The Marjorie B Kovler Viral Oncology Laboratories, University of Chicago, Chicago, IL 60637, USA.
Proc Natl Acad Sci U S A. 2012 Dec 18;109(51):E3549-57. doi: 10.1073/pnas.1218783109. Epub 2012 Dec 5.
ICP0, a key herpes simplex virus regulatory protein, functions first in the nucleus and then in the cytoplasm. The duration of its nuclear sojourn in cells transfected with DNA and then infected is related to the quantity of transfected DNA. Furthermore, ICP0 transactivates both viral genes and genes encoded by the transfected DNA. The data support the hypothesis that ICP0 is retained in the nucleus until it completes the replacement of repressive chromatin with effector proteins that enable transcription of both DNA templates.To identify the effector proteins, we transfected cells with biotinylated DNA encoding a nonviral gene and then infected the cells with wild-type virus. Proteins bound to transfected biotinylated plasmid recovered from mock-treated and infected cells were identified using mass spectrometry followed by appropriate database search. The transfected DNA from mock-infected cells yielded proteins associated with repression, whereas DNA recovered from infected cells included proteins known to enable transcription and proteins that have not been previously associated with that role. To test the hypothesis that the proteins hitherto not known to associate with viral gene expression are nevertheless essential, we tested the role of the DEAD-box helicase Ddx17. We report that Ddx17 plays a critical role in the expression of early and late viral genes. Thus, biotinylated DNA recovered from transfected infected cells can function as a surrogate for viral DNA and is a rich source of proteins that play a role in viral gene expression but which have not been previously identified in that role.
ICP0 是单纯疱疹病毒的一个关键调节蛋白，它首先在细胞核中发挥作用，然后在细胞质中发挥作用。在转染 DNA 然后感染的细胞中，其在核内停留的时间与转染的 DNA 数量有关。此外，ICP0 还可激活病毒基因和转染 DNA 编码的基因。这些数据支持了这样一种假说，即 ICP0 被保留在核内，直到它完成用效应蛋白取代抑制性染色质的过程，这些效应蛋白能够转录两种 DNA 模板。为了鉴定效应蛋白，我们用生物素化的 DNA 转染细胞，该 DNA 编码非病毒基因，然后用野生型病毒感染细胞。用质谱法鉴定从模拟处理和感染细胞中回收的转染生物素化质粒结合的蛋白，然后进行适当的数据库搜索。来自 mock 感染细胞的转染 DNA 产生与抑制相关的蛋白，而从感染细胞中回收的 DNA 包括已知可促进转录的蛋白和以前未与该作用相关的蛋白。为了测试迄今尚未与病毒基因表达相关联的蛋白仍然是必需的假说，我们测试了 DEAD-box 解旋酶 Ddx17 的作用。我们报告说，Ddx17 在早期和晚期病毒基因的表达中起着关键作用。因此，从转染感染细胞中回收的生物素化 DNA 可以作为病毒 DNA 的替代物，并且是发挥作用的蛋白质的丰富来源，但这些蛋白质以前并未在该作用中被鉴定出来。
