Mutational analysis of the membrane proximal heptad repeat of the Newcastle disease virus fusion protein

Paramyxovirus fusion proteins have two heptad repeat domains, HR1 and HR2, that have been implicated in the fusion activity of the protein. Peptides f rom these two domains form a six-stranded, coiled-coil with the HR1 sequenc es forming a central trimer and three molecules of the HR2 helix located wi thin the grooves In the central trimer (Baker et at., 1999, Mol. Cell 3, 30 9; Zhao at al. 2000, Proc. Natl. Acad. Scl, USA 97,14172). Nonconservative mutations were made in the HR2 domain of the Newcastle disease virus fusion protein in residues that are likely to form contacts with the HR1 core tri mer. These residues form the hydrophobic face of the helix and adjacent res idues ("a" and "g" positions in the HR2 helical wheel structure). Mutant pr oteins were characterized for effects on synthesis, steady-state levels, pr oteolytic, cleavage, and surface expression as well as fusion activity as m easured by syncytia formation, content mixing, and lipid mixing. While all mutant proteins were transport competent and proteolytically cleaved, these mutations did variously affect fusion activity of the protein. Nonconserva tive mutations in the "g" position had no effect on fusion. In contrast, si ngle changes in the middle "a" position of HR2 inhibited lipid mixing, cont ent mixing, and syncytia formation. A single mutation in the more carboxyl- terminal "a" position had minimal effects on lipid mixing but did inhibit c ontent mixing and syncytia formation. These results are consistent with the idea that the HR2 domain is involved In posttranslational interactions wit h HR1 that mediate the close approach of membranes. These results also sugg est that the HR2 domain, particularly the carboxyl-terminal region, plays a n additional role in fusion, a role related to content mixing and syncytia formation. (C) 2001 Academic Press.