Stereophysicochemical variability plots highlight conserved antigenic areas in Flaviviruses

Abstract

Background: Flaviviruses, which include Dengue (DV) and West Nile (WN), mutate in response to immune system pressure. Identifying escape mutants, variant progeny that replicate in the presence of neutralizing antibodies, is a common way to identify functionally important residues of viral proteins. However, the mutations typically occur at variable positions on the viral surface that are not essential for viral replication. Methods are needed to determine the true targets of the neutralizing antibodies.

Results: Stereophysicochemical variability plots (SVPs), 3-D images of protein structures colored according to variability, as determined by our PCPMer program, were used to visualize residues conserved in their physical chemical properties (PCPs) near escape mutant positions. The analysis showed 1) that escape mutations in the flavivirus envelope protein are variable residues by our criteria and 2) two escape mutants found at the same position in many flaviviruses sit above clusters of conserved residues from different regions of the linear sequence. Conservation patterns in T-cell epitopes in the NS3- protease suggest a similar mechanism of immune system evasion.

Conclusion: The SVPs add another dimension to structurally defining the binding sites of neutralizing antibodies. They provide a useful aid for determining antigenically important regions and designing vaccines.

Figure 1

The relative specific entropy (SE) function of PCPMer (Bin Zhou et al., in preparation) defines motifs even in alignments where the sequence conservation varies locally. The top of the figure shows a section of the sequence alignment for the NS3 protein. The next section shows the PCPMer output, indicating the motifs in the NS3 protease according to the sequence of DV-2 as a function of the specific entropy level (numbers to the left). PCPMer parameters were: Gap cutoff of 2, length cutoff of 5, relative entropy range between 1 and 2.5 with a step of 0.2. Note the conserved sequences around the active site residues (bold letters) of the protease are followed by variable regions that retain conservation in one of the five physical chemical property vectors. The output is colored to reflect the degree of conservation at each position.

Figure 2

Variability analysis of the envelope protein of DV-2 and illustration of how escape mutants mark cloaked conserved residues. a) PCP-motifs (blue) common to all flavivirus envelope proteins are mapped on the structure of DV-2 Env (PDB file 1OAN; the start and end residues are numbered). Note the high conservation of the fusion peptide (arrow) and two loop regions adjacent to it from other areas of the molecule. b) Stereochemical variability plot (SVP) of the DV2-Env (PDB file 1OKE), showing the per residue variation across the Flaviriridiae. Known escape mutants of DV-2 and DV-3 [10, 26] are labeled and the residue names are colored according to their variability. The boxed residues are intermediate in the conservation scale (white). c and d) Surface plots of the SVP shown in figure 2B, showing the conserved (overall blue, c) face, where the motifs of conserved areas map. The variable face (d, mostly red), which matches the orientation of the molecule where the escape mutants map to.

Figure 3

a) Local surface plot of the DV-Env SVP around residue 124 (which is highly variable but has been colored green here for clarity), illustrating how the residue forms part of a patch of variable (red) residues b) Removing part of the surface reveals how I124 lies above the highly conserved residues Cys60-121 (disulfide bonded) and Tyr59 that are distant in the sequence of the protein.

Figure 4

a) Local surface plot of the area around two escape mutant positions in domain III. Residues 307 and 311 are highly variable and have been colored green here for visibility. The faint blue area on the surface near residue 307 comes from a highly conserved aromatic residue, Phe306, which lies under the variable residue and forms a cluster with another conserved residue, Tyr326 (b).

Figure 5

The conserved essential residues in two serotype specific T-cell epitopes of the NS3 protease are followed by variable residues that will affect MHC binding. The crystal structure of the NS3 protease of DV-2 is colored according to sequence variability across the flaviviruses(see figure 1 and 2 for details). The catalytic triad sidechains are shown in neon and labeled. The T-cell epitopes (right) around the catalytic residues Asp75 (residues 71–79), and Ser135 (133–143), are shown as space filling and color coded to reflect variability, except that the 100% conserved catalytic residues are both black.