Determining how EBV episome maintenance is regulated by TIMELESS function

爱泼斯坦-巴尔病毒（EBV）每年导致20万例癌症，并导致一种非癌症但使人衰弱的疾病——腺热。EBV感染免疫系统的B淋巴细胞，导致细胞的终身潜伏感染，其中病毒DNA由少量病毒蛋白维持在细胞中。其中一种病毒蛋白是EBNA1，它与病毒DNA结合，促进其复制和细胞的持续生长。了解EBNA1的功能将有助于我们了解它的功能是如何被抵消的，病毒感染的细胞是如何被消灭的。一个似乎与EBNA1功能相关的细胞因子是DNA复制蛋白TIMELESS （TIM）。TIM不仅可以防止DNA复制过程造成DNA损伤，还可以促进DNA复制过程中检测到的DNA损伤的修复。由于TIM的缺失已被观察到导致潜伏感染细胞中EBV环状DNA基因组的断裂，TIM似乎以某种方式防止病毒DNA断裂。然而，目前尚不清楚哪些TIMs在DNA复制和修复中的几种功能参与了感染细胞中EBV的维持。了解TIM在EBV DNA维持中的作用不仅可以让我们更好地了解这种病毒如何操纵细胞蛋白来维持感染，而且还可能导致新的临床策略或治疗方法。TIM已知与其他DNA修复蛋白一起工作，其功能已在临床中成功定位。如果TIM能与这些蛋白一起维持细胞中的EBV感染，我们或许能将这些药物重新用于治疗EBV相关疾病。因此，在本提案中，我们将：通过基因控制细胞中TIM蛋白的表达，使细胞表达不同的TIM蛋白。单独来说，这些蛋白质缺乏执行TIM特定功能的能力，同时允许其他功能发生。我们将使用这些修饰的蛋白质来确定TIM的哪些功能是细胞中持续病毒感染所必需的。2. 由于TIM的丢失与病毒DNA的DNA断裂有关，我们将研究TIM相关的DNA损伤发生的时间和地点，以及TIM如何防止破碎DNA的积累。由于TIM与许多DNA修复途径相关，我们将研究这些和相关DNA修复因子的丢失如何影响EBV感染。这将使我们能够描述TIM功能如何与细胞中DNA损伤的更广泛反应相关联的更广泛的背景。

Epstein-Barr virus (EBV) causes 200,000 cancers per year and is responsible for the non-cancerous but debilitating disease, glandular fever. EBV infects the B lymphocytes of the immune system resulting in life long latent infection of the cells where the viral DNA is maintained in cells by a small number of viral proteins. One such viral protein is EBNA1 which binds to the viral DNA and promotes its replication and the continued growth of the cells. Understanding how EBNA1 functions will help us understand how its function can be counteracted and virus infected cells eliminated. One cellular factor that appears to be linked to EBNA1 function is the DNA replication protein TIMELESS (TIM). TIM is required to both prevent the process of DNA replication from causing DNA damage and also to promote the repair of DNA damage detected during DNA replication. Since loss of TIM has been observed to cause breakage of the EBV circular DNA genome in latently infected cells, it appears that TIM works to somehow prevent viral DNA breakage. However, it is unknown which of TIMs several functions in DNA replication and repair are involved in EBV maintenance in infected cells. Understanding how TIM functions in EBV DNA maintenance will not only provide us with a better understanding of how this virus manipulates cellular proteins to maintain infection, but may also lead to new clinical strategies or therapies. TIM is known to work with other DNA repair proteins whose function has being successfully targeted in the clinic. If TIM works with these proteins to maintain EBV infection in cells, we maybe able to repurpose these drugs for use in the treatment of EBV-associated diseases.Therefore, in this proposal we will;1. Genetically manipulate TIM protein expression in cells so that the cell expresses different TIM proteins. Individually these proteins lack the ability to carry out specific functions of TIM while allowing other functions to occur. We will use these modified proteins to identify which functions of TIM are required for continued viral infection in cells. 2. Since loss of TIM is associated with DNA breakage of the viral DNA, we will investigate when and where TIM linked DNA damage occurs and how TIM prevents the accumulation of broken DNA.3. Since TIM is associated with a number of DNA repair pathways, we will examine how loss of these and linked DNA repair factors affect EBV infection. This will allow us to describe the broader context of how TIM functions are linked to the wider response to DNA damage in cells.