Recognition of compartmentalized intracellular analogs of glycoprotein H of human cytomegalovirus

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Vfrology

Arch Virol (1992) 126:67-80

© Springer-Verlag 1992 Printed in Austria

Recognition of compartmentalized intracellular analogs of glycoprotein H of human cytomegalovirus E. BognerI, M. Reschke1, B. Reis1, E. ReisI, W. Britt:, and K. Radsak1 1Institut ffir Virologie der Philipps-Universit/it,Marburg, Federal Republic of Germany 2Department of Pediatrics and Microbiology, School of Medicine, Birmingham,Alabama, U.S.A. Accepted February 13, 1992

Summary. Infected cell proteins immunoprecipitated from human cytomegalovirus (HCMV)-infected fibroblasts with glycoprotein H (gH)-.specific conformation-dependent monoclonal antibody (mab 14-4 b) were found to consist of three components of 86kDa, 89kDa, and 125kDa (gp86, 89, and 125). Affinity purified antibodies from human convalescent serum reactive with an NH2-terminal epitope of gH recognized three polypeptides of comparable size in immunoblots, suggesting antigenic relatedness of these three components of the gH-complex. Using subcellular fractions for immunoblotting, gp 86 was identified as an endoglycosidase H (endo H)-sensitive gH-form present in the nuclear fraction whereas gp 89 and gp 125 were endo H-resistant and present in the membrane fraction or in virions. Incomplete endo H-digestion suggested that four of six predicted N-glycosylation sites of the gH molecule were occupied by carbohydrate side chains. Analysis under nonreducing conditions revealed that the compartmentalized as well as virion-associated gH analogs form high molecular weight complexes. The relation of the recognized gH analogs to the processing pathway of gH is discussed. Introduction The more abundant glycoproteins of human cytomegalovirus (HCMV) have been shown to form at least three high molecular weight complexes (gc I-gc III; [11, 15]) which are present in the virion envelope and in the membranes of infected cells as well [2, 7]. Glycoprotein complex I (gcI) contains disulfidelinked components of 58 kDa and 116 kDa that are derived from one viral gene product, namely the precursor form of glycoprotein B [2]. In the case of gc II and gcIII, formation of the complexes apparently involves disulfide-linked products of more than one viral gene and/or possibly host proteins [11, 14].

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This conclusion was based on analysis of the H C M V glycoprotein complexes with various monoclonal antibodies which recognized different subsets of proteins in affinity purified gcII [14] or which coprecipitated an additional glycoprotein linked by disulfide-bonds to glycoprotein H (gH) in gcIII [11]. H C M V glycoproteins B and H (gB and gH), belong to the group of gene products that are conserved among herpesviruses [20]. By analogy to their homologs in the herpes simplex virus system, both, gB and gH, contain neutralizing epitopes and are thus thought to be essential for viral infectivity [19, 25]. In support of this view, gH has been recently shown to be involved in binding to host cell receptor(s) and viral entry [16, 17]. These functions are likely to be dependent on the conformation of the proteins involved. For an understanding of the structural requirements of the functional form of gH during primary virus-host cell interaction the processing of the gH gene product(s) was analyzed. Our data suggested that gH-specific immunoprecipitates contained glycopolypeptides of 86kDa, 89 kDa, and of 125 kDa (gp 86, 89, and 125) that were immunologically related but represented differently modified forms of gH in the nuclear and cytoplasmic compartments and of virion-associated gH, respectively. Materials and methods

Cells and virus All experiments were carried out with confluent human foreskin fibroblasts (HF) between the 6th and 15 th passage. The Towne and AD 169 strains of HCMV I-9, 32] were used for experimental infection at a multiplicity of infection (moi) of 3 to ensure a > 95% infection of the cells during the first virus replication cycle. Infectivity was quantitated by the endpoint dilution method combinedwith indirect immunofluorescence using commercial monoclonal antibody (Dupont, Bad Nauheim, Federal Republic of Germany) for the detection of early viral antigen 1-3, 24]. Radioactive labelling of infected cultures For radiolabelling, uninfected or infected cells were cultured in Eagle's minimum essential medium (MEM, Gibco, Eggenstein, Federal Republic of Germany) lacking methionine plus 2% fetal calf serum (FCS) and labelled with [35S]methionine (20-50 t~Ci/ml; specific activity > 1,000Ci/mmol; Amersham Buchler, Braunschweig, Federal Republic of Germany) at various intervals postinfection (p.i.; see Results). Purification of virus and dense bodies For isolation of virions, extracellular virus and dense bodies were sedimented from the culture medium (100,000 x g for 2 h at 4 °C) of infected cultures (6 x 10 7 cells) at 72-96 h postinfection. Purification and separation of virus from dense bodies was carried out by gradient centrifugation according to Talbot and Atmeida [28]. Cell fractionation and preparation of extracts Infected HF monolayers (1.2 x 10 7 cells) were harvested by scraping and collected by sedimentation (1,000 x g for 10rain) in cold phosphate buffered salt solution (PBS) con-

