title: Use of Cre/loxP recombination to swap cell binding motifs on the adenoviral capsid protein IX.
abstract: We used Cre/loxP recombination to swap targeting ligands present on the adenoviral capsid protein IX (pIX). A loxP-flanked sequence encoding poly-lysine (pK-binds heparan sulfate proteoglycans) was engineered onto the 3'-terminus of pIX, and the resulting fusion protein allowed for routine virus propagation. Growth of this virus on Cre-expressing cells removed the pK coding sequence, generating virus that could only infect through alternative ligands, such as a tyrosine kinase receptor A (TrkA)-binding motif engineered into the capsid fibre protein for enhanced infection of neuronal cells. We used a similar approach to swap the pK motif on pIX for a sequence encoding a single-domain antibody directed towards CD66c for targeted infection of cancer cells; Cre-mediated removal of the pK-coding sequence simultaneously placed the single-domain antibody coding sequence in frame with pIX. Thus, we have developed a simple method to propagate virus lacking native viral tropism but containing cell-specific binding ligands.
authors.affiliation: Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada.
authors.forename: Kathy L
authors.initials: KL
authors.lastname: Poulin
authors.forename: Grace
authors.initials: G
authors.lastname: Tong
authors.forename: Olga
authors.initials: O
authors.lastname: Vorobyova
authors.forename: Madeline
authors.initials: M
authors.lastname: Pool
authors.forename: Rashmi
authors.initials: R
authors.lastname: Kothary
authors.forename: Robin J
authors.initials: RJ
authors.lastname: Parks
mesh_terms: D000256:Adenoviridae / Q000235:genetics* / Q000502:physiology*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000276:immunology; D036022:Capsid Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015815:Cell Adhesion Molecules / Q000276:immunology; D002460:Cell Line; D062727:Coxsackie and Adenovirus Receptor-Like Membrane Protein; D058851:GPI-Linked Proteins / Q000276:immunology; D057809:HEK293 Cells; D006801:Humans; D008024:Ligands; D011107:Polylysine / Q000737:chemistry / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D020917:Receptor, trkA / Q000378:metabolism; D011991:Receptors, Virus / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D011995:Recombination, Genetic; D053585:Virus Attachment; D014779:Virus Replication
chemical_list: D015703:Antigens, CD; C504117:CEACAM6 protein, human; C568771:CLMP protein, human; D036022:Capsid Proteins; D015815:Cell Adhesion Molecules; C568773:Clmp protein, rat; D062727:Coxsackie and Adenovirus Receptor-Like Membrane Protein; D058851:GPI-Linked Proteins; D008024:Ligands; D011991:Receptors, Virus; D011993:Recombinant Fusion Proteins; C018315:hexon capsid protein, Adenovirus; D011107:Polylysine; D020917:Receptor, trkA