title: Integration preferences of wildtype AAV-2 for consensus rep-binding sites at numerous loci in the human genome.
abstract: Adeno-associated virus type 2 (AAV) is known to establish latency by preferential integration in human chromosome 19q13.42. The AAV non-structural protein Rep appears to target a site called AAVS1 by simultaneously binding to Rep-binding sites (RBS) present on the AAV genome and within AAVS1. In the absence of Rep, as is the case with AAV vectors, chromosomal integration is rare and random. For a genome-wide survey of wildtype AAV integration a linker-selection-mediated (LSM)-PCR strategy was designed to retrieve AAV-chromosomal junctions. DNA sequence determination revealed wildtype AAV integration sites scattered over the entire human genome. The bioinformatic analysis of these integration sites compared to those of rep-deficient AAV vectors revealed a highly significant overrepresentation of integration events near to consensus RBS. Integration hotspots included AAVS1 with 10% of total events. Novel hotspots near consensus RBS were identified on chromosome 5p13.3 denoted AAVS2 and on chromsome 3p24.3 denoted AAVS3. AAVS2 displayed seven independent junctions clustered within only 14 bp of a consensus RBS which proved to bind Rep in vitro similar to the RBS in AAVS3. Expression of Rep in the presence of rep-deficient AAV vectors shifted targeting preferences from random integration back to the neighbourhood of consensus RBS at hotspots and numerous additional sites in the human genome. In summary, targeted AAV integration is not as specific for AAVS1 as previously assumed. Rather, Rep targets AAV to integrate into open chromatin regions in the reach of various, consensus RBS homologues in the human genome.
authors.affiliation: Institute of Virology, Campus Benjamin Franklin, Charité-Universitätsmedizin Berlin, Berlin, Germany.
authors.forename: Daniela
authors.initials: D
authors.lastname: Hüser
authors.forename: Andreas
authors.initials: A
authors.lastname: Gogol-Döring
authors.forename: Timo
authors.initials: T
authors.lastname: Lutter
authors.forename: Stefan
authors.initials: S
authors.lastname: Weger
authors.forename: Kerstin
authors.initials: K
authors.lastname: Winter
authors.forename: Eva-Maria
authors.initials: EM
authors.lastname: Hammer
authors.forename: Toni
authors.initials: T
authors.lastname: Cathomen
authors.forename: Knut
authors.initials: K
authors.lastname: Reinert
authors.forename: Regine
authors.initials: R
authors.lastname: Heilbronn
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D002877:Chromosomes, Human; D019295:Computational Biology; D004268:DNA-Binding Proteins / Q000378:metabolism*; D000229:Dependovirus / Q000235:genetics*; D015894:Genome, Human; D016679:Genome, Viral; D006801:Humans; D014764:Viral Proteins / Q000378:metabolism*; D016662:Virus Integration; D017735:Virus Latency / Q000235:genetics*
chemical_list: D004268:DNA-Binding Proteins; D014764:Viral Proteins; C068734:rep proteins, Adeno-associated virus 2