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BetaHPV E6 and E7 colocalize with NuMa in dividing keratinocytes

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Abstract

Human papillomaviruses (HPVs) of genus betapapillomavirus (betaHPV) are implicated in skin carcinogenesis, but their exact role in keratinocyte transformation is poorly understood. We show an interaction of HPV5 and HPV8 oncoproteins E6 and E7 with the nuclear mitotic apparatus protein 1 (NuMA). Binding of E6 or E7 to NuMA induces little aneuploidy, cell cycle alterations, or aberrant centrosomes. Intracellular localization of NuMA is not altered by E6 and E7 expression in 2D cultures. However, the localization profile is predominantly cytoplasmic in 3D organotypic skin models. Both viral proteins colocalize with NuMA in interphase cells, while only E7 colocalizes with NuMA in mitotic cells. Intriguingly, a small subset of cells shows E7 at only one spindle pole, whereas NuMA is present at both poles. This dissimilar distribution of E7 at the spindle poles may alter cell differentiation, which may in turn be relevant for betaHPV-induced skin carcinogenesis.

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Acknowledgements

This work was supported by the Canceropole Grand-Est/DKFZ Grant to AA and FA, and by the Finnish Society of Sciences and Letters Grant (EA). MK was supported by the Wilhelm-Sander Stiftung für Krebsforschung (Grant No. 2012.105.3). MH was supported by the Deutsche Krebshilfe (Grant No. 70112727). We thank Dr. Ramon Garcia-Escudero (CIEMAT, Madrid, Spain) for the HPV5 E7 mutant constructs and Professor Slawomir Majewski (Medical University in Warsaw, Poland) for providing EV skin sections.

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  1. Evelyn Oswald and Matthias Kirschberg contributed equally.

Authors and Affiliations

  1. German Cancer Research Center, Heidelberg, Germany

    Evelyn Oswald & Angel Alonso

  2. Institute of Virology, University of Cologne, Faculty of Medicine and University Hospital of Cologne, Fürst-Pückler-Str.56, 50935, Cologne, Germany

    Matthias Kirschberg, Martin Hufbauer & Baki Akgül

  3. Department of Dermatology, Université de Franche-Comté, Besançon, France

    François Aubin

  4. University of Helsinki and Helsinki University Hospital Laboratory, Helsinki, Finland

    Eeva Auvinen

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  1. Evelyn Oswald
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Contributions

EO, MK, FA, and MH performed the experiments. AA and EA conceived and designed the study. AA, BA, and EA analyzed the data and wrote the manuscript. All authors have read and approved the final manuscript.

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Correspondence to Baki Akgül.

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Ethical approval for the use of skin biopsies of epidermodysplasia verruciformis patients for scientific purposes was obtained from the Ethics Committee of the Medical University of Warsaw.

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11262_2019_1685_MOESM1_ESM.pptx

Supplementary Fig. 1. Colocalization of HPV5 E6 or wild-type E7, as well as six HPV5 E7 mutants with NuMA in interphase nuclei. COS-7 cells were transfected with expression constructs for E6, or wild-type of mutant E7 tagged with the AU1 epitope. Fixed cells were stained with anti-AU1 (green) and anti-NuMA (red) antibodies. The images show z-axis projections of 12–20 layers. PDM (pixel density mean) values [=(intensity or red color minus mean intensity of red color) × (intensity or green color minus mean intensity of green color)] of colocalizing pixels at each layer were calculated, and they are shown as z-axis projections in pseudocolor. PDM was calculated using the Intensity Correlation Analysis Plugins of ImageJ. Supplementary material 1 (PPTX 1446 kb)

11262_2019_1685_MOESM2_ESM.pptx

Supplementary Fig. 2. HPV5 or HPV8 E6E7 do not cause enhanced aneuploidy. Nonsynchronized PHK cells were transduced with the pLXSN vector, or constructs expressing E6 and E7 of either HPV5, HPV8 or HPV16, and (A) treated with DMSO only (−Noc) or (B) treated with 100 ng/ml nocodazol for 24 hours (+Noc). The cells were harvested, fixed, stained with propidium iodide, and examined by FACS analysis. Results of one representative experiment is shown. (C) Quantification of DNA contents in cells with or without nocodazole treatment. Results from n = 2 in duplicates are shown. The numbers express percentage of cells of the total cell population with DNA content exceeding 4N (%).Supplementary material 2 (PPTX 90 kb)

11262_2019_1685_MOESM3_ESM.pptx

Supplementary Fig. 3. Cell cycle profile of cells expressing HPV5, HPV8 or HPV16 E6E7. Nonsynchronized PHK cells were transduced with pLXSN vector, or constructs expressing E6 and E7 of either HPV5, HPV8 or HPV16, incubated with 100 µM BrdU for 30 minutes and fixed. DNA was visualized with propidiumiodide and BrdU was stained with a specific antibody, and the cells were analyzed using FACS. (A) Histograms of one representative experiment are shown. (B) Quantification of cell cycle profiles using BrdU and PI staining (n = 2 in duplicates), or using PI stained samples (without BrdU, n = 3 in duplicates). The cell cycle profiles of the latter were analyzed using the Dean-Jett-Fox Model of the Cell Cycle Platform in FlowJo. Supplementary material 3 (PPTX 111 kb)

Supplementary material 4 (DOCX 13 kb)

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Oswald, E., Kirschberg, M., Aubin, F. et al. BetaHPV E6 and E7 colocalize with NuMa in dividing keratinocytes. Virus Genes 55, 600–609 (2019). https://doi.org/10.1007/s11262-019-01685-9

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