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.

中文翻译：

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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。


人乳头瘤病毒将大量 DNA 损伤反应因子招募到其病毒基因组中，以促进与病毒复制因子 E1 和 E2 相关的同源重组复制。我们之前证明，SIRT1 对 WRN 的去乙酰化会促进 WRN 募集到 E1-E2 复制 DNA，并且 WRN 调节 E1-E2 复制的水平和保真度。 SIRT1 对 WRN 进行脱乙酰化，产生一种能够与复制 DNA 复合的活性蛋白，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 宫颈病变中的 SIRT1 和 WRN 之间存在负相关，这支持了我们的 SIRT1 去乙酰化破坏 WRN 蛋白稳定的模型。我们对 N/Tert-1 和 N/Tert-1+HPV16 细胞进行 CRISPR/Cas9 编辑以敲除 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、DNA 损伤和分化上皮增厚增强。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。总的来说，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，证明 WRN 控制 HPV16 E1-E2 DNA 复制的水平和保真度。结果描述了肿瘤抑制因子 WRN 在控制角质形成细胞分化和 HPV16 生命周期中的新作用。