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Journal of Virology, November 1999, p. 9508-9514, Vol. 73, No. 11
Department of Respiratory Medicine and Allergy,
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 (
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
-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
-strands E and F and the turn between them (residues
479 to 486) with the corresponding sequence from Ad3 (
EFAd3
5
mutation) resulted in the loss of receptor binding. The identical CD
spectra for
EFAd3
5 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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