A rapid procedure for the enrichment of undenaturated, antigenically active herpes simplex virus glycoproteins.

The usefulness of lentil lectin affinity chromatography for the rapid enrichment of HSV glycoproteins in an undenatured state for both research and clinical purposes was investigated. In order to compare the lentil lectin-binding characteristics and immunologic specificities of undenatured HSV-1 and HSV-2 glycoproteins, [35S]methionine-labelled extracts of virus-infected HEp-2 cells were subjected to lentil lectin affinity chromatography. Individual HSV-1 and HSV-2 glycoproteins in bound and unbound fractions were identified using monoclonal antibodies. With the exception of a portion of pgD and gD, all major viral glycoprotein species (gA, gB, gC, gD, gE and gF) and their glycosylated processive intermediates bound to lentil lectin indicating that all possess predominantly mannosyl and/or glucosyl carbohydrate moieties. Although the unbound pgD and gD species were glycosylated, no gD and only a portion of pgD bound to lentil lectin when reapplied to the column indicating that these subspecies possess alterations in factors required for efficient lectin binding. Immunoprecipitation of undenatured lectin-bound glycoproteins from infected cells using HSV-1 and HSV-2-specific rabbit and human antisera confirmed previous findings that the predominant type-specific glycoproteins of HSV-1 and HSV-2 are gC and gE/gF, respectively.

Phosphorylation of neurotropic alphaherpesvirus envelope glycoproteins: herpes simplex virus type 2 gE2 and pseudorabies virus gI.

I have examined the state of phosphorylation of the envelope glycoproteins of two neurotropic herpesviruses, HSV-2 and PRV. HSV-2 gE2 and PRV gI, which are the homologues to HSV-1 gE, were found to be phosphorylated and phosphoaminoacid analysis revealed that both contained phosphoserine. These findings are consistent with a conserved phosphorylation of the glycoprotein homologues to HSV gE among the neurotropic alphaherpesviruses.

Tyrosine sulfation of varicella-zoster virus envelope glycoprotein gpl.

Sulfation is a common post-translational modification of secreted and membrane proteins, with the sulfate attached to tyrosine residues or to glycan side-chains. I have shown that varicella-zoster virus (VZV) envelope glycoproteins gpI, gpII, and gpIII can be labeled with [35S]sulfate. The predominant VZV glycoprotein, gpI, was shown to be sulfated on asparagine-linked glycans and on tyrosine. This is the first report of tyrosine sulfation of a viral envelope glycoprotein. Examination of the predicted amino acid sequences of gpI from the Dumas and CP-5262 VZV strains revealed the presence of a single consensus sequence for tyrosine sulfation of tyr88:IWPRNDYDGFLEN. Consensus sequences are also present in the homologues of gpI in herpes simplex type 1, herpes simplex type 2, and pseudorabies virus, suggesting that tyrosine sulfation may be a general post-translational modification of the neurotropic alphaherpesviruses.

Biochemical and genetic studies on galactosamine metabolism in Neurospora crassa.

In Neurospora, galactosamine can be released from the cell wall and from an alcohol-soluble compound by acid hydrolysis. All of the detectable alcohol-soluble galactosamine was present as uridine diphospho-2-acetamido-2-deoxy-D-galactose (UDPGalNAc). The results of pulse-labeling studies and enzymatic assays indicated that UDPGalNAc was synthesized via the epimerization of uridine diphospho-2-acetamido-2-de+xy-D-glucose (UDPGlcNAc). A single-gene morphological mutant, doily (do), which grew at less than 4% the rate of the wild-type strain, had 3% of the wild-type UDPGalNAc content and 0.5% of the wild-type level of cell wall galactosamine but normal levels of UDPGlcNAc and cell wall glucosamine. Cell extracts of the doily cultures containing only 20% of the specific activity of UDPGlcNAc-4-epimerase found in the extracts of wild-type cultures. Two types of faster-growing partial revertants of the doily strain were isolated. One type had an intermediate level of both alcohol-soluble and cell wall galactosamine. A second type had an intermediate level of alcohol-soluble galactosamine but low levels of cell wass galactosamine. Genetic analyses indicated that the reverse mutations had occurred at the do locus in both types. This finding that cell wall glucosamine synthesis and growth rate can be separated genetically indicates that mutations at the do lucus lead to pleiotropic effects.

