title: Mutational analysis of the membrane proximal heptad repeat of the newcastle disease virus fusion protein.
abstract: Paramyxovirus fusion proteins have two heptad repeat domains, HR1 and HR2, that have been implicated in the fusion activity of the protein. Peptides from these two domains form a six-stranded, coiled-coil with the HR1 sequences forming a central trimer and three molecules of the HR2 helix located within the grooves in the central trimer (Baker et al., 1999, Mol. Cell 3, 309; Zhao et al. 2000, Proc. Natl. Acad. Sci. 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 trimer. These residues form the hydrophobic face of the helix and adjacent residues ("a" and "g" positions in the HR2 helical wheel structure). Mutant proteins were characterized for effects on synthesis, steady-state levels, proteolytic cleavage, and surface expression as well as fusion activity as measured 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. Nonconservative mutations in the "g" position had no effect on fusion. In contrast, single changes in the middle "a" position of HR2 inhibited lipid mixing, content mixing, and syncytia formation. A single mutation in the more carboxyl-terminal "a" position had minimal effects on lipid mixing but did inhibit content mixing and syncytia formation. These results are consistent with the idea that the HR2 domain is involved in posttranslational interactions with HR1 that mediate the close approach of membranes. These results also suggest that the HR2 domain, particularly the carboxyl-terminal region, plays an additional role in fusion, a role related to content mixing and syncytia formation.
authors.affiliation: Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, Massachusetts 01532, USA.
authors.forename: L W
authors.initials: LW
authors.lastname: McGinnes
authors.forename: T
authors.initials: T
authors.lastname: Sergel
authors.forename: H
authors.initials: H
authors.lastname: Chen
authors.forename: L
authors.initials: L
authors.lastname: Hamo
authors.forename: S
authors.initials: S
authors.lastname: Schwertz
authors.forename: D
authors.initials: D
authors.lastname: Li
authors.forename: T G
authors.initials: TG
authors.lastname: Morrison
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D005434:Flow Cytometry; D015726:Giant Cells / Q000821:virology; D008561:Membrane Fusion; D008563:Membrane Lipids / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009522:Newcastle disease virus / Q000235:genetics* / Q000378:metabolism; D017421:Sequence Analysis; D014760:Viral Fusion Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D008563:Membrane Lipids; D014760:Viral Fusion Proteins