Effect of glycosylation on the conformational epitopes of the glycoprotein of vesicular stomatitis virus (New Jersey serotype)

Abstract

The conformational epitopes reactive with neutralizing monoclonal antibodies (MAbs) appear to be clustered at the middle third of the glycoprotein (G) of the New Jersey serotype of vesicular stomatitis virus (VSV-NJ) and are flanked by two N-linked carbohydrate chains (W. Keil and R. R. Wagner, Virology 170, 392–407, 1989). We report here studies on the effect of glycosylation on the reactivity of VSV-NJ G protein with MAbs directed to conformational epitopes. When VSV-NJ was grown in the presence of tunicamycin, the unglycosylated G protein derived from released virions or immunoprecipitated from pulse-labeled cells was not significantly affected in its reactivity with MAbs directed to epitope IV mapped toward the amino-terminus, nor to the centrally located conformational epitopes VI, VIII, and IX. However, there was a 5- to 15-fold decrease in the reactivity with MAb of epitopes VI, VIII, and IX on unglycosylated G protein either isolated from a ribosome-enriched membrane fraction or immunoprecipitated from whole VSV-infected cells labeled for 15 hr in the presence of tunicamycin. In sharp contrast, epitope V and to a somewhat lesser extent epitope VII exhibited decreased reactivity with their respective MAbs when unglycosylated G protein was isolated from released viral particles or from pulse-labeled cells infected with VSV-NJ in the presence of tunicamycin. Enzymatic removal of preformed carbohydrate chains with N-glycanase had little or no effect on the MAb-reactivity of epitopes V and VII, indicating that the carbohydrate chains per se do not influence the antigenic specificity of VSV-NJ G protein. These data suggest that the formation of N-linked carbohydrate chains influences the structure of the VSV-NJ G protein in such a way that epitopes V and VII are shielded from reactivity with their specific MAbs from an early stage of G-protein processing and to a much lesser extent epitopes VI, VIII, and IX at late stages of intracellular processing. These results are compatible with, but do not prove, the hypothesis that N-linked glycosylation plays a key role in promoting the formation and the stability of the disulfide bonds that determine the epitope-specific conformational integrity of the VSV-NJ glycoprotein.