The p14 Fusion-associated Small Transmembrane (FAST) Protein Effects Membrane Fusion from a Subset of Membrane Microdomains

2009

J Virol

Self Cite

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 N exo /C cyt 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.The fusion-associated small transmembrane (FAST) proteins are a unique family of membrane fusion proteins encoded by the fusogenic reoviruses (20). At 95 to 140 amino acids in size, the FAST proteins are the smallest known viral membrane fusion proteins. Rather than mediating virus-cell fusion, the FAST proteins are nonstructural viral proteins that are expressed on the surfaces of virus-infected or -transfected cells, where they induce cell-cell fusion and the formation of multinucleated syncytia. A purified FAST protein, when reconstituted into liposome membranes, induces liposome-cell and liposome-liposome fusion, indicating the FAST proteins are bona fide membrane fusion proteins (54). In their natural biological context as cell-cell fusogens, however, the FAST proteins exploit cellular adhesins and actin remodeling to maximize their cell-cell fusion potential (40). Studies further suggest that cell-cell fusion mediated by the FAST proteins may contribute to rapid localized dissemination of the infection, followed by apoptosis-induced disruption of the syncytia, resulting in a burst of infectious-progeny-virus release (19,21,41). This two-step process for virus dissemination mediated by the FAST proteins may contribute to the natural pathogenicity of the fusogenic reoviruses. How this remarkable family of virus-encoded fusogens induce membrane fusion and syncytium formation remains unclear, but several recent studies have defined specific subdomains and structural motifs likely to be in...

Section: Vol 83 2009 Fast Protein Transmembrane Domains and Membranmentioning

confidence: 48%

Section: Methodsmentioning

confidence: 99%

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

2009

J Virol

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“…The TMDs of other viral fusion proteins such as influenza virus HA may self-associate to form a proteinaceous ring that restricts lipid flow at the fusion site. TMDs are also known to promote the clustering of fusion proteins at the fusion site via association with membrane lipid microdomains (8,41,48), and the p14 FAST protein has been shown to associate with detergent-resistant membranes (13). Based on the above considerations, it was conceivable that the loss of fusion activity resulting from p15 TMD substitutions could reflect disruption of p15 clustering at the fusion site, mediated either by disruption of potential TMD-TMD interactions or by alteration of TMD-dependent recruitment to detergent-resis- tant membrane microdomains.…”

Section: Resultsmentioning

confidence: 99%

Helix-Destabilizing, β-Branched, and Polar Residues in the Baboon Reovirus p15 Transmembrane Domain Influence the Modularity of FAST Proteins

2011

J Virol

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The fusogenic reoviruses induce syncytium formation using the fusion-associated small transmembrane (FAST) proteins. A recent study indicated the p14 FAST protein transmembrane domain (TMD) can be functionally replaced by the TMDs of the other FAST proteins but not by heterologous TMDs, suggesting that the FAST protein TMDs are modular fusion units. We now show that the p15 FAST protein is also a modular fusogen, as indicated by the functional replacement of the p15 ectodomain with the corresponding domain from the p14 FAST protein. Paradoxically, the p15 TMD is not interchangeable with the TMDs of the other FAST proteins, implying that unique attributes of the p15 TMD are required when this fusion module is functioning in the context of the p15 ecto-and/or endodomain. A series of point substitutions, truncations, and reextensions were created in the p15 TMD to define features that are specific to the functioning of the p15 TMD. Removal of only one or two residues from the N terminus or four residues from the C terminus of the p15 TMD eliminated membrane fusion activity, and there was a direct correlation between the fusion-promoting function of the p15 TMD and the presence of N-terminal, hydrophobic ␤-branched residues. Substitution of the glycine residues and triserine motif present in the p15 TMD also impaired or eliminated the fusion-promoting activity of the p15 TMD. The ability of the p15 TMD to function in an ecto-and endodomain-specific context is therefore influenced by stringent sequence requirements that reflect the importance of TMD polar residues and helix-destabilizing residues.The current membrane fusion paradigm, based on enveloped virus fusion proteins, predicts that bilayer merger is intimately linked with triggered structural changes occurring in the large, complex ectodomains of these viral fusogens, which function as autonomous, metastable fusion machines (29). Among the known viral fusogens, the fusion-association small transmembrane (FAST) proteins challenge this mechanistic paradigm (17). The FAST proteins, encoded by the nonenveloped fusogenic orthoreoviruses, are the smallest known viral fusogens. As nonstructural viral proteins, the FAST proteins do not mediate virus-cell fusion. The FAST proteins are instead expressed and trafficked to the cell surface of virusinfected or transfected cells, where their sole function is the induction of cell-cell fusion and multinucleated syncytium formation. FAST protein-induced syncytiogenesis enhances pathogenicity through a two-step dissemination process, initially serving to promote localized cell-cell viral transmission followed by an apoptosis-induced burst of progeny virus release and systemic spread of infection caused by extensive late-stage fusion events (7, 40). When reconstituted into liposomes, the purified p14 FAST protein is both necessary and sufficient to induce membrane fusion (53). However, exhibiting no inherent receptor-binding activities, the FAST proteins rely on surrogate cellular adhesins to mediate early membrane attachment e...

“…Transient or weak homotypic interactions may also explain why multimers of the p10 FAST protein cannot be detected by co-immunoprecipitation (Shmulevitz et al, 2003). Presently, it is unclear how transit through the ERGolgi complex pathway influences p14-multimer formation, but factors such as post-ER p14 localization to membrane microdomains (Corcoran et al, 2006) or the effects of the different lipid compositions of secretory membrane compartments on p14 interactions could influence this process. Additional NMR structural analysis might further define the nature of the homotypic p14 ectodomain interactions and how these interactions should be integrated into working models of how this unusual family of non-enveloped virus proteins drives cell-cell membrane fusion.…”

mentioning

confidence: 99%

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

Corcoran

Journal of General Virology

2010

The reovirus fusion-associated small transmembrane (FAST) proteins are the smallest known viral membrane-fusion proteins. How these diminutive fusogens mediate cell-cell fusion and syncytium formation is unclear. Ongoing efforts are aimed at defining the roles of the FAST protein ecto-, endo-and transmembrane domains in the membrane-fusion reaction. We now provide direct evidence for homomultimer formation by the FAST proteins by using an anti-haemagglutinin (HA) mAb to co-precipitate the untagged p14 FAST protein from cells co-transfected with HA-tagged p14. Disrupting the intracellular endoplasmic reticulum-Golgi complex vesicle transport pathway prevented p14 homomultimer formation, while lower pH disrupted p14 multimers. The p14 endodomain or transmembrane domains are not required for multimer formation, which, along with the pH sensitivity and the distribution of histidine residues, suggests the 36 aa p14 ectodomain is a multimerization motif.