The fusion of viral and cellular membranes, an event known to be promoted by a number of viral fusion proteins, is both a fundamental part of the viral life cycle and a paradigm for other membrane fusion events. The well-characterized influenza hemagglutinin protein promotes fusion after undergoing a low pH induced conformational change. However, many viral fusion events occur at neutral pH, and the mechanisms and factors involved in triggering neutral pH fusion remain elusive. It is proposed to study the neutral pH fusion event by further examination of fusion promoted by the F protein of the prototypic paramyxovirus SV5. Quantitative fusion assays measuring both lipid and cellular contents mixing will be employed to examine the effects of factors such as temperature and lipid content on the rate and extent of fusion promoted by both the wild type SV5 F protein and a mutant F protein which has been shown to promote enhanced syncytia formation. To obtain a soluble F protein for biochemical and structural characterization, an F protein in which the transmembrane and cytoplasmic tail domains has been replaced by the signal sequence for a lipid anchor will be created. The fusion characteristics of the lipid-anchored F protein will be examined, and the solubilized ectodomain fragment will be purified. Finally, the compelling hypothesis that F protein undergoes a conformational change related to fusion will be examined using a variety of methods, including protease sensitivity, CD and tyrosine fluorescence spectroscopy, and reaction with anti-peptide antibodies. A detectable conformational change will be used to assay for factors that induce these changes, and thus trigger fusion.
