HCMV-infected cells maintain efficient nucleotide excision repair of the viral genome while abrogating repair of the host genome.
HCMV-infected cells maintain efficient nucleotide excision repair of the viral genome while abrogating repair of the host genome.
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DOI：
10.1371/journal.ppat.1003038
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发表时间：
2012
期刊：
影响因子：
6.7
通讯作者：
Fortunato EA
中科院分区：
文献类型：
--
作者：
O'Dowd JM;Zavala AG;Brown CJ;Mori T;Fortunato EA
许多病毒在感染后破坏宿主细胞装载和完成各种DNA损伤反应（DDRs）的能力。HCMV感染允许型成纤维细胞在病毒沉积和复制过程中激活宿主ddr，但ddr仍未完成，没有阻滞或凋亡。我们认为这部分是由于损伤反应和双链断裂修复成分的分裂。在提取可溶性蛋白后，这些成分的定位分为三组：与病毒复制中心（RCs）特异性相关，扩散到整个核质中，并排除在RCs之外。其他研究表明，细胞在感染后无法处理外源性引入的损伤。我们假设细胞无法处理损伤可能是由于RCs内修复成分的不同关联，反过来，病毒基因组的潜在优先修复和宿主基因组的受损修复。为了验证这一假设，我们使用了多种策略来检测模拟和病毒感染的成纤维细胞中紫外线诱导的DNA损伤的修复。彗星试验表明，修复是启动的，但在感染细胞中没有完成。对环丁烷嘧啶二聚体（cyclobutane嘧啶didiine dimers， CPDs）的免疫荧光定位定量分析显示，修复24 h后，病毒DNA中的CPDs显著降低，而感染宿主DNA中的CPDs变化不显著。为了进一步量化CPD修复，我们开发了一种新的双色Southern协议，允许同时可视化宿主和病毒DNA。将这种方法与cpd特异性T4内切酶V碱性琼脂糖测定相结合，我们发现cpd可以有效地从病毒DNA中修复，而不是从宿主细胞DNA中修复。我们的数据证实，NER在hcmv感染的细胞中起作用，并且几乎完全修复病毒基因组而损害宿主基因组。人类巨细胞病毒（HCMV）是导致出生缺陷的主要原因。这可能部分是由于这种病毒能够对宿主的DNA造成特定的损伤，同时破坏受感染细胞修复损伤的能力。早期的研究发现，细胞修复机制的组成部分与细胞核中的HCMV病毒复制中心存在差异。这里的实验扩展了这一观察，包括参与紫外线损伤修复的机械部件。我们假设，病毒复制中心内DNA修复机制组件的关联可能有利于病毒DNA的修复，但更重要的是，对细胞DNA的修复是有害的。用三种不同的方法照射感染细胞并检查其修复情况。在这项研究的过程中，我们开发了一种新技术，可以同时评估感染细胞中的病毒和宿主基因组。这些实验发现，紫外线能快速、选择性地去除病毒的损伤，而不是被感染细胞内的细胞DNA。我们的研究结果表明，某些细胞修复蛋白与这种病毒的差异关联可能在HCMV感染的疾病发病机制中具有深远的意义。
Many viruses subvert the host cell's ability to mount and complete various DNA damage responses (DDRs) after infection. HCMV infection of permissive fibroblasts activates host DDRs at the time of viral deposition and during replication, but the DDRs remain uncompleted without arrest or apoptosis. We believe this was in part due to partitioning of the damage response and double strand break repair components. After extraction of soluble proteins, the localization of these components fell into three groups: specifically associated with the viral replication centers (RCs), diffused throughout the nucleoplasm and excluded from the RCs. Others have shown that cells are incapable of processing exogenously introduced damage after infection. We hypothesized that the inability of the cells to process damage might be due to the differential association of repair components within the RCs and, in turn, potentially preferential repair of the viral genome and compromised repair of the host genome. To test this hypothesis we used multiple strategies to examine repair of UV-induced DNA damage in mock and virus-infected fibroblasts. Comet assays indicated that repair was initiated, but was not completed in infected cells. Quantitative analysis of immunofluorescent localization of cyclobutane pyrimidine dimers (CPDs) revealed that after 24 h of repair, CPDs were significantly reduced in viral DNA, but not significantly changed in the infected host DNA. To further quantitate CPD repair, we developed a novel dual-color Southern protocol allowing visualization of host and viral DNA simultaneously. Combining this Southern methodology with a CPD-specific T4 endonuclease V alkaline agarose assay to quantitate repair of adducts, we found efficient repair of CPDs from the viral DNA but not host cellular DNA. Our data confirm that NER functions in HCMV-infected cells and almost exclusively repairs the viral genome to the detriment of the host's genome. Human cytomegalovirus (HCMV) is a leading cause of birth defects. This may be due in part to this virus' ability to inflict specific damage to its host's DNA, combined with the disruption of an infected cell's ability to repair damage. Earlier studies found that components of the cell's repair machinery were differentially associated with the HCMV viral replication centers in the nucleus. Experiments here extend this observation to include components of the machinery involved in UV lesion repair. We hypothesized that association of components of the DNA repair machinery within the viral replication centers could favor the repair of viral DNA, but more importantly, be detrimental to the repair of cellular DNA. Infected cells were irradiated and examined for repair by three different methods. In the course of this study, we developed a new technique allowing simultaneous evaluation of both the viral and host genomes in an infected cell. These experiments found rapid, selective removal of UV lesions from the viral and not the cellular DNA within infected cells. Our results indicate the differential association of certain cellular repair proteins with this virus may have far-reaching implications in the disease pathogenesis of HCMV infection.
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