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原因。巨细胞病毒感染的后果主要发生在中枢神经系统(CNS)，包括听力损失、视力丧失、小头畸形和智力低下。尽管付出了相当大的努力，但导致这些中枢神经系统缺陷的潜在机制仍不清楚。我们之前资助的关于HCMV与宿主细胞DNA及其DNA修复机制相互作用的研究确定了三个特别感兴趣的领域。首先，巨细胞病毒是已知的仅有的两种对宿主DNA造成部位特异性染色体损伤的病毒之一。对染色体1Q断裂点的精细定位发现了两个基因，NID1(NID1)和髓鞘蛋白零(MPZ)，与感染婴儿听力损失的发展有关。其次，对HCMV感染的容许性成纤维细胞DNA修复的研究发现，尽管DNA损伤反应在感染过程中被激活，但它们并未完成。我们怀疑，特异性和非特异性DNA损伤的修复受损可能在巨细胞病毒诱导的出生缺陷的发生中起作用。第三，我们最近开始了一种很有前途的新的体外模型--神经前体细胞及其衍生物的研究。这些来自死后新生儿脑组织的细胞，为在与人胎儿中枢神经系统直接相关的模型系统中研究HCMV感染提供了独特的机会。神经前体细胞、它们的神经胶质衍生物和它们的绝大多数神经元衍生物是完全允许的，并遭受溶血性感染。然而，一组分化的神经元，虽然允许，但表现出延长的病毒抗原表达和病毒粒子的释放。我们工作的长期目标是将从体外感染研究中获得的信息转化为了解先天性感染婴儿中枢神经系统缺陷的发展。我们建议检查临床标本，以确认我们体外实验中发现的结果。我们已经从死于巨细胞病毒感染的新生儿身上获取了样本档案脑和听觉系统组织，这将在我们拟议的实验中占据突出地位。我们将通过检验四个假设来推进我们的长期目标：1)一个或多个病毒蛋白诱导染色体1Q上的位置特异性断裂；2)HCMV诱导的断裂的受损修复导致断裂编码基因的下调；3)HCMV扰乱细胞DNA修复机制修复神经细胞的非特异性损伤的能力；以及4)在中枢神经系统内感染HCMV影响与分化、迁移和细胞功能有关的特定基因在NPC和长期神经元中的表达。这项提案中描述的实验将提供对巨细胞病毒诱导的出生缺陷发生的分子机制的详细了解，并有助于制定策略来中断这些机制，并有望防止其频繁的破坏性后果。

DESCRIPTION (provided by applicant): Human Cytomegalovirus (HCMV) is a leading cause of birth defects. Ramifications of HCMV infection are primarily observed in the central nervous system (CNS) and include hearing loss, vision loss, microcephaly and mental retardation. Despite considerable effort, the underlying mechanisms causing these CNS defects remain unknown. Our previously funded studies of HCMV interaction with the host cell DNA and its DNA repair machinery have identified three areas of particular interest to pursue. First, HCMV is one of only two viruses known to inflict site-specific chromosomal damage to the host DNA. Fine mapping of the chromosome 1q breaksites has revealed two genes, nidogen 1 (NID1) and myelin protein zero (MPZ), linked to the development of hearing loss in infected infants. Second, studies on DNA repair in HCMV infected permissive fibroblasts found that, although DNA damage responses were activated during infection, they were not completed. We suspect that compromised repair of specific and nonspecific DNA damage may play a role in the development of HCMV-induced birth defects. Third, we have recently begun working with a promising new in vitro model, the Neural Progenitor Cell (NPCs), and its derivatives. These cells, derived from post mortem neonatal brain tissue, provide a unique opportunity to investigate HCMV infection in a model system directly relevant to the human fetal CNS. NPCs, their glial derivatives, and the large majority of their neuronal derivatives, are fully permissive and suffer a lytic infection. However, a subpopulation of differentiated neurons, although permissive, exhibit extended viral antigen expression and release of virions. The long term goal of our work is to translate the information gained from studying infection in vitro, into understanding the development of CNS defects in congenitally infected infants. We propose examining clinical specimens for confirmatory evidence of the results found in our in vitro experiments. We have procured sample archival brain and auditory system tissues from neonates that have succumbed to HCMV infection, which will feature prominently in our proposed experiments. We will advance our long term goal with the testing of four hypotheses: 1) that a viral protein (or proteins) induces the site-specific breaks on Chromosome 1q; 2) that the compromised repair of HCMV-induced breaks causes downregulation of breaksite-encoded genes; 3) that HCMV disrupts the cellular DNA repair machinery's ability to repair non-specific damage in neural cells; and 4) that HCMV infection within the CNS affects expression of specific genes involved in differentiation, migration and cell function in NPCs and long-term neurons. The experiments described in this proposal will provide a detailed understanding of the molecular mechanisms underlying the genesis of HCMV-induced birth defects and contribute to the development of strategies to interrupt these mechanisms and, hopefully, prevent their frequently devastating consequences.