Enveloped viruses express transmembrane fusion proteins that undergo triggered conformation changes leading to induction of membrane fusion during host cell infection. The fusion protein of influenza virus, hemagglutinin (HA), is activated by exposure to acidic pH and undergoes irreversible refolding that relocates a membrane-active fusion peptide from a site buried in the preactivation structure. We have studied the pH-induced conformation change of fowl plague virus HA in transfected cells using conformationally sensitive antibody staining and flow cytometry. This single-cell assay indicates that HA refolding on an individual cell is an "all or nothing" phenomenon; furthermore, newly synthesized HA expressed on cells cultured following an activating pH pulse is spontaneously refolded. Spontaneous refolding is inhibited by proteolytic cleavage of the fusion peptide region of the activated HA prior to reculturing, and microscopic analysis indicates that activated HA are aggregated on the cell surface. These data indicate that HA refolding is autocatalytic and parallels the autocatalytic refolding observed for prions. We have extended this experimental system to library screening to isolate HA mutants with altered induction pH. Cells expressing library members with activation pH shifted to 5.6 and 4.8 (from 5.2 for wild-type) have been isolated. Characterization of these mutants is currently underway.

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