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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Abstract |
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Three antibody-binding sites have been identified on gB: antigenic domain 1 (AD-1), located between amino acids 552 and 635; AD-2, aa 5077; and AD-3, aa 783906 (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 815 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 552635 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 NiNTA agarose (Qiagen) under denaturing conditions (8 M urea, 100 mM NaH2PO4, 40 mM TrisHCl, pH 8) according to the manufacturers 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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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 127), AD-1 (aa 552642) and the transmembrane as well as the cytoplasmic part (aa 701907) 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 manufacturers 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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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 virushost interaction.
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Acknowledgments |
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Footnotes |
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References |
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Received 30 March 2000;
accepted 7 August 2000.