Stranglehold on the spindle assembly checkpoint: the human papillomavirus E2 protein provokes BUBR1-dependent aneuploidy

Abstract

The Human Papillomavirus (HPV) E2 protein, which inhibits the E6 and E7 viral oncogenes, is believed to have anti-oncogenic properties. Here, we challenge this view and show that HPV-18 E2 over-activates the Spindle Assembly Checkpoint (SAC) and induces DNA breaks in mitosis followed by aneuploidy. This phenotype is associated with interaction of E2 with the Mitotic Checkpoint Complex (MCC) proteins Cdc20, MAD2 and BUBR1. While BUBR1 silencing rescues the mitotic phenotype induced by E2, p53 silencing or presence of E6/E7 (inactivating p53 and increasing BUBR1 levels respectively) both amplify it. This work pinpoints E2 as a key protein in the initiation of HPV-induced cervical cancer and identifies the SAC as a target for oncogenic pathogens. Moreover, our results suggest a role of p53 in regulating the mitotic process itself and highlight SAC over-activation in a p53-negative context as a highly pathogenic event.

Keywords: APC/C, Anaphase Promoting Complex/Cyclosome; Ad, Adenovirus; BUBR1; E2; E2 TAD, E2 Transactivation Domain; E2 ΔTAD, E2 deleted of the Transactivation Domain; GFP, Green Fluorescent Protein; HPV, Human Papillomavirus; MCC, Mitotic Checkpoint Complex; MS, Mass Spectrometry; Noco, Nocodazole; SAC, Spindle Assembly Checkpoint; Thym, Thymidine; aneuploidy; m.o.i., Multiplicity of Infection; mitosis; p53; papillomavirus; spindle assembly checkpoint.

Figure 1.

E2 arrests the cell cycle in mitosis and induces DNA breaks and aneuploidy in the C33-A keratinocyte cell line. (A) Asynchronous (Asynch) C33-A cells were infected by AdGFP-E2, AdGFP-ΔTAD or AdGFP. The structure of the different proteins are shown (TAD=Transactivation Domain, DBD=DNA Binding Domain). After 36 hours, DNA was stained with propidium iodide for cell cycle analysis by flow cytometry. (B) C33-A cells were infected and synchronized at the G1/S transition using thymidine before being released for 14 hours (Thym R14). Mitotic cells labeled by anti-phosphorylated histone H3 (H3P) are shown in red. DNA was stained with DAPI. (C) Comet assays performed from cells synchronized by thymidine at the G1/S transition and released for 14 hours. Treatment with H2O2 was used as a positive control.

Figure 2.

E2 interacts with regulators of mitosis completion including the mitotic checkpoint complex, the spindle itself and the kinetochores. (A) Table listing the interactants of E2 associated with regulation of mitosis completion. See also Table S1. (B) Immunofluorescence showing co-localization of GFP-E2 with SAC proteins (BUBR1 and MAD2), kinetochores (CENP-E) and mitotic spindle (β-tubulin). (C) Immunoprecipitation confirming interactions of GFP-E2 with Cdc20, BUBR1 and MAD2 (I = Input, B = Beads).

Figure 3.

Both E2 and TAD prevent SAC inactivation but TAD is more efficient than E2 in HeLa cells. (A) Cell cycle analyses of HeLa cells expressing GFP-E2, GFP-TAD, GFP-ΔTAD or GFP released from nocodazole block. Cells were synchronized in pro-metaphase by successive thymidine/nocodazole treatments. Thym 0 = end of thymidine treatment, Thym R6 = release for 6 hours after thymidine treatment. Six hours after release from thymidine, nocodazole was added to the medium for the next 14 hours. Infection was performed during the nocodazole treatment. After mitotic shake-off (Noco 0), cells were released for 5 hours in nocodazole-free medium (Noco R5). (B) Cell cycle analyses of HeLa cells expressing GFP-E2, GFP-TAD, GFP-ΔTAD or GFP, synchronized in the middle of S phase (Thym R2), and released for 4 (Thym R6) or 12 (Thym R14) hours. Infection was performed during the thymidine treatment.

Figure 4.

E6/E7 expression and p53 inactivation potentiate E2 effects on mitosis (A) RNAs were extracted from cells used in Figure 3B, reverse transcribed and the resulting cDNAs were subjected to Real-Time PCR to detect the endogenous level of E6/E7 mRNA. (B) Western-blots were performed with extracts from cells expressing the indicated proteins. Asynch = Asynchronous cells, Thym R6 = cells released for 4 hours following thymidine treatment. The asterisk corresponds to the actin for which the membrane was probed first. (C) Western-blot showing the relative quantities of the indicated proteins at the different time-points and in the different siRNA conditions. (D) Cell cycle analyses of HeLa cells expressing GFP-E2 or GFP, transfected with either a GADPH siRNA (-) or a siRNA against p53 (+), synchronized in S phase (Thym R2), and released for 4 (Thym R6) or 12 (Thym R14) hours. Infection and tranfection were performed during the thymidine block.

Figure 5.

Silencing of BUBR1 rescues abnormal mitoses as well as mitotic cell death induced by E2. (A) Phase contrast images from time-lapse experiments. HeLa cells expressing GFP-TAD or GFP were transfected with a control siRNA (siCtrl) or a siRNA against BUBR1 (siBUBR1), synchronized by thymidine and released as indicated. (B) Time-lapse experiments performed with cells treated as in (A), but stained using Hoechst 33342. Upper panels show phase contrast pictures, lower panels show the corresponding DNA staining. Black arrows: mitotic cell death, white arrows: abnormal mitoses.

Figure 6.

Statistical analyses of time-lapse experiments. (A) Kinetics of mitotic entry and exit of GFP-TAD- and GFP-expressing HeLa cells treated with the BUBR1 siRNA or Ctrl siRNA. The figure shows both the proportion of mitoses (alive + dead, solid line graphs) and the proportion of dead mitoses only (dashed line graphs) at each indicated time-point as a percentage of the total number of mitoses observed during the 24 hours time-lapse (~300 mitotic cells). (B) Characteristics of the mitoses from (A). Complete mitoses = cells which exit mitosis either normally or abnormally. Normal = 2 daughter cells, Abnormal = more than 2 daughter cells or mitotic slippage. Metaphasic cell death = cells dying in metaphase. Duration of mitosis = time from rounding up of the cells to separation into daughter cells. The proportions shown are percentages of the total number of mitotic cells (~300), except for normal and abnormal values which are percentages of complete mitoses. See also Movies S1 to S6.