Genotypic Differences in Dengue Virus Neutralization Are Explained by a Single Amino Acid Mutation That Modulates Virus Breathing

ABSTRACT Flaviviruses sample an ensemble of virion conformations resulting from the conformational flexibility of their struc-tural proteins. To investigate how sequence variation among strains impacts virus breathing, we performed studies with the monoclonal antibody (MAb) E111, which binds an inaccessible domain III envelope (E) protein epitope of dengue virus serotype 1 (DENV1). Prior studies indicated that an observed ~200-fold difference in neutralization between the DENV1 strainsWestern Pacific-74 (West Pac-74) and 16007 could not be explained by differences in the affinity of MAb E111 for each strain. Through neutralization studies with wild-type and variant viruses carrying genes encoding reciprocal mutations at all 13 amino acid dif-ferences between the E proteins ofWest Pac-74 and 16007, we found that E111 neutralization susceptibility mapped solely to the presence of a lysine or arginine at E domain II residue 204, located distally from the E111 epitope. This same residue correlated with neutralization differences observed for MAbs specific for epitopes distinct from E111, suggesting that this amino acid dic-tates changes in the conformational ensembles sampled by the virus. Furthermore, an observed twofold difference in the stabil-ity of infectiousWest Pac-74 versus 16007 in solution also mapped to E residue 204. Our results demonstrate that neutralization susceptibility can be altered in an epitope-independent manner by natural strain variation that influences the structures sam-pled by DENV. That different conformational ensembles of flaviviruses may affect the landscape available for antibody binding, as well as virus stability, has important implications for functional studies of antibody potency, a critical aspect of vaccine devel-opment