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Human papillomavirus E7 inhibits immune responses in keratinocytes by activating HTRA1‑mediated mitophagy

  • Authors:
    • Boya Zhang
    • Defeng Kong
    • Siji Chen
    • Xuewu Sun
    • Hao Cheng
  • View Affiliations / Copyright

    Affiliations: Department of Dermatology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang 310016, P.R. China
    Copyright: © Zhang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 22
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    Published online on: November 14, 2025
       https://doi.org/10.3892/ijmm.2025.5693
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Abstract

Persistent infection with human papillomavirus (HPV) can lead to refractory disease. The HPV E7 protein causes persistent viral infection by disrupting the immune balance of keratinocytes; however, its key mechanism is not yet clear. Overexpression of the HPV E7 gene in normal human epidermal keratinocytes can promote mitophagy in the host cells and inhibit the expression of type I interferon (IFN), as previously confirmed by electron microscopy, immunofluorescence and western blot analysis. In the present study, Siha cells with stable knockdown of HPV16 E7 were constructed, and RNA sequencing at the transcription level and isobaric tags for relative and absolute quantitation analysis at the protein level were performed, with the aim of identifying genes related to mitophagy among the differentially expressed genes. Using immunohistochemistry, PCR and western blotting, significant differences were detected in the expression levels of high‑temperature requirement A serine peptidase 1 (HTRA1) between the knockdown and control groups. The results confirmed that HPV E7 could promote the expression of HTRA1. Furthermore, it was demonstrated that the HPV E7 protein interacted with HTRA1 intracellularly to activate the PTEN‑induced kinase 1 (PINK1)/Parkin pathway in keratinocytes, leading to enhanced mitophagy and reduced expression of type I IFN in host cells. In conclusion, HPV E7 could promote the expression of the HTRA1 gene in keratinocytes, thereby activating mitophagy mediated by the PINK1/Parkin pathway. Furthermore, HPV E7 could inhibit the secretion of type I IFN from cells, thus leading to persistent viral infection. These findings provide novel insights into the association between HPV infection and mitophagy, and may elucidate the mechanisms underlying persistent HPV infection.1
View Figures

Figure 1

HPV E7 induces mitophagy in
keratinocytes (A) Mitophagy in NHEKs overexpressing HPV11/16 E7 was
ascertained through transmission electron microscopy. The yellow
arrows indicate mitochondrial autophagosomes. The number of
mitochondrial autophagosomes was statistically analyzed and a total
of 100 cells were analyzed per experiment. Intracellular mitophagy
in NHEKs overexpressing (B) HPV11 E7 and (C) HPV16 E7 was analyzed
with GFP-LC3 and Mito-Tracker Red CMXRos. The yellow dots indicate
co-localized molecules. The ratio of yellow dots to all dots was
statistically analyzed and a total of 100 cells were analyzed per
experiment. Alterations in mitophagy-related proteins and pathways
were detected by western blotting in NHEKs overexpressing (D) HPV11
E7 and (F) HPV16 E7. All proteins were normalized to the loading
control GAPDH. Expression levels of mitophagy-related proteins and
pathways were investigated in NHEKs overexpressing (E) HPV11 E7 or
(G) HPV16 E7 and stimulated with the mitophagy inhibitor CsA.
*P<0.05, **P<0.01. BNIP3, Bcl-2 and
adenovirus E1B 19 kDa-interacting protein 3; BNIP3L, BNIP3-like;
CsA. CsA, cyclosporine A; NC, negative control; HPV, human
papillomavirus; NHEK, normal human epidermal keratinocyte; PINK1,
PTEN-induced kinase 1.

