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. 2009 Oct 21;28(20):3269-76.
doi: 10.1038/emboj.2009.245. Epub 2009 Aug 27.

Structural basis for the preferential recognition of immature flaviviruses by a fusion-loop antibody

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Structural basis for the preferential recognition of immature flaviviruses by a fusion-loop antibody

Mickaël V Cherrier et al. EMBO J. .

Abstract

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 antibody-dependent enhancement and flavi virus pathogenesis thereby complicating development of safe and effective vaccines.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
Organization of the E glycoprotein on the surface of (A) immature and (B) mature virus. E monomers are shown as a Cα backbone with DI, DII and DIII in red, yellow and blue, respectively. The black triangle represents an icosahedral asymmetric unit. Green spheres mark the E53 epitope residues (75, 76, 99, 106 and 107) determined by yeast surface display.
Figure 2
Figure 2
Crystal structure of E53 Fab in complex with the WNV envelope glycoprotein. On the left, ribbon diagram of the complex, with DII of E depicted in yellow, DI in red, the antibody heavy chain in purple and the light chain in cyan. The structurally defined epitope is displayed in magenta. On the right, close-up view of the E53 epitope, with the side chains of contacted residues coloured using magenta for carbon, red for oxygen, green for sulphur and blue for nitrogen atoms (<4.0 Å distance).
Figure 3
Figure 3
CryoEM reconstruction of immature WNV in complex with Fab fragments of the anti-fusion-loop antibody E53. (A) Stereoview showing the immature WNV–E53 Fab complex at 15 Å resolution. The density is coloured according to radius (red: 190 Å; yellow: 220 Å; green: 235 Å; light blue: 260 Å; blue: 305 Å). The black triangle shows an icosahedral asymmetric unit. The dashed circles mark the positions where the E53 Fab is bound to the fusion loop of molecules A and B, respectively. The scale bar represents 100 Å. Similar results were obtained for immature DENV in complex with E53 Fab (Supplementary Figure 1). (B) Stereoview showing the difference density between the immature WNV–E53 Fab complex and immature WNV (blue) superpositioned onto the surface of immature WNV (gold) at 24 Å resolution. The black triangle marks an icosahedral asymmetric unit. The scale bar represents 100 Å. (C) The difference density (blue mesh) is shown relative to the immature E protein structure coloured as in Figure 1.
Figure 4
Figure 4
Stereoview showing E53 Fab, pr and E as fitted into the cryoEM reconstruction of the immature WNV–E53 Fab complex (grey mesh). Shown is the fit into the density associated with molecule A. Domain DII of the E protein and the fusion loop are coloured in yellow and green, respectively. The E53 Fab variable and constant domains are coloured in light and dark magenta, respectively. The pr molecules are coloured in orange.
Figure 5
Figure 5
Stereoviews showing that E53 Fab cannot bind to the mature virus. (A) The crystal structure of the E53 Fab–E complex was superpositioned onto DII of molecule A' to show that severe clashes would occur between the Fab and the E molecules. The Fab is shown in grey. Each protein is shown as a Cα backbone. Domains DI, DII and DIII of the E protein are coloured in red, yellow and blue, respectively. The fusion loop is coloured in green. (B) Side view of structure. The direction of view is indicated in (A) by a cartoon eye.
Figure 6
Figure 6
CryoEM images of two different WNV samples. Some immature (red arrows) and partially immature particles (green arrows) are frequently found in preparations of mature virus. Fully mature particles are marked with blue arrows. The same type of partially immature virus is usually also present in mature DENV samples. The scale bars represent 500 Å.

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