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.

Flaviviruses, such as West Nile virus (WNV), are significant human pathogens. The humoral immune response plays an important role in the control of flavivirus infection and disease. The structure of WNV complexed with the Fab fragment of the strongly neutralizing mAb E16 was determined to 14.5-Å resolution with cryoelectron microscopy. E16, an antibody with therapeutic potential, binds to domain III of the WNV envelope glycoprotein. Because of steric hindrance, Fab E16 binds to only 120 of the 180 possible binding sites on the viral surface. Fitting of the previously determined x-ray structure of the Fab-domain III complex into the cryo-electron microscopy density required a change of the elbow angle between the variable and constant domains of the Fab. The structure suggests that the E16 antibody neutralizes WNV by blocking the initial rearrangement of the E glycoprotein before fusion with a cellular membrane.cryo-electron microscopy ͉ flavivirus ͉ neutralization W est Nile virus (WNV) causes a febrile illness in humans that can progress to encephalitis, paralysis, and death. Although endemic in parts of Africa, Asia, Europe, and the Middle East, it was first isolated in the United States in 1999 and has subsequently spread throughout North America and the Caribbean (1-3). The similar Kunjin virus is found in Australia (4). WNV, a member of the Flaviviridae family, is closely related to other arthropod-borne, medically important viruses, such as dengue, yellow fever, Japanese encephalitis, and tick-borne encephalitis viruses. These small, Ϸ500-Å-diameter, lipidenveloped viruses enter their host cells by receptor-mediated endocytosis. A low pH-triggered conformational rearrangement of the viral surface glycoproteins resulting in a fusion-active state of the virion allows the release of the single-stranded, positivesense RNA genome from the endosomes into the cytoplasm.The outer surface of mature infectious particles is formed by an icosahedral scaffold of 90 homodimers of the membraneanchored envelope glycoprotein (E). The three E monomers per icosahedral asymmetric unit each have distinctly different environments (5, 6), contrary to most other icosahedral viruses in which all subunits have at least quasi-similar environments. The ectodomain of E consists of three structural domains, DI, DII, and DIII (7-9). DI is structurally positioned between DII and DIII. The dimerization DII contains a 12-residue-long loop essential for virus-cell membrane fusion and, in dengue virus, has carbohydrate moieties that mediate receptor binding (10). The C-terminal DIII undergoes a major, pH-triggered, positional rearrangement essential for fusion and may also be involved in receptor binding (7,(11)(12)(13)(14)(15)(16)(17)(18).The humoral immune response is crucial for protection against flavivirus infection and disease (19). Antibodies can prevent infection by interference with functions mediated by viral surface proteins, such as receptor attachment, virus internalization, and membrane fusion. Indeed, the E glycoprotein is the...

Many flaviviruses are significant human pathogens, with the humoral immune response playing an essential role in restricting infection and disease. CR4354, a human monoclonal antibody isolated from a patient, neutralizes West Nile virus (WNV) infection at a postattachment stage in the viral life-cycle. Here, we determined the structure of WNV complexed with Fab fragments of CR4354 using cryoelectron microscopy. The outer glycoprotein shell of a mature WNV particle is formed by 30 rafts of three homodimers of the viral surface protein E. CR4354 binds to a discontinuous epitope formed by protein segments from two neighboring E molecules, but does not cause any detectable structural disturbance on the viral surface. The epitope occurs at two independent positions within an icosahedral asymmetric unit, resulting in 120 binding sites on the viral surface. The cross-linking of the six E monomers within one raft by four CR4354 Fab fragments suggests that the antibody neutralizes WNV by blocking the pH-induced rearrangement of the E protein required for virus fusion with the endosomal membrane.antibody | cryoelectron microscopy | flavivirus W est Nile virus (WNV) is a human pathogen that causes a febrile illness, which can progress to encephalitis, paralysis, and death. The virus is endemic in parts of Africa, Asia, and Europe, and in the past decade has spread throughout North America and into Central and South America (1). WNV is closely related to other arthropod-transmitted, medically relevant flaviviruses, such as dengue, yellow fever, Japanese encephalitis, and tick-borne encephalitis viruses. These lipid-enveloped viruses enter host cells by receptor-mediated endocytosis. The singlestranded, positive-sense RNA genome is released into the cytoplasm after low pH induces the fusion of the viral lipid envelope with the endosomal membrane.Mature WNV virions are roughly spherical with a diameter of about 500 Å. The outer viral surface is composed of an icosahedral scaffold of 180 closely packed copies of the membrane-anchored envelope (E) glycoprotein. Sets of three, nearly parallel E homodimers are associated into rafts that form a herringbone pattern on the surface of mature virions. The ectodomain of E has three structural domains, DI, DII, and DIII (2-5), with domain DI positioned structurally between DII and DIII. DII contains a fusion loop at its distal end that is indispensable for virus-cell membrane fusion. The Ig-like C-terminal domain DIII undergoes a major, pH-triggered positional rearrangement essential for fusion, and may also be involved in receptor binding (2, 6-12). During cell entry of flaviviruses, low endosomal pH triggers a proposed E protein rearrangement cascade, including the dissociation of E dimers and the outward rotation of DII during the repositioning of E monomers into fusion-active trimers (6, 9, 13).The humoral immune response is essential for protection against flavivirus infection and disease (14,15). The E glycoprotein is the principal antigen that elicits neutralizing antibodies agai...

Infection with the human parvovirus B19 usually results in a mild disease known as erythema infectiosum, or fifth disease