MEMBRANE VESICULATION FUNCTION AND EXOCYTOSIS OF WILD-TYPE AND MUTANTMATRIX PROTEINS OF VESICULAR STOMATITIS-VIRUS

Transfection of mammalian CV1 cells with a recombinant M-gene pTM1 pla smid, driven by vaccinia virus-expressed phage T7 polymerase, resulted in the expression of matrix (M) protein, which is progressively relea sed 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 posttransfec tion. Density gradient centrifugation and labeling with radioactive pa lmitic acid revealed that the M protein is released from cells in asso ciation with lipid vesicles. Expression of M-gene deletion mutants sug gests that exocytosis of M protein requires the presence of a membrane -binding site at N-terminal amino acids 1 to 50. Cells transfected wit h the pTM1 plasmid containing the M gene of the temperature-sensitive mutant tsO23 expressed ample quantities of the mutant M protein at per missive (31 degrees C) and restrictive (39 degrees C) temperatures, bu t the exocytosis of the mutant M protein occurred only at the permissi ve temperature. The tsO23 M gene has three site-specific mutations res ulting in amino acid substitutions at residues 21, 111, and 227. Expre ssion of wild-type and mutant M genes with mutations or revertants at each of these sites resulted in exocytosis of M protein at the nonperm issive temperature only when wild-type leucine was present at residue 111, but M-protein exocytosis was restricted (to some extent even at t he permissive temperature) when mutant phenylalanine was present at re sidue 111. Past and present data indicate that a specific structural c onformation of the M protein is responsible for the formation and budd ing of vesicles, a property of the M protein which probably also promo tes vesicular stomatitis virus assembly and budding of virions from ho st cells.