Fusion mechanism of Nelson Bay virus fusion-associated small transmembrane protein

Our studies focused on the fusion mechanism of the Nelson Bay Virus (NBV) Fusion-Associated Small Transmembrane (FAST) protein. Specifically, we examined the presence of a fusion peptide, the multimeric interaction of FAST protein monomers, and the fusion-inhibitory effect of synthetic peptides corresponding to the ectodomain of the NBV FAST protein, also known as p10. Furthermore, we investigated the potential of pseudotyping retroviral and lentiviral vectors with a range of viral fusion proteins in attempt to provide targeted gene therapy.; Sequence analysis and structural studies identified NBV p10 as a type I membrane protein with a central transmembrane domain, a cytoplasmic basic region, and an N-terminal hydrophobic domain (HD) that was hypothesized to function as a fusion peptide. We performed mutational analysis on this slightly hydrophobic motif to identify possible structural requirements for fusion activity. Bulky aliphatic residues were found to be essential for optimal fusion, and an aromatic or highly hydrophobic side chain was found to be required at position 12. The requirement for hydrophilic residues within the HD was also examined: substitution of 10-Ser or 14-Ser with hydrophobic residues was found to reduce cell surface expression of p10 and delayed the onset of syncytium formation. Nonconservative substitutions of charged residues in the HD did not have an effect on fusion activity. Taken together, our results suggest that the HD is involved in both syncytium formation and in determining p10 transport and surface expression.; We investigated whether multimerization occurs with the NBV p10 protein. SDS-PAGE analysis, complementation, and co-immunoprecipitation experiments indicate that interactions between NBV p10 monomers occur, which could play a role in the fusion process. Two mutants in the external domain, C5S and F12Y, were found to be dominant-negative suppressors of fusion activity. Other mutants C17S, F12G, and C5,17S were fusion-negative, but were not observed to interfere with the fusion process when coexpressed with the native p10 proteins. The dominant-negative effect seems to be linked with strong intermolecular interaction as shown by co-immunoprecipitation, while functional NBV p10 monomers exhibit much weaker association in comparison. Taken together, the results suggest a model in which oligomerization or aggregation of the p10 proteins plays a role in p10-induced syncytium formation. (Abstract shortened by UMI.)...