Understanding the mechanism of KSHV latent DNA replication

了解 KSHV 潜伏 DNA 复制机制

• 批准号: 7933873
• 负责人: Subhash C Verma
• 金额: $ 24.9万
• 依托单位: UNIVERSITY OF NEVADA RENO
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7933873
• 关键词: AT Rich Sequence; Address; Antibodies; Autonomous Replication; Biological Assay; Biotechnology; Bromodeoxyuridine; Cell Cycle; Cell Proliferation; Cells; Cloning; Consensus Sequence; DNA; DNA biosynthesis; Deoxyuridine; Doctor of Philosophy; Elements; Endothelial Cells; Environment; Episome; Evaluation; Family; Geminin; Gene Delivery; Genes; Genome; Goals; Graft Rejection; HIV; Health; Human Herpesvirus 4; Human Herpesvirus 8; Immunoprecipitation; Indium; Individual; Label; Malignant Neoplasms; Maps; Mediating; Modeling; Molecular Virology; Nature; Nucleotides; Organ Transplantation; Physiologic pulse; Plasmids; Postdoctoral Fellow; Protein Dynamics; Proteins; Replication Initiation; Replication Origin; Replicon; Research; Research Personnel; Role; Site; Techniques; Terminal Repeat Sequences; Therapeutic; Therapeutic immunosuppression; Thymidine; Transplant Recipients; Viral; Viral Proteins; Virus; Work; analog; career; chromatin immunoprecipitation; experience; human DNA; infected B cell; inhibitor/antagonist; latent infection; member; neoplastic cell; prevent; protein complex; research study; segregation; single molecule; tumor; tumorigenesis; vector; viral DNA

DESCRIPTION (provided by applicant): Long-term goals: The overall goal of this project is to elucidate the replication mechanism of KSHV during latent infection. Kaposi's sarcoma associated Herpesvirus (KSHV) predominantly infects B cells and endothelial cells and persists indefinitely with the expression of a limited number of genes. These latent genes have been shown to induce cell proliferation/tumorigenesis. Viral DNA which persists as an episome gets passage to the dividing tumor cells during tumorigenesis. Although a number of studies have shown that TR region of the genome can support replication, the mechanism of the replication is not clearly understood. Studies elucidating the mechanism of latent DNA replication will provide clues of viral and cellular molecules required for replication initiation and therefore will allow targeting of these molecules as potential therapeutics. KSHV associated tumors is a major health problem for the HIV infected individuals as well as Organ transplanted patients undergoing immunosuppressive therapies to prevent graft rejections. Specific aims:1- Identification of the replication initiation sites on the KSHV genome and cloning of the KSHV regions capable of replicating and their replicative potential in Dpnl sensitivity assay. 2- Evaluation of the role of trans acting viral protein LANA on the replication of KSHV genome fragment. Meselson and Stahl experiment with the plasmids containing these replicating elements to determine the rate and nature (semi- conservative) of replication by these replicons. Evaluation for long-term persistence by these plasmids. 3- Determination of the cellular protein dynamics at the replication origins. The candidate has a Ph.D. in Biotechnology and almost four years of post-doctoral research experience in molecular virology working on KSHV encoded LANA and the replication of KSHV genome during latency. The immediate goal of the candidate is to understand the mechanism of KSHV latent DNA replication more specifically mapping of the LANA independent replication origin, sequence requirement and protein dynamics at replication initiation sites to specifically block KSHV mediated cancers. My long-term career goal is to establish myself as a independent researcher in a an academic environment in the field of molecular virology. This research will help to understand the replication and passage of virus in tumor cells and thereby using specific inhibitors replication and passage of the virus in the tumor cells will be blocked.

描述（由申请人提供）： 长期目标：该项目的总体目标是阐明 KSHV 在潜伏感染期间的复制机制。卡波西肉瘤相关疱疹病毒 (KSHV) 主要感染 B 细胞和内皮细胞，并无限期地持续存在，表达有限数量的基因。这些潜在基因已被证明可以诱导细胞增殖/肿瘤发生。在肿瘤发生过程中，作为附加体持续存在的病毒DNA进入分裂的肿瘤细胞。尽管许多研究表明基因组的TR区可以支持复制，但复制的机制尚不清楚。阐明潜在 DNA 复制机制的研究将为复制起始所需的病毒和细胞分子提供线索，因此将允许将这些分子作为潜在的治疗药物。 KSHV 相关肿瘤是 HIV 感染者以及接受免疫抑制治疗以防止移植物排斥的器官移植患者的主要健康问题。 具体目标：1-鉴定KSHV基因组上的复制起始位点并克隆能够复制的KSHV区域及其在Dpnl敏感性测定中的复制潜力。 2-评估反式作用病毒蛋白LANA对KSHV基因组片段复制的作用。 Meselson 和 Stahl 用含有这些复制元件的质粒进行实验，以确定这些复制子的复制速度和性质（半保守）。评估这些质粒的长期持久性。 3- 确定复制起点处的细胞蛋白质动力学。 候选人拥有博士学位。拥有生物技术博士学位和近四年的分子病毒学博士后研究经验，致力于 KSHV 编码的 LANA 和潜伏期 KSHV 基因组的复制。候选人的直接目标是了解 KSHV 潜伏 DNA 复制的机制，更具体地绘制 LANA 独立复制起点、序列要求和复制起始位点的蛋白质动态，以特异性阻断 KSHV 介导的癌症。我的长期职业目标是在分子病毒学领域的学术环境中成为一名独立研究员。 这项研究将有助于了解病毒在肿瘤细胞中的复制和传代，从而利用特异性抑制剂阻断病毒在肿瘤细胞中的复制和传代。