Features of a spatially constrained cystine loop in the p10 FAST protein ectodomain define a new class of viral fusion peptides.
The reovirus fusion-associated small transmembrane (FAST) proteins are the smallest known viral membrane fusion proteins. With ectodomains of only approximately 20-40 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.
Barry
Christopher
C
Department of Microbiology and Immunology, Nova Scotia B3H 1X5, Canada.
Key
Tim
T
Haddad
Rami
R
Duncan
Roy
R
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002469:Cell Separation; D002522:Chlorocebus aethiops; D003553:Cystine / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry; D005434:Flow Cytometry / Q000379:methods; D030102:Orthoreovirus, Mammalian / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D013879:Thioredoxins / Q000737:chemistry; D014709:Vero Cells; D014760:Viral Fusion Proteins / Q000737:chemistry*
D004220:Disulfides; D010455:Peptides; D011993:Recombinant Fusion Proteins; D014760:Viral Fusion Proteins; D003553:Cystine; D013879:Thioredoxins