Characterization of a Disulphide Bridge-stabilized Antigenic Domain of Tick-borne Encephalitis Virus Structural Glycoprotein

Winkler, Günther; Heinz, Franz X.; Künz, Christian

doi:10.1099/0022-1317-68-8-2239

Cited by 35 publications

(26 citation statements)

References 21 publications

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“…Denaturation of the reduced and carboxymethylated protein irreversibly inhibits its ability to bind the neutralizing MAbs tested. Furthermore, it has been possible to isolate an E protein fragment containing epitopes of cluster b, denaturation of which is reversible if re-formation of disulphide bridges is allowed to take place during renaturation (Winkler et al, 1987). The importance of disulphide bonds and a highly organized structure for the maintenance of the epitopes of the E protein have also been described for the JE virus which belongs to the same subgroup as the WN virus (Srivastava et al, 1987).…”

Section: Institut J~r Virologie Justus-liebig-universitiit Giessen mentioning

confidence: 96%

An Analysis of the Antibody Response against West Nile Virus E Protein Purified by SDS-PAGE Indicates that This Protein Does Not Contain Sequential Epitopes for Efficient Induction of Neutralizing Antibodies

Wengler

1989

Journal of General Virology

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SUMMARYThe large envelope (E) protein of flaviviruses is the viral surface protein that contains neutralizing epitopes. We have analysed the E protein of the West Nile (WN) flavivirus for neutralizing epitopes generated from linear segments of the protein; if effective, these might allow the synthesis of peptides suitable for vaccination. For this study we used the E protein and defined fragments thereof as antigens in rabbits. The sera thus obtained, containing antibodies to E protein as shown by Western blot analyses, were tested for neutralizing activity by the plaque reduction neutralization test. If the E protein used as antigen was reduced (the native E protein contains six disulphide bridges) and denatured the resulting antibodies did not consistently demonstrate neutralizing activity. These results show that the E protein of WN virus does not contain a linear segment that is able to induce neutralizing antibodies efficiently. Studies using denatured E protein fragments containing subsets of the intact disulphide bridges showed that the local covalent primary structure of the protein involved in each of the six bridges was also insufficient for inducing the synthesis of neutralizing antibodies. The complete E protein, with all six disulphide bridges intact, purified by preparative SDS-PAGE under the conditions used in our experiments could, however, induce the synthesis of neutralizing antibodies in rabbits. These data indicate that at least one complex epitope which is able to induce neutralizing antibodies is not completely denatured or can be reformed to some extent if the complete E protein has been subjected to SDS-PAGE without prior destruction of the disulphide bridges.Flaviviruses have recently been established as a separate virus family containing more than 60 viruses (Westaway et al., 1985). Members of this family are major human pathogens, e.g. the viruses causing yellow fever, dengue, Japanese encephalitis (JE) and tick-borne encephalitis (TBE) (see Monath, 1985 for a review of flavivirus-induced diseases). Analyses of the immunological aspects of flavivirus infections have shown that the viral envelope (E) protein contains the epitopes that induce neutralizing antibodies (for recent reviews see Roehrig, 1986;Heinz, 1986). We have studied the molecular biology of the replication of the West Nile (WN) flavivirus, which causes West Nile fever, a disease which is of significance in Africa, the Middle East and some other regions (see Monath, 1985, for a review). We have determined the primary structure of the WN virus E protein (Wengler et al., 1985), have identified the six disulphide bridges which are present in this protein (Nowak & Wengler, 1987) and have identified two regions within the native virus-associated E protein that are susceptible to proteolytic cleavage (Wengler et al., 1987). A schematic representation of the WN virus E protein, which incorporates these data, is presented in Fig. 1. With this knowledge we undertook to determine whether or not neutralizing antibodies could be ind...

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Section: Institut J~r Virologie Justus-liebig-universitiit Giessen mentioning

confidence: 96%

An Analysis of the Antibody Response against West Nile Virus E Protein Purified by SDS-PAGE Indicates that This Protein Does Not Contain Sequential Epitopes for Efficient Induction of Neutralizing Antibodies

Wengler

1989

Journal of General Virology

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“…In our companion studies on DEN-l, deletion analysis was used to localize three MAb-binding sites between residues 293 and 401 of the DEN-1 E protein sequence (M. A. Zuegel, P. W. Mason, M. J. Fournier & T. L. Mason, unpublished data). In the case of TBE virus, Winkler et al (1987) identified an immune-reactive region, designated domain B, that was contained in a 9K tryptic fragment of the E protein. This fragment was stabilized by a disulphide bridge, which can re-form after reduction and SDS denaturation and was required for the binding of several domain B-specific MAbs.…”

Section: Animal Protection Assaysmentioning

confidence: 99%

Molecular Characterization of a Neutralizing Domain of the Japanese Encephalitis Virus Structural Glycoprotein

