Myristoylation, a Protruding Loop, and Structural Plasticity Are Essential Features of a Nonenveloped Virus Fusion Peptide Motif

Abstract

Members of the fusion-associated small transmembrane (FAST) protein family are a distinct class of membrane fusion proteins encoded by nonenveloped fusogenic reoviruses. The 125-residue p14 FAST protein of reptilian reovirus has an ϳ38-residue myristoylated Nterminal ectodomain containing a moderately apolar Nproximal region, termed the hydrophobic patch. Mutagenic analysis indicated sequence-specific elements in the N-proximal portion of the p14 hydrophobic patch affected cell-cell fusion activity, independent of overall effects on the relative hydrophobicity of the motif. Circular dichroism (CD) of a myristoylated peptide representing the majority of the p14 ectodomain suggested this region is mostly disordered in solution but assumes increased structure in an apolar environment. From NMR spectroscopic data and simulated annealing, the soluble nonmyristoylated p14 ectodomain peptide consists of an N-proximal extended loop flanked by two proline hinges. The remaining two-thirds of the ectodomain peptide structure is disordered, consistent with predictions based on CD spectra of the myristoylated peptide. The myristoylated p14 ectodomain peptide, but not a nonmyristoylated version of the same peptide nor a myristoylated scrambled peptide, mediated extensive lipid mixing in a liposome fusion assay. Based on the lipid mixing activity, structural plasticity, environmentally induced conformational changes, and kinked structures predicted for the p14 ectodomain and hydrophobic patch (all features associated with fusion peptides), we propose that the majority of the p14 ectodomain is composed of a fusion peptide motif, the first such motif dependent on myristoylation for membrane fusion activity.Complex, multimeric viral fusion proteins mediate the fusion of viral envelopes to target cell membranes during virus entry into cells (1). Membrane destabilization during the fusion process is dependent on a fusion peptide motif contained within these enveloped virus fusion proteins (2-4). Fusion peptides are moderately hydrophobic stretches of ϳ20 amino acids, frequently rich in glycine and alanine residues (3, 5, 6). In the case of the class I fusion proteins typified by influenza hemagglutinin (HA), 1 human immunodeficiency virus gp41, and the F proteins of paramyxoviruses, the fusion peptide motifs are located at the N terminus of the fusion polypeptide (4). Conversely, these motifs are embedded internally within the amino acid sequence of the class II fusion proteins (e.g. alphaviruses and flaviviruses) and the G protein of vesicular stomatitis virus (VSV) (7,8). Structural predictions for fusion peptides based on CD or infrared spectroscopy have yielded conflicting results (9 -12) and are influenced by the different methods used for preparation of the water-insoluble, flexible fusion peptide (13). The properties of conformational flexibility and environmentally induced structural changes may represent essential features of fusion peptides, intimately linked to their function in the fusion process (4,14,15).To deal with ...

The P2 capsid protein of the nonenveloped rice dwarf phytoreovirus induces membrane fusion in insect host cells

“…Some nonenveloped reoviruses that are fusogenic encode a distinct class of membrane fusion proteins, called fusionassociated small transmembrane (FAST) proteins, which are always N-terminal-myristoylated and function in the process of viral cell-to-cell movement but not in the process of viral entry into host cells (11)(12)(13). The VP5 of bluetongue virus acts not only as a membrane penetration protein but also as a fusion protein that induces syncytium formation when it is fused to a transmembrane anchor and expressed on the cell surface.…”

Cell-Cell Membrane Fusion Induced by p15 Fusion-associated Small Transmembrane (FAST) Protein Requires a Novel Fusion Peptide Motif Containing a Myristoylated Polyproline Type II Helix

“…7B). Although liposome aggregation is a priori for peptide-induced lipid mixing, the p10 and p14 FAST protein FPs induce both events concurrently with no lag time in lipid mixing (30,31). The same situation applies to enveloped virus FPs, where peptide addition leads to lipid mixing with little (Ͻ20 s) to no lag (47)(48)(49).…”

“…Unexpectedly, the 19-residue p15 ectodomain can be replaced by the 37-residue ectodomain of p14 with no adverse effects on cell-cell fusion activity (28), suggesting the two very different FPs present in these ectodomains may provide an equivalent function in the fusion reaction. The loop structures of the p10 and p14 FPs presumably result in solvent exposure of hydrophobic residues, particularly a Val and Phe residue at the apex of these loops (29,30). These loops share features with the fusion loops contained within several enveloped virus fusion proteins, which also expose hydrophobic anchors at their apices (8).…”

“…For structural determination, spin systems were identified through chemical shifts and characteristic TOCSY cross-peak patterns (60 ms for myristoylated p15 and 120 ms for non-myristoylated p15). Sequencespecific assignments were determined as described previously (30,34). The structure of the non-myristoylated p15 ectodomain at pH 6.0 was deposited with the Protein Data base (PDB code 2LKW).…”

Unusual Topological Arrangement of Structural Motifs in the Baboon Reovirus Fusion-Associated Small Transmembrane Protein

“…This topology results in small ectodomains of approximately 40 residues and places a membrane-proximal, polybasic region within their 40-to 70-residue endodomains, consistent with the "positive-inside rule" of membrane protein topology (26). Within their ectodomains, p10 and p14 each contain a moderately hydrophobic region of 14 to 16 residues that may serve as a fusion peptide (10,47), in a manner analogous to the fusion peptides of enveloped viruses (19,28,49). The N exo /C cyt topology also indicates that the essential palmitoylated dicysteine motif of p10 influences p10 syncytium formation from a cytosolic location (46).…”