key: cord-0004888-mgzmzeyr authors: Olofsson, S.; Norrild, Bodil; Andersen, Åse B.; Pereira, Leonore; Jeansson, S.; Lycke, E. title: Populations of herpes simplex virus glycoprotein gC with and without affinity for the N-acetyl-galactosamine specific lectin ofHelix pomatia date: 1983 journal: Arch Virol DOI: 10.1007/bf01315701 sha: 19cc656fb8259945e3c38db40e3aad39c4e6258c doc_id: 4888 cord_uid: mgzmzeyr Two fractions of herpes simplex virus glycoprotein gC were isolated and characterized by means of immunosorbent-purification with monoclonal antibodies against gC and Helix pomatia lectin (HPA) affinity chromatography. About 25 per cent of the glycoprotein gC population demonstrated affinity for the lectin, compatible with presence of N-acetylgalactosamine as terminal sugar of the oligosaccharide. The HPA-binding populations of gC appeared as two electrophoretic bands with lower molecular weights than the non-binding gC. The gC subfraction without affinity for the HPA was subjected to treatments aiming to desialylize the carbohydrate moiety. Only 5 per cent of the initially non-reactive fraction of gC became reactive to HPA after the treatments, suggesting that masking of penultimate N-acetylgalactosamine by sialic acid was not a main reason for lack of HPA affinity. Results of treatment with alkaline Na BH(4) demonstrated presence of oligosaccharide-peptide linkages sensitive to β-elimination suggesting O-glycosidic type of linkage. The subfraction of gC demonstrating affinity for HPA as well as gC devoid of HPA binding capacity both revealed affinity for Con A. Therefore N-glycosidically as well as O-glycosidically linked oligosaccharides seemed to be present on the one and same glycoprotein. On the basis of the results presented we assume that the glycosylation of HSV glycoprotein gC may lead to, at least, two populations of the glycoprotein gC, one with terminal N-acetylgalactosamine residues of oligosaccharides 0-glycosidically linked to the polypeptide and the other without affinity for HPA. However, both populations of gC contain similar proportions of oligosaccharides of the high mannose or complex types with N-glycosidic carbohydrate-peptide linkages as indicated by their affinity for Con A. All known glycoproteins of enveloped viruses contain high mannose or complex type ohgosaccharides linked to the polypeptide via an N-glycosidic linkage between asparagine and N-aeetylglueosamine (GlcNAc) (6) . Under conditions of tunicamyein (TM) inhibition of gtycosylation in virus-infected cells, aberrant polypeptides will be formed resulting in loss of viral infectivity (9, 10, 16, 17, 18, 25) . According to prevailing concepts, the main function of N-glycosidic oligosaecharides is to stabilize the polypeptide conformation and the loss of infectivity is caused only indirectly by the absence of oligosaccharides due to such secondary events as increased sensitivity to intracellutar proteolytic degradation or unspecific aggregation of underglycosylated viral glycoproteins (4) . We have found that the type-specific glycoprotein gC of herpes simplex virus type 1 (HSV-1) contains oligosaccharides with affinity for the N-acetylgalactosamine-binding Helix pomatia lectin (I-IPA). Most likely the oligosaceharide constituent of tt~e HPA-binding HSV glyeoprotein is linked to the peptide via an 0-glycosidic bond between N-aeetylgalaetosamine (GalNAc) and a serine or threonine of the peptide (19, 20) . Such O-glycosidic linkages of HSV (19) , vaecinia (26) and coronavirus (7, 15) glycoproteins have been described recently. It is tempting to compare the HPA-binding I-ISV gtycoproteins with several biologically highly active glyeoeonjugates such as blood group substances and lymphocyte differentiation antigens which also demonstrate HPA-binding activities (8, 1i) . For these types of antigens it is likely that the carbohydrate itself mediates the biological activity, a phenomenon which contrasts to the less specific biological significance of N-glycosidic oligosaceharides. Recently, the function of so called stage-specific differentiation antigens have been blocked by addition of competing carbohydrate haptens (5) . Thus, the HPA-binding oligosaccharides of HSV glyeoproteins might constitute a carbohydrate structure with a more defined biological significance, probably