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Background  Adeno-associated virus type 2 (AAV2) preferentially integrates its DNA at a ~2 kb region of human chromosome 19, designated  AAVS1  (also known as  MBS85 ). Integration at  AAVS1  requires the AAV2 replication (Rep) proteins and a DNA sequence within  AAVS1  containing a 16 bp Rep recognition sequence (RRS) and closely spaced Rep nicking site (also referred to as a terminal resolution site, or  trs ). The AAV2 genome is flanked by inverted terminal repeats (ITRs). Each ITR contains an RRS and closely spaced  trs , but the sequences differ from those in  AAVS1 . These ITR sequences are required for replication and packaging.    Results  In this study we demonstrate that the  AAVS1  RRS and  trs  can function in AAV2 replication, packaging and integration by replacing a 61 bp region of the AAV2 ITR with a 49 bp segment of  AAVS1  DNA. Modifying one or both ITRs did not have a large effect on the overall virus titers. These modifications did not detectably affect integration at  AAVS1 , as measured by semi-quantitative nested PCR assays. Sequencing of integration junctions shows the joining of the modified ITRs to  AAVS1  sequences.    Conclusions  The ability of these  AAVS1  sequences to substitute for the AAV2 RRS and  trs  provides indirect evidence that the stable secondary structure encompassing the  trs  is part of the AAV2 packaging signal.

Substitution of adeno-associated virus Rep protein binding and nicking sites with human Chromosome 19 sequences