Glycoprotein gE of herpes simplex virus type 1: effects of anti-gE on virion infectivity and on virus-induced fc-binding receptors.

An Fc-binding glycoprotein, designated gE, was detected previously in cells infected with herpes simplex virus type 1 (HSV-1) and in virion preparations isolated from infected cells. For the studies reported here, we purified gE from HSV-1 strain HFEM(syn) by affinity chromatography and preparative electrophoresis and then immunized a rabbit to produce an antiserum to glycoprotein gE. We found that this antiserum selectively precipitated gE and its precursors from detergent-solubilized extracts of HSV-1 strain HFEM(syn)-infected HEp-2 cells, from extracts of other cell lines infected with the same virus, and from extracts of HEp-2 cells infected with several other HSV-1 strains. The antiserum did not precipitate any proteins from uninfected cells. The several forms of gE detected by immunoprecipitation accumulated in variable quantities in different cells infected with the different virus strains and also varied slightly with respect to electrophoretic mobility, suggesting some differences in the gE's from different HSV-1 strains and some effects of the host cell on the nature and extent of post-translational processing. One of the electrophoretic forms of gE previously detected in purified preparations of virions could be precipitated by anti-gE from extracts of purified HSV-1 strain HFEM(syn) virions. Moreover, anti-gE neutralized HSV-1 infectivity, but only in the presence of complement. Finally, F(ab')2 fragments of the anti-gE immunoglobulin partially inhibited the binding of 125I-labeled immunoglobulin G to the Fc receptors on HSV-1-infected cells.     

Identification of a novel herpes simplex virus type 1-induced glycoprotein which complexes with gE and binds immunoglobulin.

We detected a glycoprotein on the surface of cells infected with herpes simplex virus type 1 (HSV-1) which, in conjunction with gE, binds immunoglobulin G (IgG). The novel glycoprotein, which has an apparent molecular mass of 70 kilodaltons and was provisionally named g70, was first detected in extracts of HSV-1-infected cells labeled by lactoperoxidase-catalyzed iodination and precipitated with rabbit sera or IgG and protein A-Sepharose. In subsequent experiments, g70 and gE were coprecipitated from extracts of HSV-1-infected cells labeled with [35S]methionine, [35S]cysteine, or 14C-amino acids. We were unable to precipitate a polypeptide analogous to g70 or gE from extracts of HSV-2-infected cells with rabbit IgG and protein A-Sepharose. Partial proteolytic peptide analysis indicated that g70 is structurally distinct from gE and gI). In addition, g70 was electrophoretically distinct from the HSV-1 Us4 glycoprotein gG. HSV-1 gE, expressed in mouse cells transfected with the gE gene, was not precipitated with rabbit IgG, nor could these cells bind radiolabeled IgG, suggesting that gE alone cannot act as an IgG (Fc) receptor. This result, coupled with the findings that gE and g70 are coprecipitated with IgG and with an anti-gE monoclonal antibody, suggests that gE and g70 form a complex which binds IgG. The electrophoretic mobilities of g70 molecules induced by different strains of HSV-1 differed markedly, arguing that g70 is encoded by the virus and is not a cellular protein induced by virus infection.     

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.     

Responses of herpes simplex virus type 1-infected cells to the presence of extracellular antibodies: gE-dependent glycoprotein capping and enhancement in cell-to-cell spread

Binding of anti-herpes simplex virus (HSV) immunoglobulin G (IgG) to HSV type 1 (HSV-1)-infected HEL and HEp-2 cells causes changes in surface viral glycoprotein distribution, resulting in a capping of all viral glycoproteins towards one pole of the cell. This occurs in a gE-dependent manner. In HEL cells, low concentrations of anti-HSV IgG also enhance cell-to-cell spread of wild-type HSV-1 but not of gE deletion mutant HSV-1. These observations raised the possibility that gE-dependent mechanisms exist that allow some HSV-1-infected cells to respond to the presence of extracellular antibodies by enhancing the antibody-resistant mode of virus transmission.     

