Roy Duncan

A new class of fusion-associated small transmembrane (FAST) proteins encoded by the non-enveloped fusogenic reoviruses

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.

Extensive Syncytium Formation Mediated by the Reovirus FAST Proteins Triggers Apoptosis-Induced Membrane Instability

ABSTRACT The fusion-associated small transmembrane (FAST) proteins of the fusogenic reoviruses are the only known examples of membrane fusion proteins encoded by nonenveloped viruses. While the involvement of the FAST proteins in mediating extensive syncytium formation in virus-infected and -transfected cells is well established, the nature of the fusion reaction and the role of cell-cell fusion in the virus replication cycle remain unclear. To address these issues, we analyzed the syncytial phenotype induced by four different FAST proteins: the avian and Nelson Bay reovirus p10, reptilian reovirus p14, and baboon reovirus p15 FAST proteins. Results indicate that FAST protein-mediated cell-cell fusion is a relatively nonleaky process, as demonstrated by the absence of significant [3H]uridine release from cells undergoing fusion and by the resistance of these cells to treatment with hygromycin B, a membrane-impermeable translation inhibitor. However, diminished membrane integrity occurred subsequent to extensive syncytium formation and was associated with DNA fragmentation and chromatin condensation, indicating that extensive cell-cell fusion activates apoptotic signaling cascades. Inhibiting effector caspase activation or ablating the extent of syncytium formation, either by partial deletion of the avian reovirus p10 ectodomain or by antibody inhibition of p14-mediated cell-cell fusion, all resulted in reduced membrane permeability changes. These observations suggest that the FAST proteins do not possess intrinsic membrane-lytic activity. Rather, extensive FAST protein-induced syncytium formation triggers an apoptotic response that contributes to altered membrane integrity. We propose that the FAST proteins have evolved to serve a dual role in the replication cycle of these fusogenic nonenveloped viruses, with nonleaky cell-cell fusion initially promoting localized cell-cell transmission of the infection followed by enhanced progeny virus release from apoptotic syncytia and systemic dissemination of the infection.

Reptilian Reovirus Utilizes a Small Type III Protein with an External Myristylated Amino Terminus To Mediate Cell-Cell Fusion

ABSTRACT 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 Nexoplasmic/Ccytoplasmic (Nexo/Ccyt), 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.

Sequential partially overlapping gene arrangement in the tricistronic S1 genome segments of avian reovirus and Nelson Bay reovirus: Implications for translation initiation

ABSTRACT Previous studies of the avian reovirus strain S1133 (ARV-S1133) S1 genome segment revealed that the open reading frame (ORF) encoding the ςC viral cell attachment protein initiates over 600 nucleotides distal from the 5′ end of the S1 mRNA and is preceded by two predicted small nonoverlapping ORFs. To more clearly define the translational properties of this unusual polycistronic RNA, we pursued a comparative analysis of the S1 genome segment of the related Nelson Bay reovirus (NBV). Sequence analysis indicated that the 3′-proximal ORF present on the NBV S1 genome segment also encodes a ςC homolog, as evidenced by the presence of an extended N-terminal heptad repeat characteristic of the coiled-coil region common to the cell attachment proteins of reoviruses. Most importantly, the NBV S1 genome segment contains two conserved ORFs upstream of the ςC coding region that are extended relative to the predicted ORFs of ARV-S1133 and are arranged in a sequential, partially overlapping fashion. Sequence analysis of the S1 genome segments of two additional strains of ARV indicated a similar overlapping tricistronic gene arrangement as predicted for the NBV S1 genome segment. Expression analysis of the ARV S1 genome segment indicated that all three ORFs are functional in vitro and in virus-infected cells. In addition to the previously described p10 and ςC gene products, the S1 genome segment encodes from the central ORF a 17-kDa basic protein (p17) of no known function. Optimizing the translation start site of the ARV p10 ORF lead to an approximately 15-fold increase in p10 expression with little or no effect on translation of the downstream ςC ORF. These results suggest that translation initiation complexes can bypass over 600 nucleotides and two functional overlapping upstream ORFs in order to access the distal ςC start site.

