are a group of positive-stranded RNA viruses that are of global significance due to their widespread distribution and their ability to cause a variety of diseases in humans 
. West Nile virus (WNV) is a mosquito-borne member of this genus and is the etiologic agent of West Nile encephalitis. WNV is endemic in parts of Africa, Australia, Europe, Asia, and the Middle East and has been responsible for periodic outbreaks of encephalitis in humans and horses. The introduction of WNV into North America in 1999 and its rapid spread across the United States into Canada, Mexico, and the Caribbean identifies this virus as an emerging pathogen of clinical and economic significance for the Western Hemisphere (reviewed in 
). While seroprevalence studies indicate that most WNV infections of humans are subclinical, clinically apparent infections range from a febrile illness (West Nile fever) to more severe and potentially fatal neurologic disease 
. Currently, no WNV vaccine has been approved for use in humans and treatment is supportive.
Flaviviruses are small (~50 nm diameter) spherical virions composed of three structural proteins (envelope (E), premembrane (prM), and capsid (C)), a lipid envelope, and an ~11 kilobase monocistronic RNA of positive-sense polarity 
. Crystal structures of the E protein of several related flaviviruses (WNV, dengue virus (DENV), tick-borne encephalitis virus (TBE)) reveal an organization of three domains connected by flexible hinges (reviewed in 
). Domain III (DIII) is an immunoglobulin-like fold that is thought to participate in interactions between virions and cellular factors associated with virus entry. Domain II (DII) is a long, finger-like domain that contains a stretch of 13 conserved, hydrophobic residues that form an internal fusion loop. DIII and DII are linked together by a β-barrel structure that comprises domain I (DI). The structure of prM, which forms heterodimers with the E protein during virion biogenesis, is presently unknown.
Flaviviruses assemble at the endoplasmic reticulum (ER) and bud into the lumen as immature virus particles 
. Cryoelectron microscopic reconstructions of immature virions reveal an icosahedral arrangement of 60 trimeric spikes composed of prM
E heterodimers in which the prM protein is positioned to cover the fusion peptide located at the distal end of each E protein of the trimer 
. In this position, prM may prevent conformational changes that would inactivate the E protein during virion egress through mildly acidic compartments of the secretory pathway 
. During transit through the trans
-Golgi network (TGN), prM is cleaved by a cellular furin-like protease resulting in the formation of a small virion-associated M peptide and the release of the amino-terminal “pr” portion of the protein 
. This required cleavage step promotes a rearrangement of E proteins on the surface of the virion and the formation of a mature virus particle. Mature flavivirus virions are relatively smooth and composed of 90 anti-parallel E protein dimers arranged with pseudo-icosahedral symmetry 
Antibodies are a critical component of host defenses against flavivirus infection and mediate protection via effector functions and by direct neutralization of virus (reviewed in 
). The primary target for neutralizing antibodies is the E protein, although antibodies specific for prM have been identified 
. More than twelve distinct epitopes have been identified on the surface of the E protein that elicit antibodies characterized by varying degrees of neutralization potency in vitro and efficacy in vivo 
. Neutralization of flavivirus infection is a multiple “hit” phenomenon in which virus inactivation occurs once the number of antibodies bound to a virion exceeds a required threshold 
. Previous studies with an extremely potent neutralizing mAb specific for a highly accessible epitope on an upper lateral surface of WNV DIII (DIII-lr) suggest this threshold is approximately 30 mAbs 
The pseudo-icosahedral arrangement of E proteins on the virion displays the E protein in three distinct chemical environments defined by proximity to the two-, three-, or five-fold axes of symmetry 
. Epitopes in each of these environments may be differentially accessible for antibody binding due to steric constraints imposed by adjacent E proteins on the virus particle 
. As a result, the number of sites available for binding may differ among structurally distinct epitopes on the virion. Antibodies that bind highly exposed determinants may exceed the stoichiometric threshold for neutralization by binding a small fraction of accessible epitopes on the virion (low occupancy). In contrast, epitopes predicted to be poorly exposed may require nearly complete occupancy to achieve threshold requirements for neutralization 
. Furthermore, some epitopes on the virion may not be accessible to antibody engagement with a stoichiometry that exceeds the threshold required for neutralization. Thus, antibodies that recognize such epitopes may neutralize poorly, or not at all, even at concentrations that permit saturation because too few antibodies can simultaneously dock on the virion. Paradoxically, many antibodies that recognize poorly accessible epitopes on the mature virion still show neutralizing activity in vitro and in vivo 
. How antibodies engage poorly accessible epitopes on virions with a stoichiometry that permits neutralization is difficult to reconcile using existing static models of virion structure and envelope organization. In this study, we investigate how changes in flavivirus structure associated with virion maturation impact the neutralizing activity of antibodies to WNV.