Locus of a virus neutralization epitope on the Japanese encephalitis virusenvelope protein determined by use of long PCR-based region-specific random mutagenesis

We prepared recombinant Japanese encephalitis (JE) virus populations posses sing random mutations at the envelope (E) protein region by a long PCR-base d method. Neutralization-resistant mutants were selected from these populat ions by application of JE-specific virus neutralizing monoclonal antibody ( mAb) 503, which possessed a 51,200-fold neutralization titer. We classified the mutants into three groups, each bearing two amino acid alterations at the E protein region: 52, Gln-Arg, and 136, Lys-Glu; 136, Lys-Glu, and 275, Ser-Pro; and 126, IIe-Thr, and 136, Lys-Glu, respectively. Three different genetically engineered variants, each bearing a single mutation, 126, IIe- Thr; 136, Lys-Glu; and 275, Ser-Pro, respectively, showed partial but not c omplete recovery of reactivity to mAb 503. Our results indicate that the am ino acid substitutions at amino acid positions 52, 126, 136, and 275 altere d the structure of the neutralization epitope for mAb 503 on the E protein. All these mutations were clustered at the junction of domains I and II of the E protein and it is likely that the epitope for mAb 503 is composed of at least E-o-e, D-o-a, and k strands of the E protein. We also demonstrated the efficacy of the long PCR-based recombinant virus technique as a useful tool for the creation of a variety of mutants bearing random mutations at targeted areas of the virus genome. (C) 2001 Academic Press.