Determining how a Werner helicase (WRN) tumor suppressor complex regulates the human papillomavirus 16 life cycle

确定维尔纳解旋酶 (WRN) 肿瘤抑制复合物如何调节人乳头瘤病毒 16 生命周期

• 批准号: 10615229
• 负责人: Iain Morgan
• 金额: $ 55.67万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-01 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10615229
• 关键词: Anogenital cancer; Biological Assay; CRISPR/Cas technology; Cell Death; Cell Line; Cell Proliferation; Cells; Cervical; Collaborations; Complex; Cytoplasm; DNA; DNA Damage; DNA Double Strand Break; DNA Repair; DNA biosynthesis; DNA replication fork; Data; Disease; Enzymes; Gene Activation; Gene Expression; Genetic Recombination; Genome; Goals; Head and Neck Cancer; Human Papillomavirus; Human papilloma virus 31; Human papillomavirus 16; Immune Response Genes; Immunotherapy; Innate Immune Response; Integration Host Factors; Knowledge; LacZ Genes; Lesion; Life Cycle Stages; Malignant Neoplasms; Measures; Modeling; Mutagenesis; Outcome; Pathogenicity; Pathway interactions; Phenotype; Publishing; Regulation; Reporting; Retinoblastoma Protein; Role; SIRT1 gene; Stimulator of Interferon Genes; System; TP53 gene; Testing; Time; Tumor Suppressor Proteins; Viral; Viral Genome; Virus; Virus Diseases; Virus Replication; adaptive immune response; burden of illness; endonuclease; enzyme activity; helicase; homologous recombination; human disease; innate immune mechanisms; keratinocyte; malignant mouth neoplasm; member; new therapeutic target; novel; novel strategies; novel therapeutic intervention; recruit; replication stress; response; targeted treatment; therapeutic target; transcriptome sequencing

Human papillomaviruses (HPV) are causative agents in ano-genital and head and neck cancers. Our long-term goal is to identify, and develop, novel approaches for targeting these viral diseases; to do this we must enhance our understanding of the viral life cycle and how it interacts with the host. HPV activates the DNA damage response (DDR) during the viral life cycle and uses homologous recombination (HR) to replicate its genome. The Morgan lab has identified several HR factors involved in HPV replication including TopBP1, BRD4, SIRT1 and WRN. We have demonstrated that these factors regulate the levels and fidelity of HPV16 E1-E2 DNA replication in C33a cells. Following WRN depletion, E1-E2 replication switches from high fidelity mechanisms to break induced replication (BIR). BIR is highly mutagenic and occurs when a paused fork is unable to be resolved by WRN. In the absence of WRN there is excess recruitment of MUS81 to E1-E2 replicating DNA, an endonuclease that causes DNA double strand breaks at paused forks in the absence of WRN. This resolves the fork and allows BIR to continue replication, albeit with low fidelity. We present data demonstrating that WRN is a restriction factor for the HPV16 life cycle; in the absence of WRN there is increased cell proliferation, DNA damage and viral replication in organotypic raft cultures. We observed a similar phenotype following SAMHD1 depletion, another HR factor. WRN and SAMHD1 are in the same cellular complex along with other DDR factors involved in HPV life cycles. Our first objective is to determine the roles of the WRN-SAMHD1 complex in controlling HPV life cycles. The central hypothesis is that this complex controls high fidelity replication during the viral life cycle, and that targeting components of this complex along with MUS81 may block HPV replication. Using a novel HPV16 genome, we will investigate the levels and the fidelity of viral replication during the viral life cycle. Our second objective is to determine whether there are altered host DNA replication forks in HPV16 positive cells. The central hypothesis is that differences can be exploited for differential targeting of HPV positive cells. This will be done in association with Pietro Pichierri, a WRN and DNA replication and repair expert; Morgan and Pichierri have already published together. Our third objective is to investigate the activation of the innate immune response (IIR) in HPV16 cells depleted for WRN and SAMHD1. The central hypothesis is that depletion of WRN or SAMHD1 in the presence of the active DDR in HPV16 cells results in excess cytoplasmic DNA fragments that activate the IIR. The outcomes are crucial for achieving our long-term goals. For example, targeting of WRN enzyme activities could boost the IIR in HPV16 positive cells, assisting the adaptive immune response (and immunotherapy) to eliminate HPV positive cells. If there are different factors replicating host DNA in the presence of HPV, these could be targeted to preferentially kill HPV positive cells. Double targeting of WRN and MUS81 in HPV positive cells may block viral replication resulting in cell death following E6/E7 depletion and reactivation of p53/pRb pathways.

