Regulation of DNA Damage and Innate Immunity During the Productive Phase of the HPV Life Cycle

HPV 生命周期生产阶段 DNA 损伤和先天免疫的调节

基本信息

批准号：
10392849
负责人：
CARY A MOODY
金额：
$ 19.44万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
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：
2021-04-15 至 2024-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10392849
关键词：
Antiviral Response Apoptosis Apoptotic Automobile Driving Back CASP3 gene CASP8 gene CASP9 gene Cancer Etiology Caspase Cell Cycle Cell Cycle Deregulation Cell Cycle Regulation Cell Death Cell Differentiation process Cell physiology Cells Cessation of life Clinical Treatment Country DNA DNA Damage DNA Virus Infections Data Development Disease Episome Epithelial Gene Expression Genes Genomic Instability Goals Human Human Papilloma Virus-Related Malignant Neoplasm Human Papillomavirus Human papilloma virus 31 Innate Immune Response Interferon Type I Interferon-beta Interferons JNK-activating protein kinase Knowledge Lead Life Cycle Stages Link MAPK8 gene Malignant Neoplasms Malignant neoplasm of cervix uteri Mediating Mediator of activation protein Minority Mitochondria Mitochondrial DNA Monitor Morphology Natural Immunity Outer Mitochondrial Membrane Pathway interactions Phase Prevalence Production Proliferating Proteins Proteolysis Publishing Regulation Role Signal Pathway Signal Transduction Stimulator of Interferon Genes Stress Techniques Testing Undifferentiated Vaccines Viral Viral Genome Viral Proteins Virion Virus Diseases Virus Replication Work antiviral drug development driving force helicase innovation insight mitochondrial membrane response therapeutic target

项目摘要

The productive phase of the HPV life cycle is restricted to the uppermost layer of the epithelium in cells that have normally exited the cell cycle. The E7 protein alters cell cycle regulation, pushing differentiating cells back into the cell cycle to allow for viral genome amplification to 1000s of copies per cell and virion production. Cell cycle deregulation by E7 leads to genomic instability that is a driving force in cancer development. Our long-term goal is to understand mechanisms that regulate productive viral replication, which is important to understanding how HPV causes cancer. While caspase activation is typically viewed as an anti-viral response, HPV requires low level activation of caspases belonging to the mitochondrial pathway of apoptosis for productive replication. In this apoptotic pathway, cell death is initiated by mitochondrial membrane permeabilization (MOMP), which normally induces rapid cell death. Interestingly, HPV-induced caspase activation is not accompanied by morphological features of apoptosis, suggesting that HPV may restrain MOMP to limit caspase activity. How HPV is able to take advantage of pro-viral caspase functions by blocking apoptosis is currently unclear. Caspase cleavage of the HPV E1 helicase is required for productive replication, indicating that caspases directly regulate productive replication. However, caspase activation induces DNA damage and activates DNA damage response (DDR) pathways through JNK1/2, and we have found JNK1/2 activation increases upon differentiation. While the importance of DDR pathways (e.g. ATM and ATR) in driving productive replication is well-established, the contribution of caspase activity to DDR regulation is unknown. In the absence of apoptotic caspase activity, cytosolic release of mtDNA by MOMP is sensed by the cGAS-STING pathway, leading to induction of Type I Interferons (IFN). Intriguingly, we have found that apoptotic caspase activity is necessary to block a Type I as well as Type III IFN response upon differentiation. These results indicate that caspase activity indirectly supports productive replication through effects on host cell processes in a non-lethal manner. We hypothesize that HPV exploits non-apoptotic functions of caspases upon epithelial differentiation to facilitate the productive phase of the viral life cycle. In Aim 1, we will determine if HPV induces limited caspase activation and DNA damage through minority MOMP by using innovative techniques to monitor MOMP on a single cell level; by determining if caspase activity is non-lethal in differentiating cells; and by examining the effect of caspase inhibition on DNA damage and DDR activation. In Aim 2, we will determine how HPV uses caspase activity to block the IFN response upon differentiation by examining a role for MOMP and mtDNA release in stimulating IFN production upon caspase inhibition, and by determining if caspase activity blocks the cGAS-STING DNA sensing pathway. Completion of these studies is expected to determine how caspase activity contributes to productive viral replication as well as offer insight into mechanisms of viral persistence and genomic instability. These will represent fundamental new insight into how DNA virus infection is regulated by apoptotic caspases.

HPV 生命周期的产生阶段仅限于正常退出细胞周期的细胞上皮的最上层。 E7蛋白改变细胞周期调节，将分化的细胞推回细胞周期，从而允许病毒基因组扩增至每个细胞和病毒粒子产生数千个拷贝。 E7 导致细胞周期失调导致基因组不稳定，这是癌症发展的驱动力。我们的长期目标是了解调节有效病毒复制的机制，这对于了解 HPV 如何导致癌症非常重要。虽然半胱天冬酶激活通常被视为一种抗病毒反应，但 HPV 需要低水平激活属于细胞凋亡线粒体途径的半胱天冬酶才能进行有效复制。在该凋亡途径中，细胞死亡是由线粒体膜透化（MOMP）启动的，通常会诱导细胞快速死亡。有趣的是，HPV诱导的caspase激活并不伴随细胞凋亡的形态学特征，这表明HPV可能抑制MOMP从而限制caspase活性。目前尚不清楚 HPV 如何通过阻断细胞凋亡来利用前病毒 caspase 功能。高效复制需要半胱天冬酶裂解 HPV E1 解旋酶，这表明半胱天冬酶直接调节高效复制。然而，caspase 激活会诱导 DNA 损伤，并通过 JNK1/2 激活 DNA 损伤反应 (DDR) 途径，并且我们发现 JNK1/2 激活在分化时增加。虽然 DDR 途径（例如 ATM 和 ATR）在驱动高效复制中的重要性已得到充分证实，但 caspase 活性对 DDR 调节的贡献尚不清楚。在缺乏凋亡 caspase 活性的情况下，cGAS-STING 途径可感知 MOMP 释放的 mtDNA 的胞质，从而诱导 I 型干扰素 (IFN)。有趣的是，我们发现凋亡 caspase 活性对于阻断分化时的 I 型和 III 型 IFN 反应是必需的。这些结果表明，半胱天冬酶活性通过以非致命方式影响宿主细胞过程来间接支持生产性复制。我们假设 HPV 在上皮分化时利用半胱天冬酶的非凋亡功能来促进病毒生命周期的生产阶段。在目标 1 中，我们将通过使用创新技术在单细胞水平上监测 MOMP，确定 HPV 是否通过少数 MOMP 诱导有限的 caspase 激活和 DNA 损伤；通过确定 caspase 活性在分化细胞中是否是非致命的；并通过检查 caspase 抑制对 DNA 损伤和 DDR 激活的影响。在目标 2 中，我们将通过检查 MOMP 和 mtDNA 释放在 caspase 抑制后刺激 IFN 产生中的作用，并确定 caspase 活性是否阻断 cGAS-STING DNA 传感途径，确定 HPV 如何利用 caspase 活性来阻断分化时的 IFN 反应。这些研究的完成预计将确定半胱天冬酶活性如何促进有效的病毒复制，并深入了解病毒持久性和基因组不稳定性的机制。这些将代表对凋亡 caspase 如何调节 DNA 病毒感染的基本新见解。