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

Abstract

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 goldlabeled 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.fusion protein refolding | membrane fusion | electron microscopy E nveloped viruses such as influenza virus, human immunodeficiency virus (HIV), and parainfluenza virus 5 (PIV5) encode class I fusion proteins that mediate coalescence of the viral and target membranes (1-6). Homotrimeric class I fusion proteins are synthesized as biologically inactive precursors that are activated by proteolytic cleavage, which generates a new N-terminal hydrophobic sequence known as the fusion peptide. The crystal structures of the paramyxovirus F-protein in its metastable prefusion form and the human parainfluenza virus 3 (hPIV3) F-protein in its very stable postfusion form, and also the influenza virus HA in pre-and postfusion conformations, reveal a unique protein architecture that undergoes large-scale, irreversible refolding during membrane fusion (7-10).These structures provided snapshots of the locations of the critical fusion peptide in the initial and final states: In the prefusion form, the fusion peptide is located along the protein surface, near the virus membrane; while in the postfusion state it is positioned to penetrate into the viral membrane through the formation of a hairpin conformation. Nevertheless, photochemical labeling (11) indicates that the fusion peptide engages with the target membrane in ...

Identification of a Region in the Stalk Domain of the Nipah Virus Receptor Binding Protein That Is Critical for Fusion Activation

“…To investigate whether the chimeric pro- teins G 1-180 -HN 124-571 and G 1-(2A)186 -HN 124-571 are defective at initial F triggering or at this later stage in the fusion process, we determined whether the defect is before or after insertion of the fusion protein into the target cell. Insertion of F's fusion peptide into the target cell indicates that the prehairpin intermediate has formed (18,28,(41)(42)(43); at this stage, F mediates attachment to the target cells, and disengagement of the receptor binding protein does not lead to release from the target cell (13,18,28,40,41,44). To determine whether the G 1-180 -HN 124-571 and G 1-(2A)186 -HN 124-571 chimeric proteins activate NiV F up to the stage of the prehairpin intermediate with fusion peptide inserted but then fail to complete the fusion process, we modified the assay described in Fig.…”

“…The bound RBC pool consisted only of RBCs retained via F's insertion into the target cell, forming a bridge between the glycoproteinexpressing cell and the target RBCs (Fig. 9) (40,41). If the chimeric (Fig.…”

Fusion Activation through Attachment Protein Stalk Domains Indicates a Conserved Core Mechanism of Paramyxovirus Entry into Cells

“…The attachment protein binds cellular surface receptors, initiating a sequence of events, involving interactions of the attachment protein with F, followed by activation of the fusion protein (1,4,5). Upon activation, the cleaved, metastable, prefusion form of the F protein (6,7) rearranges to insert a hydrophobic fusion peptide into the target cell membrane and through a pre-hairpin intermediate (8) ultimately refolds irreversibly into a stable, postfusion form (9)(10)(11)(12), resulting in viral and cellular membrane merger.…”

Probing the Functions of the Paramyxovirus Glycoproteins F and HN with a Panel of Synthetic Antibodies

“…Atomic structures of NDV, human PIV3 (hPIV3), and RSV F in the postfusion form reveal that a large refolding event occurs to convert prefusion F to postfusion F in which part of the globular head domain rearranges to form a six-helix bundle (36)(37)(38)(39). These structures, along with peptide inhibitory data, suggest a model for F-mediated membrane fusion where, upon activation, F1/F2 rearranges to insert a hydrophobic fusion peptide from the N terminus of F1 into the target cell membrane, forming a prehairpin intermediate (40). This relatively extended structure tethers the virus to the cell membrane and collapses to form the stable six-helix bundle of the postfusion structure.…”