人乳头瘤病毒 (HPV) 是肛门生殖器癌和头颈癌的病原体。我们的长期目标是识别和开发针对这些病毒性疾病的新方法；为此，我们必须加深对病毒生命周期及其与宿主相互作用的了解。 HPV 在病毒生命周期中激活 DNA 损伤反应 (DDR)，并使用同源重组 (HR) 来复制其基因组。 Morgan 实验室已确定了几个与 HPV 复制有关的 HR 因子，包括 TopBP1、BRD4、SIRT1 和 WRN。我们已经证明这些因素调节 C33a 细胞中 HPV16 E1-E2 DNA 复制的水平和保真度。 WRN 耗尽后，E1-E2 复制从高保真机制切换为中断诱导复制 (BIR)。 BIR 具有高度诱变性，当 WRN 无法解析暂停的分叉时，就会发生 BIR。在不存在 WRN 的情况下，MUS81 会过度招募到 E1-E2 复制 DNA，这是一种核酸内切酶，在不存在 WRN 的情况下，会导致 DNA 双链在暂停的分叉处断裂。这解决了分叉问题，并允许 BIR 继续复制，尽管保真度较低。我们提供的数据表明，WRN 是 HPV16 生命周期的限制因素；在缺乏 WRN 的情况下，器官筏培养物中的细胞增殖、DNA 损伤和病毒复制会增加。我们在另一个 HR 因素 SAMHD1 耗尽后观察到类似的表型。 WRN 和 SAMHD1 以及参与 HPV 生命周期的其他 DDR 因子位于同一细胞复合体中。我们的首要目标是确定 WRN-SAMHD1 复合物在控制 HPV 生命周期中的作用。核心假设是，该复合物在病毒生命周期中控制着高保真度复制，并且该复合物的成分与 MUS81 一起靶向可能会阻断 HPV 复制。使用新型 HPV16 基因组，我们将研究病毒生命周期中病毒复制的水平和保真度。我们的第二个目标是确定 HPV16 阳性细胞中是否存在改变的宿主 DNA 复制叉。核心假设是，可以利用差异来针对 HPV 阳性细胞进行差异化靶向。这项工作将与 WRN 和 DNA 复制与修复专家 Pietro Pichierri 合作完成；摩根和皮基耶里已经共同发表过文章。我们的第三个目标是研究 WRN 和 SAMHD1 耗尽的 HPV16 细胞中先天免疫反应 (IIR) 的激活情况。核心假设是，在 HPV16 细胞中存在活性 DDR 的情况下，WRN 或 SAMHD1 的消耗会导致细胞质 DNA 片段过多，从而激活 IIR。结果对于实现我们的长期目标至关重要。例如，针对 WRN 酶活性可以增强 HPV16 阳性细胞中的 IIR，协助适应性免疫反应（和免疫疗法）消除 HPV 阳性细胞。如果在 HPV 存在的情况下有不同的因子复制宿主 DNA，则可以针对这些因子优先杀死 HPV 阳性细胞。 HPV 阳性细胞中 WRN 和 MUS81 的双重靶向可能会阻止病毒复制，导致 E6/E7 耗尽和 p53/pRb 途径重新激活后细胞死亡。