The purposes of this study were to determine whether heparin would block human cytomegalovirus (HCMV) infection of skin fibroblast (SF) cells and to identify HCMV envelope glycoproteins which might have affinity for heparin. It was determined that soluble heparin in concentrations of 5 to 20 jg/ml was capable of blocking HCMV infection of SF cells. However, after virus had adsorbed to the SF cells, heparin lost its ability to block infection. It was also determined that treatment of SF cells with heparinase to remove cell surface heparinlike moieties prevented HCMV infection of SF cells. These data showed that HCMV, like other herpesviruses,

SUMMARYThree distinct families of glycoprotein complexes present in the envelopes of human cytomegalovirus and designated gcI, gcII and gcIII have been described recently. The synthesis of the gcI family was analysed using either inhibitors of glycoprotein processing and transport or endoglycosidase treatments of purified glycoproteins. The initial step in gcI synthesis involved the glycosylation of a 95K protein (p95) to form a high-mannose, simple N-linked glycoprotein of Mr 158K (gp158), which was detected only in the presence of the glycoprotein processing inhibitor castanospermine. This intermediate was rapidly trimmed in the virus-infected cell to form a more stable simple N-linked precursor glycoprotein of Mr 138K (gp 138). Treatment of either gp 158 or gp138 with endoglycosidase H produced p95. Both molecules, gp158 and gp138, were found in disulphide-linked complexes which are presumably infected cell precursors to gcI since they were not found in virions. The processing of these complexes involved complete cleavage of gp 138 and conversion of some but not all of its oligosaccharide to complex N-linked chains. Both processing events were inhibited by the ionophore monensin. Mature gcI contained the gp 138 cleavage product, gp55, in a disulphide-linked complex with a heterogeneous glycoprotein designated gp93-130. The latter glycoprotein could be separated into two electrophoretic forms, gp93 and gpl30. The deglycosylated form of gp55 had a discrete banding pattern with an apparent Mr of 46K (p46). In contrast, the deglycosylated forms of gp93 and gp 130 had diffuse banding patterns with apparent Mr values of 46K to 56K (p46-56) and 60K to 70K (p60-70) respectively. Peptide profiles comparing gp93 with gp 130 indicated that they have highly similar polypeptide backbones. Since the deglycosylated forms of gp55 and gpl30, 46K and 60K to 70K, respectively, together exceed the 95K precursor/deglycosylated intermediate in Mr, we propose that the above glycoproteins are derived by an alternative proteolytic cleavage of the precursor. The heterogeneous electrophoretic properties of the deglycosylated forms of gp93 and gp 130 may be due to additional post-translational modifications other than glycosylation.

Several disulfide-linked glycoprotein complexes were identified in the envelope of human cytomegalovirus (HCMV). These glycoprotein complexes were fractionated by rate-zonal centrifugation in sucrose density gradients in the presence of detergents. Fractionated glycoproteins and complexes were immunoprecipitated with three different monoclonal antibodies specific for HCMV glycoproteins and a rabbit polyclonal antiserum prepared against detergent-extracted virion and dense-body envelope glycoproteins. Three distinct families of disulfide-linked glycoprotein complexes were observed and designated glycoprotein complex gcl, gcII, and gcII1. The gcl family, recognized by monoclonal antibody 41C2 under nonreducing conditions, consisted of three complexes with approximate molecular masses of 250 to 300, 190, and 160 kilodaltons (kDa). These complexes consistently sediment more rapidly than other HCMV glycoproteins or complexes in sucrose density gradients. Upon reduction of the gcl family, two size classes of glycoproteins with average molecular masses of 93 to 130 and 55 kDa were observed. The gcII family was recognized by monoclonal antibody 9E10. Under nonreducing conditions, as many as six electrophoretic forms were observed for gcII. When reduced, the major component of the gcII family was a heterogeneous glycoprotein designated gp47-52. The gcIII family was recognized by monoclonal antibody 1G6. It consisted of a complex of approximately 240 kDa without reduction of disulfide bonds. When reduced, two glycoprotein size classes with average molecular masses of 145 and 86 kDa were observed. Polyclonal antiserum R-7 reacted strongly with the gcI and gcIII families, but weakly with the gcII family.

