The molecular basis for evasion of antibody-mediated neutralization in flaviviruses

In order to establish infection, flaviviruses induce mutations in antigenic proteins as a strategy to evade neutralizing antibodies; a phenomenon known as antibody-mediated neutralization resistance. Most of the critical mutations associated with antibody resistance are located in the envelope protein domain 3 (ED3). In West Nile virus (WNV), and in other flaviviruses, mutations in ED3 are found throughout its structure, including regions outside of the viral epitope and/or not accessible to antibodies. Besides the trivial effects of local perturbations due to mutations in the epitope, these observations are consistent with our hypothesis that there is long-range communication connecting distant residues linked to the viral epitope. Mutations at sites distant but coupled to the epitope would lead to an alteration of affinity to antibody; e.g., resistance to antibody-mediated neutralization. In support of our hypothesis, we demonstrated by multiple biophysical approaches the communications among distant sites and a network of communications of energetically coupled residues. Within this network, mutations in WNV ED3 caused perturbations only in the loop connecting strands B-C (BC loop) by changing the magnitude of energetic coupling between these distant sites. The magnitude of perturbation conveyed by the mutations is represented by a Boltzmann distribution. This suggests that neutralization resistance is the manifestation of an equilibrium process governing the distribution between ED3 conformations that are responsible for antibody neutralization-resistance and nonresistance. Indeed we observed a linear correlation between affinity for antibody and magnitude of energetic coupling on the BC loop. To test the generality of these results, we investigated the ED3 from dengue virus type 2 (DENV2), a related flavivirus. We found that only the FG loop was susceptible to mutational perturbations. Remarkably, the BC and FG loops have been shown to be the dominant epitopes in ED3 for WNV and DENV2, respectively. Evidently these distant sites are energetically coupled to their respective viral epitope. This study reveals the strategy by which flavivirus employed to evade antibody, namely, establishment of long-range communications in viral proteins to expand the mutational repertoire to perturb the epitopes and lower the affinity for antibodies resulting in evasion of antibody-mediated neutralization.