as a recognition structure. In the present study the distribution of I-IPA-binding activity among I.ISV glyeoproteins was investigated, and it was found that only a subfraetion of gC contains HPA-binding oligosaccharides while the other fra.etion contains 0-glycosidic oligosaeeharide without affinity for ItPA. The prototype virus I-ISV-1 (F) was propagated in VEI~O cells maintained as monolayer cultures in minimal essential medium (MEM) supplemented with 10 per cent foetM calf serum (FCS). The I-ISV-1 (HFEM) ts B5 (I2) was a generous gift from Dr. A. Buehan, University of Birmingham, England. This mutang, which was defective in gB at non-permissive temperature was used as a marker for gA and gB (12) . Monoclonal Antibodies ttybridoma cells were produced as previously described (22) and aseites preparations containing monoelonM antibodies against glycoproteins gC (I-IC1), gA/gB (H233) or gD (tII) 1) were used. V E R O m o n o l a y e r cells grown in 150 cm e tissue culture flasks (2 × 107 cells) were infected w i t h ItSV-1 using a m u l t i p l i c i t y of infection of 5 P F U / c e l l a n d a v o l u m e of 4 ml M E M c o n t a i n i n g 1 per cent FCS. After 1 h o u r of a d s o r p t i o n at 37 ° C t h e virus was aspirated a n d t h e cells overlayered from 5 to 18 hours postinfeetion b y t h e a d d i t i o n of 10 ml M E M w i t h 1 per cent FCS a n d t.0 y, Ci/ml m e d i u m of D-(1-14-C)-glucosamine (specific a c t i v i t y 55.5 mCi/mMol, A m e r s h a m , E n g l a n d ) . A t t h e e n d of t h e labelling period t h e cells were scraped off, washed once in P B S a n d collected as a cell pellet after e e n t r i f u g a t i o n for 5 m i n u t e s at 500 × g. The cell pellet from 1.2 × l0 s glueosamine labelled H S V infected VEI~O ceils was p r e p a r e d as previously described (20) . Briefly, p a c k e d cells were m i x e d w i t h 0.025 5~ Tris-hydrochloride, p i t 8.0, a n d homogenized in a t i g h t fitting Dounce homogenizer. After e e n t r i f u g a t i o n at 1500 × g t h e m e m b r a n e s r e m a i n i n g in t h e s u p e r n a t a n t were pelleted at 1 6 0 , 0 0 0 × g for 1 hour. The pelleted m e m b r a n e s were suspended in 0.1 g l y e i n e -N a O H buffer, p H 8.8, a n d centrifuged a t 160,000 × g for 1 hour. Finally, t h e w a s h e d m e m b r a n e s were suspended in s, g l y e i n e -N a O H buffer c o n t a i n i n g 1 per cent T r i t o n X -t 0 0 . T h e suspension was homogenized a n d centrifuged a t 1 6 0 , 0 0 0 × g for 1 hour. The s u p e r n a t a n t was used in t t P A -a f f i n i t y c h r o m a t o g r a p h y . The T r i t o n X -I 0 0 solubilized m a t e r i a l was applied on columns (diameter 16 m m ) w i t h 8 m l of H P A coupled to Sepharose 6 MB (Pharmaeia, Sweden). T h e gel was equilibrated w i t h 0.02 ~ Tris buffered saline a n d 0.5 per cent T r i t o n X -f 0 0 a n d was developed a t 4 m l per h o u r w i t h t h e same buffer. Proteins specifically b o u n d were eluted w i t h 0.02 ~ GalNAe. HSV-1 proteins w i t h or w i t h o u t affinity for H P A were p r e c i p i t a t e d w i t h TCA a n d w a s h e d in order to c o n c e n t r a t e eluted protein. After wash t h e proteins were dried a n d S. OLOFSSON et al.: solubilized in buffer and reacted with specific monoclonal antibodies added in optimal amounts. The antigen-antibody complexes were bound to fixed staphylococci (strain cowan-I) (23) . The immunopreeipitates were washed extensively in P B S supplemented with 0.5 ~ NaC1, 0.1 per cent (w/v) SDS and 0.4, per cent (v/v) TritonX-100. After further washing in the same buffer, supplemented with 1.0 ~ NaC1, the proteins were dissolved in disruption mixture for SDS-polyacrylamide geleleetrophoresis. SDS-polyacrylamide geleleetrophoresis was done according to MORSE et al. (14) . The separation gel was 9.25 percent (w/v) acrylamide cross-linked with 0.25 percent (w/v) diatyltartardiamide. The gels were stained and autoradiography was done on K o d a k X R P -1 X -C r o a t film. Desialylation To r e m o v e sialic acid H S V glycoprotein gC without affinity for H P A was treated in amounts of less t h a n 1 m g with of neuraminidase (1 unit) overnight at 37 ° C in 0.01 acetate buffer, pI-I 5.5. Alternatively, aliquots of gC without affinity for I-IPA were