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taining 100 U trasylol/ml or 0.3 mM phenylmethylsulfonylfluoride(PMSF). Total extracts of cell pellets were obtained by lysis, sonication and sedimentation of insoluble material in buffer B (20 mM Tris HC1, pH 9, 0.3 M NaC1, 10% glycerol, 1 mM CaCI2,0.5 mM MgC12, 2mM EDTA, 0.5% NP-40, 0.5mM PMSF, 100U trasolyl/ml). Cell fractionation for preparation of nuclei free of cytoplasmic contaminants was carried out at 4 °C by the following method [23]: Pellet cells were swollen with occasional vortexing for 10rain in 30 volumes of hypotonic buffer A (5 mM sodium phosphate, pH 7.5, 2 mM MgC12, 0.5 mM CaCt2, 1 mM PMSF); sedimention at 3,000 x g was followed by resuspension in 10 volumes buffer A and disruption in a Dounce homogenizer using a S-pestle until > 90% of nuclei were liberated (10 strokes) as examined by phase contrast microscopy. The homogenate was supplemented with sucrose to a final concentration of 0.3 M. Following sedimentation at 800 x g for 10min nuclei resuspended in 10 volumes of buffer A plus 0.3M sucrose were purified from residual cytoplasmic contaminants by two successive cycles of sedimentation at 2,100 x g for 15min through a 3ml cushion of 1.62M sucrose in buffer A. Nuclear pellets were subsequently washed and sedimented at 1,000 x g for 15 rain in 20 volumes of buffer A containing 0.5 % Triton X-100. Purity of isolated nuclei was monitored by transmission electron microscopy (Fig. 1). Purified muclei were resuspended in 0.5 ml of buffer B and sonified (3 x 15 s at maximum setting in a sonication bath of a Branson sonifier). "Nuclear extracts" were obtained after sedimentation of insoluble material at 1,500 x g for 15 min. For preparation of"membrane extras" postnuclear supernatants were first freed from residual nuclear fragments at 10,000 x g for 10 min; membranes were then sedimented from the resulting supernatants at 100,000 x g for 60 rain at 4 °C followed by resuspension in 0.5 ml of buffer B, sonication as above and sedimentation of insoluble materiaI.

Fig. 1. Electron micrograph of nuclei isolated from HCMV-infected fibroblasts. Cultures infected with the AD 169 strain of HCMV (moi of 3) were harvested at 72h p.i. and subjected to cell fractionation prior to purification of nuclei as described in Materials and methods. Nuclear preparations were subsequently processed for transmission electron microscopy

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Transmission electron microscopy For ultrastructural examination, purified nuclei were fixed with paraformaldehyde according to Ito and Karnovsky [-13], contrasted sequentially with osmium tetroxide and uranyl acetate prior to dehydration. The nuclei were embedded in Epon followed by ultrathin sectioning.

Recombinant plasmid construction and prokaryotic expression of the gH-gene All procedures were performed by standard methods [ 10, 26]. The complete coding sequence for the gH gene as derived from HCMV strain AD 169 [22] was isolated after a Sma T/ Hind III digestion of cosmid pCM 1075 (generously donated by B. Fleckenstein, Erlangen, Federal Republic of Germany) [8] and snbcloned into the amplification vector pUC 18 (pUC-gH). For prokaryotic expression of defined segments of the gH gene as 13-galactosidase (13-gal) fusion proteins, five defined fragments of the pUC-gH construct were transferred into pEX 2 and 3, respectively (Fig. 3): Recombinant pEX 3-gH-1 contained a Bgl II/Bam HI fragment (1710 bp) of the gH gene, pEX 3-gH-2 a Nae I/Barn HI fragment (966 bp), pEX 3gH-3 a Pvu I/Nae I fragment (411 bp), pEX 3-gH-4 the Xho II/Hind III fragment (648 bp), and pEX2-gH-5 a 389 bp Hae TIT fragment. The correct orientation of the inserts was examined by restriction analysis. The constructs were transformed into E. coli pop 2136 for the production of 13-gal-fusion protein (fuspro 1-5) which were purified from induced cultures according to Harlow and Lane [12].

