Domain structure and interactions of HSV-1 ICP27

HSV-1 ICP27 的结构域结构和相互作用

• 批准号: 6891329
• 负责人: Rozanne M Sandri-Goldin
• 金额: $ 33.19万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-05-01 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6891329
• 关键词: fluorescence microscopy; gene expression; gene mutation; genetic manipulation; herpes simplex virus 1; host organism interaction; immediate early protein; intermolecular interaction; mass spectrometry; nuclear magnetic resonance spectroscopy; protein protein interaction; protein purification; protein structure; protein structure function; virus RNA; virus genetics; virus infection mechanism; virus protein

DESCRIPTION (provided by applicant): Herpes simplex virus type 1 (HSV-1) immediate-early protein ICP27 is a multifunctional regulatory protein that is essential for viral lytic infection. ICP27 contributes to the shut off of host protein synthesis and is required for the appropriate expression of viral early and late gene products. While ICP27 has been shown to affect the transcription of late genes, it acts predominantly at the post-transcriptional level affecting RNA processing and export. ICP27 performs its activities by interacting with RNA and with a myriad of proteins. ICP27 binds viral RNAs to facilitate their export. Further, ICP27 interacts with itself to form multimers, and with HSV-1 proteins ICP4 and ICP8, as well as with cellular proteins including: SR splicing proteins, SR protein kinase 1, spliceosomal protein SAP145, hnRNP K, CK2, RNA export protein Aly/REF, mRNA export receptor TAP/NXF1 and RNA polymerase II. The regions of ICP27 involved in these interactions have been broadly mapped to protein motifs in both the N-terminal and C-terminal halves of the molecule. Although several protein motifs were identified based upon sequence comparisons, no structural information on ICP27 has been generated. The goals of this project are to elucidate the structure of the functional domains of ICP27 and to chart the complete array of its dynamic interactions during viral infection. The specific aims are: 1) To perform a structural analysis of the functional domains of ICP27 by probing the solution structure of the N-terminal leucine-rich NES-like sequence, the highly acidic region, the NLS, the RGG box RNA-binding domain and R2, a second arginine-rich region; and the C-terminal KH RNA binding domains and zinc-finger domain by nuclear magnetic resonance; 2) to create mutations in critical residues that will inactivate the structure of each domain and to test those mutations for their effects on the activities and interactions of ICP27 during infection; and 3) to chart the dynamics of the interactions of ICP27 during viral infection with wild type and mutant viruses bearing targeted structural motif mutations using fluorescence microscopy strategies to view the interactions of ICP27 with cellular and viral factors throughout the infectious cycle in living cells; and to elucidate the full array of ICP27 interacting partners and its participation in different protein complexes by purifying and analyzing these complexes biochemically and by Mass Spectrometry.

描述（由申请人提供）：单纯疱疹病毒1型（HSV-1）立即早期蛋白ICP27是一种多功能调节蛋白，对于病毒溶解感染至关重要。 ICP27 有助于关闭宿主蛋白质合成，并且是病毒早期和晚期基因产物适当表达所必需的。虽然 ICP27 已被证明会影响晚期基因的转录，但它主要在转录后水平发挥作用，影响 RNA 加工和输出。 ICP27 通过与 RNA 和多种蛋白质相互作用来发挥其活性。 ICP27 结合病毒 RNA 以促进其输出。此外，ICP27 与其自身相互作用形成多聚体，并与 HSV-1 蛋白 ICP4 和 ICP8 以及细胞蛋白相互作用，包括：SR 剪接蛋白、SR 蛋白激酶 1、剪接体蛋白 SAP145、hnRNP K、CK2、RNA 输出蛋白 Aly/REF、mRNA 输出受体 TAP/NXF1 和 RNA 聚合酶 II。参与这些相互作用的 ICP27 区域已被广泛映射到分子 N 端和 C 端半部的蛋白质基序。尽管根据序列比较鉴定了几个蛋白质基序，但尚未生成 ICP27 的结构信息。该项目的目标是阐明 ICP27 功能域的结构，并绘制其在病毒感染过程中动态相互作用的完整序列。具体目标是：1）通过探测N端富含亮氨酸的NES样序列、高酸性区域、NLS、RGG盒RNA结合结构域和R2（第二个富含精氨酸的区域）的溶液结构，对ICP27的功能域进行结构分析；核磁共振显示C端KH RNA结合结构域和锌指结构域； 2) 在关键残基中产生突变，使每个结构域的结构失活，并测试这些突变对感染期间 ICP27 的活性和相互作用的影响； 3) 使用荧光显微镜策略绘制在病毒感染野生型和带有目标结构基序突变的突变病毒期间ICP27相互作用的动态，以观察活细胞中整个感染周期中ICP27与细胞和病毒因子的相互作用；并通过生化和质谱纯化和分析这些复合物，阐明 ICP27 相互作用伙伴的全部阵列及其在不同蛋白质复合物中的参与。