subjected to t r e a t m e n t with 0.05 ~ I~eSO4 at, 85 ° C for 1 hour. The solution was neutralized with solid Na2CO~ and desalted by Sephadex G-25 gel. filtration. The alkaline borohydride t r e a t m e n t was carried out as previously described (19) . After fractionation by H P A chromatography t:he material was desalted on Sephadex G-25 and dissolved in 0.5 ~ N a O H and 0.5 ~ NaBtt4. This mixture was incubated for 48 hours at room t e m p e r a t u r e (21--23 ° C) under nitrogen in tefloncapped tubes. The reaction was stopped by adding glacial acetic acid and the results of the ~-elimination reaction were assayed by Sephadex G50 gel filtration. Preparations of membrane proteins of HSV-1 infected Vero cells were subjected to H P A affinity chromatography (Fig. 1) . Two fractions one with (bound) and the other without affinity (unbound) for the leetin were collected. The HPA binding material was eluted with 0.02 M GalNAc. Approximately 10 per cent of the HSV glycoprotein preparation demonstrated affinity for HPA. In order to identify the glycoproteins present both fractions were immunoprecipitated with monoclona] antibodies directed against gA/gB, gC or gD. Precipitated proteins were subsequently characterized in SDS-polyacrylamide gels. The glycoprotein with affinity for H P A demonstrated gC specificity (Fig. 2, slot 4 ), but with a higher electrophoretic mobility than the nonbinding gC (Fig. 2 , slot 2). The precursor to gC was also identified. Presence of glycoprotein gA/gB was not observed (Fig. 2, slot 5) . The glycoproteins without affinity for H P A contained gA/gB, gC and gD (Fig. 2, slots 2 and 3) . The data on gD is not shown. Purification of gC was made possible by binding extracts of 14C-glucosamine labelled infected cells on Sepharose immunosorbent columns containing monoclonal antibodies reactive with gC (Fig. 3) . Material passing through the column and designated pool I was identified as a mixture of gA/gB, gD and their precursors as based on the eleetrophoretic mobilities observed. Pool I I i.e. the specifically bound protein represented 30 to 4:0 per cent of the radioactive material loaded on the column, was identified as gC and precursors gC. Two additional potypeptide bands were detected corresponding to proteins with molecular weights of 76 K and 72 K (Fig. 5, slot 4) . The lectin affinity chromatography with immunosorbent-purified gC demonstrated that about 25 per cent of the isolated gC was biospecifically bound to HPA (Fig. 4) . Both unbound and HPA-bound immunosorbent purified gC were subjected to SDS-PAGE. Two polypeptide bands with affinity for ItPA were identified and both proteins had electrophoretic mobilities that were significantly higher than that of the major band identified without affinity for the leetin (Fig. 5, slots 2 and 3) . These data together with those presented in Fig. 2 therefore indicate that among the HSV glyeoproteins gA, gB, gC and gD only two subspecies of gC have affinity for HPA. If the HPA-binding subfraction comprises an underglycosylated precursor to gC, one would expect that the non-binding fraction may consist of sialylized g~Teoproteins with masked HPA-binding oligosaccharides. For removal of such masking structures tt~e gC fraction without HPA affinity was treated with neuraminidase or dilute It2S04 before HPA chromatography (Fig. 6) . However, only about 5 per cent of the originally HPA negative gC fraction became HPA positive after treatment with neuraminidase or H 2 S O 4. Therefore the fraction of gC without affinity for the HPA seemed not to contain sialylized glyeoprotein gC with GalNAe as penultimate sugars. 5 . The autoradiographie image of the immunosorbent purified, 14C glucosamine labelled gC with affinity for ttPA. Identified on SDS polyaerylamide gel. Slot 1, total extracts from HSV-1 (HEFM) t s B 5 infected cells grown at 34 ° C. Slot 2, gC isolated from pool W of an t t P A column without a purification on immunosorbent. The protein was precipitated with monoeloanl antibodies to gC (analogous to Fig. 2, slot 2) . Slot 3, the fraction of immunosorbent purified gC which could bind to HPA. Slot 4, immunosorbent purified gC (analogous to Fig. 4, slot 3 ). Notiee the difference in the electrophoretie mobility between g C isolated by immunosorbent chromatography (slot 4) and