Renewal: HPV and the DNA Damage Response

更新：HPV 和 DNA 损伤反应

• 批准号: 10612428
• 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
• 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/10612428
• 关键词: 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; HPV-High Risk; 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; Productivity; Proteins; RNA; Role; Site; Stratified Squamous Epithelium; Stratum Basale; Stress; Time; Topoisomerase; Topoisomerase II; Undifferentiated; Viral; Viral Genome; Viral Physiology; Viral Proteins; Virus Integration; Virus Replication; Work; ataxia telangiectasia mutated protein; cell motility; cytokine; experimental study; 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

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。虽然已经开发了预防HPV感染的预防性疫苗，但目前还没有有效的治疗方法来治疗现有的HPV病变。因此，了解如何调节高危HPV的生产生命周期，以确定潜在的新治疗靶点是至关重要的。HPV感染复层鳞状上皮，并将其生产生命周期与感染细胞的分化联系起来。我的实验室证明，HPV基因组在分化细胞中的扩增依赖于两条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。我们已经确定了这些通路中对HPV生命周期重要的成员，并表征了许多关键活动。我们确定在HPV阳性细胞中，ATR结合蛋白TopBP1激活了DNA损伤修复因子和p73的表达，而另外的ATR因子p62/GATA4控制了细胞因子和干扰素κ的表达。进一步的研究表明，HPV蛋白在细胞和病毒DNA中都诱导了高比例的DNA断裂。病毒基因组中的断裂被证明是通过优先招募同源重组修复因子如RAD51和BRCA1到外体上而远离细胞损伤部位而迅速修复的。这种优先修复导致了基因组的放大，同时允许细胞基因座的断裂积累。拓扑异构酶TOP2DNA通过诱导β断裂来缓解转录和复制引起的扭应力。我们确定，在人乳头瘤病毒阳性细胞中，TOP2β水平显著增加，这是导致人乳头状瘤病毒阳性细胞中大多数断裂产生的原因。重要的是，TOP2DDR基因的敲除削弱了β通路的激活，阻断了病毒的复制。进一步的研究表明，HPV阳性细胞含有水平增强的R-环，这是RNA和DNA的三聚体复合体，会导致复制分叉停滞和DNA断裂。在未分化的细胞中，在HPV基因组上检测到R-环，但在分化后分解，这表明它们可能有助于调节病毒的功能。在这一应用中，我们将研究ATR如何调节未分化细胞中病毒异构体的稳定维持，以及是什么决定了哪些细胞在分化时经历了扩增。其他工作将检查哪些DDR和复制因子与放大基因组有关，以及这些途径是如何被激活的。我们研究的总体目标是了解DNA损伤修复途径的成员如何调节依赖于分化的HPV生命周期。