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 已知为卡波西肉瘤（KS）病原体的致癌γ疱疹病毒，和 有助于体腔淋巴瘤（BCBL）或胸腔积液淋巴瘤（PEL）， 艾滋病患者。它也与多中心Castleman病（MCD）有关。KSHV感染 内皮细胞和人B细胞，其表达有限的基因库，所述基因库与 潜伏感染，包括主要潜伏相关核抗原（拉娜）。KSHV 经历两种主要的复制模式;裂解模式和潜在复制模式，并且在一些实施例中， 在某些情况下，在延迟期间可以看到潜在的低水平裂解复制。这 可能是病毒致病的关键KSHV潜伏复制是 依赖于拉娜的表达并起始于末端重复序列（TR）。拉娜绑定 在TR元件上定位了一个位点，这些位点募集复制蛋白ORC 和MCMs。我们已经证明，KSHV基因组上的其他位点可以启动复制， 其他地区也显示招募ORC和MCM。一种独特的技术， 复制DNA的分子分析（SMARD）被用来鉴定其他能够 在细胞增殖期间掺入荧光核苷类似物。我们还表明， 复制起始区不依赖于拉娜的存在，这表明 KSHV基因组能够在潜伏期期间在沿着细胞的多个位点启动复制。 基因组在这个提议的应用中，我们将把我们的努力集中在理解基因组上 KSHV病毒的复制从头感染后，通过集中在主要区域的 在感染原代细胞时被激活用于复制的基因组。康贝特人将以 基因组的表观遗传编程，以及决定基因组的高阶构象 含有成功复制基因组的发射能力的位点。受感染的细胞将 在感染的不同时间点收获，并通过SMARD监测复制区。 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 使用Meselson斯塔尔改良方法定量半保守复制， 实时荧光定量PCR ChIP/ChIP-Seq和ChIA-PET测序将用于鉴定基因组 与复制蛋白ORC、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后的时间点， 核获得的过渡表观遗传标记，是决定因素的时间图片 用于复制。此外，我们还将监测长程相互作用和构象 在从头感染过程中病毒基因组发生的变化，以了解 表观遗传学，高阶相互作用和病毒和细胞抗原所需的 KSHV基因组在从头感染后的复制和潜伏期的建立。这将 确定KSHV治疗的潜在目标和制定干预策略 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.