Molecular Characterization of Herpes Simplex Virus ICP27

单纯疱疹病毒 ICP27 的分子表征

• 批准号: 6679451
• 负责人: STEPHEN A RICE
• 金额: $ 23.96万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-07-01 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6679451
• 关键词: RNA binding protein; gene induction /repression; gene mutation; genetic regulation; glycoproteins; herpes simplex virus 1; heterogeneous nuclear ribonucleoprotein; intracellular transport; methylation; posttranslational modifications; protein structure function; protein transport; recombinant virus; tissue /cell culture; transcription factor; virus genetics; virus infection mechanism; virus protein

DESCRIPTION (provided by applicant): The infection of mammalian cells with herpes simplex virus type 1 (HSV-1) results in dramatic alterations to the host cell nucleus, so that viral genes are expressed at high levels while cellular genes are nearly completely suppressed. These changes are mediated by a small set of viral regulatory proteins that are amenable to biochemical and genetic analysis. This proposal focuses on ICP27, one of the key HSV-1 regulators. We will focus on understanding how ICP27 post-transcriptionally activates viral gene expression during infection. In Specific Aim 1, we will use the viral glycoprotein C (gC) gene as an experimental model to study how ICP27 induces responsive genes. A transfection assay will be used to map ICP27-response elements in the gC gene. We will also test whether ICP27 binds to sequences in gC mRNA. Potential ICP27 response elements or RNA-binding sites in the gC gene will be mutated in the context of recombinant viruses to ask whether they function during viral infection. In Specific Aim 2, we will characterize the nuclear export of ICP27, which is likely crucial for its ability to activate viral genes. We hypothesize that ICP27 interacts with and utilizes two distinct cellular export systems: CRM1 and REF/TAP. We will use a genetic approach to test this, by engineering and characterizing viral ICP27 mutants that have lost the ability to interact with CRM1 and/or REF. We will also study how certain ICP27 mutations confer viral resistance to leptomycin B, a CRM1inhibitor. In Specific Aim 3, we will identify other HSV-1 genes that modulate ICP27 function and expression. To do this, we will characterize two second-site suppressor mutants that we have isolated, M16R and dLeuR. Both viruses appear to have extragenic mutations that suppress ICP27defects. By identifying the genes involved, we will define viral factors which regulate ICP27 function, or compensate for its loss. Overall, this research will increase our understanding of pathogenic herpesviruses and provide novel insight into post-transcriptional gene regulation.

描述（由申请人提供）：用单纯疱疹病毒1型（HSV-1）感染哺乳动物细胞会导致对宿主细胞核的急剧改变，因此病毒基因在高水平中表达，而细胞基因几乎被完全抑制。这些变化是由一系列病毒调节蛋白适应生化和遗传分析的介导的。该提案重点介绍了ICP27，这是HSV-1关键监管机构之一。我们将专注于了解ICP27转录后如何激活感染过程中病毒基因的表达。在特定的目标1中，我们将使用病毒糖蛋白C（GC）基因作为实验模型，以研究ICP27如何诱导反应基因。转染测定法将用于绘制GC基因中的ICP27响应元件。我们还将测试ICP27是否与GC mRNA中的序列结合。 GC基因中潜在的ICP27反应元件或RNA结合位点将在重组病毒的背景下突变，以询问它们在病毒感染过程中是否起作用。在特定目标2中，我们将表征ICP27的核出口，这对于它激活病毒基因的能力可能至关重要。我们假设ICP27与两个不同的蜂窝出口系统相互作用：CRM1和REF/TAP。我们将使用一种遗传方法来测试这一点，通过工程和表征失去与CRM1和/或Ref相互作用的病毒ICP27突变体。我们还将研究某些ICP27突变如何赋予CRM1抑制剂Leptomycin B的病毒抗性。在特定目标3中，我们将确定其他调节ICP27功能和表达的HSV-1基因。为此，我们将表征我们已经隔离的两个第二站点抑制突变体M16R和dleur。两种病毒似乎都有抑制ICP27FEFTS的外部突变。通过识别所涉及的基因，我们将定义调节ICP27功能或弥补其损失的病毒因子。总体而言，这项研究将增加我们对病原疱疹病毒的理解，并为转录后基因调节提供新的见解。