Using Cell-Free Assembled Virus-Like Proteoliposome Complexes to Deliver Cargo Directly to Cellular Cytosol

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. In this work, we propose that viral fusion machinery can be repurposed for delivering user-defined cargo to cells containing the appropriate receptors. Hemagglutinin (HA), which is a protein found on the surface of influenza viruses and is known to mediate the virus-cell fusion mechanisms, is one of the proteins explored for this goal, along with several other viral membrane proteins including NiV-F and Spike (SARS 2). To circumvent challenges associated with using infectious viruses or isolating/reconstituting membrane proteins, we use cell-free synthesis techniques to directly insert the proteins into our delivery vehicles of choice- liposomes. Not only do we show that we can successfully assemble folded and functional viral membrane proteins, we also show that our virus-proteoliposome complexes can deliver both genomic and intact proteins directly to the cell cytosol.