Recombinant adeno-associated viral vectors are deficient in provoking a DNA damage response
Recombinant adeno-associated viral vectors are deficient in provoking a DNA damage response
复制标题
DOI：
10.1128/jvi.00358-08
复制
发表时间：
2008-08-01
影响因子：
5.4
通讯作者：
Beard, Peter
中科院分区：
文献类型：
--
作者：
Fragkos, Michalis;Breuleux, Madlaina;Beard, Peter
腺相关病毒2型（AAV 2）引起DNA损伤反应，模拟停滞的复制叉。我们以前已经表明，这种反应是依赖于共济失调毛细血管扩张症突变和Rad3相关的激酶，并涉及招募DNA修复蛋白到与AAV2 DNA相关的病灶。在这里，我们研究了重组AAV2（rAAV2）载体是否能够产生类似的反应。令人惊讶的是，结果表明表达单链和双链绿色荧光蛋白的rAAV 2载体在产生这种应答方面都有缺陷。我们表明，由AAV2启动的DNA损伤信号不是由于病毒编码的Rep或病毒衣壳蛋白。UN灭活的AAV2诱导与未处理的AAV2相似的应答。这种类型的DNA损伤反应不是由其他DNA分子引起的，例如单链噬菌体M13或质粒DNA。相反，结果表明，AAV 2产生DNA损伤反应的能力可归因于顺式作用的AAV 2 DNA序列的存在，其在rAAV 2载体中不存在，并且可以作为复制起点发挥作用，产生停滞的复制复合物。通过使用含有p5 AAV 2序列的单链rAAV 2载体来测试该假设，所述p5 AAV 2序列先前已显示出增强AAV 2复制。这种载体确实能够触发DNA损伤信号。这些发现支持了这样的结论，即AAV 2复制复合物的有效形成是这种AAV 2诱导的DNA损伤反应所必需的，并为rAAV 2感染细胞中的不良反应提供了解释。
Adeno-associated virus type 2 (AAV2) provokes a DNA damage response that mimics a stalled replication fork. We have previously shown that this response is dependent on ataxia telangiectasia-mutated and Rad3-related kinase and involves recruitment of DNA repair proteins into foci associated with AAV2 DNA. Here, we investigated whether recombinant AAV2 (rAAV2) vectors are able to produce a similar response. Surprisingly, the results show that both single-stranded and double-stranded green fluorescent protein-expressing rAAV2 vectors are defective in producing such a response. We show that the DNA damage signaling initiated by AAV2 was not due to the virus-encoded Rep or viral capsid proteins. UN-inactivated AAV2 induced a response similar to that of untreated AAV2. This type of DNA damage response was not provoked by other DNA molecules, such as single-stranded bacteriophage M13 or plasmid DNAs. Rather, the results indicate that the ability of AAV2 to produce a DNA damage response can be attributed to the presence of cis-acting AAV2 DNA sequences, which are absent in rAAV2 vectors and could function as origins of replication creating stalled replication complexes. This hypothesis was tested by using a single-stranded rAAV2 vector containing the p5 AAV2 sequence that has previously been shown to enhance AAV2 replication. This vector was indeed able to trigger DNA damage signaling. These findings support the conclusion that efficient formation of AAV2 replication complexes is required for this AAV2-induced DNA damage response and provide an explanation for the poor response in rAAV2-infected cells.