Characterization of two different human cytomegalovirus glycoproteins which are targets for virus neutralizing antibody.
In previous studies we have identified two viral polypeptides detected by murine monoclonal antibodies which neutralize the infectivity of human cytomegalovirus (CMV) AD169. One is an 86,000-Da polypeptide (p86) and the second is a complex of two major coimmunoprecipitating polypeptides of 130,000 and 55,000 Da (p130/55). In this study we have shown that the two viral polypeptides are immunologically unrelated and have distinct peptide cleavage patterns. We have characterized these polypeptides as glycoproteins and studied their biosynthesis in human embryonic lung cells. The oligosaccharides found on both the p86 and the p130/55 were characterized by endoglycosidase digestion as N-linked high-mannose carbohydrates. Inhibitors of glycosylation were used to further characterize the oligosaccharides. Tunicamycin, which inhibits the biosynthesis of N-linked oligosaccharides on the endoplasmic reticulum, inhibited both the infectivity and biosynthesis of the p86 and p130/55. The underglycosylated forms in tunicamycin-treated cultures could be detected only under conditions of pulse-labeling with L-[35S]methionine. Monensin, which inhibits the modification of glycoproteins from simple to complex forms in the Golgi, reduced viral infectivity at concentrations which had no effect on viral protein synthesis, but did not alter the apparent molecular weight of either the p86 or the p130/55. The oligosaccharides were critical for the in vitro immunologic reactivity of the p86 in immunoblots. However, endoglycosidase F-treated p86 was comparable to the native form in inducing virus neutralizing antibody in guinea pigs. Endoglycosidase F-treated p130/55 retained its ability to bind antibody in Western blots.
Rasmussen
L
L
Stanford University School of Medicine, Division of Infectious Diseases, California 94305.
Nelson
M
M
Neff
M
M
Merigan
T C
TC
D000911:Antibodies, Monoclonal / Q000276:immunology; D000914:Antibodies, Viral / Q000276:immunology*; D003587:Cytomegalovirus / Q000276:immunology*; D006023:Glycoproteins / Q000276:immunology*; D006031:Glycosylation; D008985:Monensin / Q000494:pharmacology; D009500:Neutralization Tests; D010455:Peptides / Q000276:immunology / Q000302:isolation & purification; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D014415:Tunicamycin / Q000494:pharmacology; D014764:Viral Proteins / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D006023:Glycoproteins; D010455:Peptides; D014764:Viral Proteins; D014415:Tunicamycin; D008985:Monensin