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 Angstrom resolution for the CPMV/F-ab complexes and 30 Angstrom 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 582 and 10B7 bound to nearly identical positions. The refined 2.8 Angstrom 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 582 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. (C) 1994 Academic Press, Inc.