Liposome reconstitution of a minimal protein-mediated membrane fusion machine

Biological membrane fusion is dependent on protein catalysts to mediate localized restructuring of lipid bilayers. A central theme in current models of protein‐mediated membrane fusion involves the sequential refolding of complex homomeric or heteromeric protein fusion machines. The structural features of a new family of fusion‐associated small transmembrane (FAST) proteins appear incompatible with existing models of membrane fusion protein function. While the FAST proteins function to induce efficient cell–cell fusion when expressed in transfected cells, it was unclear whether they function on their own to mediate membrane fusion or are dependent on cellular protein cofactors. Using proteoliposomes containing the purified p14 FAST protein of reptilian reovirus, we now show via liposome–cell and liposome–liposome fusion assays that p14 is both necessary and sufficient for membrane fusion. Stoichiometric and kinetic analyses suggest that the relative efficiency of p14‐mediated membrane fusion rivals that of the more complex cellular and viral fusion proteins, making the FAST proteins the simplest known membrane fusion machines.

Palmitoylation, Membrane-Proximal Basic Residues, and Transmembrane Glycine Residues in the Reovirus p10 Protein Are Essential for Syncytium Formation

ABSTRACT Avian reovirus and Nelson Bay reovirus are two unusual nonenveloped viruses that induce extensive cell-cell fusion via expression of a small nonstructural protein, termed p10. We investigated the importance of the transmembrane domain, a conserved membrane-proximal dicysteine motif, and an endodomain basic region in the membrane fusion activity of p10. We now show that the p10 dicysteine motif is palmitoylated and that loss of palmitoylation correlates with a loss of fusion activity. Mutational and functional analyses also revealed that a triglycine motif within the transmembrane domain and the membrane-proximal basic region were essential for p10-mediated membrane fusion. Mutations in any of these three motifs did not influence events upstream of syncytium formation, such as p10 membrane association, protein topology, or surface expression, suggesting that these motifs are more intimately associated with the membrane fusion reaction. These results suggest that the rudimentary p10 fusion protein has evolved a mechanism of inducing membrane merger that is highly dependent on the specific interaction of several different motifs with donor membranes. In addition, cross-linking, coimmunoprecipitation, and complementation assays provided no evidence for p10 homo- or heteromultimer formation, suggesting that p10 may be the first example of a membrane fusion protein that does not form stable, higher-order multimers.

Myristoylation, a Protruding Loop, and Structural Plasticity Are Essential Features of a Nonenveloped Virus Fusion Peptide Motif

Members of the fusion-associated small transmembrane (FAST) protein family are a distinct class of membrane fusion proteins encoded by nonenveloped fusogenic reoviruses. The 125-residue p14 FAST protein of reptilian reovirus has an ∼38-residue myristoylated N-terminal ectodomain containing a moderately apolar N-proximal region, termed the hydrophobic patch. Mutagenic analysis indicated sequence-specific elements in the N-proximal portion of the p14 hydrophobic patch affected cell-cell fusion activity, independent of overall effects on the relative hydrophobicity of the motif. Circular dichroism (CD) of a myristoylated peptide representing the majority of the p14 ectodomain suggested this region is mostly disordered in solution but assumes increased structure in an apolar environment. From NMR spectroscopic data and simulated annealing, the soluble nonmyristoylated p14 ectodomain peptide consists of an N-proximal extended loop flanked by two proline hinges. The remaining two-thirds of the ectodomain peptide structure is disordered, consistent with predictions based on CD spectra of the myristoylated peptide. The myristoylated p14 ectodomain peptide, but not a nonmyristoylated version of the same peptide nor a myristoylated scrambled peptide, mediated extensive lipid mixing in a liposome fusion assay. Based on the lipid mixing activity, structural plasticity, environmentally induced conformational changes, and kinked structures predicted for the p14 ectodomain and hydrophobic patch (all features associated with fusion peptides), we propose that the majority of the p14 ectodomain is composed of a fusion peptide motif, the first such motif dependent on myristoylation for membrane fusion activity.

