Abstract
The p15 fusion-associated small transmembrane (FAST) protein is a nonstructural viral protein that induces cell-cell fusion and syncytium formation. The exceptionally small, myristoylated N-terminal ectodomain of p15 lacks any of the defining features of a typical viral fusion protein. NMR and CD spectroscopy indicate this small fusion module comprises a left-handed polyproline type II (PPII) helix flanked by small, unstructured N and C termini. Individual prolines in the 6-residue proline-rich motif are highly tolerant of alanine substitutions, but multiple substitutions that disrupt the PPII helix eliminate cell-cell fusion activity. A synthetic p15 ectodomain peptide induces lipid mixing between liposomes, but with unusual kinetics that involve a long lag phase before the onset of rapid lipid mixing, and the length of the lag phase correlates with the kinetics of peptide-induced liposome aggregation. Lipid mixing, liposome aggregation, and stable peptide-membrane interactions are all dependent on both the N-terminal myristate and the presence of the PPII helix. We present a model for the mechanism of action of this novel viral fusion peptide, whereby the N-terminal myristate mediates initial, reversible peptide-membrane binding that is stabilized by subsequent amino acid-membrane interactions. These interactions induce a biphasic membrane fusion reaction, with peptide-induced liposome aggregation representing a distinct, rate-limiting event that precedes membrane merger. Although the prolines in the proline-rich motif do not directly interact with membranes, the PPII helix may function to force solvent exposure of hydrophobic amino acid side chains in the regions flanking the helix to promote membrane binding, apposition, and fusion.
Highlights
The p15 fusion-associated small transmembrane (FAST) protein mediates cell-cell fusion using a 19-residue ectodomain
Microscopic examination of Giemsa-stained QM5 cells transfected with p15 or p15pro1 revealed that Ala substitutions of the proline-rich motif (PRM) abrogated the ability of p15 to induce cell-cell membrane fusion and multinucleated syncytium formation (Fig. 1B)
The extent of syncytiogenesis induced by the various mutants was variable at a given time point, all of the Pro substitution constructs retained substantial levels of cell-cell fusion activity and induced extensive syncytium formation, including a mutant that contained two Ala substitutions (PPAPPP converted to APAPAP) (Fig. 1D)
Summary
The p15 FAST protein mediates cell-cell fusion using a 19-residue ectodomain. Results: The p15 ectodomain requires both an N-terminal myristate and a polyproline type II helix for membrane fusion activity. Studies over the past few years have identified the orthoreovirus and aquareovirus proteins responsible for syncytiogenesis (2, 4 –7) These fusion-associated small transmembrane (FAST) proteins define a new family of viral fusogens whose structural and functional features distinguish them from the well characterized fusion proteins of enveloped viruses, a para-. It is not at all apparent how this fusion module could function to promote cell-cell membrane fusion To examine this issue, we used a combination of NMR and CD spectroscopy, syncytium formation, peptide-induced lipid mixing assays, and peptide-liposome binding studies. Results indicate the p15 ectodomain functions as a novel FP motif to induce lipid mixing, dependent on both the N-terminal myristate and the presence of a polyproline type II (PPII) helix. Residues in the PPII helix do not directly associate with membranes, this structure appears to be required to force exposure of flanking hydrophobic residues that do interact with membranes, stabilizing the weak myristate-membrane interactions and driving the fusion reaction
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