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Journal of Virology, November 1999, p. 9508-9514, Vol. 73, No. 11
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Mutations in the DG Loop of Adenovirus Type 5 Fiber Knob Protein Abolish High-Affinity Binding to Its Cellular Receptor CAR

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, Guy's Hospital, London SE1 9RT,1 and The Randall Institute, King's College London, London WC2B 5RL,2 United Kingdom, and GenVec, Inc., Rockville, Maryland 208523

Received 15 April 1999/Accepted 9 July 1999

The amino acid residues in adenovirus type 5 (Ad5) fiber that interact with its cellular receptor, the coxsackie B virus and Ad receptor (CAR), have not been defined. To investigate this, multiple mutations were constructed in the region between residues 479 and 497 in Ad5 fiber (beta -strands E and F and the adjacent region of the DG loop). The effects of these mutations on binding to CAR were determined by use of cell-binding competition experiments, surface plasmon resonance, and direct binding studies. The mutation effects on the overall folding and secondary structure of the protein were assessed by circular dichroism (CD) spectroscopy. Deletions of two consecutive amino acids between residues 485 and 493 abolished high-affinity binding to CAR; the CD spectra indicated that although there was no disruption of the overall folding and secondary structure of the protein, local conformational changes did occur. Moreover, single site mutations in this region of residues with exposed, surface-accessible side chains, such as Thr492, Asn493, and Val495, had no effect on receptor binding, which demonstrates that these residues are not in contact with CAR themselves. This implies the involvement of residues in neighboring loop regions. Replacement of the segment containing the two very short beta -strands E and F and the turn between them (residues 479 to 486) with the corresponding sequence from Ad3 (beta EFAd3right-arrow5 mutation) resulted in the loss of receptor binding. The identical CD spectra for beta EFAd3right-arrow5 and wild-type proteins suggest that these substitutions caused no conformational rearrangement and that the loss of binding may thus be due to the substitution of one or more critical contact residues. These findings have implications for our understanding of the interaction of Ad5 fiber with CAR and for the construction of targeted recombinant Ad5 vectors for gene therapy purposes.

* Corresponding author. Mailing address: Department of Respiratory Medicine and Allergy, The Guy's, King's College, and St. Thomas' Hospitals School of Medicine, 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, November 1999, p. 9508-9514, Vol. 73, No. 11
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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