The non‐enveloped fusogenic avian and Nelson Bay reoviruses encode homologous 10 kDa non‐structural transmembrane proteins. The p10 proteins localize to the cell surface of transfected cells in a type I orientation and induce efficient cell–cell fusion. Mutagenic studies revealed the importance of conserved sequence‐predicted structural motifs in the membrane association and fusogenic properties of p10. These motifs included a centrally located transmembrane domain, a conserved cytoplasmic basic region, a small hydrophobic motif in the N‐terminal domain and four conserved cysteine residues. Functional analysis indicated that the extreme C‐terminus of p10 functions in a sequence‐independent manner to effect p10 membrane localization, while the N‐terminal domain displays a sequence‐dependent effect on the fusogenic property of p10. The small size, unusual arrangement of structural motifs and lack of any homologues in previously described membrane fusion proteins suggest that the fusion‐associated small transmembrane (FAST) proteins of reovirus represent a new class of membrane fusion proteins.

Reptilian reovirus is one of a limited number of nonenveloped viruses that are capable of inducing cell-cell fusion. A small, hydrophobic, basic, 125-amino-acid fusion protein encoded by the first open reading frame of a bicistronic viral mRNA is responsible for this fusion activity. Sequence comparisons to previously characterized reovirus fusion proteins indicated that p14 represents a new member of the fusion-associated small transmembrane (FAST) protein family. Topological analysis revealed that p14 is a representative of a minor subset of integral membrane proteins, the type III proteins N exoplasmic /C cytoplasmic (N exo /C cyt ), that lack a cleavable signal sequence and use an internal reverse signal-anchor sequence to direct membrane insertion and protein topology. This topology results in the unexpected, cotranslational translocation of the essential myristylated N-terminal domain of p14 across the cell membrane. The topology and structural motifs present in this novel reovirus membrane fusion protein further accentuate the diversity and unusual properties of the FAST protein family and clearly indicate that the FAST proteins represent a third distinct class of viral membrane fusion proteins.Biological membrane fusion is an essential cellular process mediated by specific fusion proteins (22,57,61). Extensive analysis of a number of enveloped virus fusion proteins has contributed to a model of protein-mediated membrane fusion. Enveloped virus fusion proteins are complex, multimeric, type I N exoplasmic /C cytoplasmic (N exo /C cyt ) integral membrane proteins that facilitate virus entry into cells by mediating fusion between the viral envelope and the target cell membrane. Two distinct classes of enveloped virus fusion proteins have been identified: the class I fusion proteins exemplified by influenza virus and human immunodeficiency virus proteins and the class II proteins of the alpha-and flaviviruses (25,47,56,57). For both classes, triggered conformational changes and/or multimer reorganization of their complex ectodomains are essential aspects of the fusion reaction (25,56). This transition from a metastable to a low-energy form is believed to provide the energy to overcome the thermodynamic barriers that inhibit spontaneous membrane mergers (30, 56). However, the necessity and/or precise role of structural remodeling as a thermodynamic mediator of the fusion reaction remains unresolved (3,13,14,37).Since nonenveloped viruses lack a lipid bilayer, virus entry is not dependent on membrane fusion. As a result, nonenveloped viruses do not encode membrane fusion proteins. The rare exceptions to this generalization are the fusogenic reoviruses, an unusual group of syncytium-inducing nonenveloped viruses with segmented double-stranded RNA genomes (9, 32). Unlike those of enveloped viruses, the reovirus fusion proteins are not components of the virus particle and therefore are not involved in virus entry (7, 45). The reovirus fusion-associated small transmembrane (FAST) proteins are the only known example...

