Aquareovirus Effects Syncytiogenesis by Using a Novel Member of the FAST Protein Family Translated from a Noncanonical Translation Start Site

Racine, Trina; Hurst, Tara; Barry, Christopher; Shou, Jingyun; Kibenge, Frederick S. B.; Duncan, Roy

doi:10.1128/jvi.00171-09

Cited by 46 publications

(52 citation statements)

References 31 publications

(44 reference statements)

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“…As with all FAST proteins, the p10 proteins assume a bitopic N exoplasmic /C cytoplasmic topology in the plasma membrane, using their single transmembrane domain as a reverse signal-anchor to direct membrane insertion (10,(17)(18)(19). In contrast to the enveloped virus fusion proteins that position the majority of their mass external to the membrane in which they reside, the p10 FAST proteins have very small, approximately equally sized ecto-and endodomains (40 and 36 residues, respectively, for ARV p10).…”

Section: * This Research Was Supported In Part By a Grant From The Camentioning

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: * This Research Was Supported In Part By a Grant From The Camentioning

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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“…For instance, Hubei grass carp disease reovirus (HGDRV) induces a detectable cytopathic effect (CPE) in Ctenopharyngodon idellus kidney (CIK) cells and causes hemorrhagic disease with a high mortality rate in grass carp [8,9]. In contrast, grass carp reovirus (GCRV), a representative strain of fish reovirus, produces obvious CPE in infected CIK cells, but its ability to cause disease and lesions in grass carp is weak [10,11]. Other GCRV strains, such as GCRV-V, GCRV-HZ08, GCRV-GD108 and GCRV-JX02, can cause high mortality in grass carp but fail to produce CPE in CIK cells [12][13][14][15][16].…”

Section: Introductionmentioning

confidence: 99%

Fish reovirus GCReV-109 VP33 protein elicits protective immunity in rare minnows

et al. 2015

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Grass carp reovirus strain 109 (GCReV-109) was previously isolated from a grass carp (Ctenopharyngodon idellus) with hemorrhagic disease, and its complete genome has been sequenced. However, the infectivity of GCReV-109 has not been studied, and the viral protein VP33, encoded on genome segment S11, had no detectable sequence homology to other known reovirus proteins. In this study, we characterized GCReV-109 infections in vivo and in vitro, as well as the VP33 protein.Infectivity analysis showed that GCReV-109 caused severe hemorrhagic disease and 100 % mortality at dilutions up to 10 -4 in rare minnows (Gobiocypris rarus) by 8 days postinfection, but no visible cytopathic effect was observed in GCReV-109-infected subcultured grass carp muscle (GCM) cells. To confirm that GCReV-109 could be propagated in GCM cells, three virus genome segments were detected by RT-PCR, and large numbers of virus particles were observed by transmission electron microscopy in samples from the infected GCM cells. The suspension of GCReV-109-infected GCM cells was pathogenic to rare minnows. VP33 protein was expressed and purified for generation of an anti-VP33 antiserum. In western blot analysis of purified GCReV-109 particles, the antiserum specifically recognized a protein band (approximately 33 kDa). This revealed that VP33 is a major structural protein of GCReV-109 that might have immunogenic properties. The protective efficacy of the anti-VP33 antiserum against GCReV-109 infection was tested. The death of infected fish was delayed and the mortality fell to 10 % when fish were treated with the anti-VP33 antiserum, suggesting that it might be useful for the prevention and control of fish reoviral disease.

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“…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) 6 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- 3 Supported by scholarships from the CIHR and NSHRF and an Eliza Ritchie scholarship. 4 Supported by scholarships from the Natural Sciences and Engineering…”

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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

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Dawe

et al. 2012

Journal of Biological Chemistry

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Background: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. Conclusion:The p15 ectodomain functions as a novel fusion peptide motif. Significance: This novel fusion peptide provides new insights into the essential biological process of protein-mediated membrane fusion.

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Aquareovirus Effects Syncytiogenesis by Using a Novel Member of the FAST Protein Family Translated from a Noncanonical Translation Start Site

Cited by 46 publications

References 31 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

Fish reovirus GCReV-109 VP33 protein elicits protective immunity in rare minnows

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

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