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Journal of Virology, July 2001, p. 6625-6634, Vol. 75, No. 14
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.14.6625-6634.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

A Monoclonal Antibody Specific for Reovirus Outer-Capsid Protein sigma 3 Inhibits sigma 1-Mediated Hemagglutination by Steric Hindrance

Emma L. Nason,1 J. Denise Wetzel,2,3 S. K. Mukherjee,1 Erik S. Barton,3,4 B. V. Venkataram Prasad,1,* and Terence S. Dermody2,3,4,*

Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030,1 and Departments of Pediatrics2 and Microbiology and Immunology4 and Elizabeth B. Lamb Center for Pediatric Research,3 Vanderbilt University School of Medicine, Nashville, Tennessee 37232

Received 20 November 2000/Accepted 13 April 2001

Reovirus virions are nonenveloped icosahedral particles consisting of two concentric protein shells, termed outer capsid and core. Outer-capsid protein sigma 1 is the viral attachment protein and binds carbohydrate molecules on the surface of host cells. Monoclonal antibody (MAb) 4F2, which is specific for outer-capsid protein sigma 3, blocks the binding of sigma 1 protein to sialic acid and inhibits reovirus-induced hemagglutination (HA). To determine whether MAb 4F2 inhibits HA by altering sigma 1-sigma 3 interactions or by steric hindrance, we analyzed the effect of 4F2 immunoglobulin G (IgG) and Fab fragments (Fabs) on HA induced by reovirus strain type 3 Dearing (T3D). The concentration of 4F2 IgG sufficient to inhibit T3D-induced HA was 12.5 µg per ml, whereas that of Fabs was >200 µg per ml. Dynamic light scattering analysis showed that at the concentration of IgG sufficient to inhibit HA, virion-antibody complexes were monodispersed and not aggregated. The affinity of 4F2 Fabs for T3D virions was only threefold less than that of intact IgG, which suggests that differences in HA inhibition titer exhibited by 4F2 IgG and Fabs are not attributable to differences in the affinity of these molecules for T3D virions. We used cryoelectron microscopy and three-dimensional image analysis to visualize T3D virions alone and in complex with either IgG or Fabs of MAb 4F2. IgG and Fabs bind the same site at the distal portion of sigma 3, and binding of IgG and Fabs induces identical conformational changes in outer-capsid proteins sigma 3 and µ1. These results suggest that MAb 4F2 inhibits reovirus binding to sialic acid by steric hindrance and provide insight into the conformational flexibility of reovirus outer-capsid proteins.

* Corresponding author. Mailing address for B. V. Venkataram Prasad: Department of Biochemistry and Molecular Biology, Baylor College of Medicine, One Baylor Plaza, Room N410, Houston, TX 77030-3498. Phone: (713) 798-5686. Fax: (713) 798-1625. E-mail: vprasad{at}bcm.tmc.edu. Mailing address for Terence S. Dermody: Elizabeth B. Lamb Center for Pediatric Research, Vanderbilt University School of Medicine, D-7235 MCN, Nashville, TN 37232-2581. Phone: (615) 322-2250. Fax: (615) 343-9723. E-mail: terry.dermody{at}mcmail.vanderbilt.edu.

Journal of Virology, July 2001, p. 6625-6634, Vol. 75, No. 14
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.14.6625-6634.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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Copyright © 2001 by the American Society for Microbiology. All rights reserved.