Institute of Virology, Campus Benjamin Franklin, Charité-Universitätsmedizin Berlin, Berlin, Germany.
PLoS Pathog. 2010 Jul 8;6(7):e1000985. doi: 10.1371/journal.ppat.1000985.
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.
腺相关病毒 2 型（AAV）通过优先整合到人类染色体 19q13.42 中而建立潜伏状态。AAV 非结构蛋白 Rep 似乎通过同时结合 AAV 基因组和 AAVS1 内存在的 Rep 结合位点（RBS）来靶向称为 AAVS1 的位点。在没有 Rep 的情况下，就像 AAV 载体一样，染色体整合很少且随机。为了对野生型 AAV 整合进行全基因组调查，设计了一种连接体选择介导（LSM）-PCR 策略来检索 AAV-染色体连接。DNA 序列测定揭示了野生型 AAV 整合位点散布在整个人类基因组中。这些整合位点与 rep 缺陷型 AAV 载体的整合位点进行生物信息学分析表明，在共识 RBS 附近，整合事件的代表性过高。整合热点包括 AAVS1，占总事件的 10%。在染色体 5p13.3 上发现了靠近共识 RBS 的新热点，命名为 AAVS2，在染色体 3p24.3 上发现了新热点，命名为 AAVS3。AAVS2 显示了七个独立的连接，簇集在一个共识 RBS 内仅 14 个碱基对内，该 RBS 被证明在体外与 AAVS3 中的 RBS 相似地结合 Rep。在 rep 缺陷型 AAV 载体存在的情况下表达 Rep，使靶向偏好从随机整合转变为热点处的共识 RBS 附近，以及人类基因组中的许多其他位点。总之，靶向 AAV 整合并不像以前假设的那样特异性针对 AAVS1。相反，Rep 使 AAV 靶向整合到人类基因组中各种共识 RBS 同源物可及的开放染色质区域。
