Liu Xuefeng, Roberts Jeffrey, Dakic Aleksandra, Zhang Yiyu, Schlegel Richard
Department of Pathology and Oncology, Georgetown University Medical School, Washington, DC 20057, USA.
Virology. 2008 Jun 5;375(2):611-23. doi: 10.1016/j.virol.2008.02.025. Epub 2008 Mar 26.
The E6 and E7 proteins of high-risk HPVs are both required for the immortalization of primary human keratinocytes and the maintenance of the malignant phenotype of HPV-positive cancer cell lines. Our previous studies have shown that E6 protein binds Myc protein and that both E6 and Myc associate with and cooperatively activate the hTERT promoter, thereby increasing cellular telomerase activity. In this study, we evaluated the role of E7 in the maintenance and activation of telomerase in immortalized and tumorigenic cells. siRNA knockdown of either E6 or E7 (or both) in HPV-immortalized cells or an HPV-positive cancer cell line reduced hTERT transcription and telomerase activity. Since telomerase was inhibited by E7 siRNA in cells that independently expressed the E6 and E7 genes, our results reveal an independent role for E7 in the maintenance of telomerase activity. However, E7 alone was insufficient to increase endogenous hTERT mRNA or telomerase activity, although it significantly augmented E6-induced hTERT transcription and telomerase activity. To further explore this apparent E7-induced promoter augmentation, we analyzed an exogenous hTERT core promoter in transduced keratinocytes. E7 alone induced the wt hTERT promoter and augmented E6-induced hTERT promoter activity. Mutation of the E2F site in the hTERT promoter abrogated the ability of E7 to induce the hTERT promoter or to enhance the ability of E6 to induce the promoter. Correspondingly, keratinocytes expressing E6 and a mutant E7 (defective for binding pRb pocket proteins) showed lower telomerase activity than cells expressing wt E6 and wt E7. Thus, HPV E7 plays a role in the maintenance of telomerase activity in stable cell lines and augments acute, E6-induced hTERT promoter activity.
高危型人乳头瘤病毒（HPV）的E6和E7蛋白对于原代人角质形成细胞的永生化以及HPV阳性癌细胞系恶性表型的维持均是必需的。我们之前的研究表明，E6蛋白与Myc蛋白结合，且E6和Myc均与hTERT启动子结合并协同激活该启动子，从而增加细胞端粒酶活性。在本研究中，我们评估了E7在永生化细胞和致瘤细胞中端粒酶维持和激活过程中的作用。在HPV永生化细胞或HPV阳性癌细胞系中，使用小干扰RNA（siRNA）敲低E6或E7（或两者）可降低hTERT转录和端粒酶活性。由于在独立表达E6和E7基因的细胞中，E7 siRNA可抑制端粒酶，我们的结果揭示了E7在维持端粒酶活性方面具有独立作用。然而，尽管E7可显著增强E6诱导的hTERT转录和端粒酶活性，但单独的E7不足以增加内源性hTERT mRNA或端粒酶活性。为了进一步探究这种明显的E7诱导的启动子增强作用，我们分析了转导角质形成细胞中的外源性hTERT核心启动子。单独的E7可诱导野生型hTERT启动子并增强E6诱导的hTERT启动子活性。hTERT启动子中E2F位点的突变消除了E7诱导hTERT启动子的能力或增强E6诱导该启动子的能力。相应地，表达E6和突变型E7（与pRb口袋蛋白结合缺陷）的角质形成细胞比表达野生型E6和野生型E7的细胞显示出更低的端粒酶活性。因此，HPV E7在稳定细胞系中端粒酶活性的维持中发挥作用，并增强急性E6诱导的hTERT启动子活性。
