Mechanistic Contributions of the p10 FAST Protein Ectodomains to Membrane Fusion and Syncytiogenesis

Abstract

The homologous p10 fusion-associated small transmembrane (FAST) proteins of the fusogenic avian (ARV) and Nelson Bay (NBV) reoviruses are the smallest known proteins capable of mediating syncytiogenesis. Their extremely small size precludes them from following the paradigmatic membrane fusion pathway proposed for enveloped viral fusion proteins. I exploited the sequence conservation/divergence and differential syncytiogenic rates between ARV and NBV to define functional motifs in the p10 ectodomains. Using chimeric p10 constructs, I determined the 40-residue ectodomain (sizes refer to ARV) comprises two distinct functional motifs essential for syncytiogenesis. Cellular syncytiogenic and surface biotinylation assays identified an indivisible, 25- residue, N-terminal ectodomain motif required for cystine loop fusion peptide formation. I further determined the roles of this cystine loop in promoting lipid binding and cholesterol-dependent lipid destabilization. Immunofluorescence staining, FRET analysis and cholesterol depletion/repletion studies identified a second motif comprising the 13 membrane-proximal ectodomain residues (MPER). This motif governs the reversible, cholesterol-dependent assembly of p10 multimers in the plasma membrane. I demonstrate that ARV and NBV homomultimers segregate to separate foci in the plasma membrane, and the four juxtamembrane residues present in the multimerization motif dictate species- specific homomultimerization. I also discovered the novel codependency of p10 multimerization and cholesterol-dependent microdomain localization. The majority of enveloped virus membrane fusion proteins function as stable multimers, which nonetheless must undergo dramatic, irreversible, tertiary structure rearrangements to mediate membrane fusion. Cholesterol-rich membrane microdomains have also been implicated in the function of several enveloped virus fusion proteins, and a limited number of studies have investigated the role of cholesterol in multimerization. My results reveal cholesterol-dependent p10 homomultimerization is an essential aspect of p10- mediated syncytium formation, and I identify the motifs responsible for this process. The reversible nature of p10 cholesterol-dependent multimerization at the plasma membrane is in line with several other studies suggesting that the dynamic clustering and dispersion of cholesterol microdomains, as well as protein transitioning from multimeric to monomeric intermediates, are essential phenomena of protein mediated membrane fusion.