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

• 批准号: 10609807
• 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
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-05-21 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10609807
• 关键词: Binding; C-terminal; Cause of Death; Cell Death; Cells; Code; Complex; Cytoplasm; DNA Polymerase II; DNA Sequence; Data; Disease; Dissociation; 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; Polyadenylation; 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; 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

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： 该项目旨在确定单纯疱疹病毒蛋白和机制，负责改变 宿主转录组，以及选择RNA聚合酶II（RNAP II）复合物在病毒晚期 基因.使用深度测序技术的初步数据显示，单纯疱疹病毒（HSV）可以去除 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， 导致表达不佳。同时，RNAP II复合物在HSV的所有动力学类型上积累 基因.重要的是，虽然在病毒早期立即感染中从暂停到延长的进展是快速的（5分钟）， 基因，它被延迟在晚期基因上，这些基因在这个时候表达很差。这表明了一个新颖的概念 延伸是调节病毒晚期基因表达的重要机制。我们有 显示一种或多种立即早期或早期病毒蛋白负责对细胞基因的影响， 以及RNAP II在感染后早期加载在晚期基因上。为了确定病毒基因， 这些效应和释放到延伸期对病毒基因的影响，我们将确定RNAP II占有率， 持续合成能力、RNA稳定性和mRNA产生在基因和基因特征特异性的基础上， （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的结构域。结果将与被携带恢复基因的病毒感染的细胞进行比较。在 此外，我们将测试两个非排他性的假设，以解释转录终止是如何有效的病毒 （i）沿着病毒基因的延伸速率沿着是否比在宿主上慢 基因，和（ii）病毒是否改变终止核酸内切酶XRN2，以重定向其活性， 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及其结合伴侣 如RNA切割和多聚腺苷酸化因子将在感染细胞中检查适当的修饰， 活化、相互作用、在细胞中的定位以及与细胞或病毒序列的关联。细胞内分析 感染了上述突变体的病毒应指明哪种病毒蛋白负责任何XRN2重定向 或伸长率的变化。基因位置交换实验也将进行，以确定 是否有病毒DNA序列固有的东西或它们在基因组中的背景决定了 病毒基因的有效转录终止。