Bruce E. Kari

Structure, composition and heparin binding properties of a human cytomegalovirus glycoprotein complex designated gC-II

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 [3H]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 of gB 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-NaCl 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.

Epitope analysis of human cytomegalovirus glycoprotein complexes using murine monoclonal antibodies

A panel of 10 monoclonal antibodies reactive with human cytomegalovirus (HCMV) glycoproteins was generated. These antibodies immunoprecipitated disulfide-linked complexes which contained glycoproteins with molecular weights of 130,000, 93,000, and 52,000. These complexes were designated gC-I. Epitope analysis of gC-I was done with a simultaneous two antibody binding assay. A network of epitopes was revealed which clustered in three major domains designated I, II, and III. Antibodies within individual domains I and II showed strong mutual inhibition of each others binding. However, there were multiple antibody interactions between domains I and II. For example, the binding of most antibodies in domain I was augmented to some extent by antibodies from domain II. However, the binding of only one antibody from domain II was augmented by all antibodies from domain I. The augmentation in binding between two antibodies was dependent on the native structure of gC-I and was sensitive to conformational changes due to nonionic detergent extraction of gC-I and/or disruption of disulfide bonds. A synergistic effect was also observed between antibodies in domains I and II in a virus neutralization assay. A neutralizing antibody had a much greater neutralizing activity in the presence of a nonneutralizing antibody, which also enhanced the binding of the neutralizing antibody in the simultaneous two antibody binding assay. Also, two antibodies which were nonneutralizing individually were neutralizing when used in combination. Such antibodies also augmented each others binding in the simultaneous two antibody binding assay. Finally, domain III consisted of a nonneutralizing antibody that inhibited the binding of all antibodies in domains I and II. This antibody also inhibited the neutralizing activity of a neutralizing antibody in a virus neutralizing assay.

STRUCTURE AND COMPOSITION OF A FAMILY OF HUMAN CYTOMEGALOVIRUS GLYCOPROTEIN COMPLEXES DESIGNATED GC-I (GB)

Murine monoclonal antibodies (MAbs) were made to the 52,000 (gp52) and the 93,000 to 130,000 Mr (gp93-130) glycoproteins from a human cytomegalovirus (HCMV) glycoprotein complex designated gC-I or the gB homologue. MAbs recognizing either gp52 or gp93-130 could immunoprecipitate unreduced gC-I complexes from non-ionic detergent extracts of HCMV. Western blotting was performed with immunoaffinity-purified gC-I complexes which were reduced prior to analysis. MAbs made against gp52 recognized gp52 and a 158,000 Mr glycoprotein (gp158). MAbs which recognized gp93-130 in a Western blot also reacted with gp158, which is a gC-I precursor glycoprotein. The origin of gp93-130 was demonstrated by the reactivity of our gp93-130 MAbs with a recombinant protein containing the N-terminal portion of the gB gene. These data are consistent with the hypothesis that gp52 and gp93-130 are generated from the same high Mr precursor by proteolysis. MAbs recognizing either gp52 or gp93-130 neutralized Towne strain HCMV, but MAbs recognizing gp52 required complement to neutralize whereas MAbs recognizing gp93-130 did not. It was also determined that gp93-130 and gp 158 have detectable amounts of O-linked glycans but gp52 does not, showing a difference in the glycosylation of these glycoproteins. Analysis of gC-I disulphide bonds showed that two types were present, one which was very susceptible to reduction and a second which was less susceptible. These complexes could consist of very susceptible inter-complex disulphide bonds and less susceptible intra-complex disulphide bonds.

The human cytomegalovirus UL100 gene encodes the gC-II glycoproteins recognized by group 2 monoclonal antibodies.

In human cytomegalovirus (HCMV) the envelope glycoprotein complexes designated gC-II contain two immunologically and biochemically distinct glycoproteins. Monoclonal antibodies (MAbs) recognizing the gC-II glycoproteins have been divided into two groups based on the M(r) of the glycoproteins they recognize. We have now identified the HCMV UL100 gene as the gene encoding the gC-II glycoprotein recognized by the Group 2 MAbs. To do this, gC-II complexes were immunoaffinity purified and cleaved with cyanogen bromide (CNBr). CNBr peptides were separated by reverse phase high performance liquid chromatography (RPHPLC). Amino acid sequences which matched sequences found in the protein encoded by the HCMV UL100 gene were obtained from three purified peptides. To confirm the assignment we made synthetic peptides using amino acid sequence from the carboxyl terminus of the protein encoded by the UL100 gene. These peptides were used to make murine antibodies. The anti-UL100 antibodies immunoprecipitated gC-II complexes and were reactive with gC-II glycoproteins recognized by Group 2 MAbs in Western blotting. Several overlapping UL100 fusion proteins were expressed in E. coli. Only one of these fusion proteins was recognized by gC-II Group 2 MAbs. None of these UL100 fusion proteins were recognized by gC-II Group 1 MAbs. These data showed that the UL100 gene encoded the gC-II glycoprotein recognized by the Group 2 MAbs and that the epitope recognized by these antibodies was located between amino acids 315 to 372 at the carboxyl terminus.