Attachment of Giardia lamblia to rat intestinal epithelial cells.

The human enteric protozoan, Giardia lamblia, has surface membrane lectin activity which mediates parasite adherence to erythrocytes. To determine whether an intestinal binding site exists for this lectin we have studied the interaction in vitro between axenically cultured Giardia trophozoites and isolated rat intestinal epithelial cells. Scanning electron microscopy showed that Giardia attached to the apical microvillus membrane and basolateral membrane of rat enterocytes. Any location on the parasite surface could mediate attachment without predeliction for the ventral disc. Trophozoites attached more avidly to jejunal compared with colonic epithelial cells. Attachment was inhibited at 4 degrees C, by sugars and glycoproteins containing D-mannosyl residues and by subagglutinating concentrations of anti-Giardia rabbit serum and two monoclonal antibodies, all with reactivity to parasite surface membrane determinants. Trypsinisation of trophozoites also reduced attachment but the ability to attach was rapidly restored after returning trophozoites to TYI-S culture medium for 4 h at 37 degrees C. Attachment was unaltered by the presence of the microfilament inhibitor cytochalasin B and in the absence of Ca++ and Mg++ ions. These findings support previous work that Giardia possesses a surface membrane mannose binding lectin and indicate that appropriate binding sites are present on rat intestinal epithelial cells. This lectin may play a part in mediating adherence of Giardia to mammalian intestine and could be a target for host immune defence.

Varicella-zoster virus gpI and herpes simplex virus gE: phosphorylation and Fc binding.

gpI, the predominant varicella-zoster virus (VZV) envelope glycoprotein, was shown to be phosphorylated exclusively on serine and threonine residues, and phosphorylated gpI was detected in isolated virions. In cells infected with herpes simplex virus type 1 (HSV-1), a related neurotropic alpha-herpesvirus, HSV gE, the homolog to VZV gpI, and HSV gB, the homolog to VZV gpII, were also phosphorylated. The phosphate on gB and gE was alkali labile and resistant to endo H, suggesting linkage to serine and/or threonine. Although VZV gpI and HSV gE share sequence homology and similar post-translational modifications, no Fc-binding activity similar to that associated with gE was detected for gpI or any of the VZV glycoproteins.

Changes in glucosamine and galactosamine levels during conidial germination in Neurospora crassa.

The levels of glucosamine and galactosamine were determined in conidia, germinating conidia, and vegetative mycelia of Neurospora crassa. In the vegetative mycelia about 90% of the amino sugars were shown to be components of the cell wall. The remaining 10% of the amino sugars were tentatively identified as the nucleotide sugars uridine diphospho-2-acetamido-2-deoxy-D-glucose and uridine diphospho-2-acetamido-2-deoxy-D-galactose. Conidia and vegetative mycelia contained about the same levels of glucosamine. During the first 9 h after the initiation of germination, the total glucosamine content had increased 3.1-fold, whereas the residual dry weight of the culture had increased 7.7-fold. This led to a drop in the glucosamine concentration from 100 mumol/g of residual dry weight to 42 mumol/g. During this time, all of the conidia had germinated and the surface area of the new germ tubes had increased to 10 times that of the conidia. Either germ tubes were initially produced without glucosamine-containing polymers, or these polymers (probably chitin) were deposited only at low densities in the germ tube cell walls. The chitin precursor uridine diphospho-2-acetamido-2-deoxy-D-glucose was present at all times during conidial germination. Conida contained very low levels of galactosamine. During germination, galactosamine could not be detected until the culture had reached a cell density of about 0.6 mg of residual dry weight per ml of growth medium. This was observed regardless of the time required to reach this cell density or the fold increase in dry weight. The accumulation of galactosamine-containing polymers does not appear to be necessary for germ tube formation. The levels of soluble galactosamine (uridine diphospho-2-actamido-2-deoxy-D-galatose) were very low in conidia and increased during germination at the same time that galactosamine appeared in the cellular polymers. In addition, under certain culture conditions, the appearance of galactosamine and the increase in the glucosamine concentration occurred simultaneously.

Identification of the products of a varicella-zoster virus glycoprotein gene.

