Designing addressable proteoliposomes by using viral transmembrane proteins and biomimetic cell-free synthesis techniques.

Project Summary /Abstract Viruses present one of the most efficient mechanisms for intracellular cargo (i.e. viral genome) delivery in which interactions at the virus-host cell interface dictate the delivery pathway. For instance, enveloped viruses- those that are 'wrapped' in a lipid bilayer, deliver their genetic cargo by first interacting with extracellular receptors, triggering a reaction cascade that results in fusion of the virus- and host cell lipid membranes and cargo release into the cytosol. Harnessing the efficiency of this translocation mechanism would drastically improve cellular uptake of therapeutic and bioactive cargo, currently a major obstacle in both agricultural and pharmaceutical communities, each with major impact on human health. The research proposed in this fellowship aims to repurpose viral fusion machinery for delivering user-defined cargo to cells containing the appropriate receptors. More specifically, several virus-derived proteins have been chosen including Hemagglutinin (HA) - from Influenza, glycoprotein G (NiV-G) and fusion protein F (NiV- F) - from Nipah virus, and Spike protein - from SARS-CoV-2. These proteins represent a small selection of model proteins, all of which interact with different receptor types found on the cellular surface, providing a potential handle for targeting cells that abundantly display the specific receptors. To circumvent challenges associated with using infectious viruses or isolating membrane proteins, we will concurrently adapt existing cell-free synthesis (CFPS) techniques to produce membrane proteins and efficiently insert them into our delivery vehicles of choice- liposomes. The short-term goals of this project are to demonstrate 1) virus fusion-protein activity and delivery, and 2) improved efficiency of virus membrane insertion into liposomes using the adapted CFPS methodologies. The long-term goals include tuning the biodistribution capabilities, afforded by the virus-derived proteins, to deliver cargo to discrete and specific locations within the human body or other organism. This fellowship will provide the applicant with the financial support needed to design and test the proteoliposome-based delivery system and develop ideas that will aid the applicant's independent research program in the field of virus-inspired biomaterials.