Date Published: February 16, 2017
Publisher: Public Library of Science
Author(s): Leslie Goo, Laura A. VanBlargan, Kimberly A. Dowd, Michael S. Diamond, Theodore C. Pierson, Alexander A. Khromykh.
The structural flexibility or ‘breathing’ of the envelope (E) protein of flaviviruses allows virions to sample an ensemble of conformations at equilibrium. The molecular basis and functional consequences of virus conformational dynamics are poorly understood. Here, we identified a single mutation at residue 198 (T198F) of the West Nile virus (WNV) E protein domain I-II hinge that regulates virus breathing. The T198F mutation resulted in a ~70-fold increase in sensitivity to neutralization by a monoclonal antibody targeting a cryptic epitope in the fusion loop. Increased exposure of this otherwise poorly accessible fusion loop epitope was accompanied by reduced virus stability in solution at physiological temperatures. Introduction of a mutation at the analogous residue of dengue virus (DENV), but not Zika virus (ZIKV), E protein also increased accessibility of the cryptic fusion loop epitope and decreased virus stability in solution, suggesting that this residue modulates the structural ensembles sampled by distinct flaviviruses at equilibrium in a context dependent manner. Although the T198F mutation did not substantially impair WNV growth kinetics in vitro, studies in mice revealed attenuation of WNV T198F infection. Overall, our study provides insight into the molecular basis and the in vitro and in vivo consequences of flavivirus breathing.
Flaviviruses are enveloped, positive-stranded RNA viruses typically transmitted to humans via infected ticks or mosquitoes. As many members of the flavivirus genus are emerging, they constitute a significant threat to global health. For example, approximately 390 million humans worldwide are infected annually with one of the four serotypes of dengue virus (DENV) . West Nile virus (WNV) was introduced into North America in 1999  and rapidly became the leading cause of arbovirus-related encephalitis in the United States ; Zika virus (ZIKV) emerged from Asia and Africa for the first time in 2007 and has since caused epidemics in French Polynesia , Oceania , and most recently, the Americas [6, 7]. Despite causing significant morbidity, licensed vaccines or therapeutic agents to protect humans against many flaviviruses are lacking. However, highly effective vaccines for some flaviviruses such as yellow fever virus, Japanese encephalitis virus, and tick-borne encephalitis virus are in use. The induction of a neutralizing antibody (NAb) response is a correlate of protection for these vaccines [8–12]. While a live-attenuated tetravalent DENV vaccine was recently licensed, its efficacy and durability varied by DENV serotype, pre-existing flavivirus immune status, and age of vaccine recipient [13–15]; the relationship between neutralization titer and protection for this vaccine is less clear. Because of the importance of antibodies for flavivirus immunity, a detailed understanding of flavivirus antigenic structure as well as the mechanisms of antibody-mediated neutralization is critical .
Our study demonstrates that a single residue in the E protein DI-DII hinge regulates conformational dynamics in distinct flaviviruses, with relevant consequences in vivo for WNV infection and pathogenesis. Although conformational flexibility has been described for different virus families , the first evidence of the dynamic properties of flaviviruses came from structural studies of mAb 1A1D-2, which is capable of neutralizing multiple DENV serotypes, yet binds to an epitope in the β-strand of DIII that is not predicted to be fully accessible on the mature virus particle . Monoclonal antibody 1A1D-2 can bind to DENV particles at 37°C, but not at 4°C, suggesting that at an elevated temperature, this mAb trapped a conformation on which the DIII β-strand epitope was otherwise not accessible at lower temperatures . Consistent with the conformational flexibility of flaviviruses, subsequent studies showed that exposure of DENV2 virions to physiological temperatures in the absence of antibody results in the formation of an expanded ‘bumpy’ structure, on which E protein dimers are more loosely arranged and are rotated outwards relative to their orientation on fully mature particles [80, 81]. However, this ‘bumpy’ structure was not observed for all DENV strains or serotypes [81, 82], suggesting that sequence variation contributes to the structural pathways sampled by virus breathing at equilibrium.