Capture and imaging of a prehairpin fusion intermediate of the paramyxovirus PIV5

• Published in: Proceedings of the National Academy of Sciences - PNAS 2011-12, Vol.108 (52), p.20992-20997
• Main Authors: Kim, Yong Ho, Donald, Jason E, Grigoryan, Gevorg, Leser, George P, Fadeev, Alexander Y, Lamb, Robert A, DeGrado, William F
• Format: Article
• Language: English
• Subjects: Binding sites; Biochemistry; Biological Sciences; Cell Membrane - metabolism; Cell membranes; Chromatography, High Pressure Liquid; Computational modeling; Computed tomography; Crystallography; Electron microscopy; Fusion protein; Hemagglutinins; Hydrophobicity; image analysis; Immunohistochemistry; Influenza; Influenza A virus; Influenza virus; Lipids; Mathematical models; Membranes; Microscopy, Electron, Transmission; Models, Biological; Orthomyxoviridae; Paramyxoviridae - metabolism; Paramyxovirus; plasma membrane; Protein Conformation; Protein Folding; Proteins; Respirovirus; spatial distribution; Ultracentrifugation; Viral Fusion Proteins - metabolism; Viral morphology; Virions; Virus Attachment; Viruses
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1116034108

During cell entry, enveloped viruses fuse their viral membrane with a cellular membrane in a process driven by energetically favorable, large-scale conformational rearrangements of their fusion proteins. Structures of the pre- and postfusion states of the fusion proteins including paramyxovirus PIV5 F and influenza virus hemagglutinin suggest that this occurs via two intermediates. Following formation of an initial complex, the proteins structurally elongate, driving a hydrophobic N-terminal "fusion peptide" away from the protein surface into the target membrane. Paradoxically, this first conformation change moves the viral and cellular bilayers further apart. Next, the fusion proteins form a hairpin that drives the two membranes into close opposition. While the pre- and postfusion hairpin forms have been characterized crystallographically, the transiently extended prehairpin intermediate has not been visualized. To provide evidence for this extended intermediate we measured the interbilayer spacing of a paramyxovirus trapped in the process of fusing with solid-supported bilayers. A gold-labeled peptide that binds the prehairpin intermediate was used to stabilize and specifically image F-proteins in the prehairpin intermediate. The interbilayer spacing is precisely that predicted from a computational model of the prehairpin, providing strong evidence for its structure and functional role. Moreover, the F-proteins in the prehairpin conformation preferentially localize to a patch between the target and viral membranes, consistent with the fact that the formation of the prehairpin is triggered by local contacts between F- and neighboring viral receptor-binding proteins (HN) only when HN binds lipids in its target membrane.