Identification and characterization of an adeno-associated virus integration site in CV-1 cells from the XXXX .

@entity1 @entity778 @entity3176 @entity4700 @entity6989 @entity21481
@entity778 ( @entity778 ) is a classification given to a group of nonpathogenic, single-stranded DNA viruses known to reside latently in primates. During latency in @entity1 , @entity778 type 2 ( @entity4700 ) preferentially integrates at a site on chromosome 19q13.3ter by targeting a sequence composed of an @entity778 Rep binding element (RBE), a spacer, and a nicking site. Here, we report the DNA sequence of an @entity3176 @entity778 integration site isolated from CV-1 cells. Overall, it has 98% homology to the analogous @entity1 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 @entity778 targeted integration. Using electromobility shift assays it was demonstrated that @entity21481 Rep68 bound the expanded RBE with a sixfold-greater affinity than the @entity1 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 @entity1 and simian RBEs revealed nearly identical protection; however, MI analysis suggested greater interaction with the @entity6989 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 @entity1 RBE and provide further evidence for a developing model that proposes individual roles for the RBE and the spacer and nicking site elements.
