Repair of HCMV-Induced DNA Damage in Infected Cells

描述(申请人提供)：人类巨细胞病毒(HCMV)是导致出生缺陷的主要病毒原因，每年感染1-2%的新生儿。这些先天感染的婴儿中，大约有5%-10%在出生时会表现出严重的神经损伤迹象，包括耳聋、失明、智力低下和小头畸形。另有10%-15%的人将在生命的头10年内患上感觉神经性听力损失和/或学习障碍。我们的长期目标是了解先天性感染HCMV的婴儿发病率和死亡率发展的机制。在过去的几年里，我们研究了巨细胞病毒与允许感染细胞的细胞周期和DNA修复机制的相互作用。我们已经发现，HCMV将许多关键的调节和修复蛋白隔离到其病毒复制中心。然而，它似乎分割了几个复合体的成分，使所有的蛋白质都存在于复制中心，但并不是所有的蛋白质都可用于细胞基因组。我们还发现，在S期，人巨细胞病毒可以引起1号染色体的特异性损伤。当结合关于感染后晚期诱导的非特异性损伤的文献时，我们的数据更清楚地突出了HCMV的遗传毒性效应。我们假设，如果1)最初的特定损伤被传播，或者2)感染后后期发生的损伤由于修复机制的隔离而没有修复，则可能发生对细胞基因组的长期有害后果。为了检验我们的假设，我们提出了三个具体目标。首先，我们将从诱导的快速性和感染时的细胞周期时相两个方面，彻底定义HCMV感染细胞中染色体LQ断裂的参数。我们认为，我们的结果必须转移到更多临床相关的细胞类型，特别是神经系细胞，因为这些细胞在先天性感染期间受病毒影响最严重。其次，我们还将确定这些临床相关细胞中1号染色体损伤的后果，以及在半允许的环境中，我们是否可以观察到1号染色体损伤的传播，而不是修复断裂或细胞走向凋亡。最后，我们将表征在病毒复制中心组装后，感染细胞在感染后期修复外源性损伤的能力。

DESCRIPTION (provided by applicant): Human Cytomegalovirus (HCMV) is the major viral cause of birth defects, infecting 1-2% of all newborns annually. Approximately 5-10% of these congenitally infected infants will manifest signs of serious neurological damage at birth, which can include deafness, blindness, mental retardation and microcephaly. Another 10-15% will develop sensori-neural hearing loss and/or learning disabilities within the first 10 years of life. Our long-term goal is to understand the mechanism behind the development of morbidity and mortality in infants congenitally infected with HCMV. Over the last several years we have studied the interaction of HCMV with the cell cycle and DNA repair machinery of the permissively infected cell. We have discerned that HCMV sequesters many key regulatory and repair proteins into its viral replication centers. However, it appears to partition the components of several complexes so that all the proteins are present within the replication centers, but not all are available to the cellular genome. We have also determined that HCMV can induce specific damage on chromosome 1 during S-phase. When coupled with the literature regarding nonspecific damage induced at late times post infection our data more clearly highlights the genotoxic effects of HCMV. We hypothesize that long-term detrimental consequences to the cellular genome may occur if 1) the initial specific damage is propagated or 2) damage incurred at late times post infection is not repaired due to sequestration of the repair machinery. To test our hypothesis, we propose three specific aims. First, we will thoroughly define the parameters of chromosome lq breakage in HCMV-infected cells with regard to rapidity of induction and cell cycle phase at time of infection. We think it is imperative that our results be moved into more clinically relevant cell types, especially cells of neural lineage, as these are the cells most severely affected by the virus during congenital infection. Second, we also will determine the consequences of chromosome 1 damage in these clinically relevant cells, and whether in a semi-permissive environment we can observe propagation of the chromosome 1 damage instead of healing of the break or movement of the cell toward apoptosis. Lastly, we will characterize the ability of HCMV-infected cells to repair exogenously introduced damage at late times post infection, after viral replication centers are assembled.