Use of monoclonal antibodies against two 75,000-molecular-weight glycoproteins specified by herpes simplex virus type 2 in glycoprotein identification and gene mapping

We produced two monoclonal antibodies that precipitate different glycoproteins of similar apparent molecular weight (70,000 to 80,000) from extracts of cells infected with herpes simplex virus type 2. Evidence is presented that one of these glycoproteins is the previously characterized glycoprotein gE, whereas the other maps to a region of the herpes simplex virus type 2 genome collinear with the region in herpes simplex virus type 1 DNA that encodes gC.     

gA and gB glycoproteins of herpes simplex virus type 1: two forms of a single polypeptide.

Utilizing a combination of preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sodium dodecyl sulfate-hydroxylapatite column chromatography, we have separated and purified the gA and gB glycoproteins of the major virus-specific glycoprotein region from herpes simplex virus type 1-infected cells. By using purified antigen preparations, antisera specific to each of these glycoproteins were produced. Immunoprecipitation from detergent extracts of infected cells and radioimmune precipitation of the purified antigens have shown that the anti-gA and anti-gB sera each recognize both the gA and the gB glycoproteins. The anti-gA serum was also shown to neutralize virus despite the presence of only minute quantities of the gA glycoprotein in virions. Pulse-chase studies have indicated that the gA and gB glycoproteins are synthesized from a common precursor polypeptide. Together, these data demonstrate that the gA and gB glycoproteins of herpes simplex virus type 1 are antigenically similar but not identical and probably represent two different forms of the same polypeptide which differ in their degree of glycosylation.     

Fine structure analysis of type-specific and type-common antigenic sites of herpes simplex virus glycoprotein D

The fine structure of the antigenic determinants of herpes simplex virus type 1 and 2 glycoprotein D (gD) was analyzed to determine whether structural differences underlie the differential immunogenicity of these glycoproteins. A region common to herpes simplex virus type 1 and 2 gD (amino acid residues 11 to 19) and two sites specific for herpes simplex virus type 2 gD (one determined by proline at position 7, the other determined by asparagine at position 21) were localized within the N-terminal 23 amino acids of gD by synthesis of peptides and comparison of their cross-reactivity with antisera raised to herpes simplex virus type 1 and 2 gD. The secondary structure of these peptides, as predicted by computer analysis, is discussed in relation to their immunogenicity.     

Glycoprotein C of herpes simplex virus type 1 plays a principal role in the adsorption of virus to cells and in infectivity.

The purpose of this study was to identify the herpes simplex virus glycoprotein(s) that mediates the adsorption of virions to cells. Because heparan sulfate moieties of cell surface proteoglycans serve as the receptors for herpes simplex virus adsorption, we tested whether any of the viral glycoproteins could bind to heparin-Sepharose in affinity chromatography experiments. Two glycoproteins, gB and gC, bound to heparin-Sepharose and could be eluted with soluble heparin. In order to determine whether virions devoid of gC or gB were impaired for adsorption, we quantitated the binding of wild-type and mutant virions to cells. We found that at equivalent input concentrations of purified virions, significantly fewer gC-negative virions bound to cells than did wild-type or gB-negative virions. In addition, the gC-negative virions that bound to cells showed a significant delay in penetration compared with wild-type virus. The impairments in adsorption and penetration of the gC-negative virions can account for their reduced PFU/particle ratios, which were found to be about 5 to 10% that of wild-type virions, depending on the host cell. Although gC is dispensable for replication of herpes simplex virus in cell culture, it clearly facilitates virion adsorption and enhances infectivity by about a factor of 10.     

Simultaneous triple-immunogold staining of virus and host cell antigens with monoclonal antibodies of virus and host cell antigens in ultrathin cryosections

The mechanism of intracellular maturation and sorting of herpes simplex virus type I glycoproteins is not known in details. To elucidate the intracellular sorting of viral glycoproteins and their possible interaction with the cytoskeleton, a method for simultaneous immunogold staining of three antigens in ultrathin cryosections is described. Each antigen is stained by an indirect technique using mouse monoclonal IgG as first layer, rabbit anti-mouse IgG as second and gold-conjugated goat anti-rabbit IgG as third layer antibody. After each staining cycle the sections are covered by methyl cellulose and exposed to paraformaldehyde vapour at 80 degrees C for 30 min. This destroys the free antigen combining sites of the second and the third layer IgG and abolish contaminating staining. Simultaneous triple-staining is documented with three mouse monoclonal antisera specific for 1) herpes simplex virus type 1 glycoprotein C, 2) glycoprotein D and 3) alpha- and beta-tubulin as primary antibodies. Labelling for virus glycoproteins was found in some Golgi vesicles and close to the cytoplasmic microtubules as well as on the cell surface and on intracytoplasmic and extracellular virus particles.     

