title: Engineered intermonomeric disulfide bonds in the globular domain of Newcastle disease virus hemagglutinin-neuraminidase protein: implications for the mechanism of fusion promotion.
abstract: The promotion of membrane fusion by Newcastle disease virus (NDV) requires an interaction between the viral hemagglutinin-neuraminidase (HN) and fusion (F) proteins, although the mechanism by which this interaction regulates fusion is not clear. The NDV HN protein exists as a tetramer composed of a pair of dimers. Based on X-ray crystallographic studies of the NDV HN globular domain (S. Crennell et al., Nat. Struct. Biol. 7:1068-1074, 2000), it was proposed that the protein undergoes a significant conformational change from an initial structure having minimal intermonomeric contacts to a structure with a much more extensive dimer interface. This conformational change was predicted to be integral to fusion promotion with the minimal interface form required to maintain F in its prefusion state until HN binds receptors. However, no evidence for such a conformational change exists for any other paramyxovirus attachment protein. To test the NDV model, we have engineered a pair of intermonomeric disulfide bonds across the dimer interface in the globular domain of an otherwise non-disulfide-linked NDV HN protein by the introduction of cysteine substitutions for residues T216 and D230. The disulfide-linked dimer is formed both intracellularly and in the absence of receptor binding and is efficiently expressed at the cell surface. The disulfide bonds preclude formation of the minimal interface form of the protein and yet enhance both receptor-binding activity at 37 degrees C and fusion promotion. These results confirm that neither the minimal interface form of HN nor the proposed drastic conformational change in the protein is required for fusion.
authors.affiliation: Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, MA 01655, USA.
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Mahon
authors.forename: Anne M
authors.initials: AM
authors.lastname: Mirza
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Musich
authors.forename: Ronald M
authors.initials: RM
authors.lastname: Iorio