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. 2020 Oct 14;12(10):1162.
doi: 10.3390/v12101162.

Merkel Cell Polyomavirus Large T Antigen is Dispensable in G2 and M-Phase to Promote Proliferation of Merkel Cell Carcinoma Cells

Roland Houben  1 , Marlies Ebert  1 , Sonja Hesbacher  1 , Thibault Kervarrec  2 , David Schrama  1
Affiliations

Merkel Cell Polyomavirus Large T Antigen is Dispensable in G2 and M-Phase to Promote Proliferation of Merkel Cell Carcinoma Cells

Roland Houben et al. Viruses. .

Abstract

Merkel cell carcinoma (MCC) is an aggressive skin cancer frequently caused by the Merkel cell polyomavirus (MCPyV), and proliferation of MCPyV-positive MCC tumor cells depends on the expression of a virus-encoded truncated Large T antigen (LT) oncoprotein. Here, we asked in which phases of the cell cycle LT activity is required for MCC cell proliferation. Hence, we generated fusion-proteins of MCPyV-LT and parts of geminin (GMMN) or chromatin licensing and DNA replication factor1 (CDT1). This allowed us to ectopically express an LT, which is degraded either in the G1 or G2 phase of the cell cycle, respectively, in MCC cells with inducible T antigen knockdown. We demonstrate that LT expressed only in G1 is capable of rescuing LT knockdown-induced growth suppression while LT expressed in S and G2/M phases fails to support proliferation of MCC cells. These results suggest that the crucial function of LT, which has been demonstrated to be inactivation of the cellular Retinoblastoma protein 1 (RB1) is only required to initiate S phase entry.

Keywords: Merkel cell carcinoma; Merkel cell polyomavirus; cell cycle; large T antigen.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Merkel cell polyomavirus (MCPyV)- Large T antigen (LT) fusion proteins expressed in different phases of the cell cycle. (a) Schematic illustration of different Large T proteins analyzed in this study. To make SV40 Large T antigen (LT) detectable by flow cytometry and fluorescence microscopy, the red fluorescent protein mRuby3 was fused to the C-terminus of a truncated MCPyV-LT. To achieve cell cycle-dependent expression, either amino acids 30–120 of CDT1 or amino acids 1–110 of GMNN (also known as Geminin) were added at the C-terminus. (b) HeLa cells were stably transduced with retroviral vector pIH encoding the indicated LT variants depicted in (a). Time lapse fluorescence microscopy was performed, and the presence of mRuby fluorescence in the course of time is depicted.
Figure 2
Figure 2
LT-mRuby-CDT1 is largely lacking in S and G2/M phases of the cell cycle in MKL-1 cells. MKL-1 TA.shRNA.tet cells were transduced with retroviral pIH vectors coding for the different indicated LT fusion proteins. Using Vybrant™ DyeCycle Violet, the DNA of viable cells was stained, and combined analyses of the DNA stain and mRuby fluorescence was performed by flow cytometry. (a) mRuby fluorescence is depicted in the upper, the cell cycle distribution of all cells in the middle, and of only mRuby-positive cells in the lower row. (b) The percentages of cells in S and G2/M phases of the total population and of only mRuby-positive cells are depicted as bar graphs (mean values (±SD) of at least three measurements).
Figure 3
Figure 3
MCPyV-LT present only in G1 is capable of rescuing the growth arrest induced by knockdown of endogenous LT. MKL-1 cells transduced with a vector (TA.shRNA.tet) allowing Dox-inducible expression of a T antigen-targeting shRNA as well as constitutive expression of GFP were additionally transduced with retroviral vectors coding for the indicated LT fusion proteins. Notably, these ectopically-expressed LT variants had been rendered shRNA-insensitive by six silent nucleotide exchanges in the shRNA target sequence. (a) Following five days in the absence or presence of Dox (1 µg/mL) expression of endogenous and ectopic LT were analyzed by immunoblot. (b) The ratios of a mixed population of green-fluorescent double infected cells with uninfected, non-fluorescent parental cells were determined over time. Relative ratios based in each case on the measurement of the first time point were calculated and mean values (±SD) of three independent experiments are depicted. Statistical significance was evaluated by calculating the area under the curve (AUC) for each setting of the Dox-treated cells and then comparing the AUC of each group by one-way-ANOVA. Since the group means were significantly different (p = 0.2204), the Tukey post hoc test was performed to identify significant between-group differences. To this end, the comparison of LT-mRuby/vector, LT-mRuby/LT-mRuby-GMNN, LT-mRuby-CDT1/vector and LT-mRuby-CDT1/LT-mRuby-GMNN were statistically significant (* = adjusted p < 0.05; ** = adjusted p < 0.01).
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