West Nile virus in complex with the Fab fragment of a neutralizing monoclonal antibody

Abstract

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-Angstrom resolution with cryo-electron 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.

Fig. 1.

Cryo-EM reconstruction of WNV in complex with the Fab of the neutralizing anti-DIII mAb E16. (a) Cryo-EM micrograph of vitrified WNV particles alone (Left) and complexed with Fab E16 (Right). (Scale bar: 500 Å.) (b) Central cross-section of the 3D image reconstruction of the complex viewed down an icosahedral twofold axis. The positions of icosahedral twofold, threefold, and fivefold axes are indicated. (Scale bar: 100 Å.)

Fig. 2.

Stereoview showing the surface rendering of the 3D image reconstruction of WNV (green) in complex with Fab E16 (blue) at 14.5-Å resolution, viewed down an icosahedral twofold axis. The black triangles mark an icosahedral asymmetric unit.

Fig. 3.

Asymmetric utilization of binding sites by Fab E16. (a) Arrangement of the E glycoproteins on the viral surface. One icosahedral asymmetric unit is outlined in black. DI, DII, and DIII of each E momomer are colored red, yellow, and blue, respectively. The fusion loop is shown in green. (b) E protein arrangement within a defined icosahedral asymmetric unit. The shadows outline three E monomers. The three independent positions of DIII are labeled DIII-A, DIII-B, and DIII-C. (c) Difference density (gray) between the WNV+Fab E16 complex and WNV superpositioned onto the E protein arrangement, viewed down an icosahedral twofold axis. (d) As in c, but viewed down a fivefold axis of symmetry. Fab E16 binds to DIII-B and DIII-C, but not to the fivefold-proximal position DIII-A because of steric hindrance.

Fig. 4.

Fit of Fab E16 into the cryo-EM difference density (gray). (a) Crystal structure of the Fab E16+DIII complex shown after superpositioning of the x-ray DIII portion (blue) onto DIII-C of the fitted WNV E protein. The variable domains are shown in magenta; the constant domains are in green. The black axes represent the pseudodyads between light and heavy chain for the variable and the constant parts. (b) As in a, but the constant domains were adjusted to fit to the cryo-EM density, resulting in an increase of the elbow angle by 40°.

Fig. 5.

Stereoview showing the steric hindrance between a potentially bound Fab E16 and a neighboring E molecule close to the icosahedral fivefold axis. Part of the C_α backbone of one DIII-A is shown in dark blue with the E16 epitope in green and the corresponding bound Fab molecule in magenta. A severe clash can be observed between the Fab and a symmetry-related DIII-A (blue), verifying that E16 cannot bind to any of the DIII-As.

Fig. 6.

A computer-generated model, shown as a stereo diagram, suggests that the binding of the whole E16 antibody to WNV would leave surface areas accessible to putative receptor binding. The antibody density was calculated from the x-ray coordinates of an IgG (PDB ID code 1IGY) after superpositioning one Fab of the IgG on the fitted Fab E16. The WNV surface is shown in green, and Fab E16 is in blue. The unbound Fab portion of the IgG is colored red, and the Fc portion is yellow. The black triangle marks an icosahedral asymmetric unit. A large portion of the WNV surface is accessible, including the glycan at Asn-154 (red arrow). Severe clashes between antibodies bound to DIII-Bs (forming the outer circle around the icosahedral fivefolds) suggest that only some of the possible binding sites can be occupied simultaneously by the whole E16 antibody. Partial occupancy would leave the area close to the fivefold axes accessible to putative receptor molecules.

Fig. 7.

Inhibition of the low pH-triggered conformational rearrangement to a putative prefusion T = 3 structure during flavivirus cell entry. (a) Dengue virus T = 3 particle suggested to be an intermediate in the rearrangement of the E glycoproteins (5). DI, DII, and DIII of each E momomer are colored red, yellow, and blue, respectively. The fusion loop is shown in green. The black triangle marks an icosahedral asymmetric unit. (b) Arrangement of DIIIs at the icosahedral threefold (quasi-sixfold) axis of the T = 3 particle. The shadows outline symmetry-related asymmetric units. DIIIs originating from position DIII-B of the mature virion are colored blue, and DIII-Cs are light blue. (c) Arrangement of the variable domains of the docked Fab E16 at the icosahedral threefold axis of the T = 3 particle. Each of the six Fabs is colored differently. Significant clashes between neighboring Fabs can be observed, potentially inhibiting the fusion pathway. (d) Arrangement of the constant domains of Fab E16 at the icosahedral threefold axis also results in major clashes.