Adenovirus type 9 fiber knob binds to the coxsackie B virus-adenovirus receptor (CAR) with lower affinity than fiber knobs of other CAR-binding adenovirus serotypes

The coxsackie B virus and adenovirus (Ad) receptor (CAR) functions as an at tachment receptor for multiple Ad serotypes, Here we show that the Ad serot ype 9 (Ad9) fiber knob binds to CAR with much reduced affinity compared to the binding by Ad5 and Ad12 fiber knobs as well as the knob of the long fib er of Ad41 (Ad41L), Substitution of Asp222 in Ad9 fiber knob with a lysine that is conserved in Ad5, Ad12, and Ad41L substantially improved Ad9 fiber knob binding to CAR, while the corresponding substitution in Ad5 (Lys442Asp ) significantly reduced Ad5 binding. The presence of an aspartic acid resid ue in Ad9 therefore accounts, at least in part, for the reduced CAR binding affinity of the Ad9 fiber knob. Site-directed mutagenesis of CAR revealed that CAR residues Leu73 and Lys121 and/or Lys123 are critical contact resid ues, with Tyr80 and Tyr83 being peripherally involved in the binding intera ction with the Ad5, Ad9, Ad12, and Ad41L fiber knobs. The overall affinitie s and the association and dissociation rate constants for wild-type CAR as well as Tyr80 and Tyr83 CAR mutants differed between the serotypes, indicat ing that their binding modes, although similar, are not identical.