Role of Membrane Curvature and Exosome Secretion during FAST-Mediated Cell-Cell Fusion

Abstract

The fusogenic reoviruses are a unique group of viruses that encode Fusion-Associated Small Transmembrane (FAST) proteins, a family of non-structural membrane proteins that induce cell-cell membrane fusion in infected cells. The compact FAST proteins range in size from 98 to 148 amino acids, making them an ideal model for studying the process of cell-cell fusion. In this work, we have examined the mechanisms by which the FAST proteins modulate membrane curvature during membrane fusion through the examination of (1) a novel cytosolic fusion-inducing lipid packing sensor (FLiPS) element, and (2) the induction of exosome secretion by the FAST proteins during syncytiogenesis. Unlike the archetypal enveloped virus fusion proteins, the FAST proteins position the majority of the protein within the cytoplasm. We now show a unique 20 amino acid motif in the endodomain of baboon reovirus p15 functions as a novel FLiPS element. We show p15FLiPS is essential for pore formation during cell-cell fusion and reliant on a hydrophobic helix-loop-helix architecture. Furthermore, we show that p15FLiPS preferentially partitions into membranes fraught with lipid packing defects, and this partitioning is impeded when packing defects are blocked. Finally, p15FLiPS can be functionally replaced with other heterologous lipid packing sensor motifs. Therefore, we propose p15FLiPS binds to the highly curved rim of the nascent fusion pore, thereby lowering the energy to pore formation. Exosomes are secreted membrane vesicles that function in intercellular communication. The role of exosome secretion in cell-cell fusion has not yet been determined. Here, we show that FAST proteins are secreted in exosomes during cell-cell fusion, and their incorperation does not significantly alter the exosome proteome. We show that export of p14 to exosomes is dependent on TSG101 and the cytosolic endodomain of p14. Finally, we demonstrate that exogenous p14-exosomes are sufficient to induce cell-cell fusion. Taken together, we suggest that the FAST proteins hijack the exosome secretion pathway to enhance cell-cell fusion, thereby augmenting the overall spread of virus. Taken together, these results suggest that FAST proteins modulate membrane curvature during cell-cell fusion through use of specific lipid packing sensors and interactions with host proteins such as the ESCRT complex.