the sub-population of gC which had H P A affinity (Not 3, marked with arrows) Binding of gC to HPA suggests presence of terminal GalNAe and also presence of O-linked oligosaccharides. The finding that only a subfraetion of gC contained HPA binding activity even after desialylation raised the question whether or not the subfraction which primarily did not bind to HPA contained ohgosaccharides O-glycosidically linked to the peptide. Immunosorbent purified gC was therefore fractionated by HPA affinity chromatography and the subfraction not binding to HPA subsequently subjected to treatment with alkaline NaBH4. Before and after treatment with NaBtt4 the fractions of 14C-glucosamine labelled gC was studied on Sephadex G50, Untreated or mocktreated material was found in the void volume exclusively (panel A, Fig. 7 ). After treatment with alkaline NaBH4 release of labelled material was demonstrated. The molecular weights of these structures corresponded to 3000 or more. The results were interpreted as ~elimination of O-linkages between oligosaccharides and peptides of gC. Experiment, s with pronase-digested underglycosylated HSV glycoprotein produced in the presence of tunieamycin indicate existence of at least two classes of relatively large tunicamycin-resistent oligosaccharides; with and without affinity for HPA (OnoFsso~ et al., submitted for publication). These data support the conclusion that more than one type of O-linked sugar may be attached to HSV glycoproteins. Oligosaceharides with N-glycosidical linkages of the high mannose type or moderately branched complex type exhibit affinity to Con A (13) . Testing of the HPA binding and non-binding fractions of gC with Con A affinity chromatography revealed approximately the same abundance of biospecific binding of both the gC fractions to Con A (Fig. 8) . These data indicate that N-glycosidieally linked The viral glycoproteins (gA/gB, gC and gD) synthesized in HSV-1 infected cells are glycosylated in several discrete steps (2, 3, 27) . We have found that the type-specific glyeoprotein, gC, of HSV-1 contains oligosaccharides binding to HPA, a lectin with specific affinity for GalNAc (11) . This sugar is genera~Ily absent in normal N-glycosidic oligosaccharides but is present in oligosaccharides linked O-glycosidically to a serine or a threonine residue of the poIypeptide. Recent findings that glycoprotein gC is sensitive to weak alkaline borohydride treatment (19) and contains TM resistent oligosaccharides (OLo~sso~ et al., submitted for publication) further supports that gC, in addition to the N-glycosidic carbohydrate chains, contains O-glycosidically linked oligosaccharides. In the present study the immunochemical specificity of the HSV glycoproteins without affinity for HPA and those with affinity for the lectin were identified by immunoprecipitation with monoclonal antibodies against HSV specified glyeoproteins. The glycoprotcins without affinity for HPA contained gA/gB, gD and the majority of gC, whereas only glycoproteins with the immunological specificity of gC were found in the fraction binding to HPA. The data thus emphasized that of the HSV-glycoproteins only gC contains oligosaccharides with affinity for HPA. Using immunosorbent-purified glycoprotein it could be clearly demonstrated that glyeoproteins with HPA-binding oligosaccharides constituted a subfraction comprising about 25 per cent of the glycosylatcd gC population (Fig. 4) . However, our data also show that gC without affinity for HPA contains O-linked carbohydrates, and that O-glycosidic as well ~s N-glycosidic linkages exist on the one and same gC molecule (Figs. 7 and 8) . The eleetrophoretic analysis of the HPA-binding gC subfraction revealed two polypeptides, one with a molecular weight of 108 K which corresponds to the partially glycosylated precursor of gC (pgC) and one with a molecular weight of 12t K which is intermediate between that of the fully glycosylated gC and that of pgC. The low molecular weights of HPA-binding gC molecules relative to the bulk of glyeoproteins with gC specificity suggest that gC with affinity for HPA constitute partially glycosylated gC intermediates. If we assume that, the HPAbinding oligosaecharides represent such precursors to larger O-glycosidic ohgosaccha.rides lacking HPA-affinity it