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

细胞 DNA 损伤反应与 HPV 生命周期之间的相互作用

基本信息

批准号：
10734394
负责人：
CARY A MOODY
金额：
$ 42.11万
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：
2023
资助国家：
美国
起止时间：
2023-05-18 至 2028-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10734394
关键词：
ATM Signaling Pathway Address Affect Antiviral Agents Antiviral Therapy BRCA1 gene Basal Cell Binding Cell Differentiation process Cells Chromatin Clinical Treatment DNA DNA Damage DNA Double Strand Break DNA Repair DNA Repair Gene DNA Repair Pathway DNA-dependent protein kinase Data Disease Double Strand Break Repair Ensure Environment Episome Epithelium G2 Phase Genetic Recombination Genome HPV E7 HPV-High Risk Head and Neck Cancer Histones Human Human Papilloma Virus Vaccine Human Papillomavirus Human papilloma virus infection Infection Knowledge Life Cycle Stages Link Maintenance Malignant Neoplasms Malignant neoplasm of cervix uteri Mediating Monitor Nonhomologous DNA End Joining Oncoproteins Pathogenesis Pathway interactions Phase Phosphotransferases Plasmids Play Process Productivity Proteins Publishing Repression Role Shapes Stratified Epithelium Surveys Testing Ubiquitination Undifferentiated Viral Viral Genome Viral Pathogenesis Virus Replication Work chronic infection extracellular homologous recombination insight new therapeutic target novel therapeutic intervention p53-binding protein 1 recombinational repair recruit repaired response therapeutic development ubiquitin ligase viral DNA virus host interaction

项目摘要

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.

高危 HPV 的持续感染会导致多种人类癌症；然而，没有抗病毒药物可以治疗这些疾病。对调节病毒生命周期的病毒与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 基因组会短暂扩增至每个细胞 50-100 个游离拷贝，并稳定维持。上皮分化触发高效复制，导致病毒基因组扩增至每个细胞 100-1000 个拷贝。我们已经证明，ATM 依赖性 DNA 损伤反应 (DDR) 促进同源重组 (HR) 修复因子（例如 BRCA1、Rad51）向 HPV 基因组的募集，以促进高效复制。尽管分化时细胞双链断裂 (DSB) 增加，但 HPV 基因组会优先修复，但其机制基础尚不清楚。我们最近证明，DDR 泛素连接酶 RNF168 是分化时病毒基因组扩增所特别需要的。 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，这两种蛋白均定位于 HPV 复制灶），从而在 DDR 中发挥关键作用。尽管最近的几项研究表明 RNF168 参与 HR 修复，但 HPV 如何利用 RNF168 活性驱动病毒复制尚不清楚。 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，将 RNF168 活性引导至病毒染色质，从而促进分化时 HR 因子优先募集至病毒 DNA。然而，RNF168 介导的 53BP1 向 DSB 的募集对 HR 启动造成了阻碍，而 HPV 必须克服这一障碍。 BRCA1 在 S/G2 期招募至复制后染色质，通过重新分配 53BP1 来拮抗该阻断。我们发现 53BP1 在分化时在 HPV DNA 焦点处重新分布，表明 BRCA1 促进病毒染色质上的 HR 起始。有趣的是，我们的初步研究表明，DNA-PK（一种对易错非同源末端连接（NHEJ）至关重要的激酶）可能通过抑制细胞 DSB 修复途径（可能干扰 HR 因子招募到病毒 DNA）来促进病毒基因组扩增。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。在本提案中，我们将测试 RNF168 是否通过向病毒染色质招募 HR 因子来促进高效复制，并确定 E7-RNF168 相互作用是否促进这一过程。我们还将确定 HPV 介导的 RNF168 蛋白稳定性增加是否会影响病毒复制。我们将确定 BRCA1 是否监控复制后病毒 DNA 的状态，以通过去除 53BP1 来直接修复 HR。此外，我们将通过确定 DNA-PK 活性是否提供保护 HR 因子招募到病毒 DNA 的细胞环境来定义高效复制过程中 HR 和 NHEJ 之间的相互作用。了解 HPV 如何以细胞 DNA 修复为代价将 HR 活性引导至病毒 DNA，将有助于深入了解病毒复制和发病机制，并可能确定破坏病毒生命周期的新治疗靶点。