Herpes simplex virus type 2 glycoprotein gF and type 1 glycoprotein gC have related antigenic determinants.

The 104-S monoclonal antibody immunoprecipitated from herpes simplex virus type 2 (HSV-2)-infected cell extracts the 75,000-molecular-weight glycoprotein gF and its 65,000-molecular-weight precursor (pgF). The precursor pgF was sensitive to endoglycosidase H digestion, indicating the presence of high mannose-type oligosaccharides, whereas the stable gF product was sensitive to neuraminidase digestion, indicating the presence of sialic acid residues. The 104-S antibody also weakly precipitated the 130,000-molecular-weight herpes simplex virus type 1 (HSV-1) glycoprotein gC from both infected cell extracts and purified preparations obtained through the use of monoclonal antibody-containing immunoadsorbent columns. Immunofluorescence tests demonstrated that the 104-S antibody reacted with antigen present in cells infected with HSV-2 strain 333 and HSV-1 strain 14012 but not with antigen present in cells infected with HSV-1 strain MP, a strain deficient in HSV-1 gC production. These findings indicate that HSV-1 gC and HSV-2 gF have antigenic determinants that are related.     

Application of antibody to synthetic peptides for characterization of the intact and truncated alpha 22 protein specified by herpes simplex virus 1 and the R325 alpha 22- deletion mutant.

The alpha 22 protein is one of five proteins synthesized immediately after infection of permissive cells with herpes simplex virus 1 and 2 (HSV-1 and HSV-2). On the basis of the reported nucleotide sequence of the HSV-1 gene, we synthesized two peptides containing the predicted amino acids 12 through 23 (12 residues) and 21 through 36 (16 residues) in two hydrophilic domains near the N terminus of the protein. Rabbit antisera made against these peptides were then used to characterize the alpha 22 protein made by wild-type HSV-1(F) strain and by an HSV-1 mutant, R325, carrying a 500-base-pair deletion within the coding domain of the gene. The results were as follows. (i) Both antisera reacted with HSV-1(F) alpha 22 protein in lysates electrophoretically separated in denaturing polyacrylamide gels and electrically transferred to a nitrocellulose sheet; neither antiserum reacted with the corresponding HSV-2 protein. The protein accumulated at 34 and 39 degrees C in the nucleus of infected permissive HEp-2 and baby hamster kidney (BHK) cells. The protein formed at least five spots differing in charge, mobility, and extent of phosphorylation on two-dimensional electrophoretic separation. (ii) The antisera reacted with a truncated nuclear protein (33,700 apparent molecular weight) in permissive HEp-2 and restrictive BHK cells infected with R325 and incubated at 39 degrees C but not at 34 degrees C. The truncated protein represents, therefore, the product of the undeleted 5' domain of the alpha 22 gene in R325. (iii) The presence of identical as well as slower migrating, reactive proteins in infected BHK cell lysates indicated that wild-type and truncated alpha 22 proteins are processed differently in BHK and HEp-2 cells.     

Identification and characterization of a novel herpes simplex virus glycoprotein, gK, involved in cell fusion.

Antipeptide sera were used to identify a novel glycoprotein encoded by the UL53 gene of herpes simplex virus type 1 (HSV-1). The UL53 gene product is thought to play a central role in regulating membrane fusion because mutations giving rise to the syncytial phenotype, wherein cells are extensively fused, frequently map to this gene. A single 40-kDa protein, designated gK (the ninth HSV-1 glycoprotein to be described), was detected with antipeptide sera in cells infected with both wild-type and syncytial strains of HSV-1 which were labelled with [35S]methionine and [35S]cysteine or with [3H]glucosamine, and this protein was sensitive to treatment of cells with tunicamycin. With all other HSV glycoproteins studied to date, at least two glycosylated species, often differing substantially in electrophoretic mobility, have been observed in infected cells; thus, gK is unusual in this respect. The 40-kDa gK protein was also immunoprecipitated from cells infected with a recombinant adenovirus vector carrying the UL53 gene. Two glycosylated species of 39 and 41 kDa were produced when UL53 mRNA was translated in vitro in the presence of microsomes, and these proteins differed from gK produced in infected cells not only because they possessed different electrophoretic mobilities but also because they were unable to enter gels after being heated. In addition, a 36-kDa protein was detected in extracts from cells infected with HSV-2 with use of these sera.     

