The persistence of the current epidemic is in part a result of the lack of any vaccine, and any approved post- exposure therapies. Progress in the development of such preventative and therapeutic agents has been slow due to a limited understanding of the Ebola viral life cycle. Like all enveloped viruses, entry of Ebolaviruses into target cells requires fusion of the viral membrane to a cell membrane. This process is catalyzed by the envelope glycoprotein (GP). GP is the only viral protein exposed on the surface of Ebola virions, making it an attractive target for vaccines and antiviral therapies. The significant energetic barrier to fusing viral and cell membranes is surmounted by transition of GP to successively lower energy conformations. But virtually no information presently exists on the structural dynamics of GP, the impact of receptor binding on GP conformation, or on the mechanism by which GP catalyzes the fusion of viral and cell membranes. Given the central role of envelope glycoprotein conformational changes in the mechanism of enveloped viral entry, an understanding of Ebolavirus entry requires a deep insight into the conformational landscape of GP. Here, we propose the application of an integrated framework involving smFRET imaging, and kinetic and thermodynamic analysis. We will apply this framework to probe the conformational dynamics of individual trimeric GP molecules on the surface of intact pseudovirions, interrogate their interaction with cellular receptors and antibodies, and elucidate the impact of changes in pH. This approach will bring new light to our understanding of Ebolavirus entry specifically, and enveloped virus entry in general. In addition, we will design GP variants that stably expose antigenic surfaces, and use our smFRET-based framework as a robust and high-throughput means of assaying the conformational landscape of these potential immunogens. We will thereby develop smFRET as a tool for structural vaccine design.
This proposal aims to apply single-molecule fluorescence resonance energy transfer (smFRET) imaging to understand how Ebolaviruses enter target cells in terms of the structural dynamics of their envelope glycoprotein (GP). It further aims to engineer GP variants that stably express antigenic hotspots, and use smFRET to assay their conformation. These variants could provide novel immunogens for structural vaccine design.
| Das, Dibyendu Kumar; Govindan, Ramesh; Niki?-Spiegel, Ivana et al. (2018) Direct Visualization of the Conformational Dynamics of Single Influenza Hemagglutinin Trimers. Cell 174:926-937.e12 |