Structural and Functional Properties of an Unusual Internal Fusion Peptide in a Nonenveloped Virus Membrane Fusion Protein

ABSTRACT The avian and Nelson Bay reoviruses are two of only a limited number of nonenveloped viruses capable of inducing cell-cell membrane fusion. These viruses encode the smallest known membrane fusion proteins (p10). We now show that a region of moderate hydrophobicity we call the hydrophobic patch (HP), present in the small N-terminal ectodomain of p10, shares the following characteristics with the fusion peptides of enveloped virus fusion proteins: (i) an abundance of glycine and alanine residues, (ii) a potential amphipathic secondary structure, (iii) membrane-seeking characteristics that correspond to the degree of hydrophobicity, and (iv) the ability to induce lipid mixing in a liposome fusion assay. The p10 HP is therefore predicted to provide a function in the mechanism of membrane fusion similar to those of the fusion peptides of enveloped virus fusion peptides, namely, association with and destabilization of opposing lipid bilayers. Mutational and biophysical analysis suggested that the internal fusion peptide of p10 lacks alpha-helical content and exists as a disulfide-stabilized loop structure. Similar kinked structures have been reported in the fusion peptides of several enveloped virus fusion proteins. The preservation of a predicted loop structure in the fusion peptide of this unusual nonenveloped virus membrane fusion protein supports an imperative role for a kinked fusion peptide motif in biological membrane fusion.

Bioinformatics of recent aqua- and orthoreovirus isolates from fish: evolutionary gain or loss of FAST and fiber proteins and taxonomic implications.

Family Reoviridae, subfamily Spinareovirinae, includes nine current genera. Two of these genera, Aquareovirus and Orthoreovirus, comprise members that are closely related and consistently share nine homologous proteins. Orthoreoviruses have 10 dsRNA genome segments and infect reptiles, birds, and mammals, whereas aquareoviruses have 11 dsRNA genome segments and infect fish. Recently, the first 10-segmented fish reovirus, piscine reovirus (PRV), has been identified and shown to be phylogenetically divergent from the 11-segmented viruses constituting genus Aquareovirus. We have recently extended results for PRV by showing that it does not encode a fusion-associated small transmembrane (FAST) protein, but does encode an outer-fiber protein containing a long N-terminal region of predicted α-helical coiled coil. Three recently characterized 11-segmented fish reoviruses, obtained from grass carp in China and sequenced in full, are also divergent from the viruses now constituting genus Aquareovirus, though not to the same extent as PRV. In the current study, we reexamined the sequences of these three recent isolates of grass carp reovirus (GCRV)–HZ08, GD108, and 104–for further clues to their evolution relative to other aqua- and orthoreoviruses. Structure-based fiber motifs in their encoded outer-fiber proteins were characterized, and other bioinformatics analyses provided evidence against the presence of a FAST protein among their encoded nonstructural proteins. Phylogenetic comparisons showed the combination of more distally branching, approved Aquareovirus and Orthoreovirus members, plus more basally branching isolates GCRV104, GCRV-HZ08/GD108, and PRV, constituting a larger, monophyletic taxon not suitably recognized by the current taxonomic hierarchy. Phylogenetics also suggested that the last common ancestor of all these viruses was a fiber-encoding, nonfusogenic virus and that the FAST protein family arose from at least two separate gain-of-function events. In addition, an apparent evolutionary correlation was found between the gain or loss of NS-FAST and outer-fiber proteins among more distally branching members of this taxon.

Reovirus FAST Protein Transmembrane Domains Function in a Modular, Primary Sequence-Independent Manner To Mediate Cell-Cell Membrane Fusion

ABSTRACT The FAST proteins are a unique family of virus-encoded cell-cell membrane fusion proteins. In the absence of a cleavable N-terminal signal peptide, a single-pass transmembrane domain (TMD) functions as a reverse signal-anchor to direct the FAST proteins into the plasma membrane in an Nexo/Ccyt topology. There is little information available on the role of the FAST protein TMD in the cell-cell membrane fusion reaction. We show that in the absence of conservation in the length or primary amino acid sequence, the p14 TMD can be functionally exchanged with the TMDs of the p10 and p15 FAST proteins. This is not the case for chimeric p14 proteins containing the TMDs of two different enveloped viral fusion proteins or a cellular membrane protein; such chimeric proteins were defective for both pore formation and syncytiogenesis. TMD structural features that are conserved within members of the FAST protein family presumably play direct roles in the fusion reaction. Molecular modeling suggests that the funnel-shaped architecture of the FAST protein TMDs may represent such a conserved structural and functional motif. Interestingly, although heterologous TMDs exert diverse influences on the trafficking of the p14 FAST protein, these TMDs are capable of functioning as reverse signal-anchor sequences to direct p14 into lipid rafts in the correct membrane topology. The FAST protein TMDs are therefore not primary determinants of type III protein topology, but they do play a direct, sequence-independent role in the membrane fusion reaction.