The antigenic domain 1 of human cytomegalovirus glycoprotein B contains an intramolecular disulphide bond

Andrea Specknerb,1, Barbara Kropff1, Susanne Knör1 and Michael Mach1

Institut für Klinische und Molekulare Virologie, Universität Erlangen-Nürnberg, Schloßgarten 4, 91054 Erlangen, Germany1

Author for correspondence: Michael Mach. Fax +49 9131 8522101. e-mail mlmach{at}viro.med.uni-erlangen.de


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Glycoprotein B (gB, gpUL55) is the major antigen recognized by the neutralizing humoral immune response against human cytomegalovirus (HCMV). The immunodominant region on gB is the antigenic domain 1 (AD-1), a complex structure that requires a minimal continuous sequence of more than 75 amino acids (aa 552–635) for antibody binding. In this study, the structural requirements for antibody binding to AD-1 have been determined. The domain was expressed in prokaryotic and eukaryotic systems and analysed in immunoblots under reducing and non-reducing conditions. In addition, AD-1 was purified in an immunologically active form and the concentration of sulphydryl groups was determined. The data clearly show that the only form that is recognized by antibodies is a disulphide-linked monomer of AD-1. The disulphide bond is formed between cysteines at amino acid positions 573 and 610 of gB.


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Glycoprotein B (gB) is the dominant antigenic protein within the envelope of human cytomegalovirus (HCMV). The 906 amino acid gB polypeptide of HCMV strain AD169 is translated into a 160 kDa glycosylated precursor molecule that is subsequently cleaved proteolytically into the two subunits gp116 and gp58 (Britt & Mach, 1996 ). The subunits remain covalently linked by disulphide bonds. Disulphide-linked gB homodimers represent the mature intracellular as well as the virion form of gB (Britt & Vugler, 1992 ). Nearly all HCMV-infected individuals develop antibodies against this protein (Kniess et al., 1991 ; Marshall et al., 1992 ; Schoppel et al., 1997 ). Several studies have demonstrated that a considerable fraction of the virus-neutralizing activity found in human serum following natural infection is directed against gB (Britt et al., 1990 ; Gonczol et al., 1991 ; Marshall et al., 1992 ). Consequently, gB has been proposed as a potential candidate for the development of a subunit vaccine and trials have been initiated (Wang et al., 1996 ).

Three antibody-binding sites have been identified on gB: antigenic domain 1 (AD-1), located between amino acids 552 and 635; AD-2, aa 50–77; and AD-3, aa 783–906 (Kniess et al., 1991 ; Meyer et al., 1992 ; Wagner et al., 1992 ). The protein also contains a number of additional non-linear or assembled epitopes (Kari et al., 1990 ; Qadri et al., 1992 ). AD-1 represents the dominant antibody-binding site on gB. In fact, nearly all infected individuals who are seropositive for gB have antibodies against AD-1 (Schoppel et al., 1997 ). This domain is unusually complex, consisting of more than 75 amino acids between residues 552 and 635 of gB. Antibody binding requires the entire AD-1 sequence. Attempts to define conventional 8–15 aa epitopes within AD-1 have been unsuccessful (Ohlin et al., 1993 ). A recent study has provided evidence that AD-1 induces a multitude of different antibodies during natural infection (Speckner et al., 1999 ).

Analysis of AD-1 has provided conflicting data so far. On the one hand, the only mutations within AD-1 that have resulted uniformly in complete loss of binding of all antibodies have involved the cysteines at positions 573 and 610 (Schoppel et al., 1996 ). Considering the multitude of individual antibody-binding structures on AD-1, the most likely interpretation of these results is that the cysteines are essential for building the correct AD-1 structure via formation of disulphide bonds, rather than representing essential contact residues. On the other hand, the previous results were obtained using immunoblots under conditions where the protein was reduced immediately before the analysis, thereby disrupting existing disulphide bonds. A potential explanation for this discrepancy could be the formation of disulphide bonds during late stages of the analysis, e.g. during or after blotting of the proteins. In this case, the cysteines could be involved in formation of disulphide-linked monomers, thereby creating a loop structure or various forms of dimers and/or multimers via intermolecular disulphide bonds.

In order to analyse the structures that can bind antibody, three plasmids were constructed for the expression of different His6-tagged fusion proteins: Xa-AD-1, containing aa 552–635 of gB from strain AD169, represented AD-1, whereas pAD1-C573S and pAD1-C610S contained single cysteine to serine mutations at positions 573 and 610, respectively (Fig. 1E). To construct these plasmids, coding sequences were amplified by PCR with plasmids gig 58-2 (Kniess et al., 1991 ), ATH-Cys573 and ATH-Cys610 (Schoppel et al., 1996 ) as templates. PCR products were ligated into the vector pQE9 (Qiagen). The Xa-AD-1 construct also contained a nucleotide sequence encoding a factor Xa protease cleavage motif (GSIEGRKGS) to enable removal of the His6 tag. Correct insertion of the respective DNA fragments was monitored by nucleotide sequence analysis. Recombinant proteins were purified from E. coli lysates via Ni–NTA agarose (Qiagen) under denaturing conditions (8 M urea, 100 mM NaH2PO4, 40 mM Tris–HCl, pH 8) according to the manufacturer’s instructions. Elution of protein was performed by decreasing the pH to 5·9 in the same buffer. The method achieved an estimated purity of fusion proteins of >95%. Recombinant proteins were analysed in immunoblots. Analysis in the presence of 2-mercaptoethanol (2-ME) was modified in that, in contrast to conventional immunoblot assays, the reducing agent (1% final concentration) was also present during blotting of the antigen as well as blocking of the nitrocellulose filters.