Immunoglobulin G(Fc)-binding receptors on virions of herpes simplex virus type 1 and transfer of these receptors to the cell surface by infection

We have found that virions of herpes simplex virus type 1 have surface receptors capable of binding to the Fc region of immunoglobulin G and have also shown that the previously identified Fc-binding glycoprotein (designated gE) is present in extracts prepared from purified virions. The technique of affinity chromatography was used to detect gE. Two approaches were used to demonstrate the presence of Fc-binding receptors on intact virions. First, it was shown that [125I]-immunoglobulin G, but not [125I]F(ab')2 fragments, cosedimented with virions in sucrose gradients and, second, electron microscopy was done to show that soluble peroxidase-antiperoxidase complexes bound to virions. The use of peroxidase-antiperoxidase complexes also permitted us to demonstrate that Fc-binding receptors from input (parental) virions become incorporated into the surface membranes of infected cells. This occurs presumably as a consequence of fusion between the virion envelope and cell surface membrane.     

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.     

Specificity of human natural killer cells in limiting dilution culture for determinants of herpes simplex virus type 1 glycoproteins.

The frequency and specificity of human cells with natural killer (NK) cytotoxic activity for herpes simplex virus type 1 (HSV-1)-infected targets was measured by limiting dilution culture. The frequency of NK cell precursors (NK-p) reactive with HSV-1-infected cells was 2- to 11-fold higher than that of NK-p reactive with mock-infected cells. The frequency of NK-p reactive with infected target cells lacking viral glycoprotein C or presenting an antigenically altered glycoprotein B was approximately twofold lower than that with wild-type virus-infected cells. Specificity analysis demonstrated that NK cells with a high statistical probability of being monoclonal were reactive with either glycoprotein B or C. These results provide the first evidence that cells with human NK activity possess clonal specificity for HSV-1-infected target cells.     

Type-Common CP-1 Antigen of Herpes Simplex Virus Is Associated with a 59,000-Molecular-Weight Envelope Glycoprotein

The CP-1 antigen of herpes simplex virus type 1 (HSV-1) is a glycoprotein found in the soluble portion of infected cells, in detergent extracts of infected cell membranes, and in the envelope of purified virus. Antisera were prepared against a further purified form of CP-1 prepared from HSV soluble antigen mix; a glycoprotein, gp52, isolated from detergent-treated infected cells; and detergent extracts of purified virus. Each of the antisera reacted with CP-1 to give a single immunoprecipitin band of identity, and each antiserum neutralized the infectivity of HSV-1 and HSV-2. Our results suggested that the type-common determinants involved in the stimulation of neutralizing antibody resided on a 52,000-molecular-weight (52K) glycoprotein. The envelope of HSV contains several glycoproteins: one component at 59K and a complex of two or three components at 130K, none of which corresponds in molecular weight to gp52. Using the antisera as immunological probes, we performed pulse-chase experiments with [(35)S]methionine-labeled HSV-1-infected cells and followed the disposition of the glycoproteins during the infectious cycle. Each antiserum immunoprecipitated a (35)S-labeled 52K protein from lysates of cells pulse-labeled at 5 h after infection. By 10 h, the label was chased into a 59K protein also precipitable by each of the three antisera. The results suggest that gp52 is a precursor of gp59 and that the latter corresponds in molecular weight to one of the major glycoproteins of the virion envelope.     

Specificities of monoclonal and polyclonal antibodies that inhibit adsorption of herpes simplex virus to cells and lack of inhibition by potent neutralizing antibodies.

