Disulfide-bonded discontinuous epitopes on the glycoprotein of vesicular stomatitis virus (New Jersey serotype).
Intrachain disulfide bonds between paired cysteines in the glycoprotein (G) of vesicular stomatitis virus (VSV) are required for the recognition of discontinuous epitopes by specific monoclonal antibodies (MAbs) (W. Keil and R. R. Wagner, Virology 170:392-407, 1989). Cleavage by Staphylococcus aureus V8 protease of the 517-amino-acid VSV-New Jersey G protein, limited to the glutamic acid at residue 110, resulted in a protein (designated GV8) with greatly retarded migration by polyacrylamide gel electrophoresis (PAGE) under nonreducing conditions. By Western blot (immunoblot) analysis, protein GV8 was found to lose discontinuous epitope IV, which maps within the first 193 NH2-terminal amino acids. These data, coupled with those obtained by PAGE migration of a vector-expressed, truncated protein (G1-193) under reducing and nonreducing conditions, lead us to postulate the existence of a major loop structure within the first 193 NH2-terminal amino acids of the G protein, possibly anchored by a disulfide bond between cysteine 108 and cysteine 169, encompassing epitope IV. Site-directed mutants in which 10 of the 12 cysteines were individually converted to serines in vaccinia virus-based vectors expressing these single-site mutant G proteins were also constructed, each of which was then tested by immunoprecipitation for its capacity to recognize epitope-specific MAbs. These results showed that mutations in NH2-terminal cysteines 130, 174, and, to a lesser extent, 193 all resulted in the loss of neutralization epitope VIII. A mutation at NH2-terminal cysteine 130 also resulted in the loss of neutralization epitope VII, as did a mutation at cysteine 108 to a lesser extent. Both epitopes VII and VIII disappeared when mutations were made in COOH-distal cysteine 235, 240, or 273, the general map locations of epitopes VII and VIII. These studies also reveal that distal, as well as proximal, cysteine residues markedly influence the disulfide-bond secondary structure, which ostensibly determines the conformational structure of the VSV-New Jersey G protein required for presentation of the major discontinuous epitopes recognized by neutralizing MAbs.
Grigera
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PR
Department of Microbiology and Cancer Center, University of Virginia School of Medicine, Charlottesville 22908.
Keil
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W
Wagner
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D000818:Animals; D000911:Antibodies, Monoclonal; D003545:Cysteine / Q000737:chemistry; D004252:DNA Mutational Analysis; D004220:Disulfides / Q000737:chemistry; D000939:Epitopes / Q000737:chemistry* / Q000276:immunology; D006023:Glycoproteins / Q000737:chemistry* / Q000276:immunology; D008562:Membrane Glycoproteins; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D010449:Peptide Mapping; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology; D012694:Serine / Q000737:chemistry; D018116:Vesiculovirus / Q000737:chemistry* / Q000276:immunology; D014759:Viral Envelope Proteins / Q000737:chemistry* / Q000276:immunology
D000911:Antibodies, Monoclonal; D004220:Disulfides; D000939:Epitopes; C028391:G protein, vesicular stomatitis virus; D006023:Glycoproteins; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; D012694:Serine; D003545:Cysteine