Enveloped viruses (those surrounded by a lipid bilayer membrane,) are covered with numerous copies of a unique molecular machine, the envelope fusion protein, that is responsible for targeting the virus to a specific cell, sensing the appropriate environment for infection, and efficiently transporting the viral genetic payload across the barrier of the target cell membrane -- the key functions in all forms of delivery. The investigators recently discovered a novel intermolecular communication phenomenon associated with the activity of influenza hemagglutinin, leading to "prion-like" autocatalysis of a major conformational change involved in driving membrane fusion. It is proposed to study the mechanism of autocatalytic cross-talk and to apply directed evolution to gain control over the biosensing capabilities of this prototypical viral fusion protein, potentiating use of these engineered proteins in lipid vesicle-based platforms for a variety of applications. Using conditions that perturb cell membranes and in vitro reconstitution of hemagglutinin into synthetic lipid bilayers, the hypothesis will be tested that modulation of local membrane properties plays a role in autocatalytic communication. Both structure-directed and random mutagenesis will be applied, the latter combined with library screening and high-throughput sequencing, to map the mutational plasticity of hemagglutinin. Finally, the knowledge of mutationally tolerant structural sites in hemagglutinin will be used to design libraries in directed evolution experiments, aiming to isolate novel mutants capable of activation and fusogenic activity in response to novel stimuli.

This project will enhance the understanding of molecular mechanisms critical to the life cycle of many viruses, with possible implications in designing future therapeutic interventions. In addition, new molecules to be generated in this project and the tools and methods used to identify them may be immediately useful in creating new components to be used in cell-specific drug or gene delivery. Other applications for such molecular components, such as development of membrane-based "smart" materials that respond to a variety of stimuli by fusing to mix their contents, may be possible, as well. Finally, the student personnel educated within this research project will be uniquely equipped with interdisciplinary skills and will be poised to lead STEM education and practice of the future.

This award by the Biotechnology, Biochemical, and Biomass Engineering Program of the CBET Division is co-funded by the Biomaterials Program of the Division of Materials Research.

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University of Tennessee Knoxville
United States
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