title: Identification and mutagenesis of the adeno-associated virus 5 sialic acid binding region.
abstract: As a genus, the dependoviruses use a diverse group of cell surface carbohydrates for attachment and entry. Despite the fact that a majority of adeno-associated viruses (AAVs) utilize sialic acid (SIA) for binding and transduction, this virus-carbohydrate interaction is poorly understood. Utilizing X-ray crystallography, two SIA binding regions were mapped for AAV5. The first site mapped to the depression in the center of the 3-fold axis of symmetry, while the second site was located under the βHI loop close to the 5-fold axis. Mutagenesis of amino acids 569 and 585 or 587 within the 3-fold depression resulted in elimination or alteration in SIA-dependent transduction, respectively. This change in SIA binding was confirmed using glycan microarrays. Mutagenesis of the second site identified a role in transduction that was SIA independent. Further studies of the mutants at the 3-fold site demonstrated a change in transduction activity and cell tropism in vivo as well as resistance to neutralization by a polyclonal antibody raised against the wild-type virus.
  
  
  OBJECTIVE
  Despite the fact that a majority of AAVs utilize sialic acid for binding and transduction, this virus-carbohydrate interaction is poorly understood. Utilizing X-ray crystallography, the sialic acid binding regions of AAV5 were identified and studied using a variety of approaches. Mutagenesis of this region resulted in elimination or alteration in sialic acid-dependent transduction in cell lines. This change in sialic acid glycan binding was confirmed using glycan arrays. Further study also demonstrated a change in transduction and activity and cell tropism in vivo as well as resistance to neutralization by antibodies raised against the wild-type virus.
authors.affiliation: NIDCR, NIH, Molecular Physiology and Therapeutics Branch, Bethesda, Maryland, USA.
authors.forename: Sandra
authors.initials: S
authors.lastname: Afione
authors.affiliation: University of Florida, Department of Biochemistry and Molecular Biology, Gainesville, Florida, USA.
authors.forename: Michael A
authors.initials: MA
authors.lastname: DiMattia
authors.affiliation: University of Florida, Department of Biochemistry and Molecular Biology, Gainesville, Florida, USA.
authors.forename: Sujata
authors.initials: S
authors.lastname: Halder
authors.affiliation: NIDCR, NIH, Molecular Physiology and Therapeutics Branch, Bethesda, Maryland, USA.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Di Pasquale
authors.affiliation: University of Florida, Department of Biochemistry and Molecular Biology, Gainesville, Florida, USA mckenna@ufl.edu jchiorini@dir.nidcr.nih.gov.
authors.forename: Mavis
authors.initials: M
authors.lastname: Agbandje-McKenna
authors.affiliation: NIDCR, NIH, Molecular Physiology and Therapeutics Branch, Bethesda, Maryland, USA mckenna@ufl.edu jchiorini@dir.nidcr.nih.gov.
authors.forename: John A
authors.initials: JA
authors.lastname: Chiorini
mesh_terms: D000818:Animals; D057134:Antibodies, Neutralizing / Q000276:immunology; D000914:Antibodies, Viral / Q000276:immunology; D001665:Binding Sites; D036022:Capsid Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D018360:Crystallography, X-Ray; D004252:DNA Mutational Analysis; D000229:Dependovirus / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000502:physiology*; D006801:Humans; D008958:Models, Molecular; D016296:Mutagenesis; D019158:N-Acetylneuraminic Acid / Q000378:metabolism*; D014161:Transduction, Genetic; D056189:Viral Tropism; D053585:Virus Attachment
chemical_list: D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D036022:Capsid Proteins; D019158:N-Acetylneuraminic Acid