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Journal of Virology, November 2006, p. 10346-10356, Vol. 80, No. 21
0022-538X/06/$08.00+0     doi:10.1128/JVI.00841-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Host Cell DNA Repair Pathways in Adeno-Associated Viral Genome Processing

Vivian W. Choi,1,2,{dagger} Douglas M. McCarty,2,{ddagger} and R. Jude Samulski1,2*

Department of Pharmacology,1 Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 275992

Received 24 April 2006/ Accepted 31 July 2006

Recentstudies have shown that wild-type and recombinant adeno-associated virus (AAV and rAAV) genomes persist in human tissue predominantly as double-stranded (ds) circular episomes derived from input linear single-stranded virion DNA. Using self-complementary recombinant AAV (scAAV) vectors, we generated intermediates that directly transition to ds circular episomes. The scAAV genome ends are palindromic hairpin-structured terminal repeats, resembling a double-stranded break repair intermediate. Utilizing this substrate, we found cellular DNA recombination and repair factors to be essential for generating circular episomal products. To identify the specific cellular proteins involved, the scAAV circularization-dependent vector was used as a reporter in 19 mammalian DNA repair-deficient cell lines. The results show that RecQ helicase family members (BLM and WRN), Mre11 and NBS1 of the Mre11-Rad50-Nbs1 (MRN) complex, and ATM are required for efficient scAAV genome circularization. We further demonstrated that the scAAV genome requires ATM and DNA-PKCS, but not NBS1, to efficiently convert to a circular form in nondividing cells in vivo using transgenic mice. These studies identify specific pathways involved for further elucidating viral and cellular mechanisms of DNA maintenance important to the viral life cycle and vector utilizations.

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

{dagger} Present address: CBR Institute for Biomedical Research, Children's Hospital, Department of Genetics, Harvard Medical School, Boston, MA 02115.

{ddagger} Present address: Center for Gene Therapy, Columbus Children's Research Institute, The Ohio State University, Columbus, OH 43205.

Journal of Virology, November 2006, p. 10346-10356, Vol. 80, No. 21
0022-538X/06/$08.00+0     doi:10.1128/JVI.00841-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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