Direct imaging of interactions between an icosahedral virus and conjugate F(ab) fragments by cryoelectron microscopy and X-ray crystallography.
The binding properties of seven mouse monoclonal antibodies (McAbs) raised against cowpea mosaic virus (CPMV) were characterized by conventional and inhibition enzyme-linked immunosorbent assay (ELISA) technique. McAb binding to CPMV on electron microscope (EM) grids was also assayed with gold-labeled anti-mouse antibodies. Two of the seven McAbs (5B2 and 10B7) were found to bind tighter to CPMV than the others in the inhibition ELISA and the EM assay. F(ab) fragments from both of these McAbs were prepared and complexed with CPMV in solution. Electron micrographs of flash frozen (vitrified) samples of native CPMV and CPMV complexed with F(ab) fragments from McAbs 5B2 and 10B7 as well as IgGs from 5B2 were recorded and reconstructions were computed at 23 A resolution for the CPMV/F(ab) complexes and 30 A resolution for the CPMV/IgG complex. Structures of all three complexes clearly displayed the F(ab) fragments distributed with icosahedral symmetry on the surface of CPMV. The IgG bound in a monodentate fashion with only one F(ab) attached to the virus surface. F(ab) fragments from 5B2 and 10B7 bound to nearly identical positions. The refined 2.8 A X-ray structure of CPMV was used to identify the roughly 30 amino acids covered by the F(ab) fragments. The "footprint" spans a subunit interface and appears spatially similar to antigenic site 3B on poliovirus. In a previous, preliminary report of the CPMV/F(ab) 5B2 complex (Wang et al., 1992, Nature 355, 275-278) the wrong enantiomorph of the reconstruction was chosen. This was corrected and, since the F(ab) binds close to a mirror plane, the change in the footprint was minor.
Porta
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C
Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907-1392.
Wang
G
G
Cheng
H
H
Chen
Z
Z
Baker
T S
TS
Johnson
J E
JE
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001666:Binding Sites, Antibody; D017798:Comovirus / Q000737:chemistry* / Q000648:ultrastructure; D018360:Crystallography, X-Ray; D004797:Enzyme-Linked Immunosorbent Assay; D007140:Immunoglobulin Fab Fragments / Q000032:analysis*; D007074:Immunoglobulin G / Q000032:analysis; D051379:Mice; D008807:Mice, Inbred BALB C; D008854:Microscopy, Electron; D011817:Rabbits
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G