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Journal of Virology, September 2000, p. 8472-8479, Vol. 74, No. 18
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Identification of Carbohydrate-Binding Domains in the Attachment Proteins of Type 1 and Type 3 Reoviruses

James D. Chappell,1,2 Joy L. Duong,2 Benjamin W. Wright,2 and Terence S. Dermody1,2,3,*

Departments of Pediatrics1 and Microbiology and Immunology3 and Elizabeth B. Lamb Center for Pediatric Research,2 Vanderbilt University School of Medicine, Nashville, Tennessee 37232

Received 1 March 2000/Accepted 20 June 2000

The reovirus attachment protein, sigma 1, is responsible for strain-specific patterns of viral tropism in the murine central nervous system and receptor binding on cultured cells. The sigma 1 protein consists of a fibrous tail domain proximal to the virion surface and a virion-distal globular head domain. To better understand mechanisms of reovirus attachment to cells, we conducted studies to identify the region of sigma 1 that binds cell surface carbohydrate. Chimeric and truncated sigma 1 proteins derived from prototype reovirus strains type 1 Lang (T1L) and type 3 Dearing (T3D) were expressed in insect cells by using a baculovirus vector. Assessment of expressed protein susceptibility to proteolytic cleavage, binding to anti-sigma 1 antibodies, and oligomerization indicates that the chimeric and truncated sigma 1 proteins are properly folded. To assess carbohydrate binding, recombinant sigma 1 proteins were tested for the capacity to agglutinate mammalian erythrocytes and to bind sialic acid presented on glycophorin, the cell surface molecule bound by type 3 reovirus on human erythrocytes. Using a panel of two wild-type and ten chimeric and truncated sigma 1 proteins, the sialic acid-binding domain of type 3 sigma 1 was mapped to a region of sequence proposed to form the more amino terminal of two predicted beta -sheet structures in the tail. This unit corresponds to morphologic region T(iii) observed in computer-processed electron micrographs of sigma 1 protein purified from virions. In contrast, the homologous region of T1L sigma 1 sequence was not implicated in carbohydrate binding; rather, sequences in the distal portion of the tail known as the neck were required. Results of these studies demonstrate that a functional receptor-binding domain, which uses sialic acid as its ligand, is contained within morphologic region T(iii) of the type 3 sigma 1 tail. Furthermore, our findings indicate that T1L and T3D sigma 1 proteins contain different arrangements of receptor-binding domains.

* Corresponding author. Mailing address: Lamb Center for Pediatric Research, D7235 MCN, Vanderbilt University School of Medicine, Nashville, TN 37232. Phone: (615) 343-9943. Fax: (615) 343-9723. E-mail: terry.dermody{at}mcmail.vanderbilt.edu.

Journal of Virology, September 2000, p. 8472-8479, Vol. 74, No. 18
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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