GSBS Student Publications

Title

Evidence for mixed membrane topology of the newcastle disease virus fusion protein

UMMS Affiliation

Program in Virology/Immunology; Department of Molecular Genetics and Microbiology

Date

1-15-2003

Document Type

Article

Medical Subject Headings

Amino Acid Sequence; Blotting, Western; Cell Membrane; Cytoplasm; Glycosylation; Membrane Fusion; Molecular Sequence Data; Newcastle disease virus; Protein Biosynthesis; Protein Transport; Viral Fusion Proteins

Disciplines

Life Sciences | Medicine and Health Sciences

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.

Rights and Permissions

Citation: J Virol. 2003 Feb;77(3):1951-63.

Related Resources

Link to article in PubMed

Journal Title

Journal of virology

PubMed ID

12525629