An siRNA Screen Identifies the U2 snRNP Spliceosome as a Host Restriction Factor for Recombinant Adeno-associated Viruses.
An siRNA Screen Identifies the U2 snRNP Spliceosome as a Host Restriction Factor for Recombinant Adeno-associated Viruses.
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DOI：
10.1371/journal.ppat.1005082
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发表时间：
2015-08
期刊：
影响因子：
6.7
通讯作者：
Ikeda Y
中科院分区：
文献类型：
--
作者：
Schreiber CA;Sakuma T;Izumiya Y;Holditch SJ;Hickey RD;Bressin RK;Basu U;Koide K;Asokan A;Ikeda Y
腺相关病毒（AAV）已经进化到在腺病毒和其他辅助病毒共同感染期间利用宿主细胞机器的动态重组。在没有辅助病毒的情况下，蛋白酶体和 DNA 损伤反应机制等宿主因素已被证明可以通过限制从核进入到第二链 DNA 合成的过程来有效抑制 AAV 转导。为了确定可能影响 AAV 感染其他关键步骤的宿主因素，我们筛选了一个 siRNA 文库，该文库揭示了几个候选基因，包括 PHD 指状结构域蛋白 5A (PHF5A)（一种 U2 snRNP 相关蛋白）。 PHF5A 表达的破坏通过增加转录物水平选择性地增强 AAV 的转基因表达，并且似乎以血清型和细胞类型无关的方式影响第二链合成后的步骤。 U2 snRNP 和相关蛋白（如 SF3B1 和 U2AF1）的遗传破坏也增加了 AAV 载体的表达，表明 U2 snRNP 剪接体复合物在这种宿主介导的限制中发挥着关键作用。值得注意的是，腺病毒共感染和 U2 snRNP 抑制似乎针对增加 AAV 载体表达的共同途径。此外，meayamycin B（一种有效的 SF3B1 抑制剂）对 U2 snRNP 的药理抑制显着增强了临床相关细胞类型的 AAV 载体转导。进一步分析表明，U2 snRNP 蛋白通过直接识别完整 AAV 衣壳来抑制 AAV 载体转基因表达。总之，我们确定 U2 snRNP 和相关剪接因子（已知在腺病毒感染期间受到影响）作为有效限制 AAV 转基因表达的新型宿主限制因子。同时，我们假设对剪接体机制组件的药理学/遗传操作可能使重组 AAV 载体的基因转移方式更加有效。哺乳动物细胞已经发展出多种先天/内在免疫策略来对抗病毒感染。单链 DNA 病毒、腺相关病毒 (AAV) 的入境后感染步骤受到此类限制。在这里，我们筛选了一个 siRNA 文库，以确定一种参与 AAV 限制的新型细胞因子。我们发现 PHF5A（U2 snRNP mRNA 剪接因子的一个组成部分）可阻断重组 AAV 载体的表达。 PHF5A 表达的破坏特别增强了 AAV 载体的性能。此外，对其他U2 snRNP蛋白的遗传和药理学抑制，但对参与其他剪接步骤的剪接体蛋白的抑制，强烈增加了AAV载体的转基因表达。进一步的研究表明，U2 snRNP 蛋白识别传入的 AAV 衣壳，在第二链合成后的步骤中介导这种细胞限制。总之，我们将 U2 snRNP 剪接体复合物鉴定为有效限制重组 AAV 载体的新型宿主因子。考虑到DNA病毒感染中宿主剪接机制的频繁重组，可以想象U2 snRNP发挥了广谱抗病毒因子的作用，并且辅助病毒已经进化到通过隔离snRNP蛋白来抵消这种限制。
Adeno-associated viruses (AAV) have evolved to exploit the dynamic reorganization of host cell machinery during co-infection by adenoviruses and other helper viruses. In the absence of helper viruses, host factors such as the proteasome and DNA damage response machinery have been shown to effectively inhibit AAV transduction by restricting processes ranging from nuclear entry to second-strand DNA synthesis. To identify host factors that might affect other key steps in AAV infection, we screened an siRNA library that revealed several candidate genes including the PHD finger-like domain protein 5A (PHF5A), a U2 snRNP-associated protein. Disruption of PHF5A expression selectively enhanced transgene expression from AAV by increasing transcript levels and appears to influence a step after second-strand synthesis in a serotype and cell type-independent manner. Genetic disruption of U2 snRNP and associated proteins, such as SF3B1 and U2AF1, also increased expression from AAV vector, suggesting the critical role of U2 snRNP spliceosome complex in this host-mediated restriction. Notably, adenoviral co-infection and U2 snRNP inhibition appeared to target a common pathway in increasing expression from AAV vectors. Moreover, pharmacological inhibition of U2 snRNP by meayamycin B, a potent SF3B1 inhibitor, substantially enhanced AAV vector transduction of clinically relevant cell types. Further analysis suggested that U2 snRNP proteins suppress AAV vector transgene expression through direct recognition of intact AAV capsids. In summary, we identify U2 snRNP and associated splicing factors, which are known to be affected during adenoviral infection, as novel host restriction factors that effectively limit AAV transgene expression. Concurrently, we postulate that pharmacological/genetic manipulation of components of the spliceosomal machinery might enable more effective gene transfer modalities with recombinant AAV vectors. Mammalian cells have developed diverse innate/intrinsic immune strategies to counteract viral infections. Post-entry infection steps of a single-strand DNA virus, adeno-associated virus (AAV), are subject to such restrictions. Here, we screened an siRNA library to identify a novel cellular factor involved in AAV restriction. We found PHF5A, a component of the U2 snRNP mRNA splicing factor, blocks expression from recombinant AAV vectors. Disruption of PHF5A expression specifically enhanced AAV vector performance. Moreover, genetic and pharmacological inhibition of other U2 snRNP proteins, but not spliceosome proteins involved in other splicing steps, strongly increased transgene expression from AAV vectors. Further study demonstrated that U2 snRNP proteins recognize incoming AAV capsids to mediate this cellular restriction at the step after second-strand synthesis. In summary, we identify the U2 snRNP spliceosome complex as novel host factors that effectively restrict recombinant AAV vectors. Considering frequent reorganization of host splicing machinery in DNA virus infections, it is conceivable that U2 snRNP plays a role as a broad spectrum antiviral factor and helper viruses have evolved to counteract this restriction through sequestration of snRNP proteins.