Repair of HCMV-Induced DNA Damage in Infected Cells

描述（由申请人提供）：人类巨细胞病毒（HCMV）是导致出生缺陷的主要原因。HCMV感染的后果主要见于中枢神经系统（CNS），包括听力丧失、视力丧失、小头畸形和智力迟钝。尽管付出了巨大的努力，但导致这些中枢神经系统缺陷的潜在机制仍然未知。我们之前资助的HCMV与宿主细胞DNA相互作用及其DNA修复机制的研究已经确定了三个特别感兴趣的领域。首先，HCMV是已知仅有的两种对宿主DNA造成位点特异性染色体损伤的病毒之一。染色体1q断裂位点的精细定位揭示了两个基因，nidogen 1 （NID1）和髓鞘蛋白零（MPZ），与受感染婴儿听力损失的发展有关。其次，对HCMV感染的受容性成纤维细胞DNA修复的研究发现，虽然DNA损伤反应在感染期间被激活，但并没有完成。我们怀疑特异性和非特异性DNA损伤的修复受损可能在hcmv诱导的出生缺陷的发展中起作用。第三，我们最近开始研究一种有前景的新型体外模型——神经祖细胞（Neural Progenitor Cell, npc）及其衍生物。这些细胞来源于死后新生儿脑组织，为在与人类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。npc及其神经胶质衍生物和绝大多数神经元衍生物是完全允许的，并遭受溶解性感染。然而，分化的神经元亚群，虽然允许，表现出延长病毒抗原表达和释放病毒粒子。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，以证实我们在体外实验中发现的结果。我们已经从死于HCMV感染的新生儿身上获得了大脑和听觉系统组织样本，这将在我们提出的实验中发挥重要作用。我们将通过测试四个假设来推进我们的长期目标：1)一种病毒蛋白（或多种蛋白质）诱导染色体1q上的位点特异性断裂；2) hcmv诱导的断裂修复受损导致断裂位点编码基因下调；3) HCMV破坏了细胞DNA修复机制修复神经细胞非特异性损伤的能力；4)中枢神经系统内的HCMV感染会影响npc和长时神经元中参与分化、迁移和细胞功能的特定基因的表达。本提案中描述的实验将提供对hcmv诱导出生缺陷发生的分子机制的详细理解，并有助于开发中断这些机制的策略，并有希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.