Affinity purification of gH-specific fractions from human convalescent sera Approximately 10mg of t3-gal-fusion protein 5 were purified from inclusion bodies of bacterial recombinants by SDS-PAGE and subsequent electro-elution [12] and bound to a mixture of affigel t0 and 15 according to the instructions of the mamffacturer (Biorad Lab., Richmond, CA, U.S.A). Subsequent affinity chromatography was used to prepare gH-specific fractions (pab gH) from human convalescent serum [12]. Bound antibodies were eluted with 0.2M glycine-HC1, pH2.5, neutralized with 2.5M Tris-HC1, pH8, and dialyzed against PBS prior to use in immunoreactions.

Immunoprecipitation and immunoblotting For immunoprecipitation [23] aliquots of nuclear and membrane extracts, respectively, of comparable protein content were precleared by incubation with protein A-sepharose CL4B beads (Sigma, Deisenhofen, Federal Republic of Germany) prior to incubation overnight at 4 °C with mab 14-4 b (raised and characterized by W. Britt) and adsorption of immunocomplexes for 2 h at room temperature to protein A sepharose CL4B beads which had been subjected to coating with rabbit anti-human IgG (Dakopatts, Hamburg, Federal Republic of Germany) prior to use. Following five washing cycles of the beads with PBS plus 0.1% NP-40 the precipitates were subjected to SDS-polyacrylamide gel etectrophoresis (PAGE), and consecutive fixation and fluorography [23] of the dried slab gels. For immunoblotting [23] cellular or viral extracts containing comparable amounts of protein (approx. 20-70 ~tg per sample and slot), or aliquots of 13-gal-fusion proteins were separated by SDS-PAGE, electrotransferred to nitrocellulose sheets (BA 85; Schleicher & Schtill, Dassel, Federal Republic of Germany) and subjected to indirect immunostaining using pab gH as the first antibody and horseradish peroxidase-conjugated rabbit antihuman as the second antibody with either 4-chloronaphthol or N-3, 3'-diaminobenzidine as chromogens.

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Glycosidase digestion Digestion with endoglycosidaseH or N-glycosidaseF was performed on cellular, nuclear, membrane, or viral extracts according to the instructions of the manufacturer (Boehringer, Mannheim, Federal Republic of Germany) [18].

Determination of protein content Protein content in cellular extracts was estimated by the method of Lowry etal. [211. Chemicals for SDS-PAGE were purchased from Merck, Darmstadt, Federal Republic of Germany, and restriction endonucleases from Boehringer, Mannheim, Federal Republic of Germany. Results

Kinetics of gc III formation In order to examine the time course of gc III formation, parallel cultures of HCMV (strain Towne)-infected HF were pulse labelled with [35S]methionine for 60 rain at 72 h p.i. prior to chase periods with excess unlabelled methionine. Cellular extracts prepared from pulse labelled and chased cultures were immunoprecipitated with the conformation-dependent gH-specific monoclonal antibody (mab) 14-4 b and the precipitates analyzed by SDS-PAGE and fluorography (Fig. 2). Separation of the precipitated polypeptides under reducing conditions revealed an 86 kDa protein (gp 86) in all samples; a 125 kDa protein (gp t25) was detected following 60 min of the chase period. After the 6 h chase period, the initially discrete band migrating at 86 kDa became more diffuse and upon reduced exposures of the fluorogram, a third species migrating with an estimated molecular weight of 89 kDa was noted (Fig. 2 B). In nonreducing gels, low molecular weight polypeptides of 86 kDa and 125 kDa were present already after 30 min of chase; high molecular weight gc III was readily observed only in samples later than 60 min of chase. Appearance of gp 89 was again delayed until 6 h of chase (Fig. 2 A). It is evident in Fig. 2 that the efficiency of immunoprecipitation increased during the course of the chase. This phenomenon could be due to conformation-dependent binding of mab 14-4b which may increase with progressive maturation of gH.

