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

• 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
• 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

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.

高风险HPV的持续感染导致多种人类癌症;然而，没有抗病毒药物来治疗这些疾病。对调节病毒生命周期的病毒-宿主相互作用的更多理解可能揭示治疗开发的新方法。感染后，HPV基因组瞬时扩增至每个细胞50- 100个附加型拷贝，并稳定维持。上皮分化触发生产性复制，导致病毒基因组扩增至100 - 1000个拷贝/细胞。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BRCA 1、Rad 51）向HPV基因组的募集以促进生产性复制。尽管分化后细胞双链断裂（DSB）增加，但HPV基因组优先修复，尽管其机制基础尚不清楚。我们最近证明了DDR泛素连接酶RNF 168是分化后病毒基因组扩增所特别需要的。RNF 168通过ATM信号被募集到DSB。RNF 168通过催化组蛋白泛素化来募集DNA修复蛋白，包括53 BP 1和BRCA 1，两者都定位于HPV复制灶，在DDR中起关键作用。HPV如何利用RNF 168活性来驱动病毒复制尚不清楚，尽管最近的几项研究表明RNF 168参与了HR修复。HPV E7蛋白直接与RNF 168相互作用，破坏细胞DSB的修复。E7可以从细胞DSB隔离RNF 168以将RNF 168活性引导至病毒染色质，促进HR因子在分化时优先募集至病毒DNA。然而，RNF 168介导的53 BP 1向DSB的募集对HPV必须克服的HR起始造成阻断。BRCA 1在S/G2期向复制后染色质的募集通过重新分配53 BP 1来拮抗这种阻断。我们已经发现，53 BP 1在分化后在HPV DNA灶处重新分布，暗示BRCA 1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因子向病毒DNA的募集）来促进病毒基因组扩增。我们假设HPV在分化后重塑细胞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RNF 168是否通过HR因子募集到病毒染色质来促进生产性复制，并确定E7-RNF 168相互作用是否促进了这一过程。我们还将确定HPV介导的RNF 168蛋白稳定性增加是否影响病毒复制。我们将确定BRCA 1是否监测复制后病毒DNA的状态，以通过去除53 BP 1来指导修复HR。此外，我们将通过确定DNA-PK活性是否提供保护HR因子募集至病毒DNA的细胞环境，来定义生产性复制期间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，并可能确定新的治疗靶点以破坏病毒生命周期。