Identifying functional targets of HSV-1 ICP0-directed degradation

识别 HSV-1 ICP0 定向降解的功能目标

• 批准号: 10043320
• 负责人: David J Davido
• 金额: $ 22.17万
• 依托单位: UNIVERSITY OF KANSAS LAWRENCE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-01 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10043320
• 关键词: Affect; Amino Acids; Antiviral Therapy; Biochemical Pathway; Biology; Cells; Data; Detection; Disease; DsRed; Encephalitis; Event; Exploratory/Developmental Grant; Expression Library; Eye Infections; Flow Cytometry; Fluorescence; Future; Gene Expression; Genes; Genetic Transcription; Genital system; Goals; Health; Herpes Labialis; Herpes Simplex Infections; Herpesvirus 1; Human; Immediate-Early Proteins; Impairment; Individual; Infection; Knowledge; Life; Life Cycle Stages; Lytic; Lytic Phase; Mediating; Methodology; Mission; Molecular; Monitor; Neurons; Nuclear; Oral; Pathway interactions; Phase; Phosphoproteins; Play; Polymers; Population; Positioning Attribute; Posttranslational Amino Acid Modification; Production; Proteins; Proteolysis; Proteomics; Public Health; Research; Research Project Grants; Role; Screening procedure; Simplexvirus; Solubility; System; Time; Transactivation; Ubiquitin; Ubiquitination; United States National Institutes of Health; Viral; Viral Genes; Viral Pathogenesis; Viral Proteins; Virus; Virus Diseases; Virus Replication; cDNA Library; cellular targeting; gene product; improved; innovation; knock-down; latent infection; lytic gene expression; lytic replication; multicatalytic endopeptidase complex; novel; novel therapeutics; pathogenic microbe; prevent; protein degradation; therapeutic target; ubiquitin-protein ligase; virus host interaction

Herpes simplex virus (HSV-1) causes diseases in humans, which can result in oral, genital, or ocular sores, or in rare occasions, life-threatening encephalitis. HSV-1 has two distinct phases of its viral life cycle: lytic and latent. An HSV-1 immediate-early protein, infected cell protein 0 (ICP0), is a key determinant as to whether an infection will be lytic or latent. ICP0 is a 110-KDa protein that potently transactivates viral gene expression via its E3 ubiquitin (Ub) ligase activity. Typically, E3 Ub ligases are components of pathways that attach and polymerize Ub (a 76 amino acid protein) to target proteins, marking them for degradation by the proteasome. Available evidence suggests that ICP0 mediates the degradation of several cellular proteins required for its transactivating activity. Efforts to identify specific targets of ICP0-directed degradation by proteomic approaches have been challenging because of limitations in the purity, solubility, and amount of a given protein required for its detection. While several functions of ICP0 during viral infection have been discovered, the identities of cellular proteins marked for degradation by ICP0 are largely unknown. Until these novel targets have been identified, it is unclear the exact role ICP0’s E3 Ub ligase activity plays in its transactivating activity. The long-term goal of our studies is to understand at the molecular level how virus-host interactions regulate the HSV-1 life cycle. The objective of this application is to a use a unique screening procedure to identify cellular proteins whose degradation is directed ICP0, determining the role these ICP0 targets play in HSV-1 gene expression and lytic replication. Our central hypothesis is that ICP0 mediates the degradation of novel cellular proteins; we propose that the loss of these proteins promotes efficient viral gene expression and replication. Our approach is to identify functional targets of ICP0-mediated degradation using a cutting edge screen. Our rationale for carrying out these studies is that by understanding how ICP0 facilitates productive infection via ubiquitination, targets of ICP0 degradation could be used to develop new therapies to impair HSV replication and its associated diseases. For this purpose, the following specific aims are proposed: Aim #1: Identify targets of the HSV-1 E3 Ub ligase, ICP0; Aim #2: Determine the contribution ICP0 targets play in HSV- 1 gene expression and replication. From this proposal we expect to identify cellular proteins involved in the biology of ICP0 using an innovative approach, which is a significant contribution that can used to isolate and identify genes and pathways that play important roles involved proteolysis and viral pathogenesis.

单纯疱疹病毒（HSV-1）引起人类疾病，可导致口腔、生殖器或眼部溃疡，或在罕见情况下，危及生命的脑炎。HSV-1的病毒生命周期有两个不同的阶段：裂解期和潜伏期。HSV-1的一种立即早期蛋白，即感染细胞蛋白0（ICP 0），是决定感染是裂解性的还是潜伏性的关键决定因素。ICP 0是一种110-KDa蛋白，通过其E3泛素（Ub）连接酶活性有效反式激活病毒基因表达。通常，E3 Ub连接酶是将Ub（一种76个氨基酸的蛋白质）附着并固定到靶蛋白上的途径的组分，标记它们用于被蛋白酶体降解。现有证据表明，ICP 0介导其反式激活活性所需的几种细胞蛋白质的降解。由于其检测所需的给定蛋白质的纯度、溶解度和量的限制，通过蛋白质组学方法鉴定ICP 0定向降解的特定靶标的努力一直具有挑战性。虽然已经发现了ICP 0在病毒感染过程中的几种功能，但标记为ICP 0降解的细胞蛋白的身份在很大程度上是未知的。直到这些新的目标已经确定，目前还不清楚的确切作用ICP 0的E3 Ub连接酶活性在其反式激活活性中发挥作用。我们研究的长期目标是在分子水平上了解病毒-宿主相互作用如何调节HSV-1生命周期。本申请的目的是使用独特的筛选程序来鉴定其降解受ICP 0指导的细胞蛋白，确定这些ICP 0靶标在HSV-1基因表达和裂解复制中的作用。我们的中心假设是，ICP 0介导的新的细胞蛋白质的降解，我们建议，这些蛋白质的损失，促进有效的病毒基因表达和复制。我们的方法是使用最先进的屏幕来识别ICP 0介导的降解的功能靶标。我们进行这些研究的基本原理是，通过了解ICP 0如何通过泛素化促进生产性感染，ICP 0降解的靶点可用于开发新的治疗方法来削弱HSV复制及其相关疾病。为此，提出了以下具体目标：目标#1：鉴定HSV-1 E3 Ub连接酶（ICP 0）的靶标;目标#2：确定ICP 0靶标在HSV- 1基因表达和复制中的作用。从这个提议中，我们期望使用创新的方法来鉴定参与ICP 0生物学的细胞蛋白，这是一个重大的贡献，可以用于分离和鉴定在蛋白水解和病毒发病机制中起重要作用的基因和途径。