Polyclonal and monoclonal antibodies to individual herpes simplex virus (HSV) glycoproteins were tested for ability to inhibit adsorption of radiolabeled HSV type 1 (HSV-1) strain HFEMsyn [HSV-1(HFEM)syn] to HEp-2 cell monolayers. Polyclonal rabbit antibodies specific for glycoprotein D (gD) or gC and three monoclonal mouse antibodies specific for gD-1 or gC-1 most effectively inhibited HSV-1 adsorption. Antibodies of other specificities had less or no inhibitory activity despite demonstrable binding of the antibodies to virions. Nonimmune rabbit immunoglobulin G and Fc fragments partially inhibited adsorption when used at relatively high concentrations. These results suggest involvement of gD, gC, and perhaps gE (the Fc-binding glycoprotein) in adsorption. The monoclonal anti-gD antibodies that were most effective at inhibiting HSV-1 adsorption had only weak neutralizing activity. The most potent anti-gD neutralizing antibodies had little effect on adsorption at concentrations significantly higher than those required for neutralization. This suggests that, although some anti-gD antibodies can neutralize virus by blocking adsorption, a more important mechanism of neutralization by anti-gD antibodies may be interference with a step subsequent to adsorption, possibly penetration.     

Herpes simplex virus immunoglobulin G Fc receptor activity depends on a complex of two viral glycoproteins, gE and gI.

Evidence was recently presented that herpes simplex virus type 1 (HSV-1) immunoglobulin G (IgG) Fc receptors are composed of a complex containing a previously described glycoprotein, gE, and a novel virus-induced polypeptide, provisionally named g70 (D. C. Johnson and V. Feenstra, J. Virol. 61:2208-2216, 1987). Using a monoclonal antibody designated 3104, which recognizes g70, in conjunction with antipeptide sera and virus mutants unable to express g70 or gE, we have mapped the gene encoding g70 to the US7 open reading frame of HSV-1 adjacent to the gE gene. Therefore, g70 appears to be identical to a recently described polypeptide which was named gI (R. Longnecker, S. Chatterjee, R. J. Whitley, and B. Roizman, Proc. Natl. Acad. Sci. USA 84:147-151, 1987). Under mildly denaturing conditions, monoclonal antibody 3104 precipitated both gI and gE from extracts of HSV-1-infected cells. In addition, rabbit IgG precipitated the gE-gI complex from extracts of cells transfected with a fragment of HSV-1 DNA containing the gI, gE, and US9 genes. Cells infected with mutant viruses which were unable to express gE or gI did not bind radiolabeled IgG; however, cells coinfected with two viruses, one unable to express gE and the other unable to express gI, bound levels of IgG approaching those observed with wild-type viruses. These results further support the hypothesis that gE and gI form a complex which binds IgG by the Fc domain and that neither polypeptide alone can bind IgG.     

Release of a virus coded glycoprotein from herpes simplex virus type 1 infected cells

HEp-2 cells, which were infected with HSV-1, excrete besides other proteins a soluble glycoprotein (Mr 125000–130000) related to the virus protein gC. The excretion of the glycoprotein and the production of extracellular virus particles is reduced to a similar extent when the cells were treated with monensin. Possible consequences of the excretion of soluble viral proteins to a modulation of the immune response are discussed.Abbreviations HSV-1 Herpes simplex virus type 1 - PAGE Polyacrylamide gel electrophoresis - SDS Sodium dodecylsulfate     

Identification of an 80 kDa glycoprotein located at sites of close apposition between skeletal muscle cells

An 80 kDa glycoprotein was isolated from adult frog skeletal muscle by concanavalin (Con A) affinity chromatography and electrophoretic separation by molecular mass. Characteristics of the 80 kDa glycoprotein are that it: 1) binds non-covalently to gelatin-agarose or some other protein(s) bound to gelatin-agarose, 2) does not bind wheat germ agglutinin, 3) appears only at 80 kDa in both reducing and non-reducing electrophoretic separations, 4) is present in skeletal muscle but absent in smooth muscle and cardiac muscle, 5) is not collagenase or hyaluronidase-sensitive, and 6) is antigenically similar to a protein in embryonic chicken skeletal muscle. It was used to generate a polyclonal antiserum which was affinity-purified and used for immunolocalization. Indirect immunofluorescence procedures showed the antigen to be present on the surface of the skeletal muscle cells and concentrated at sites where cells are closely apposed to one another. Preparations in which adult muscle cells were depleted of basement membrane and endomysial proteins did not reduce the amount of 80 kDa protein present in skeletal muscle. These data indicate that this is a cell surface glycoprotein that may mediate attachment of the cell to extracellular proteins at sites where adjacent skeletal muscle cells are apposed.     

