Defining how the DNA- and RNA-binding protein SFPQ represses Epstein-Barr Virus lytic reactivation

定义 DNA 和 RNA 结合蛋白 SFPQ 如何抑制 Epstein-Barr 病毒裂解再激活

• 批准号: 10537257
• 负责人: Laura Murray-Nerger
• 金额: $ 6.99万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-11-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10537257
• 关键词: 3-Dimensional; Address; Adult; Architecture; B-Lymphocytes; Binding; Biology; Biopsy; Burkitt Lymphoma; CRISPR screen; Carcinoma; Cell Compartmentation; Cells; DNA; DNA Binding; DNA-Binding Proteins; Detection; Development; Disease; Down-Regulation; EBV-associated disease; Environment; Epithelial; Epithelial Cells; Epstein-Barr Virus Infections; Epstein-Barr Virus latency; Epstein-Barr pathogenesis; Equilibrium; Foundations; Gene Expression; Genetic Transcription; Genome; Genomic Segment; Genomics; Glutamine; Goals; HIV; Histones; Hodgkin Disease; Hospitals; Human; Human Herpesvirus 4; Immune; Immune Evasion; Infectious Mononucleosis; Integration Host Factors; Knock-out; Knowledge; Lead; Life; Location; Lymphoma; Lytic; Lytic Phase; Lytic Virus; Malignant Neoplasms; Malignant neoplasm of nasopharynx; Mediating; Memory B-Lymphocyte; Methods; Microscopy; Molecular Conformation; Molecular Virology; Nasopharynx Carcinoma; Nuclear; Nuclear Protein; Oral; Oropharyngeal; Population; Proline; Protein Splicing; Proteins; RNA; RNA Binding; RNA Splicing; RNA-Binding Proteins; Repression; Research; Resources; Role; Running; Saliva; Salivary; Site; Stomach Carcinoma; Structure; Testing; Therapeutic; Tissues; Tonsil; Tonsillar Tissue; Training; Transcriptional Regulation; Untranslated RNA; Viral Genes; Viral Genome; Virus Diseases; Virus Latency; Woman; Work; collaborative environment; craniofacial; gammaherpesvirus; insight; interdisciplinary approach; live cell imaging; lytic gene expression; lytic replication; medical schools; nervous system disorder; novel therapeutic intervention; oral cavity epithelium; pathogen; pathogenic virus; post-transplant; programs; promoter; reactivation from latency; scientific atmosphere; transcription factor; transcriptomics; transmission process; viral genomics; virus host interaction

PROJECT SUMMARY/ABSTRACT Epstein-Barr virus (EBV) is spread through saliva, infects oropharyngeal tissues including the tonsillar epithelium, and establishes life-long latency in the B-cell compartment in over 95% of the global adult human population. The oral transmission of EBV can result in infectious mononucleosis and can lead to several B-cell and epithelial cancers, including Burkitt lymphoma. This frequently presents as craniofacial and nasopharyngeal carcinomas. EBV uses both latent and lytic phases of its replication cycle to colonize the oropharynx and tonsils. Reactivation from latency is closely tied to EBV pathogenicity; however, the mechanisms that maintain latency and regulate reactivation in tonsillar memory B-cells and in EBV-associated diseases remain incompletely understood. Because EBV can evade immune detection while latent, most currently available therapies cannot harness the presence of the latent EBV genome. Developing a detailed understanding of the switch from latency to lytic reactivation may lay the foundation for lytic induction therapeutic strategies. One of the challenges to understanding the EBV lytic switch is to define the host factors necessary for maintaining EBV latency and how these factors are circumvented by EBV to allow reactivation. To begin to address this question, a human CRISPR/Cas9 screen was performed in Burkitt B-cells that were originally derived from a craniofacial biopsy. It showed that knockout of the human nuclear protein splicing factor proline and glutamine rich (SFPQ) strongly induces EBV lytic reactivation. SFPQ binds both DNA and RNA and is known to regulate both transcription and RNA splicing. Thus, SFPQ is poised as a master regulator of human and viral gene expression and viral genomic conformation during EBV latency. The goal of this study is to test the hypothesis that SFPQ suppresses EBV lytic reactivation in both DNA- and RNA- dependent manners and that EBV relieves this suppression by redistributing SFPQ to paraspeckles. Aim 1 is to determine the mechanism by which SFPQ represses EBV lytic reactivation. Aim 2 is to define the factors that mediate SFPQ subnuclear redistribution and function upon lytic reactivation. Molecular virology, transcriptomic, genomic, and microscopy approaches will be integrated to test this hypothesis. Understanding how SFPQ regulates the EBV and host genomes during latency and how SFPQ responds during lytic reactivation will contribute to a fundamental knowledge gap in how EBV subverts host factors to colonize oropharyngeal tissues and the B-cell compartment. This study may lay the foundation for lytic induction therapeutic strategies. The Brigham and Women’s Hospital at Harvard Medical School will provide an environment rich in both physical and intellectual resources for completion of this training. Furthermore, working with Dr. Gewurz in the collaborative atmosphere of his lab will enhance training in multi- disciplinary approaches to study EBV-host interactions. Altogether, this is an excellent environment for scientific training for development towards running an independent research program.