Purification of a human antibody monospecific for gH To study further the nature of the various components of the gc III complex, affinity purified antibodies recognizing a linear gH epitope were prepared from human convalescent sera. For this purpose defined restriction fragments of the gH gene derived from the HCMV AD 169 strain, were cloned into the prokaryotic expression vector pEX (Fig. 3) as described in Materials and methods and the ~-gal-fusion proteins obtained were characterized by immunoblotting with human convalescent sera. Of the five gH-specific [3-gal-fusion proteins, only one (fuspro 5) containing 129 NH2-terminal amino acid residues of gH, showed a pronounced reaction with eight out of twenty sera tested (data not

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Fig. 2. Immunoprecipitation with mab 14-4b from cell extracts of uninfected (Co) and of HCMV (Towne)-infected HF (P and Chase) following labelling with [35S]methionine (50 gCi/ml) under pulse-chase conditions. Extracts were prepared 72-96 h p.i. after a 1h pulse (Co, b and P, 60') or after pulse and subsequent chase with excess unlabelled methionine for various time intervals (chase, 30', 60', 3 h, 6 h). Immunoprecipitates of the extracts were separated by SDS-PAGE (6% polyacrylamide) under nonreducing (A) or reducing (B) conditions and analyzed by fluorography. The arrows on the right indicate the positions of gp 125 and gH. Molecular weight markers (M) on the left were a-2macroglobulin (340 kDa) and phosphorylase b (97 kDa)

shown; a comparable observation has been made by M. Mach, manuscript in press). Fuspro 5 carrying this H C M V gH epitope, was used to prepare an affinity-purified fraction from one of the sera (pab gH). Use of pab gH in immunoblot analysis of the chimeric proteins verified its specificity (Fig. 4). Furthermore, when pab gH was used for immunobtot analysis of mab 14-4bimmunoprecipitated infected cell proteins, it reacted only with the previously identified gH-specific polypeptides. Interestingly, pab gH (which was obtained by use of an AD 169 derived ]3-gal-fusion protein) did not recognize the corresponding gH epitope in gH-specific precipitates of Towne-infected extracts (data not shown). H C M V strains A D 169 and Towne have been shown to exhibit obvious sequence deviations directly downstream of the potential signal peptide sequence [5, 22]. The NH2-terminal linear epitope for pab gH should

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5GG-bp-NaeI/BanHI

IiG48-bp-PvuII/Hael_ml i ~58-bp-HaeIII

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Fig. 3. Subcloning of fragments of the gH gene for prokaryotic expression of ~-galactosidase fusion proteins. The relative positions of the five fragments (I-V) used for subcloning into pEX are projected onto the linearized coding sequence of gH. The size of the fragments (bp) as well as the relevant restriction enzymes used in this procedure are indicated therefore be localized between amino acid (aa) 19 and the sequon (aa 56-58) for the first potential N-glycosylation site.

Recognition of compartment-specificforms of gH In order to examine whether gp 86, gp 89, and gp 125 represented differentially modified gH forms in different cellular compartments, AD 169-infected cultures (1.2 x 107 cells) were subjected to cell fractionation at 72 h p.i. to obtain nuclear and membrane preparations (see Materials and methods). Electrophoretic separation under reducing conditions of extracts of nuclei, membranes and from extracellular virions was followed by immunoblotting with pab gH (Fig. 5). In infected cell homogenates two prominent bands of 86 kDa and 89 kDa were stained under these conditions; bands of higher molecular weight were usually less evident (Fig. 5). In membrane fractions, prominent staining between 89100 kDa was obtained (Fig. 5). On the other hand, in nuclear fractions a polypeptide of clearly faster migration, i.e., 86 kDa, was recognized (Fig. 5). Virion extracts yielded, in addition to strong reactions for possibly two polypeptides between 89 and 100 kDa, weaker staining also for polypeptides of 110-125 kDa (Fig. 5).

