How HSV repurposes host transcriptional machinery for viral gene expression

项目总结： 该项目旨在确定单纯疱疹病毒的蛋白和导致改变的机制。 病毒晚期寄主转录组和RNA聚合酶II(RNAP II)复合体的筛选 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复合体在HSV的所有动力学类别上积聚 基因。重要的是，虽然从暂停到延长的进展很快(5分钟)，但在病毒早期立即 基因，它被延迟在晚基因，在这个时候表达很差的基因。这让人想到了新奇的概念 这种伸长过程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的影响负责， RNAP II在感染后早期加载晚期基因。确定致病基因(S) 这些作用和释放到病毒基因的延伸期，我们将确定RNAP II的占有率， 在感染细胞中基于基因和基因特征的处理能力、RNA稳定性和信使核糖核酸产生 与(I)野生型病毒，(Ii)缺乏候选即刻早期基因的病毒突变体，或(Iii)缺乏激活 VP16的结构域。结果将与携带恢复基因的病毒感染的细胞进行比较。在……里面 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 对宿主基因无效的基因：(I)病毒基因的延伸率是否比宿主基因慢 基因，以及(Ii)病毒是否改变终止内切酶XRN2，以将其活性从 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及其结合体 例如RNA裂解和多聚腺苷化因子将在受感染的细胞中进行适当的修饰， 激活、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 感染上述突变体应该表明哪种病毒蛋白(S)负责任何XRN2重定向 或延伸率的变化。还将进行基因位置互换实验，以确定 病毒DNA序列或它们在基因组中的上下文是否固有的东西决定了 病毒基因的高效转录终止。

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.