Mutational analysis of heptad repeats in the membrane-proximal region of Newcastle disease virus HN protein

For most paramyxoviruses, syncytium formation requires the expression of bo th surface glycoproteins (HN and Fl in the same cell, and evidence suggests that Fusion involves a specific interaction between the HN and F proteins (X. Hu et al,, J, Virol, 66:1528-1533, 1992). The stalk region of the Newca stle disease virus (NDV) HN protein has been implicated in both fusion prom otion and virus specificity of that activity, The NDV F protein contains tw o heptad repeat motifs which have been shown by site-directed mutagenesis t o be critical for fusion (R. Buckland et al.. J. Gen. Virol, 73:1703-1707, 1992; T, Sergel-Germano ct al,, J. Virol, 68:7654-7658, 1994; J, Reitter et al,, J. Virol, 69:5995-6004, 1995), Heptad repeat motifs mediate protein-p rotein interactions by enabling the formation of coiled coils, Upon analysi s of the stalk region of the NDV HN protein, we identified two heptad repea ts, Secondary structure analysis of these repeats suggested the potential f or these regions to form alpha helices, To investigate the importance of th is sequence motif for fusion promotion, we mutated the hydrophobic a-positi on amino acids of each heptad repeat to alanine or methionine, In addition, hydrophobic amino acids in other positions were also changed to alanine, E very mutant protein retained levels of attachment activity that was greater than or equal to the wild-type protein activity and bound to conformation- specific monoclonal as well as polyclonal antisera, Neuraminidase activity was variably affected. Every mutation, however, showed a dramatic decrease in fusion promotion activity. The phenotypes of these mutant proteins indic ate that individual amino acids within the heptad repeat region of the stal k domain of the I-IN protein are important for the fusion promotion activit y of the protein. These data are consistent with the idea that the HN prote in associates with the F protein via specific interactions between the hept ad repeat regions of both proteins.