MUTATIONAL ANALYSIS OF A NEUTRALIZATION EPITOPE ON THE DENGUE TYPE-2 VIRUS (DEN2) ENVELOPE PROTEIN - MONOCLONAL-ANTIBODY RESISTANT DEN2 DEN4 CHIMERAS EXHIBIT REDUCED MOUSE NEUROVIRULENCE/

The antigenic site of dengue type 2 virus (DEN2)-neutralizing monoclon al antibody (mab) 3H5 was investigated by mutational analysis. Sequenc e comparisons indicated that much of the 12-amino-acid sequence extend ing from position 386 to 397 of the DEN2 envelope glycoprotein (E) pre viously thought to represent the DEN2-specific mab 3H5 binding site wa s also present in some dengue type 1, 3, or 4 virus strains. However, the region occupied by the Glu-Pro-Gly sequence at upstream positions 383 to 385 was completely conserved among DEN2 strains, but divergent in other serotype viruses, suggesting that this sequence might be part of the antigenic site of malc 3H5. We investigated this possibility b y employing the previously constructed chimeric DEN2(PreM-E)/DEN4 cDNA clone to produce viable mutants bearing DEN2 PreM and E sequences tha t could be analyzed for binding to and neutralization by mab 3H5. We c onstructed 13 such DEN2 mutants that contained a single amino acid sub stitution in the region between positions 383 and 393 of DEN2 E. Each single substitution in the region spanning positions 386 through 393 o f DEN2 yielded a virus that was as reactive with mab 3H5 as the parent al chimeric virus. These results are consistent with the extent of seq uence conservation in the region. In contrast, 5 of 6 mutants that sus tained an amino acid substitution at position 383, 384, or 385 failed to react with mab 3H5 as detected by immunofluorescence assay and fail ed to be neutralized by the mab. Interestingly, each of the 5 mab-resi stant DEN2 mutants also exhibited reduced mouse neurovirulence compare d to parental chimeric DEN2 when inoculated intracerebrally. These obs ervations suggest that the Glu-Pro-Gly sequence at positions 383-385 o f the DEN2 E is a component of the site against which mab 3H5 is direc ted. In the recently determined three-dimensional structure of the rel ated tick-borne encephalitis virus E, the Glu-Pro-Gly sequence would b e located on the lateral surface of the immunoglobulin-likedomain that is proposed to bind to the host cell receptor. (C) 1996 Academic Pres s, Inc.