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Fig. 1. Influence of disulphide bonding on recognition of AD-1 by antibodies. (A)–(D) Recombinant His6-tagged AD-1 proteins (aa 552–635) containing either intact AD-1 (Xa-AD-1) or AD-1 with substitutions C573S and C610S were purified by Ni-chelation chromatography and subjected to immunoblot analysis. Electrophoresis, transfer to nitrocellulose filters and the blocking procedure were performed in the presence (+2-ME) or absence (-2-ME) of 2-ME. Blots were developed with an antibody against the His6 tag, RGS-His (A), the murine monoclonal antibody 9–3 (B) or human HCMV-positive sera (C, D). Oligomerization of AD-1 is indicated in (A) as monomeric (mo), dimeric (di) and trimeric (tri). (E) Schematic representation of gB and the recombinant proteins used in this study. Abbreviations: sig, authentic signal sequence; His, His6 tag; Xa, recognition sequence for factor Xa protease; TM, membrane anchor; cyto, cytosolic part of gB.

 
Under non-reducing conditions, an antibody directed against the His6 tag (RGS-His, Qiagen) reacted with a protein of 9 kDa, representing the Xa-AD-1 monomer, as well as proteins with molecular masses of multiples of 9, representing all possible forms of disulphide-linked AD-1 complexes (Fig. 1A). Addition of 2-ME to the preparation resulted in the exclusive formation of monomers, demonstrating that the various forms of AD-1 in our preparation were linked by disulphide bonds. The comparable signal intensities obtained in the presence or absence of 2-ME also proved that addition of the reducing agent during the late stages of the analysis did not affect the sensitivity of the assay. Proteins AD1-C573S and AD1-C610S gave signals corresponding to monomers and dimers, since the absence of a second cysteine in the recombinant polypeptide prevented formation of multimers (Fig. 1A). In the case of AD1-C573S and AD1-C610S, a slightly different mobility was observed as compared with Xa-AD1. In the case of monomers, this was due to the presence of additional sequences within Xa-AD1 encoding the recognition site for factor Xa protease. The different mobilities of the dimers were probably caused by the different locations of the disulphide bond, resulting in protein structures with divergent migration in PAGE.

When the proteins were analysed with gB-specific monoclonal antibodies (Fig. 1B) or sera from HCMV-seropositive donors (Fig. 1C, D), positive reactions were observed only with AD-1 monomers in the absence of 2-ME. No reactivity was seen when the immunoblots were developed under reducing conditions or when AD1-C573S or AD1-C610S were analysed. These results demonstrated that (i) antibody binding to AD-1 is dependent on an intramolecular disulphide bond, (ii) formation of this disulphide linkage takes place in conventional immunoblots during late stages of the analysis and (iii) antibodies induced during natural infection as well as after immunization of mice exclusively recognize the disulphide-linked monomer of AD-1.

In order to confirm these results in eukaryotic cells, the plasmid AD-1co was constructed. It contains the signal sequence (aa 1–27), AD-1 (aa 552–642) and the transmembrane as well as the cytoplasmic part (aa 701–907) of gB (Fig. 1E). Fragments encoding the respective gB sequences were amplified by PCR with DNA from HCMV AD169 as template and were inserted sequentially into the vector pcDNA3 (Invitrogen). The correct sequence was confirmed by nucleotide sequence analysis. Indirect immunofluorescence analysis with AD-1-specific antibodies indicated that the protein product from AD-1co was transported to the cell surface after transfection of the DNA in COS-7 cells, indicating normal processing and transport (data not shown).

In order to determine whether AD-1co formed a complex within cells, COS-7 cells were transfected with DNA by using Lipofectamine Plus (Gibco) according to the manufacturer’s instructions. Lysates were prepared 48 h later and conventional immunoblots were performed with antibody 27-287, which is specific for AD-1 (Schoppel et al., 1996 ). In both the presence and absence of 2-ME, a 40 kDa protein was detected, which was in agreement with the theoretical molecular mass for AD-1co (35 kDa) (Fig. 2). No higher molecular mass forms were seen. Virions were used as the control antigen and showed the expected reaction pattern; i.e. the 58 kDa subunit of gB was recognized by the antibody in the presence of reducing agents and the higher molecular mass forms corresponding to gB monomer and dimer were detected in the absence of 2-ME. Identical results were obtained with fibroblasts as expressing cells and the human monoclonal antibody ITC52 (data not shown).



