FIG 7.

Limited proteolysis of ΔP27 furin_mut F_ecto induces protein trimerization and association by the fusion peptide. (A) Sodium dodecyl sulfate-polyacrylamide gel showing limited proteolysis of RSV F constructs. RSV ΔP27 furin_mut F_ecto prior to digestion with trypsin is labeled “Monomer” and produces the F0 band. Two lots of RSV ΔFP furin_wt F_ecto are labeled “Trimer” and produce F1 and F2 bands in the absence of trypsin. RSV ΔP27 furin_mut F_ecto is labeled “Rosettes” and produces F1 and F2 bands after digestion with trypsin. (B) Electron microscopy image of the RSV F_ecto rosettes formed spontaneously after trypsin treatment of ΔP27 furin_mut F_ecto. The rosettes have the elongated crutch shape of postfusion RSV F_ecto, and proteins are associated at the end of the stalk where the hydrophobic fusion peptide is located. (C) Hypothetical model of ΔP27 furin_mut F_ecto as it undergoes conformational rearrangement after cleavage into the F1/F2 species. (Top) Model of uncleaved ΔP27 furin_mut F_ecto based on prefusion structure with Site A (sites II), Site C (site IV) and site Ø formed on the protein surface. HRB in blue is likely unfolded and is represented as a dashed line. A black arrow represents trypsin digestion of the monomer into F1/F2, leading to a conformational change of the prefusion-like monomer into the postfusion trimer. (Bottom) A model of RSV F_ecto rosettes formed by postfusion trimers associated by interactions between their fusion peptides (green). Two neutralizing epitopes, Sites A and C, remain on the protein surface, while the prefusion site Ø epitope is lost as the HRA (red) is largely buried by the HRB (blue).