ABSTRACT
Dengue fever is a re-emerging tropical disease and its severe form is caused by cross-reactivity between its four serotypes (DEN1, DEN2, DEN3 and DEN4). The third domain of the viral envelope protein (ED3) contains the two major putative epitopes and is a highly suitable model protein for examining the molecular determinants of a virus' sero-specificity. Here we examine d the sero-specificity and cross-reactivity of the immune response against DEN3 and DEN4 ED3 using six epitope grafted ED3 variants where the surface-exposed epitope residues from DEN3 ED3 were switched to those of DEN4 ED3 and vice versa. We prepared anti-DEN3 and anti-DEN4 ED3 serum by immunizing Swiss albino mice and measured their reactivities against all six grafted mutants. As expected, both sera exhibited strong reactivity against its own serotype's ED3, and little cross-reactivity against their counterpart serotype's ED3s. E2 played a major role in the sero-specificity of anti-DEN3 serum, whereas E1 was important for DEN4 ED3's sero-specificity. Next, the reactivity patterns corroborated our working hypothesis that sero-specificity could be transferred by grafting the surface exposed epitope residues from one serotype to the other. To analyze the above results from a structural viewpoint, we determined the crystal structure of a DEN4 ED3 variant, where E2 was grafted from DEN3 ED3, at 2.78Å resolution and modeled the structures of the five remaining grafted variants by assuming that the overall backbone remained unchanged. The examination of the electrostatic and molecular surfaces of the variants suggested some further rationale for the sero-specificity of the immune responses.
Subject(s)
Amino Acids/chemistry , Dengue Virus/chemistry , Epitopes/chemistry , Immune Sera/chemistry , Viral Envelope Proteins/chemistry , Amino Acid Sequence , Amino Acids/immunology , Animals , Cross Reactions , Crystallography, X-Ray , Dengue Virus/genetics , Dengue Virus/immunology , Epitopes/genetics , Epitopes/immunology , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genetic Engineering , Immunization , Mice , Models, Molecular , Molecular Sequence Data , Mutation , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Sequence Alignment , Serotyping , Viral Envelope Proteins/genetics , Viral Envelope Proteins/immunologyABSTRACT
Dengue viruses (DEN) are classified into four serotypes (DEN1-DEN4) exhibiting high sequence and structural similarities, and infections by multiple serotypes can lead to the deadly dengue hemorrhagic fever. Here, we aim at characterizing the thermodynamic stability of DEN envelope protein domain III (ED3) during its evolution, and we report a structural analysis of DEN4wt ED3 combined with a systematic mutational analysis of residues 310 and 387. Molecular modeling based on our DEN3 and DEN4 ED3 structures indicated that the side-chains of residues 310/387, which are Val(310)/Ile(387) and Met(310)/Leu(387) in DEN3wt and DEN4wt, respectively, could be structurally compensated, and that a "size switch type repacking" might have occurred at these sites during the evolution of DEN into its four serotypes. This was experimentally confirmed by a 10°C and 5°C decrease in the thermal stability of, respectively, DEN3 ED3 variants with Met(310)/Ile(387) and Val(310)/Leu(387), whereas the variant with Met(310)/Leu(387), which contains a double mutation, had the same stability as the wild type DEN3. Namely, the Met310Val mutation should have preceded the Leu387Ile mutation in order to maintain the tight internal packing of ED3 and thus its thermodynamic stability. This view was confirmed by a phylogenetic reconstruction indicating that a common DEN ancestor would have Met(310)/Leu(387), and the intermediate node protein, Val(310)/Leu(387), which then mutated to the Val(310)/Ile(387) pair found in the present DEN3. The hypothesis was further confirmed by the observation that all of the present DEN viruses exhibit only stabilizing amino acid pairs at the 310/387 sites.
Subject(s)
Dengue Virus/chemistry , Viral Envelope Proteins/chemistry , Amino Acid Sequence , Circular Dichroism , Dengue Virus/classification , Enzyme-Linked Immunosorbent Assay , Models, Molecular , Molecular Sequence Data , Phylogeny , Protein Stability , Sequence Homology, Amino Acid , Thermodynamics , Viral Envelope Proteins/isolation & purificationABSTRACT
Dengue viruses are classified into four serotypes. Here, we report a 1.7 Å crystal structure of a recombinant dengue-3 envelope protein domain III (ED3), which contains most of the putative epitopes. Although the fold was well conserved, we found that a local backbone deformation in the first ß-strand, which contains the putative epitope-1, occurred upon domain isolation. Furthermore, a comparison with dengue-2 ED3 indicated a large structural change by as much as 4.0 Å at Asp(662), located in epitope-2. These minute structural and surface properties changes observed in the high resolution ED3 structure represent potential determinants for serospecificity and epitope recognition by antibodies.