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Journal of Virology, August 2008, p. 7379-7387, Vol. 82, No. 15
0022-538X/08/$08.00+0     doi:10.1128/JVI.00358-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Recombinant Adeno-Associated Viral Vectors Are Deficient in Provoking a DNA Damage Response

Michalis Fragkos,¹ Madlaina Breuleux,^1, Nathalie Clément,² and Peter Beard^1*

Ecole Polytechnique Fédérale de Lausanne, Swiss Institute for Experimental Cancer Research, CH-1066 Epalinges, Switzerland,¹ Department of Gene and Cell Medicine, Mount Sinai School of Medicine, New York, New York 10029²

Received 19 February 2008/ Accepted 30 April 2008

Adeno-associated virus type 2 (AAV2) provokes a DNA damage response that mimics a stalled replication fork. We have previously shown that this response is dependent on ataxia telangiectasia-mutated and Rad3-related kinase and involves recruitment of DNA repair proteins into foci associated with AAV2 DNA. Here, we investigated whether recombinant AAV2 (rAAV2) vectors are able to produce a similar response. Surprisingly, the results show that both single-stranded and double-stranded green fluorescent protein-expressing rAAV2 vectors are defective in producing such a response. We show that the DNA damage signaling initiated by AAV2 was not due to the virus-encoded Rep or viral capsid proteins. UV-inactivated AAV2 induced a response similar to that of untreated AAV2. This type of DNA damage response was not provoked by other DNA molecules, such as single-stranded bacteriophage M13 or plasmid DNAs. Rather, the results indicate that the ability of AAV2 to produce a DNA damage response can be attributed to the presence of cis-acting AAV2 DNA sequences, which are absent in rAAV2 vectors and could function as origins of replication creating stalled replication complexes. This hypothesis was tested by using a single-stranded rAAV2 vector containing the p5 AAV2 sequence that has previously been shown to enhance AAV2 replication. This vector was indeed able to trigger DNA damage signaling. These findings support the conclusion that efficient formation of AAV2 replication complexes is required for this AAV2-induced DNA damage response and provide an explanation for the poor response in rAAV2-infected cells.

Published ahead of print on 7 May 2008.

Present address: Basilea Pharmaceutica, Grenzacherstrasse 487, CH-4005 Basel, Switzerland.