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: 10408136
• 负责人: Iain Morgan
• 金额: $ 55.11万
• 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/10408136
• 关键词: Anogenital cancer; Biological Assay; CRISPR/Cas technology; Cell Death; Cell Line; Cell Proliferation; Cells; Cervical; Collaborations; Complex; DNA; DNA Damage; DNA Double Strand Break; DNA Repair; DNA biosynthesis; DNA replication fork; Data; Deoxyribonuclease I; 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）是肛门生殖器和头颈部癌症的病原体。我们的长期目标是确定并开发针对这些病毒性疾病的新方法；要做到这一点，我们必须加强对病毒生命周期以及它如何与宿主相互作用的理解。HPV在病毒生命周期中激活DNA损伤反应（DDR），并利用同源重组（HR）复制其基因组。摩根实验室已经确定了几个参与HPV复制的HR因子，包括TopBP1、BRD4、SIRT1和WRN。我们已经证明这些因子调节C33a细胞中hpv16e1 - e2 DNA复制的水平和保真度。在WRN耗尽后，E1-E2复制从高保真机制切换到中断诱导复制（BIR）。BIR是高度诱变的，当一个暂停的分叉无法被WRN解决时发生。在缺乏WRN的情况下，过量的MUS81招募到E1-E2复制DNA，这是一种内切酶，在缺乏WRN的情况下，在暂停分叉处导致DNA双链断裂。这解决了分叉问题，并允许BIR继续复制，尽管保真度较低。我们目前的数据表明，WRN是HPV16生命周期的一个限制因素；在没有WRN的情况下，器官型筏培养中细胞增殖、DNA损伤和病毒复制增加。我们观察到SAMHD1缺失（另一个HR因子）后也出现了类似的表型。WRN和SAMHD1与其他参与HPV生命周期的DDR因子位于相同的细胞复合体中。我们的第一个目标是确定WRN-SAMHD1复合物在控制HPV生命周期中的作用。核心假设是，该复合体在病毒生命周期中控制着高保真复制，并且靶向该复合体的成分和MUS81可能会阻止HPV复制。利用新的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。中心假设是差异可以用于HPV阳性细胞的不同靶向。这将与WRN和DNA复制和修复专家Pietro Pichierri合作完成；Morgan和Pichierri已经共同发表了文章。我们的第三个目标是研究在WRN和SAMHD1缺失的HPV16细胞中先天免疫反应（IIR）的激活。核心假设是，在HPV16细胞中，在活性DDR存在的情况下，WRN或SAMHD1的耗竭会导致过量的细胞质DNA片段激活IIR。其结果对于实现我们的长期目标至关重要。例如，靶向WRN酶活性可以提高HPV16阳性细胞的IIR，帮助适应性免疫反应（和免疫治疗）消除HPV阳性细胞。如果有不同的因子在HPV存在的情况下复制宿主DNA，这些因子可以被靶向优先杀死HPV阳性细胞。在HPV阳性细胞中，双重靶向WRN和MUS81可能会阻断病毒复制，导致E6/E7耗竭和p53/pRb通路的再激活后细胞死亡。