A Virus-Encoded Cell–Cell Fusion Machine Dependent on Surrogate Adhesins

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Abstract
The reovirus fusion-associated small transmembrane (FAST) proteins function as virus-encoded cellular fusogens, mediating efficient cell–cell rather than virus–cell membrane fusion. With ectodomains of only ∼20–40 residues, it is unclear how such diminutive viral fusion proteins mediate the initial stages (i.e. membrane contact and close membrane apposition) of the fusion reaction that precede actual membrane merger. We now show that the FAST proteins lack specific receptor-binding activity, and in their natural biological context of promoting cell–cell fusion, rely on cadherins to promote close membrane apposition. The FAST proteins, however, are not specifically reliant on cadherin engagement to mediate membrane apposition as indicated by their ability to efficiently utilize other adhesins in the fusion reaction. Results further indicate that surrogate adhesion proteins that bridge membranes as close as 13 nm apart enhance FAST protein-induced cell–cell fusion, but active actin remodelling is required for maximal fusion activity. The FAST proteins are the first example of membrane fusion proteins that have specifically evolved to function as opportunistic fusogens, designed to exploit and convert naturally occurring adhesion sites into fusion sites. The capacity of surrogate, non-cognate adhesins and active actin remodelling to enhance the cell–cell fusion activity of the FAST proteins are features perfectly suited to the structural and functional evolution of these fusogens as the minimal fusion component of a virus-encoded cellular fusion machine. These results also provide a basis for reconciling the rudimentary structure of the FAST proteins with their capacity to fuse cellular membranes. Much of our current understanding of how proteins mediate membrane fusion derives from the study of enveloped virus fusion proteins. These fusion protein complexes function autonomously to co-ordinately regulate virus–cell attachment and subsequent membrane merger. In contrast, the reovirus Fusion-Associated Small Transmembrane (FAST) proteins are the only example of virus-encoded cellular fusogens, specifically designed to mediate cell–cell rather than virus–cell membrane fusion. In view of their small size, it was unclear if, or how, the FAST proteins are responsible for promoting the membrane attachment and close apposition stages of the fusion reaction. We now show that the FAST proteins have specifically evolved to function as the fusion component in a biphasic cell–cell fusion reaction, where the membrane attachment and membrane merger stages represent two distinct, uncoupled phases. Exploiting cadherins as surrogate adhesins, the FAST proteins have retained within their rudimentary structures the minimal determinants required to convert pre-existing adherens junctions into sites of cell–cell membrane fusion. These results raise the interesting possibility that other, yet to be identified cellular fusion proteins may resemble the FAST proteins, using separate adhesins and less complex fusion proteins in a similar biphasic membrane fusion reaction.