Mutational analysis of the leucine zipper motif in the Newcastle disease virus fusion protein

• Published in: Journal of Virology Vol. 69; no. 10; pp. 5995 - 6004
• Main Authors: Reitter, J.N. (University of Massachusetts Medical School, Worcester, MA.), Sergel, T, Morrison, T.G
• Format: Journal Article
• Language: English
• Published: United States American Society for Microbiology 01.10.1995
• Subjects: ALANINA; ALANINE; Amino Acid Sequence; Animals; Base Sequence; Cell Line; Cell Membrane - metabolism; Cercopithecus aethiops; COMPOSICION QUIMICA; COMPOSITION CHIMIQUE; DNA Mutational Analysis; Gene Expression; GLICOPROTEINAS; GLYCOPROTEINE; Kidney; LEUCINA; LEUCINE; Leucine Zippers; Membrane Fusion; Methionine - metabolism; Models, Structural; Molecular Sequence Data; MUTACION; MUTACION INDUCIDA; Mutagenesis, Site-Directed; MUTANT; MUTANTES; MUTATION; MUTATION PROVOQUEE; Newcastle disease virus; Newcastle disease virus - genetics; Newcastle disease virus - metabolism; Oligodeoxyribonucleotides - chemical synthesis; paramyxovirus; Protein Conformation; PROTEINAS; PROTEINE; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Sulfur Radioisotopes; Viral Fusion Proteins - biosynthesis; Viral Fusion Proteins - chemistry; Viral Fusion Proteins - metabolism; VIRUS DE LA ENFERMEDAD DE NEWCASTLE; VIRUS DE LOS ANIMALES; VIRUS DES ANIMAUX; VIRUS MALADIE DE NEWCASTLE
• ISSN: 0022-538X; 1098-5514
• DOI: 10.1128/JVI.69.10.5995-6004.1995

The paramyxovirus fusion proteins have a highly conserved leucine zipper motif immediately upstream from the transmembrane domain of the F1 subunit (R. Buckland and F. Wild, Nature [London] 338:547,1989). To determine the role of the conserved leucines in the oligomeric structure and biological activity of the Newcastle disease virus (NDV) fusion protein, the heptadic leucines at amino acids 481, 488, and 495 were changed individually and in combination to an alanine residue. While single amino acid changes had little effect on fusion, substitution of two or three leucine residues abolished the fusogenic activity of the protein, although cell surface expression of the mutants was higher than that of the wild-type protein. Substitution of all three leucine residues with alanine did not alter the size of the fusion protein oligomer as determined by sedimentation in sucrose gradients. Furthermore, deletion of the C-terminal 91 amino acids, including the leucine zipper motif and transmembrane domain, resulted in secretion of an oligomeric polypeptide. These results indicate that the conserved leucines are not necessary for oligomer formation but are required for the fusogenic ability of the protein. When the polar face of the potential alpha helix was altered by nonconservative changes of serine to alanine (position 473), glutamic acid to lysine or alanine (position 482), asparagine to lysine (position 485), or aspartic acid to alanine (position 489), the fusogenic ability of the protein was not significantly disrupted. In addition, a double mutant (E482A,D489A) which removed negative charges along one side of the helix had negligible effects on fusion activity