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: 7226001
• 负责人: Rozanne M Sandri-Goldin
• 金额: $ 37.09万
• 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/7226001
• 关键词: Acidic Region; Affect; Amino Acid Motifs; Arginine; Binding; Boxing; C-terminal; Cells; Complex; Cysteine; Fluorescence Microscopy; Fluorescence Resonance Energy Transfer; Gel; Goals; Herpesvirus 1; Heterogeneous-Nuclear Ribonucleoprotein K; Histidine; Image; Infection; Late Gene Transcriptions; Leucine; Life; Lytic Phase; Maps; Mass Spectrum Analysis; Mutagenesis; Mutation; N-terminal; Nuclear Magnetic Resonance; Phosphorus 32; Protein Biosynthesis; Protein Export Pathway; Protein Kinase; Protein Splicing; Proteins; RNA; RNA Polymerase II; RNA Processing; RNA Recognition Motif; RNA Splicing; RNA-Binding Proteins; Site; Solutions; Structure; Testing; Time; Viral; Viral Genome; Virus; Virus Diseases; Zinc Fingers; base; genetic regulatory protein; herpes simplex virus type 1 immediate early protein; herpes simplex virus, type 1 protein ICP4; mRNA Export; mutant; receptor; viral RNA

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）即时早期蛋白ICP27是一种多功能调节蛋白，对病毒裂解感染至关重要。ICP27有助于关闭宿主蛋白合成，是病毒早期和晚期基因产物适当表达所必需的。虽然ICP27已被证明影响晚期基因的转录，但它主要在转录后水平作用，影响RNA的加工和输出。ICP27通过与RNA和无数蛋白质相互作用来发挥其活性。ICP27结合病毒rna促进其输出。此外，ICP27与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区域已广泛定位于分子n端和c端一半的蛋白质基序。虽然通过序列比较确定了几个蛋白质基序，但没有产生关于ICP27的结构信息。该项目的目标是阐明ICP27的功能域结构，并绘制其在病毒感染期间动态相互作用的完整阵列。具体目的是：1)通过探究富含亮氨酸的n端NES-like序列、高酸性区域、NLS、RGG盒rna结合区域和第二富含精氨酸区域R2的溶液结构，对ICP27的功能域进行结构分析；以及c端KH RNA结合域和锌指结构域的核磁共振分析；2)在关键残基上产生突变，使每个结构域失活，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)利用荧光显微镜观察ICP27在活细胞感染周期中与细胞因子和病毒因子的相互作用，绘制ICP27与携带靶向结构基序突变的野生型和突变型病毒感染过程中的相互作用动态图；并通过纯化和生化及质谱分析这些复合物，阐明ICP27相互作用伙伴的全阵列及其在不同蛋白质复合物中的参与。