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

HPV生命周期的产生期仅限于正常情况下退出细胞周期的细胞中最上层的上皮。E7蛋白改变细胞周期调节，将分化的细胞推回到细胞周期，允许病毒基因组放大到每个细胞1000个拷贝和产生病毒粒子。E7对细胞周期的失控导致基因组不稳定，而基因组不稳定是癌症发展的驱动力。我们的长期目标是了解调节病毒复制的机制，这对了解HPV如何导致癌症很重要。虽然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线粒体凋亡途径的caspase才能进行有效复制。在这一凋亡途径中，细胞死亡是由线粒体膜通透性(MOMP)启动的，这通常会导致细胞快速死亡。有趣的是，HPV诱导的caspase激活并不伴随着细胞凋亡的形态特征，提示HPV可能通过抑制MOMP来限制caspase的活性。目前尚不清楚HPV是如何通过阻断细胞凋亡来利用亲病毒的caspase功能的。Caspase对HPV E1解旋酶的切割是生产性复制所必需的，这表明caspase直接调节生产性复制。然而，caspase的激活通过JNK1/2诱导DNA损伤并激活DNA损伤反应(DDR)通路，我们发现JNK1/2的激活随着分化的增加而增加。虽然DDR途径(如ATM和ATR)在驱动生产性复制中的重要性已经得到证实，但caspase活性对DDR调控的贡献尚不清楚。在缺乏凋亡caspase活性的情况下，MOMP细胞内mtDNA的释放是通过cGAS-STING途径感受到的，从而导致I型干扰素(I型干扰素)的诱生。有趣的是，我们发现凋亡的caspase活性是在分化时阻断I型和III型干扰素反应所必需的。这些结果表明，caspase活性通过对宿主细胞过程的非致命性影响间接支持生产性复制。我们假设HPV在上皮分化时利用半胱氨酸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，我们将通过在单细胞水平上监测MOMP的创新技术，通过确定caspase活性在分化细胞中是否非致死性，以及通过检测caspase抑制对DNA损伤和DDR激活的影响，来确定HPV是否通过少数MOMP诱导有限的caspase激活和DNA损伤。在目标2中，我们将通过检测MOMP和mtDNA的释放在caspase抑制时刺激干扰素的产生中的作用，以及通过确定caspase活性是否阻止cGAS-sting DNA传感途径，来确定HPV在分化时如何利用caspase活性来阻断干扰素反应。这些研究的完成有望确定caspase活性如何有助于病毒的高效复制，并为病毒持续和基因组不稳定的机制提供洞察力。这些将代表对DNA病毒感染如何由凋亡的caspase调控的根本新见解。

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.