Restriction of Oncogenic Herpesviruses by Host Cell Factors

描述（由申请人提供）： EB病毒（EBV）和卡波西肉瘤相关病毒（KSHV）是人类致癌疱疹病毒，可引起多种上皮和内皮来源的淋巴瘤和恶性肿瘤。潜伏感染的裂解再激活和裂解周期基因的表达在两种病毒的发病机制中都是重要的。虽然对复制重要的病毒因子已经被广泛研究，但我们对控制KSHV和EBV再活化的宿主因子的认识存在重大差距。我们已经表明，粘附素和CTCF，两种细胞蛋白，结合染色质和调节转录，有效地抑制KSHV裂解复制和病毒的生产。当KSHV基因组开始复制时，粘附素从KSHV基因组中去除，CTCF或粘附素的消耗导致KSHV基因转录的广泛去抑制。CTCF和粘着蛋白是染色体重塑剂，介导DNA成环和构象的三维变化。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拓扑约束，这些约束阻止有效的转录和DNA复制，直到它们被去除。 相反，当粘附素或CTCF耗尽时，KSHV基因的选择子集表达差。这些基因中的许多具有暂停的RNA pol II的启动子，这是受粘着蛋白正调控的启动子的特征。这簇KSHV基因还编码具有独特的免疫逃避和生长促进功能的蛋白质。它们中的一些是免疫调节性的，并作为病毒细胞因子，减弱宿主的抗病毒反应。其他下调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。这些蛋白质在初次感染早期迅速表达。我们假设KSHV利用粘附素和CTCF在感染和再激活的早期有效表达这些特定的基因。 我们首先建议将我们的研究扩展到EB病毒，从而建立这些cohesin/CTCF调节机制作为宿主控制γ疱疹病毒再激活的一般范例。这些机制将在来自粘附素途径突变患者的独特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。我们将研究从潜伏病毒基因组中去除粘附素以允许裂解复制的分子途径-系统地研究粘附素的蛋白质的作用，并从人类染色体中加载和释放它。将采用抑制粘附素的去除或增强粘附素的加载的化合物来验证这些途径作为治疗靶点。最后，我们将确定的贡献，凝聚素和CTCF的KSHV的能力，表达免疫逃避基因。我们将确定CTCF和粘附素的消耗在多大程度上损害KSHV免疫调节剂的表达和功能。我们将通过构建和表征KSHV突变病毒来证实这些发现，这些突变病毒不能在免疫调节基因位点和复制起点结合粘附素和CTCF。这种多方面的方法来调查宿主控制KSHV和EBV应该产生许多新的见解宿主-病原体平衡的病毒再激活和免疫逃避的广泛领域。

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.