Host determinants of Epstein-Barr virus lytic cycle activation

Epstein-Barr 病毒裂解循环激活的宿主决定因素

基本信息

批准号：
9542943
负责人：
SUMITA BHADURI-MCINTOSH
金额：
$ 32.79万
依托单位：
UNIVERSITY OF FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-05-15 至 2019-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9542943
关键词：
Address Affect B-Cell Lymphomas B-Lymphocytes BZLF1 gene Binding Binding Sites Biological Assay Blood Carcinoma Cell Count Cell physiology Cells Chemicals Chromatin Complex DNA-Binding Proteins Development Disease Effectiveness Epstein-Barr Virus latency Equilibrium Gene Expression Gene Expression Profiling Genes Genetic Transcription Genomics Goals Hand Herpesviridae Heterochromatin Human Human Herpesvirus 4 Immunoglobulins In Vitro Infectious Mononucleosis Knowledge Laboratories Life Cycle Stages Location Lytic Lytic Phase Lytic Virus Malignant Neoplasms Maps Mediating Messenger RNA Methods Modeling Molecular Nucleotides Oncolytic Pathogenesis Patients Periodicity Physiological Population Predisposition Primary Infection Proteins RNA Polymerase II Recruitment Activity Refractory Regulation Research Resistance Resolution STAT3 gene Signal Transduction Site Stat3 protein System Techniques Testing Transcript Transcription Repressor Transcriptional Regulation Treatment Efficacy Viral Viral Cancer Viral Genome Virus Work Zinc Fingers cancer cell cellular targeting crosslink gene repression genome-wide histone modification improved infected B cell killings knock-down latent virus activation lytic gene expression mutant neoplastic cell novel public health relevance small molecule success therapeutic target tool transcription factor virus host interaction

项目摘要

DESCRIPTION (provided by applicant): Persistence of a herpesvirus - in both the host and population - requires a balance between latent and lytic phases. This balance is also important for herpesvirus-mediated pathogenesis, including EBV-related diseases. EBV provides a powerful system for studying human herpesvirus latency (and lytic activation), due to its ability to establish latency in vitro. While latent EBV can be (re-)activated into the lytic phase by chemicals or immunoglobulin crosslinking, not every EBV-infected B cell is susceptible to lytic activation. This refractory state, while crucial for viral persistence, negatively impacts success f viral oncolytic therapy for EBV-cancers. Unfortunately, determinants of susceptibility to lytic activation signals are poorly understood. To address this critical issue at the single cell level, e pioneered a method for separating lytic and refractory B cells, and made the fundamental discovery that cellular STAT3, a transcription factor overactive in many human cancers, regulates susceptibility to lytic activation. We propose to test the hypothesis that STAT3 does this by employing cellular DNA-binding protein SZF1 to recruit the transcriptional co-repressor KAP1 to simultaneously repress multiple EBV lytic genes, thereby promoting the refractory state. In Aim 1, we will identify and validate SZF1-binding sites on the EBV genome in infected cells, using ChIP-exo to achieve single nucleotide resolution. In Aim 2, we will elucidate the mechanism of SZF1-mediated transcriptional repression of EBV lytic genes - using STAT3, SZF1, and KAP1 mutants, as well as purified lytic and refractory cells. The proposed research is expected to significantly impact our understanding of 1) how 2 major host transcriptional mechanisms (mediated via STAT3 and KAP1) regulate expression of EBV lytic genes to affect susceptibility to lytic activation, and thereby viral persistence, 2) how STAT3, SZF1, and KAP1 function may be exogenously manipulated to increase the number of lytic cells, and 3) how KAP1 repressor identifies targets in a genomic context - promoting discovery of cellular targets and physiologic functions of SZF1. Importantly, by providing tools to devise approaches (e.g., small molecules) to enhance lytic activation, our work promises to cripple viral persistence, and improve effectiveness of therapies for EBV-associated cancers.

描述（由申请人提供）：疱疹病毒在宿主和人群中的持续存在需要潜伏期和裂解期之间的平衡。这种平衡对于疱疹病毒介导的发病机制也很重要，包括 EBV 相关疾病。 EBV 为研究人类疱疹病毒潜伏期（和裂解激活）提供了一个强大的系统，因为它能够在体外建立潜伏期。虽然潜伏的 EBV 可以通过化学物质或免疫球蛋白交联（重新）激活进入裂解阶段，但并非每个 EBV 感染的 B 细胞都对裂解激活敏感。这种难治性状态虽然对于病毒持久性至关重要，但会对 EBV 癌症的病毒溶瘤治疗的成功产生负面影响。不幸的是，人们对裂解激活信号敏感性的决定因素知之甚少。为了在单细胞水平上解决这一关键问题，e 首创了一种分离裂解性 B 细胞和难治性 B 细胞的方法，并做出了基本发现：细胞 STAT3（一种在许多人类癌症中过度活跃的转录因子）可调节裂解激活的易感性。我们建议测试 STAT3 通过利用细胞 DNA 结合蛋白 SZF1 招募转录共阻遏物 KAP1 以同时抑制多个 EBV 裂解基因，从而促进难治状态来实现这一点的假设。在目标 1 中，我们将使用 ChIP-exo 来识别和验证受感染细胞中 EBV 基因组上的 SZF1 结合位点，以实现单核苷酸分辨率。在目标 2 中，我们将使用 STAT3、SZF1 和 KAP1 突变体以及纯化的裂解细胞和难治性细胞，阐明 SZF1 介导的 EBV 裂解基因转录抑制机制。拟议的研究预计将显着影响我们对以下方面的理解：1）两种主要宿主转录机制（通过 STAT3 和 KAP1 介导）如何调节 EBV 裂解基因的表达，从而影响裂解激活的易感性，从而影响病毒持久性，2）STAT3、SZF1 如何，并且 KAP1 功能可以通过外源操作来增加裂解细胞的数量，以及 3) KAP1 阻遏蛋白如何识别基因组背景中的靶标 - 促进发现SZF1 的细胞靶点和生理功能。重要的是，通过提供工具来设计增强裂解激活的方法（例如小分子），我们的工作有望削弱病毒的持久性，并提高 EBV 相关癌症的治疗效果。