Processing of human cytomegalovirus envelope glycoproteins in and egress of cytomegalovirus from human astrocytoma cells

The synthesis of human cytomegalovirus (HCMV) envelope glycoproteins and the production of infectious HCMV in human astrocytoma and skin fibroblast (SF) cells were analysed. HCMV envelope glycoproteins synthesized in astrocytoma cells had lower Mrs than the same glycoproteins synthesized in SF cells regardless of the strain of HCMV used, showing that the differences observed were due to differences in processing by the host cell and not the strain of HCMV used. HCMV envelope glycoproteins synthesized in astrocytoma cells were found to contain less galactosamine. Moreover, when synthesized in SF cells some HCMV glycoproteins contained a protease-resistant fragment owing to the presence of a cluster of O-linked oligosaccharides on the polypeptide. This fragment was not present when these HCMV glycoproteins were synthesized in astrocytoma cells. These data suggested that HCMV glycoproteins synthesized in astrocytoma cells contain fewer O-linked oligosaccharides. In contrast, other post-translational events such as proteolytic cleavage of the HCMV gB glycoprotein and the formation of disulphide-linked complexes did occur. The virus produced in astrocytoma cells was capable of infecting SF cells, suggesting that complete O-glycosylation is not needed to produce infectious HCMV. However, astrocytoma cells were slow to release virus into the culture medium, suggesting that a fully functional Golgi network is needed for efficient egress of HCMV from the host cell.

Biochemical and immunological analysis of discontinuous epitopes in the family of human cytomegalovirus glycoprotein complexes designated gC-I.

The envelope of human cytomegalovirus contains a family of disulphide-linked glycoprotein complexes designated gC-I which contain two glycoproteins of 52,000 Mr (gp52) and 93,000 to 130,000 Mr (gp93-130). Epitopes recognized by several of our gC-I gp52-specific monoclonal antibodies (MAbs) were previously assigned to three domains based on reactivity with gC-I in a competitive binding assay. In this report, we have used additional gC-I MAbs to characterize three distinct discontinuous epitopes in the gC-I complexes. Two of these epitopes were in Domain I and one in Domain III. These epitopes were resistant to proteolysis, heat denaturation and SDS treatment. However, the discontinuous epitopes were lost after reduction of disulphide bonds. After digestion of gC-I complexes with chymotrypsin, two fragments of 43,000 (43K) Mr and 34,000 (34K) Mr were obtained which contained all discontinuous and continuous epitopes recognized by our gp52 MAbs. The Mr of these fragments could not be reduced further by longer digestion or by use of other proteases such as trypsin or pronase. The 43K fragment contained N-linked oligosaccharides not detected in the 34K fragment. These oligosaccharides may have prevented a complete proteolytic digestion so that the 34K fragment was not always obtained. It was established that 80 to 90% of the mass of these fragments was contributed by gp52. Thus the discontinuous epitopes were composed primarily of gp52 and not gp93-130.

A combination of human cytomegalovirus (HCMV)-specific murine monoclonal antibodies exhibits synergistic antiviral activity in vitro.

A combination of HCMV-specific monoclonal antibodies (MAbs) reactive with glycoproteins in gcI complexes which exhibit synergistic antiviral activity in vitro is described. MAbs directed against different structural and biological properties of HCMV have been selected to increase the antiviral activity against all possible strains, and to reduce the likelihood that resistant strains will emerge with prolonged exposure. Furthermore, in vitro analysis demonstrates that certain of the MAbs in the combination augment the virus-neutralizing activity of other component antibodies, thereby decreasing the amount of total antibody protein required to inhibit HCMV infection. Certain MAbs have been selected to inactivate extracellular virus during the early phase of HCMV infection, whereas others have been selected to prevent its spread once cells have been infected. These data suggest that a MAb cocktail may be useful for prophylaxis and treatment of patients at risk of life-threatening HCMV infections.

Effects of the synthetic glucocorticoid betamethasone on the survival of neurons in fetal rabbit brain cell culture

Dissociated fetal rabbit brain cells were grown on petri dishes coated with collagen. Culture medium consisted of Dulbeccos Modified Eagles Medium plus 10% serum. The mitotic inhibitor 1-beta-D-arabinofuranosylcytosine was added at 6 days for a 2 day period to inhibit over-growth by glial cells and fibroblasts. In some cases cultures were chronically exposed to 0.5, 1.0 or 2.0 microM betamethasone. Examination of cultures by phase microscopy and acetylcholinesterase (AChE) staining demonstrated that cultures incubated with 1.0 and 2.0 microM betamethasone contained 2-2.5 times as many neurons as compared to control cultures. Furthermore, there was an increase in the specific activities of both AChE and choline acetyltransferase (ChAT) which were proportional to the increase in neuronal cell numbers obtained from phase microscopy and AChE staining. These results suggested that betamethasone enhanced survival of cholinergic neurons. Cultures were also examined for neuron specific gamma-aminobutyric acid (GABA) uptake. Again GABA uptake was approximately 2-2.5 times as great in cultures incubated with 1-2 microM betamethasone when compared to controls. Thus, the increase in GABA uptake paralleled the increase in neurons observed by phase microscopy and AChE staining, suggesting that the survival effect of betamethasone was not specific to cholinergic neurons. While betamethasone treated cultures always contained greater numbers of neurons the percentage of neurons lost from all cultures after 2 weeks was the same.(ABSTRACT TRUNCATED AT 250 WORDS)