Epigenetic Regulation of KSHV Genome Replication

卡波西肉瘤相关疱疹病毒(KSHV)或人类疱疹病毒8(HHV-8)是一种 已知致癌的伽马疱疹病毒是卡波西氏肉瘤(KS)的病原体，以及 导致体腔淋巴瘤(BCBL)或胸腔积液淋巴瘤(PEL) 艾滋病患者。它还与多中心性Castleman病(MCD)有关。KSHV感染 内皮细胞和人类B细胞表达有限的与之相关的基因 潜伏感染包括主要潜伏相关核抗原(LANA)。KSHV 经历两种主要的复制模式；裂解模式和潜伏复制模式，在某些情况下 例如，在延迟期间可以看到潜在的低水平裂解复制。这 可能在与病毒相关的发病机制中起关键作用。KSHV潜伏复制是 依赖于LANA的表达，并在末端启动重复(TRs)。LANA绑定 这些位点已经被定位到tr元件，并且这些位点招募复制蛋白orcs。 和MCMS。我们已经证明，KSHV基因组上的其他位点可以在 其他地区也招募了ORC和MCM。一种独特的技术，称为Single 复制DNA的分子分析(SMARD)用于识别其他能够 在细胞增殖过程中加入荧光核苷类似物。我们还表明， 复制起始区与LANA的存在无关，这表明 KSHV基因组能够在潜伏期内在沿线的多个位置启动复制 基因组。在这项拟议的应用中，我们将集中精力了解基因组 新感染后KSHV病毒的复制通过集中在主要区域 在原代细胞感染时被激活以供复制的基因组。我们将确定 基因组的表观遗传编程，以及决定基因组的更高阶构象 包含成功复制基因组的激发能力的位置。被感染的细胞将是 在SMARD监测的不同感染时间点和复制区收获。 我们将比较感染原代B细胞和内皮细胞后的这些区域。我们还将 用改进的Meselson Stahl方法定量半保守复制 实时定量聚合酶链式反应。CHIP/CHIP-SEQ和CHIA-PET-Sequence将用于鉴定基因组 与复制蛋白orcs、mcms、染色质修饰因子和 病毒抗原。分析将确定病毒基因组进入病毒基因组后的时间点 细胞核以获得作为决定因素的过渡性表观遗传标记的时间图像 用于复制。此外，我们将监测远程相互作用和构象 病毒基因组在从头感染期间发生的变化，以了解其贡献 表观遗传学、高级相互作用以及病毒和细胞抗原所需的 新感染后KSHV基因组的复制和潜伏期的建立。这将是 确定治疗KSHV的潜在靶点和干预策略的发展 相关疾病。

Kaposi's sarcoma Associated Herpesvirus (KSHV) or Human Herpesvirus 8 (HHV-8) is an oncogenic gammaherpesvirus known to be the causative agent of Kaposi's sarcoma (KS), and contributes to body cavity based lymphomas (BCBLs) or pleural effusion lymphomas (PELs) in AIDS patients. It is also associated with Multicentric Castleman's Disease (MCD). KSHV infects endothelial and human B cells with expression of a limited repertoire of genes that are linked to latent infection including the major latency associated nuclear antigen (LANA). KSHV undergoes two major replication modes; a lytic mode and a latent replication mode and in some instances there is an underlying low level of lytic replication that is seen during latency. This may be critical for the pathogenesis associated with the virus. KSHV latent replication is dependent on expression of LANA and initiates at the terminal repeats (TRs). LANA binding sites have been mapped to the TR elements and these sites recruit replication proteins ORCs and MCMs. We have shown that additional sites on the KSHV genome can initiate replication at other regions shown to also recruit ORC and MCMs. A unique technology referred to as single molecule analysis of replicated DNA (SMARD) was used to identify other regions capable of incorporating fluorescent nucleoside analogs during cell proliferation. We have also shown that the replication initiation zone is independent of the presence of LANA demonstrating that the KSHV genome is capable of initiating replication during latency at multiple sites along the genome. In this proposed application we will focus our efforts on understanding genome replication of the KSHV virus after de novo infection by focusing on the major regions of the genome that are activated for replication on infection of primary cells. We will determine the epigenetic programming of the genome, and higher order conformations which dictates genome sites containing firing capabilities for successful replication of the genome. Infected cells will be harvested at different time points of infection and the replication zones monitored by SMARD. We will compare these zones after infection of primary B- and endothelial cells. We will also quantitate the semi-conservative replication using a Meselson Stahl modified approach with real-time PCR. ChIP/ChIP-Seq and ChIA-PET-sequencing will be used to identify the genome regions associated with replication proteins ORCs, MCMs, chromatin modifying factors, and viral antigens. The analysis will determine the time points after the viral genome enters the nucleus to obtain a temporal picture of the transitional epigenetic marks that are determinants for replication. Furthermore, we will monitor the long range interactions, and conformation changes that occur on the viral genome during de novo infection to understand the contribution of epigenetics, higher order interactions and the viral and cellular antigens required for replication of the KSHV genome after de novo infection and establishment of latency. This will identify potential targets and development of intervention strategies for treatment of KSHV associated diseases.