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Journal of Virology, February 2003, p. 1904-1915, Vol. 77, No. 3
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.3.1904-1915.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Identification and Characterization of an Adeno-Associated Virus Integration Site in CV-1 Cells from the African Green Monkey

Terry J. Amiss,1,2 Doug M. McCarty,2 Anna Skulimowski,2 and R. Jude Samulski1,2,3*

Department of Pharmacology,1 Gene Therapy Center,2 Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 275993

Received 26 July 2002/ Accepted 4 November 2002

Adeno-associated virus (AAV) is a classification given to a group of nonpathogenic, single-stranded DNA viruses known to reside latently in primates. During latency in humans, AAV type 2 (AAV2) preferentially integrates at a site on chromosome 19q13.3ter by targeting a sequence composed of an AAV Rep binding element (RBE), a spacer, and a nicking site. Here, we report the DNA sequence of an African green monkey AAV integration site isolated from CV-1 cells. Overall, it has 98% homology to the analogous human site, including identical spacer and nicking sequences. However, the simian RBE is expanded, having five perfect directly repeated GAGC tetramers. We carried out a number of in vitro and in vivo assays to determine the effect of this expanded RBE sequence on the Rep-RBE interaction and AAV targeted integration. Using electromobility shift assays it was demonstrated that AAV4 Rep68 bound the expanded RBE with a sixfold-greater affinity than the human RBE. To determine the basis for the affinity increase, DNase I protection and methylation interference (MI) assays were performed. Comparison of footprints on both the human and simian RBEs revealed nearly identical protection; however, MI analysis suggested greater interaction with the guanine nucleotides of the expanded RBE, thus providing a biochemical basis for the increased binding activity. In vivo, integration targeted to the simian RBE was demonstrated by PCR analysis of latently infected Cos-7 cells. Interestingly, the frequency of site-specific integration was twofold greater in Cos-7 cells than in HeLa cells. Overall, these experiments establish that the simian RBE, identified in CV-1 cells, functions analogously to the human RBE and provide further evidence for a developing model that proposes individual roles for the RBE and the spacer and nicking site elements.

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

Journal of Virology, February 2003, p. 1904-1915, Vol. 77, No. 3
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.3.1904-1915.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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