Different activities of the reovirus FAST proteins and influenza hemagglutinin in cell–cell fusion assays and in response to membrane curvature agents

Clancy, Eileen K.; Barry, Christopher; Ciechonska, Marta; Duncan, Roy

doi:10.1016/j.virol.2009.10.039

Cited by 18 publications

(26 citation statements)

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“…FAST protein-mediated cell-cell membrane fusion is refractory to standard assays used to detect hemifusion, the earliest event in membrane fusion (28). However, a quantitative fluorescent cell-cell pore formation assay was recently developed for use with the FAST proteins (29).…”

Section: Conserved Cysteine Residues In the Arv P10 Ectodomain Arementioning

confidence: 99%

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

Barry¹,

Key²,

Haddad³

et al. 2010

Journal of Biological Chemistry

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The reovirus fusion-associated small transmembrane (FAST) proteins are the smallest known viral membrane fusion proteins. With ectodomains of only ϳ20 -40 residues, it is unclear how such diminutive fusion proteins can mediate cell-cell fusion and syncytium formation. Contained within the 40-residue ectodomain of the p10 FAST protein resides an 11-residue sequence of moderately apolar residues, termed the hydrophobic patch (HP). Previous studies indicate the p10 HP shares operational features with the fusion peptide motifs found within the enveloped virus membrane fusion proteins. Using biotinylation assays, we now report that two highly conserved cysteine residues flanking the p10 HP form an essential intramolecular disulfide bond to create a cystine loop. Mutagenic analyses revealed that both formation of the cystine loop and p10 membrane fusion activity are highly sensitive to changes in the size and spatial arrangement of amino acids within the loop. The p10 cystine loop may therefore function as a cystine noose, where fusion peptide activity is dependent on structural constraints within the noose that force solvent exposure of key hydrophobic residues. Moreover, inhibitors of cell surface thioreductase activity indicate that disruption of the disulfide bridge is important for p10-mediated membrane fusion. This is the first example of a viral fusion peptide composed of a small, spatially constrained cystine loop whose function is dependent on altered loop formation, and it suggests the p10 cystine loop represents a new class of viral fusion peptides.Membrane fusion is an essential process involved in an abundance of biological events, including sperm-egg fusion, multinucleated myotube formation, exocytosis, and enveloped virus entry (1). The fusion reaction is catalyzed by specialized membrane fusion proteins designed to alter bilayer structure and bring about membrane merger. The membrane fusion proteins of various enveloped viruses represent some of the best characterized examples of such fusion catalysts (2). Detailed structural and functional analysis of diverse enveloped virus membrane fusion proteins reveals a common pathway of protein-mediated membrane fusion. Triggered exposure of a hydrophobic fusion peptide (FP), 3 normally sequestered within the complex pre-fusion ectodomain structure of these proteins, results in FP insertion into the donor and/or target membranes. Subsequent structural remodeling of these large ectodomains generates a trimeric hairpin structure that draws the two membranes together, driving lipid mixing (also referred to as hemifusion) and pore formation and expansion (3, 4). Despite a wealth of information, the precise mechanisms by which FPs and conformational remodeling of enveloped virus fusion proteins mediate lipid bilayer rearrangement and fusion remain unclear.The reovirus fusion-associated small transmembrane (FAST) proteins are a singular family of viral membrane fusion proteins. Although many nonenveloped viruses encode proteins involved in membrane permeabilization (...

