Host determinants of Epstein-Barr virus lytic cycle activation

描述（由申请方提供）：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，包括EBV相关疾病也很重要。EBV提供了一个强大的系统，用于研究人类疱疹病毒的潜伏期（和裂解活化），由于它的能力，建立潜伏期在体外。虽然潜伏的EBV可以通过化学品或免疫球蛋白交联（再）活化进入裂解期，但不是每个EBV感染的B细胞都对裂解活化敏感。这种难治性状态虽然对病毒持久性至关重要，但对EBV癌症的病毒溶瘤疗法的成功产生负面影响。不幸的是，裂解活化信号的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开创了一种分离溶解性和难治性B细胞的方法，并发现了细胞STAT 3（一种在许多人类癌症中过度活跃的转录因子）调节对溶解性激活的敏感性。我们建议测试的假设，STAT 3这样做，采用细胞DNA结合蛋白SZF 1招募转录辅阻遏物KAP 1，同时抑制多个EBV裂解基因，从而促进不应状态。在目标1中，我们将使用ChIP-exo识别和验证感染细胞中EBV基因组上的SZF 1结合位点，以实现单核苷酸分辨率。在目标2中，我们将阐明SZF 1介导的EBV裂解基因的转录抑制机制-使用STAT 3，SZF 1和KAP 1突变体，以及纯化的裂解和难治细胞。预计拟议的研究将显著影响我们对1）2种主要宿主转录机制的理解（通过STAT 3和KAP 1介导）调节EBV裂解基因的表达以影响对裂解活化的易感性，从而影响病毒持久性，2）STAT 3、SZF 1和KAP 1的功能如何可以被外源操纵以增加裂解细胞的数量，以及3）KAP 1阻遏物如何在基因组背景下识别靶点-促进SZF 1的细胞靶点和生理功能的发现。重要的是，通过提供设计方法的工具（例如，小分子）来增强裂解活化，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。

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.