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

Abstract

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...

Trimeric Transmembrane Domain Interactions in Paramyxovirus Fusion Proteins

“…Information on factors that stabilize or destabilize F protein TM interactions may therefore provide critical new insight into the triggering process. TM domain ␤-branched residues such as valine or isoleucine have been implicated in backbone dynamics and fusogenicity of peptides (68) and the fusion function of the reovirus fusion-associated small transmembrane proteins (61). ␤-Branched residues are present throughout the paramyxovirus F protein TM domains, and we have recently demonstrated that mutation of these residues can alter membrane fusion without affecting surface expression (64); however, the effect of these mutations on TM interactions remains to be determined.…”

Beyond Anchoring: the Expanding Role of the Hendra Virus Fusion Protein Transmembrane Domain in Protein Folding, Stability, and Function

“…Combining these data with the data from the C-terminal insertions (which immediately preceded the VIV sequence) suggests that ␤-branched residues in the sequence FVIV could be important modulators of F protein-promoted membrane fusion. Recent work with the baboon reovirus p15 TMD (15) and model peptides (34,35,60,61) suggests that ␤-branched residues are important in membrane fusion in a variety of systems. Nonpolar ␤-branched residues (valine and isoleucine) are traditionally thought of as being disruptive of ␣-helical structures; however, studies of synthetic peptides in detergent micelles demonstrated that these residues are incorporated into hydrophobic ␣-helices as easily as leucine (39,40).…”

Efficient Reovirus- and Measles Virus-Mediated Pore Expansion during Syncytium Formation Is Dependent on Annexin A1 and Intracellular Calcium

“…They assume a bitopic, N exoplasmic /C cytoplasmic topology in membranes, positioning very small (ϳ20 to 40 residues) fusion peptide-containing domains external to the plasma membrane (17)(18)(19)(20) and equalsized or considerably larger (ϳ36 to 141 residues) domains in the cytoplasm (21). The ecto-, endo-, and transmembrane domains all function as fusion modules and play an active role in the membrane fusion process (22,23).…”