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Journal of Virology, September 2006, p. 9093-9103, Vol. 80, No. 18
0022-538X/06/$08.00+0     doi:10.1128/JVI.00895-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

{alpha}2,3 and {alpha}2,6 N-Linked Sialic Acids Facilitate Efficient Binding and Transduction by Adeno-Associated Virus Types 1 and 6

Zhijian Wu,1 Edward Miller,2 Mavis Agbandje-McKenna,2 and Richard Jude Samulski1*

Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599,1 Department of Biochemistry and Molecular Biology, Center for Structural Biology, The McKnight Brain Institute, College of Medicine, University of Florida, Gainesville, Florida 326102

Received 2 May 2006/ Accepted 9 June 2006

Recombinant adeno-associated viruses (AAVs) are promising vectors in the field of gene therapy. Different AAV serotypes display distinct tissue tropism, believed to be related to the distribution of their receptors on target cells. Of the 11 well-characterized AAV serotypes, heparan sulfate proteoglycan and sialic acid have been suggested to be the attachment receptors for AAV type 2 and types 4 and 5, respectively. In this report, we identify the receptor for the two closely related serotypes, AAV1 and AAV6. First, we demonstrate using coinfection experiments and luciferase reporter analysis that AAV1 and AAV6 compete for similar receptors. Unlike heparin sulfate, enzymatic or genetic removal of sialic acid markedly reduced AAV1 and AAV6 binding and transduction. Further analysis using lectin staining and lectin competition assays identified that AAV1 and AAV6 use either {alpha}2,3-linked or {alpha}2,6-linked sialic acid when transducing numerous cell types (HepG2, Pro-5, and Cos-7). Treatment of cells with proteinase K but not glycolipid inhibitor reduced AAV1 and AAV6 infection, supporting the hypothesis that the sialic acid that facilitates infection is associated with glycoproteins rather than glycolipids. In addition, we determined by inhibitor (N-benzyl GalNAc)- and cell line-specific (Lec-1) studies that AAV1 and AAV6 require N-linked and not O-linked sialic acid. Furthermore, a resialylation experiment on a deficient Lec-2 cell line confirmed a 2,3 and 2,6 N-linked sialic acid requirement, while studies of mucin with O-linked sialic acid showed no inhibition effect for AAV1 and AAV6 transduction on Cos-7 cells. Finally, using a glycan array binding assay we determined that AAV1 efficiently binds to NeuAc{alpha}2-3GalNAcß1-4GlcNAc, as well as two glycoproteins with {alpha}2,3 and {alpha}2,6 N-linked sialic acids. Taken together, competition, genetic, inhibitor, enzymatic reconstitution, and glycan array experiments support {alpha}2,3 and {alpha}2,6 sialic acids that are present on N-linked glycoproteins as primary receptors for efficient AAV1 and AAV6 viral infection.

* Corresponding author. Mailing address: CB # 7352, Gene Therapy Center, 7119 Thurston Building, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7352. Phone: (919) 962-3285. Fax: (919) 966-0907. E-mail: rjs{at}med.unc.edu.

Journal of Virology, September 2006, p. 9093-9103, Vol. 80, No. 18
0022-538X/06/$08.00+0     doi:10.1128/JVI.00895-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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