Glycosidase sensitivityof gH analogs To establish that differential modification was an explanation for the polymorphism of the gH forms, homogenates and nuclear as well as virion extracts

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Fig. 4. Analysis of gH-specific [3-gal-fusion proteins. Bacterial cultures transformed with recombinant constructs pEX-gH-1 to -gH-5 (fuspro 1-5) or with vector pEX 3 ([3-gal)were induced by temperature elevation for 90 min. Aliquots of the cultures (approx. 50 gg of protein each) were separated by SDS-PAGE (7% polyacrylamide) in parallel gels; one gel was subjected to coommassie blue staining (A), the other to immunoblotting with pab gH (B). Molecular weight markers on the left were: a-2-macroglobulin (170 kDa), phosphorylase b (97 kDa), bovine serum albumin (68 kDa), glutamate dehydrogenase (55 kDa). The asterisk marks fuspro 5, the arrowhead on the right indicates the position of the Coommassie stained (A) product and that recognized by pab gH

were treated with endoglycosidase H (endo H) or with N-glycosidase F (glyco F) prior to immunoblotting with pab gH (Fig. 6). Endo H-sensitivity was observed for the nuclear gH form; the molecular weight of digested gH was reduced by approximately 8 kDa (Fig. 6 B). In homogenates the gp 86 was equally endo H-sensitive (Fig. 6 A) whereas gp 89 representing membrane gH was endo Hresistant as were the virion-associated gH forms (Fig. 6 A and C). All polypeptides recognized by pab gH, except for the high molecular weight virion-associated polypeptides, exhibited obvious sensitivity to glyco F. Glyco F-treated gH consistently exhibited a retarded migration (by approx. 2 kDa) compared to that digested with endo H (Fig. 6 A-C) suggesting that one of the four Nglycosylation sites (see below) was possibly not accessible to the action of glyco F. To determine the number of N-linked oligosaccharide side chains, mannoserich nuclear gH was subjected to partial digestion with endo H E18] prior to immunoblotting with pab gH. This treatment resulted in three intermediate products in addition to the completely deglycosylated form (Fig. 7) suggesting

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Fig. 5. Identification of compartmentalized gH analogs. AD 169-infected cultures (1.2 x 107 cells) were harvested at 72 h p.i. and subjected to cell fractionation as described in Materials and methods to obtain membrane and nuclear fractions. Total cellular extracts (Horn), those from membranes (Mere) and nuclei (Nuc) as well as from extracellular virions (V0 were analyzed by immunoblotting with pab gH after SDS-PAGE (6% polyacrylamide) under reducing conditions. Arrowheads on the right indicate the relative migration of the membrane (gH memb) and nuclear (gH nuc) forms of gH, respectively. The molecular weight marker (M) used was phosphorylase B (97 kDa)

Fig. 6. Glycosidase sensitivity of gH analogs. Total cellular extracts (A), those from isolated nuclei (B) as well as virion extracts (C) were subjected to digestion with endo H (b) or glyco F (c) or were incubated without enzyme (a) prior to SDS-PAGE (6% polyacrylamide) and immunoblotting with pab gH. The large arrowheads on the right indicate the positions of endo H-resistant virion and membrane analogs of gH, the small arrowhead that of glycosidase-sensitive gH forms. The molecular weight markers (M) used were phosphorylase B (97 kDa) and bovine serum albumin (68 kDa)

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Fig. 7. Partial endo H digestion of the nuclear gH analog. Extracts of nuclei isolated from infected cultures at 72 h p.i. were incubated for 30 min with endo H (b) or without enzyme (a) as described in Materials and methods followed by SDS-PAGE (6% polyacrylamide) under reducingconditions and immunoblottingwith pab gH. The positions of intermediate products with no (0), 1, 2, 3, or 4 carbohydrate side chains resulting from incomplete digestion are indicated on the right. The molecular weightmarkers (M) used were the same as those given in the legend of Fig. 6

that four of the six potential N-glycosylation sites of the nuclear gH analog were occupied. This was consistent with the reduction by about 8 kDa following complete endo H digestion as shown in Fig. 6. Immunoblotting with pab gH after separation of the cell fractions under nonreducing conditions suggested that the various gH forms existed as disulfidelinked complexes in the nuclear and membrane compartments as well as in virions (Fig. 8). In addition, a 150 kDa nonglycosylated polypeptide was observed (Fig. 8). The origin of this polypeptide is unknown. Discussion Maturation of viral membrane glycoproteins is known to occur via processing intermediates as precursors of the biologically active form [6]. This complex process requires vectorial intracellular transport which is intimately related to sequential modifying events in different cellular compartments, e.g., translocation of the peptide backbone and co-translational N-glycosylation in the rough endoplasmic reticulum (RER) followed by passage through the Golgi complex where trimming is succeeded by additional post-translational modification events. Distinction of precursor intermediates from the fully processed mature form is a prerequisite for the understanding of the biological function of the final product at the molecular level. The experiments described in this report suggested that the early steps of biosynthesis of HCMV gH include a mannose-rich 86 kDa intermediate which is eventually converted into later endo H-resistant forms of higher molecular weight. Gp 86 was found to be confined to the nuclear compartment. This was not surprising considering that the outer nuclear membrane constitutes part of

