title: Evidence for mixed membrane topology of the newcastle disease virus fusion protein.
abstract: The synthesis of the Newcastle disease virus (NDV) fusion (F) protein in a cell-free protein-synthesizing system containing membranes was characterized. The membrane-associated products were in at least two different topological forms with respect to the membranes. The properties of one form were consistent with the expected membrane insertion as a classical type 1 glycoprotein. This form of the protein was fully glycosylated, and sequences amino terminal to the transmembrane domain were protected from protease digestion by the membranes. The second form of membrane-associated F protein was partially glycosylated and partially protected from protease digestion by the membranes. Protease digestion resulted in a 23-kDa protease-protected polypeptide derived from F2 sequences and sequences from the amino-terminal end of the F1 domain. Furthermore, a 10-kDa polypeptide derived from the cytoplasmic domain (CT) was also protected from protease digestion by the membranes. Protease resistance of the 23- and 10-kDa polypeptides suggested that this second form of F protein inserted in membranes in a polytopic conformation with both the amino-terminal end and the carboxyl-terminal end translocated across membranes. To determine if this second form of the fusion protein could be found in cells expressing the F protein, two different approaches were taken. A polypeptide with the size of the partially translocated F protein was detected by Western analysis of proteins in total-cell extracts of NDV strain B1 (avirulent)-infected Cos-7 cells. Using antibodies raised against a peptide with sequences from the cytoplasmic domain, CT sequences were detected on surfaces of F protein-expressing Cos-7 cells by immunofluorescence and by flow cytometry. This antibody also inhibited the fusion of red blood cells to cells expressing F and HN proteins. These results suggest that NDV F protein made both in a cell-free system and in Cos-7 cells may exist in two topological forms with respect to membranes and that the second form of the protein may be involved in cell-cell fusion.
authors.affiliation: Program in Virology/Immunology, Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA.
authors.forename: Lori W
authors.initials: LW
authors.lastname: McGinnes
authors.forename: Julie N
authors.initials: JN
authors.lastname: Reitter
authors.forename: Kathy
authors.initials: K
authors.lastname: Gravel
authors.forename: Trudy G
authors.initials: TG
authors.lastname: Morrison
mesh_terms: D000595:Amino Acid Sequence; D015153:Blotting, Western; D002462:Cell Membrane / Q000737:chemistry*; D003593:Cytoplasm / Q000378:metabolism; D006031:Glycosylation; D008561:Membrane Fusion; D008969:Molecular Sequence Data; D009522:Newcastle disease virus / Q000737:chemistry*; D014176:Protein Biosynthesis; D021381:Protein Transport; D014760:Viral Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D014760:Viral Fusion Proteins