Disulfide-bonded discontinuous epitopes on the glycoprotein of vesicular stomatitis virus (New Jersey serotype)

Grigera, Pablo R.; Keil, Walter; Wagner, Robert R.

doi:10.1128/jvi.66.6.3749-3757.1992

Cited by 16 publications

(15 citation statements)

References 26 publications

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“…Site-directed mutagenesis of cysteine residues has been previously employed in the characterization of envelope glycoproteins of vesicular stomatitis virus [24], herpes simplex virus [25] and human immunodeficiency virus [26]. In all these cases, elimination of a disulfide bond provokes pro- found functional and antigenic alterations.…”

Section: Discussionmentioning

confidence: 99%

Site‐directed mutagenesis of cysteine residues of hepatitis B surface antigen Analysis of two single mutants and the double mutant

Antoni

Rodrı́guez-Crespo

Gómez-Gutiérrez

et al. 1994

European Journal of Biochemistry

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The structure of hepatitis B surface antigen (HBsAg) is mainly maintained by an intricate disulfide network responsible for most of its structural and antigenic properties. Characterization of three cysteine-replacement mutants of HBsAg has been performed by both structural and immunological methods. Replacement of Cysl21 or Cys124 with serine results in mutant proteins that show diminished binding titres to both monoclonal antibodies and to a polyclonal serum, indicating that a structural change has taken place. Circular dichroism analysis shows that the substitution of either of these two residues also diminishes the helical content of the protein. However, the double mutant, in which both cysteine residues have been simultaneously changed, reverts the properties of the single mutations, and shows similar behaviour to the wild-type protein. Both the single and double cysteine mutants are efficiently glycosylated and secreted from Chinese hamster ovary cells and, in all cases, the mutant proteins assemble into spherical particles of similar buoyant density to both the wild-type and serum-derived HBsAg.Hepatitis B surface antigen (HBsAg), the envelope of the hepatitis B virus, is a complex macromolecular structure composed of proteins (75% by mass), carbohydrates (in the form of glycoproteins) and host-derived lipids (25% by mass) [ 11. During infection, hepatocytes synthesize and secrete HBsAg in large excess, chiefly in the form of lipoprotein particles of 22-nm diameter [2, 31. SDSPAGE of HBsAg reveals the existence of two proteins with molecular masses 25 kDa and 28 kDa, designated S and gS respectively, which account for more than 90% of the protein content. These two proteins have identical amino acid sequence and differ only by the presence of carbohydrates attached to gS at As11146 [4, 51.Chemical-modification studies have demonstrated the importance of disulfide bridges in terms of maintaining the native HBsAg structure. The observation that reduction and alkylation of HBsAg particles leads to a complete loss of immunological properties [6], corroborates the model of the surface antigen as a highly cross-linked protein aggregate held together by disulfide bonds. Thus, this type of covalent linkage might be essential for maintaining the structural and antigenic properties of the particle.When HBsAg particles are subjected to SDSPAGE under non-reducing conditions almost no polypeptides enter the gel, with most of the protein remaining in the stacking gel [7]. In an attempt to identify the specific cysteine residues involved in the intermolecular disulfide bonds, HBsAg was reduced under increasingly strong reducing conditions, and alkylated with I4C-labelled iodoacetamide. The results of these experiments indicated the presence of at least two and as many as four intermolecular disulfide bridges probably involving cysteine residues at positions 121, 124 and 221 [7]. However, recent studies have suggested that disulfide bonds might be equally important in terms of establishing the appropiate three-dimension...

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Section: Discussionmentioning

confidence: 99%

Site‐directed mutagenesis of cysteine residues of hepatitis B surface antigen Analysis of two single mutants and the double mutant

Antoni

Rodrı́guez-Crespo

Gómez-Gutiérrez

et al. 1994

European Journal of Biochemistry

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“…4B). The disulfide bond between the distant Cys44 and Cys295 in the VHSV G protein may thus be a general feature, and interestingly the existence of such a major loop in the VSV G protein anchored by at least one disulfide bond has also been proposed by Grigera and coworkers (6). The remaining six cysteines form three bonds in the VHSV G protein situated in a region characterized by discontinuous neutralizing epitopes in VHSV, IHNV, VSV, and rabies virus (1,2,10,24).…”

Section: Discussionmentioning

confidence: 87%

Characterization of Intramolecular Disulfide Bonds and Secondary Modifications of the Glycoprotein from Viral Hemorrhagic Septicemia Virus, a Fish Rhabdovirus

Einer-Jensen¹,

Krogh²,

Roepstorff³

et al. 1998

J Virol

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Viral hemorrhagic septicemia virus (VHSV) infections cause high losses in cultured rainbow trout in Europe. Attempts to produce a recombinant vaccine based on the transmembrane glycoprotein (G protein) have indicated that proper folding is important for the antigenicity and immunogenicity of the protein. The present study was initiated to identify the disulfide bonds and other structural aspects relevant to vaccine design. The N-terminal amino acid residue was identified as being a pyroglutamic acid, corresponding to Gln21 of the primary transcript. Peptides from endoproteinase-degraded G protein were analyzed by mass spectrometry before and after chemical reduction, and six disulfide bonds were identified: Cys29-Cys339, Cys44-Cys295, Cys90-Cys132, Cys172-Cys177, Cys195-Cys265, and Cys231-Cys236. Mass spectrometric analysis in combination with glycosidases allowed characterization of the glycan structure of the G protein. Three of four predicted N-linked oligosaccharides were found to be predominantly biantennary complex-type structures. Furthermore, an O-linked glycan near the N terminus was identified. Alignment of the VHSV G protein with five other rhabdovirus G proteins indicates that eight cysteine residues are situated at conserved positions. This finding suggests that there might be some common disulfide bonding pattern among the six rhabdoviruses.

