Immunogenicity of herpes simplex virus glycoproteins gC and gB and their role in protective immunity.

The relative antigenicity of the individual herpes simplex virus type 1 (KOS) glycoproteins gC and gB was analyzed in BALB/c mice by using KOS mutants altered in their ability to present these antigens on cell surface membranes during infection. The mutants employed were as follows: syn LD70 , a non-temperature-sensitive mutant defective in the synthesis of cell surface membrane gC; tsF13 , a temperature-sensitive mutant defective in the processing of the precursor form of gB to the mature cell surface form at 39 degrees C; and ts606 , an immediate early temperature-sensitive mutant defective in the production of all early and late proteins including the glycoproteins. By comparing the relative susceptibility to immunolysis of mouse 3T3 cells infected at 39 degrees C with wild-type virus, presenting the full complement of the glycoprotein antigens, gC, gB, and gD, with target cells infected with mutants presenting only subsets of these antigens, we determined that a major portion of cytolytic antibody contained in hyperimmune anti-herpes simplex virus type 1 (KOS) mouse antiserum was directed against glycoproteins gC and gB. The relative immunogenicity of wild-type and mutant virus-infected cells also was compared in BALB/c mice. Immunogen lacking the mature form of gB induced a cytolytic antibody titer comparable to that of the wild-type virus, whereas that lacking the mature form of gC showed a 70% reduction in titer. The absence of the mature cell surface forms of gB and gC in immunogen preparations resulted in a 4- to 15-fold reduction in in virus neutralizing titer. Animals immunized with ts606 -infected cells (39 degrees C) induced relatively little virus-specific cytolytic and neutralizing antibody. Analysis of the glycoprotein specificities of these antisera by radioimmunoprecipitation showed that the antigens immunoprecipitated reflected the viral plasma membrane glycoprotein profiles of the immunogens. The absence of the mature forms of gC or gB in the immunizing preparation did not appreciably affect the immunoprecipitating antibody response to other antigens. Mice immunized with wild-type and mutant virus-infected cells were tested for their resistance to intracranial and intraperitoneal challenge with the highly virulent WAL strain of herpes simplex virus type 1. Despite the observed alterations in serum virus-specific antibody induced with the individual immunogens, all animals survived an intraperitoneal challenge of 10 50% lethal doses. However, differences in the survival of animals were obtained upon intracranial challenge.(ABSTRACT TRUNCATED AT 400 WORDS)

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

The surface membranes of cells infected with herpes simplex virus type 1 (HSV-1), strain KOS, contain three principal glycoproteins, gC (apparent Mr 129k), gB (apparent Mr 120k), and gD (apparent Mr 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.

Mutant analysis of herpes simplex virus-induced cell surface antigens: resistance to complement-mediated immune cytolysis.

BHK-21 cells infected with temperature-sensitive mutants of herpes simplex virus type 1 strain KOS representing 16 complementation groups were tested for susceptibility to complement-mediated immune cytolysis at permissive (34 degrees C) and nonpermissive (39 degrees C) temperatures. Only cells infected by mutants in complementation group E were resistant to immune cytolysis in a temperature-sensitive manner compared with wild-type infections. The expression of group E mutant cell surface antigens during infections at 34 and 39 degrees C was characterized by a combination of cell surface radioiodination, specific immunoprecipitation, and gel electrophoretic analysis of immunoprecipitates. Resistance to immune lysis at 39 degrees C correlated with the absence of viral antigens exposed at the cell surface. Intrinsic radiolabeling of group E mutant infections with [14C]glucosamine revealed that normal glycoproteins were produced at 34 degrees C but none were synthesized at 39 degrees C. The effect of 2-deoxy-D-glucose on glycosylation of group E mutants at 39 degrees C suggested that the viral glycoprotein precursors were not synthesized. The complementation group E mutants failed to complement herpes simplex virus type 1 mutants isolated by other workers. These included the group B mutants of strain KOS, the temperature-sensitive group D mutants of strain 17, and the LB2 mutant of strain HFEM. These mutants should be considered members of herpes simplex virus type 1 complementation group 1.2, in keeping with the new herpes simplex virus type 1 nomenclature.

Regulation of a metabolic system in vitro: synthesis of threonine from aspartic acid.

Six enzymes involved in the conversion of aspartate to threonine have been extracted from Escherichia coli and separated from each other. Two of these enzymes, aspartokinase and homoserine dehydrogenase, have also been partially purified from Rhodopseudomonas spheroides. In an attempt to determine whether small changes in the kinetic properties of individual enzymes are important to the regulation of metabolic flux through a coupled reaction system, the partially purified enzymes were recombined in a variety of ways under reaction conditions designed to resemble the in vivo situation. These conditions include: use of an entire metabolic system rather than a single reaction; high enzyme concentrations at the same relative concentrations as found in the cell; and low, steady-state concentrations of substrates and products. Metabolic flux was followed spectrophotometrically and the concentrations of aspartic semialdehyde, hemoserine, O-phosphohomoserine, and threonine were measured. The results indicate that the threonine concentration is of major importance in regulating metabolic flux by inhibiting aspartokinase, the first reaction in threonine in the pathway. When threonine-insensitive aspartokinases were used, concentrations reached higher levels and the rate of NADPH oxidation remained higher. The fact that neither aspartic semialdehyde nor homoserine accumulated as the threonine concentration increased and the lack of correlation between changes in metabolic flux and ADP/ATP or NADPH/NADP ratios indicate that more subtle forms of metabolic regulation, such as "reverse cascade", secondary feedback sites, or "energy charge", are of little regulatory importance in this isolated, metabolic system. The results also emphasize the need for caution in projecting in vivo control mechanisms from in vitro experiments.