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Journal of Virology, July 2003, p. 7517-7526, Vol. 77, No. 13
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.13.7517-7526.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Kinetic Analysis of Binding Interaction between the Subgroup A Rous Sarcoma Virus Glycoprotein SU and Its Cognate Receptor Tva: Calcium Is Not Required for Ligand Binding

Department of Microbiology and Immunology,¹ Department of Biochemistry and Molecular Biology, College of Medicine, University of Illinois at Chicago, Chicago, Illinois 60612,² McArdle Laboratory for Cancer Research, University of Wisconsin—Madison, Madison, Wisconsin 53706³

Received 24 October 2002/ Accepted 1 April 2003

Tva is the receptor for subgroup A Rous sarcoma virus, and it contains a single LDL-A module which is the site of virus interaction. In this study, we expressed the entire extracellular region of Tva (referred to as Ecto-Tva) as a GST fusion protein and characterized its refolding properties. We demonstrated that the correct folding of the Ecto-Tva protein, like that of the Tva LDL-A module, is calcium dependent. We used the IAsys system to measure the kinetics of binding between the surface (SU) subunit of the viral glycoprotein and Tva in real time. We found that the Ecto-Tva protein and the Tva LDL-A module displayed similar affinities for SU, providing direct evidence that the LDL-A module of Tva is the only viral interaction domain of the receptor. Furthermore, misfolded Tva proteins displayed lower binding affinities to SU, largely due to a decrease in their association rates, suggesting that a high association rate between SU and Tva is crucial for efficient virus-host interaction. Furthermore, we found that calcium did not influence the overall binding affinity between Tva and SU. These results indicate that, although calcium is important in facilitating correct folding of the LDL-A module of Tva, it is not essential for ligand binding. Thus, these results may have broad implications for the mechanism of protein folding and ligand recognition of the LDL receptor and other members of the LDL receptor superfamily.

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