Sequence requirements for proteolytic processing of glycoprotein B of human cytomegalovirus strain Towne.
Truncated versions of the human cytomegalovirus (CMV) strain Towne glycoprotein B (gB) gene were stably expressed in CHO cell lines. The calcium-specific ionophore A23187 inhibited proteolytic cleavage of C-terminal-truncated gB expressed by cell line 67.77. These inhibition studies also showed that the 93-kilodalton cleavage product most likely represents the N-terminal cleavage fragment of gB. The ionophore carboxyl cyanide m-chlorophenyl-hydrazone was used to show that proteolytic cleavage of gB did not occur in the endoplasmic reticulum. Two-dimensional polyacrylamide gel electrophoresis demonstrated that the N- and C-terminal cleavage products of gB remained associated by disulfide linkages after cleavage. Expression studies using constructs in which 80% or all of the N terminus was deleted demonstrated that the N terminus was required for secretion of the gB molecule. The amino acid sequence at the site of cleavage was shown to be critical for cleavage by a cellular protease. Our results indicate that an arginine-to-threonine change at either amino acid 457 or 460, a lysine-to-glutamine change at amino acid 459, or all three substitutions together block gB cleavage. The effect on proteolysis of the arginine-to-threonine amino acid change at residue 457 (position -4 relative to the cleavage site) demonstrated that a basic pair of amino acids at the endoproteolytic processing site is not the only requirement in cis for gB cleavage.
Spaete
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Chiron Corporation, Emeryville, California 94608-2916.
Saxena
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Scott
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Song
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Probert
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Britt
W J
WJ
Gibson
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Rasmussen
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Pachl
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D000001:Calcimycin / Q000494:pharmacology; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002460:Cell Line; D003587:Cytomegalovirus / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional; D005822:Genetic Vectors; D006026:Glycoside Hydrolases; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000302:isolation & purification; D015183:Restriction Mapping; D014759:Viral Envelope Proteins / Q000235:genetics* / Q000302:isolation & purification
D004279:DNA, Viral; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; C040495:glycoprotein B, Simplexvirus; D000001:Calcimycin; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D006026:Glycoside Hydrolases