Werner Syndrome Protein (WRN) Regulates Cell Proliferation and the Human Papillomavirus 16 Life Cycle during Epithelial Differentiation.
Werner Syndrome Protein (WRN) Regulates Cell Proliferation and the Human Papillomavirus 16 Life Cycle during Epithelial Differentiation.
复制标题
维尔纳综合征蛋白 (WRN) 在上皮分化过程中调节细胞增殖和人乳头瘤病毒 16 生命周期。
DOI：
10.1128/msphere.00858-20
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发表时间：
2020
期刊：
影响因子：
4.8
通讯作者：
Morgan,IainM
中科院分区：
文献类型：
--
作者：
James,ClaireD;Das,Dipon;Morgan,EthanL;Otoa,Raymonde;Macdonald,Andrew;Morgan,IainM
人乳头瘤病毒在其病毒基因组中招募大量DNA损伤反应因子，以促进与病毒复制因子E1和E2相关的同源重组复制。我们之前已经证明SIRT1去乙酰化的WRN促进了WRN向E1-E2复制DNA的招募，并且WRN调节了E1-E2复制的水平和保真度。SIRT1对WRN的去乙酰化导致一种活性蛋白能够与复制DNA复合，但这种蛋白不太稳定。在这里，我们证明了与正常对照组织相比，CIN宫颈病变中SIRT1和WRN之间存在负相关，支持我们的SIRT1去乙酰化破坏WRN蛋白稳定的模型。我们通过CRISPR/Cas9编辑N/Tert-1和N/Tert-1+HPV16细胞敲除WRN蛋白表达，并将细胞进行器官型筏培养。在没有WRN表达的N/Tert-1细胞中，基底细胞增殖增强，DNA损伤，分化上皮增厚。在N/Tert-1+HPV16细胞中，基底细胞增殖增强，整个上皮细胞DNA损伤增加，病毒DNA复制增加。综上所述，这些结果表明WRN的表达是控制N/Tert-1细胞增殖和控制这些细胞中HPV16生命周期所必需的。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。这些结果描述了肿瘤抑制因子WRN在控制角化细胞分化和HPV16生命周期中的新作用。hpv16是主要的人类病毒性致癌物，约占全球所有癌症的3%至4%。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的复制，在这里我们证明了该轴在HPV16诱导的临床宫颈病变中持续存在。在这里，我们描述了WRN消耗对有或没有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是病毒生命周期的一个限制因素，因为在没有WRN的情况下复制会中断。未来的研究将集中于加强我们对WRN如何调节病毒复制的理解。我们的目标是最终确定对HPV16复制至关重要的细胞因子，这些细胞因子可以靶向治疗。
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.IMPORTANCEHPV16 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.