Two of the genes identified from the previously published DNA sequence of the Us component of the varicella-zoster virus (VZV) genome were predicted to encode membrane proteins with polypeptide molecular weights of 39000 (39K) and 70K. A rabbit antiserum directed against a unique peptide containing the seven amino acid residues at the carboxy terminus of the 39K gene product specifically precipitated glycoproteins with apparent molecular weights of 55K and 45K from VZV-infected cells labelled with [3H]mannose. The complete inhibition of precipitation of gp55 by free peptide and the partial inhibition of precipitation of gp45 support the conclusion that the 39K gene encodes gp55 and perhaps gp45. The number of VZV genes currently thought to encode glycoproteins is discussed in view of this finding.

Synthesis and processing of the three major envelope glycoproteins of Epstein-Barr virus.

In pulse-chase experiments, the three major Epstein-Barr virus envelope glycoproteins, gp350/300, gp250/200, and gp85, were shown to be synthesized from separate precursors of 190,000, 160,000, and 83,000 daltons, respectively. These three pulse-labeled species were chased into the mature forms of the glycoproteins between 1 and 3 h after transfer to nonradioactive medium. Digestion of precursor forms with endo-beta-N-acetylglucosaminidase H (endo H) yielded polypeptides of 160,000, 120,000, and 75,000 daltons. Comparison of these results with those from experiments with tunicamycin, which specifically blocks N-linked glycosylation, indicated that some other post-translational modification(s), probably O-linked glycosylation, contributes about 100,000 and 60,000 daltons of apparent molecular mass to gp350/300 and gp250/200, respectively. Experiments with endo H showed that mature gp350/300 and gp250/200 contain complex-type (endo H-resistant) N-linked glycosyl chains, whereas gp85 contains both high-mannose (endo H-sensitive)- and complex-type oligosaccharides. In contrast to the results obtained with the three envelope glycoproteins, no precursor forms of the two unglycosylated protein, p160 (the major Epstein-Barr virus capsid antigen) and p140 (an envelope protein), were detected. The partial proteolytic maps of gp350/300 and gp250/200 were quite similar, suggesting that polypeptide sequence homology could account for at least part of the observed serological cross-reactivity of the two proteins. Taken together, these results demonstrate that the polypeptide portions of gp350/300 and gp250/200 are closely related but not derived from a common precursor. Furthermore, the polypeptide portions comprise half or less of the apparent molecular weight of the mature glycoproteins on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Epstein-Barr virus membrane antigens: characterization, distribution, and strain differences.

The Epstein-Barr virus (EBV)-associated membrane antigen polypeptides (350,000, 220,000, 140,000, and 85,000 daltons) are recognized by a rabbit anti-EBV serum and are present on the plasma membranes of producer cell lines, as we demonstrated previously. In this report, we show that these polypeptides are present on intact virus particles. Subcellular fractionation revealed that these antigens are distributed throughout the cell, except for the 85,000-dalton protein, which was poorly represented in the nuclear fraction. In addition, an EBV-associated protein of 160,000 daltons, which comigrates with a major component of the viral capsid, was detected in the cytoplasmic and nuclear fractions. The immunoprecipitation patterns of 13 different EBV isolates were similar, with two exceptions. First, the 350,000- and 220,000-dalton polypeptides from marmoset cell lines had slightly larger molecular sizes than the corresponding polypeptides from human cell lines. Second, B95-8 virus and B95-8-derived human and marmoset cell lines contained little of the 220,000-dalton protein; however, 883L, the human parent line of B95-8, has a normal amount of the 220,000-dalton protein. Thus, the B95-8 strain of EBV appears to be a structurally defective variant. We have not observed any variation in protein patterns associated with different EBV disease states. The 350,000-, 220,000-, and 85,000-dalton polypeptides were shown to be glycoproteins by incorporation of [3H]mannose and [3H]glucosamine and to contain N-asparagine-linked glycosyl groups by their sensitivity to tunicamycin. To simplify future work, the following nomenclature for these EBV-associated polypeptides is suggested: 350,000 (gp350), 220,000 (gp220), 160,000 (p160), 140,000 (p140), and 85,000 (gp85).

Polypeptides of the Epstein-Barr virus membrane antigen complex.