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Section: Conserved Cysteine Residues In the Arv P10 Ectodomain Arementioning

confidence: 99%

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

Barry¹,

Key²,

Haddad³

et al. 2010

Journal of Biological Chemistry

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“…The funnel-shaped architecture of the p15 TMD is predicted to induce positive curvature of the inner leaflet, promoting the rapid transition from lipid mixing to pore formation. (19,50) and may be an attractive possibility for the FAST proteins, consistent with the inability of membrane curvature agents to detect this hemifusion state during p14-mediated cell-cell fusion (9). As with p14, the funnel shape of the p15 TMD could increase the positive curvature of the inner leaflet, promoting the direct transition from lipid mixing to fusion pore formation and completion of the membrane fusion reaction (Fig.…”

Section: Discussionmentioning

confidence: 67%

“…The direct correlation between the syncytiogenic and pore-forming activities of the various p15 constructs further implies that the TMD is required for stable pore formation. Whether this reflects a direct role for the p15 TMD in pore formation or in membrane merger events that precede pore formation is unclear, since assays and conditions that allow detection of hemifused structures induced by influenza virus HA are not applicable to the FAST proteins and/or are unable to detect a hemifusion intermediate (9). While no conclusive role for the TMDs of fusion proteins has emerged, several hypotheses converge upon a model in which elastic stresses generated by conformational rearrangements in the ectodomain are transferred to the membrane by the TMD, with the resulting membrane tensions serving to rupture the hemifusion diaphragm and drive pore formation (21,28,31).…”

Section: Discussionmentioning

confidence: 99%

“…The region of the TMD that resides in the inner membrane leaflet is composed of bulky aromatic residues, while the region lying in the outer leaflet is composed primarily of residues with smaller side chains. Juxtaposition of the FAST protein TMDs due to ecto-or endodomain interactions through protein clustering would amplify the asymmetric distribution of the bulk of the TMD across the bilayer, promoting curvature of the donor membrane toward the target membrane (9,10). Following close apposition of the target membrane, the concerted effects of the ectodomain and TMD on the lamellar structure of the outer leaflet would induce lipid mixing (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…It was proposed that a funnel-shaped TMD, with bulky aromatic residues clustering toward the C-terminal half of the TMD, may help the FAST proteins induce positive curvature of the inner leaflet to promote pore formation (10). Further support for this model derives from recent results indicating that chlorpromazine (CPZ), which induces the transition from hemifusion to pore formation by promoting positive curvature of the inner leaflet, has no effect on p14-mediated membrane fusion (9). These results are consistent with the possibility that the role of CPZ in promoting curvature of the inner leaflet may instead be fulfilled by the funnel-shaped p14 transmembrane domain.…”

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Helix-Destabilizing, β-Branched, and Polar Residues in the Baboon Reovirus p15 Transmembrane Domain Influence the Modularity of FAST Proteins

Clancy

Duncan

2011

J Virol

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The fusogenic reoviruses induce syncytium formation using the fusion-associated small transmembrane (FAST) proteins. A recent study indicated the p14 FAST protein transmembrane domain (TMD) can be functionally replaced by the TMDs of the other FAST proteins but not by heterologous TMDs, suggesting that the FAST protein TMDs are modular fusion units. We now show that the p15 FAST protein is also a modular fusogen, as indicated by the functional replacement of the p15 ectodomain with the corresponding domain from the p14 FAST protein. Paradoxically, the p15 TMD is not interchangeable with the TMDs of the other FAST proteins, implying that unique attributes of the p15 TMD are required when this fusion module is functioning in the context of the p15 ecto-and/or endodomain. A series of point substitutions, truncations, and reextensions were created in the p15 TMD to define features that are specific to the functioning of the p15 TMD. Removal of only one or two residues from the N terminus or four residues from the C terminus of the p15 TMD eliminated membrane fusion activity, and there was a direct correlation between the fusion-promoting function of the p15 TMD and the presence of N-terminal, hydrophobic ␤-branched residues. Substitution of the glycine residues and triserine motif present in the p15 TMD also impaired or eliminated the fusion-promoting activity of the p15 TMD. The ability of the p15 TMD to function in an ecto-and endodomain-specific context is therefore influenced by stringent sequence requirements that reflect the importance of TMD polar residues and helix-destabilizing residues.The current membrane fusion paradigm, based on enveloped virus fusion proteins, predicts that bilayer merger is intimately linked with triggered structural changes occurring in the large, complex ectodomains of these viral fusogens, which function as autonomous, metastable fusion machines (29). Among the known viral fusogens, the fusion-association small transmembrane (FAST) proteins challenge this mechanistic paradigm (17). The FAST proteins, encoded by the nonenveloped fusogenic orthoreoviruses, are the smallest known viral fusogens. As nonstructural viral proteins, the FAST proteins do not mediate virus-cell fusion. The FAST proteins are instead expressed and trafficked to the cell surface of virusinfected or transfected cells, where their sole function is the induction of cell-cell fusion and multinucleated syncytium formation. FAST protein-induced syncytiogenesis enhances pathogenicity through a two-step dissemination process, initially serving to promote localized cell-cell viral transmission followed by an apoptosis-induced burst of progeny virus release and systemic spread of infection caused by extensive late-stage fusion events (7, 40). When reconstituted into liposomes, the purified p14 FAST protein is both necessary and sufficient to induce membrane fusion (53). However, exhibiting no inherent receptor-binding activities, the FAST proteins rely on surrogate cellular adhesins to mediate early membrane attachment e...

