Epigenetic Regulation of KSHV Genome Replication

KSHV 基因组复制的表观遗传调控

基本信息

批准号：
10208828
负责人：
ERLE S. ROBERTSON
金额：
$ 50.18万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-08-01 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10208828
关键词：
3-Dimensional Acquired Immunodeficiency Syndrome Address Antibodies Antigens Area B-Lymphocytes Binding Sites Biological Biological Assay Cell Line Cell Nucleus Cell Proliferation Cells ChIP-on-chip ChIP-seq Chromatin Chromatin Interaction Analysis by Paired-End Tag Sequencing Coupled Coupling DNA DNA Viruses DNA analysis Data Dermal Disease Dyes Elements Endothelial Cells Endothelium Epigenetic Process Event Fluorescence Microscopy Fostering Generations Genes Genome Genomics Goals HIV Harvest Health Herpesviridae Infections Human Human Herpesvirus 8 Individual Infection Kaposi Sarcoma Knock-out Life Cycle Stages Link Lymphoma Lytic Malignant Neoplasms Maps Modeling Modification Molecular Molecular Conformation Monitor Multicentric Angiofollicular Lymphoid Hyperplasia Mutate Mutation Oncogenic Organ Transplantation Pathogenesis Patients Pattern Physiologic pulse Pleural effusion disorder Population Pre-Replication Complex Primary Infection Proliferating Publishing Replication Initiation Replication Origin Role Signal Transduction Site Stains Stretching Techniques Technology Terminal Repeat Sequences Testing Therapeutic Therapeutic immunosuppression Time Transplant Recipients Viral Viral Antigens Viral Genes Viral Genome Virus Virus Latency Virus Replication base body cavity chromatin modification daughter cell epigenetic regulation epigenome established cell line gammaherpesvirus human pathogen imprint insight latency-associated nuclear antigen latent infection lytic replication mutant novel nucleoside analog nucleotide analog origin recognition complex protein complex recruit single molecule therapy development treatment strategy tumor tumorigenesis

项目摘要

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.

卡波西肉瘤相关疱疹病毒 (KSHV) 或人类疱疹病毒 8 (HHV-8) 是一种已知致癌性伽马疱疹病毒是卡波西肉瘤 (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。它还与多中心卡斯尔曼病 (MCD) 有关。 KSHV 感染内皮细胞和人类 B 细胞表达有限的基因库，这些基因与潜伏感染包括主要潜伏相关核抗原（LANA）。科什病毒经历两种主要的复制模式；裂解模式和潜在复制模式，在某些情况下在某些情况下，在潜伏期会出现潜在的低水平裂解复制。这可能对于与病毒相关的发病机制至关重要。 KSHV 潜在复制是依赖于 LANA 的表达并在末端重复 (TR) 处起始。 LANA绑定位点已被映射到 TR 元件，这些位点招募复制蛋白 ORC 和 MCM。我们已经证明 KSHV 基因组上的其他位点可以在以下位置启动复制：其他地区也招募 ORC 和 MCM。称为单一技术的独特技术复制 DNA 分子分析 (SMARD) 用于识别其他能够在细胞增殖过程中掺入荧光核苷类似物。我们还表明复制启动区域独立于 LANA 的存在，这表明 KSHV 基因组能够在潜伏期间在沿基因组的多个位点启动复制。基因组。在这个拟议的应用程序中，我们将集中精力了解基因组通过集中于主要区域，从头感染后 KSHV 病毒的复制原代细胞感染后被激活复制的基因组。我们将确定基因组的表观遗传编程，以及决定基因组的高阶构象具有成功复制基因组的发射能力的位点。被感染的细胞将会在感染的不同时间点和 SMARD 监控的复制区域收获。我们将在原代 B 细胞和内皮细胞感染后比较这些区域。我们也会使用 Meselson Stahl 修改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、MCM、染色质修饰因子相关的区域，以及病毒抗原。分析将确定病毒基因组进入后的时间点细胞核以获得作为决定因素的过渡表观遗传标记的时间图像用于复制。此外，我们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其贡献表观遗传学、高阶相互作用以及病毒和细胞抗原所需的从头感染并建立潜伏期后 KSHV 基因组的复制。这将确定 KSHV 治疗的潜在目标并制定干预策略相关疾病。