Characterization of the N-terminal part of the neutralizing antigenic site I of coxsackievirus B4 by mutation analysis of antigen chimeras.
Coxsackievirus B3 (CVB3) as a potential RNA virus vector for the presentation of foreign antigenic epitopes was further characterized. Insertion mutagenesis of infectious CVB3 cDNA yielded viable antigen chimeras containing variant BC loops of VP1 of coxsackievirus B4 (CVB4). Analysis of three antigen chimeras allowed the mapping of the N-terminal part of the neutralizing antigenic site 1 (N-Ag1) of CVB4 which is located in the BC loop of the structural protein VP1. A significant neutralization of a viable chimera with the deletion of CVB4-specific amino acid Ser-83 at the amino terminus of the VP1 BC loop was obtained with CVB4 serotype-specific polyclonal antisera. This neutralization was reduced after further deletion of the adjacent Ala-84, suggesting that this amino acid either constitutes the beginning of N-Ag1 of CVB4 or is essential for the conformation of the adjacent epitope. In contrast, exchange of amino acid Ser-86 to alanine, in the middle of the BC loop, led to complete loss of reactivity with CVB4-specific antibodies, demonstrating the importance of this residue for binding of CVB4 neutralizing antisera. Furthermore, we observed that manipulations of the VP1 BC loop resulted in increased thermolability of the viable chimeras in comparison to CVB3, although replication efficiencies were similar.
McPhee
F
F
Max-Planck-Institut für Biochemie, Abteilung Virusforschung, Martinsried, Germany.
Zell
R
R
Reimann
B Y
BY
Hofschneider
P H
PH
Kandolf
R
R
D000595:Amino Acid Sequence; D000956:Antigens, Viral / Q000737:chemistry / Q000276:immunology*; D001483:Base Sequence; D017931:DNA Primers; D029822:Enterovirus B, Human / Q000235:genetics / Q000276:immunology / Q000502:physiology*; D000939:Epitopes / Q000032:analysis; D014644:Genetic Variation; D016679:Genome, Viral; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D009500:Neutralization Tests; D011993:Recombinant Fusion Proteins / Q000276:immunology; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology; D017384:Sequence Deletion; D014162:Transfection; D015678:Viral Structural Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology*; D014779:Virus Replication
D000956:Antigens, Viral; D017931:DNA Primers; D000939:Epitopes; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D015678:Viral Structural Proteins