Effect of tunicamycin on the synthesis of herpes simplex virus type 1 glycoproteins and their expression on the cell surface.

Herpes simplex virus specifies five glycoproteins which have been found on the surface of both the intact, infected cells and the virion envelope. In the presence of the drug tunicamycin, glycosylation of the herpes simplex virus type 1 glycoproteins is inhibited. We present in this report evidence that the immunologically specificity of the glycoproteins designated gA, gB, and gD resides mainly in the underglycosylated "core" proteins, as demonstrated by the immunoblotting technique. We showed also that tunicamycin prevented exposure of the viral glycoproteins on the cell surface, as the individual glycoproteins lost their ability to participate as targets for the specific antibodies applied in the antibody-dependent, cell-mediated cytotoxicity test. Immunocytolysis was reduced between 73 and 97%, depending on the specificity of the antibodies used. The intracellular processing of the herpes simplex virus type 1-specific glycoprotein designated gC differed from the processing of gA, gB, and GD, as evidenced by the identification of an underglycosylated but immunochemically modified form of gC on the surface of infected cells grown in the presence of tunicamycin.     

Galactose-rich glycoproteins are on the cell surface of herpes virus-infected cells. 1. Surface labeling and serial lectin binding studies of Asn-linked oligosaccharides of glycoprotein gC

Cell-surface glycoproteins of mock-infected and herpes simplex virus type 1 (HSV-1)-infected BHK-21 and HEp-2 cells were radiolabeled by incubation with galactose oxidase followed by reduction with NaB3H4. The incorporation of radiolabel into glycoconjugates in both BHK-21 and HEp-2 cells was increased several fold following infection with HSV, showing an increase in surface-exposed Gal residues in the infected cells. This was further confirmed by an increase in binding of cell-surface-labeled glycoproteins gC and gB from HSV-infected BHK-21 cells to Ricinus communis agglutinin I, which is specific for beta-D-Gal residues. Prior treatment of cells with Clostridium perfringens neuraminidase enhanced the surface radiolabeling by the galactose oxidase/NaB3H4 method: HEp-2 cells exhibited over sixfold enhancement in labeling, while BHK-21 cells showed only a slight increase. HSV glycoprotein gC was the predominant cell-surface glycoprotein radiolabeled by the galactose oxidase/NaB3H4 method in virus-infected BHK-21 cells. The glycoprotein gC was purified by immunoaffinity column chromatography on monoclonal anti-gC-antibody-Sepharose. The radiolabel in the glycopeptides of gC was resistant to beta elimination, showing that it was associated only with Asn-linked oligosaccharides. A serial lectin affinity chromatography of glycopeptides on columns of concanavalin A-Sepharose, lentil (Lens culinaris) lectin-Sepharose, and Ricin I-agarose allowed the assignment of minimal oligosaccharide structures bearing terminal Gal residues in gC.     

Role of the membrane anchoring and cytoplasmic domains in intracellular transport and localization of viral glycoproteins.

The role of the transmembrane and the cytoplasmic regions of viral glycoproteins namely, the envelope glycoprotein gD of herpes simplex virus and the integral membrane glycoprotein E3-11.6 K of the nonenveloped adenovirus that are localized in the nuclear envelope has been studied. Chimeras of the cell surface glycoprotein G of vesicular stomatitis virus containing the transmembrane and (or) the cytoplasmic-tail domains of either herpes simplex virus gD or adenovirus E3-11.6 K protein were examined for their intracellular transport and localization. The results show that hybrids containing the membrane anchoring and (or) the cytoplasmic tail domains of either herpes simplex virus gD or adenovirus E3-11.6 K glycoprotein were localized in the nuclear envelope as well as in the endoplasmic reticulum and the Golgi complex. These results suggest that the membrane anchoring and the cytoplasmic domains of herpes simplex virus glycoproteins gD, as well as the adenovirus integral membrane protein E3-11.6 K, were necessary for localization in the nuclear envelope and could influence retention in the endoplasmic reticulum and the Golgi complex.     

