The structure of a neutralized virus: canine parvovirus complexed with neutralizing antibody fragment

Abstract

Background: Members of the Parvovirus genus cause a variety of diseases in mammals, including humans. One of the major defences against viral infection is the presence of neutralizing antibodies that prevent virus particles from infecting target cells. The mechanism of neutralization is not well understood. We therefore studied the structure of canine parvovirus (CPV) complexed with the Fab fragment of a neutralizing antibody, A3B10, using image reconstruction of electron micrographs of vitrified samples, together with the already known structure of CPV from X-ray crystallographic data.

Results: The structure of the complex of CPV with Fab A3B10 has been determined to 23 A resolution. The known CPV atomic structure was subtracted from the electron density of the complex, and the difference map was used to fit the atomic coordinates of a known Fab fragment, HyHEL-5. The long axis of each Fab molecule is oriented in a near radial direction, inclined away from the two-fold axes. The viral epitope consists of 14 amino acid residues found in loops 1, 2 and 3 on the capsid surface, which include previously identified escape mutations.

Conclusions: The mode of Fab binding suggests that the A3B10 neutralizing antibody cannot bind bivalently to the capsid across the two-fold axes, consistent with the observation that whole A3B10 antibody readily precipitates CPV. Since Fab A3B10 can also neutralize the virus, mechanisms of neutralization such as interference with cell attachment, cell entry, or uncoating, must be operative.

Fig. 1

(opposite). Roadmap showing the surface amino acids of CPV for one asymmetric unit of the icosahedral particle. The radial distance of the surface from the viral center is color-coded at the top. Boundaries between symmetry-related polypeptides are indicated with a heavy black line. The lower figure shows (shaded) epitopes identified by pepscan or peptide mapping. Epitopes on the amino terminus of VP1 identified by pepscan and not present in the atomic structure are indicated as a line from the five-fold axis. Escape mutations to Mab A3B10 are indicated in red (epitope A) and blue (epitope B). (The figure was produced in part by the program Roadmap [46]).

Fig. 2

Micrograph of a frozen-hydrated sample of CPV complexed with Fab A3B10. The boxed region in the top image is shown enlarged three times in the bottom image. The scale bars indicate 1000 Å.

Fig. 3

Stereoviews of the image reconstruction of the CPV:Fab A3B10 complex (a) and of the CPV capsid atomic structure (b), both represented at 23Å resolution. One CPV asymmetric unit, or 1/60 of the T = 1 virus, is outlined. The five-fold axis is indicated with a filled pentagon, the three-fold axes are indicated by filled triangles and the two-fold axis is indicated by a filled ellipse. The Fab region is colored red and the capsid region is gray. The scale bar indicates 100 Å.

Fig. 4

Results of docking the HyHEL-5 Fab molecule into the electron density of the CPV:Fab A3B10 complex. (a) Section through the capsid containing approximately a two-fold axis (indicated with a black line) and a five-fold axis (not shown). The electron density is green, the Fab heavy chain is blue and the light chain is red. (The figure was produced by the programs O [44] and Macinplot [47].) (b) As (a) but viewed from a different angle. (c) Ribbon diagram, on a larger scale than (a) and (b), showing the interaction between the Fab and one CPV subunit. The orientation is identical to (a). Assuming the preferred Fab orientation, the heavy chain is blue and the light chain is red. The β-barrel domain of CPV is purple while the remainder of the structure is green. The site of escape mutations at residues 299, 300 and 302 are indicated as gray spheres. Two-fold and five-fold axes are indicated. The approximate virus surface is indicated with a white line. (The figure was produced by the programs MOLSCRIPT [48] and Raster3D [49]).

Fig. 5

Central section through CPV showing the external fit of EM density to the known atomic structure of the C_α backbone. Symmetry axes are indicated by numbered lines. The orientation is perpendicular to that of Figs 4a and 4c.

Fig. 6

Comparison of the number of antigen residues in contact between the fitted HyHEL-5 model and CPV (squares) with that of Fab HyHEL-5 complexed with lysozyme (circles). HyHEL-5 is translated radially by 1.00 Å (triangles) and 1.56 Å (diamonds) away from the lysozyme surface. In the latter case, the number of contacts is similar to that found in the CPV:Fab complex.

Fig. 7

Alignment of Fab A3B10 and HyHEL-5 heavy and light chain variable domains. Identical regions are indicated in bold faced type. The H1, H2, H3 and L1, L2, L3 CDR loop regions of the heavy and light chains, respectively, are boxed. Those residues of HyHEL-5 which, when fitted into the A3B10 density, are in contact with the CPV surface are marked (●).

Fig. 8

Enlarged portion of the footprint region on the CPV surface showing the distance of the closest approach between CPV residues and the fitted HyHEL-5 model (left). The distance is indicated by shading, up to a maximum of 5 Å. The location of these residues on the CPV surface is outlined on the icosahedral asymmetric unit (middle inset). The shading of any one amino acid corresponds to the closest approach any non-hydrogen atom within the residue makes to the CPV surface. The separate footprints of each Fab CDR (H1, H2, H3, L1 and L3) are also shown (middle). CPV residues in contact with more than one CDR loop contain the combination of hatching or shading from each of the contacting CDR loops. The conjunction of three symmetry-related subunits (see heavy black lines in Fig. 1) within the footprint is shown on the right. (The figure was produced in part by the program ′Roadmap′ [46]).

Fig. 9

Plaque neutralization by A3B10 IgG and Fab. Titers of each sample are shown with a triangle for IgG and a circle for Fab. The dashed and solid lines join the mean titers at different dilutions for IgG and Fab, respectively.