Fusion proteins facilitate the combining of two membranes in a process such as viral infection. Hemagglutinin (HA) is viral fusion protein that undergoes an irreversible conformational change upon acidification to catalyze the fusion of endosomal and viral membranes during influenza infection. This rearrangement, which is essential for fusion activity, exposes a 20 amino acid sequence, the fusion peptide, which is previously buried within the protein’s core. The conformational change of HA to the fusion active state has been associated with a cell-wide autocatalytic activation. Other viral fusion proteins go through similar rearrangements in order to deliver viral DNA to the host cell. Understanding and engineering the mechanism of viral fusion protein activity will lend insight toward the design of efficient delivery systems for transport of drugs or genes across cell membranes. Here we probe the kinetics of autocatalytic activation and aggregation. We also suggest an alternative pathway to activation can be created by engineering the fusion peptide region of HA and that a ligand binding to this region can activate the conformational change.  <br/>We demonstrate, using epitope-tagged HA mutants that enable observation of the conformational change, that HA expressed on the cell surface showed activation of hemagglutinin even in the absence of low pH. The engineered HA have the ability to bind a fluorescent dye upon refolding.   Both samples incubated with the dye at pH 5 and neutral pH showed similar fluorescence. This suggests that hemagglutinin activation can be driven by agents that bind the fusion peptide. The binding curve indicates a first-order plus delay phenomenon, which supports the need for multiple HA trimers for activation. The dye presumably binds the tag in the preactivation state, changes its position, and destabilizes the preactivation conformation, allowing the HA timer to refold to the activated conformation. This result suggests it may be possible to create fusogenic HA proteins responding to a variety of stimuli.    <br/>The fusion peptide region of HA was also epitope tagged with c-myc to enable simple observation of the conformational state by immunofluorescence. Cells co-expressing dye sensitive and c-myc mutants showed full activation of HA trimers in the presence of the dye.  This suggests that trimers activated by a ligand binding the fusion peptide region can activate c-myc trimers through an autocatalysis.

Characterization of an engineered, ligand-induced, viral membrane fusion protein

251st National Meeting (2016)

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