Regulation of HPV Replication.

HPV 复制的调节。

基本信息

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
项目类别：
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

项目摘要

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.

项目概要人乳头瘤病毒 (HPV) 感染复层鳞状上皮并将其生产生命周期与 HPV 感染复层上皮基底层的细胞并建立基因组。当 HPV 感染的细胞从基底层迁移时，它们会重新进入 S/G2 期。在最分化的层中，允许植物病毒 DNA 在称为的过程中复制 HPV 复制受到病毒蛋白以及控制细胞周期的细胞因子的调节。我们最近的研究表明，激活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 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，我们已经鉴定了许多我们的进一步研究表明，这些途径的成员提供了重要的功能。 HPV 阳性细胞中的 DNA 断裂是这些途径激活的原因，但其背后的机制这种增加尚未被证实是由于暴露于外源 DNA 损伤而引起的。药物或通过内源性途径，例如通过拓扑异构酶的作用，我们最近发现。证明 II 型拓扑异构酶 TOP2 负责产生超过 50% 的 DNA 断裂 HPV 阳性细胞通过瞬时断裂调节高级染色质结构。我们的研究表明，双链 DNA 的磷酸二酯主链的一条或两条链的重新连接。重要的是，具有高危 HPV 基因组的细胞中 TOP2 的水平增加了 3 至 5 倍。 TOP2 的敲除可阻断 HPV 基因组复制并适度减少病毒转录，但没有作用此外，敲低 TOP2 减少了 HPV 中 DNA 断裂的数量。阳性细胞导致 DDR 核修复灶减少，而我们的检测表明超过一半。 HPV 阳性细胞中总 DNA 断裂是由 TOP2 引起的，其他因素必须负责此活动的剩余断裂点是另外两个拓扑异构酶，TOP2 和 TOP1，以及研究这种可能性以及检查这些酶在病毒复制中的作用是这些研究将为了解分化依赖性如何提供重要见解。 HPV 生命周期受到调控。