Understanding the mechanism of KSHV latent DNA replication

描述(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，并通过有限数量的基因表达而无限期地持续存在。这些潜伏的基因已被证明可以诱导细胞增殖/肿瘤发生。在肿瘤形成过程中，病毒DNA以EB病毒的形式存在，并通过肿瘤细胞分裂。虽然许多研究表明基因组的TR区可以支持复制，但复制的机制还不清楚。阐明潜在DNA复制机制的研究将提供复制启动所需的病毒和细胞分子的线索，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反应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点，克隆KSHV具有复制能力的区域及其在DpnI敏感性试验中的复制潜力。2-评价反式病毒蛋白LANA在KSHV基因组片段复制中的作用。梅塞尔森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度和性质(半保守)。通过这些质粒对长期持久性的评估。3-确定复制起始处的细胞蛋白质动力学。 该候选人拥有生物技术博士学位，并在KSHV编码的LANA和潜伏期KSHV基因组复制方面拥有近四年的分子病毒学博士后研究经验。候选者的近期目标是了解KSHV潜在DNA复制的机制，更具体地说，定位不依赖于LANA的复制起点、序列要求和复制起始点的蛋白质动力学，以特异性地阻断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确立自己作为一名独立研究员的地位。 这项研究将有助于了解病毒在肿瘤细胞中的复制和传递，从而使用特定的抑制剂将阻断病毒在肿瘤细胞中的复制和传递。

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.