KSHV genome modification in KS tissue

描述(申请人提供)：KSHV是已知的两种已知的人类致癌伽马疱疹病毒之一，可在受感染的敏感细胞中诱导肿瘤形成。在HIV阳性免疫缺陷患者中，胸腔积液淋巴瘤(PEL)、卡波西肉瘤(KS)和多中心性Castleman病(MCD)较为常见。相关的人类恶性肿瘤是导致发展中国家艾滋病毒阳性人群死亡率上升的主要原因。 病人。本研究将探索KSHV在内皮细胞和KS感染组织中的表观遗传调控机制。我们将与上海和西北部新疆中国的调查人员合作，这两个地区的KS发病率相当高。我们将集中于三个主要目的来探索KSHV感染的KS组织中的基因调控是由感染的人内皮细胞纺锤体细胞中KSHV基因组上发生的特定表观遗传变化控制的假说。我们期望基因组修饰是内皮细胞特异性的，并将为KS组织和感染的内皮细胞中不同的表达谱提供线索。我们将利用组蛋白上的乙酰化和甲基化标记的特异性抗体，通过染色质免疫沉淀来确定代表KS组织表观遗传格局的特定标记。我们还将确定与LANA、RTA和CSL/RBP-JK表达相关的整体基因表达谱，以及病毒和细胞水平的调控机制。将使用条件表达和RNAi策略来调节LANA、RTA和CSL/RBP-JK的表达，以及病毒基因组中的基因突变，以确定用于驱动受感染的KS细胞增殖的控制机制。表达谱将由RNA-Seq确定，并分析在受病毒感染的细胞中调节的独特基因。然后，将使用特定mRNA转录本的实时PCR分析，以及KS组织和感染的内皮细胞的免疫组织化学和免疫荧光分析来验证这些结果。这些研究将进一步加深我们对KSHV潜伏期机制的理解，并通过靶向KS病中激活的通路为治疗潜力提供见解。

DESCRIPTION (provided by applicant): KSHV is one of the two known human oncogenic gammaherpesvirus which induces oncogenesis in infected susceptible cells. In the HIV positive immunocompromised patients, pleural effusion lymphoma (PELs), Kaposi's sarcoma (KS) and Multicentric Castleman's Disease (MCD) are common. The associated human malignancy is a major contributor to increased mortality in developing countries in this population of HIV positive patients. This study will explore epigenetic mechanism by which KSHV is regulated in endothelial cells and KS infected tissue. We will be collaborating with investigators in Shanghai and Northwest China Xinjiang province where KS incidence is quite high. We will focus on three major aims to explore the hypothesis that gene regulation in KSHV infected KS tissue is controlled by specific epigenetic changes that occur on the KSHV genome in the infected human endothelial spindle cells. We expect that genomic modifications are endothelial cell specific, and would provide clues as to the distinct profiles of expression in the KS tissue and infected endothelial cells. We will determine the specific markers which represent the epigenetic landscape in KS tissue by chromatin immunoprecipitation using specific antibodies to acetylation and methylation marks on histones. We will also identify the overall gene expression profiles related to LANA, Rta and CSL/RBP-Jk expression and the regulatory mechanisms at the viral and cellular level. Conditional expression as well as RNAi strategies for modulation of expression of LANA, Rta and CSL/RBP-Jk will be used as well as genetic mutations in the viral genomes to determine the control mechanisms that are utilized to drive proliferation of the infected KS cells. The expression profiles will be determined by RNA-Seq and analyzed for unique genes that are regulated in the viral infected cells. These will then be validated using real-time PCR analysis of the specific mRNA transcripts, as well as immunohistochemistry and immunofluorescence analysis in KS tissue and infected endothelial cells. These studies will further our understanding of KSHV latency mechanisms and provide insights for therapeutic potential by targeting the pathways activated in the KS disease.