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

HPV生命周期的生产阶段仅限于正常退出细胞周期的细胞中上皮的最上层。E7蛋白改变细胞周期调节，将分化细胞推回到细胞周期中，以允许病毒基因组扩增到每个细胞1000个拷贝和病毒体产生。E7导致的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生产性病毒复制的机制，这对于了解HPV如何导致癌症非常重要。虽然半胱天冬酶活化通常被视为抗病毒应答，但HPV需要属于细胞凋亡线粒体途径的半胱天冬酶的低水平活化以进行生产性复制。在该凋亡途径中，细胞死亡由线粒体膜透化（MOMP）启动，其通常诱导快速细胞死亡。有趣的是，HPV诱导的caspase激活并不伴随凋亡的形态学特征，这表明HPV可能抑制MOMP以限制caspase活性。HPV如何能够通过阻断细胞凋亡来利用前病毒胱天蛋白酶功能目前尚不清楚。HPV E1解旋酶的半胱天冬酶切割是生产性复制所需的，表明半胱天冬酶直接调节生产性复制。然而，caspase激活诱导DNA损伤并通过JNK 1/2激活DNA损伤反应（DDR）途径，并且我们发现JNK 1/2激活在分化后增加。虽然DDR途径（例如ATM和ATR）在驱动生产性复制中的重要性是公认的，但是半胱天冬酶活性对DDR调节的贡献是未知的。在不存在凋亡半胱天冬酶活性的情况下，通过cGAS-STING途径感测MOMP的mtDNA的胞质释放，导致I型干扰素（IFN）的诱导。有趣的是，我们已经发现，凋亡半胱天冬酶活性是必要的，以阻止I型以及III型IFN分化后的反应。这些结果表明，半胱天冬酶活性通过以非致死方式影响宿主细胞过程间接支持生产性复制。我们假设HPV利用了caspase在上皮分化时的非凋亡功能，以促进病毒生命周期的生产阶段。在目标1中，我们将确定HPV是否通过少数MOMP诱导有限的caspase激活和DNA损伤，方法是使用创新技术在单细胞水平上监测MOMP;确定caspase活性在分化细胞中是否是非致死性的;以及检查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释放在半胱天冬酶抑制后刺激IFN产生中的作用，以及通过确定半胱天冬酶活性是否阻断cGAS-STING DNA传感途径，来确定HPV如何使用半胱天冬酶活性来阻断分化后的IFN应答。这些研究的完成预计将确定半胱天冬酶活性如何有助于生产性病毒复制，以及提供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DNA病毒感染如何受凋亡半胱天冬酶调控的基本新见解。

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.