CHARACTERIZATION OF THE N-TERMINAL PART OF THE NEUTRALIZING ANTIGENICSITE-I OF COXSACKIEVIRUS B4 BY MUTATION ANALYSIS OF ANTIGEN CHIMERAS

Coxsackievirus B3 (CVB3) as a potential RNA virus vector for the prese ntation of foreign antigenic epitopes was further characterized. Inser tion mutagenesis of infectious CVB3 cDNA yielded viable antigen chimer as containing variant BC loops of VP1 of coxsackievirus B4 (CVB4). Ana lysis of three antigen chimeras allowed the mapping of the N-terminal part of the neutralizing antigenic site 1 (N-Ag1) of CVB4 which is loc ated in the BC loop of the structural protein VP1. A significant neutr alization 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 wit h 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 lo op, led to complete loss of reactivity with CVB4-specific antibodies, demonstrating the importance of this residue for binding of CVB4 neutr alizing antisera. Furthermore, we observed that manipulations of the V P1 BC loop resulted in increased thermolability of the viable chimeras in comparison to CVB3, although replication efficiencies were similar .