Flaviviruses are a group of human pathogens causing severe encephalitic or hemorrhagic diseases that include West Nile, dengue and yellow fever viruses. Here, using X-ray crystallography we have defined the structure of the flavivirus cross-reactive antibody E53 that engages the highly conserved fusion loop of the West Nile virus envelope glycoprotein. Using cryo-electron microscopy, we also determined that E53 Fab binds preferentially to spikes in noninfectious, immature flavivirions but is unable to bind significantly to mature virions, consistent with the limited solvent exposure of the epitope. We conclude that the neutralizing impact of E53 and likely similar fusion-loop-specific antibodies depends on its binding to the frequently observed immature component of flavivirus particles. Our results elucidate how fusion-loop antibodies, which comprise a significant fraction of the humoral response against flaviviruses, can function to control infection without appreciably recognizing mature virions. As these highly cross-reactive antibodies are often weakly neutralizing they also may contribute to antibodydependent enhancement and flavi virus pathogenesis thereby complicating development of safe and effective vaccines.

The envelope glycoprotein (E) of West Nile virus (WNV) undergoes a conformational rearrangement triggered by low pH that results in a class II fusion event required for viral entry. Herein we present the 3.0-Å crystal structure of the ectodomain of WNV E, which reveals insights into the flavivirus life cycle. We found that WNV E adopts a three-domain architecture that is shared by the E proteins from dengue and tick-borne encephalitis viruses and forms a rod-shaped configuration similar to that observed in immature flavivirus particles. Interestingly, the single N-linked glycosylation site on WNV E is displaced by a novel ␣-helix, which could potentially alter lectin-mediated attachment. The localization of histidines within the hinge regions of E implicates these residues in pH-induced conformational transitions. Most strikingly, the WNV E ectodomain crystallized as a monomer, in contrast to other flavivirus E proteins, which have crystallized as antiparallel dimers. WNV E assembles in a crystalline lattice of perpendicular molecules, with the fusion loop of one E protein buried in a hydrophobic pocket at the DI-DIII interface of another. Dimeric E proteins pack their fusion loops into analogous pockets at the dimer interface. We speculate that E proteins could pivot around the fusion loop-pocket junction, allowing virion conformational transitions while minimizing fusion loop exposure. West Nile virus (WNV), a member of the Flavivirus genus, is closely related to several arthropod-borne human pathogens, including Japanese encephalitis virus, St. Louis encephalitis virus, Yellow fever virus, Dengue virus (DENV), and Tick-borne encephalitis virus (TBEV). Endemic to parts of Africa, Asia, Europe, and the Middle East, WNV has recently spread to the Western Hemisphere (38). The virus cycles enzootically between birds and mosquitoes but can infect mammals, including humans. Most human infections are asymptomatic or mild, but in a small subset of individuals the infection develops into a severe neuroinvasive disease (18). Treatment for WNV infection is supportive, as there is currently no vaccine or specific therapy for use in humans (15).The WNV genome encodes 10 proteins, including three structural (capsid, premembrane [prM], and envelope [E]) and seven nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins. These are translated as a single polypeptide, which is subsequently cleaved by viral and cellular proteases (6). The initial step toward virion generation occurs when the 11-kb positive-strand RNA genome, in complex with capsid protein, buds through the endoplasmic reticulum membrane. A lipid envelope coats the nascent flavivirus particles and contains 180 molecules each of E and prM organized into 60 asymmetric trimeric spikes consisting of prM-E heterodimers (45,46). At the apices of the spikes, prM caps the fusion loop of E (34), presumably to prevent premature fusion as the virus passes through the acidic secretory pathway (19). A furin-catalyzed membrane-proximal cleavage releases the Nterminal...

Nanobodies (Nbs) or single-domain antibodies are among the smallest and most stable binder scaffolds known. In vitro display is a powerful antibody discovery technique used worldwide. We describe the first adaptation of in vitro mRNA/cDNA display for the rapid, automatable discovery of Nbs against desired targets, and use it to discover the first ever reported nanobody against the human full-length glucose transporter, GLUT-1. We envision our streamlined method as a bench-top platform technology, in combination with various molecular evolution techniques, for expedited Nb discovery.