title: Membrane vesiculation function and exocytosis of wild-type and mutant matrix proteins of vesicular stomatitis virus.
abstract: Transfection of mammalian CV1 cells with a recombinant M-gene pTM1 plasmid, driven by vaccinia virus-expressed phage T7 polymerase, resulted in the expression of matrix (M) protein, which is progressively released from the exterior surface of the transfected-cell plasma membrane. Exocytosis of M protein begins 2 to 4 h posttransfection and reaches a peak by 10 to 16 h posttransfection; dye uptake studies reveal that > 97% of cells are alive and have intact membranes at 16 h posttransfection. Density gradient centrifugation and labeling with radioactive palmitic acid revealed that the M protein is released from cells in association with lipid vesicles. Expression of M-gene deletion mutants suggests that exocytosis of M protein requires the presence of a membrane-binding site at N-terminal amino acids 1 to 50. Cells transfected with the pTM1 plasmid containing the M gene of the temperature-sensitive mutant tsO23 expressed ample quantities of the mutant M protein at permissive (31 degrees C) and restrictive (39 degrees C) temperatures, but the exocytosis of the mutant M protein occurred only at the permissive temperature. The tsO23 M gene has three site-specific mutations resulting in amino acid substitutions at residues 21, 111, and 227. Expression of wild-type and mutant M genes with mutations or revertants at each of these sites resulted in exocytosis of M protein at the nonpermissive temperature only when wild-type leucine was present at residue 111, but M-protein exocytosis was restricted (to some extent even at the permissive temperature) when mutant phenylalanine was present at residue 111. Past and present data indicate that a specific structural conformation of the M protein is responsible for the formation and budding of vesicles, a property of the M protein which probably also promotes vesicular stomatitis virus assembly and budding of virions from host cells.
authors.affiliation: Department of Microbiology, University of Virginia School of Medicine, Charlottesville 22908, USA.
authors.forename: P A
authors.initials: PA
authors.lastname: Justice
authors.forename: W
authors.initials: W
authors.lastname: Sun
authors.forename: Y
authors.initials: Y
authors.lastname: Li
authors.forename: Z
authors.initials: Z
authors.lastname: Ye
authors.forename: P R
authors.initials: PR
authors.lastname: Grigera
authors.forename: R R
authors.initials: RR
authors.lastname: Wagner
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism / Q000821:virology; D003600:Cytosol / Q000378:metabolism / Q000821:virology; D005089:Exocytosis; D009154:Mutation; D017354:Point Mutation; D013696:Temperature; D014162:Transfection; D014721:Vesicular stomatitis Indiana virus / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D014763:Viral Matrix Proteins / Q000235:genetics* / Q000378:metabolism*
chemical_list: D014763:Viral Matrix Proteins