Giraud C, Winocour E, Berns K I
Department of Microbiology, Hearst Microbiology Research Center, Cornell University Medical College, New York, New York 10021, USA.
J Virol. 1995 Nov;69(11):6917-24. doi: 10.1128/JVI.69.11.6917-6924.1995.
A model system using an episomal Epstein-Barr virus shuttle vector was recently developed to study the adeno-associated virus (AAV) site-specific integration event in chromosome 19q13.3-qter (C. Giraud, E. Winocour, and K.I. Berns, Proc. Natl. Acad. Sci. USA 91:10039-10043, 1994). In this study, we analyze the recombinant junctions generated after integration of the AAV genome into an Epstein-Barr virus shuttle vector carrying 8.2, 1.6, or 0.51 kb of the chromosome 19 preintegration sequence (AAVS1 locus). In most of the recombinants, one end of the viral genome was joined to a portion of the AAVS1 DNA previously shown to be a minimum target for AAV integration. Within this AAVS1 segment, the AAV insertion points were strikingly clustered around a binding site for the AAV regulatory protein. In all cases, the second junction with AAV occurred with vector DNA outside of the AAVS1 segment. With respect to the viral genome, one junction with the shuttle vector DNA occurred either within the AAV inverted terminal repeat (itr), or near the P5 promoter, approximately 100 nucleotides distal to a modified itr. The modified itr in 5 of 11 recombinants involved a head-to-tail organization. In one such instance, the AAV insert contained slightly more than one genome equivalent arranged in a head-to-tail manner with a junction close to the P5 promoter; the AAV insert in this recombinant episome could be rescued by adenovirus infection and replicated to virus particles. The significance of the head-to-tail organization is discussed in terms of the possible circularization of AAV DNA before or during integration.
最近开发了一种使用附加型爱泼斯坦-巴尔病毒穿梭载体的模型系统，以研究腺相关病毒（AAV）在19号染色体19q13.3-qter上的位点特异性整合事件（C.吉罗、E.维诺库尔和K.I.伯恩斯，《美国国家科学院院刊》91:10039-10043，1994）。在本研究中，我们分析了AAV基因组整合到携带19号染色体整合前序列（AAVS1位点）的8.2、1.6或0.51 kb爱泼斯坦-巴尔病毒穿梭载体后产生的重组连接点。在大多数重组体中，病毒基因组的一端与先前显示为AAV整合最小靶标的AAVS1 DNA的一部分相连。在这个AAVS1片段内，AAV插入点惊人地聚集在AAV调节蛋白的结合位点周围。在所有情况下，与AAV的第二个连接点发生在AAVS1片段之外的载体DNA处。就病毒基因组而言，与穿梭载体DNA的一个连接点发生在AAV反向末端重复序列（itr）内，或靠近P5启动子，距离修饰的itr约100个核苷酸。11个重组体中有5个的修饰itr涉及头对头排列。在一个这样的例子中，AAV插入片段包含略多于一个基因组当量，以头对头的方式排列，连接点靠近P5启动子；该重组附加体中的AAV插入片段可通过腺病毒感染拯救并复制成病毒颗粒。根据整合前或整合过程中AAV DNA可能的环化情况讨论了头对头排列的意义。
