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The ubiquitin specific protease 7 stabilizes HPV16E7 to promote HPV-mediated carcinogenesis

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Abstract

Human papillomavirus (HPV) encoded E7 oncoprotein plays an important role in supporting the viral productive cycle and inducing cancer phenotypes. The ability of E7 to exercise these functions, partly, depends upon its steady-state level. HPV manipulates the host de-ubiquitination pathway to maintain the stability of its viral proteins. In this study, we uncovered that HPV interacts with the host ubiquitin specific protease 7 (USP7), a universal de-ubiquitinating enzyme, leading to the stabilization of E7 oncoprotein. We observed that HPV16E7 complexes with USP7 via the E7-CR3 domain, and this E7-USP7 complex exists predominantly in the nucleus. Our results showed that USP7 stabilizes and prolongs the half-life of HPV16E7 by antagonizing ubiquitination and proteasomal degradation. Consistently, when we inhibited USP7 activity using HBX 19818, HPV16E7 protein level was reduced and its turnover was increased. We also provide evidence that HBX 19818-induced USP7 inhibition can halt HPV-mediated carcinogenesis, including cell proliferation, invasion, migration and transformation. These findings indicate that USP7 plays an essential role in stabilizing E7. The specific and potent inhibitory effects of HBX 19818 on HPV-induced carcinogenesis provide a molecular insight, suggesting the potential of targeting USP7 as a new therapeutic approach for the treatment of HPV-associated cancers.

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Acknowledgements

We gratefully acknowledge Prof. Lawrence Banks and Dr. Miranda Thomas, International Centre for Genetic Engineering and Biotechnology, for generously providing us with the GST-E7 constructs, as well as editing this manuscript and giving us constructive comments. We also express sincere appreciation to Professor HUEN, Michael Shing Yan, University of Hong Kong, for the kind gift of the USP7 expression construct.

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Authors and Affiliations

  1. Department of Microbiology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR

    Chichao Xia, Chuanyun Xiao, Ho Yin Luk, Paul K. S. Chan & Siaw Shi Boon

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  1. Chichao Xia
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  2. Chuanyun Xiao
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  3. Ho Yin Luk
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  4. Paul K. S. Chan
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  5. Siaw Shi Boon
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Contributions

SSB and PKSC conceived and supervised the study. SSB and PKSC were involved in the conceptualization of the study. CCX performed experiments, collected data and analyzed results with help from CYX, HYL and SSB. CCX and SSB wrote the manuscript. All authors reviewed and edited the manuscript.

Corresponding author

Correspondence to Siaw Shi Boon.

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Conflict of interest

Part of the findings of this study had been submitted for the U.S. Patent application (no. 18/333872).

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18_2023_4941_MOESM1_ESM.tif

Figure S1. The association between E7-encoded by HPV16 and USP7 in H1299 cells. A. Schematic diagram shows the alignment of amino acid sequences between HPV oncoproteins E6 and E7, plus EBNA1 from Epstein-Barr virus (EBV) and ICP0 from herpes simplex virus-1 (HSV-1), using Clustal Omega online tool (http://www.ebi.ac.uk/Tools/msa/clustalo/). The amino acid sequence 436 to 450 of EBNA-1 that is important for association with USP7, particularly the "PXE" motif (underlined), was found to align within HPV16-E6 at amino acids 12 to 14 (as highlighted by *), but not with HPV18-E6. Interestingly, the C-terminal ends of HPV-6 and HPV16-E7 (amino acids 94 to 97) appear to share close homology with amino acids 621 to 624 within ICP0 (as highlighted by *). B. Human non-small lung carcinoma cell lines, H1299 cells were either mock-transfected or transiently transfected with plasmids expressing Flag-tagged USP7 (FL-USP7) alone (+) or co-transfected with HA-tagged HPV16-E6 (HA-16 E6) (+) or -E7 (HA-16 E7) (+), as indicated. After 24 h, cell lysates were collected. HPV16-E6 and -E7 were immunoprecipitated using HA-conjugated agarose beads for 2 h at 4 °C. The samples were then subjected to SDS-PAGE and Western blotting to detect FL-USP7. The levels of HPV16-E6 and -E7 obtained by immunoprecipitation were ascertained using HA antibody and beta (β)-actin was included as a loading control. “-” indicates the relevant plasmid is not transfected. C. The levels of bound FL-USP7 or endogenous USP7 relative to their respective amount of input and precipitated E6 or E7 were quantitated from at least triplicates. Note that FL-USP7 (mean ± SD, p < 0.001) confers strong interaction with HA-16 E7 (TIF 17011 KB)

18_2023_4941_MOESM2_ESM.tif

Effect of HBX 19818 treatment on oncogenic signaling pathway. CaSki cells were treated with DMSO (a vehicle control) or HBX 19818 (3 μM or 6 μM) for 24 h. The total cell lysate was collected and analyzed by western blotting. The representative immunoblots show the levels of USP7, both phospho- and total AKT and ERK, Ras and HPV16E7 in CaSki cells. β-actin was included as a loading control (TIF 6524 KB)

18_2023_4941_MOESM3_ESM.tif

HBX 19818 destabilizes MDM2 and restores the p53 protein level. A. The representative immunoblotting images show the protein level of apoptotic pathways. CaSki cells were treated with DMSO or HBX 19818 (3 or 6 μM, as indicated) overnight. The protein level of p21, p53, MDM2, HPV16E7, USP7, caspase-3, -7 and -9 were examined via western blotting. B. The bar chart compares the relative protein level of MDM2 and p53 normalized by β-actin (n=3). Data are indicated as mean ± SEM. *p < 0.05; **p < 0.01 (TIF 11962 KB)

18_2023_4941_MOESM4_ESM.tif

USP7 co-localizes with HPV16E7 in the cell nucleus. Immunofluorescent images show the subcellular localization pattern of HPV16E7 and USP7. CaSki cells were incubated with anti-USP7 and anti-16E7 antibodies. Cellular localization of USP7 and E7 were visualized by fluorescent microscope under 1000x magnification (TIF 942 KB)

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Xia, C., Xiao, C., Luk, H.Y. et al. The ubiquitin specific protease 7 stabilizes HPV16E7 to promote HPV-mediated carcinogenesis. Cell. Mol. Life Sci. 80, 278 (2023). https://doi.org/10.1007/s00018-023-04941-2

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