is known about Sudan ebolavirus (SUDV), which is emerging with increasing frequency. Here we describe monoclonal antibodies containing a human framework that potently inhibit infection by SUDV and protect mice from lethal challenge. The murine antibody 16F6, which binds the SUDV envelope glycoprotein (GP), served as the starting point for design. Sequence and structural alignment revealed similarities between 16F6 and YADS1, a synthetic antibody with a humanized scaffold. A focused phage library was constructed and screened to impart 16F6-like recognition properties onto the YADS1 scaffold. A panel of 17 antibodies were characterized and found to have a range of neutralization potentials against a pseudotype virus infection model. Neutralization correlated with GP binding as determined by ELISA. Two of these clones, E10 and F4, potently inhibited authentic SUDV and conferred protection and memory immunity in mice from lethal SUDV challenge. E10 and F4 were further shown to bind to the same epitope on GP as 16F6 with comparable affinities. These antibodies represent strong immunotherapeutic candidates for treatment of SUDV infection