Mapping and characterization of antigenic epitopes and the nucleic acid-binding domains of the VP6 protein of bluetongue viruses.
Heterologously expressed VP6 and truncated VP6 proteins of bluetongue virus (BTV) serotype 11 purified to near homogeneity were used for structure and function analyses. The yield of the expressed VP6 was host cell dependent. Six antigenic epitopes of VP6 of BTV were identified and mapped by immunoblot analyses and enzyme-linked immunosorbent assay with oligoclonal antibodies. These determinants were surface accessible and conserved among the cognate VP6 proteins of five U.S. BTV serotypes. The amino acid sequences and sizes of these six antigenic epitopes were determined, and their precise locations were also mapped and confirmed by deletion analyses. The nucleic acid binding activities of VP6, confirmed by electrophoretic mobility shift assay, were concentration dependent. The binding activities and affinities of the purified expressed VP6 protein towards double-stranded RNA and double-stranded DNA were similar. Two domains of VP6, corresponding to three of the six antigenic epitopes, were responsible for the nucleic acid binding activities and have been mapped within 28 amino acids near the middle and 11 residues near the carboxyl terminus of VP6 by electrophoretic mobility shift assay and deletion mutant analyses. Synthetic oligopeptides corresponding to these three regions also exhibited similar concentration-dependent nucleic acid binding activities.
Hayama
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Program in Molecular Biology, Utah State University, Logan 84322-5500.
Li
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JK
D000595:Amino Acid Sequence; D000818:Animals; D000956:Antigens, Viral / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D001820:Bluetongue virus / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D002213:Capsid / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D036022:Capsid Proteins; D002460:Cell Line; D017124:Conserved Sequence; D006224:Cricetinae; D004247:DNA / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D000939:Epitopes / Q000235:genetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D012330:RNA, Double-Stranded / Q000378:metabolism; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid
D000956:Antigens, Viral; D036022:Capsid Proteins; D017931:DNA Primers; D004279:DNA, Viral; D000939:Epitopes; D010446:Peptide Fragments; D012330:RNA, Double-Stranded; C061332:VP6 protein, Rotavirus; D004247:DNA