Membrane proteins specified by herpes simplex viruses. V. Identification of an Fc-binding glycoprotein.

A glycoprotein with affinity for the Fc region of immunoglobulin was isolated from extracts of cultured cells infected with herpes simplex virus type 1, and experiments were done to characterize its properties and to investigate whether it could account for the Fc-binding activity previously demonstrated on the surfaces of intact herpes simplex virus-infected cells. The technique of affinity chromatography was used to identify and isolate the Fc-binding glycoprotein and to demonstrate the specificity of its interaction with immunoglobulin G-Fc. Although three electrophoretically distinguishable Fc-binding polypeptides were identified by affinity chromatography, these three species appear to be different forms of the same translation product, based on comparisons of proteolytic digestion products and on the kinetics of appearance of each form after a brief pulse with radioactive amino acids. The results suggest that one polypeptide, designated pE, is processed to yield gE1, which is in turn processed to yield gE2. Both gE1 and gE2 are glycosylated membrane proteins and both can be labeled by the lactoperoxidase-catalyzed radioiodination of intact infected cells, indicating the presence of these proteins in surface membranes of the cells. Increases in the amounts of gE1 and gE2 at the cell surface were found to parallel the increase in Fc-binding activity of intact infected cells.     

Identification and characterization of a novel structural glycoprotein in pseudorabies virus, gL.

Herpesvirus envelope glycoproteins play important roles in the interaction between virions and target cells. In the alphaherpesvirus pseudorabies virus (PrV), seven glycoproteins that all constitute homologs of glycoproteins found in herpes simplex virus type 1 (HSV-1) have been characterized, including a homolog of HSV-1 glycoprotein H (gH). Since HSV-1 gH is found associated with another essential glycoprotein, gL, we analyzed whether PrV also encodes a gL homolog. DNA sequence analysis of a corresponding part of the UL region adjacent to the internal inverted repeat in PrV strains Kaplan and Becker revealed the presence of two open reading frames (ORF). Deduced proteins exhibited homology to uracil-DNA glycosylase encoded by HSV-1 ORF UL2 (54% identity) and gL encoded by HSV-1 ORF UL1 (24% identity), respectively. To identify the PrV UL1 protein, rabbit antisera were prepared against two synthetic oligopeptides that were predicted by computer analysis to encompass antigenic epitopes. Sera against both peptides reacted in Western blots of purified virions with a 20-kDa protein. The specificity of the reaction was demonstrated by peptide competition. Since the PrV UL1 sequence did not reveal the presence of a consensus N-linked glycosylation site, concanavalin A affinity chromatography and enzymatic deglycosylation of virion glycoproteins were used to ascertain that the PrV UL1 product is O glycosylated. Therefore, we designated this protein PrV gL. Analysis of mutant PrV virions lacking gH showed that concomitantly with the absence of gH, gL was also missing in purified virions. In summary, we identified and characterized a novel structural PrV glycoprotein, gL, which represents the eighth PrV glycoprotein described. In addition, we show that virion location of PrV gL is dependent on the presence of PrV gH.     

Herpes Simplex Virus Glycoproteins: Participation of Individual Herpes Simplex Virus Type 1 Glycoprotein Antigens in Immunocytolysis and Their Correlation with Previously Identified Glycopolypeptides

