(320b) Engineering Ligand-Activated Fusogens from Influenza Hemagglutinin

The envelope protein of influenza virus, hemagglutinin (HA), represents one of the best studied paradigms among protein machines that facilitate fusion of lipid bilayer membranes. These proteins demonstrate environmentally-triggered conformational changes that activate their fusogenic function. While understanding the structure and function of these systems would benefit efforts to design and engineer regulated membrane fusogens for a range of applications, including drug or non-viral gene delivery, many details regarding both the sensing and actuating functions of these molecular switch proteins remain to be elucidated. Here, we have engineered mutants of fowl plague virus HA that respond to alternative environmental stimuli compared to the wild-type protein. We created a mutant designed to exhibit conditional binding to a fluorescent ligand (FlAsH) following activation and discovered that FlAsH binding is sufficient to induce the HA conformational transition. Analysis of engineered HA demonstrated that allmutants were unable to induce membrane fusion despite activation of the fusion-associated conformational change; however, co-expressing these mutants with fusion-competent, wild-type HA enabled recovery of fusion triggered by the alternative stimuli, indicating that modular fusogenic systems can be created in which HAs engineered for novel sensing functions can be mixed with other HAs that provide the actuating component of the system. Finally, studies of both conformational activation and fusion indicate that cholesterol depletion induces a significant delay in the kinetics of conformational activation and the associated extent of fusion, and exogenous, soluble HA fusion peptide triggers the HA conformation change in the absence of any other stimulus. Taken together, these results suggest that membrane properties play an important role in HA function. Our results motivate further protein engineering efforts to develop membrane fusogens responding to additional environmental stimuli and compel further studies of HA behavior in synthetic systems with defined phospholipid composition.