Inhibition of glycosylation of herpes simplex virus glycoproteins: Identification of antigenic and immunogenic partially glycosylated glycopeptides on the cell surface membrane

Abstract

The surface membranes of cells infected with herpes simplex virus type 1 (HSV-1), strain KOS, contain three principal glycoproteins, gC (apparent M r 129k), gB (apparent M r 120k), and gD (apparent M r 58k). Infections carried out in the presence of the glycosylation inhibitor 2-deoxy- d-glucose result in the loss of the mature species with the concurrent appearance of lower-molecular-weight polypeptides which are presumably partially glycosylated forms of the fully processed glycoproteins. Specific immunoprecipitation of radiolabeled cytoplasmic extracts of 2-deoxy- d-glucose-inhibited infections identified partially glycosylated proteins designated DG92, DG88, and DG53, which are antigenically related to the corresponding mature forms gB, gC, and gD. Cell surface radioiodination, in combination with specific immunoprecipitation, revealed that DG88 and DG53 were the principal species transported to the cell surface in 2-deoxy- d-glucose-inhibited infections. DG92 was readily detected in the cytoplasm but not on the plasma membrane. Cells infected with the KOS mutant, syn LD70, did not synthesize glycoprotein gC. In glycosylation-inhibited syn LD70 infections, DG88 was not detected in either the cytoplasm or plasma membrane, demonstrating a genetic relationship between DG88 and gC. Polyclonal and monoclonal antibodies directed against the glycoproteins gC, gB, and gD sensitized infected cells to complement-mediated immune cytolysis. Cells infected in the presence of the inhibitor were sensitized to lysis only by antibody specific for gC and gD. The glycosylation-inhibited cells were insensitive to immunolysis by anti-gB monoclonal antibody. These findings confirm that the glycosylation-deficient forms of gC and gD, but not gB reach the cell surface in the presence of inhibitor and that the inhibitor-induced alterations in glycosylation do not cause a complete loss of antigenicity. Inoculation of mice with syngeneic 3T3 cells infected in the presence or absence of inhibitor-induced cytolytic and neutralizing antibody. A major portion of the cytolytic antibody was directed against gC, but anti-gC antibody appeared to play a minor role in virus neutralization. While the serum induced by the control infected cells contained precipitating antibodies for gC, gB, and gD, the serum derived from mice inoculated with inhibitor-treated infected cells had only weak immunoprecipitating activity against gB. Together, these findings have identified partially glycosylated forms of the major HSV glycoproteins and show that complete glycosylation is not required for transport of some of these partially glycosylated polypeptides to the cell surface. Moreover, complete glycosylation of the glycopeptides is not essential for maintenance of antigenicity or immunogenicity, indicating that at least some determinants recognized by antibodies directed against the mature glycoproteins are not affected by 2-deoxy- d-glucose-induced carbohydrate alterations.