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: 10499199
• 负责人: JOEL D. BAINES
• 金额: $ 25.61万
• 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 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10499199
• 关键词: How; HSV; repurposes; host; transcriptional

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.

项目摘要： 该项目旨在识别疱疹单纯性病毒蛋白质和负责改变的机制 宿主转录组和RNA聚合酶II（RNAP II）复合物的选择，以延长病毒延长 基因。使用深层测序技术的初步数据表明，单纯疱疹病毒（HSV）去除 RNA聚合酶II（RNAP II），并将大多数细胞基因的转录终止区扩展3小时， 导致表达不佳。同时，RNAP II复合物在HSV的所有动力学类别上积累 基因。重要的是，虽然从暂停到伸长的发展是迅速的（5分钟）的早期病毒 基因，它延迟了晚期基因，该基因目前表达不佳。这暗示了新颖的概念 向伸长的进展是调节病毒晚期基因表达的重要机制。我们有 表明一种或多种早期或早期病毒蛋白是对细胞基因的影响的原因， 以及在感染后早期基因上的RNAP II加载。确定负责的病毒基因 这些影响并释放到病毒基因的伸长阶段，我们将确定RNAP II占用率， 在感染的细胞中，基因和基因特异性基础的加工性，RNA稳定性和mRNA产生 （i）野生型病毒，（ii）缺乏候选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，我们还将检验两个非判定假设，以解释转录终止如何有效地对病毒有效 基因但对宿主基因无效：（i）沿病毒基因的伸长率是否比宿主慢。 基因，（ii）病毒是否改变终止核酸内切酶XRN2，以重定向其活性从 细胞基因与病毒基因。该假设得到了初步数据的支持。 XRN2及其具有约束力的合作伙伴 例如，将在感染细胞中检查RNA裂解和聚腺苷酸化因子，以进行适当的修饰， 激活，相互作用，细胞中的定位以及与细胞或病毒序列的关联。细胞分析 被上述突变体感染应表明哪种病毒蛋白是任何XRN2重定向的原因 或伸长率的变化。基因位置交换实验也将进行确定 病毒DNA序列固有的东西还是基因组中的上下文 病毒基因的有效转录终止。