Assuming the RSV F structure is closely related to the PIV5 and hPIV3 prefusion and postfusion structures, it seems possible that the uncleaved cone-shaped RSV F may represent a postfusion conformation that folded spontaneously in the absence of a TM domain. form as judged by its golf tee morphology in the electron microscope. Heating of uncleaved F also causes conversion of F to a morphologically similar form. The reactivity of the F protein with conformation-specific mAbs and peptide binding suggest that soluble F-GCNt and membrane-bound F proteins refold through a comparable pathway. and was the RSV activation event. In light of the AZ191 evidence that PIV5 and hPIV3 F proteins can convert to the postfusion form in the absence of cleavage, the interpretation of the RSV F morphologic data is open to an alternative explanation. Assuming the RSV F structure is closely related to the PIV5 and hPIV3 prefusion and postfusion structures, it seems possible that the uncleaved cone-shaped RSV F may represent a postfusion conformation that folded spontaneously in the absence of a TM domain. Cleavage may trigger rosette formation simply by releasing the fusion peptide and the transition from a cone shape to a lollipop shape may reflect the movement after cleavage of the fusion peptide and some of the HRA residues from the side of the molecule to the tip. The precise role of HN in triggering F remains to be understood but the emerging picture indicates a regulated complex biological machine. HN Edg3 could exert its effects, for example, by influencing the stability of the F prefusion stalk in a receptor-dependent manner. It is postulated that one of the first steps in fusion after HN binding sialic acid is the dissociation of the HRB 3HB in prefusion F that makes available the binding site for the HRA peptide AZ191 (14, 19). A model for Newcastle disease virus F-mediated fusion has been proposed in which HN binding to sialic acid alters the oligomeric arrangement of HN, creates a second sialic acid binding site and in the process activates F and tethers HN to the membrane (45, 46). However, the lack of the second sialic acid binding site in PIV5 and hPIV3 HN (31, 47) makes it unlikely to be a general model of paramyxovirus HN activation of F. For PIV5, an alternative model for HN involvement in membrane fusion was proposed that involves ligand-dependent changes in the HN oligomer that are driven by subunit-subunit interactions (31). In this model, the HN dimer of dimers forms in the absence of ligand and can interact with the F protein, potentially through lateral interactions on two sides of the tetramer. Binding of cell surface receptors could trigger the partial disassembly of the HN tetramer, driven by the energy of receptor engagement, and could lead to changes in both the HN stalk region and the interaction with F, thus activating F for membrane fusion (31). Materials and Methods Proteins and Peptides. The soluble proteins PIV5 F-GCNt and hPIV3 solF0 were expressed in High Five cells by using a recombinant baculovirus as described previously (18, 19). Both proteins carry altered cleavage sites such that addition of exogenous trypsin is required for cleavage. Secreted F proteins were purified by Ni2+-chelating chromatography (18). Protein concentrations were determined by using the BCA protein AZ191 assay (Pierce, Rockford, IL). The HAt-C1 peptide (14) contains the amino acid residues of the PIV5 W3A F protein HRB and extended chain region, with an HA tag (YPYDVPDYASL) at its N terminus. C1 peptide was expressed in and purified as described previously (48). Antibodies. Ascites fluid containing PIV5 F-specific mAb F1a (25) and hybridoma supernatant containing mAb 6-7 (26), a mAb raised against the PIV5 F hyperfusogenic mutant S443P, were used. Hybridoma supernatant containing 12CA5, a mAb specific for the HA tag, was also used. IgG from F1a and 14C2 (49), a mAb specific for influenza virus M2 protein, was purified by using AZ191 AffinityPak protein A columns (Pierce), and its concentration was determined by OD280. Polyclonal antibody PAb245 was raised against a PIV5 peptide (residues 388C402) (24). Preparation of Liposomes. 1-palmitoyl-2-oleoyl- em sn /em -glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl- em sn /em -glycero-3-phosphoethanolamine, and cholesterol in chloroform (Avanti Polar Lipids, Alabaster, AL) were mixed at an 8:2:5 molar ratio, and the chloroform was evaporated under argon. The resulting lipid films were dried under vacuum overnight and resuspended in PBS at 40 mM total lipid. After five freezeCthaw cycles, the lipids were vortexed and extruded 21 times through two 100-m filters by using a miniextruder (Avanti Polar Lipids). Liposome Association Assay. For each sample,.