Virginia Commonwealth University (VCU), Philips Institute for Oral Health Research, School of Dentistry, Richmond, Virginia, USA.
School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK.
mSphere. 2020 Sep 16;5(5):e00858-20. doi: 10.1128/mSphere.00858-20.
Human papillomaviruses recruit a host of DNA damage response factors to their viral genome to facilitate homologous recombination replication in association with the viral replication factors E1 and E2. We previously demonstrated that SIRT1 deacetylation of WRN promotes recruitment of WRN to E1-E2 replicating DNA and that WRN regulates both the levels and fidelity of E1-E2 replication. The deacetylation of WRN by SIRT1 results in an active protein able to complex with replicating DNA, but a protein that is less stable. Here, we demonstrate an inverse correlation between SIRT1 and WRN in CIN cervical lesions compared to normal control tissue, supporting our model of SIRT1 deacetylation destabilizing WRN protein. We CRISPR/Cas9 edited N/Tert-1 and N/Tert-1+HPV16 cells to knock out WRN protein expression and subjected the cells to organotypic raft cultures. In N/Tert-1 cells without WRN expression, there was enhanced basal cell proliferation, DNA damage, and thickening of the differentiated epithelium. In N/Tert-1+HPV16 cells, there was enhanced basal cell proliferation, increased DNA damage throughout the epithelium, and increased viral DNA replication. Overall, the results demonstrate that the expression of WRN is required to control the proliferation of N/Tert-1 cells and controls the HPV16 life cycle in these cells. This complements our previous data demonstrating that WRN controls the levels and fidelity of HPV16 E1-E2 DNA replication. The results describe a new role for WRN, a tumor suppressor, in controlling keratinocyte differentiation and the HPV16 life cycle. HPV16 is the major human viral carcinogen, responsible for around 3 to 4% of all cancers worldwide. Our understanding of how the viral replication machinery interacts with host factors to control/activate the DNA damage response to promote the viral life cycle remains incomplete. Recently, we demonstrated a SIRT1-WRN axis that controls HPV16 replication, and here we demonstrate that this axis persists in clinical cervical lesions induced by HPV16. Here, we describe the effects of WRN depletion on cellular differentiation with or without HPV16; WRN depletion results in enhanced proliferation and DNA damage irrespective of HPV16 status. Also, WRN is a restriction factor for the viral life cycle since replication is disrupted in the absence of WRN. Future studies will focus on enhancing our understanding of how WRN regulates viral replication. Our goal is to ultimately identify cellular factors essential for HPV16 replication that can be targeted for therapeutic gain.
人乳头瘤病毒 (HPV) 将许多 DNA 损伤反应因子募集到其病毒基因组中，以促进与病毒复制因子 E1 和 E2 相关的同源重组复制。我们之前的研究表明，SIRT1 对 WRN 的去乙酰化作用促进了 WRN 与 E1-E2 复制 DNA 的募集，而 WRN 调节了 E1-E2 复制的水平和保真度。SIRT1 对 WRN 的去乙酰化作用导致一种能够与复制 DNA 复合的活性蛋白，但这种蛋白的稳定性较差。在这里，与正常对照组织相比，我们在 CIN 宫颈病变中观察到 SIRT1 和 WRN 之间存在负相关，这支持了我们的 SIRT1 去乙酰化作用使 WRN 蛋白不稳定的模型。我们使用 CRISPR/Cas9 编辑了 N/Tert-1 和 N/Tert-1+HPV16 细胞以敲除 WRN 蛋白表达，并将细胞进行器官样筏培养。在没有 WRN 表达的 N/Tert-1 细胞中，基础细胞增殖、DNA 损伤和分化上皮增厚。在 N/Tert-1+HPV16 细胞中，基础细胞增殖增加，整个上皮的 DNA 损伤增加，病毒 DNA 复制增加。总的来说，这些结果表明，WRN 的表达是控制 N/Tert-1 细胞增殖所必需的，并控制这些细胞中的 HPV16 生命周期。这补充了我们之前的数据，表明 WRN 控制 HPV16 E1-E2 DNA 复制的水平和保真度。这些结果描述了 WRN（一种肿瘤抑制因子）在控制角质形成细胞分化和 HPV16 生命周期中的新作用。HPV16 是主要的人类病毒致癌物质，占全球所有癌症的 3%至 4%左右。我们对病毒复制机制如何与宿主因子相互作用以控制/激活 DNA 损伤反应以促进病毒生命周期的理解仍然不完整。最近，我们证明了 SIRT1-WRN 轴控制 HPV16 复制，在这里我们证明该轴在 HPV16 诱导的临床宫颈病变中仍然存在。在这里，我们描述了 WRN 耗竭对有或没有 HPV16 的细胞分化的影响；无论 HPV16 状态如何，WRN 耗竭都会导致增殖增强和 DNA 损伤。此外，WRN 是病毒生命周期的限制因子，因为在没有 WRN 的情况下复制会中断。未来的研究将集中于增强我们对 WRN 如何调节病毒复制的理解。我们的目标是最终确定 HPV16 复制所必需的细胞因子，以便为治疗增益进行靶向。
