title: Rescue of vector-expressed fowl plague virus hemagglutinin in biologically active form by acidotropic agents and coexpressed M2 protein.
abstract: The hemagglutinin of the Rostock strain of fowl plague virus was expressed in CV-1 cells by a simian virus 40 vector, and its stability in the exocytotic transport process was examined by a fusion assay. A 50-fold increase in the fusion activity of the hemagglutinin was observed when expression occurred in the presence of ammonium chloride, Tris-HCl, or high doses of amantadine. When chloroquine, another acidotropic agent, was used, the hemagglutinin exposed at the cell surface had to be activated by trypsin, because intracellular cleavage was inhibited by this compound. Hemagglutinin mutants resistant to intracellular cleavage did not require acidotropic agents for full expression of fusion activity, when treated with trypsin after arrival at the cell surface. These results indicate that fowl plague virus hemagglutinin expressed by a simian virus 40 vector is denatured in the acidic milieu of the exocytotic pathway and that cleavage is a major factor responsible for the pH instability. Coexpression with the M2 protein also markedly enhanced the fusion activity of the hemagglutinin, and this effect was inhibited by low doses of amantadine. These results support the concept that M2, known to have ion channel function, protects the hemagglutinin from denaturation by raising the pH in the exocytotic transport system. The data also stress the importance of acidotropic agents or coexpressed M2 for the structural and functional integrity of vector-expressed hemagglutinin.
authors.affiliation: Institut für Virologie, Philipps-Universität Marburg, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Ohuchi
authors.forename: A
authors.initials: A
authors.lastname: Cramer
authors.forename: M
authors.initials: M
authors.lastname: Vey
authors.forename: R
authors.initials: R
authors.lastname: Ohuchi
authors.forename: W
authors.initials: W
authors.lastname: Garten
authors.forename: H D
authors.initials: HD
authors.lastname: Klenk
mesh_terms: D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D002459:Cell Fusion / Q000187:drug effects; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D005455:Fluorescent Antibody Technique; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D009980:Influenza A virus / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000096:biosynthesis; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis; D013539:Simian virus 40 / Q000235:genetics; D014760:Viral Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D014763:Viral Matrix Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology*; D000086082:Viroporin Proteins
chemical_list: D000643:Ammonium Chloride; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral; D008565:Membrane Proteins; D011994:Recombinant Proteins; D014760:Viral Fusion Proteins; D014763:Viral Matrix Proteins; C031540:M-protein, influenza virus; C488937:M2 protein, Influenza A virus; D000086082:Viroporin Proteins