Figure 2

HPV E7 facilitates the expression of
HTRA1 within keratinocytes. (A) Heatmap of RNA-seq and iTRAQ
analyses. (B) Examination of differentially expressed genes via
RNA-seq and iTRAQ analyses. 'None' indicates that there was no
difference in the expression of these genes between groups;
'opposite' indicates that the expression trend of the gene was
opposite in RNA-seq and iTRAQ analyses; 'same' indicates that the
expression trend of the gene was consistent in RNA-seq and iTRAQ
analyses; 'transcript only' indicates that these alterations were
observed exclusively in the RNA-seq data; 'protein only' indicates
that these alterations were observed exclusively in the iTRAQ data.
(C) Heatmap presenting the potential genes related to mitophagy
derived from the analysis (PODXL, CEBPD, HTRA1, S10A6 and ANXA8).
(D) qPCR was used to verify the expression of potential
mitophagy-related genes in the HPV16 E7 KD group. (E) GO analysis
of RNA-seq data indicated that HTRA1 was significantly enriched in
GO terms associated with mitophagy. (F) qPCR was used to detect the
expression of HTRA1 in HPV11 E7-overexpressing NHEKs. (G) qPCR was
used to detect the expression of HTRA1 in HPV16 E7-overexpressing
NHEKs and in stable Siha cells with knockdown of HPV16 E7. (H)
Western blotting was used to confirm the expression of
intracellular and extracellular HTRA1 in HPV11 E7-overexpressing
NHEKs. (I) Western blotting was used to determine the expression of
intracellular and extracellular HTRA1 in HPV16 E7-overexpressing
NHEK cells and HPV16 E7 KD Siha cells. (J) Examination of the
expression of HTRA1 and HPV11 E7 in CA and normal foreskin samples
by IHC. (K) Examination of the expression of HTRA1 and HPV16 E7 in
HPV-negative cervical cancer and HPV 16-positive cervical cancer
tissues by IHC. Intracellular expression levels of HTRA1 protein
were assessed and compared between the CTRL group and the (L) HPV11
E7 and (M) HPV16 E7 overexpression groups using immunofluorescence
analysis. *P<0.05, **P<0.01. CA,
condyloma acuminatum; HPV, human papillomavirus; HTRA1,
high-temperature requirement A serine peptidase 1; GO, Gene
Ontology; IHC, immunohistochemistry; iTRAQ, isobaric tags for
relative and absolute quantitation; NC, negative control; KD,
knockdown; NHEK, normal human epidermal keratinocyte; qPCR,
quantitative PCR; RNA-seq, RNA sequencing.

Figure 3

HPV E7 activates mitophagy in
keratinocytes through modulating the expression of HTRA1. (A)
Transmission electron microscopy was adopted to observe the
mitophagy status within HPV11/16 E7-overexpressing NHEKs with HTRA1
knockdown. The yellow arrows indicate mitochondrial autophagosomes.
The number of mitochondrial autophagosomes was statistically
analyzed and a total of 100 cells were analyzed per experiment.
Western blotting was employed to detect the expression of
mitophagy-related proteins and pathways in NHEKs with
overexpression of (B) HPV11 E7 and (D) HPV16 E7 after HTRA1
knockdown. Co-localization of GFP-LC3 and Mito-Tracker Red CMXRos
was employed to observe mitophagy within (C) HPV11
E7-overexpressing and (E) HPV16 E7-NHEKs with HTRA1 knockdown. The
yellow dots represent the co-localized areas. The ratio of yellow
dots to all dots was calculated and a total of 100 cells were
analyzed for each experiment. All proteins were normalized to the
loading control GAPDH. *P<0.05,
**P<0.01. BNIP3, Bcl-2 and adenovirus E1B 19
kDa-interacting protein 3; BNIP3L, BNIP3-like; CTRL, control; HPV,
human papillomavirus; HTRA1, high-temperature requirement A serine
peptidase 1; NHEK, normal human epidermal keratinocyte; PINK1,
PTEN-induced kinase 1; sh, short hairpin.

