1.G.13 The Orthoreovirus Fusion-associated Small Transmembrane (FAST) Family
The Orthoreovirus fusion-associated small transmembrane (FAST) proteins are dedicated cell-cell fusogens responsible for multinucleated syncytia formation, and are virulence determinants of the fusogenic reoviruses (Ciechonska et al. 2014). FAST proteins and enveloped viral fusogens have delineated steps involved in membrane fusion and pore formation which involves eventual pore expansion needed for syncytiogenesis. The 3-D NMR structure of a 32 aas active peptide of FAST p14 has been solved (Corcoran et al. 2004).
Ciechonska et al. 2014 reported that RNAi knockdown of annexin A1 (AX1) expression dramatically reduced both reptilian reovirus p14 and measles virus F and H protein- mediated pore expansion during syncytiogenesis, but had no effect on p14-induced pore formation. A similar effect was obtained by chelating intracellular calcium, which dramatically decreased syncytiogenesis in the absence of detectable effects on p14-induced pore formation. Co- immunoprecipitation revealed calcium-dependent interactions between AX1 and p14 or measles virus F and H proteins, and fluorescence resonance energy transfer (FRET) demonstrated calcium-dependent p14-AX1 interactions in cellulo. Furthermore, antibody inhibition of extracellular AX1 had no effect on p14-induced syncytium formation, but did impair cell-cell fusion mediated by the endogenous muscle cell fusion machinery in C2C12 mouse myoblasts. AX1 can therefore exert diverse, fusogen- specific effects on cell-cell fusion, functioning as an extracellular mediator of differentiation-dependent membrane fusion or as an intracellular promoter of post-fusion pore expansion and syncytium formation following viral-mediated cell-cell fusion.
Numerous enveloped viruses, and nonenveloped fusogenic orthoreoviruses, encode membrane fusion proteins that induce syncytium formation linked to viral pathogenicity. Ciechonska et al. 2014 identified intracellular calcium and annexin A1 (AX1) as key factors required for efficient pore expansion during syncytium formation mediated by the reptilian reovirus p14 and measles virus F and H fusion protein complexes. Involvement of intracellular AX1 in syncytiogenesis directly correlates with a requirement for intracellular calcium in p14-AX1 interactions and pore expansion, but not membrane fusion and pore formation. Lateral propagation of stable fusion pores leading to syncytiogenesis mediated by diverse viral fusogens is inhibited by promotion of positive membrane curvature in the outer leaflets of the lipid bilayer surrounding intercellular fusion pores. Positive membrane curvature is induced by lysophosopholipids (Ciechonska and Duncan 2014).
Reptilian reovirus is one of a limited number of nonenveloped viruses that are capable of inducingcell-cell fusion. A small, hydrophobic, basic, 125-amino-acid fusion protein encoded by the first open reading frame of a bicistronic viral mRNA is responsible for this fusion activity (Corcoran and Duncan 2004). Topological analysis revealed that p14 is a representative of a minor subset of integral membrane proteins, the type III proteins N(exoplasmic)/C(cytoplasmic) (N(exo)/C(cyt)), that lack a cleavable signal sequence and use an internal reverse signal-anchor sequence to direct membrane insertion and protein topology. This topology results in the unexpected, cotranslational translocation of the essential myristylated N-terminal domain of p14 across the cell membrane. The topology and structural motifs present in this reoviral membrane fusion protein accentuate the diversity and unusual properties of the FAST protein family (Corcoran and Duncan 2004).