Analysis of the interaction between RGD-expressing adenovirus type 5 fiber knob domains and alphavbeta3 integrin reveals distinct binding profiles and intracellular trafficking.
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 alphavbeta3 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 alphavbeta3 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 alphavbeta3 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 alphavbeta3 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.
Lord
Rosie
R
Division of Asthma Allergy and Lung Biology, King's College London School of Medicine at Guy's Kings and St Thomas' Hospitals, Fifth Floor Thomas Guy House, Guy's Hospital, St Thomas Street, London SE1 9RT, UK.
Parsons
Maddy
M
The Randall Division of Cell and Molecular Biophysics, King's College London School of Medicine at Guy's Kings and St Thomas' Hospitals, Fifth Floor Thomas Guy House, Guy's Hospital, St Thomas Street, London SE1 9RT, UK.
Kirby
Ian
I
Division of Asthma Allergy and Lung Biology, King's College London School of Medicine at Guy's Kings and St Thomas' Hospitals, Fifth Floor Thomas Guy House, Guy's Hospital, St Thomas Street, London SE1 9RT, UK.
Beavil
Andrew
A
The Randall Division of Cell and Molecular Biophysics, King's College London School of Medicine at Guy's Kings and St Thomas' Hospitals, Fifth Floor Thomas Guy House, Guy's Hospital, St Thomas Street, London SE1 9RT, UK.
Hunt
James
J
The Randall Division of Cell and Molecular Biophysics, King's College London School of Medicine at Guy's Kings and St Thomas' Hospitals, Fifth Floor Thomas Guy House, Guy's Hospital, St Thomas Street, London SE1 9RT, UK.
Sutton
Brian
B
The Randall Division of Cell and Molecular Biophysics, King's College London School of Medicine at Guy's Kings and St Thomas' Hospitals, Fifth Floor Thomas Guy House, Guy's Hospital, St Thomas Street, London SE1 9RT, UK.
Santis
George
G
Division of Asthma Allergy and Lung Biology, King's College London School of Medicine at Guy's Kings and St Thomas' Hospitals, Fifth Floor Thomas Guy House, Guy's Hospital, St Thomas Street, London SE1 9RT, UK.
D000260:Adenoviruses, Human / Q000235:genetics* / Q000502:physiology*; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D045744:Cell Line, Tumor; D004279:DNA, Viral / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D039302:Integrin alphaVbeta3 / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D016254:Mutagenesis, Insertional; D041681:NIH 3T3 Cells; D009842:Oligopeptides; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020349:Surface Plasmon Resonance; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D004279:DNA, Viral; D039302:Integrin alphaVbeta3; D009842:Oligopeptides; D011994:Recombinant Proteins; D014764:Viral Proteins; C047981:arginyl-glycyl-aspartic acid