Low-risk human papillomaviruses (LR-HPVs) are the causative agents of genital warts, which are a widespread sexually transmitted disease. How LR-HPVs affect autophagy and the specific proteins involved are unknown. In the current study, we investigated the impact of LR-HPV11 early protein 6 (E6) on the activity of the autophagy pathway. We transfected an HPV11 E6 (11E6) plasmid into HaCaT cells, H8 cells, and NHEK cells and established a stable cell line expressing the HPV11 E6 protein. The differences in autophagy activity and upstream regulatory pathways compared with those in the parent cell lines were investigated using a Western blot analysis of the total and phosphorylated protein levels and confocal microscopy of immunostained cells and cells transfected with an mCherry-green fluorescent protein-LC3 expression plasmid. We used short hairpin RNA (shRNA) to knock down 11E6 and showed that these effects require continued 11E6 expression. Compared with its expression in the control cells, the expression of HPV11 E6 in the cells activated the autophagy pathway. The increased autophagy activity was the result of the decreased phosphorylation levels of the canonical autophagy repressor mammalian target of rapamycin (mTOR) at its Ser2448 position (the mTOR complex 1 [mTORC1] phosphorylation site) and decreased AKT and Erk phosphorylation. Therefore, these results indicate that HPV11 E6 activates autophagy through the AKT/mTOR and Erk/mTOR pathways. Our findings provide novel insight into the relationship between LR-HPV infections and autophagy and could help elucidate the pathogenic mechanisms of LR-HPV.IMPORTANCE We transfected an HPV11 E6 plasmid into HaCaT cells, H8 cells, and NHEK cells and established a stable cell line expressing the HPV11 E6 protein. Then, we confirmed that HPV11 E6 induces autophagy by suppressing the AKT/mTOR and Erk/mTOR pathways. In contrast to the high-risk HPV E6 genes, HPV11 E6 did not affect the expression of p53. To the best of our knowledge, this study represents the first direct in-depth investigation of the relationship between the LR-HPV E6 gene and autophagy, which may help to reveal the pathogenesis of LR-HPV infection.

中文翻译：

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人类乳头瘤病毒11早期蛋白E6通过抑制AKT / mTOR和Erk / mTOR激活自噬。

低风险的人乳头瘤病毒（LR-HPV）是尖锐湿疣的病原体，尖锐湿疣是一种广泛的性传播疾病。LR-HPV如何影响自噬，涉及的特定蛋白质尚不清楚。在当前的研究中，我们调查了LR-HPV11早期蛋白6（E6）对自噬途径活性的影响。我们将HPV11 E6（11E6）质粒转染到HaCaT细胞，H8细胞和NHEK细胞中，并建立了表达HPV11 E6蛋白的稳定细胞系。使用总蛋白和磷酸化蛋白水平的蛋白质印迹分析以及免疫染色细胞和转染了mCherry-绿色荧光蛋白-LC3表达的细胞的共聚焦显微镜研究了自噬活性和上游调节途径与亲本细胞系相比的差异质粒。我们使用短发夹RNA（shRNA）敲低11E6，并显示这些作用需要持续的11E6表达。与在对照细胞中的表达相比，HPV11 E6在细胞中的表达激活了自噬途径。自噬活性增加是雷帕霉素（mTOR）的Ser2448位置（mTOR复合体1 [mTORC1]磷酸化位点）的雷帕霉素经典自噬抑制因子哺乳动物靶标的磷酸化水平降低以及AKT和Erk磷酸化降低的结果。因此，这些结果表明，HPV11 E6通过AKT / mTOR和Erk / mTOR途径激活自噬。我们的发现为LR-HPV感染与自噬之间的关系提供了新颖的见解，并可能有助于阐明LR-HPV的致病机制。重要信息我们将HPV11 E6质粒转染了HaCaT细胞，H8细胞和NHEK细胞建立了表达HPV11 E6蛋白的稳定细胞系。然后，我们证实HPV11 E6通过抑制AKT / mTOR和Erk / mTOR途径诱导自噬。与高风险的HPV E6基因相反，HPV11 E6不影响p53的表达。据我们所知，这项研究代表了对LR-HPV E6基因与自噬之间关系的首次直接深入研究，这可能有助于揭示LR-HPV感染的发病机理。