Rheumatoid factors may bear the internal image of the Fc gamma-binding protein of herpes simplex virus type 1

Affinity-purified rheumatoid factors (RF) from 20 patients with rheumatoid arthritis were tested for their reactivity with the mAb II-481 against glycoprotein E (gE), the Fc gamma-binding protein of HSV-1, as well as with a panel of mAb against human Fc gamma R. All RF bound to mAb II-481 in preference to mAb IV.3 (anti-human Fc gamma RII) or MOPC 141 (control mAb) which belong to the same IgG2b subclass. Five RF showed strong reactivity with II-481. No significant reactivity was observed between RF and mAb against human Fc gamma R. Non-RF human IgM did not react with any of the mAb. Clear-cut binding to II-481 was also seen with monoclonal IgM-RF derived from MRL/1 mice (mRF-2). The reaction between RF and II-481 was completely inhibited by human IgG. It was also inhibited by BHK cell extract infected with HSV-1, and with purified gE. II-481 inhibited the binding of human IgG Fc to the infected cell extract, confirming that II-481 recognizes the Fc-binding site on gE. II-481 did not react directly with human IgG or Fc of IgG. mAb to human IgG2 showed stronger binding to II-481 than to MOPC 141, suggesting II-481 has conformational similarity to human IgG H chain. These results suggest that at least some RF bear the "internal image" of HSV-1 Fc gamma-binding protein and support the hypothesis that some RF may be generated as anti-idiotype antibodies against antiviral antibodies.     

Characterization of oligosaccharides of highly purified glycoprotein gC of herpes simplex virus type 1 (HSV-1)

The envelope membrane glycoprotein gC of HSV-1 was purified from Triton X-100 extracts of virus-infected BHK-21 or HEp-2 cells by a single step immuno-affinity column using monoclonal anti-gC antibody. The analysis of the purified [³H]G1cN labeled glycoprotein gC (by gel filtration on Bio-Gel P4) before and after digestion with endo-β-N-acetylglucosaminidase (endo D) indicated that gC contains Asn-linked “complex type” oligosaccharides. No “high mannose” type oligosaccharides were detected. Fractionation of radio-labeled glycopeptides of gC on a column of concanavalin A-sepharose suggested that glycopeptides have “diantennary” and “triantennary” and/or “tetra antennary” structures. Tunicamycin inhibited the incorporation of [¹⁴C]GalN or [³H]GlcN into gC in HSV-1 infected BHK-21 or HEp-2 cells. Gel filtration analysis of [³H]GlcN labeled gC following β-elimination reaction failed to indicate O-glycosidically linked oligosaccharides.     

Localization of two cellular forms of the vesicular stomatitis viral glycoprotein.

Two cell-associated forms of the glycoprotein (G) of vesicular stomatitis virus, termed G1 and G2, have been resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. G1 has the higher electrophoretic mobility, but both forms migrate more slowly than G protein synthesized in a wheat germ cell-free system (G0), which presumably is the unglycosylated form. G1 is a kinetic precursor of the G2 form, and the apparent cause of the electrophoretic difference between the two species is the presence of N-acetylneuraminic acid on the G2 form. Conversion of G1 to G2 occurs 10 to 20 min prior to the appearance of the G2 form of the protein on the cell surface. This suggests that the G protein may be completely glycosylated several minutes prior to its migration to the cell surface and that glycosylation is not the limiting step in its maturation. No glycoprotein comigrating with G0 can be detected in the infected cells, even after 5-min labeling periods; this suggests that partial clycosylation of G occurs concomitantly with or immediately after its synthesis.