Direct Imaging of Interactions between an Icosahedral Virus and Conjugate Fab Fragments by Cryoelectron Microscopy and X-Ray Crystallography

Claudine Porta, Guoji Wang, Holland Cheng, Zhongguo Chen, Timothy S. Baker, John E. Johnson

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

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, Fab 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 Fab fragments from McAbs 5B2 and 10B7 as well as IgGs from 5B2 were recorded and reconstructions were computed at 23 Å resolution for the CPMV/Fab complexes and 30 Å resolution for the CPMV/IgG complex. Structures of all three complexes clearly displayed the Fab fragments distributed with icosahedral symmetry on the surface of CPMV. The IgG bound in a monodentate fashion with only one Fab attached to the virus surface. Fab fragments from 5B2 and 10B7 bound to nearly identical positions. The refined 2.8 Å X-ray structure of CPMV was used to identify the roughly 30 amino acids covered by the Fab 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/Fab 5B2 complex (Wang et al., 1992, Nature 355, 275-278) the wrong enantiomorph of the reconstruction was chosen. This was corrected and, since the Fib binds close to a mirror plane, the change in the footprint was minor.

Original languageEnglish (US)
Pages (from-to)777-788
Number of pages12
JournalVirology
Volume204
Issue number2
DOIs
StatePublished - Nov 1 1994
Externally publishedYes

Fingerprint

Comovirus
Cryoelectron Microscopy
Immunoglobulin Fab Fragments
X Ray Crystallography
Viruses
Monoclonal Antibodies
Electrons
Immunoglobulin G
Enzyme-Linked Immunosorbent Assay
Immunosorbent Techniques
Poliovirus
Gold

ASJC Scopus subject areas

  • Infectious Diseases
  • Virology

Cite this

Direct Imaging of Interactions between an Icosahedral Virus and Conjugate Fab Fragments by Cryoelectron Microscopy and X-Ray Crystallography. / Porta, Claudine; Wang, Guoji; Cheng, Holland; Chen, Zhongguo; Baker, Timothy S.; Johnson, John E.

In: Virology, Vol. 204, No. 2, 01.11.1994, p. 777-788.

Research output: Contribution to journalArticle

Porta, Claudine ; Wang, Guoji ; Cheng, Holland ; Chen, Zhongguo ; Baker, Timothy S. ; Johnson, John E. / Direct Imaging of Interactions between an Icosahedral Virus and Conjugate Fab Fragments by Cryoelectron Microscopy and X-Ray Crystallography. In: Virology. 1994 ; Vol. 204, No. 2. pp. 777-788.
@article{6dfe075c94e7490fbc5e82bb7535755d,
title = "Direct Imaging of Interactions between an Icosahedral Virus and Conjugate Fab Fragments by Cryoelectron Microscopy and X-Ray Crystallography",
abstract = "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, Fab 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 Fab fragments from McAbs 5B2 and 10B7 as well as IgGs from 5B2 were recorded and reconstructions were computed at 23 {\AA} resolution for the CPMV/Fab complexes and 30 {\AA} resolution for the CPMV/IgG complex. Structures of all three complexes clearly displayed the Fab fragments distributed with icosahedral symmetry on the surface of CPMV. The IgG bound in a monodentate fashion with only one Fab attached to the virus surface. Fab fragments from 5B2 and 10B7 bound to nearly identical positions. The refined 2.8 {\AA} X-ray structure of CPMV was used to identify the roughly 30 amino acids covered by the Fab 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/Fab 5B2 complex (Wang et al., 1992, Nature 355, 275-278) the wrong enantiomorph of the reconstruction was chosen. This was corrected and, since the Fib binds close to a mirror plane, the change in the footprint was minor.",
author = "Claudine Porta and Guoji Wang and Holland Cheng and Zhongguo Chen and Baker, {Timothy S.} and Johnson, {John E.}",
year = "1994",
month = "11",
day = "1",
doi = "10.1006/viro.1994.1593",
language = "English (US)",
volume = "204",
pages = "777--788",
journal = "Virology",
issn = "0042-6822",
publisher = "Academic Press Inc.",
number = "2",

}

TY - JOUR

T1 - Direct Imaging of Interactions between an Icosahedral Virus and Conjugate Fab Fragments by Cryoelectron Microscopy and X-Ray Crystallography

AU - Porta, Claudine

AU - Wang, Guoji

AU - Cheng, Holland

AU - Chen, Zhongguo

AU - Baker, Timothy S.

AU - Johnson, John E.

PY - 1994/11/1

Y1 - 1994/11/1

N2 - 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, Fab 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 Fab fragments from McAbs 5B2 and 10B7 as well as IgGs from 5B2 were recorded and reconstructions were computed at 23 Å resolution for the CPMV/Fab complexes and 30 Å resolution for the CPMV/IgG complex. Structures of all three complexes clearly displayed the Fab fragments distributed with icosahedral symmetry on the surface of CPMV. The IgG bound in a monodentate fashion with only one Fab attached to the virus surface. Fab fragments from 5B2 and 10B7 bound to nearly identical positions. The refined 2.8 Å X-ray structure of CPMV was used to identify the roughly 30 amino acids covered by the Fab 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/Fab 5B2 complex (Wang et al., 1992, Nature 355, 275-278) the wrong enantiomorph of the reconstruction was chosen. This was corrected and, since the Fib binds close to a mirror plane, the change in the footprint was minor.

AB - 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, Fab 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 Fab fragments from McAbs 5B2 and 10B7 as well as IgGs from 5B2 were recorded and reconstructions were computed at 23 Å resolution for the CPMV/Fab complexes and 30 Å resolution for the CPMV/IgG complex. Structures of all three complexes clearly displayed the Fab fragments distributed with icosahedral symmetry on the surface of CPMV. The IgG bound in a monodentate fashion with only one Fab attached to the virus surface. Fab fragments from 5B2 and 10B7 bound to nearly identical positions. The refined 2.8 Å X-ray structure of CPMV was used to identify the roughly 30 amino acids covered by the Fab 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/Fab 5B2 complex (Wang et al., 1992, Nature 355, 275-278) the wrong enantiomorph of the reconstruction was chosen. This was corrected and, since the Fib binds close to a mirror plane, the change in the footprint was minor.

UR - http://www.scopus.com/inward/record.url?scp=0027974229&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0027974229&partnerID=8YFLogxK

U2 - 10.1006/viro.1994.1593

DO - 10.1006/viro.1994.1593

M3 - Article

C2 - 7941346

AN - SCOPUS:0027974229

VL - 204

SP - 777

EP - 788

JO - Virology

JF - Virology

SN - 0042-6822

IS - 2

ER -