Abstract
Pore formation is the most energy-demanding step during virus-induced membrane fusion, where high curvature of the fusion pore rim increases the spacing between lipid headgroups, exposing the hydrophobic interior of the membrane to water. How protein fusogens breach this thermodynamic barrier to pore formation is unclear. We identified a novel fusion-inducing lipid packing sensor (FLiPS) in the cytosolic endodomain of the baboon reovirus p15 fusion-associated small transmembrane (FAST) protein that is essential for pore formation during cell-cell fusion and syncytiogenesis. NMR spectroscopy and mutational studies indicate the dependence of this FLiPS on a hydrophobic helix-loop-helix structure. Biochemical and biophysical assays reveal the p15 FLiPS preferentially partitions into membranes with high positive curvature, and this partitioning is impeded by bis-ANS, a small molecule that inserts into hydrophobic defects in membranes. Most notably, the p15 FLiPS can be functionally replaced by heterologous amphipathic lipid packing sensors (ALPS) but not by other membrane-interactive amphipathic helices. Furthermore, a previously unrecognized amphipathic helix in the cytosolic domain of the reptilian reovirus p14 FAST protein can functionally replace the p15 FLiPS, and is itself replaceable by a heterologous ALPS motif. Anchored near the cytoplasmic leaflet by the FAST protein transmembrane domain, the FLiPS is perfectly positioned to insert into hydrophobic defects that begin to appear in the highly curved rim of nascent fusion pores, thereby lowering the energy barrier to stable pore formation.
Highlights
The fusogenic ortho- and aquareoviruses are the only known examples of nonenveloped viruses that induce syncytium formation
We determined that the baboon reovirus p15 fusion-associated small transmembrane (FAST) protein endodomain contains a novel type of helix-loop-helix lipid packing sensor that partitions into hydrophobic defects present in highly curved membranes
By masking hydrophobic defects appearing in the highly curved rim of nascent fusion pores, the FliPS would make the forward reaction to pore formation a more energetically favored means of resolving an unstable hemifusion intermediate. These results define a new role for curvature sensing motifs, and reveal how viral fusion proteins can drive pore formation without having to rely on membrane stresses induced by complex refolding of large ectodomains
Summary
The fusogenic ortho- and aquareoviruses are the only known examples of nonenveloped viruses that induce syncytium formation. Reovirus-induced syncytiogenesis is mediated by a novel family of viral fusogens, the fusion-associated small transmembrane (FAST) proteins [1, 2]. And functionally, FAST proteins differ dramatically from the three well-characterized classes of enveloped virus fusion proteins [3]. FAST protein-mediated membrane fusion must proceed in the absence of the complex ectodomain refolding used by enveloped virus fusogens to drive the fusion process [10]. Unlike enveloped virus fusogens, FAST proteins are nonessential for reovirus replication, and as nonstructural viral proteins are not involved in virus entry. The unique structural and functional attributes of the FAST proteins and their link to virulence underscore the need for a better understanding of how these diminutive viral fusogens induce syncytium formation
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