The orthoreoviruses can be divided into subgroups based on either their restricted host range or the unusual ability of certain members of this group of nonenveloped viruses to induce cell-cell fusion from within. Phylogenetic relationships cannot be inferred based on these biological properties because fusogenic reoviruses are present in both the avian and mammalian subgroups. To address this issue, the complete nucleotide sequences of the three S-class genome segments encoding the major sigma-class core, outer capsid, and nonstructural proteins of four fusogenic reoviruses were determined and used to establish the phylogeny of the orthoreoviruses. The viruses analysed included two strains of avian reovirus and the only known fusogenic mammalian reoviruses, Nelson Bay virus and baboon reovirus. Comparative sequence analysis of these fusogenic reoviruses and the prototypical nonfusogenic mammalian reoviruses indicated a highly diverged genus with both conserved and unique sequence-predicted structural motifs in the major sigma-class proteins. Phylogenetic analysis provided the basis for the first taxonomic subdivision of the orthoreoviruses into species classes based on inferred evolutionary relationships. It is proposed that the orthoreoviruses consist of at least four species that separate into three clades. The nonfusogenic mammalian reovirus species represent a single clade, and the fusogenic reoviruses separate into two distinct clades. The first clade of fusogenic reoviruses contains the avian reovirus- and Nelson Bay virus-type species, with the second clade being occupied by the single baboon reovirus isolate that represents a fourth orthoreovirus species.

We demonstrate that the S4 genome segment of baboon reovirus (BRV) contains two sequential partially overlapping open reading frames (ORFs), both of which are functional in vitro and in virus-infected cells. The 15-kDa gene product (p15) of the 5-proximal ORF induces efficient cell-cell fusion when expressed by itself in transfected cells, suggesting that p15 is the only viral protein required for induction of syncytium formation by BRV. The p15 protein is a small, hydrophobic, basic, integral membrane protein, properties shared with the p10 fusion-associated small transmembrane (FAST) proteins encoded by avian reovirus and Nelson Bay reovirus. As with p10, the BRV p15 protein is also a nonstructural protein and, therefore, is not involved in virus entry. Sequence analysis indicates that p15 shares no significant sequence similarity with the p10 FAST proteins and contains a unique repertoire and arrangement of sequence-predicted structural and functional motifs. These motifs include a functional N-terminal myristylation consensus sequence, an N-proximal prolinerich motif, two potential transmembrane domains, and an intervening polybasic region. The unique structural properties of p15 suggest that this protein is a novel member of the new family of FAST proteins.Membrane fusion is an essential cellular event, the precise mechanism of which remains poorly understood. Much of our understanding of membrane fusion is derived from studies of enveloped virus fusion proteins. Structure-function analysis of numerous enveloped virus fusion proteins has led to a generalized model of protein-mediated membrane fusion. This model suggests that triggered conformational changes in the metastable viral fusion proteins expose a previously concealed hydrophobic fusion peptide for insertion into, and destabilization of, the target lipid bilayer (16,43,50,51). Further extensive conformational changes are predicted to be involved in supplying the energy required to pull the donor and target bilayers together and allow the fusion reaction to proceed 1 (6, 50, 51). Based on the nature of the structural rearrangements, the enveloped virus fusion proteins fall into two general classes. Class I proteins, such as the influenza virus hemagglutinin, human immunodeficiency virus gp41, and Ebola virus GP2, use coiled-coil rearrangements to drive the fusion process while class II proteins, such as those of the alphaviruses, flaviviruses, and rhabdoviruses, undergo extensive multimer rearrangements in the absence of apparent coiled-coil interactions (19,34,42,43,44).Our recent characterization of a fusion-associated small transmembrane (FAST) protein encoded by certain fusogenic reoviruses (39) suggests that extensive conformational changes in large, complex, multimeric viral fusion proteins may not be a universal requirement for protein-mediated membrane fusion. The only examples of nonenveloped viruses that induce syncytium formation are all members of the family Reoviridae, a diverse group of nonenveloped viruses with double-stranded RNA geno...

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The genome segment B sequence of infectious pancreatic necrosis virus was determined for both the Jasper and Sp serotypes. The sequences are 2784 and 2630 bp long, respectively, and contain a single large open reading frame encoding the VP1 protein, the putative RNA-dependent RNA polymerase (RdRp) of IPNV. The proteins exhibit an 88% homology with each other, but only 41% with infectious bursal disease virus (IBDV) VP1, another member of the Birnaviridae. Despite the low overall homology between the IPNV and IBDV VP1 proteins, homologous regions were detected within the central portion of the proteins. The carboxy-proximal regions of the VP1, which contain very low amino acid homology, displayed evidence of conservation in structural features such as a hydrophilic, highly basic domain. Consensus sequences associated with GTP-binding proteins and RdRps were also detected in VP1. However, unlike the RdRps associated with single-stranded plus RNA viruses, the birnavirus RdRp lacks the Gly-Asp-Asp motif characteristic of this enzyme family.