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Evolution of programmed cell fusion: Common mechanisms and distinct functions

Oren‐Suissa

Podbilewicz

2010

Developmental Dynamics

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Eukaryotic cells have evolved diverged mechanisms to merge cells. Here, we discuss three types of cell fusion: (1) Non-self-fusion, cells with different genetic contents fuse to start a new organism and fusion between enveloped viruses and host cells; (2) Self-fusion, genetically identical cells fuse to form a multinucleated cell; and (3) Auto-fusion, a single cell fuses with itself by bringing specialized cell membrane domains into contact and transforming itself into a ring-shaped cell. This is a new type of selfish fusion discovered in C. elegans. We divide cell fusion into three stages: (1) Specification of the cell-fusion fate; (2) Cell attraction, attachment, and recognition; (3) Execution of plasma membrane fusion, cytoplasmic mixing and cytoskeletal rearrangements. We analyze cell fusion in diverse biological systems in development and disease emphasizing the mechanistic contributions of C. elegans to the understanding of programmed cell fusion, a genetically encoded pathway to merge specific cells. Developmental Dynamics 239:1515-1528, 2010. V C 2010 Wiley-Liss, Inc.Key words: cell fusion; non-self fusion; self-fusion; auto-fusion; sperm; egg; osteoclast; macrophage; placenta; epithelia; muscles; vulva; eye lens; tumor; stem cells; enveloped viruses; hybridoma; hybrid; syncytia; hemifusion; Caenorhabditis elegans; Plasmodium; Chlamydomonas; Neurospora crassa; Drosophila; yeast; sponges; plants; EFF-1; AFF-1; Syncytins; Prm1; CD9; IgSF; Izumo; SPE-9; SPE-38; SPE-41; SPE-42; EGG-1; EGG-2; HAP2/GCS1; FAST; Hox; FOS-1; Notch; Ras; Wnt; SCAR/WAVE; WASp; Actin; LIM2; MT1-MMP Accepted 28 February 2010The only living units that seem to have no sense of privacy at all are the eukaryotic cells that have been detached from the parent organism and isolated in a laboratory dish. Given the opportunity, under the right conditions, two cells from widely different sources, a yeast cell, say, and a chicken erythrocyte, will touch, fuse, and the two nuclei will then fuse as well, and the new hybrid cell will now divide into monstrous progeny. Naked cells, lacking self-respect, do not seem to have any sense of self.-Lewis Thomas, The Medusa and the Snail (Thomas, 1977)

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Different activities of the reovirus FAST proteins and influenza hemagglutinin in cell–cell fusion assays and in response to membrane curvature agents

Cited by 18 publications

References 60 publications

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

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

Helix-Destabilizing, β-Branched, and Polar Residues in the Baboon Reovirus p15 Transmembrane Domain Influence the Modularity of FAST Proteins

Evolution of programmed cell fusion: Common mechanisms and distinct functions

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