Herpesvirus-induced telomerase dysregulation and tumor formation

疱疹病毒诱导的端粒酶失调和肿瘤形成

• 批准号: 7617576
• 负责人: Keith William Jarosinski
• 金额: $ 22.87万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-06-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7617576
• 关键词: Address; Animal Model; Apoptosis; Birds; Boxing; Cell Aging; Cell hybridization; Cells; Chickens; Chromosomal Stability; Chromosome abnormality; Chromosomes; Cloning; Complement 3d Receptors; Complex; DNA; Development; Digestion; Event; Exhibits; Fluorescent in Situ Hybridization; Functional disorder; Genes; Genome; Genomics; Goals; Harvest; Herpesviridae; Homologous Gene; Human Herpesvirus 4; Human Herpesvirus 6; Human Herpesvirus 7; Incidence; Individual; Lead; Lymphocyte Activation; Lymphoma; Lymphomagenesis; Macrophage-1 Antigen; Maintenance; Malignant - descriptor; Malignant Neoplasms; Marek Disease; Marek' s Disease-like Viruses; Mutate; Mutation; Neoplasm Metastasis; Oncogenic; Paint; Point Mutation; Public Health; Recombinants; Research; Retinoblastoma Protein; Ribonucleoproteins; Role; Sequence Homologs; Shotgun Sequencing; Site; Specific qualifier value; Structure; System; T-Cell Lymphoma; T-Lymphocyte; Telomerase; Telomerase RNA Component; Testing; Vaccines; Viral; Viral Genes; Virulent; Virus; Virus Integration; base; cell transformation; in vivo; latent infection; lymphoblastoid cell line; lytic replication; member; mutant; neoplastic cell; novel strategies; nucleocytoplasmic transport; prevent; programs; promoter; restriction enzyme; stem; telomerase reverse transcriptase; telomere; tumor; tumorigenesis; vector; viral DNA

DESCRIPTION (provided by applicant): A number of viruses, including members of the Herpesviridae, can cause tumors in their hosts. Tumorigenesis requires the continued survival of once transformed cells, which is critically dependent on telomerase activity to avoid cellular senescence and apoptosis. Telomerase is a ribonucleoprotein complex with its core constituents being telomerase reverse transcriptase (TERT) and telomerase RNA (TR). Telomerase dysfunction can have tumor-promoting functions independently of net telomere elongation and maintenance of chromosomal integrity, which is consistent with the short telomeres usually present in malignantly transformed tumor cells. Marek's disease virus (MDV) is a herpesvirus of chickens that causes a highly malignant T cell lymphoma. MDV harbors two copies of a viral TR gene (vTR) that exhibits 88% sequence identity to chicken TR (chTR) and was presumably pirated from the host genome. Our long-term goal is to elucidate in detail the mechanisms of the tumor-promoting effects of telomerase. In this proposal we shall test the hypothesis that the structural integrity of vTR is critical for transformation and metastasis, as well as integration of the MDV DNA into the host genome. The hypothesis will be tested by two specific aims: Specific Aim 1: To test the effect of mutations in conserved regions of vTR on MDV-induced tumor formation and dissemination. Using an infectious clone of the highly oncogenic MDV strain RB-1B (pRB-1B), we shall replace vTR or its promoter with the chicken homologues, and also introduce mutations into vTR that have been shown to render the molecule nonfunctional. We shall target the template region (conserved region 1, CR1) specifying the telomeric repeat sequence TTAGGG and the so-called H box, in which a single point mutation was identified in avirulent MDV vaccine strain CVI988. Recombinant MDV carrying the mutations will be tested in vivo for lytic replication, establishment of latency and tumorigenesis. Specific Aim 2: To analyze the contributions of vTR and telomeric repeats to MDV genome integration and host cell chromosome stability. MDV, like other herpesviruses such as Epstein-Barr virus (EBV) or human herpesviruses 6 and 7 (HHV-6, HHV-7), is capable of integrating its DNA genome into host chromosomes without any obvious hotspot of integration. The MDV genome contains telomeric repeats at both ends of the genome, suggesting that viral telomeres may be utilized for specific integration into homologous sequences within chicken chromosomes. Mutant MDV lacking telomeric repeats will be generated and their ability for integration and induction chromosome aberrations will be analyzed by using fluorescent in situ hybridization using MDV probes and chicken chromosome paints. Virus integration sites will be determined by cloning and sequencing of cloned cellular DNA, which are positive for MDV genomic termini by colony PCR. The relevance of the proposed research to public health is that it will provide a better understanding of the role of telomerase in cancer development and could lead to novel approaches for the treatment of a variety of tumors by specifically targeting telomerase RNA. It will also address the mechanism of herpesvirus integration into the host genome and its importance for latent infection and transformation of lymphocytes.

描述（由申请人提供）：许多病毒，包括疱疹病毒科的成员，可能在其宿主中引起肿瘤。肿瘤发生需要曾经转化的细胞的持续存活，这在关键上取决于端粒酶活性，以避免细胞衰老和凋亡。端粒酶是一种核糖核蛋白复合蛋白，其核心成分是端粒酶逆转录酶（TERT）和端粒酶RNA（TR）。端粒酶功能障碍可以独立于终止伸长率伸长和维持染色体完整性的肿瘤功能，这与通常存在于恶性转化的肿瘤细胞中的短端粒一致。 Marek病毒病毒（MDV）是导致高度恶性T细胞淋巴瘤的鸡疱疹病毒。 MDV拥有两个病毒TR基因（VTR）的副本，该副本对鸡TR（CHTR）具有88％的序列身份，并且可能是从宿主基因组中盗用的。我们的长期目标是详细阐明端粒酶肿瘤促进作用的机制。在该提案中，我们应检验以下假设：VTR的结构完整性对于转化和转移以及将MDV DNA整合到宿主基因组中至关重要。该假设将通过两个具体目的进行检验： 具体目的1：测试VTR保守区域中突变对MDV诱导的肿瘤形成和传播的影响。使用高度致癌的MDV菌株RB-1B（PRB-1B）的传染性克隆，我们将用鸡同源物代替VTR或其启动子，并将突变引入VTR中，这些突变已被证明可以呈现分子非功能。我们将针对指定端粒重复序列TTAGGG和所谓的H框的模板区域（保守区域1，CR1），其中在Aviruity MDV疫苗CVI988中鉴定了单点突变。携带突变的重组MDV将在体内进行裂解复制，延迟和肿瘤发生的测试。 特定目的2：分析VTR和端粒重复序列对MDV基因组整合和宿主细胞染色体稳定性的贡献。与其他疱疹病毒（例如爱泼斯坦 - 巴尔病毒（EBV））或人类疱疹病毒6和7（HHV-6，HHV-7）一样，MDV能够将其DNA基因组整合到宿主染色体中而没有任何明显的整合热点。 MDV基因组在基因组的两端包含端粒重复序列，这表明病毒端粒可用于特定整合到鸡染色体内的同源序列中。将产生缺乏端粒重复序列的突变体MDV，并通过使用MDV探针和鸡染色体涂料使用荧光原位杂交来分析其整合和诱导染色体畸变的能力。病毒整合位点将通过克隆的细胞DNA的克隆和测序来确定，这对MDV基因组末端呈阳性。拟议的研究与公共卫生的相关性是，它将更好地理解端粒酶在癌症发展中的作用，并通过专门针对端粒酶RNA来治疗各种肿瘤的新方法。它还将解决疱疹病毒整合到宿主基因组中的机制及其对潜在感染和淋巴细胞转化的重要性。