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.

项目总结/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， 并在全球超过95%的成年人群中建立了B细胞区室的终身潜伏期。 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 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。再活化 潜伏期与EBV致病性密切相关;然而，维持潜伏期和调节EBV致病性的机制， 扁桃体记忆B细胞和EBV相关疾病中的再活化仍然不完全清楚。 由于EB病毒在潜伏时可以逃避免疫检测，因此目前大多数可用的疗法不能利用EB病毒。 潜伏的EBV基因组的存在。详细了解从延迟到裂解的转变 再活化可能为裂解诱导治疗策略奠定基础。的挑战之一 理解EBV裂解开关是为了定义维持EBV潜伏期所必需的宿主因素，以及如何 这些因素被EBV绕过以允许再活化。为了开始解决这个问题， CRISPR/Cas9筛选在最初来源于颅面活检的伯基特B细胞中进行。它 结果表明，敲除人核蛋白剪接因子脯氨酸和谷氨酰胺丰富（SFPQ）， 诱导EBV裂解性再活化。SFPQ结合DNA和RNA两者，并且已知调节转录和转录。 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。 在EBV潜伏期的构象。本研究的目的是检验SFPQ抑制 EB病毒以DNA和RNA依赖性方式裂解再激活，并且EB病毒缓解了这种抑制 把SFPQ重新分配给副斑目的1是确定SFPQ抑制EB病毒的机制 裂解再活化目的2是确定介导SFPQ亚核再分布的因子， 裂解再活化分子病毒学、转录组学、基因组学和显微镜方法将被整合， 测试这个假设。了解SFPQ在潜伏期期间如何调节EBV和宿主基因组以及如何 SFPQ在裂解再激活期间的反应将有助于EBV如何颠覆 宿主因子定殖口咽组织和B细胞区室。这项研究可能会奠定基础 用于裂解诱导治疗策略。哈佛医学院的布里格姆妇女医院将 为完成这项培训提供一个物质和智力资源丰富的环境。 此外，与Gewurz博士在他的实验室的合作氛围中工作将加强多方面的培训， 研究EBV-宿主相互作用的学科方法。总之，这是一个很好的环境， 培训发展走向运行一个独立的研究计划。