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.

项目总结 人乳头瘤病毒(HPV)感染复层鳞状上皮，并将其生产生命周期与 宿主细胞的分化。人乳头瘤病毒感染复层上皮基底层细胞并建立基因组 作为低拷贝的核表现体。当HPV感染细胞从基底层迁移时，它们重新进入S/G2期 在最分化的层允许营养病毒DNA复制的过程称为 放大。HPV的复制受病毒蛋白和控制细胞周期的细胞因子的调节 进展、分化和DNA损伤修复途径。我们最近的研究表明，激活 共济失调毛细血管扩张(ATM)途径以及共济失调毛细血管扩张和RAD3相关(ATR) 途径是依赖分化的病毒基因组扩增所必需的，我们已经鉴定了许多 这些提供重要功能的通路的成员。我们的研究进一步表明，增强的水平 HPV阳性细胞的DNA断裂是激活这些途径的原因，但背后的机制 这一增长尚未确定。暴露于外源DNA损伤可导致DNA断裂 或通过内源性途径，如通过拓扑异构酶的作用。我们最近做了 研究表明，2型拓扑异构酶TOP2dna断裂的产生占的50%以上。 HPV阳性细胞。拓扑异构酶通过瞬时断裂调节高阶染色质结构 以及重新连接双链DNA的磷酸二酯骨架的一条或两条。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到5倍。重要的是 敲除TOP2可阻断HPV基因组复制，并适度减少病毒转录，但没有 对细胞增殖的影响。此外，敲除TOP2DNA减少了HPVDNA断裂的数量 阳性细胞，导致DDR核修复灶减少。而我们的化验结果显示超过一半的人 在总DNA断裂中，TOP2诱导的HPV阳性细胞DNA断裂中，其他因素可能是导致 剩下的休息时间。这种活性的有力候选者是另外两种拓扑异构酶，Top2和Top1，以及 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 此R21应用程序的焦点。这些研究将提供重要的见解，以了解分化是如何依赖的 HPV的生命周期是受调控的。