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Journal of Virology, March 2000, p. 2804-2813, Vol. 74, No. 6
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Identification of Contact Residues and Definition of the CAR-Binding Site of Adenovirus Type 5 Fiber Protein

Ian Kirby,1 Elizabeth Davison,1 Andrew J. Beavil,2 Cecilia P. C. Soh,1 Thomas J. Wickham,3 Peter W. Roelvink,3 Imre Kovesdi,3 Brian J. Sutton,2 and George Santis1,*

Department of Respiratory Medicine and Allergy, The Guy's, King's College, and St. Thomas' Hospitals School of Medicine,1 and The Randall Institute, King's College London,2 London SE1 9RT, United Kingdom, and GenVec Inc., Rockville, Maryland 208523

Received 30 September 1999/Accepted 13 December 1999

The binding of adenovirus (Ad) fiber knob to its cellular receptor, the coxsackievirus and Ad receptor (CAR), promotes virus attachment to cells and is a major determinant of Ad tropism. Analysis of the kinetics of binding of Ad type 5 (Ad5) fiber knob to the soluble extracellular domains of CAR together (sCAR) and each immunoglobulin (Ig) domain (IgV and IgC2) independently by surface plasmon resonance demonstrated that the IgV domain is necessary and sufficient for binding, and no additional membrane components are required to confer high-affinity binding to Ad5 fiber knob. Four Ad5 fiber knob mutations, Ser408Glu and Pro409Lys in the AB loop, Tyr477Ala in the DG loop, and Leu485Lys in beta  strand F, effectively abolished high-affinity binding to CAR, while Ala406Lys and Arg412Asp in the AB loop and Arg481Glu in beta  strand E significantly reduced the level of binding. Circular dichroism spectroscopy showed that these mutations do not disorder the secondary structure of the protein, implicating Ser408, Pro409, Tyr477, and Leu485 as contact residues, with Ala406, Arg412, and Arg481 being peripherally or indirectly involved in CAR binding. The critical residues have exposed side chains that form a patch on the surface, which thus defines the high-affinity interface for CAR. Additional site-directed mutagenesis of Ad5 fiber knob suggests that the binding site does not extend to the adjacent subunit or toward the edge of the R sheet. These findings have implications for our understanding of the biology of Ad infection, the development of novel Ad vectors for targeted gene therapy, and the construction of peptide inhibitors of Ad infection.

* Corresponding author. Mailing address: Department of Respiratory Medicine and Allergy, 5th Floor, Thomas Guy House, Guy's Hospital, St. Thomas St., London SE1 9RT, United Kingdom. Phone: 44-171-9552758. Fax: 44-171-4038640. E-mail: george.santis{at}kcl.ac.uk.

Journal of Virology, March 2000, p. 2804-2813, Vol. 74, No. 6
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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