Structural and Functional Properties of an Unusual Internal Fusion Peptide in a Nonenveloped Virus Membrane Fusion Protein

Abstract

The avian and Nelson Bay reoviruses are two of only a limited number of nonenveloped viruses capable of inducing cell-cell membrane fusion. These viruses encode the smallest known membrane fusion proteins (p10). We now show that a region of moderate hydrophobicity we call the hydrophobic patch (HP), present in the small N-terminal ectodomain of p10, shares the following characteristics with the fusion peptides of enveloped virus fusion proteins: (i) an abundance of glycine and alanine residues, (ii) a potential amphipathic secondary structure, (iii) membrane-seeking characteristics that correspond to the degree of hydrophobicity, and (iv) the ability to induce lipid mixing in a liposome fusion assay. The p10 HP is therefore predicted to provide a function in the mechanism of membrane fusion similar to those of the fusion peptides of enveloped virus fusion peptides, namely, association with and destabilization of opposing lipid bilayers. Mutational and biophysical analysis suggested that the internal fusion peptide of p10 lacks alpha-helical content and exists as a disulfidestabilized loop structure. Similar kinked structures have been reported in the fusion peptides of several enveloped virus fusion proteins. The preservation of a predicted loop structure in the fusion peptide of this unusual nonenveloped virus membrane fusion protein supports an imperative role for a kinked fusion peptide motif in biological membrane fusion.The physical properties of phospholipid bilayers impose an energy barrier to spontaneous membrane fusion, thereby maintaining the compartmentalized nature of cells (48, 61). Nonetheless, regulated membrane merger is an essential cellular process. Cellular fusion proteins facilitate membrane merger for diverse processes, including intracellular vesicle transport and the formation of zygotes, multinucleated myotubes, and osteoclasts (44,58,59). Furthermore, the entry of all enveloped viruses involves membrane fusion induced by viral fusion proteins (29,52,58). Understanding the mechanisms of membrane fusion mediated by proteins from diverse sources, therefore, has broad implications.The best-characterized fusion proteins are those involved in the entry of enveloped viruses. The fusion machinery of enveloped viruses frequently consists of a single large multimeric protein (18,50). A hallmark feature of enveloped viral fusion proteins is the presence of two hydrophobic sequences, the transmembrane (TM) domain and the fusion peptide, that simultaneously anchor the protein in both the donor and target membranes (58). Numerous studies have demonstrated the significance of enveloped virus fusion peptides in destabilizing target membranes and favoring membrane merger (17, 25). Fusion peptides are short membrane-seeking motifs that frequently assume helical structures in association with membranes. These fusion peptides lie sequestered within the tertiary structure of the prefusion conformation of the fusion protein. Fusion protein activation follows receptor binding-or low pH-induced remodeling of th...

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.…”

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

“…Lipid Mixing Assay for Membrane Fusion-The ability of p14 peptides to promote lipid mixing was assessed using the resonance energy transfer assay of Struck et al (26) with large (0.1 m) unilamellar vesicles (LUVs) composed of 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), and cholesterol (1:1:1 molar ratio) (Avanti Polar Lipids) as described previously (27). One population of LUVs was labeled with 2 mol % each of N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine and N- (7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine, and a 9:1 molar ratio of unlabeled to labeled liposomes was used in the assay.…”

Features of a Spatially Constrained Cystine Loop in the p10 FAST Protein Ectodomain Define a New Class of Viral Fusion Peptides

“…We previously noted similarities between the p10 HPs and the FPs present in all enveloped virus fusion proteins (13). FPs are small (ϳ20 -30 residues) regions of mostly apolar residues, frequently enriched in glycine, and usually highly conserved within the fusion proteins of different strains of the same virus (21).…”

“…Following their expression in virus-infected cells, the FAST proteins traffic to the plasma membrane where they induce cell-cell fusion and multinucleated syncytium formation, promoting virus dissemination (11). Although the FAST proteins (95-198 residues) approximate the size of the soluble NSF attachment protein receptor proteins involved in intracellular vesicle fusion (12), the FAST proteins need only be present in one of the two membranes undergoing fusion (13). The FAST proteins are also both necessary and sufficient to mediate the actual merger of lipid bilayers (14), although they exploit or recruit cellular cofactors to enhance the pre-fusion (i.e.…”