Human Papillomavirus 11 Early Protein E6 Activates Autophagy by Repressing AKT/mTOR and Erk/mTOR

Abstract

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.

Autophagy is induced in human keratinocytes during human papillomavirus 11 pseudovirion entry

“…Some studies showed that HPV16 entry requires clathrin-dependent endocytosis [ 22 – 25 ], however, other studies showed that HPV16 internalization requires tetraspanin-enriched microdomains for endocytosis, which was independent of clathrin [ 26 , 27 ]. Our previous study has revealed that HPV11 E6 induces autophagy by suppressing the AKT/mTOR and Erk/mTOR pathways, suggesting elevated autophagy level is beneficial for the survival of HPV11 in host cells after successful infection [ 28 ]. However, the host autophagic response during HPV11 entry has not been reported thus far.…”

“…Cells were incubated with 1,000 vge/cell of HPV11 PsVs for indicated times and approximately 30 μg of cell lysate protein were subjected to western blotting protocols as described previously [ 28 ], using the following antibodies: polyclonal rabbit anti-LC3B (Cat#: L7543) and monoclonal mouse anti-actin (Sigma, Cat#: A9357), monoclonal rabbit anti-p62 (MBL Life science, Cat#: PM045), monoclonal rabbit anti-phospho-mTOR (Ser2448, Cat#: 2971), monoclonal rabbit anti-mTOR (Cat#: 2983), anti-phospho-70S6 kinase (Cat#: 9234), monoclonal rabbit anti-70S6 kinase (Cat#: 2708), monoclonal rabbit anti-phospho-4E-BP1 (Thr37/46, Cat#: 2855), monoclonal rabbit anti-4E-BP1 (Cat#: 9452), monoclonal rabbit anti-Erk (Cat#: 4695), monoclonal rabbit anti-phospho-Erk (Thr202/Tyr204, Cat#: 4370), monoclonal rabbit anti-Akt (Cat#: 4691), monoclonal rabbit anti-phospho-Akt (Ser473, Cat#: 4060), monoclonal rabbit anti-ULK1 (Cat#: 8054), monoclonal rabbit anti-phospho-ULK1 (Ser555, Cat#: 5869), monoclonal rabbit anti-phospho-ULK1 (Ser757, Cat#: 14202), monoclonal rabbit anti-AMPK (Cat#: 2603) and monoclonal rabbit anti-phospho-AMPK (Ser172, Cat#: 2531) (Cell Signaling Technology).…”

Molecular pathways in the development of HPV-induced cervical cancer

“…HPV can use different mechanisms to evade the proper autoimmune response of the host, through its oncoproteins (Tindle, 2002[ 151 ]). During infection two proteins (E6 and E7) are involved in the process of pathogenicity (Androphy et al, 1987[ 5 ]; Mesri et al, 2014[ 103 ]; Zhang et al, 2019[ 169 ]) (Figure 2 (Fig. 2) ).…”

The Role of Ataxia Telangiectasia Mutant and Rad3-Related DNA Damage Response in Pathogenesis of Human Papillomavirus

“…HPV16 E7 has been shown to upregulate autophagy marker LC3B [ 135 ]. E6 is capable of activating autophagy by suppressing the mTOR (mammalian target of rapamycin) signaling [ 136 ]. The combination of E6 and E7 can increase the autophagosome cycle rate via ATG9B(Autophagy-related protein 9B) and LAMP1 (Lysosome-associated membrane glycoprotein 1) [ 137 ].…”