Wang X S, Srivastava A
Department of Medicine, Walther Oncology Center, Indiana University School of Medicine, Indianapolis 46202, USA.
J Virol. 1997 Feb;71(2):1140-6. doi: 10.1128/JVI.71.2.1140-1146.1997.
The adeno-associated virus 2 (AAV) contains a single-stranded DNA genome of which the terminal 145 nucleotides are palindromic and form T-shaped hairpin structures. These inverted terminal repeats (ITRs) play an important role in AAV DNA replication and resolution, since each of the ITRs contains a terminal resolution site (trs) that is the target site for the AAV rep gene products (Rep). However, the Rep proteins also interact with the AAV DNA sequences that lie outside the ITRs, and the ITRs also play a crucial role in excision of the proviral genome from latently infected cells or from recombinant AAV plasmids. To distinguish between Rep-mediated excision of the viral genome during rescue from recombinant AAV plasmids and the Rep-mediated resolution of the ITRs during AAV DNA replication, we constructed recombinant AAV genomes that lacked either the left or the right ITR sequence and one of the Rep-binding sites (RBSs). No rescue and replication of the AAV genome occurred from these plasmids following transfection into adenovirus type 2-infected human KB cells, as expected. However, excision and abundant replication of the vector sequences was clearly detected from the plasmid that lacked the AAV left ITR, suggesting the existence of an additional putative excision site in the left end of the AAV genome. This site was precisely mapped to one of the AAV promoters at map unit 5 (AAV p5) that also contains an RBS. Furthermore, deletion of this RBS abolished the rescue and replication of the vector sequences. These studies suggest that the Rep-mediated cleavage at the RBS during viral DNA replication may, in part, account for the generation of the AAV defective interfering particles.
腺相关病毒2型（AAV）含有单链DNA基因组，其末端145个核苷酸是回文序列，并形成T形发夹结构。这些反向末端重复序列（ITR）在AAV DNA复制和拆分过程中发挥重要作用，因为每个ITR都包含一个末端拆分位点（trs），该位点是AAV rep基因产物（Rep）的靶位点。然而，Rep蛋白也与ITR之外的AAV DNA序列相互作用，并且ITR在从潜伏感染细胞或重组AAV质粒中切除前病毒基因组的过程中也起着关键作用。为了区分重组AAV质粒拯救过程中Rep介导的病毒基因组切除与AAV DNA复制过程中Rep介导的ITR拆分，我们构建了缺少左或右ITR序列以及一个Rep结合位点（RBS）的重组AAV基因组。正如预期的那样，将这些质粒转染到2型腺病毒感染的人KB细胞后，未发生AAV基因组的拯救和复制。然而，从缺少AAV左ITR的质粒中清楚地检测到载体序列的切除和大量复制，这表明在AAV基因组左端存在一个额外的假定切除位点。该位点被精确地定位到图谱单位5处的一个AAV启动子（AAV p5），该启动子也包含一个RBS。此外，删除该RBS消除了载体序列的拯救和复制。这些研究表明，病毒DNA复制过程中Rep介导的RBS切割可能部分解释了AAV缺陷干扰颗粒的产生。
