Piscine reovirus (PRV) is a tentative new member of the family Reoviridae and has been linked to heart and skeletal muscle inflammation in farmed Atlantic salmon (Salmo salar L.). Recent sequence-based evidence suggests that PRV is about equally related to members of the genera Orthoreovirus and Aquareovirus. Sequence similarities have also suggested that PRV might encode a fusion-associated small transmembrane (FAST) protein, which in turn suggests that PRV might be the prototype of a new genus with syncytium-inducing potential. In previous support of this designation has been the absence of identifiable PRV-encoded homologues of either the virion outer-clamp protein of ortho-and aquareoviruses or the virion outer-fibre protein of most orthoreoviruses. In the current report, we have provided experimental evidence that the putative p13 FAST protein of PRV lacks the defining feature of the FAST protein family -the ability to induce syncytium formation. Instead, p13 is the first example of a cytosolic, integral membrane protein encoded by ortho-or aquareoviruses, and induces cytotoxicity in the absence of cell-cell fusion. Sequence analysis also identified signature motifs of the outer-clamp and outer-fibre proteins of other reoviruses in two of the predicted PRV gene products. Based on these findings, we conclude that PRV does not encode a FAST protein and is therefore unlikely to be a new fusogenic reovirus. The presence of a novel integral membrane protein and two previously unrecognized, essential outer-capsid proteins has important implications for the biology, evolution and taxonomic classification of this virus.

Arrestins regulate the signaling and endocytosis of many G protein-coupled receptors (GPCRs). It has been suggested that the functions of arrestins are dependent upon both the number and pattern of phosphorylation sites present in an activated GPCR. However, little is currently known about the relationships between the sites of receptor phosphorylation, the resulting affinities of arrestin binding, and the ensuing mechanisms of receptor regulation for any given GPCR. To investigate these interactions, we used an active truncated mutant of arrestin (amino acids 1-382) and phosphorylation-deficient mutants of the N-formyl peptide receptor (FPR). In contrast to results with wild type arrestins, the truncated arrestin-2 protein bound to the unphosphorylated wild type FPR, although with lower affinity and a low affinity for the agonist as revealed by competition studies with heterotrimeric G proteins. Using FPR mutants, we further demonstrated that the phosphorylation status of serines and threonines between residues 328 -332 is a key determinant that regulates the affinity of the FPR for arrestins. Furthermore, we found that the phosphorylation status of serine and threonine residues between amino acids 334 and 339 regulates the affinity of the receptor for agonist when arrestin is bound. These results suggest that the agonist affinity state of the receptor is principally regulated by phosphorylation at specific sites and is not simply a consequence of arrestin binding as has previously been proposed. Furthermore, this is the first demonstration that agonist affinity of a GPCR and the affinity of arrestin binding to the phosphorylated receptor are regulated by distinct receptor phosphodomains.

Although heptahelical chemoattractant and chemokine receptors are known to play a significant role in the host immune response and the pathophysiology of disease, the molecular mechanisms and transient macroassemblies underlying their activation and regulation remain largely uncharacterized. We report herein real time analyses of molecular assemblies involving the formyl peptide receptor (FPR), a well described member of the chemoattractant subfamily of G protein-coupled receptors (GPCRs), with both arrestins and heterotrimeric G proteins. In our system, the ability to define and discriminate distinct, in vitro receptor complexes relies on quantitative differences in the dissociation rate of a fluorescent agonist as well as the guanosine 5-3-O-(thio)-triphosphate (GTP␥S) sensitivity of the complex, as recently described for FPR-G protein interactions. In the current study, we demonstrate a concentration-and timedependent reconstitution of liganded, phosphorylated FPR with exogenous arrestin-2 and -3 to form a high agonist affinity, nucleotide-insensitive complex with EC 50 values of 0.5 and 0.9 M, respectively. In contrast, neither arrestin-2 nor arrestin-3 altered the ligand dissociation kinetics of activated, nonphosphorylated FPR. Moreover, we demonstrated that the addition of G proteins was unable to alter the ligand dissociation kinetics or induce a GTP␥S-sensitive state of the phosphorylated FPR. The properties of the phosphorylated FPR were entirely reversible upon treatment of the receptor preparation with phosphatase. These results represent to our knowledge the first report of the reconstitution of a detergent-solubilized, phosphorylated GPCR with arrestins and, furthermore, the first demonstration that phosphorylation of a nonvisual GPCR is capable of efficiently blocking G protein binding in the absence of arrestin. The significance of these results with respect to receptor desensitization and internalization are discussed.

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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 (...