How HSV repurposes host transcriptional machinery for viral gene expression

HSV 如何重新利用宿主转录机制来表达病毒基因

基本信息

批准号：
10392392
负责人：
JOEL D. BAINES
金额：
$ 39.25万
依托单位：
CORNELL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-05-21 至 2024-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10392392
关键词：
Address Binding C-terminal Cause of Death Cell Death Cells Code Complex Cytoplasm DNA Polymerase II DNA Sequence Data Disease Excision Gene Expression Gene Expression Regulation Gene Order Genes Genetic Transcription Genital Genitalia Genome Herpes Labialis Herpes Simplex Infections Hour Immediate-Early Genes Immunocompromised Host Infection Kinetics Lead Life Measurable Measures Messenger RNA Methods Modification Movement Nuclear Oral Phase Phosphorylation Poly A Process Production Proteins RNA RNA Polymerase II RNA Stability Recurrence Reverse Transcriptase Polymerase Chain Reaction Role Running Serine Signal Transduction Simplexvirus Site Techniques Testing Time Transcription Initiation Site Translations VP 16 Viral Viral Genes Viral Genome Viral Proteins Virus Virus Replication combinatorial deep sequencing drug development endonuclease experimental study gene synthesis global run on sequencing mRNA Cleavage and Polyadenylation Factors mRNA Export mRNA Expression mutant negative elongation factor novel prevent promoter recruit response termination factor transcription factor transcription termination transcriptome transcriptome sequencing viral DNA

项目摘要

Project Summary: This project seeks to identify the herpes simplex virus proteins and mechanisms responsible for alteration of the host transcriptome, and selection of RNA polymerase II (RNAP II) complexes for elongation on viral late genes. Preliminary data using deep sequencing techniques show that herpes simplex virus (HSV) removes RNA polymerase II (RNAP II) and extends transcriptional termination zones of most cellular genes by 3 hours, leading to poor expression. Simultaneously, RNAP II complexes accumulate on all kinetic classes of HSV genes. Importantly, while progression from pausing to elongation is rapid (5 minutes) on viral early immediate genes, it is delayed on late genes which are expressed poorly at this time. This suggests the novel concept that progression to elongation is an important mechanism to regulate viral late gene expression. We have shown that one or more immediate early or early viral proteins are responsible for the effects on cellular genes, and for RNAP II loading on late genes at early times post infection. To identify the viral gene(s) responsible for these effects and release to the elongation phase on viral genes, we will determine RNAP II occupancy, processivity, RNA stability, and mRNA production on a gene- and gene feature-specific basis in cells infected with (i) wild type virus, (ii) viral mutants lacking candidate immediate early genes, or (iii) lacking the activation domains of VP16. Results will be compared with cells infected with viruses bearing restored genes. In addition, we will test two nonexclusive hypotheses to explain how transcriptional termination is effective on viral genes but ineffective on host genes: (i) whether elongation rates along viral genes are slower than on host genes, and (ii) whether the virus alters the termination endonuclease XRN2, to redirect its activities from cellular genes to viral genes. This hypothesis is supported by preliminary data. XRN2 and its binding partners such as RNA cleavage and polyadenylation factors will be examined in infected cells for proper modification, activation, interactions, localization in the cell, and association with cellular or viral sequences. Analysis in cells infected with the above mutants should indicate which viral protein(s) is responsible for any XRN2 redirection or change in elongation rates. Gene position swapping experiments will also be conducted to determine whether there is something inherent to viral DNA sequences or their context in the genome that dictates efficient transcriptional termination of viral genes.

项目概要：该项目旨在鉴定单纯疱疹病毒蛋白和负责改变的机制宿主转录组，以及选择用于病毒晚期延伸的 RNA 聚合酶 II (RNAP II) 复合物基因。使用深度测序技术的初步数据表明，单纯疱疹病毒（HSV）可消除 RNA 聚合酶 II (RNAP II) 并将大多数细胞基因的转录终止区延长 3 小时，导致表达能力差。同时，RNAP II 复合物在 HSV 的所有动力学类别上积累基因。重要的是，虽然病毒早期立即从暂停到延长的进展很快（5 分钟）基因，它在此时表达不佳的晚期基因上被延迟。这表明了新颖的概念延伸的进展是调节病毒晚期基因表达的重要机制。我们有表明一种或多种直接早期或早期病毒蛋白对细胞基因产生影响，以及感染后早期对晚期基因的 RNAP II 加载。确定负责的病毒基因这些影响和病毒基因延伸阶段的释放，我们将确定 RNAP II 占用，受感染细胞中基于基因和基因特征特异性的持续合成能力、RNA 稳定性和 mRNA 生成 (i) 野生型病毒，(ii) 缺乏候选立即早期基因的病毒突变体，或 (iii) 缺乏激活 VP16 的域。结果将与感染带有恢复基因的病毒的细胞进行比较。在此外，我们将测试两个非排他性假设来解释转录终止如何对病毒有效基因但对宿主基因无效：（i）病毒基因的延伸率是否比宿主慢基因，以及 (ii) 病毒是否改变终止核酸内切酶 XRN2，以将其活性从细胞基因到病毒基因。这一假设得到了初步数据的支持。 XRN2 及其结合伙伴例如RNA切割和聚腺苷酸化因子将在受感染的细胞中进行检查以进行适当的修饰，激活、相互作用、细胞内定位以及与细胞或病毒序列的关联。细胞内分析感染上述突变体应指出哪种病毒蛋白负责任何 XRN2 重定向或伸长率的变化。还将进行基因位置交换实验以确定病毒 DNA 序列或其在基因组中的背景是否存在某些固有的东西病毒基因的有效转录终止。