Restriction of Oncogenic Herpesviruses by Host Cell Factors

描述(由申请人提供)： Epstein-Barr病毒(EBV)和卡波西肉瘤相关病毒(KSHV)是人类致癌疱疹病毒，可引起多种淋巴瘤和上皮性和内皮源性恶性肿瘤。潜伏感染的裂解再激活和裂解周期基因的表达在这两种病毒的发病机制中都是重要的。虽然对复制很重要的病毒因素已经被广泛研究，但我们对控制KSHV和EBV重新激活的宿主因素的了解存在重大差距。我们已经证明了粘附素和CTCF这两种与染色质结合并调节转录的细胞蛋白，有效地抑制了KSHV的裂解复制和病毒生产。当粘附素开始复制时，粘附素就会从KSHV基因组中移除，而CTCF或粘附素的耗尽会导致KSHV基因转录的广泛下调。CTCF和粘附素是介导DNA环和三维构象变化的染色体重构体。基于CTCF和粘附素的这些已知功能，我们假设它们对KSHV和EBV环状潜伏基因组施加了拓扑限制，阻止了有效的转录和DNA复制，直到它们被移除。相反，当凝集素或CTCF耗尽时，KSHV基因的一个精选子集表达很差。其中许多基因的启动子带有停顿的RNA PolII，这是启动子的一个特征，受到凝集素的正向调控。这组KSHV基因还编码具有独特的免疫逃避和促进生长功能的蛋白质。它们中的几个具有免疫调节作用，并充当病毒细胞因子，削弱宿主的抗病毒反应。其他人则下调KSHV感染细胞的表面分子，使它们不太容易被细胞介导的免疫识别。这些蛋白在初次感染的早期迅速表达。我们假设KSHV利用粘附素和CTCF在感染和再激活的早期有效地表达这些特定的基因。我们首先建议将我们的研究扩展到EBV，从而建立这些粘附素/CTCF调节机制作为宿主控制伽马疱疹病毒重新激活的通用范例。这些机制将在来自粘附素途径突变患者的独特的EBV感染细胞系中得到证实和表征。我们将确定粘附素和CTCF抑制KSHV转录的分子机制，以及它们直接抑制病毒DNA复制的物理过程的程度。我们将研究从潜伏的病毒基因组中移除粘附素以允许裂解复制的分子途径--系统地研究修饰粘附素的蛋白质的作用，并从人类染色体中加载和释放粘附素。抑制粘附素去除或增加粘附素负荷的化合物将被用于验证这些途径是否为治疗靶点。最后，我们将确定粘附素和CTCF对KSHV表达免疫逃避基因能力的贡献。我们将确定CTCF和粘附素的缺失对KSHV免疫调节剂表达和功能的损害程度。我们将通过构建和鉴定不能在免疫调节基因位点和复制起点结合粘附素和CTCF的KSHV突变病毒来证实这些发现。这种多方面的方法来研究KSHV和EBV的宿主控制，应该会对病毒重新激活和免疫逃避等广泛领域的宿主-病原体平衡产生许多新的见解。

DESCRIPTION (provided by applicant): Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated virus (KSHV) are human oncogenic herpesviruses that cause a wide variety of lymphomas and malignancies of epithelial and endothelial origin. Lytic reactivation from latent infection and expression of lytic cycle genes ar important in pathogenesis of both viruses. While viral factors important for replication have been extensively studied, major gaps exist in our knowledge of host factors that control KSHV and EBV reactivation. We have shown that cohesin and CTCF, two cellular proteins that bind to chromatin and modulate transcription, potently repress KSHV lytic replication and virus production. Cohesin is removed from the KSHV genome when it begins to replicate and depletion of either CTCF or cohesin leads to widespread de-repression of KSHV gene transcription. CTCF and cohesin are chromosome remodelers that mediate DNA looping and three-dimensional changes in conformation. Based on these known functions of CTCF and cohesin, we hypothesize that they impose topological constraints on KSHV and EBV circular latent genomes that prevent efficient transcription and DNA replication until they are removed. In contrast, a select subset of KSHV genes is poorly expressed when cohesin or CTCF is depleted. Many of these genes have promoters with paused RNA pol II, a characteristic of promoters that are positively regulated by cohesin. This cluster of KSHV genes also encodes proteins that have unique immunoevasive and growth promoting functions. Several of them are immunomodulatory and act as viral cytokines that blunt the host antiviral response. Others downregulate surface molecules on KSHV-infected cells rendering them less prone to cell-mediated immune recognition. These proteins are rapidly expressed early during primary infection. We hypothesize that KSHV utilizes cohesin and CTCF to efficiently express these particular genes early in infection and reactivation. We first propose to expand our studies to EBV, thus establishing these cohesin/CTCF regulatory mechanisms as a general paradigm for host control of gammaherpes virus reactivation. These mechanisms will be confirmed and characterized in unique EBV-infected cell lines from patients with mutations in the cohesin pathway. We will determine the molecular mechanisms by which cohesin and CTCF inhibit KSHV transcription and the extent to which they directly inhibit the physical process of viral DNA replication. We will investigate the molecular pathways by which cohesin is removed from latent viral genomes to permit lytic replication - systematically studying the role of proteins that modif cohesin and load and release it from human chromosomes. Compounds that inhibit removal or enhance loading of cohesin will be employed to validate these pathways as therapeutic targets. Finally, we will define the contribution of cohesin and CTCF to KSHV's ability to express immune evasion genes. We will determine the extent to which depletion of CTCF and cohesin impairs KSHV immune modulator expression and function. We will confirm these findings by constructing and characterizing KSHV mutant viruses which cannot bind cohesin and CTCF at the immunonomodulatory gene locus and the origin of replication. This multi-faceted approach to investigating host control of KSHV and EBV should yield many novel insights into the host-pathogen balance in the broad areas of viral reactivation and immune evasion.