Domain structure and interactions of HSV-1 ICP27

描述（由申请人提供）：单纯疱疹病毒1型（HSV-1）立即早期蛋白ICP 27是一种多功能调节蛋白，对病毒裂解性感染至关重要。ICP 27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的适当表达所需的。虽然ICP 27已被证明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。ICP 27通过与RNA和无数蛋白质相互作用来发挥其活性。ICP 27结合病毒RNA以促进其输出。此外，ICP 27与自身相互作用以形成多聚体，并与HSV-1蛋白ICP 4和ICP 8相互作用，以及与细胞蛋白相互作用，所述细胞蛋白包括：SR剪接蛋白、SR蛋白激酶1、剪接体蛋白SAP 145、hnRNP K、CK 2、RNA输出蛋白Aly/REF、mRNA输出受体TAP/NXF 1和RNA聚合酶II。参与这些相互作用的ICP 27区域已广泛映射到分子N-末端和C-末端两半的蛋白质基序。尽管基于序列比较鉴定了几种蛋白基序，但尚未产生关于ICP 27的结构信息。该项目的目标是阐明ICP 27的功能结构域的结构，并绘制病毒感染过程中其动态相互作用的完整阵列。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结合结构域和第二富含亮氨酸的区域R2的溶液结构，以及通过核磁共振探测C端KH RNA结合结构域和锌指结构域，来进行ICP 27的功能结构域的结构分析; 2）在关键残基中产生突变，其将破坏每个结构域的结构，并测试这些突变在感染期间对ICP 27的活性和相互作用的影响;和3）使用荧光显微镜策略观察ICP 27与细胞和病毒的相互作用，绘制ICP 27在病毒感染期间与携带靶向结构基序突变的野生型和突变型病毒的相互作用的动力学。在整个活细胞的感染周期中的因子;并阐明ICP 27相互作用的合作伙伴和其参与不同的蛋白质复合物的完整阵列，通过纯化和分析这些复合物的生化和质谱。

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.