Intermediates of Filovirus Membrane Fusion Explored Through Antibody Engineering and Protein Design
Koellhoffer, Jayne F.
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Ebolavirus and Marburgvirus (MARV) belong to the highly pathogenic Filoviridae family of viruses that cause severe hemorrhagic fever. I have employed two general strategies to study the envelope glycoprotein (GP) of Ebolavirus, MARV, and a novel virus, the California Academy of Science Virus (CASV) that contains a filoviruslike GP despite being predominantly arenavirus-like.;In Chapter 2, we strive to develop a cross-neutralizing monoclonal antibody that can be used therapeutically to protect against the two most lethal Ebolavirus strains, Zaire (EBOV) and Sudan (SUDV). Our approach utilizes protein engineering to extend the neutralization capabilities of two known monoclonal antibodies, 16F6 and KZ52, which specifically bind SUDV and EBOV GP, respectively. Current work has focused on the humanization of 16F6, a murine antibody, and engineering of the humanized 16F6 scaffolds in order to identify cross-strain binders. Chapter 3 describes the development of synthetic antibodies targeting intermediates of EBOV GP along the viral entry pathway.;Fusion of the viral and host cell membranes is a necessary first step for infection by enveloped viruses. The "Class I viruses" use their envelope glycoproteins to mediate this membrane fusion process via the formation of a stable six-helix bundle in the ectodomain of the transmembrane subunit. In the second half of my thesis, I present our studies on the envelope glycoprotein transmembrane subunit, GP2, of MARV (Chapter 4) and CASV (Chapter 5). For both proteins, we observed that the core domain backbone conformation was essentially identical to that of the previously described EBOV GP2, forming a six alpha-helix bundle. We also observed that both MARV and CASV GP2 show a pattern of pH-dependent stability, with the proteins being significantly more stable at lower pH. We hypothesize that this pH-dependent stability provides a mechanism for conformational control such that the post-fusion six-helix bundle is promoted in the environments of appropriately matured endosomes. We provide a structural rationale for this pH-dependent stability, involving a high-density array of core and surface acidic side chains at the midsection of the both structures. These studies yield insights into the entry mechanisms for both viruses.