title: Functional analysis of the putative integrin recognition motif on adeno-associated virus 9.
abstract: Adeno-associated viruses (AAVs) display a highly conserved NGR motif on the capsid surface. Earlier studies have established this tripeptide motif as being essential for integrin-mediated uptake of recombinant AAV serotype 2 (AAV2) in cultured cells. However, functional attributes of this putative integrin recognition motif in other recombinant AAV serotypes displaying systemic transduction in vivo remain unknown. In this study, we dissect the biology of an integrin domain capsid mutant derived from the human isolate AAV9 in mice. The AAV9/NGA mutant shows decreased systemic transduction in mice. This defective phenotype was accompanied by rapid clearance of mutant virions from the blood circulation and nonspecific sequestration by the spleen. Transient vascular hyperpermeability, induced by histamine coinjection, exacerbated AAV9/NGA uptake by the spleen but not the liver. However, such treatment did not affect AAV9 virions, suggesting a potential entry/post-entry defect for the mutant in different tissues. Further characterization revealed modestly decreased cell surface binding but a more pronounced defect in the cellular entry of mutant virions. These findings were corroborated by the observation that blocking multiple integrins adversely affected recombinant AAV9 transduction in different cell types, albeit with variable efficiencies. From a structural perspective, we observed that the integrin recognition motif is located in close proximity to the galactose binding footprint on AAV9 capsids and postulate that this feature could influence cell surface attachment, cellular uptake at the tissue level, and systemic clearance by the reticuloendothelial system.
authors.affiliation: From the Gene Therapy Center.
authors.forename: Shen
authors.initials: S
authors.lastname: Shen
authors.affiliation: From the Gene Therapy Center, Department of Genetics, and.
authors.forename: Garrett E
authors.initials: GE
authors.lastname: Berry
authors.affiliation: From the Gene Therapy Center.
authors.forename: Ruth M
authors.initials: RM
authors.lastname: Castellanos Rivera
authors.affiliation: From the Gene Therapy Center.
authors.forename: Roland Y
authors.initials: RY
authors.lastname: Cheung
authors.affiliation: From the Gene Therapy Center.
authors.forename: Andrew N
authors.initials: AN
authors.lastname: Troupes
authors.affiliation: From the Gene Therapy Center.
authors.forename: Sarah M
authors.initials: SM
authors.lastname: Brown
authors.affiliation: From the Gene Therapy Center.
authors.forename: Tal
authors.initials: T
authors.lastname: Kafri
authors.affiliation: From the Gene Therapy Center, Department of Genetics, and Department of Biochemistry and Biophysics, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27516 aravind@med.unc.edu.
authors.forename: Aravind
authors.initials: A
authors.lastname: Asokan
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D001667:Binding, Competitive; D016466:CHO Cells; D002213:Capsid / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D000229:Dependovirus / Q000235:genetics / Q000502:physiology*; D005260:Female; D006367:HeLa Cells; D006801:Humans; D016023:Integrins / Q000737:chemistry*; D051379:Mice; D008807:Mice, Inbred BALB C; D008958:Models, Molecular; D009154:Mutation; D010641:Phenotype; D011134:Polysaccharides / Q000737:chemistry; D011485:Protein Binding; D014764:Viral Proteins / Q000378:metabolism; D014771:Virion / Q000502:physiology; D053585:Virus Attachment
chemical_list: D016023:Integrins; D011134:Polysaccharides; D014764:Viral Proteins
keywords: Adeno-Associated Virus; Cell Surface Receptor; Gene Therapy; Integrin; Viral Protein; Virus Entry