might be suspected that HPA-binding sites were blocked by terminal sialic acids as observed for HPA-binding oligosaccharides of T lymphoeytes (1, 8) . However, our experiments with two different procedures for elimination of sialic acids only marginally increased the HPA-binding activity of gC, suggesting that sialylation of HPA-binding sites was not a main reason for absence of HPA binding propert:ies. The HPA-binding glycoprotein might therefore represent a fully glycosylated fraction of gC, that is glycosylated differently than the bulk of gC. One possible explanation would be that the biosynthesis of the gC-associated O-glycosidic oligosaecharides at some point is branched and follows different 3* pathways, one leading to HPA-binding oligosaccharides and the other to oligosaceharides with terminal structural properties without affinity for HPA. This diversity m a y be caused either solely by host cell glyeosyl transferases or by one or more virus specified or virus modified enzymes acting in concert with the cell specified glycosyl transferases. We have previously pointed out the changes of kinetic properties of glyeosyl transferases after HSV infection of cells (21) . Presence of competing glycosyl transferases leading to biosynthetic branching of a core structure into different types of otigosaccharides with and without blood group A activity has been demonstrated with 0-1inked carbohydrates of the sheep submaxillary mucin (24) . In presence of TM only trace amounts of glycoprotcins gA/gB and gC were demonstrable on the surface of the infected cells as shown by iodination of the intact cells (16) and by indirect immunofluorescence with monoclonal Ab HC-1 of fixed cells (unpublished data). In fact gC was the only glycoprotein transported to and inserted into the plasmamembrane in significant amounts in presence of TM. The gC exposed on the cell surface lacked reactivity in the antibodydependent cell-mediated c)~otoxicity test a finding which suggests that presence of N-glycosidic oligosaccharides were essential for the ADCC reaction (16) . On the other hand the presence of 0-1inked carbohydrates seems sufficient for transport of glycoprotcins to the plasmamembrane and different functions of the two classes of oligosaccharidcs on the ItSV glycoprotcin gC might thus be discerneable. 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Mapping of viral genes by analysis of polypeptides and functions specified by ttSV1 × t I S V 2 recombinants Coronavirus glyeoprotein E 1, a new type of viral glyeoprotein The effect of tunieamycin on the synthesis of Herpes simplex virus type 1 glyeoproteins and their expression of the cell surface Effects of glueosamine 2-deoxyglueose and tunicamycin on glyeosylation, sulfation and assembly of influenza viraI proteins Effect of tunicamycin on the morphogenesis of Semlild-Forest virus and I~ous Sarcoma virus O-glycosidic carbohydrate-peptide linkages of herpes simplex virus glycoproteins Unusual lectin binding properties of a herpes simplex virus type 1-specific glycoprotein Altered kinetic properties of sialyl and galactosyl transferases associated with herpes simplex virus infection of GMK and B t l K cells 12). : Serological analysis of herpes simplex virus types 1 and 2 with monoclonal antibodies. Inf. I m m u n Differential immunological reactivity and processing of glyeoproteins Biology and Chemistry of euearyotic cell surfaces. Miami ~ri:nter Symposia Supression of glyeoprotein formation of Semliki Forest, influenza, and Avian Sarcoma virus b~-tunicamyein Biogenesis of vaccinia: carbohydrate of the hemagglutinin molecule Membrane proteins specified by herpes simplex virus. I. Identification of four glycoprotein precursors and their products in type 1-infected cells We thank Dr. Richard Emmons for support of the work done at the California Department of Health Services and Dale Dondero for excellent technical assistance.The work done at University of GSteborg was sponsored by the Swedish Medical Research Council (grant no 4514), The National Swedish Board Development (project Kb 5112304-0/2601-3) and the Medical Faculty of G6teborg.The work done at the University of Copenhagen was sponsored by the Carlsberg foundation, the Novo foundation and King Christian the tenth's foundation. Received October 6, 1982