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“…Cells and viruses. Growth of CV1 cells in monolayer cultures and production of the vaccinia virus recombinant expressing the bacteriophage T7 polymerase (vTF1-6,2; a gift of Bernard Moss) have been described previously (13). Native FMDV used as an antigen for the enzyme-linked immunosorbent assay (ELISA) and neutralization studies was obtained by infecting BHK-21 cell monolayers with the FMDV C3 serotype as described by Denoya et al (7).…”

Section: Methodsmentioning

confidence: 99%

“…For example, binding sites of four independent neutralizing monoclonal antibodies (MAbs) have been mapped to a region between amino acids 193 and 289 of the G protein of the VSV New Jersey serotype (VSV-NJ), suggesting the existence of a major antigenic region exposed within the central segment of the 517-amino-acid protein (16). In addition, gel electrophoresis and amino acid sequence analyses after protease V8 exposure of purified VSV-NJ G protein led us to postulate the existence of a second exposed loop region located upstream and flanking amino acid 110 (13). This latter region is also the location of the disulfide bond-dependent discontinuous epitope IV; unlike the more centrally located discontinuous epitopes between amino acids 193 to 289, epitope IV is not neutralizing and is shared by G proteins of both the Indiana and New Jersey serotypes (16).…”

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confidence: 99%

Immunogenicity of an aphthovirus chimera of the glycoprotein of vesicular stomatitis virus

Grigera

García-Briones

Periolo

et al. 1996

J Virol

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An oligodeoxynucleotide coding for amino acids 139 through 149 of antigenic site A (ASA) of the VP 1 capsid protein of the foot-and-mouth disease virus C3 serotype (FMDV C3) was inserted into three different in-frame sites of the vesicular stomatitis virus New Jersey serotype (VSV-NJ) glycoprotein (G) gene cDNA present in plasmid pKG97 under control of the bacteriophage T7 polymerase promoter. Transfection of these plasmids into CV1 cells coinfected with the T7 polymerase-expressing vaccinia virus recombinant vTF1-6,2 resulted in expression of chimeric proteins efficiently reactive with both anti-FMDV and anti-VSV G antibodies. However, in vitro translation of transcripts of these VSV-G/FMDV-ASA chimeric plasmids resulted in proteins that were recognized by anti-G serum but not by anti-FMDV serum, indicating a requirement for in vivo conformation to expose the ASA antigenic determinant. Insertion of DNA coding for a dimer of the ASA unidecapeptide between the VSV-NJ G gene region coding for amino acids 160 and 161 gave rise to a chimeric ASA-dimer protein designated GF2d, which reacted twice as strongly with anti-FMDV antibody as did chimeric proteins in which the ASA monomer was inserted in the same position or two other G-gene positions. For even greater expression of chimeric VSV-G/FMDV-ASA proteins, plasmid pGF2d and a deletion mutant p⌬GF2d (G protein deleted of 324 C-terminal amino acids) were inserted into baculovirus vectors expressing chimeric proteins GF2d-bac and ⌬GF2d-bac produced in Sf9 insect cells. Mice vaccinated with three booster injections of 30 g each of partially purified GF2d-bac protein responded by enzyme-linked immunosorbent assay with FMDV antibody titers of 1,000 units, and those injected with equivalent amounts of ⌬GF2d-bac protein showed serum titers of up to 10,000 units. Particularly impressive were FMDV neutralizing antibody titers in serum of mice vaccinated with ⌬GF2d-bac protein, which approached those in the sera of mice vaccinated with three 1-g doses of native FMDV virions. Despite excellent reactivity with native FMDV, the anti-⌬GF2d-bac antibody present in vaccinated mouse serum showed no capacity to bind to sodium dodecyl sulfate (SDS)-denatured FMDV virions and only minimal reactivity with VP 1 protein by Western blotting (immunoblotting) after SDS-polyacrylamide gel electrophoresis. It was also shown in a competitive binding assay that a synthetic ASA unidecapeptide, up to concentrations of 200 g/ml, was quite limited in its ability to inhibit binding of anti-⌬GF2-bac antibody to native FMDV virions. These results suggest that the chimeric VSV-G/FMDV-ASA proteins mimic the capacity of FMDV to raise and react with neutralizing antibodies to a restricted number of ASA conformations present on the surface of native FMDV particles.

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Disulfide-bonded discontinuous epitopes on the glycoprotein of vesicular stomatitis virus (New Jersey serotype)

Abstract: Intrachain disulfide bonds between paired cysteines in the glycoprotein (G) of vesicular stomatitis virus (VSV) are required for the recognition of discontinuous epitopes by specific monoclonal antibodies (MAbs) (W.

Cited by 16 publications

References 26 publications

Site‐directed mutagenesis of cysteine residues of hepatitis B surface antigen Analysis of two single mutants and the double mutant

Site‐directed mutagenesis of cysteine residues of hepatitis B surface antigen Analysis of two single mutants and the double mutant

Characterization of Intramolecular Disulfide Bonds and Secondary Modifications of the Glycoprotein from Viral Hemorrhagic Septicemia Virus, a Fish Rhabdovirus

Immunogenicity of an aphthovirus chimera of the glycoprotein of vesicular stomatitis virus

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