Wang X S, Qing K, Ponnazhagan S, Srivastava A
Department of Medicine, Indiana University School of Medicine, Indianapolis 46202, USA.
J Virol. 1997 Apr;71(4):3077-82. doi: 10.1128/JVI.71.4.3077-3082.1997.
The adeno-associated virus type 2 (AAV) genome contains inverted terminal repeats (ITRs) of 145 nucleotides. The terminal 125 nucleotides of each ITR form palindromic hairpin (HP) structures that serve as primers for AAV DNA replication. These HP structures also play an important role in integration as well as rescue of the proviral genome from latently infected cells or from recombinant AAV plasmids. Each ITR also contains a stretch of 20 nucleotides, designated the D sequence, that is not involved in HP structure formation. We have recently shown that the D sequence plays a crucial role in high-efficiency rescue, selective replication, and encapsidation of the AAV genome and that a host cell protein, designated the D sequence-binding protein (D-BP), specifically interacts with this sequence (X.-S. Wang, S. Ponnazhagan, and A. Srivastava, J. Virol. 70:1668-1677, 1996). We have now performed mutational analyses of the D sequences to evaluate their precise role in viral DNA rescue, replication, and packaging. We report here that 10 nucleotides proximal to the HP structure in each of the D sequences are necessary and sufficient to mediate high-efficiency rescue, replication, and encapsidation of the viral genome in vivo. In in vitro studies, the same 10 nucleotides were found to be required for specific interaction with D-BP, but viral Rep protein-mediated cleavage at the functional terminal resolution site is independent of these sequences. These data suggest that AAV replication and terminal resolution functions can be uncoupled and that the lack of efficient replication of AAV DNA may not be a consequence of impaired resolution of the viral ITRs. These studies further illustrate that the D sequence-D-BP interaction plays an important role in the AAV life cycle and indicate that it may be possible to develop the next generation of AAV vectors capable of encapsidating larger pieces of DNA.
2型腺相关病毒（AAV）基因组包含145个核苷酸的反向末端重复序列（ITR）。每个ITR的末端125个核苷酸形成回文发夹（HP）结构，作为AAV DNA复制的引物。这些HP结构在整合以及从潜伏感染细胞或重组AAV质粒中拯救前病毒基因组方面也发挥着重要作用。每个ITR还包含一段20个核苷酸的序列，称为D序列，它不参与HP结构的形成。我们最近表明，D序列在AAV基因组的高效拯救、选择性复制和包装中起着关键作用，并且一种宿主细胞蛋白，称为D序列结合蛋白（D-BP），与该序列特异性相互作用（X.-S. Wang、S. Ponnazhagan和A. Srivastava，《病毒学杂志》70:1668-1677，1996年）。我们现在对D序列进行了突变分析，以评估它们在病毒DNA拯救、复制和包装中的精确作用。我们在此报告，每个D序列中靠近HP结构的10个核苷酸对于在体内介导病毒基因组的高效拯救、复制和包装是必要且充分的。在体外研究中，发现相同的10个核苷酸是与D-BP特异性相互作用所必需的，但病毒Rep蛋白在功能性末端切割位点的切割与这些序列无关。这些数据表明，AAV复制和末端切割功能可以分离，并且AAV DNA缺乏有效复制可能不是病毒ITR切割受损的结果。这些研究进一步说明，D序列-D-BP相互作用在AAV生命周期中起着重要作用，并表明有可能开发出能够包装更大片段DNA的下一代AAV载体。
