Modification of RNA polymerase II by HSV-1 proteins

HSV-1 蛋白对 RNA 聚合酶 II 的修饰

• 批准号: 6726190
• 负责人: STEPHEN A RICE
• 金额: $ 22.59万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6726190
• 关键词: DNA directed RNA polymerase; genetic transcription; herpes simplex virus 1; host organism interaction; immediate early protein; phosphorylation; tissue /cell culture; transcription factor; transfection; virus genetics; virus infection mechanism; virus protein

DESCRIPTION (provided by applicant): Infection of cells by herpes simplex virus type 1 (HSV-1) results in dramatic changes in RNA polymerase II (RNAP II) function, such that viral genes are highly transcribed in a regulated cascade, while host genes are transcriptionally silenced. This subversion of the host transcription machinery is mediated by a small set of viral regulatory proteins that are amenable to genetic and biochemical analysis. HSV-1 thus provides a powerful system to understand how RNAP II can be globally regulated by trans-acting factors. The fundamental hypothesis of this research is that HSV-1-encoded regulatory proteins induce physical and functional modifications to the RNAP II machinery which result in the preferential transcription of viral genes. To date we have identified three physical modifications to the RNAP II apparatus that are induced by HSV-1. These are: (a) the aberrant phosphorylation of the C-terminal domain (CTD) of RNAP II; (b) the loss of transcription factor TFIIE from the RNAP II holoenzyme, and (c) the association of several HSV-1 immediate-early proteins with RNAP II complexes. Our first specific aim is to use genetic and biochemical approaches to characterize the functions of the viral proteins ICP22 and UL13, focusing on their roles in CTD modification. We will engineer and analyze ICP22 mutants and an ICP22/UL13 mutant, and ask whether CTD modification is mediated by virion or newly-expressed UL13. We will also investigate whether ICP22 and UL13 are sufficient for RNAP II modification, and whether together they comprise a CTD kinase. Our second aim is to study the mechanisms and consequences of the HSV-1 -induced loss of TFIIE from the RNAP II holoenzyme. In vitro transcription assays will be used to test the functionality of the post-infection holoenzyme. We will also investigate if the post infection holoenzyme has an altered CTD kinase activity. Our third aim is to understand how the viral IE proteins inhibit cellular transcription. We will use transfection assays and mutant viruses to identify the IE proteins which mediate transcriptional repression, and we will study the mechanisms involved. In addition, we will investigate the hypothesis that HSV-1 inhibits the cellular transcriptional pathway by disrupting the normal association between the RNAP Il CTD and cellular cofactors that interact with it, specifically mRNA splicing factors and the transcriptional mediator complex. Together, the experiments outlined in this proposal will further our understanding of mechanisms by which trans-acting proteins can globally regulate transcription. This research will also help to elucidate the fundamental replication mechanisms of HSV-1 and other pathogenic herpesviruses.

描述（由申请人提供）：单纯疱疹病毒感染细胞 1型（HSV-1）导致RNA聚合酶II（RNAP II）的急剧变化 功能，使病毒基因高度转录在受调节的级联反应中， 而宿主基因在转录基因上沉默。主机的颠覆 转录机械是由一小部分病毒调节蛋白介导的 适合遗传和生化分析。因此，HSV-1提供了 强大的系统了解如何在全球范围内进行RNAP II的监管 跨作用因素。这项研究的基本假设是 HSV-1编码的调节蛋白诱导物理和功能修饰 到RNAP II机械，导致优先转录 病毒基因。迄今为止，我们已经确定了三个物理修改 由HSV-1诱导的RNAP II设备。这些是：（a）异常 RNAP II的C末端结构域（CTD）的磷酸化； （b）损失 来自RNAP II全酶的转录因子TFIIE，（c） 在具有RNAP II复合物的几种HSV-1即时蛋白质中。我们的第一个 具体目的是使用遗传和生化方法来表征 病毒蛋白ICP22和UL13的功能，重点是它们在CTD中的作用 修改。我们将设计和分析ICP22突变体和ICP22/UL13 突变体，并询问CTD修饰是由病毒体介导的还是 新表达的UL13。我们还将调查ICP22和UL13是否是 足以进行RNAP II修改，以及它们是否共同组成CTD 激酶。我们的第二个目的是研究HSV-1的机制和后果 - RNAP II Holoenzyme诱导的TFIIE丢失。体外转录 测定将用于测试感染后全酶的功能。 我们还将调查感染后全酶是否改变CTD 激酶活性。我们的第三个目标是了解病毒IE蛋白如何 抑制细胞转录。我们将使用转染测定和突变体 鉴定介导转录抑制的IE蛋白的病毒， 我们将研究所涉及的机制。此外，我们将调查 HSV-1抑制细胞转录途径的假设 破坏RNAP IL CTD与细胞之间的正常关联 与之相互作用的辅助因子，特别是mRNA剪接因子和 转录介质复合体。一起概述了实验 提案将进一步理解反式作用的机制 蛋白质可以在全球调节转录。这项研究也将有助于 阐明HSV-1和其他致病性的基本复制机制 疱疹病毒。