Selective recruitment of nuclear factors to productively replicating herpes simplex virus genomes.
Selective recruitment of nuclear factors to productively replicating herpes simplex virus genomes.
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DOI：
10.1371/journal.ppat.1004939
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发表时间：
2015-05
期刊：
影响因子：
6.7
通讯作者：
DeLuca NA
中科院分区：
文献类型：
--
作者：
Dembowski JA;DeLuca NA
HSV-1生命周期的大部分在细胞核中进行，包括病毒基因组的表达、复制、修复和包装。病毒蛋白质以及细胞因子在这些过程中发挥重要作用。开发了新生DNA上的蛋白质分离（iPOND）以标记和纯化细胞复制叉。我们调整了这种方法的各个方面，以标记病毒基因组，以同时成像和纯化复制的HSV-1基因组，用于鉴定相关蛋白。许多病毒和细胞因子在病毒基因组上富集，包括介导DNA复制、修复、染色质重塑、转录和RNA加工的因子。随着感染的进行，包装和结构成分在更大程度上被富集。在与基因组共纯化的更丰富的蛋白质中，有病毒转录因子ICP4和复制蛋白ICP8。此外，所有7种病毒复制蛋白都富集在病毒基因组上，沿着细胞PCNA和拓扑异构酶，而其他细胞复制蛋白未检测到。存在于病毒基因组上的染色质重塑复合物包括INO 80、SWI/SNF、FACT D和FACT复合物，其可以防止基因组的染色质化。与这一结论一致，组蛋白不容易与纯化的病毒基因组回收，成像研究显示，组蛋白在病毒基因组上的代表性不足。RNA聚合酶II，介体复合物，TFIID，TFIIH，和其他几种转录激活因子和抑制因子也用病毒DNA进行亲和纯化。INO 80、CNOD、SWI/SNF、介体、TFIID和TFIIH组分的存在与先前的研究一致，其中这些复合物与ICP 4共纯化。因此，ICP4可能参与这些关键细胞染色质重塑和转录因子向病毒基因组的募集。总之，iPOND是研究感染期间病毒基因组动力学的有价值的方法，并提供了HSV-1如何选择性利用细胞资源的全面观点。HSV-1是一种人类病原体，感染超过50%的人口。该病毒在受感染宿主的神经节中以潜伏感染的形式持续存在，并且在应激条件下被重新激活至裂解状态，其中其在初始感染部位引起复发性溃疡。在裂解感染期间，HSV劫持宿主细胞以繁殖其基因组并产生新的病毒颗粒。然而，对于哪些细胞蛋白与病毒基因组相互作用并在病毒基因组上发挥作用的知识有限。因此，我们开发了从生产性感染的细胞中纯化病毒基因组以鉴定相关病毒和细胞蛋白的方法。我们发现了以前参与HSV感染的蛋白质和蛋白质复合物在病毒基因组上富集，以及可能参与生产性感染的几种新蛋白质。这些数据为HSV生物学提供了有价值的见解。此外，这些方法可以适用于研究其他病毒，以及HSV生命周期的其他方面。
Much of the HSV-1 life cycle is carried out in the cell nucleus, including the expression, replication, repair, and packaging of viral genomes. Viral proteins, as well as cellular factors, play essential roles in these processes. Isolation of proteins on nascent DNA (iPOND) was developed to label and purify cellular replication forks. We adapted aspects of this method to label viral genomes to both image, and purify replicating HSV-1 genomes for the identification of associated proteins. Many viral and cellular factors were enriched on viral genomes, including factors that mediate DNA replication, repair, chromatin remodeling, transcription, and RNA processing. As infection proceeded, packaging and structural components were enriched to a greater extent. Among the more abundant proteins that copurified with genomes were the viral transcription factor ICP4 and the replication protein ICP8. Furthermore, all seven viral replication proteins were enriched on viral genomes, along with cellular PCNA and topoisomerases, while other cellular replication proteins were not detected. The chromatin-remodeling complexes present on viral genomes included the INO80, SWI/SNF, NURD, and FACT complexes, which may prevent chromatinization of the genome. Consistent with this conclusion, histones were not readily recovered with purified viral genomes, and imaging studies revealed an underrepresentation of histones on viral genomes. RNA polymerase II, the mediator complex, TFIID, TFIIH, and several other transcriptional activators and repressors were also affinity purified with viral DNA. The presence of INO80, NURD, SWI/SNF, mediator, TFIID, and TFIIH components is consistent with previous studies in which these complexes copurified with ICP4. Therefore, ICP4 is likely involved in the recruitment of these key cellular chromatin remodeling and transcription factors to viral genomes. Taken together, iPOND is a valuable method for the study of viral genome dynamics during infection and provides a comprehensive view of how HSV-1 selectively utilizes cellular resources. HSV-1 is a human pathogen that infects over 50% of the population. The virus persists as a latent infection in the ganglia of an infected host and upon stressful conditions is reactivated to a lytic state in which it causes recurrent sores at the initial site of infection. During lytic infection, HSV highjacks the host cell to propagate its genome and produce new virus particles. However, there is limited knowledge of what cellular proteins interact with and function on the viral genome. We therefore developed methods to purify viral genomes from productively infected cells to identify associated viral and cellular proteins. We found proteins and protein complexes that have previously been implicated in HSV infection to be enriched on viral genomes, as well as several novel proteins that are likely involved in productive infection. These data provide valuable insight into HSV biology. Furthermore, these methods can be adapted to study other viruses, as well as other aspects of the HSV life cycle.
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DOI：
10.1007/978-1-4939-1680-1_10
发表时间：
2015
期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
--
作者：
Dungrawala, Huzefa;Cortez, David
通讯作者：
Cortez, David
影响因子：
5.4
作者：
Balasubramanian, Nandakumar;Bai, Ping;Weller, Sandra K.
通讯作者：
Weller, Sandra K.
影响因子：
3.1
作者：
Gierasch, William W.;Zimmerman, David L.;Leib, David A.
通讯作者：
Leib, David A.
影响因子：
6.7
作者：
Boyne, James R.;Colgan, Kevin J.;Whitehouse, Adrian
通讯作者：
Whitehouse, Adrian
影响因子：
5.4
作者：
Everett, RD;Sourvinos, G;Orr, A
通讯作者：
Orr, A