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

Abstract

The antigenic site of dengue type 2 virus (DEN2)-neutralizing monoclonal antibody (mab) 3H5 was investigated by mutational analysis. Sequence comparisons indicated that much of the 12-amino-acid sequence extending from position 386 to 397 of the DEN2 envelope glycoprotein (E) previously thought to represent the DEN2-specific mab 3H5 binding site was 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 mab 3H5. We investigated this possibility by employing the previously constructed chimeric DEN2(PreM-E)/DEN4 cDNA clone to produce viable mutants bearing DEN2 PreM and E sequences that could be analyzed for binding to and neutralization by mab 3H5. We constructed 13 such DEN2 mutants that contained a single amino acid substitution in the region between positions 383 and 393 of DEN2 E. Each single substitution in the region spanning positions 386 through 393 of DEN2 yielded a virus that was as reactive with mab 3H5 as the parental chimeric virus. These results are consistent with the extent of sequence conservation in the region. In contrast, 5 of 6 mutants that sustained an amino acid substitution at position 383, 384, or 385 failed to react with mab 3H5 as detected by immunofluorescence assay and failed to be neutralized by the mab. Interestingly, each of the 5 mab-resistant DEN2 mutants also exhibited reduced mouse neurovirulence compared to parental chimeric DEN2 when inoculated intracerebrally. These observations suggest that the Glu-Pro-Gly sequence at positions 383-386 of the DEN2 E is a component of the site against which mab 3H5 is directed. In the recently determined three-dimensional structure of the related tick-borne encephalitis virus E, the Glu-Pro-Gly sequence would be located on the lateral surface of the immunoglobulin-like domain that is proposed to bind to the host cell receptor.

Identification of a major determinant of mouse neurovirulence of dengue virus type 2 using stably cloned genomic-length cDNA.

“…A second determinant of mouse neurovirulence is the epitope of DEN-2 E recognized by monoclonal antibody 3H5 (Trirawatanapong et al, 1992 ;Hiramatsu et al, 1996). In a neurovirulent DEN-2(prM-E)-DEN-4 hybrid virus, Hiramatsu et al (1996) made a number of point mutations in this epitope from E383 to E393.…”

West Nile virus in complex with the Fab fragment of a neutralizing monoclonal antibody

“…Indeed, the E glycoprotein is the principal antigen that elicits neutralizing antibodies against flaviviruses. Many of the neutralizing epitopes are located in DIII of E and in close proximity to the conserved fusion loop in DII (20)(21)(22)(23)(24)(25)(26)(27)(28). E16 is a strongly neutralizing mAb against WNV that recognizes four polypeptide loops at the tip of DIII and inhibits infection primarily subsequent to cell attachment.…”

Residues in domain III of the dengue virus envelope glycoprotein involved in cell-surface glycosaminoglycan binding

“…This arrangement results in the loops of DIII forming the most distal projections from the virion surface, ideally positioned for interaction with a host-cell receptor. Antibody neutralization-and peptide-based studies have further supported a critical role for DIII in host-cell binding (Abd-Jamil et al, 2008;Hiramatsu et al, 1996;Lin et al, 1994;Roehrig et al, 1998;Thullier et al, 2001). In addition to these studies, investigation of sequence variation among cell cultureadapted, serially passed and mutant viruses has also supported a receptor-binding role for DIII (Erb et al, 2010;Lee & Lobigs, 2000, 2002Lee et al, 2004).…”