Structural analysis of adeno-associated virus transduction circular intermediates
Structural analysis of adeno-associated virus transduction circular intermediates
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DOI：
10.1006/viro.1999.9821
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发表时间：
1999-08-15
期刊：
影响因子：
3.7
通讯作者：
Engelhardt, JF
中科院分区：
文献类型：
--
作者：
Duan, DS;Yan, ZY;Engelhardt, JF
重组腺相关病毒（rAAV）最近被证明在肌肉组织和细胞系中转导后形成环状中间体。虽然限制性内切酶和Southern blot分析显示了一致的单体和多个头尾构象，但由于ITR阵列的高二级结构，缺乏详细的结构序列分析。为了进一步了解AAV环状基因组由线性单链病毒DNA形成的潜在机制，我们对从原代成纤维细胞和Hela细胞中独立分离的7个环状中间体中的ITR阵列进行了化学测序。这些研究的结果表明，几种类型的圆形中间体具有镶嵌的ITR元件，两侧是两个D序列。最主要的形式是由类似于先前产生的AAV双d质粒的结构组成，一个完整的ITR两侧有两个d区元件。然而，中间删除的ITR数组也有多个完整的ITR。基于这一结构信息，我们提出了一种AAV环状中间体通过在长柄单链AAV基因组的ITR端之间重组/连接形成的模型。(C) 1999学术出版社。
Recombinant adeno-associated virus (rAAV) has recently been demonstrated to form circular intermediates following transduction in muscle tissue and cell lines. Although restriction enzyme and Southern blot analysis has revealed a consistent monomer and multimer head-to-tail conformation, detailed structural sequence analysis has been lacking due to the high secondary structure of the ITR arrays. To gain further insight into potential mechanisms by which AAV circular genomes are formed from linear single-stranded viral DNA, we have performed chemical sequencing of ITR arrays within seven circular intermediates independently isolated from primary fibroblasts and Hela cells. Results from these studies demonstrated several types of circular intermediates with mosaic ITR elements flanked by two D sequences. The most predominant form consisted of a structure similar to that of previously generated AAV double-D plasmids, with one complete ITR flanked by two D-region elements. However, intermediately deleted ITR arrays with more than one complete ITR were also seen. Based on this structural information, we have proposed a model for formation of AAV circular intermediates by recombination/ligation between ITR ends of panhandle single-stranded AAV genomes. (C) 1999 Academic Press.