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Three monoclonal antibodies were characterized by examining their reactivity to human cytomegalovirus (HCMV) glycoproteins under reducing and nonreducing conditions and their reactivity to glycoproteins and disulfide-linked glycoprotein complexes isolated by ion-exchange high-performance liquid chromatography. One monoclonal antibody, 9E10, reacted with glycoprotein complexes which had molecular weights of 93,000 and 450,000 and eluted from the ion-exchange column at 0.3 and 0.9 M NaCl, respectively. All glycoproteins associated in these complexes could be immunoprecipitated under reducing conditions by 9E10, suggesting that they were related to one another. The most abundant glycoproteins immunoprecipitated by 9E10 had molecular weights of 50,000 to 52,000. In contrast to this antibody, two other monoclonal antibodies, 9B7 and 41C2, reacted with glycoprotein complexes which had molecular weights of 130,000 and >200,000 and eluted from the ion-exchange column at 0.6 M NaCl. All glycoproteins associated in these complexes could be immunoprecipitated by 9B7 or 41C2 under reducing conditions, suggesting that they were also related to one another. The most abundant glycoprotein immunoprecipitated by 41C2 or 9B7 had a molecular weight of 93,000. In addition, it was also determined that a 93,000-molecular-weight glycoprotein which was not associated with other glycoproteins by disulfide bonds could not be precipitated by any of the three antibodies, suggesting that it was different from the other glycoproteins. The monoclonal antibodies were also examined for specificity and neutralizing activity. Monoclonal antibodies 41C2 and 9B7 were specific to HCMV as determined by immunofluorescent staining of skin fibroblast cells infected with several different viruses. However, 41C2 did not neutralize Towne strain HCMV, while 9B7 did. The neutralizing activity of 9B7 did require compliment. These results suggested that 41C2 and 9B7 reacted with different antigenic sites on the same glycoproteins. Unlike 41C2 and 9B7, monoclonal antibody 9E10 was found to cross-react with adenovirus and herpes simplex virus as determined by immunofluorescent staining of infected skin fibroblast cells. Furthermore, 9E10 neutralized the Towne and Toledo strains of HCMV in the absence of complement.

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The structure and heparin binding properties of a family of human cytomegalovirus (HCMV) disulphide-linked glycoprotein complexes designated gC-II were analysed. gC-II complexes contain two groups of glycoproteins designated Group 1 and Group 2. These glycoproteins were separated from each other by short exposure of virions to a reducing agent. This showed that the disulphide bonds between these glycoproteins were on the external surface of the virion. Although these glycoproteins were no longer associated they were not released from the virion, suggesting that they were transmembrane glycoproteins, Approximately 75 to 90 % of the gC-II complexes and 18 % of the complexes containing the HCMV gB glycoprotein obtained from the virion envelope bound immobilized heparin. When virions were incubated with [~H]heparin, gC-II complexes bound more heparin than gB complexes, by approximately threefold. These data showed that gC-II complexes had a greater heparin-binding capacity. After treatment of virions with a reducing agent the affinity of gC-II glycoproteins for heparin was greatly reduced whereas the affinity ofgB glycoproteins was only slightly reduced. Thus, higher order structure was important for heparin binding by gC-II complexes but not by those of gB. Relative to gC-II Group 1 glycoproteins, a greater portion of gC-II Group 2 glycoproteins still bound to heparin after reduction, suggesting that Group 2 glycoproteins may be the important heparin binding component of the gC-II complexes. Both gB and gC-II complexes were eluted from immobilized heparin with soluble heparin or 0.65 M-NaC1 suggesting that both formed ionic bonds with heparin. Chondroitin sulphate was not effective at eluting HCMV envelope glycoproteins from immobilized heparin. Thus, the structure of the glucosaminoglycan backbone is important to the binding of HCMV glycoproteins to heparin.

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