Transmembrane orientation and possible role of the fusogenic peptide from parainfluenza virus 5 (PIV5) in promoting fusion

Abstract

Membrane fusion is required for diverse biological functions ranging from viral infection to neurotransmitter release. Fusogenic proteins increase the intrinsically slow rate of fusion by coupling energetically downhill conformational changes of the protein to kinetically unfavorable fusion of the membrane-phospholipid bilayers. Class I viral fusogenic proteins have an N-terminal hydrophobic fusion peptide (FP) domain, important for interaction with the target membrane, plus a C-terminal transmembrane (C-term-TM) helical membrane anchor. The role of the water-soluble regions of fusogenic proteins has been extensively studied, but the contributions of the membrane-interacting FP and C-term-TM peptides are less well characterized. Typically, FPs are thought to bind to membranes at an angle that allows helix penetration but not traversal of the lipid bilayer. Here, we show that the FP from the paramyxovirus parainfluenza virus 5 fusogenic protein, F, forms an N-terminal TM helix, which self-associates into a hexameric bundle. This FP also interacts strongly with the C-term-TM helix. Thus, the fusogenic F protein resembles SNARE proteins involved in vesicle fusion by having water-soluble coiled coils that zipper during fusion and TM helices in both membranes. By analogy to mechanosensitive channels, the force associated with zippering of the water-soluble coiled-coil domain is expected to lead to tilting of the FP helices, promoting interaction with the C-term-TM helices. The energetically unfavorable dehydration of lipid headgroups of opposing bilayers is compensated by thermodynamically favorable interactions between the FP and C-term-TM helices as the coiled coils zipper into the membrane phase, leading to a pore lined by both lipid and protein.T he basic mechanisms of viral membrane fusion have been studied extensively, but major gaps remain in our understanding of the relative roles of lipidic intermediates and viral fusogenic proteins in lowering the energy barrier for the overall process (1-4). The most common mechanistic hypothesis concerning enveloped viral fusion is that fusogenic proteins primarily serve to bring the target cell and viral membranes into proximity. Fusion occurs in a multistep process, in which the virus first binds to a specific receptor; this event and/or other environmental cues then cause a conformational change in the protein, leading to a metastable state with an exposed hydrophobic fusion peptide (FP) that binds to the target membrane. Once engaged with the bilayer, a second energetically favorable conformational change in the fusogenic protein then exerts a force pulling the FP toward the viral membrane, in effect reeling the host and viral membranes together.The conformational changes involved in the water-soluble portions of viral fusogenic proteins have been largely elucidated, but the roles of the membrane-binding FP and the C-terminal transmembrane (C-term-TM) anchor are less clear. After the crystal structure of the prefusogenic form of influenza hemagglutinin (HA) was solved...

pH-triggered, activated-state conformations of the influenza hemagglutinin fusion peptide revealed by NMR

“…In its final stage, fusion is then believed to progress through a mechanism where the N-and C-terminal helices of multiple hemagglutinin trimers zipper together in an antiparallel fashion to form a competent fusion pore (20). Opening of the fusion peptide therefore appears a requisite step for adopting a helical structure that is sufficiently long to traverse the lipid bilayer, which is needed to allow formation of the stipulated oligomeric fusion pore structure, assembled from fusion peptides and HA2 C-terminal transmembrane helices (38).…”

A bundling of viral fusion mechanisms

“…In their study, Donald et al (5) present analytical ultracentrifugation results showing that the parainfluenza virus 5 (PIV5) fusion peptide assembles into hexamers in dodecylphosphocholine micelles. Minimal evidence was found for other multimeric states.…”

Efficient replication of a paramyxovirus independent of full zippering of the fusion protein six-helix bundle domain

“…Studies with class I vFMGs have shown that insertion of synthetic, soluble fusion peptides into the membrane can deform the lipid bilayer (48) and induce membrane fusion (49). Paramyxovirus fusion peptides derived from the PIV5 F protein were demonstrated to associate into a helical transmembrane bundle in lipid bilayers; this bundle interacts tightly with the corresponding PIV5 F transmembrane domains and forms a thermodynamically favorable coiled-coil in the lipid phase (13). In analogy to cellular SNARE proteins (50), this zippering of the F-protein helix in the bilayer -equivalent to the formation of the four-helix bundle in the SNARE transmembrane domain-may extend the water-soluble 6HB fusion core into the membrane and contribute part of the energy required to drive fusion (13).…”

“…Paramyxovirus fusion peptides derived from the PIV5 F protein were demonstrated to associate into a helical transmembrane bundle in lipid bilayers; this bundle interacts tightly with the corresponding PIV5 F transmembrane domains and forms a thermodynamically favorable coiled-coil in the lipid phase (13). In analogy to cellular SNARE proteins (50), this zippering of the F-protein helix in the bilayer -equivalent to the formation of the four-helix bundle in the SNARE transmembrane domain-may extend the water-soluble 6HB fusion core into the membrane and contribute part of the energy required to drive fusion (13). Critical functional interactions between fusion peptides and transmembrane domains also were reported for influenza virus HA (45) and HIV-1 gp41 (47).…”

“…An alternative model assumes that the fusion peptide and transmembrane domains may assemble directly into an antiparallel bundle that extends the water-soluble 6HB core into the lipid phase, functioning as a fusion pinprick that expands into a fusion pore (13). Importantly, each of these models proposes a mechanistic link between full 6HB assembly and fusion pore opening, and, accordingly, several classes of virus-entry inhibitors, including peptides and small-molecule drug candidates, are thought to interfere with 6HB formation (14).…”