Characterization of a cleavage mutant of the measles virus fusion protein defective in syncytium formation.
Membrane fusion caused by measles virus (MV) is a function of the fusion (F) protein. This process is essential for penetration into the host cell and subsequent initiation of the virus replicative cycle. The biological activity of the MV F protein is generated by endoproteolytic cleavage of a precursor protein (F0) into a large F1 subunit and a smaller F2 subunit held together by disulfide bonds. The cleavage site consists of a cluster of five basic amino acids (amino acids 108 to 112) within the predicted primary structure of the F protein. To investigate the role of the arginine residue at the carboxy terminus of the F2 subunit (arginine 112), site-directed mutagenesis was used to construct a cleavage mutant of the MV F protein in which this arginine residue was changed to a leucine residue. The mutated F gene, encoding four out of the five basic amino acids at the cleavage site, was inserted into the genome of vaccinia virus. The resulting recombinant virus was used to study expression of the mutant F protein in infected cells. Analysis of the Leu-112 mutant protein made in infected cells demonstrated that this single-amino-acid substitution resulted in a reduced rate of transport of the mutant protein to the cell surface, despite its efficient cleavage to yield F1 and F2 subunits. However, the electrophoretic mobilities of the Leu-112 polypeptides suggested that the protein was cleaved incorrectly. This aberrant cleavage appears to have abolished the ability of the F protein to cause syncytium formation. The data indicate that the arginine 112 residue is critical for the correct proteolytic cleavage that is required for the membrane fusion activity of the MV F protein.
Alkhatib
G
G
Division of Molecular Immunology and Neurobiology, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.
Roder
J
J
Richardson
C
C
Briedis
D
D
Weinberg
R
R
Smith
D
D
Taylor
J
J
Paoletti
E
E
Shen
S H
SH
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D017931:DNA Primers; D005814:Genes, Viral; D015726:Giant Cells / Q000502:physiology*; D046911:Macromolecular Substances; D008459:Measles virus / Q000235:genetics / Q000502:physiology*; D008561:Membrane Fusion; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D014760:Viral Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*
D017931:DNA Primers; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D014760:Viral Fusion Proteins