Schnepp Bruce C, Jensen Ryan L, Clark K Reed, Johnson Philip R
The Research Institute, Nationwide Children's Hospital, Columbus, Ohio, USA.
J Virol. 2009 Feb;83(3):1456-64. doi: 10.1128/JVI.01686-08. Epub 2008 Nov 19.
Adeno-associated virus (AAV) replication and biology have been extensively studied using cell culture systems, but there is precious little known about AAV biology in natural hosts. As part of our ongoing interest in the in vivo biology of AAV, we previously described the existence of extrachromosomal proviral AAV genomes in human tissues. In the current work, we describe the molecular structure of infectious DNA clones derived directly from these tissues. Sequence-specific linear rolling-circle amplification was utilized to isolate clones of native circular AAV DNA. Several molecular clones containing unit-length viral genomes directed the production of infectious wild-type AAV upon DNA transfection in the presence of adenovirus help. DNA sequence analysis of the molecular clones revealed the ubiquitous presence of a double-D inverted terminal repeat (ITR) structure, which implied a mechanism by which the virus is able to maintain ITR sequence continuity and persist in the absence of host chromosome integration. These data suggest that the natural life cycle of AAV, unlike that of retroviruses, might not have genome integration as an obligatory component.
腺相关病毒（AAV）的复制和生物学特性已在细胞培养系统中得到广泛研究，但对于其在天然宿主中的生物学特性却知之甚少。作为我们对AAV体内生物学特性持续关注的一部分，我们之前描述了人类组织中存在染色体外前病毒AAV基因组。在当前的研究中，我们描述了直接从这些组织中获得的感染性DNA克隆的分子结构。利用序列特异性线性滚环扩增技术分离天然环状AAV DNA的克隆。几个包含单位长度病毒基因组的分子克隆在腺病毒辅助下进行DNA转染时可指导产生感染性野生型AAV。对分子克隆的DNA序列分析揭示了双D型反向末端重复（ITR）结构的普遍存在，这暗示了一种机制，通过该机制病毒能够维持ITR序列的连续性并在不整合到宿主染色体的情况下持续存在。这些数据表明，与逆转录病毒不同，AAV的自然生命周期可能并不以基因组整合作为必需组成部分。
