Gene Therapy Center, University of North Carolina, Chapel Hill, North Carolina, USA.
Gene Therapy Center, University of North Carolina, Chapel Hill, North Carolina, USA Department of Pharmacology, University of North Carolina, Chapel Hill, North Carolina, USA
J Virol. 2015 Jan;89(1):181-94. doi: 10.1128/JVI.01990-14. Epub 2014 Oct 15.
Adeno-associated virus (AAV) is a dependent virus of the family Parvoviridae. The gene expression and replication of AAV and derived recombinant AAV (rAAV) vectors are severely limited (>10-fold) by the cellular DNA damage-sensing complex made up of Mre11, Rad50, and Nbs1 (MRN). The AAV genome does not encode the means to circumvent this block to productive infection but relies on coinfecting helper virus to do so. Using adenovirus helper proteins E1B55k and E4orf6, which enhance the transduction of AAV via degradation of MRN, we investigated the mechanism through which this DNA damage complex inhibits gene expression from rAAV. We tested the substrate specificity of inhibition and the contribution of different functions of the MRN complex. Our results demonstrate that both single- and double-stranded rAAV vectors are inhibited by MRN, which is in contrast to the predominant model that inhibition is the result of a block to second-strand synthesis. Exploring the contribution of known functions of MRN, we found that inhibition of rAAV does not require downstream DNA damage response factors, including signaling kinases ATM and ATR. The nuclease domain of Mre11 appears to play only a minor role in inhibition, while the DNA binding domain makes a greater contribution. Additionally, mutation of the inverted terminal repeat of the rAAV genome, which has been proposed to be the signal for interaction with MRN, is tolerated by the mechanism of inhibition. These results articulate a model of inhibition of gene expression in which physical interaction is more important than enzymatic activity and several key downstream damage repair factors are dispensable.
Many viruses modulate the host DNA damage response (DDR) in order to create a cellular environment permissive for infection. The MRN complex is a primary sensor of damage in the cell but also responds to invading viral genomes, often posing a block to infection. AAV is greatly inhibited by MRN and dependent on coinfecting helper virus, such as adenovirus, to remove this factor. Currently, the mechanism through which MRN inhibits AAV and other viruses is poorly understood. Our results reform the predominant model that inhibition of rAAV by MRN is due to limiting second-strand DNA synthesis. Instead, a novel mechanism of inhibition of gene expression independent of a block in rAAV DNA synthesis or downstream damage factors is indicated. These findings have clear implications for understanding this restriction to transduction of AAV and rAAV vectors, which have high therapeutic relevance and likely translate to other viruses that must navigate the DDR.
腺相关病毒 (AAV) 是细小病毒科的一种依赖病毒。AAV 和衍生的重组 AAV(rAAV) 载体的基因表达和复制受到由 Mre11、Rad50 和 Nbs1 (MRN) 组成的细胞 DNA 损伤感应复合物的严重限制（超过 10 倍）。AAV 基因组没有编码绕过这种有效感染的方法，而是依赖于辅助病毒进行感染。我们使用腺病毒辅助蛋白 E1B55k 和 E4orf6，它们通过降解 MRN 增强 AAV 的转导，研究了这种 DNA 损伤复合物抑制 rAAV 基因表达的机制。我们测试了抑制的底物特异性和 MRN 复合物不同功能的贡献。我们的结果表明，MRN 抑制单链和双链 rAAV 载体，这与主要模型相反，该模型认为抑制是第二链合成受阻的结果。探索 MRN 已知功能的贡献，我们发现 rAAV 的抑制不需要下游 DNA 损伤反应因子，包括信号激酶 ATM 和 ATR。Mre11 的核酸酶结构域似乎只在抑制中起次要作用，而 DNA 结合结构域的贡献更大。此外，rAAV 基因组的反向末端重复突变，该突变已被提议作为与 MRN 相互作用的信号，在抑制机制中是可以耐受的。这些结果阐明了一种基因表达抑制模型，其中物理相互作用比酶活性更重要，并且几个关键的下游损伤修复因子是可有可无的。
许多病毒调节宿主 DNA 损伤反应 (DDR) 以创造有利于感染的细胞环境。MRN 复合物是细胞中损伤的主要传感器，但也会对入侵的病毒基因组做出反应，通常会对感染造成阻碍。AAV 受到 MRN 的强烈抑制，并依赖于辅助腺病毒等辅助病毒来去除这种因子。目前，MRN 抑制 AAV 和其他病毒的机制知之甚少。我们的结果改变了 MRN 抑制 rAAV 是由于限制第二链 DNA 合成的主要模型。相反，表明了一种与 rAAV DNA 合成或下游损伤因子的阻断无关的新型基因表达抑制机制。这些发现对理解这种对 AAV 和 rAAV 载体转导的限制具有明确的意义，这些载体具有很高的治疗相关性，并且可能转化为其他必须在 DDR 中导航的病毒。