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Fig. 2. Immunoblot analysis of AD-1 expressed in mammalian cells. COS-7 cells were transfected with DNA from the vector (pcDNA3) or AD-1co and lysates were analysed in immunoblots under reducing (+2-ME) or non-reducing (-2-ME) conditions. Blots were developed with the AD-1-specific antibody 27–287. Extracellular HCMV particles (virus) were used as control antigen.

 
Finally, we determined the concentration of free thiol groups in AD-1. Monomeric Xa-AD-1 protein was purified by Ni chelation and reverse-phase chromatography to yield a protein of >95% purity (data not shown). This polypeptide was reactive with a set of previously described human AD-1-specific monoclonal antibodies as well as human sera from HCMV-seropositive donors, indicating that it retained its antigenic structure (data not shown). Thiol groups were determined quantitatively by using the method of Singh et al. (1995) . In this assay, an inactive disulphide derivative of papain (papain–S–SCH3) is activated stoichiometrically by thiol groups to give active papain (papain–SH). The amount of active papain is assayed by measuring its catalytic activity with the substrate N-benzoyl-L-arginine-p-nitroanilide, which releases the chromophoric product p-nitroaniline. This assay is approximately 100-fold more sensitive for the detection of free thiol groups than the classical Ellman reaction (Ellman, 1959 ). Thiol groups from cysteine and the reduced form of glutathione gave linear dose–response curves at 0·5–1·7 nmol, whereas the oxidized form of glutathione was non-reactive (Fig. 3). Purified AD-1 protein did not give a signal in this assay at amounts of up to 15 nmol thiol (corresponding to 7·5 nmol protein). AD-1 that was reduced prior to analysis by treatment with 2-ME gave an intermediate response. The lower concentration of thiol groups detected in reduced AD-1 compared with cysteine or glutathione was due to the fact that the reducing agent had to be removed from the protein preparation by dialysis, which allowed partial refolding and oxidation of AD-1. Considering the detection limit of the assay, which was well below 0·5 nmol thiol, it can be concluded that more than 96·5% of cysteines are engaged in disulphide bonds in monomeric AD-1 peptide.



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Fig. 3. Determination of thiol groups in AD-1. Samples were incubated with papain–S–SCH3 for 60 min, substrate was added and the activity of papain was measured by monitoring the change in absorbance between 30 and 60 min after addition of substrate. Substrates tested were oxidized ({square}) and reduced ({blacksquare}) AD-1, oxidized ({triangleup}) and reduced ({blacktriangleup}) glutathione and cysteine ({circ}).

 
In conclusion, our data show that formation of an intramolecular disulphide bond between Cys-573 and Cys-610 is essential for antibody binding to AD-1. The fact that neutralizing antibodies, developed during natural infection, exclusively recognize monomeric AD-1 suggests strongly that oligomerization of gB, as it occurs in the viral envelope, leaves the monomeric AD-1 structure intact. Thus, AD-1 can be included in the list of disulphide-linked loops on the surface of viruses, which represent highly antigenic structures (Earl et al., 1997 ; Grigera et al., 1992 ; Megret et al., 1992 ).

Our results also provide an assignment for a disulphide linkage between two cysteine residues within HCMV gB. Glycoprotein B is perhaps the most highly conserved envelope component of all members of the herpesvirus family. One remarkable feature of this homology is the conservation of most of the cysteine residues. When disulphide linkages were analysed between the cysteine residues in herpes simplex virus (HSV) type 2 gB, a disulphide bridge was identified between the AD-1 positional homologues Cys-571 and Cys-608 (Norais et al., 1996 ). Thus, the bridging of the AD-1 cysteines represents a conserved structural element in these two gB molecules. This conservation would not necessarily be expected, since gB molecules from HSV and HCMV differ in that: (i) HCMV gB contains two additional cysteine residues not found in HSV gB (positions 246 and 778 in gB from strain AD169); (ii) HCMV-gB molecules form disulphide-linked homodimers whereas HSV gB does not (Laquerre et al., 1996 ); and (iii) HCMV gB is proteolytically cleaved between residues 458 and 459 in the subunits gp116 and gp58, which are held together by disulphide bonds. In contrast, HSV gB is not cleaved proteolytically. Moreover, a previous publication has linked Cys-572 and Cys-610 to HCMV gB dimerization (Eickmann et al., 1998 ). The apparent discrepancy from our data could result from the fact that, in this case, point mutations in cysteine residues were analysed in the context of the entire gB, thus making it difficult to exclude effects secondary to misfolding of the mutated protein.

In summary, we have shown that the most immunogenic domain of HCMV gB represents a monomer with an intramolecular disulphide bond between Cys-573 and Cys-610. The potent neutralizing capacity of some AD-1-specific antibodies highlights the importance of this domain for the virus–host interaction.


   Acknowledgments
 
We are grateful to W. Britt, University of Birmingham, AL, USA, and Mats Ohlin, University of Lund, Sweden, for the supply of monoclonal antibodies. This work was supported by grants from the Deutsche Forschungsgemeinschaft (MA 929/4-1) and the Wilhelm Sander-Stiftung.


   Footnotes
 
b Present address: November AG, Ulrich-Schalk-Str. 3, 91056 Erlangen, Germany.


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Received 30 March 2000; accepted 7 August 2000.