Regulation of HPV Replication.

HPV 复制的调节。

• 批准号: 10495235
• 负责人: Laimonis A. LAIMINS
• 金额: $ 20万
• 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 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10495235
• 关键词: ATR gene; Affect; Ataxia Telangiectasia; Biological Assay; Cell Cycle Progression; Cell Cycle Regulation; Cell Proliferation; Cells; 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）感染复层鳞状上皮细胞，并将其生产生命周期与细胞周期联系起来。 宿主细胞的分化。HPV感染复层上皮基底层细胞并建立基因组 作为低拷贝核游离体。当HPV感染的细胞从基底层迁移时，它们重新进入S/G2期 在最分化的层中，以允许在称为 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， 共济失调毛细血管扩张（ATM）通路以及共济失调毛细血管扩张和Rad 3相关（ATR） 这条途径对于病毒基因组的分化依赖性扩增是必需的，我们已经鉴定了许多 这些途径的成员提供重要的功能。我们的研究进一步表明， HPV阳性细胞中的DNA断裂负责这些途径的激活，但其背后的机制 这一增长尚未确定。外源性DNA损伤可诱导DNA断裂 试剂或通过内源性途径如通过拓扑异构酶的作用。我们最近 证明II型拓扑异构酶TOP2激酶负责产生超过50%的DNA断裂， HPV阳性细胞。拓扑异构酶通过瞬时断裂调节染色质的高级结构 和双链体DNA的磷酸二酯骨架的一条或两条链的重新连接。我们的研究显示 在具有高危HPV基因组的细胞中，TOP 2水平增加了3至5倍。重要的是， 敲低TOP2抑制剂可阻断HPV基因组复制，并适度降低病毒转录，但没有 影响细胞增殖。此外，TOP2基因敲低减少了HPV中DNA断裂的数量， 阳性细胞，导致DDR核修复灶减少。虽然我们的分析表明，超过一半的 HPV阳性细胞中总DNA断裂量的10%是由TOP 2诱导的，但其他因素可能是导致HPV阳性细胞中总DNA断裂的原因。 剩下的休息。这种活性的强有力的候选者是另外两种拓扑异构酶，TOP2拓扑异构酶和TOP1， 对这种可能性的研究以及对这些酶在病毒复制中的作用的检查是 这是R21应用的重点。这些研究将提供重要的见解如何分化依赖 HPV的生命周期是有规律的。