Bohenzky R A, LeFebvre R B, Berns K I
Department of Immunology and Medical Microbiology, College of Medicine, University of Florida, Gainesville 32610.
Virology. 1988 Oct;166(2):316-27. doi: 10.1016/0042-6822(88)90502-8.
The defective parvovirus, adeno-associated virus (AAV), contains a single-stranded DNA genome of 4681 bases with inverted terminal repeats of 145 bases. The distal 125 bases of the repeat are palindromic allowing a hairpin to form for initiation of DNA synthesis. The palindromic region contains three palindromes, two smaller internal palindromes flanked by a larger palindrome, which allow the hairpinned DNA to assume a T-shaped conformation during DNA replication. Deletion of an internal palindrome forming one of the crossarms of the T results in the inability of the AAV genome to be rescued from plasmid sequences and replicated. Restoration of the crossarm sequences with DNA that differs in primary sequence but maintains the symmetry of the palindrome results in viable AAV and propagation of the mutant sequences. In this paper we report further studies on the nature of mutants made within the crossarm of the T. Two types of substitution mutants were analyzed. Symmetrical sequence substitution mutants were viable as previously reported. An analysis of the kinetics of AAV DNA accumulation showed that the symmetrical sequence substitution mutants were indistinguishable from wild-type AAV. This was true if the AAV DNA was introduced into the cells either as plasmid DNA or as DNA extracted from virions. In contrast, intermolecular competition experiments showed either a dominance of the wild-type sequence or codominance of both sequences when both alleles were cotransfected into helper virus-infected cells. A preference for the wild-type sequence may also exist but is not required for efficient AAV replication. The second type of mutation studied was an asymmetrical sequence substitution mutant. This mutant was replicated but at a level too low to be propagated. These data suggest that symmetry is required in the internal palindromic region, presumably for the formation of the crossarm structure in the T-shape.
缺陷细小病毒——腺相关病毒（AAV），含有一个4681个碱基的单链DNA基因组，其末端有145个碱基的反向重复序列。重复序列远端的125个碱基是回文序列，可形成发夹结构以启动DNA合成。回文区域包含三个回文结构，两个较小的内部回文结构两侧是一个较大的回文结构，这使得发夹状DNA在DNA复制过程中呈现T形构象。删除形成T形交叉臂之一的内部回文结构会导致AAV基因组无法从质粒序列中拯救并复制。用一级序列不同但保持回文对称性的DNA恢复交叉臂序列，可产生有活力的AAV并传播突变序列。在本文中，我们报告了对T形交叉臂内产生的突变体性质的进一步研究。分析了两种类型的取代突变体。如先前报道，对称序列取代突变体是有活力的。对AAV DNA积累动力学的分析表明，对称序列取代突变体与野生型AAV没有区别。无论AAV DNA是以质粒DNA形式还是从病毒粒子中提取的DNA形式引入细胞，都是如此。相比之下，分子间竞争实验表明，当两个等位基因共转染到辅助病毒感染的细胞中时，要么野生型序列占优势，要么两个序列共显性。可能也存在对野生型序列的偏好，但高效AAV复制并不需要。研究的第二种突变类型是不对称序列取代突变体。该突变体可以复制，但水平过低无法传播。这些数据表明，内部回文区域需要对称性，大概是为了在T形中形成交叉臂结构。
