Department of Dermatology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, China.
Department of Dermatology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, China
J Virol. 2019 May 29;93(12). doi: 10.1128/JVI.00172-19. Print 2019 Jun 15.
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. 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.
低危型人乳头瘤病毒（LR-HPV）是引起生殖器疣的病原体，生殖器疣是一种广泛传播的性传播疾病。LR-HPV 如何影响自噬以及涉及的特定蛋白质尚不清楚。在本研究中，我们研究了 LR-HPV11 早期蛋白 6（E6）对自噬途径活性的影响。我们将 HPV11 E6（11E6）质粒转染入 HaCaT 细胞、H8 细胞和 NHEK 细胞，并建立了稳定表达 HPV11 E6 蛋白的细胞系。通过 Western blot 分析总蛋白和磷酸化蛋白水平以及免疫染色细胞和转染 mCherry-绿色荧光蛋白-LC3 表达质粒的细胞的共聚焦显微镜观察，研究了与亲本细胞系相比自噬活性和上游调节途径的差异。我们使用短发夹 RNA（shRNA）敲低 11E6，并表明这些效应需要持续表达 11E6。与对照细胞相比，细胞中 HPV11 E6 的表达激活了自噬途径。自噬活性的增加是由于经典自噬抑制剂哺乳动物雷帕霉素靶蛋白（mTOR）在其 Ser2448 位（mTOR 复合物 1[mTORC1]磷酸化位点）的磷酸化水平降低以及 AKT 和 Erk 磷酸化降低所致。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的见解，并有助于阐明 LR-HPV 的致病机制。我们将 HPV11 E6 质粒转染入 HaCaT 细胞、H8 细胞和 NHEK 细胞，并建立了稳定表达 HPV11 E6 蛋白的细胞系。然后，我们通过抑制 AKT/mTOR 和 Erk/mTOR 途径证实 HPV11 E6 诱导自噬。与高危型 HPV E6 基因不同，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 感染的发病机制。
