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目的人乳头瘤病毒(Humanpapilomavirus,HPV)16型早期启动子P97控制着病毒癌基因的表达。为了观测长控制区(Longcontrolregion,LCR)上YY1结合位点的破坏对病毒转化能力的影响。方法构建了带有自然发生突变LCR序列的HPV16全基因质粒,利用EMSA和荧光素酶实验检测小鼠纤维细胞胞核内源性YY1蛋白存在和功能状态;将标准及突变的HPV16毒株转染至C127细胞进行软琼脂糖培养基生长实验。结果与上皮类细胞相似,在C127胞核提取物中可检出YY1蛋白,并且具有良好的DNA结合功能和P97活性抑制作用。转化实验显示YY1位点突变HPV16毒株可在软琼脂糖培养基上形成2~10倍量多的生长集落。结论细胞转录调节因子YY1存在于啮齿类动物纤维细胞胞核中;LCR上YY1结合位点的破坏可在完整基因组范围内明显增强病毒对小鼠纤维细胞的转化能力。
Objective Human papillomavirus (HPV) 16 type early promoter P97 controls the expression of viral oncogenes. In order to observe the effect of the disruption of the YY1 binding site on the long-term control region (LCR) on the virus transformation ability. Methods HPV16 genomic plasmid with naturally occurring LCR sequence was constructed. The presence and functional status of endogenous YY1 protein in the nuclei of mouse fibroblasts were detected by EMSA and luciferase assays. The standard and mutant HPV16 strains were transfected C127 cells were grown in soft agar medium. Results Similar to epithelial cells, YY1 protein was detected in C127 nuclear extracts and had good DNA binding and P97 activity inhibition. Transformation experiments showed that the YY1 mutant HPV16 strain can grow 2-10 times more colonies in soft agarose medium. Conclusions The transcriptional regulator YY1 is found in the nuclei of rodent fibroblasts. The disruption of the YY1 binding site on LCR can significantly enhance the ability of the virus to transfect mouse fibroblasts in the complete genome.