title: Epitope mapping by deletion mutants and chimeras of two vesicular stomatitis virus glycoprotein genes expressed by a vaccinia virus vector.
abstract: Deletion mutants and chimeras of the glycoprotein (G) genes of vesicular stomatitis virus serotypes Indiana (VSV-Ind) and New Jersey (VSV-NJ) were cloned in plasmids and vaccinia virus vectors under control of the bacteriophage T7 polymerase promoter for expression in CV-1 cells co-infected with a T7 polymerase-expressing vaccinia virus recombinant. Truncated and chimeric G proteins expressed by these vectors were tested for their capacity to react with VSV-Ind and VSV-NJ epitope-specific monoclonal antibodies (MAbs) by Western blot analysis for those antigenic determinants not affected by disulfide-bond reducing conditions or by immuno dotblot analysis for those that are. These experiments allowed us to create putative epitope maps for glycoproteins of both serotypes based on binding affinity and cross-reactivity of VSV-Ind and VSV-NJ MAbs for truncated or chimeric G proteins of known amino acid sequences. Seven of the 9 VSV-NJ G epitopes, including all 4 epitopes involved in virus neutralization by MAbs, mapped to the center (amino acid sequence 193-289) of the 517 amino acid VSV-NJ G protein. Four of the 11 VSV-Ind G epitopes, including 2 neutralizable epitopes, mapped to the cysteine-rich amino-terminal domain (amino acid sequence 80-183) of the 511 amino acid VSV-Ind G protein; the remaining 7 VSV-Ind G epitopes, including 2 involved in virus neutralization, were clustered in the cysteine-poor carboxy-terminal domain (amino acid sequence 286-428). In site-specific mutants of the VSV-Ind G gene defective in one or both glycosylation sites, only the amino-terminal epitopes of the VSV-Ind G protein were affected by deletion of the carbohydrate chain at residue 179; deletion of the carbohydrate chain at residue 336 did not alter reactivity of the G protein with any of the relevant monoclonal antibodies. These results are discussed in relation to earlier attempts to map the antigenic determinants of VSV-NJ and VSV-Ind G proteins by proteolysis of the G protein and by sequencing the G genes of mutant viruses selected for their resistance to neutralization by epitope-specific monoclonal antibodies.
authors: Department of Microbiology and Cancer Center, University of Virginia School of Medicine, Charlottesville 22908.
authors: W
authors: W
authors: Keil
authors: R R
authors: RR
authors: Wagner
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000956:Antigens, Viral / Q000032:analysis* / Q000235:genetics; D015153:Blotting, Western; D002460:Cell Line; D002678:Chimera; D000939:Epitopes / Q000032:analysis / Q000235:genetics; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D015151:Immunoblotting; D008562:Membrane Glycoproteins; D009154:Mutation; D010957:Plasmids; D015183:Restriction Mapping; D014616:Vaccinia virus / Q000235:genetics; D014721:Vesicular stomatitis Indiana virus / Q000235:genetics* / Q000276:immunology; D018116:Vesiculovirus; D014759:Viral Envelope Proteins / Q000235:genetics* / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D000956:Antigens, Viral; D000939:Epitopes; C028391:G protein, vesicular stomatitis virus; D008562:Membrane Glycoproteins; D014759:Viral Envelope Proteins