Systems biology analysis of herpes simplex virus 1 induced host shut-off

单纯疱疹病毒1型(HSV-1)是常见唇疱疹的病原体，但也会导致严重的、危及生命的疾病，包括脑炎、肺炎、肝炎和一般性皮肤感染。HSV-1是一种在RNA水平上实现病毒诱导的宿主关闭的范例。这被认为是由两种病毒蛋白介导的，即病毒宿主关闭蛋白(VHS)和ICP27。我们使用了两种前沿方法，即新转录的RNA的4sU标签和核糖体图谱，以研究转录和RNA加工的全球变化以及它们在整个感染过程中对翻译的影响。出乎意料的是，我们发现HSV-1感染会引发细胞基因转录终止的广泛中断，但不是病毒基因。这导致广泛的转录活性绕过聚(A)位点，通常延伸到数万个核苷酸并进入下游基因。读入转录解释了令人惊讶的观察结果，即数百个细胞基因是转录诱导的，但不是翻译的。与以前的报道不同，我们发现HSV-1并不是普遍地抑制剪接，而是只抑制转录后剪接，而不是共转录剪接。关于这些令人着迷的发现的手稿目前正在《自然》杂志上修改。确定负责任的病毒基因产物的工作正在进行中。我们现在试图研究VHS、ICP27和转录终止中断在HSV-1诱导的宿主关闭中的作用。这将包括以下三个工作包：i)我们将通过分析亚细胞RNA组分(总细胞质、核和染色质相关RNA)来研究共转录和转录后RNA的加工和核输出。ii)我们将详细说明染色质状态、单个核小体、DNA结合蛋白的变化以及由于中断转录终止而导致的基因间隔区的大量转录在细胞和病毒基因表达调控中的作用(使用ATAC-seq(使用测序分析转座酶可访问的染色质)。iii)我们将确定介导HSV-1宿主关闭的两个关键病毒蛋白，即ICP27和VHS，使用PAR-CLIP(光激活的核糖核苷酸增强的交联免疫沉淀)在核苷酸分辨率下与细胞和病毒RNA结合。这项工作的结果将详细说明一种重要的人类病原体使用的一种有趣的新宿主关闭机制，并建立HSV-1作为一个有趣的新模型来研究哺乳动物细胞中耦合RNA合成、加工、输出和翻译的分子机制。

Herpes simplex virus 1 (HSV-1) is the causative agent of the common cold sores but also responsible for severe, life-threatening diseases including encephalitis, pneumonia, hepatitis and generalised skin infections. HSV-1 is a paradigm for virus-induced host shut-off exerted at RNA level. This is thought to be mediated by two viral proteins, namely the virus host shut-off protein (vhs) and ICP27. We employed two cutting-edge methodologies, i.e. 4sU-tagging of newly transcribed RNA and ribosome profiling, to study global changes in transcription and RNA processing as well as their impact on translation throughout the full course of infection. Unexpectedly, we found HSV-1 infection to trigger widespread disruption of transcription termination of cellular but not viral genes. This results in extensive transcription activity bypassing poly(A) sites, commonly extending for tens-of-thousands of nucleotides and into downstream genes. Read-in transcription explained the surprising observation that hundreds of cellular genes were transcriptionally induced but not translated. In contrast to previous reports, we found that HSV-1 does not install a general inhibition of splicing but rather only inhibits post-transcriptional but not co-transcriptional splicing. A manuscript on these fascinating findings is currently in revision with Nature. Work is ongoing to identify the responsible viral gene product. We now seek to study the role of vhs, ICP27 and disruption of transcription termination in HSV-1 induced host-shut-off. This will include the following three work packages:i) We will study co- and post-transcriptional RNA processing and nuclear export by analysing subcellular RNA fractions (total cytoplasmic, nuclear and chromatin-associated RNA).ii) We will detail the role of chromatin status, individual nucleosomes, alterations in DNA-binding proteins and the massive transcription of intergenic regions resulting from disrupted transcription termination in the modulation of cellular and viral gene expression using ATAC-seq (Assay for Transposase-Accessible Chromatin using Sequencing).iii) We will define the interaction of the two key viral proteins mediating HSV-1 host shut-off, namely ICP27 and vhs, with both cellular and viral RNAs at nucleotide resolution using PAR-CLIP (Photo-Activated Ribonucleotide-enhanced Cross-Linking ImmunoPrecipitation). Results obtained from this work will detail a fascinating new host-shut-off mechanism employed by an important human pathogen and establish HSV-1 as an interesting new model to study the molecular mechanisms coupling RNA synthesis, processing, export and translation in mammalian cells.