项目概要/摘要 EB病毒（EBV）通过唾液传播，感染口咽组织，包括扁桃体上皮， 并在全球 95% 以上的成年人口中建立了 B 细胞区室的终生潜伏期。 EB 病毒的口腔传播可导致传染性单核细胞增多症，并可导致多种 B 细胞和上皮细胞感染 癌症，包括伯基特淋巴瘤。这通常表现为颅面癌和鼻咽癌。 EBV 利用其复制周期的潜伏期和裂解期来定殖口咽和扁桃体。重新激活 潜伏期与 EBV 致病性密切相关；然而，维持潜伏期和调节的机制 扁桃体记忆 B 细胞和 EBV 相关疾病中的重新激活仍不完全清楚。 由于 EBV 可以在潜伏时逃避免疫检测，因此目前大多数可用的疗法无法利用 潜在 EBV 基因组的存在。详细了解从潜伏期到裂解期的转变 再激活可能为裂解诱导治疗策略奠定基础。挑战之一 了解 EBV 裂解开关的目的是定义维持 EBV 潜伏期所需的宿主因素以及如何 EBV 规避了这些因素以允许重新激活。为了开始解决这个问题，人类 CRISPR/Cas9 筛选是在 Burkitt B 细胞中进行的，这些细胞最初来源于颅面活检。它 研究表明，人类核蛋白剪接因子脯氨酸和富含谷氨酰胺（SFPQ）的敲除强烈 诱导 EBV 裂解再激活。 SFPQ 结合 DNA 和 RNA，已知可调节转录和 RNA剪接。因此，SFPQ 有望成为人类和病毒基因表达以及病毒基因组的主要调节因子 EBV潜伏期的构象。本研究的目的是检验 SFPQ 抑制的假设 EBV 以 DNA 和 RNA 依赖性方式裂解再激活，并且 EBV 缓解了这种抑制 通过将 SFPQ 重新分配给 paraspeckles。目标 1 是确定 SFPQ 抑制 EBV 的机制 裂解重新激活。目标 2 是定义介导 SFPQ 亚核重新分布和功能的因素 裂解重新激活。分子病毒学、转录组学、基因组学和显微镜方法将被整合到 检验这个假设。了解 SFPQ 如何在潜伏期调节 EBV 和宿主基因组以及如何 SFPQ 在裂解重新激活过程中的反应将有助于弥补 EBV 如何颠覆的基本知识差距 定植口咽组织和 B 细胞区室的宿主因子。这项研究可能会奠定基础 用于裂解诱导治疗策略。哈佛医学院布莱根妇女医院将 为完成本次培训提供丰富的体力和智力资源的环境。 此外，与 Gewurz 博士在实验室的协作氛围中合作将加强多方面的培训 研究 EBV 与宿主相互作用的学科方法。总而言之，这里为科研提供了良好的环境。 开展独立研究项目的发展培训。