Intracellular processing, glycosylation, and cell-surface expression of the measles virus fusion protein (F) encoded by a recombinant adenovirus.
The membrane fusion protein of measles virus (MVF) is a surface glycoprotein which is essential for initiation of viral infection. The F protein mediates penetration of the host cell through a process of membrane fusion between the viral envelope and the host cell plasma membrane. To study the structure-function relationship of the MVF protein, a recombinant adenovirus, Ad5MVF, was constructed which expressed the F protein in mammalian cells. The MVF gene was inserted into the Ad5 genome by homologous recombination, which resulted in replacement of most of the E1 region. This recombinant virus was stable and replicated efficiently in the 293 cell line which complemented the deleted E1 functions. Human 293 cells infected with Ad5MVF synthesized an authentic MVF protein precursor (F0) which appeared to be cleaved efficiently to the F1 and F2 polypeptides. This recombinant F protein was glycosylated, transported to the cell surface, and found to be capable of inducing syncytia formation and hemolysis of monkey erythrocytes. The hemagglutinin protein (HA), provided by a coinfecting adenovirus, was not able to increase the biological activity of the F protein. Treatment of MV or Ad5MVF-infected cells with tunicamycin, an inhibitor of N-linked glycosylation, abolished processing of the F protein. This observation suggests that glycosylation might play an important role in cleavage-dependent activation of the precursor F0 protein or in its transport to the subcellular region where proteolytic cleavage occurs.
Alkhatib
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Genetic Engineering Section, National Research Council Canada, Biotechnology Research Institute, Montreal, Quebec.
Richardson
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Shen
S H
SH
D000260:Adenoviruses, Human / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D006031:Glycosylation; D006367:HeLa Cells; D006461:Hemolysis; D006801:Humans; D008459:Measles virus / Q000235:genetics*; D008562:Membrane Glycoproteins / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids; D011499:Protein Processing, Post-Translational; D011995:Recombination, Genetic; D015183:Restriction Mapping; D014415:Tunicamycin / Q000494:pharmacology; D014709:Vero Cells; D014760:Viral Fusion Proteins / Q000235:genetics*
D008562:Membrane Glycoproteins; D014760:Viral Fusion Proteins; D014415:Tunicamycin