title: Role of the simian virus 5 fusion protein N-terminal coiled-coil domain in folding and promotion of membrane fusion.
abstract: Formation of a six-helix bundle comprised of three C-terminal heptad repeat regions in antiparallel orientation in the grooves of an N-terminal coiled-coil is critical for promotion of membrane fusion by paramyxovirus fusion (F) proteins. We have examined the effect of mutations in four residues of the N-terminal heptad repeat in the simian virus 5 (SV5) F protein on protein folding, transport, and fusogenic activity. The residues chosen have previously been shown from study of isolated peptides to have differing effects on stability of the N-terminal coiled-coil and six-helix bundle (R. E. Dutch, G. P. Leser, and R. A. Lamb, Virology 254:147-159, 1999). The mutant V154M showed reduced proteolytic cleavage and surface expression, indicating a defect in intracellular transport, though this mutation had no effect when studied in isolated peptides. The mutation I137M, previously shown to lower thermostability of the six-helix bundle, resulted in an F protein which was properly processed and transported to the cell surface but which had reduced fusogenic activity. Finally, mutations at L140M and L161M, previously shown to disrupt alpha-helix formation of isolated N-1 peptides but not to affect six-helix bundle formation, resulted in F proteins that were properly processed. Interestingly, the L161M mutant showed increased syncytium formation and promoted fusion at lower temperatures than the wild-type F protein. These results indicate that interactions separate from formation of an N-terminal coiled-coil or six-helix bundle are important in the initial folding and transport of the SV5 F protein and that mutations that destabilize the N-terminal coiled-coil can result in stimulation of membrane fusion.
authors.affiliation: Department of Molecular and Cellular Biochemistry, University of Kentucky, 800 Rose St., UKMC MN606, Lexington, KY 40536-0298, USA.
authors.forename: Dava S
authors.initials: DS
authors.lastname: West
authors.forename: Michael S
authors.initials: MS
authors.lastname: Sheehan
authors.forename: Patrick K
authors.initials: PK
authors.lastname: Segeleon
authors.forename: Rebecca Ellis
authors.initials: RE
authors.lastname: Dutch
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D006224:Cricetinae; D015726:Giant Cells; D006367:HeLa Cells; D006801:Humans; D008561:Membrane Fusion; D008969:Molecular Sequence Data; D045402:Parainfluenza Virus 5 / Q000378:metabolism* / Q000472:pathogenicity; D011487:Protein Conformation; D017510:Protein Folding; D011993:Recombinant Fusion Proteins; D014709:Vero Cells; D014760:Viral Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D014760:Viral Fusion Proteins