Renewal: HPV and the DNA Damage Response

更新：HPV 和 DNA 损伤反应

基本信息

批准号：
10439190
负责人：
Laimonis A. LAIMINS
金额：
$ 38万
依托单位：
NORTHWESTERN UNIVERSITY AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-08-01 至 2027-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10439190
关键词：
ATM Signaling Pathway ATM activation ATR gene Anus Ataxia Telangiectasia BRCA1 gene Binding Binding Proteins CHEK1 gene Cell Differentiation process Cells Cervical Cessation of life Chromosomes Complex Country DNA DNA Damage DNA Repair DNA biosynthesis DNA replication fork Episome Event Fanconi Anemia pathway GATA4 gene Generations Genetic Recombination Genetic Transcription Genome Goals Human Papillomavirus Human papilloma virus infection Hybrids Impairment Laboratories Lead Lesion Life Cycle Stages Link Maintenance Malignant Neoplasms Malignant neoplasm of cervix uteri Mitosis Mutation Pathway interactions Phosphotransferases Play Preventive vaccine Process Proteins RNA Role Site Stratified Squamous Epithelium Stratum Basale Stress Time Topoisomerase Topoisomerase II Torsion Undifferentiated Viral Viral Genome Viral Physiology Viral Proteins Virus Integration Virus Replication Work ataxia telangiectasia mutated protein cytokine experimental study high risk homologous recombination in vivo insight knock-down malignant mouth neoplasm member new therapeutic target prevent recombinational repair recruit repaired response single-cell RNA sequencing therapeutically effective viral DNA virus episome maintenance

项目摘要

Human papillomaviruses (HPV) are the causative agents of cervical, anal and many oral cancers. While prophylactic vaccines have been developed to prevent HPV infections, there is no effective therapeutic treatment for existing HPV lesions. It is therefore of critical importance to understand how the productive life cycle of high- risk HPVs is regulated to identify potential new therapeutic targets. HPVs infect stratified squamous epithelia and link their productive life cycles to the differentiation of the infected cell. My laboratory demonstrated that the amplification of HPV genomes in differentiating cells is dependent on activation of two DNA damage repair (DDR) pathways: the ataxia-telangiectasia mutated (ATM) kinase pathway as well as the ataxia telangiectasia and Rad3-related (ATR) pathway. We have identified members of these pathways that are important for the HPV life cycle and characterized many critical activities. We determined that in HPV positive cells the ATR binding protein, TopBP1, activated expression of DNA damage repair factors along with p73 while additional ATR factors, p62/GATA4, controlled expression of cytokines as well as IFNκ. Further work demonstrated that HPV proteins induced high rates of DNA breaks in both cellular and viral DNAs. The breaks in viral genomes were shown to be rapidly repaired through the preferential recruitment of homologous recombination repair factors such as RAD51 and BRCA1 to episomes and away from cellular sites of damage. This preferential repair resulted in genome amplification while at the same time permitting accumulation of breaks in cellular loci. The topoisomerase TOP2β induces DNA breaks to relieve torsional stress caused by transcription and replication. We determined that TOP2β levels were substantially increased in HPV positive cells and that this was responsible for the generation of a majority of breaks in HPV positive cells. Importantly knockdown of TOP2β impaired activation of DDR pathways and blocked viral replication. Additional work showed that HPV positive cells contain enhanced levels of R-loops which are trimeric complexes of RNA and DNA that lead to stalled replication forks and DNA breaks. R-loops were detected on HPV genomes in undifferentiated cells but resolved upon differentiation suggesting they may help in regulating viral functions. In this application, we will investigate how ATR regulates the stable maintenance of viral episomes in undifferentiated cells along with what determines which cells that undergo amplification upon differentiation. Additional work will examine which DDR and replication factors associate with amplifying genomes and how these pathways are activated. The overall goal of our studies is to understand how members of the DNA damage repair pathways regulate the differentiation- dependent HPV life cycle.

人乳头瘤病毒 (HPV) 是宫颈癌、肛门癌和许多口腔癌的病原体，虽然已经开发出预防性疫苗来预防 HPV 感染，但目前还没有针对现有 HPV 病变的有效治疗方法。调节高危 HPV 的生产生命周期，以确定潜在的新治疗靶标，并将其生产生命周期与受感染细胞的分化联系起来。分化细胞中 HPV 基因组的扩增依赖于两条 DNA 损伤修复 (DDR) 途径的激活：共济失调毛细血管扩张突变 (ATM) 激酶途径以及共济失调毛细血管扩张和 Rad3 相关 (ATR) 途径。这些途径对于 HPV 生命周期很重要，并且具有许多关键活性，我们确定在 HPV 阳性细胞中，ATR 结合蛋白 TopBP1 激活 DNA 损伤修复因子的表达。 p73 和其他 ATR 因子 p62/GATA4 控制细胞因子以及 IFNκ 的表达。进一步的研究表明，HPV 蛋白可诱导细胞和病毒 DNA 中的高 DNA 断裂率。病毒基因组中的断裂可以快速修复。通过优先将同源重组修复因子（例如 RAD51 和 BRCA1）招募到附加体并远离细胞损伤位点，这种优先修复导致基因组扩增，同时允许积累。拓扑异构酶 TOP2β 诱导 DNA 断裂，以缓解转录和复制引起的扭转应力，我们确定 HPV 阳性细胞中 TOP2β 水平显着增加，这导致了 HPV 阳性细胞中大部分断裂的产生。重要的是，敲低 TOP2β 会损害 DDR 通路的激活并阻止病毒复制。另外的研究表明，HPV 阳性细胞含有增强水平的 R 环，R 环是 RNA 和 DNA 的三聚体复合物，可导致病毒复制。在未分化细胞中的 HPV 基因组上检测到停滞的复制叉和 DNA 断裂，但在分化后消失，表明它们可能有助于调节病毒功能。在本应用中，我们将研究 ATR 如何调节未分化细胞中病毒附加体的稳定维持。以及决定哪些细胞在分化时进行扩增的其他工作将检查哪些 DDR 和复制因子与扩增基因组相关以及这些途径如何被激活。我们研究的总体目标是了解 DNA 损伤修复的成员。途径调节分化依赖性 HPV 生命周期。