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Journal of Virology, October 2004, p. 10695-10705, Vol. 78, No. 19
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.19.10695-10705.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Protective Immunoglobulin A and G Antibodies Bind to Overlapping Intersubunit Epitopes in the Head Domain of Type 1 Reovirus Adhesin {sigma}1

Anna Helander,1,2,{dagger} Cathy L. Miller,3 Kimberly S. Myers,3,4 Marian R. Neutra,1,2 and Max L. Nibert3,4*

GI Cell Biology Laboratory, Children's Hospital,1 Departments of Pediatrics,2 Microbiology and Molecular Genetics, Harvard Medical School, Boston,3 Ph.D. Training Program in Virology, Division of Medical Sciences, Harvard University, Cambridge, Massachusetts4

Received 17 March 2004/ Accepted 24 May 2004

Nonfusogenic mammalian orthoreovirus (reovirus) is an enteric pathogen of mice and a useful model for studies of how an enteric virus crosses the mucosal barrier of its host and is subject to control by the mucosal immune system. We recently generated and characterized a new murine immunoglobulin A (IgA)-class monoclonal antibody (MAb), 1E1, that binds to the adhesin fiber, {sigma}1, of reovirus type 1 Lang (T1L) and thereby neutralizes the infectivity of that strain in cell culture. 1E1 is produced in hybridoma cultures as a mixture of monomers, dimers, and higher polymers and is protective against peroral challenges with T1L either when the MAb is passively administered or when it is secreted into the intestines of mice bearing subcutaneous hybridoma tumors. In the present study, selection and analysis of mutants resistant to neutralization by 1E1 identified the region of T1L {sigma}1 to which the MAb binds. The region bound by a previously characterized type 1 {sigma}1-specific neutralizing IgG MAb, 5C6, was identified in the same way. Each of the 15 mutants isolated and analyzed was found to be much less sensitive to neutralization by either 1E1 or 5C6, suggesting the two MAbs bind to largely overlapping regions of {sigma}1. The tested mutants retained the capacity to recognize specific glycoconjugate receptors on rabbit M cells and cultured epithelial cells, even though viral binding to epithelial cells was inhibited by both MAbs. S1 sequence determinations for 12 of the mutants identified {sigma}1 mutations at four positions between residues 415 and 447, which contribute to forming the receptor-binding head domain. When aligned with the {sigma}1 sequence of reovirus type 3 Dearing (T3D) and mapped onto the previously reported crystal structure of the T3D {sigma}1 trimer, the four positions cluster on the side of the {sigma}1 head, across the interface between two subunits. Three such interface-spanning epitopes are thus present per {sigma}1 trimer and require the intact quaternary structure of the head domain for MAb binding. Identification of these intersubunit epitopes on {sigma}1 opens the way for further studies of the mechanisms of antibody-based neutralization and protection with type 1 reoviruses.

* Corresponding author. Mailing address: Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Ave., Boston, MA 02115. Phone: (617) 645-3680. Fax: (617) 738-7664. E-mail: mnibert{at}hms.harvard.edu.

{dagger} Present address: Department of Histology, Microbiology, and Medical Biotechnologies, University of Padua, 35121 Padua, Italy.

Journal of Virology, October 2004, p. 10695-10705, Vol. 78, No. 19
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.19.10695-10705.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

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