Cutaneous papillomavirus E6 oncoproteins associate with MAML1 to repress transactivation and NOTCH signaling

Abstract

Papillomavirus E6 oncoproteins associate with LXXLL motifs on target cellular proteins to alter their function. Using a proteomic approach, we found the E6 oncoproteins of cutaneous papillomaviruses Bovine Papillomavirus Type 1 (BPV-1) E6 and human papillomavirus (HPV) types 1 and 8 (1E6 and 8E6) associated with the MAML1 transcriptional co-activator. All three E6 proteins bind to an acidic LXXLL motif at the carboxy-terminus of MAML1 and repress transactivation by MAML1. MAML1 is best known as the co-activator and effector of NOTCH-induced transcription, and BPV-1 E6 represses synthetic NOTCH-responsive promoters, endogenous NOTCH-responsive promoters, and is found in a complex with MAML1 in stably transformed cells. BPV-1-induced papillomas show characteristics of repressed NOTCH signal transduction, including suprabasal expression of integrins, talin and basal type keratins, and delayed expression of the NOTCH-dependent HES1 transcription factor. These observations give rise to a model whereby papillomavirus oncoproteins, including BPV-1 E6, and the cancer-associated HPV-8 E6 repress NOTCH-induced transcription, thereby delaying keratinocyte differentiation.

Fig. 1. Cutaneous E6 proteins associate with MAML1

Plasmids for FLAG-tagged MAML1 and MAML1Δ were transfected together with HA-tagged E6 and beta-galactosidase into HEK-293T cells as indicated in the figure and harvested by lysis in NP40 lysis buffer 24 hours later. Clarified lysates were immune precipitated with mouse anti-FLAG coupled to agarose beads and western blots probed with rabbit anti-HA and anti-FLAG antibody.

Fig. 2. BE6 and MAML1 are in a complex in stably transduced cells

Murine C127 cells were sequentially retrovirally transduced and selected for FLAG-MAML1, and FLAG-MAML1Δ, and then drug selected cells transduced with BE6 retrovirus with puromycin selection; resulting cell lines (1.5 × 10⁷ cells per sample) were lysed with NP40 lysis buffer and immune precipitated with anti-flag antibody beads, blots probed with rabbit anti-BE6 and then re-probed with rabbit anti-FLAG. In the exposure shown, FLAG-MAML1 in the lysate samples is too faint to be seen in comparison to the signal in the immune precipitated sample.

Fig. 3. E6 proteins repress transactivation by MAML1

Plasmids for GAL4 fusions to full length MAML1 (black bars) or LXXLL deleted MAML1Δ (grey bars) were co- transfected with plasmids for the indicated amounts of native E6 proteins, a luciferase reporter and internal control lacZ expression plasmid into HEK-293T cells that were harvested 24 hours later and assayed for lacZ activity and luciferase activity. Results shown on the vertical axis are relative luciferase activity and are the average and standard deviation of five separate experiments, normalized first to lacZ activity and then to GAL4-MAML1Δ activity in the absence of E6 (expressed as 100). Error bars are standard deviations. Asterisk denotes P<0.01 by student t-test.

Fig. 4. Repression of Notch dependent transcriptional activation by BE6

CV-1 cells were transfected and analyzed for luciferase and beta-galactosidase activity 48 hrs later. A. Cells were co-transfected with 600 ng of a luciferase reporter with 4 copies of CBF1 (also termed RBP-J) binding sites upstream of a minimal SV40 early promoter (CBFRE1-Luc, abbreviated as CBF-Luc in the figure (72)) and as indicated, 300 ng Notch intracellular domain (NID) and 120 ng MAML1 or 120 ng MAML1Δ. White bars had no BE6, and increasingly darker grey bars were transfected with 25, 200, or 600 ng of BE6 expression plasmid, balanced with empty vector. pCMV-lacZ was co-transfected as an internal transfection control. Results shown are relative luciferase activity, and are the average and standard deviation of five separate transfection experiments, with results normalized to lacZ expression and CBFRE1-Luc(mt) to normalize for variation in transfection efficiency. B. CV-1 cells were transfected with the HES1-luciferase reporter and expression plasmids as indicated in the figure and as described in part A. C. Cutaneous E6 proteins repress endogenous HES1 RNA expression. Human diploid lung fibroblasts were retrovirally transduced with the indicated vector or E6 genes and total RNA from drug-selected cells analyzed by qRT-PCR as detailed in the methods. Bars show the average of duplicate experiments with the lines showing the range of values obtained, normalized to vector control cells.

Fig. 5. BE6 but not 16E6 repressed Notch dependent transcription

Monkey CV-1 cells were transfected with 600 ng of either CBFRE1-Luc, (black bars) or by CBFRE(mt)-Luc, (grey bars), 300 ng of an expression plasmid for Notch1 intracellular domain, 120 ng of a MAML1 expression plasmid, and from 10 ng to 1.2 ug of the indicated E6 protein expression plasmid. Results shown on the vertical axis are relative luciferase activity, and are the average and standard deviation of seven separate experiments, normalized to the CBF1-Luc(mt) reporter in the absence of E6, set to 1.0. Asterisk denotes P<0.01 by student t-test.

Fig. 6. BE6 represses basal Notch signaling from chromosomal reporters

Human keratinocytes were transduced at an MOI of 5 with a self-inactivating lentiviral Notch-responsive lentiviral reporter, and then passaged for 2 weeks. Feeder cells were then detached, and the keratinocytes transduced overnight with either empty lentiviral vector, lentiviral vector expressing 16E6, or lentiviral vector expressing BE6 at an MOI of 3, then feeder cells were added the next morning and the cells harvested for luciferase assay 3 days later. Results shown on the vertical axis are the average and standard deviation of four experiments.

Fig. 7. Bovine fibropapillomas express basal cell markers suprabasally

Frozen sections from BPV-1 induced fibropapillomas and adjacent normal skin were stained with monoclonal antibodies to talin or β1-integrin as indicated and nuclei counter-stained with DAPI. Normal and papilloma images were captured with the same exposure time. A dashed white line shows the dermal-epidermal boundary.

Fig. 8. Expression of MAML1 and HES1 in bovine skin and bovine fibropapillomas

Frozen sections of a BPV-1 induced fibropapilloma that also included normal skin at the margin of the tumor were stained as indicated with rabbit anti-MAML1 or HES1 (green) and the DNA counter-stain DAPI (blue). A dashed white line shows the dermal-epidermal boundary. Normal and papilloma images were captured with the same exposure time. A. Expression of MAML1 and HES1 in normal skin. B. Expression of MAML1 and HES1 in the fibropapilloma.