Host determinants of Epstein-Barr virus lytic cycle activation

描述(由申请人提供)：疱疹病毒在宿主和种群中的持久性需要潜伏期和裂解期之间的平衡。这种平衡对疱疹病毒介导的发病机制也很重要，包括EBV相关疾病。由于EBV能够在体外建立潜伏期，它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。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，但并不是每个EBV感染的B细胞都对裂解激活敏感。这种难治性状态，虽然对病毒的持久性至关重要，但对EBV癌症的病毒溶瘤治疗的成功具有负面影响。不幸的是，对裂解激活信号易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开创了一种分离裂解B细胞和难治性B细胞的方法，并取得了基本发现，细胞STAT3是一种在许多人类癌症中过度激活的转录因子，调节裂解激活的易感性。我们建议通过使用细胞DNA结合蛋白SZF1招募转录共抑制因子KAP1来同时抑制多个EBV裂解基因，从而促进难治状态，从而检验STAT3做到这一点的假设。在目标1中，我们将在感染细胞中识别和验证EBV基因组上的SZF1结合位点，使用CHIP-EXO实现单核苷酸解析。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细胞来阐明SZF1介导的EBV裂解基因转录抑制的机制。这项拟议的研究有望显著影响我们对以下问题的理解：1)两种主要的宿主转录机制(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敏感性，从而影响病毒的持久性；2)STAT3、SZF1和KAP1的功能如何被外源操纵以增加裂解细胞的数量；以及3)KAP1抑制子如何在基因组背景下识别靶点-促进SZF1细胞靶点和生理功能的发现。重要的是，通过提供工具来设计方法(例如小分子)来增强裂解激活，我们的工作有望削弱病毒的持久性，并提高EBV相关癌症治疗的有效性。

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.