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Fig. 8. Complexation of compartmentalized gH analogs. Total infected cell extracts (Hom), extracts from isolated membranes (Mem) and nuclei (Nuc) prepared at 72 h p.i. as well as extracts from extracellular virions (Vi) were separated by SDS-PAGE (6% polyacrylamide) under nonreducing conditions and subjected to immunoblotting with pab gH. The arrow on the right indicates the position of high molecular weight glycoprotein complex III (gc 111"). The molecular weight markers (M) used were a-2-macroglobulin (340 kDa) and phosphorylase B (97 kDa) the RER. However, this gH form was comparatively stable persisting beyond 6 h of chase, and, in addition, nuclear association of gp 86 was resistant to treatment of isolated nuclei with Triton X-100. These observations suggested the possibility that HCMV-gH was localized in the inner nuclear membrane of infected fibroblasts where it may participate in transport of virions through the nuclear envelope. Such a function has been previously suggested for HCMVgB [23] and gB homologs of other herpesviruses [1, 30]. It would be of interest in this context to define the gH form of intracetlular virions isolated from the nuclear cisterna. Brefeldin A which traps herpesviruses in this particular location and prevents maturational envelopment in the trans-Golgi-network (TGN) [4, 31] should be an experimental tool to approach this problem. Partial endo H digestion of gp 86 produced three products of intermediate molecular weight in addition to the completely deglycosylated form of 78 kDa suggesting that four of six potential N-glycosylation sites were occupied by mannose-rich carbohydrate side chains. The calculated molecular weight for the polypeptide backbone after cleavage of the potential signal peptide amounts to approximately 80 kDa [22] which is in close agreement with the observed molecular weight of the completely deglycosylated product. The endo H-resistant 89 kDa intracellular form of gH was observed exclusively in membrane preparations from infected cells and in virions. This is consistent with the notion that this form underwent modification by trimming

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and subsequent addition of terminal sugar residues during passage through the Golgi complex and thus was modified by complex oligosaccharide side chains. There was evidence in several immunoblotting experiments for the existance of additional membrane-associated endo H-resistant gH analogs of higher molecular weight migrating between 90-100 kDa. In extracts of extracellular virions this latter form was consistently as prominent as gp 89. Both gH analogs were equally glyco F-sensitive suggesting that late addition of O-linked carbohydrates was not an explanation for the higher molecular weight of these forms. The presence of polypeptides of about 125 kDa in our gH-specific immunoprecipitations as well as immunoblots remained intriguing. Gretch et al. reported co-precipitation of a 145 kDa glycopeptide with gH using monoclonal antibody 1G6 [11]. Our experiments are essentially in agreement with his observation: The conformation-dependent monoclonal used here, mab 14-4 b, also co-precipitated a second polypeptide of somewhat faster migration, i.e., of 125kDa, after labelling with radioactive glucosamine/galactose (unpubl. obs.); furthermore, pab gH stained a polypeptide of similar molecular weight in immunoblots of membrane and virion extracts. It is tempting to speculate that this polypeptide represented a highly modified glyco F-resistant O-glycosylated form of gH. O-glycanase treatment of the preparations, however, was not consistent with this view (unpubl. obs.). In addition, the notion that gp t25 was a fully processed form of gH was not supported by the kinetics of its appearance in our pulse-chase experiments where gp 125 was observed after shorter chase intervals than gp 89, the membrane form ofgH. Final clarification as to whether gp 125 is a gH derivative or an unrelated complex ligand must await production of gp 125-specific antibodies and identification of the gene encoding this protein. On the basis of the data presented, processing of HCMV-gH to the presumably functional virion-associated form involves apparently the following steps: (/) Biosynthesis of gp 86 by co-translational N-glycosylation of the nascent polypeptide backbone at four sites with mannose-rich carbohydrates and subsequent complexation with gp 125 in the RER. These events take place within 30-60 min of biosynthesis of gH. A portion of this complex may be translocated to the inner nuclear membrane possibly by lateral diffusion [31] and retained in the nuclear/RER compartment. (i/) Transport and posttranslational modification of the carbohydrate side chains during passage through the Golgi complex which leads to endo H-resistant gp 89. This process appears to be relatively slow, requiring at least three hours. (iii) Higher molecular weight forms of gH (90-100 kDa) may subsequently be produced by further but undefined modifications prior to virion-association of gH during maturational envelopment of viral nucleocapsids in the trans-Golgi-network [27].

