MOLECULAR CHARACTERIZATION OF HERPES SIMPLEX VIRUS ICP27

单纯疱疹病毒的分子特征 ICP27

• 批准号: 6510800
• 负责人: STEPHEN A RICE
• 金额: $ 23.05万
• 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 2003-09-17
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6510800
• 关键词: RNA binding protein; gene mutation; 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 infection mechanism; virus protein

DESCRIPTION: 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 number of viral regulatory proteins that are amenable to biochemical and genetic analysis. This proposal focuses on ICP27, one of the key HSV-1 regulators. ICP27 is an unusual regulator that modulates genes post-transcriptionally, affecting multiple aspects of pre-mRNA metabolism. However, the specific molecular mechanisms used by ICP27 are unknown. In this application, genetic, biochemical and cell biological approaches are proposed to elucidate these mechanisms. The first aim is to characterize ICP27 by engineering and analyzing novel HSV-1 mutants. A comprehensive set of mutants that contain in-frame deletions in the ICP27 gene will be constructed and characterized. In addition, a recently isolated viral revertant, M16R, will be analyzed to understand how a truncated amino-terminal form of ICP27 can carry out essential replicative activities. The second aim is to study ICP27's recently discovered RNA-binding activity. In vitro RNA-binding assays and selection-amplification technology will be used to identify specific RNA sequences or structures which are recognized by ICP27. Additional experiments will test the hypothesis that post-translational methylation of ICP27 alters its interaction with RNA. The third aim is to understand the biological relevance of ICP27's nuclear shuttling activity. Viral ICP27 mutants will be surveyed to see which are defective in shuttling. Experiments will also be carried out to determine whether ICP27's nuclear export is an active, energy-dependent process. If so, ICP27's nuclear export signal will be mapped. The final aim is to test whether ICP27 is a virus-encoded hnRNP protein, a hypothesis which could explain ICP27's diverse effects on gene expression. To address this, the protein components of infected cell-hnRNP complexes will be characterized. The research in this proposal will increase our understanding of the basic replication pathways of herpesviruses, and will provide insight into how specific trans-acting factors can regulate genes post-transcriptionally.

描述：用单纯疱疹病毒感染哺乳动物细胞 1型（HSV-1）导致对宿主细胞核的急剧改变，因此 该病毒基因在高水平中表达，而细胞基因是 几乎完全被压制。 这些变化是由少数数字介导的 病毒调节蛋白可与生化和遗传 分析。 该提案重点介绍了ICP27，这是HSV-1关键监管机构之一。 ICP27是一种不寻常的调节剂，在转录后调节基因， 影响MRNA代谢的多个方面。 但是，具体 ICP27使用的分子机制尚不清楚。 在此应用程序中 提出了遗传，生化和细胞生物学方法 阐明这些机制。 第一个目的是表征ICP27 工程和分析新型HSV-1突变体。 一组综合 ICP27基因中包含框内缺失的突变体将是 构造和表征。 另外，最近分离的病毒 将分析恢复M16R，以了解如何截断 ICP27的氨基末端形式可以开展基本的复制活动。 第二个目的是研究ICP27最近发现的RNA结合活性。 体外RNA结合测定和选择扩增技术将是 用于识别识别的特定RNA序列或结构 由ICP27。 其他实验将检验以下假设 ICP27的翻译后甲基化会改变其与RNA的相互作用。 第三个目的是了解ICP27核的生物学相关性 穿梭活动。 将调查病毒ICP27突变体以查看哪些是 穿梭缺陷。 还将进行实验以确定 ICP27的核出口是否是一个积极的能量依赖性过程。 如果 因此，ICP27的核出口信号将被映射。 最终目标是测试 ICP27是否是病毒编码的HNRNP蛋白，这是一种假设 解释ICP27对基因表达的各种影响。 为了解决这个问题， 将表征感染细胞-HNRNP复合物的蛋白质成分。 该提案中的研究将增加我们对基本的理解 疱疹病毒的复制途径，并将提供有关如何 特定的跨作用因子可以在转录后调节基因。