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Journal of Virology, June 2003, p. 6995-7006, Vol. 77, No. 12
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.12.6995-7006.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Identification of Amino Acid Residues in the Capsid Proteins of Adeno-Associated Virus Type 2 That Contribute to Heparan Sulfate Proteoglycan Binding{dagger}

Shaun R. Opie,1,2 Kenneth H. Warrington, Jr.,1,2 Mavis Agbandje-McKenna,1,3,4,5 Sergei Zolotukhin,1,2 and Nicholas Muzyczka1,2,3*

Department of Molecular Genetics and Microbiology,1 Department of Biochemistry and Molecular Biology,5 Center for Structural Biology,3 UF Brain Institute,4 Powell Gene Therapy Center, College of Medicine, University of Florida, Gainesville, Florida 32610-02662

Received 20 December 2002/ Accepted 28 March 2003

The adeno-associated virus type 2 (AAV2) uses heparan sulfate proteoglycan (HSPG) as its primary cellular receptor. In order to identify amino acids within the capsid of AAV2 that contribute to HSPG association, we used biochemical information about heparin and heparin sulfate, AAV serotype protein sequence alignments, and data from previous capsid studies to select residues for mutagenesis. Charged-to-alanine substitution mutagenesis was performed on individual residues and combinations of basic residues for the production and purification of recombinant viruses that contained a green fluorescent protein (GFP) reporter gene cassette. Intact capsids were assayed for their ability to bind to heparin-agarose in vitro, and virions that packaged DNA were assayed for their ability to transduce normally permissive cell lines. We found that mutation of arginine residues at position 585 or 588 eliminated binding to heparin-agarose. Mutation of residues R484, R487, and K532 showed partial binding to heparin-agarose. We observed a general correlation between heparin-agarose binding and infectivity as measured by GFP transduction; however, a subset of mutants that partially bound heparin-agarose (R484A and K532A) were completely noninfectious, suggesting that they had additional blocks to infectivity that were unrelated to heparin binding. Conservative mutation of positions R585 and R588 to lysine slightly reduced heparin-agarose binding and had comparable effects on infectivity. Substitution of AAV2 residues 585 through 590 into a location predicted to be structurally equivalent in AAV5 generated a hybrid virus that bound to heparin-agarose efficiently and was able to package DNA but was noninfectious. Taken together, our results suggest that residues R585 and R588 are primarily responsible for heparin sulfate binding and that mutation of these residues has little effect on other aspects of the viral life cycle. Interactive computer graphics examination of the AAV2 VP3 atomic coordinates revealed that residues which contribute to heparin binding formed a cluster of five basic amino acids that presented toward the icosahedral threefold axis from the surrounding spike protrusion. Three other kinds of mutants were identified. Mutants R459A, H509A, and H526A/K527A bound heparin at levels comparable to that of wild-type virus but were defective for transduction. Another mutant, H358A, was defective for capsid assembly. Finally, an R459A mutant produced significantly lower levels of full capsids, suggesting a packaging defect.

* Corresponding author. Mailing address: Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, P.O. Box 100266 JHMHSC, Gainesville, FL 32610-8541. Phone: (352) 392-5914. Fax: (352) 392-5914. E-mail: muzyczka{at}ufl.edu.

{dagger} This study is dedicated to the memory of Wu Xiao.

Journal of Virology, June 2003, p. 6995-7006, Vol. 77, No. 12
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.12.6995-7006.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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