Understanding the mechanism of KSHV latent DNA replication

了解 KSHV 潜伏 DNA 复制机制

• 批准号: 7917080
• 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/7917080
• 关键词: AT Rich Sequence; Address; Antibodies; Autonomous Replication; Biological Assay; Bromodeoxyuridine; Cell Cycle; Cell Proliferation; Cells; Consensus Sequence; DNA; DNA biosynthesis; Deoxyuridine; Elements; Endothelial Cells; Episome; Evaluation; Family; Geminin; Gene Delivery; Genes; Genome; Goals; Graft Rejection; HIV; Health; Human Herpesvirus 4; Human Herpesvirus 8; Immunoprecipitation; Indium; Individual; Label; Mediating; Modeling; Nucleotides; Organ Transplantation; Physiologic pulse; Plasmids; Proteins; Replication Initiation; Replication Origin; Replicon; Role; Site; Techniques; Terminal Repeat Sequences; Therapeutic; Therapeutic immunosuppression; Thymidine; Transplant Recipients; Viral; Viral Proteins; analog; chromatin immunoprecipitation; 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：本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，并无限期持续表达有限数量的基因。这些潜在基因已被证明诱导细胞增殖/肿瘤发生。在肿瘤发生过程中，作为附加体存在的病毒DNA进入分裂的肿瘤细胞。虽然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。阐明潜伏DNA复制机制的研究将提供复制起始所需的病毒和细胞分子的线索，因此将允许靶向这些分子作为潜在的治疗剂。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。 具体目的：1-鉴定KSHV基因组上的复制起始位点和克隆能够复制的KSHV区域及其在DpnI敏感性测定中的复制潜力。2-反式作用病毒蛋白拉娜对KSHV基因组片段复制作用的评价Meselson和斯塔尔用含有这些复制元件的质粒进行实验，以确定这些复制子复制的速率和性质（半保守）。这些质粒的长期持久性评价。3-在复制起点测定细胞蛋白质动力学。 候选人拥有博士学位。在生物技术和近四年的博士后研究经验，在分子病毒学工作的KSHV编码的拉娜和复制的KSHV基因组在潜伏期。候选人的直接目标是了解KSHV潜伏DNA复制的机制，更具体地定位拉娜独立复制起点、复制起始位点的序列要求和蛋白质动力学，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的研究人员。 本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。