Domain structure and interactions of HSV-1 ICP27

描述(申请人提供)：单纯疱疹病毒1型(HSV-1)即刻早期蛋白ICP27是一种多功能调节蛋白，对病毒裂解感染是必不可少的。ICP27有助于阻断宿主蛋白的合成，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上影响RNA的加工和输出。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以促进它们的输出。此外，ICP27与自身相互作用形成多聚体，与HSV-1蛋白ICP4和ICP8以及与细胞蛋白包括：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相互作用。虽然根据序列比较确定了几个蛋白质基序，但尚未产生ICP27的结构信息。该项目的目标是阐明ICP27功能结构域的结构，并绘制其在病毒感染过程中动态相互作用的完整阵列。其具体目的是：1)通过探测N末端富含亮氨酸的NES样序列、高酸性区域、NLS、RGG盒RNA结合域和第二富含精氨酸区域R2的溶液结构，以及C末端KH RNA结合域和锌指域的核磁共振，对ICP27的功能结构域进行结构分析；2)在关键残基中产生使每个结构域的结构失活的突变，并测试这些突变在感染过程中对ICP27的活性和相互作用的影响；以及3)利用荧光显微镜策略绘制ICP27在病毒感染过程中与携带靶向结构基序突变的野生型和突变型病毒相互作用的动态，以观察ICP27与细胞和病毒因子在活细胞感染周期中的相互作用；并通过生化和质谱学纯化和分析ICP27相互作用伙伴的完整阵列及其在不同蛋白质复合体中的参与。

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.