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

单纯疱疹病毒1型（HSV-1）是普通唇疱疹的病原体，但也会导致严重的、危及生命的疾病，包括脑炎、肺炎、肝炎和全身皮肤感染。HSV-1是病毒诱导宿主在RNA水平上关闭的范例。这被认为是由两个病毒蛋白介导的，即病毒宿主关闭蛋白（vhs）和ICP27。我们采用了两种前沿方法，即新转录RNA的4su标记和核糖体分析，来研究转录和RNA加工的全局变化及其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感染会引发细胞而不是病毒基因转录终止的广泛破坏。这导致广泛的转录活性绕过poly(A)位点，通常延伸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，而不是翻译的。与之前的报道相反，我们发现HSV-1不安装剪接的一般抑制，而只是抑制转录后剪接，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》杂志上修订。工作正在进行中，以确定负责的病毒基因产物。我们现在寻求研究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（细胞质、细胞核和染色质相关的总RNA）来研究共转录和转录后RNA加工和核输出。ii)我们将使用ATAC-seq（转座酶可及染色质测序法）详细说明染色质状态、单个核小体、dna结合蛋白的改变以及由于转录终止中断而导致的基因间区域的大量转录在细胞和病毒基因表达调节中的作用。iii)我们将使用PAR-CLIP（光活化核糖核苷酸增强交联免疫沉淀）在核苷酸分辨率上定义介导HSV-1宿主关闭的两个关键病毒蛋白，即ICP27和vhs，与细胞和病毒rna的相互作用。这项工作的结果将详细描述一种重要的人类病原体所采用的令人感兴趣的新的宿主关闭机制，并将HSV-1建立为研究哺乳动物细胞中RNA合成、加工、输出和翻译的分子机制的有趣新模型。

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.