Abstract
The reovirus fusion-associated small transmembrane (FAST) proteins are virus-encoded membrane fusion proteins that function as dedicated cell–cell fusogens. The topology of these small, single-pass membrane proteins orients the majority of the protein on the distal side of the membrane (i.e., inside the cell). We now show that ectopic expression of the endodomains of the p10, p14, and p15 FAST proteins enhances syncytiogenesis induced by the full-length FAST proteins, both homotypically and heterotypically. Results further indicate that the 68-residue cytoplasmic endodomain of the p14 FAST protein (1) is endogenously generated from full-length p14 protein expressed in virus-infected or transfected cells; (2) enhances syncytiogenesis subsequent to stable pore formation; (3) increases the syncytiogenic activity of heterologous fusion proteins, including the differentiation-dependent fusion of murine myoblasts; (4) exerts its enhancing activity from the cytosol, independent of direct interactions with either the fusogen or the membranes being fused; and (5) contains several regions with protein–protein interaction motifs that influence enhancing activity. We propose that the unique evolution of the FAST proteins as virus-encoded cellular fusogens has allowed them to generate a trans-acting, soluble endodomain peptide to harness a cellular pathway or process involved in the poorly understood process that facilitates the transition from microfusion pores to macrofusion and syncytiogenesis.
Highlights
The formation of multi-nucleated syncytia is an essential feature of a diverse range of biological processes [1]
We have been interested in reconciling the donor membrane-biased structural features of the fusion-associated small transmembrane (FAST) proteins with their ability to orchestrate the multi-step cell–cell membrane fusion process that leads to syncytium formation
We show that the FAST proteins generate a soluble endodomain fragment that functions in trans from the cytosol, enhancing the capacity of diverse viral and cellular fusogens to drive the conversion of fusion pores into syncytia
Summary
The formation of multi-nucleated syncytia is an essential feature of a diverse range of biological processes [1]. Our current understanding of protein-mediated membrane fusion derives largely from the study of enveloped virus proteins designed to promote virus–cell fusion [4,5], and from the SNARE proteins involved in intracellular vesicle fusion [6]. These studies converge on what may be a unifying model of membrane fusion involving a multi-step fusionthrough-hemifusion pathway mediated by dynamic remodelling of the fusion protein complex [7,8]. While mechanisms by which membrane fusion proteins promote membrane merger and the formation of focal fusion pores are beginning to emerge, relatively little is known about the processes that drive expansion of these fusion apertures, an essential step for those cell–cell fusion events that result in syncytium formation [9,10]
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