Mason

Dalrymple

Gentry³

et al. 1989

Journal of General Virology

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SUMMARYExpression of antigenic fragments of the Japanese encephalitis virus envelope protein (E) in Escherichia coli has been used to define the boundaries of an antigenic domain that contains the binding sites for 10 anti-E monoclonal antibodies (MAbs). All of these antibodies neutralized the virus in vitro and some of them passively protected mice from a fatal virus challenge. We have shown previously that nine of these antibodies react with the antigenic determinants encoded by a 405 bp fragment of viral cDNA. To determine the amino acid sequences of specific determinants, truncated polypeptides were expressed as fusion proteins in E. coli following progressive Bal 31 exonuclease digestion of the 5' and 3' ends of the cDNA fragment. Examination of the immunoreactivity of these polypeptides revealed that the region from methionine 303 to tryptophan 396 was the shortest sequence capable of reacting with any of the I0 MAbs or with a polyclonal, antiviral hyperimmune mouse ascitic fluid. Biochemical tests showed that an intramolecular disulphide cross-linkage between cysteine 304 and cysteine 335 of the E protein sequence was required for presentation of the binding site(s) for these MAbs. Although this 95 amino acid antigenic domain appeared to be capable of forming several conformational neutralizing epitopes, it was not an effective immunogen for inducing neutralizing or protective antibodies in mice.

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“…SS6 is of particular interest because of experimental evidence implicating DIII in cell attachment (5). By using proteolytic fragments of the TBE virus E protein, it was shown that SS6 spontaneously reformed following reduction and oxidation, suggesting that the surrounding secondary structure favored SS6 bond formation (25). This bond was also critical for the correct antigenic structure of the E proteins of DEN1 and DEN2 viruses (9,12).…”

mentioning

confidence: 99%

Contribution of Disulfide Bridging to Epitope Expression of the Dengue Type 2 Virus Envelope Glycoprotein

Roehrig

Volpe

Squires

et al. 2004

J Virol

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The flavivirus envelope (E) glycoprotein encodes important phenotypic and immunogenic properties of the virion (7,17). This protein elicits virus-neutralizing antibodies, mediates virus-cell membrane fusion, and initiates infection through binding to cell surfaces. The high-resolution crystal structures of the ectodomain of the tick-borne encephalitis (TBE) virus and dengue type 2 (DEN2) virus E-protein homodimers have been solved (13, 16). The three structural domains (DI, DII, and DIII) identified in the E protein were roughly analogous to the previously defined antigenic domains (C, A, and B) (Fig. 1). The amino acid sequence of the ectodomain of all flavivirus E proteins contains 12 strictly conserved cysteine residues that form six disulfide (SS) bonds (14). For DEN2 virus, the SS1 bond (Cys 3 bonded to Cys 30) stabilizes an amino-terminal loop (Fig. 1). The SS2 (Cys 60-Cys 121), SS3 (Cys 74-Cys 105), and SS4 (Cys 92-Cys 116) bonds stabilize the amino-proximal part of DII that harbors the virus membrane fusion sequence located at amino acids (aa) 98 to 110 (17, 19). SS5 (Cys 185-Cys 285) stabilizes the carboxy-proximal loop of DII, which is separated from the amino-proximal DII loop by DI. The SS6 bond makes the only bridge in DIII (Cys 302-Cys 333).Reduction of all six of the West Nile virus E-protein SS bonds resulted in a protein that was unable to elicit virusneutralizing antibody in mice (23). To date, only DIII SS6 has been investigated individually for its contribution to the structure and function of the E protein. SS6 is of particular interest because of experimental evidence implicating DIII in cell attachment (5). By using proteolytic fragments of the TBE virus E protein, it was shown that SS6 spontaneously reformed following reduction and oxidation, suggesting that the surrounding secondary structure favored SS6 bond formation (25). This bond was also critical for the correct antigenic structure of the E proteins of DEN1 and DEN2 viruses (9, 12).The antigenic structure of the DEN2 virus E protein has been investigated previously using monoclonal antibodies (MAbs) (8,17). Not unexpectedly, the DEN2 virus E-protein antigenic structure was similar to the TBE virus E-protein antigenic structure. Three antigenic regions (A, B, and C) with unique biochemical characteristics were identified, and these regions correlated well with their analogous regions in the TBE virus E protein. The locations of many of the epitopes were mapped on the DEN2 E-protein three-dimensional structure using MAb competition binding assays, binding of MAb to peptide fragments, and serological testing of MAbs (17).In this study we used site-directed mutagenesis of a chimeric plasmid expressing the prM and E proteins of DEN2 virus strain 16681 to determine the individual contributions of the six SS bonds to E-protein epitope expression. This chimeric plasmid, pCB8D2-2J-2-9-1, expressed modified DEN2 proteins (4). The DEN2 prM signal sequence was replaced by the Japanese encephalitis virus prM signal sequence. The last 20% (aa 39...

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Characterization of a Disulphide Bridge-stabilized Antigenic Domain of Tick-borne Encephalitis Virus Structural Glycoprotein

Cited by 35 publications

References 21 publications

An Analysis of the Antibody Response against West Nile Virus E Protein Purified by SDS-PAGE Indicates that This Protein Does Not Contain Sequential Epitopes for Efficient Induction of Neutralizing Antibodies

An Analysis of the Antibody Response against West Nile Virus E Protein Purified by SDS-PAGE Indicates that This Protein Does Not Contain Sequential Epitopes for Efficient Induction of Neutralizing Antibodies

Molecular Characterization of a Neutralizing Domain of the Japanese Encephalitis Virus Structural Glycoprotein

Contribution of Disulfide Bridging to Epitope Expression of the Dengue Type 2 Virus Envelope Glycoprotein

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