1887

Abstract

Adenovirus (Ad) vectors are used widely for experimental and therapeutic gene transfer. Ad-mediated gene delivery is often inefficient and, thus, there is considerable interest in developing Ad vectors that overcome biological barriers to efficient virus uptake. For this strategy to succeed, it is imperative that the interaction between such Ad vectors and their novel receptors is well understood. In this study, three surface-exposed loops (HI, CD and IJ loops) on the Ad5 fiber knob domain were selected as sites for insertion of an v3 integrin-binding RGD sequence. Three RGD-containing Ad5 fiber knob-domain mutants were produced as recombinant proteins and all were shown to interact with soluble v3 integrin by using biomolecular cell-free assays. Cell adsorption and subsequent internalization and intracellular trafficking of each of these proteins were assessed by confocal microscopy. Whilst the Ad5 fiber knob domain expressing the RGD sequence in the HI and CD loops bound with similar association and dissociation profiles, the fiber knob domain expressing the RGD sequence in the IJ loop bound with slower association and faster dissociation rates. By using molecular modelling, it was shown that the Ad5 fiber knob domain in which the RGD peptide was expressed in the IJ loop was only capable of binding to one v3 integrin molecule per trimer. In contrast, fiber knob domains in which the RGD peptide was expressed in the HI and CD loops were capable of binding to one integrin molecule per monomer. These differences in the interactions between each mutant and v3 may explain our observation that the three RGD-bearing Ad5 fiber knob domains demonstrated similar internalization rates, but distinct patterns of endosomal transport and escape.

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2006-09-01
2019-11-18
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