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

项目摘要/摘要 EB病毒(EBV)通过唾液传播，感染包括扁桃体上皮在内的口咽组织， 并在全球95%以上的成年人口中建立了B细胞室的终身潜伏期。 EBV经口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。这通常表现为颅面和鼻咽癌。 EBV利用其复制周期的潜伏期和裂解期来定植口咽和扁桃体。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相关疾病的重新激活仍不完全清楚。 由于EBV在潜伏时可以逃避免疫检测，目前大多数可用的治疗方法都无法利用 潜在的EB病毒基因组的存在。详细了解从延迟到解析的转换 再激活可能为溶栓诱导治疗策略奠定基础。挑战之一是 了解EBV裂解开关是定义维持EBV潜伏期所需的主机因素以及如何 EBV绕过了这些因素，允许重新激活。为了开始解决这个问题，一个人类 CRISPR/Cas9筛查是在Burkitt B细胞中进行的，这些细胞最初来自头面部活检。它 显示敲除人类核蛋白剪接因子富含脯氨酸和谷氨酰胺(SFPQ)的能力很强 诱导EBV裂解重新激活。SFPQ既能与DNA结合又能与RNA结合，已知既能调节转录，又能调节RNA 核糖核酸拼接。因此，SFPQ被认为是人类和病毒基因表达以及病毒基因组的主要调节器。 EBV潜伏期间的构象。这项研究的目标是检验SFPQ抑制 EBV裂解以DNA和RNA依赖方式重新激活，EBV解除这种抑制 通过将SFPQ重新分配给paraspeckles。目的1是确定SFPQ抑制EBV的机制 裂解复活。目标2是定义影响SFPQ亚核再分布和功能的因素。 裂解复活。分子病毒学、转录学、基因组学和显微技术将被整合到 检验这一假设。了解SFPQ如何在潜伏期调节EBV和宿主基因组，以及如何 SFPQ在溶血重新激活期间的反应将有助于在EBV如何颠覆方面的基本知识缺口 宿主因素对口咽组织和B细胞室的定植。本研究可为下一步的研究奠定基础 用于溶栓诱导治疗策略。哈佛医学院布里格姆妇女医院将 为完成本次培训提供丰富的物质和智力资源环境。 此外，与格伍兹博士在他的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，这是一个极好的科学环境 为发展而进行的培训，以管理独立的研究计划。

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.