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生命周期的生产阶段仅限于通常退出细胞周期的细胞中上皮的最上层。 E7蛋白会改变细胞周期的调节，将分化细胞重新恢复到细胞周期中，以使病毒基因组扩增至每个细胞和病毒粒子产生的1000份。细胞周期对E7的管制导致基因组不稳定性，这是癌症发展的驱动力。我们的长期目标是了解调节生产性病毒复制的机制，这对于了解HPV如何引起癌症很重要。 caspase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凋亡的线粒体途径的胱天蛋白酶以进行生产式复制。在这种凋亡途径中，细胞死亡是由线粒体膜通透性（MOMP）引发的，该通透性通常会诱导快速细胞死亡。有趣的是，HPV诱导的caspase激活不伴有凋亡的形态特征，这表明HPV可能会限制MOMP以限制caspase活性。目前尚不清楚HPV如何通过阻断凋亡来利用促病毒caspase功能。 HPV E1解旋酶的caspase裂解是生产性复制所必需的，这表明caspase直接调节生产性复制。但是，胱天蛋白酶激活会诱导DNA损伤，并通过JNK1/2激活DNA损伤响应（DDR）途径，我们发现在分化时JNK1/2激活增加。尽管DDR途径（例如ATM和ATR）在驱动生产复制方面的重要性是良好的，但caspase活动对DDR调节的贡献尚不清楚。在没有凋亡的caspase活性的情况下，MOMP的胞质释放被CGAS刺激途径感知，从而导致I型干扰素的诱导（IFN）。有趣的是，我们发现凋亡性caspase活性对于阻断I型和III型IFN响应是必要的。这些结果表明，caspase活性通过以非致命方式对宿主细胞过程的影响间接支持生产性复制。我们假设HPV在上皮分化时利用了胱天蛋白酶的非凋亡功能，以促进病毒生命周期的生产阶段。在AIM 1中，我们将通过使用创新技术在单个细胞水平上监测MOMP来确定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非致命；通过检查caspase抑制对DNA损伤和DDR激活的影响。在AIM 2中，我们将确定HPV如何使用caspase活性来通过检查MOMP和mtDNA释放在刺激caspase抑制时刺激IFN产生的作用以及确定capase活性是否阻止CGAS刺激DNA感应途径来阻止IFN响应。这些研究的完成有望确定caspase活性如何促进生产性病毒复制，并提供对病毒持久性和基因组不稳定性机制的见解。这些将代表对DNA病毒感染如何受凋亡胱天蛋白酶调节的基本新见解。