Interplay between the cellular DNA damage response and the HPV life cycle

持续感染高危HPV会导致多种人类癌症；然而，目前还没有治疗这些疾病的抗病毒药物。对调节病毒生命周期的病毒-宿主相互作用的进一步了解可能会揭示治疗开发的新方法。感染后，HPV基因组瞬间扩增到每个细胞50-100个附体拷贝，并稳定保持。上皮分化触发生产性复制，导致病毒基因组扩增到100s-1000s拷贝/细胞。我们已经证明，ATM依赖的DNA损伤反应(DDR)促进同源重组(HR)修复因子(例如BRCA1、RAD51)招募到HPV基因组，以促进生产性复制。尽管分化后细胞双链断裂(DSB)增加，但HPV基因组优先修复，尽管其机制尚不清楚。我们最近证明了DDR泛素连接酶RNF168是病毒分化时基因组扩增所必需的。RNF168通过ATM信令被招募到DSB。RNF168通过催化组蛋白泛素化来招募DNA修复蛋白，包括53BP1和BRCA1，这两种蛋白都定位于HPV复制点，在DDR中发挥关键作用。HPV如何利用RNF168活性来驱动病毒复制尚不清楚，尽管最近的几项研究表明RNF168参与HR修复。HPV E7蛋白直接与RNF168相互作用，破坏细胞DSB的修复。E7可以将RNF168从细胞DSB中隔离出来，将RNF168的活性导向病毒染色质，促进HR因子在分化后优先招募到病毒DNA。然而，RNF168介导的53BP1募集到DSB构成了HPV必须克服的HR启动的障碍。BRCA1在S/G2期重新分布到复制后染色质，通过重新分配53BP1拮抗这一阻断。我们发现，53BP1在分化后重新分布在HPV DNA焦点上，暗示BRCA1促进了病毒染色质上的HR启动。有趣的是，我们的初步研究表明，DNA-PK是一种对容易出错的非同源末端连接(NHEJ)至关重要的激酶，它可能通过抑制细胞DSB修复途径而促进病毒基因组的扩增，而DSB修复途径可能会干扰HR因子重新募集到病毒DNA。我们假设HPV在分化时重塑细胞DNA损伤反应，以支持病毒染色质上的HR。在这项提案中，我们将测试RNF168是否通过将HR因子招募到病毒染色质来促进生产性复制，并确定E7-RNF168相互作用是否促进了这一过程。我们还将确定HPV介导的RNF168蛋白稳定性增加是否影响病毒复制。我们将确定BRCA1是否监控复制后病毒DNA的状态，以通过移除53BP1来指导HR修复。此外，我们将通过确定DNA-PK活性是否提供保护HR因子重新招募到病毒DNA的细胞环境来定义HR和NHEJ在生产性复制过程中的相互作用。了解HPV如何以细胞DNA修复为代价将HR活性导向病毒DNA，将有助于深入了解病毒复制和发病机制，并可能确定扰乱病毒生命周期的新的治疗靶点。

Persistent infection with high-risk HPVs cause multiple human cancers; however there are no antivirals to treat these diseases. An increased understanding of the virus-host interactions that regulate the viral life cycle may reveal novel approaches for therapeutic development. Upon infection, HPV genomes transiently amplify to 50- 100 episomal copies per cell that are stably maintained. Epithelial differentiation triggers productive replication, resulting in amplification of viral genomes to 100s-1000s of copies/cell. We have shown that the ATM-dependent DNA damage response (DDR) promotes recruitment of homologous recombination (HR) repair factors (e.g. BRCA1, Rad51) to HPV genomes to facilitate productive replication. Despite an increase in cellular double-strand breaks (DSBs) upon differentiation, HPV genomes are preferentially repaired, though the mechanistic basis for this is unknown. We recently demonstrated that the DDR ubiquitin ligase RNF168 is specifically required for viral genome amplification upon differentiation. RNF168 is recruited to DSBs by ATM signaling. RNF168 plays a critical role in the DDR by catalyzing histone ubiquitination to recruit DNA repair proteins, including 53BP1 and BRCA1, both of which localize to HPV replication foci. How HPV uses RNF168 activity to drive viral replication is unclear, although several recent studies have implicated RNF168 in HR repair. The HPV E7 protein interacts directly with RNF168, disrupting repair at cellular DSBs. E7 may sequester RNF168 from cellular DSBs to direct RNF168 activity to viral chromatin, promoting preferential recruitment of HR factors to viral DNA upon differentiation. However, RNF168-mediated 53BP1 recruitment to DSBs poses a block to HR initiation that HPV must overcome. BRCA1 recruitment to post-replicative chromatin in S/G2 phases antagonizes this block by redistributing 53BP1. We have found that 53BP1 is redistributed at HPV DNA foci upon differentiation, implicating BRCA1 in promoting HR initiation on viral chromatin. Interestingly, our preliminary studies indicate that DNA-PK, a kinase critical for error-prone non-homologous end joining (NHEJ), may contribute to viral genome amplification by repressing cellular DSB repair pathways that could interfere with HR factor recruitment to viral DNA. We hypothesize HPV reshapes the cellular DNA damage response upon differentiation to support HR on viral chromatin. In this proposal, we will test if RNF168 promotes productive replication through HR factor recruitment to viral chromatin and determine if the E7-RNF168 interaction facilitates this process. We will also determine if the HPV-mediated increase in RNF168 protein stability influences viral replication. We will determine if BRCA1 monitors the state of post-replicative viral DNA to direct repair to HR by removing 53BP1. Additionally, we will define the interplay between HR and NHEJ during productive replication by determining if DNA-PK activity provides a cellular environment that protects HR factor recruitment to viral DNA. Understanding how HPV directs HR activity to viral DNA at the expense of cellular DNA repair will provide insight into mechanisms of viral replication and pathogenesis and may identify novel therapeutic targets to disrupt the viral life cycle.