Tissue culture cells infected with herpes simplex type 1 virus express virus-specified glycoprotein antigens on the plasma membrane. Three of these have been previously identified and have been designated as Ag-11, Ag-8, and Ag-6. In the present study, immunoglobulins to each of the antigens were shown to be capable of mediating immunocytolysis in the presence of either complement (antibody-dependent complement-mediated cytotoxicity) or peripheral blood mononuclear cells (antibody-dependent cell-mediated cytotoxicity [ADCC]). Two herpes simplex virus type 1 strains, VR-3 and F, reacted similarly in the ADCC test in the presence of immunoglobulins to Ag-11, Ag-8, and Ag-6 in both infected Chang liver cells and HEp-2 cells. Anti-Ag-6, however, produced a lower ADCC reaction in HEp-2 cells than in Chang liver cells, suggesting differences in the Ag-6 surface expression in, or release from, these cells. Chang liver and HEp-2 cells infected with the MP mutant strain of herpes simplex virus type 1 showed reduced ADCC in the presence of anti-Ag-11 and anti-Ag-8, but no reactivity at all with anti-Ag-6. Crossed immunoelectrophoretic analysis showed that MP-infected cell extracts contain Ag-11 and Ag-8, but lack Ag-6. Polypeptide analysis of herpes simplex virus type 1 strains F, VR-3, and MP showed that Ag-11 consists of the glycoproteins gA and gB, that Ag-8 consists of gD, and that Ag-6 consists of gC. In conclusion, the present study demonstrates that either one of the glycoproteins (gC, gD, and a mixture of gA and gB) can function as a target for immunocytolysis and that the antibody preparation to gC (Ag-6) does not cross-react with any of the other glycoproteins.     

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.     

Physical mapping of the mutation in an antigenic variant of herpes simplex virus type 1 by use of an immunoreactive plaque assay.

Two mutations affecting herpes simplex virus type 1 glycoprotein B were mapped by marker rescue using cloned sequences of wild-type herpes simplex virus type 1 strain KOS DNA. One mutant, tsB5, is a temperature-sensitive mutant which does not express mature, functional glycoprotein B at the nonpermissive temperature. The other mutant, marB1.1, expresses an antigenic variant of glycoprotein B and was selected for resistance to neutralization by a monoclonal antibody. The mutation in tsB5 mapped to a 1.2-kilobase segment of the herpes simplex virus type 1 genome between coordinates 0.361 and 0.368, whereas the mutation in marB1.1 mapped to a 1.6-kilobase segment between coordinates 0.350 and 0.361. An in situ enzyme immunoassay was used to detect plaques of recombinant wild-type virus among the progeny of transfections with mutant marB1.1 DNA and wild-type DNA fragments.     

Variation in herpes simplex virus type 1 glycoproteins produced in kidney cells from different species

Viral glycoproteins from herpes simplex virus, type 1 (HSV-1) infected NBL-1, Vero, and BHK-21 cells were labelled with 14C-glucosamine and studied by SDS-PAGE and Con-A chromatography. SDS-PAGE analysis demonstrated differences in the number and molecular weight of glycoproteins from these cells. Con-A chromatography resulted in similar binding of glycoproteins from NBL-1 and Vero cells of 10.5 and 18.6%, respectively, whereas BHK-21 cells showed binding of 65%. These studies indicate that HSV-1 glycoprotein oligosaccharide processing is variable in kidney cells of different species.     

Separation of Herpes Simplex Virus-Induced Antigens by Concanavalin A Affinity Chromatography

Biologically active herpes simplex virus (HSV)-induced antigens were selectively removed from extracts of infected BHK cells by affinity chromatography by utilizing an insoluble form of concanavalin A (Con A). Soluble extracts of (3)H-glucosamine-labeled, HSV-infected cells were absorbed to a Con A column. Bound material was eluted with alpha-methyl-d-mannoside (alphaMM) and NaCl. The specific activity of the eluted glycoproteins increased by 10-fold. Two broad groups of viral-induced antigens were isolated from Con A. Group I includes two antigens which bind to Con A by a specific mechanism because the antigens are dissociated by alphaMM. Group II contains three antigens which bind to Con A but apparently by a nonspecific or electrolytic mechanism. One antigen in group I was identified as the glycoprotein antigen, CP-1, described previously.     

Preparation and characterization of specific antisera to individual glycoprotein antigens comprising the major glycoprotein region of herpes simplex virus type 1.

The major glycoprotein complex (VP123) of herpes simplex virus type 1 resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was purified and further fractionated into two major and two minor components by chromatography of the isolated VP123 region on SDS-hydroxylapatite columns. The two major components (gC and gA/gB) were purified free of other polypeptides and used to prepare specific antisera to these glycoproteins. Radioimmune precipitation demonstrated that these antisera were specific for the antigens used in their production. These two antisera as well as an anti-VP123 serum were further characterized by immunoprecipitation, neutralization, and membrane immunofluorescence techniques. Results indicate that both of the major glycoprotein antigens are expressed on the surface of virions as well as on the surface of infected cells.     

