Unusual Topological Arrangement of Structural Motifs in the Baboon Reovirus Fusion-Associated Small Transmembrane Protein

Abstract

Select members of the Reoviridae are the only nonenveloped viruses known to induce syncytium formation. The fusogenic orthoreoviruses accomplish cell-cell fusion through a distinct class of membrane fusioninducing proteins referred to as the fusion-associated small transmembrane (FAST) proteins. The p15 membrane fusion protein of baboon reovirus is unique among the FAST proteins in that it contains two hydrophobic regions (H1 and H2) recognized as potential transmembrane (TM) domains, suggesting a polytopic topology. However, detailed topological analysis of p15 indicated only the H1 domain is membrane spanning. In the absence of an N-terminal signal peptide, the H1 TM domain serves as a reverse signal-anchor to direct p15 membrane insertion and a bitopic N exoplasmic /C cytoplasmic topology. This topology results in the translocation of the smallest ectodomain (ϳ20 residues) of any known viral fusion protein, with the majority of p15 positioned on the cytosolic side of the membrane. Mutagenic analysis indicated the unusual presence of an N-terminal myristic acid on the small p15 ectodomain is essential to the fusion process. Furthermore, the only other hydrophobic region (H2) present in p15, aside from the TM domain, is located within the endodomain. Consequently, the p15 ectodomain is devoid of a fusion peptide motif, a hallmark feature of membrane fusion proteins. The exceedingly small, myristoylated ectodomain and the unusual topological distribution of structural motifs in this nonenveloped virus membrane fusion protein necessitate alternate models of proteinmediated membrane fusion.The baboon reovirus (BRV) p15 protein is a novel member of the recently described fusion-associated small transmembrane (FAST) protein family (9,11,45). The FAST proteins are unusual membrane fusion proteins encoded by the fusogenic subgroup of orthoreoviruses, one of the few examples of nonenveloped viruses that induce cell-cell fusion and syncytium formation (15,18). At 10 to 15 kDa, the reovirus FAST proteins are the smallest known viral membrane fusion proteins and are unlikely to undergo the types of extensive structural rearrangements required for enveloped virus fusion protein activity (27,48). The FAST proteins are also the only examples of nonstructural viral proteins that induce membrane fusion (11, 45). As a result of their nonstructural nature, the FAST proteins play no role in reovirus entry. Their sole purpose appears to reflect enhanced dissemination of the infection via syncytium formation, following FAST protein expression in reovirus-infected cells (16,17). The unusual structural features of the FAST proteins and their unique role in the virus replication cycle suggest the mechanism of FAST-mediated membrane fusion is unlikely to adhere to the existing paradigm, which is derived from studies of the enveloped virus fusion proteins (5,48,52).In addition to BRV p15, FAST proteins have been recently characterized from avian reovirus (ARV), Nelson Bay reovirus (NBV), and reptilian reovirus (RRV) (9, 45). Although th...

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

“…The preceding Gln residue at position 9 (Gln-9) was also changed to Ala, or the intervening Ala residue at position 12 (Ala-12) was converted to Pro. Each construct was transfected into cells and the extent of syncytium formation was quantified using a syncytial index based on the average number of syncytial nuclei present in random microscopic fields (32). Although the extent of syncytiogenesis induced by the various mutants was variable at a given time point, all of the Pro substitution constructs retained substantial levels of cell-cell fusion activity and induced extensive syncytium formation, including a mutant that contained two Ala substitutions (PPAPPP converted to APAPAP) (Fig.…”

“…1A). The p15 FAST protein has the smallest ectodomain (19 residues), and this fusion module has a proline-rich motif (PRM) and an overall hydrophilic nature with no apparent FP motif (5,32). It is not at all apparent how this fusion module could function to promote cell-cell membrane fusion.…”

“…The generation and specificity of the rabbit polyclonal p15-specific antisera has been previously described (32).…”

“…Immunostaining and Immunoblotting-Transfected monolayers were methanol-fixed and immunostained using primary rabbit polyclonal anti-p15 antiserum as previously described (32). For immunoblot analysis, cells were grown in 10-cm dishes and lysed in RIPA buffer 8 h post-transfection.…”

Homomultimerization of the reovirus p14 fusion-associated small transmembrane protein during transit through the ER-Golgi complex secretory pathway

“…The FAST proteins lack significant sequence similarity but share a subset of structural features. These include a single transmembrane domain flanked on the cytosolic side by a cluster of basic residues, a small region of moderate hydrophobicity (termed the hydrophobic patch) that can reside on either side of the transmembrane domain and a fatty acid modification (either an N-terminal myristate or a palmitoylated dicysteine motif), all of which are required for membrane fusion activity Dawe et al, 2005;Shmulevitz et al, 2003). Defining the roles of these motifs in the fusion reaction requires a more detailed understanding of the structure-function relationships of the FAST proteins.…”