Epstein-Barr virus (EBV)-associated membrane antigens have been purified from the plasma membranes of the producer cell line P3HR-1 NONO. The antigens were assayed with a specific rabbit anti-ebv antiserum using an 125I-labeled staphylococcal protein A binding assay. The antigens have been shown to be present on purified plasma membranes. Treatment of the plasma membranes with Triton X-100 allows the separation of two antigenically distinct classes of antigens, one soluble and one insoluble in the detergent. Immunoprecipitates of [125I5- and [35S]methionine-labeled, detergent-soluble antigens contained three major polypeptides of molecular weights of 350,000, 140,000, and 75,003 (on 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and several minor components. These polypeptides were all specifically precipitated from four EBV-producer cell lines, P3HR-1, P3HR-1 NONO, B95-8, and 7744. They could not be precipitated from producer cell lines treated with phosphonoacetic acid, which inhibits late viral functions, nor could they be precipitated from nonproducer cell lines. The 350,000 and 75,000 molecular weight polypeptides bound to Ricin and lentil lectin columns; however, most of the 140,000 molecular weight material did not. A component of molecular weight 220,000 (prominent only in P3HR-1 NONO) was probably a degradation product of the 350,000 molecular weight polypeptide.

Biochemical analysis suggests distinct functional roles for the BLAST-1 and BLAST-2 antigens.

The biochemical processing of the BLAST-1 and BLAST-2 activation antigens has been studied. Both are glycoproteins that derive from different precursors of the same apparent m.w. on SDS-PAGE. BLAST-1 is synthesized as a 43,000 m.w. light chain in association with a second heavier chain of 55,000 m.w. The light chain acquires sialylated O-linked glycans and is stably expressed at the cell surface with a half-life of 14 hr. BLAST-2 is also synthesized as a 43,000 m.w. precursor, but it acquires only unsialylated N-linked glycans. The mature glycoprotein is only expressed briefly at the cell surface (half-life of 1 to 2 hr), and is then shed into the culture supernatant as a soluble 33,000 m.w. derivative. The different fates of these molecules, one stably expressed at the cell surface and one shed, suggest disparate roles for these two antigens in B cell activation.

An 88,000-Mr Giardia lamblia surface protein which is immunogenic in humans.

Human anti-Giardia lamblia sera specifically immunoprecipitated an 88,000-Mr surface protein from radioiodinated trophozoites, establishing this protein as a potentially important immunogen in humans. A mouse monoclonal antibody (GL-1) was isolated which immunoprecipitated the same 88,000-Mr surface protein recognized by the human sera. GL-1 gave uniform fluorescent staining of the cell surface and flagella of G. lamblia trophozoites from the Portland 1 and WB strains as well as fresh clinical isolates, but not of Giardia muris, suggesting that the surface antigen is specific to G. lamblia. Other human parasites, including Entamoeba histolytica, Trichomonas vaginalis, and Trichomonas hominis, were not stained. A second mouse monoclonal antibody (GL-2) gave weaker immunofluorescent staining of living G. lamblia trophozoites but intense staining of fixed cells. None of the other parasites tested were stained, with the exception of E. histolytica, which may contain a cross-reactive antigen. No proteins were recognized in immunoprecipitation studies with iodinated trophozoites.

A ligand for human CD48 on epithelial cells.

CD48 is a member of the Ig superfamily with a high degree of sequence homology to CD58 (LFA-3). In rodents, CD48 is the ligand for CD2 whereas in humans, CD58 is the ligand for CD2. Despite intensive efforts, no ligand for human CD48 has been convincingly demonstrated. We now show that a ligand for human CD48 is present on epithelial cells. The ligand was detected based on the ability of epithelial cells to bind both a decameric, soluble CD48 IgM fusion protein and monomeric CD48 immobilized on plastic dishes. mAbs raised to the ligand completely block binding of CD48 to all epithelial cells tested. We further show that the cell surface proteoglycan CD44 plays an auxiliary role in the binding of epithelial cells to CD48 and that this interaction involves the glycosaminoglycan binding site of CD44. No interaction of human CD48 with CD2 was detected. This is the first clear demonstration that human CD48 can function as an adhesion molecule and suggests a role for CD48 in lymphocyte epithelial cell interactions.

Purification and properties of poly(adenosine diphosphate ribose) synthetase.