Acknowledgements This investigation was supported by the Deutsche Forschungsgemeinschaft(KI-ll/1 and SFB 286), and by the National Institutes of Health, NIAID (A130105) and the March of

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Dimes Birth Defects Foundation (6-490). The authors are indebted to Prof. Dr. H. D. Klenk, Marburg, for constructive criticism during the course of the study, to Ms. B. Agricola for the electron micrographs, and to Mr. B. Becket for assistance with the photographical reproduction.

References 1. Ali M, Butcher M, Gosh H (1987) Expression and nuclear localization of biologically active fusion glycoprotein gB of herpes simplex virus in mammalian cells using cloned DNA. Proc Natl Acad Sci USA 84:5675-5679 2. Britt W (1984) Neutralizing antibodies detect a disulfide-linked glycoprotein complex within the envelope of human cytomegalovirus. Virology 135:369-378 3. Brficher KH, Garten W, Klenk HD, Shaw E, Radsak K (1990) Inhibition of endoproteolytic cleavage of cytomegalovirus (HCMV) glycoprotein B by palmitoyl-peptidylchloromethyl ketone. Virology 178:617-620 4. Cheung P, Banfietd BW, Tufaro F (1991) Brefeldin A arrests the maturation and egress of herpes simplex virus particles during infection. J Virol 65:1893-1904 5. Cranage M, Smith G, Bell S, Hart H, Brown C, Bankier A, Tomlinson P, Barrell B, Minson T (1988) Identification and expression of a human cytomegalovirus glycoprorein with homology to the Epstein-Barr virus BXLF 2 product, varizella-zoster virus gp III and herpes simplex virus type glycoprotein H. J Virol 62:1416-1422 6. Elbein A (1987) Inhibitors of the biosynthesis and processing of N-linked oligosaccaride chains. Ann Rev Biochem 56:497-534 7. Farrar O, Oram J (1984) Characterization of human cytomegalovirus envelope glycoproteins. J Gen Virol 65:1991-2001 8. Fleckenstein B, Mfiller I, Collins J (1982) Cloning of the complete human cytomegalovirus genome in cosmids. Gene 18:39-46 9. Furukawa T, Fioretti A, Plotkin S (1973) Growth characteristics of cytomegalovirus in human fibroblasts with the demonstration of protein synthesis early in viral replecation. J Virol 11:99t-997 10. Glover D (1986) DNA cloning. A practical approach. IRL Press Oxford 11. Gretch D, Kari B, Rasmussen L, Gehrz R, Stinski M (1988) Identification and characterization of three distinct families of glycoprotein complexes in the envelopes of human cytomegalovirus. J Virol 62:875-881 12. Harlow E, Lane D (1988) Antibodies. A laboratory manual. Cold Spring Harbor Laboratory, New York 13. Ito S, Karnovsky M (1968) Formaldehyde-glutaratdehyde fixation containing trinitro compounds. J Cell Biol 39:168 a-169 a 14. Kari B, Goertz R, Gehrz R (1990) Characterization of cytomegalovirus glycoproteins in a family of complexes designated gc II with murine monoclonal antibodies. Arch Virol 112:55-65 15. Karl B, Lussenhop N, Goertz R, Wabuke-Bunoti M, Radeke R, Gehrz R (1986) Characterization of monoclonal antibodies reactive to several biochemically distinct human cytomegalovirus glycoprotein complexes. J Virol 60:345-352 16. Keay S, Baldwin B (1991) Anti-idiotype antibodies that mimic gp 86 of human cytomegalovirus inhibit viral fusion but not attachment. J Virol 65:5124-5128 17. Keay S, Merigan T, Rasmussen L (1989) Identification of cell surface receptors for the 86-kilodalton glycoprotein of human cytomegalovirus. Proc Natl Acad Sci USA 86:10100-10103 18. Klenk HD, Schwarz R (1987) Variations in glycosylation of the influenza virus hemagglutinin of subtype H 7. Arch Virol 97:359-363

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