Role of cooperative interactions of HSV multifunctional protein ICP27

HSV多功能蛋白ICP27协同相互作用的作用

• 批准号: 8849833
• 负责人: Alexander P. Golovanov
• 金额: $ 32.32万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-06-15 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8849833
• 关键词: Affect; Alzheimer' s Disease; Antiviral Agents; Arginine; Binding; Biological Assay; Blindness; C-terminal; Calorimetry; Cells; Chimera organism; Circular Dichroism; Complex; Coupled; Cyclic AMP-Dependent Protein Kinases; Cytomegalovirus; Development; Disease; Distant; Drug Targeting; Encephalitis; Family; Fluorescence; Fluorescence Resonance Energy Transfer; Fluorescent in Situ Hybridization; Future; Goals; HIV; Head; Health; Herpesviridae; Herpesvirus 1; Homologous Gene; Human; Human Herpesvirus 4; Human Herpesvirus 8; In Vitro; Infection; Intervention; Keratitis; Learning; Macromolecular Complexes; Measures; Mediating; Messenger RNA; Methylation; Methyltransferase; Microarray Analysis; Molecular; Morbidity - disease rate; Mutate; Mutation; N-terminal; NMR Spectroscopy; Nuclear; Pain; Phenotype; Phosphorylation; Phosphotransferases; Point Mutation; Post-Translational Protein Processing; Production; Proteins; RNA; RNA Binding; RNA-Protein Interaction; Recurrence; Risk; Role; Simplexvirus; Site; Structure; Tail; Testing; Titrations; Viral; Viral Proteins; Virus; Virus Diseases; X-Ray Crystallography; biophysical techniques; design; genetic regulatory protein; in vivo; mRNA Export; mortality; mutant; novel; prevent; protein complex; recombinant virus; skin lesion; structural biology; viral RNA

DESCRIPTION (provided by applicant): Herpes simplex virus 1 (HSV-1) causes diseases that range from painful skin lesions to keratitis and encephalitis. HSV-1 encodes an essential protein called ICP27 that is involved in a diversity of functions during viral infection. ICP27 has the intriguing ability to interact with viral mRNA and a multiplicity of host cell proteins, hijacking hem to benefit virus production. Thus, ICP27 could conceivably serve as a target for antiviral intervention. The future development of antivirals requires an understanding of how the cooperative assembly of these essential multicomponent complexes occurs and how the assembly is regulated. We will study the role of cooperativity in protein-protein and protein-RNA interactions mediated by ICP27 and characterize the binding interfaces of its complexes to determine how ICP27 is assembled in different complexes during viral infection, with the goal of revealing the molecular mechanism of its function. The structural information will be used to explore the role of interaction interface residues in ICP27 function during infection. ICP27 consists of a number of structured as well as intrinsically-unstructured domains, which participate in a large number of diverse protein-protein and protein-RNA interactions. We hypothesize that these interactions may be multi-site and therefore cooperative, mediated by regions that may be distant in the primary sequence. We postulate that transient interactions also are possible, especially when unfolded regions are involved. We hypothesize that at least some of ICP27's interactions are regulated by phosphorylation and/or arginine methylation. We will test these hypotheses in two specific aims: 1) To study the direct binding interfaces of ICP27 with cellular proteins and viral mRNA by generating shorter protein constructs in which structural stability is not perturbed, and expressing them in amounts sufficient for structural biology studie including traditional and novel Isotopically-Discriminated (IDIS) NMR spectroscopy as well as biophysical approaches. Cooperativity will be analyzed in interactions between short functional fragments of intrinsically unstructured N-terminal ICP27 and partners that interact within this region, including viral RNA and host cell proteins. In vitro post-translational modifications of ICP27 will be performed to explore how ICP27-multi-protein complex assembly is affected by phosphorylation and arginine methylation. We will also endeavor to perform structural studies of folded domains from the C-terminal part of ICP27 and to characterize interactions with its cellular protein partners. Because ICP27 undergoes a head-to-tail intramolecular interaction in vivo, a chimera containing appropriate fragments of the N- and C-termini could be created to study how these regions interact. 2) To explore the role of interaction interface residues in ICP27 function during viral infection, recombinant viruses will be constructed bearing point mutations at interaction interfaces and will be characterized for in vivo interactions and ICP27 functional activities and effects on viral infection.

描述(申请人提供)：单纯疱疹病毒1型(HSV-1)可引起各种疾病，从疼痛的皮肤病到角膜炎和脑炎。HSV-1编码一种名为ICP27的基本蛋白质，在病毒感染过程中参与多种功能。ICP27具有与病毒mRNA和多种宿主细胞蛋白相互作用的耐人寻味的能力，劫持HEM以利于病毒生产。因此，可以想象ICP27可以作为抗病毒干预的靶点。抗病毒药物的未来发展需要了解这些重要的多组分复合体如何进行合作组装，以及如何对组装进行调控。我们将研究协作性在ICP27介导的蛋白质-蛋白质和蛋白质-RNA相互作用中的作用，并表征其复合体的结合界面，以确定ICP27在病毒感染过程中如何组装在不同的复合体中，目的是揭示其作用的分子机制。这些结构信息将被用来探索相互作用界面残基在ICP27感染过程中的作用。ICP27由许多结构化和内在非结构化结构域组成，参与大量不同的蛋白质-蛋白质和蛋白质-RNA相互作用。我们假设这些相互作用可能是多位点的，因此是合作的，由初级序列中可能相距较远的区域介导。我们假设瞬时相互作用也是可能的，特别是当涉及展开区域时。我们推测，至少部分ICP27‘S相互作用受磷酸化和/或精氨酸甲基化的调节。我们将在两个特定的目标上检验这些假设：1)研究ICP27的直接结合界面 通过产生结构稳定性不受干扰的较短的蛋白质结构，并以足够的数量表达它们，以足够的数量进行结构生物学研究，包括传统的和新型的同位素识别(IDIS)核磁共振波谱以及生物物理方法。将分析内在非结构化N端ICP27的短功能片段与该区域内相互作用的伙伴之间的相互作用，包括病毒RNA和宿主细胞蛋白。在体外，ICP27的翻译后修饰将被进行，以探索ICP27-多蛋白复合体组装如何受到磷酸化和精氨酸甲基化的影响。我们还将努力进行ICP27 C-末端折叠结构域的结构研究，并表征与其细胞蛋白伙伴的相互作用。由于ICP27在体内经历了头到尾的分子内相互作用，因此可以创建包含适当N-末端和C-末端片段的嵌合体来研究这些区域是如何相互作用的。2)为了探讨相互作用界面残基在病毒感染过程中ICP27功能中的作用，构建了在相互作用界面具有点突变的重组病毒，并对其体内相互作用、ICP27功能活性和对病毒感染的影响进行了表征。