Figure 4

Knockout of HTRA1 can suppress
mitophagy. (A) Heatmap illustrating the differential gene
expression RNA-seq analysis of the skin of HTRA1(−/−) mice. For
each sample, 1 μg total RNA was utilized to assess RNA
integrity and construct libraries. A poly(A) mRNA selection system
was employed to eliminate ribosomal RNA contamination. The purified
RNA underwent reverse transcription, end-repairing, adapter
ligation and PCR amplification prior to sequencing on the Illumina
HiSeq 2500 platform. (B) KEGG pathway analysis of differentially
expressed genes from the RNA-seq results of HTRA1(−/−) mouse skin.
(C) Immunohistochemical assessment of PINK1, Parkin, IFN-β, IL-1β,
IL-6 and MCP-1 expression in the ear tissues of WT and HTRA1(−/−)
mice. (D) Mann-Whitney U test was employed to perform statistical
analysis of the immunohistochemistry results. (E) Quantitative PCR
and ELISA was employed to assess the levels of IFN-β in HPV11/16
E7-overexpressing NHEKs following HTRA1 gene knockdown. (F) Western
blotting was performed to evaluate the expression levels of
mitophagy-related markers and their associated signaling pathways
in NHEK cells with HTRA1 overexpression (Flag tag). All protein
expression levels were normalized to the loading control GAPDH. (G)
IP was utilized to investigate the interaction between HPV E7 and
HTRA1 in NHEKs. (H) Truncated HTRA1 plasmids were constructed and
transfected into 293 cells overexpressing HPV E7. Next, IP was
applied to identify the specific binding sites between HPV E7 and
HTRA1. *P<0.05, **P<0.01. BNIP3, Bcl-2
and adenovirus E1B 19 kDa-interacting protein 3; BNIP3L,
BNIP3-like; CTRL, control; HPV, human papillomavirus; HTRA1,
high-temperature requirement A serine peptidase 1; IFN, interferon;
IP, immunoprecipitation; KEGG, Kyoto Encyclopedia of Genes and
Genomes; NHEK, normal human epidermal keratinocyte; PINK1,
PTEN-induced kinase 1; RNA-seq, RNA sequencing; sh, short hairpin;
WT, wild-type.
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Spandidos Publications style
Zhang B, Kong D, Chen S, Sun X and Cheng H: Human papillomavirus E7 inhibits immune responses in keratinocytes by activating HTRA1‑mediated mitophagy. Int J Mol Med 57: 22, 2026.
APA
Zhang, B., Kong, D., Chen, S., Sun, X., & Cheng, H. (2026). Human papillomavirus E7 inhibits immune responses in keratinocytes by activating HTRA1‑mediated mitophagy. International Journal of Molecular Medicine, 57, 22. https://doi.org/10.3892/ijmm.2025.5693
MLA
Zhang, B., Kong, D., Chen, S., Sun, X., Cheng, H."Human papillomavirus E7 inhibits immune responses in keratinocytes by activating HTRA1‑mediated mitophagy". International Journal of Molecular Medicine 57.1 (2026): 22.
Chicago
Zhang, B., Kong, D., Chen, S., Sun, X., Cheng, H."Human papillomavirus E7 inhibits immune responses in keratinocytes by activating HTRA1‑mediated mitophagy". International Journal of Molecular Medicine 57, no. 1 (2026): 22. https://doi.org/10.3892/ijmm.2025.5693
Copy and paste a formatted citation
x
Spandidos Publications style
Zhang B, Kong D, Chen S, Sun X and Cheng H: Human papillomavirus E7 inhibits immune responses in keratinocytes by activating HTRA1‑mediated mitophagy. Int J Mol Med 57: 22, 2026.
APA
Zhang, B., Kong, D., Chen, S., Sun, X., & Cheng, H. (2026). Human papillomavirus E7 inhibits immune responses in keratinocytes by activating HTRA1‑mediated mitophagy. International Journal of Molecular Medicine, 57, 22. https://doi.org/10.3892/ijmm.2025.5693
MLA
Zhang, B., Kong, D., Chen, S., Sun, X., Cheng, H."Human papillomavirus E7 inhibits immune responses in keratinocytes by activating HTRA1‑mediated mitophagy". International Journal of Molecular Medicine 57.1 (2026): 22.
Chicago
Zhang, B., Kong, D., Chen, S., Sun, X., Cheng, H."Human papillomavirus E7 inhibits immune responses in keratinocytes by activating HTRA1‑mediated mitophagy". International Journal of Molecular Medicine 57, no. 1 (2026): 22. https://doi.org/10.3892/ijmm.2025.5693
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