Mutations in the fusion peptide and adjacent heptad repeat inhibit foldingor activity of the Newcastle disease virus fusion protein

Paramyxovirus fusion proteins have two heptad repeat domains, HR1 and HR2, which have been implicated in the fusion activity of the protein. Peptides with sequences from these two domains form a six-stranded coiled coil, with the HR1 sequences forming a central trimer (K. A. Baker, R. E. Dutch, R. A . Lamb, and T. S. Jardetzky, Mol. Cell 3:309-319,1999; X. Zhao, M. Singh, V . N. Malashkevich, and P. S. Kim, Proc. Natl. Acad. Sci. USA 97:14172-14177 , 2000). We have extended our previous mutational analysis of the HR1 domai n of the Newcastle disease virus fusion protein, focusing on the role of th e amino acids forming the hydrophobic core of the trimer, amino acids in th e "a" and "d" positions of the helix from amino acids 123 to 182. Both cons ervative and nonconservative point mutations were characterized for their e ffects on synthesis, stability, proteolytic cleavage, and surface expressio n. Mutant proteins expressed on the cell surface were characterized for fus ion activity by measuring syncytium formation, content mixing, and lipid mi xing. We found that all mutations in the "a" position interfered with prote olytic cleavage and surface expression of the protein, implicating the HR1 domain in the folding of the F protein. However, mutation of five of seven "d" position residues had little or no effect on surface expression but, wi th one exception at residue 175, did interfere to various extents with the fusion activity of the protein. One of these "d" mutations, at position 154 , interfered with proteolytic cleavage, while the rest of the mutants were cleaved normally. That most "d" position residues do affect fusion activity argues that a stable HR1 trimer is required for formation of the six-stran ded coiled coil and, therefore, optimal fusion activity. That most of the " d" position mutations do not block folding suggests that formation of the c ore trimer may not be required for folding of the prefusion form of the pro tein. We also found that mutations within the fusion peptide, at residue 12 8, can interfere with folding of the protein, implicating this region in fo lding of the molecule. No characterized mutation enhanced fusion.