Isolation of the membrane glycoproteins of human blood platelets by lectin affinity chromatography

The major platelet membrane glycoproteins have been solubilized in 1.0% sodium deoxycholate and subjected to affinity chromatography on the lectins from Lens culinaris, wheat germ and Abrus precatorius. Polyacrylamide gel electrophoresis in the presence and absence of a reducing agent together with the differential binding of the lectins to the glycoproteins permitted the distinction of at least seven separate glycoprotein entities. A new nomenclature for the glycoproteins is proposed to accomodate the additional data.Using combinations of lectin columns, glycoproteins Ia and Ib could be prepared in a pure state and IIb and IIIa could be greatly purified. The binding of lectins to glycoprotein Ib has been strongly implicated as a necessary step in the aggregation response of platelets to lectins.     

New common nomenclature for glycoprotein genes of varicella-zoster virus and their glycosylated products.

The accumulation of recent data concerning the reactivity of monoclonal antibodies with particular varicella-zoster virus (VZV) glycoproteins and the mapping of several of their respective genes on the VZV genome has led to a unified nomenclature for the glycoprotein genes of VZV and their mature glycosylated products. Homologs to herpes simplex virus glycoprotein genes are noted.     

Analysis of cell surface glycoprotein changes related to hematopoietic differentiation

A high-resolution technique has been used to study differentiation-related and leukemia-associated glycoproteins. Cells are labeled with the membrane-impermeable probe sulfo-N-hydroxysuccinimidyl-biotin. Nonionic detergent extracts are subjected to affinity chromatography on a number of immobilized lectins and after polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE) and western transfer, the biotin-labeled glycoproteins are visualized by using avidin-horseradish peroxidase and 4-chloronaphthol. With the aid of the lectins concanavalin A, Dolichos biflouros agglutinin, Lens culinaris hemagglutinin, peanut agglutinin, pokeweed mitogen, Ricinus communus agglutinin I, soybean agglutinin, Ulex europeus agglutinin I (UEA), and wheat germ agglutinin, each purifies different glycoprotein subsets from the same cell type. Mature cells of distinct hematopoietic lineages differ considerably in their cell surface glycoprotein patterns. This technique was used to analyze the glycoproteins of human leukemia cells before and after the induction of differentiation. K562 cells differentiated along different lineages after treatment with phorbol 12-myristate 13-acetate, sodium butyrate, dimethyl sulfoxide, or hemin. Limited specific alterations were observed with a number of lectins when K562 erythroleukemia cells were induced to differentiate. Among these, a number of bands were identified that were either lost or appeared after induction of differentiation with all four agents. In contrast, the glycoproteins bound by UEA were drastically diminished after induction of differentiation, and the remaining UEA-bound glycoproteins bore little resemblance to those of the cells before treatment. This high-resolution technique may be useful as a general method for the examination of cell surface glycoprotein differences. Once specific glycoprotein alterations are detected, lectin affinity chromatography and SDS-PAGE allow purification of antigens for the production of monoclonal antibodies.     

Simultaneous triple-immunogold staining of virus and host cell antigens with monoclonal antibodies of virus and host cell antigens in ultrathin cryosections

Summary The mechanism of intracellular maturation and sorting of herpes simplex virus type I glycoproteins is not known in details. To elucidate the intracellular sorting of viral glycoproteins and their possible interaction with the cytoskeleton, a method for simultaneous immunogold staining of three antigens in ultrathin cryosections is described. Each antigen is stained by an indirect technique using mouse monoclonal IgG as first layer, rabbit antimouse IgG as second and gold-conjugated goat anti-rabbit IgG as third layer antibody. After each staining cycle the paraformaldehyde vapour at 80° C for 30 min. This destroys the free antigen combining sites of the second and the third layer IgG and abolish contaminating staining. Simultaneous triple-staining is documented with three mouse monoclonal antisera specific for 1) herpes simplex virus type 1 glycoprotein C, 2) glycoprotein D and 3) - and -tubulin as primary antibodies. Labelling for virus glycoproteins was found in some Golgi vesicles and close to the cytoplasmic microtubules as well as on the cell surface and on intracytoplasmic and extracellular virus particles.Presented in part at the 9th European Congress on Electron Microscopy, York, England, September 4–9, 1988