Regulation of HPV Replication.

HPV 复制的调节。

• 批准号: 10347959
• 负责人: Laimonis A. LAIMINS
• 金额: $ 24万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-24 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10347959
• 关键词: ATR gene; Affect; Ataxia Telangiectasia; Biological Assay; Cell Cycle Progression; Cell Cycle Regulation; Cell Proliferation; Cells; Cleaved cell; Complex; DNA; DNA Damage; DNA Double Strand Break; DNA Repair; DNA Repair Pathway; DNA biosynthesis; DNA replication fork; DNA-Directed DNA Polymerase; DNA-Directed RNA Polymerase; DNA-protein crosslink; Enzymes; Episome; Exposure to; Failure; G2 Phase; Genetic Transcription; Genome; Higher Order Chromatin Structure; Human Papillomavirus; Hybrids; Investigation; Lead; Lentivirus; Life Cycle Stages; Ligation; Link; Maintenance; Measures; Nuclear; Pathway interactions; Play; Process; Production; Proliferating; Proteins; RNA; Regulation; Resolution; Role; Rotation; Single-Stranded DNA; Stratified Epithelium; Stratified Squamous Epithelium; Stratum Basale; Stress; TOP1 gene; TOP2A gene; Time; Topoisomerase; Topoisomerase II; Torsion; Type I DNA Topoisomerases; Vertebral column; Viral; Viral Genome; Viral Proteins; Virion; Virus Replication; cell growth; high risk; insight; knock-down; member; phosphodiester; prevent; repaired; small hairpin RNA; viral DNA

PROJECT SUMMARY Human papillomaviruses (HPV) infect stratified squamous epithelia and link their productive life cycles to the differentiation of the host cell. HPVs infect cells in the basal layer of stratified epithelia and establish genomes as low copy nuclear episomes. When HPV infected cells migrate from the basal layer, they re-enter S/G2 phases in the most differentiated layers to allow for vegetative viral DNA replication in a process referred to as amplification. HPV replication is regulated by viral proteins as well as cellular factors that control cell cycle progression, differentiation and DNA damage repair pathways. Our recent studies have shown that activation of both the ataxia telangiectasia (ATM) pathway as well as the ataxia telangiectasia and Rad3-related (ATR) pathway is necessary for differentiation-dependent amplification of viral genomes and we have identified many members of these pathways that provide important functions. Our studies further indicate that enhanced levels of DNA breaks in HPV positive cells are responsible for activation of these pathways but the mechanism behind this increase has not been identified. DNA breaks can be induced by exposure to exogenous DNA damaging agents or through endogenous pathways such as through the action of topoisomerases. We have recently demonstrated that the type II topoisomerase TOP2is responsible for generating over 50% of DNA breaks in HPV positive cells. Topoisomerases regulate higher order chromatin structures through the transient breaking and re-ligation of one or both strands of the phosphodiester backbone of duplex DNA. Our studies have shown that the levels of TOP2 are increased by a 3 to 5-fold in cells with high-risk HPV genomes. Importantly, knockdown of TOP2 blocks HPV genome replication and moderately reduces viral transcription but has no effect on cell proliferation. Furthermore, knockdown of TOP2reduced the amount of DNA breaks in HPV positive cells which results in decreased DDR nuclear repair foci. While our assays demonstrated that over half of total DNA breaks in HPV positive cells were induced by TOP2 other factors must be responsible for the remaining breaks. Strong candidates for this activity are two other topoisomerases, TOP2and TOP1, and investigation of this possibility as well as an examination of the role of these enzymes in viral replication is the focus of this R21 application. These studies will provide important insights into how the differentiation-dependent HPV life cycle is regulated.

项目摘要 人乳头瘤病毒（HPV）感染了分层的鳞状上皮，并将其生产寿命联系起来 宿主细胞的分化。 HPV在分层上皮的基本层中感染了细胞并建立基因组 作为低复制的核促进。当HPV感染的细胞从基本层迁移时，它们会重新进入S/G2阶段 在最差异化的层中，可以在称为蔬菜病毒DNA复制的过程中被称为 放大。 HPV复制受病毒蛋白以及控制细胞周期的细胞因子调节 进展，分化和DNA损伤修复途径。我们最近的研究表明，激活 双开性毛道症（ATM）途径以及共济失调的毛细血管扩张和RAD3相关（ATR） 途径对于病毒基因组的分化依赖性扩增是必要的，我们已经确定了许多 这些提供重要功能的途径的成员。我们的研究进一步表明了增强的水平 HPV阳性细胞中DNA断裂的原因是激活这些途径，但背后的机制 这种增加尚未确定。 DNA断裂可以通过暴露于外源性DNA损伤来诱导 试剂或通过内源性途径，例如通过拓扑异构酶的作用。我们最近有 证明II型拓扑异构酶TOP2负责产生超过50％的DNA断裂 HPV阳性细胞。拓扑异构酶通过瞬态破裂调节高阶染色质结构 并重新连接双链DNA的磷酸二酯骨架链。我们的研究表明 在具有高危HPV基因组的细胞中，TOP2的水平增加了3至5倍。重要的是， TOP2的敲低阻断HPV基因组复制并适度降低病毒转录，但没有 对细胞增殖的影响。此外，TOP2的敲低降低了HPV中的DNA断裂量 阳性细胞可改善DDR核修复灶。而我们的测定表明超过一半 TOP2诱导HPV阳性细胞中的总DNA断裂，其他因素必须负责 剩余的休息。该活动的强大候选人是另外两个拓扑异构酶，top2和top1，和 研究这种可能性以及对这些酶在病毒复制中的作用的检查是 此R21应用程序的重点。这些研究将为差异化依赖性如何提供重要的见解 HPV生命周期受到调节。