Poly(ADP-ribose) synthetase has been purified approximately 5000-fold from rat liver nuclei. The activity of the purified enzyme is absolutely dependent upon the presence of native or synthetic DNA, and the further addition of histone(s) stimulates the activity 3- to 5-fold. When the ADP-ribosylated material synthesized in the absence or presence of various histones is analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the major product in all cases migrates between histones H1 and H3-H2B with the same RF value of 0.58 relative to the marker dye. No ADP-ribose was found to co-electrophorese with any of thehistones. The addition of histones does not affect the chain number of the poly(ADP-ribose) synthesized but does result in an increase in the average chain length of the polymer. In the presence of histones, the Km for NAD+ decreases from 80 micron to 25 micron and the Vmax doubles. These results indicate that, in the purified poly(ADP-ribose) synthetase system, histones are not ADP-robosylated but act as allosteric activators.

An unusually high-titer human anti-Epstein Barr virus (EBV) serum and its use in the study of EBV-specific proteins synthesized in vitro and in vivo.

Sera from a patient with a chronic Epstein Barr virus (EBV) infection contained unusually high anti-EBV antibody titers (1:2560 to 1:10,240 for EA(D) and 1:5,120 to 1:40,960 for VCA). One of these serum samples was shown by immunoprecipitation to recognize at least 11 EBV-specific proteins from virus producer cells labeled in vivo and 10 EBV-specific proteins from in vitro translations of producer cell mRNA. Six of the in vivo labeled proteins (135,000, 89,000, 50,000 to 55,000 doublet, 46,000, and 34,000 daltons) are "early" by their resistance to phosphonoacetic acid, and five (350,000, 220,000, 160,000, 140,000, and 85,000 daltons) are "late" membrane and capsid proteins. The EBV-specific proteins immunoprecipitated from in vitro translations had molecular masses of 150,000, 140,000, 115,000, 52,000, 50,000, 45,000, 34,000, 29,000, 17,000, and 15,000. Subcellular fractionation studies of cells labeled in vivo revealed that the 135,000-dalton protein and part of the 50,000 to 55,000 dalton protein doublet were found in both the nuclear and the cytoplasmic fractions, and thus are good candidates to be components of the EA(D) diffuse-type immunofluorescence observed with many EA-positive sera.

Schistosoma mansoni: genetic restriction and cytokine profile of the CD4 + T helper cell response to dominant epitope peptide of major egg antigen Sm-p40.

Granuloma formation in schistosomiasis is mediated by MHC class II-restricted CD4 + T helper lymphocytes sensitized to egg antigens. We previously reported that C3H mice, which develop large granulomas, display strong CD4 + T helper cell responses to the major egg antigen Sm-p40. Moreover, all members of a panel of egg antigen-specific T cell hybridomas responded to the Sm-p40 antigen. Given the significance of the Sm-p40 molecule in the C3H T cell repertoire against schistosomal egg antigens, the current work was undertaken to map its immunogenic epitopes, using a library of 15 synthetic overlapping 30-mer peptides. The dominant epitope recognized by polyclonal CD4 + Th cells was located in peptide 10 (amino acids 229-258); subdominant epitopes were detected in peptides 8 (amino acids 179-208) and 12 (amino acids 279-308). The anti-Sm-p40 T cell hybridomas variously responded to any one of the same three stimulatory peptides. Furthermore, studies with various mouse strains demonstrated that a strong anti-Sm-p40 response was restricted by H-2(k). Interestingly, the cells responding to peptide 10 and to the Sm-p40 antigen only secreted IL-2 and IFN-gamma, but not IL-4 and IL-10, indicating that they are entirely of the Th-1-type, a subset with demonstrated capacity to mediate egg granuloma formation. The identification of dominant epitopes within key egg antigens offers opportunities for desensitization of the CD4 + Th cells that mediate pathology in schistosomia sis.

Cross-reactivity between herpes simplex virus glycoprotein B and a 63,000-dalton varicella-zoster virus envelope glycoprotein.

Cross-reactive monoclonal antibodies recognizing both herpes simplex virus (HSV) glycoprotein B and a major 63,000-dalton varicella-zoster virus (VZV) envelope glycoprotein were isolated and found to neutralize VZV infection in vitro. None of the other VZV glycoproteins was recognized by any polyclonal anti-HSV serum tested. These results demonstrate that HSV glycoprotein B and the 63,000-dalton VZV glycoprotein share antigenic epitopes and raise the possibility that these two proteins have a similar function in infection.

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.