Cutaneous β-human papillomavirus E6 proteins bind Mastermind-like coactivators and repress Notch signaling - PubMed (original) (raw)

Cutaneous β-human papillomavirus E6 proteins bind Mastermind-like coactivators and repress Notch signaling

Min Jie Alvin Tan et al. Proc Natl Acad Sci U S A. 2012.

Abstract

The Notch signaling pathway is a key determinant in keratinocyte differentiation and growth cycle arrest, and has been reported to have a tumor suppressor function in skin. The papillomavirus life cycle is intricately linked to the differentiation status of keratinocytes. Papillomaviruses are associated with benign proliferative epithelial lesions in their respective hosts. Although human papillomaviruses (HPVs) associated with genital tract lesions have been extensively studied, studies of the cutaneous HPVs are more limited. In particular, it is well established that the E6 proteins of high-risk HPVs of the α-genus such as HPV16 and HPV18 mediate the degradation of p53 by its association with the ubiquitin ligase E6AP. In contrast, less is known about the cellular activities of the cutaneous HPVs of the β-genus. By using an unbiased proteomic approach, we identify MAML1 and other members of the Notch transcription complex as high-confidence cellular interacting proteins of E6 proteins of the β-genus HPVs and of the bovine papillomavirus type 1 associated with cutaneous fibropapillomas. We show that bovine papillomavirus type 1 and β-HPV E6 repress Notch transcriptional activation, and that this repression is dependent on an interaction with MAML1. Finally, we show that the expression levels of endogenous Notch target genes are repressed by β-HPV E6 proteins. These findings elucidate a mechanism of viral antagonism of Notch signaling, and suggest that Notch signaling is an important epithelial cell pathway target for the β-HPVs.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

MAML1 is an HCIP of BPV-1 E6 and the β-HPV E6 proteins. (A) Plot of NWD score against z-score of candidate interacting proteins of BPV-1 E6. The z-score reflects the abundance of the protein whereas the NWD score is calculated by CompPASS software and takes into account its uniqueness and reproducibility. (B) Interaction map of BPV-1 E6 with components of the Notch transcription complex identified by CompPASS. The thickness of the solid lines is proportional to the indicated NWD score of that interactor determined through the CompPASS software. Dashed lines denote known interactions from the STRING database. The NWD score for each interactor is as labeled. In this analysis, an HCIP has an NWD score of at least 1. (C) NWD scores of MAML1, other components of the Notch transcription complex, and E6AP identified by CompPASS for different HPV E6 proteins. The genus and species of each E6 protein is indicated.

Fig. 2.

Fig. 2.

MAML1 and MAML3 interact with BPV-1 and β-HPV E6 proteins. (A) MAML1 interacts with BPV-1 and the β-HPV17a E6. Lysates from 293T cells and 293T cells stably expressing BPV-1 E6 (BE6) and BPV-1 E7 (BE7; Left) were subjected to HA IP and blotted for HA, MAML1, ICN1, RBPJ, and actin. Right: Lysates from N/Tert-1 cells and N/Tert-1 cells stably expressing HPV17a E6 and E7 were similarly subjected to HA IP and blotted for HA, MAML1, and actin. In each experiment, 5% of cell lysate used for IP is loaded as input and 50% of the IP samples is loaded as IP. (B) MAML1 interacts with the β-HPV (HPV17a and HPV38) E6 proteins but not with the α-HPV16 E6 protein. Lysates from N/Tert-1 cells stably expressing the indicated HPV E6 proteins were subjected to HA IP and blotted as in A. (C) BPV-1 E6 does not interact with ICN1 and RBPJ in the absence of MAML1. Lysates from stable 293T-BE6-HA cells transfected with control or MAML1-specific siRNAs were subjected to HA IP and blotted for HA, MAML1, ICN1, RBPJ, and actin. (D) BPV-1 E6 interacts with MAML1 and MAML3, but not MAML2. Lysates from stable 293T-BE6-HA cells transfected with FLAG-tagged MAML1, MAML2, and MAML3 were subjected to HA IP and blotted for HA, FLAG, and actin.

Fig. 3.

Fig. 3.

The C-terminal LDDLL motif of MAML1 is necessary for its interaction with E6. (A) Schematic of the N-terminally tagged FLAG- or V5-tagged MAML1 constructs, together with an alignment of the C-termini of MAML1, MAML2, and MAML3. (B) A C-terminal fragment (842–1016) of MAML1 is sufficient for its interaction with BPV-1 E6. 293T-BE6-HA cell lysates transiently expressing the indicated FLAG-tagged fragments of MAML1 were subjected to HA IP and blotted for HA, FLAG, and actin. (C) C-terminal LDDLL motif is necessary for interaction of MAML1 with BPV-1 and the β-HPV8 E6. Lysates from stable 293T-BE6-HA or C33A-8E6-HA cells expressing the indicated V5-tagged MAML1 plasmids were subjected to HA IP and blotted for HA, V5, and actin.

Fig. 4.

Fig. 4.

β-HPV E6 repression of Notch transcriptional activation requires their interaction with MAML1. (A and B) BPV-1 E6 and β-HPV (HPV8 and HPV17a17a) E6 repress ICN1-mediated transcriptional activation in a dose-dependent manner. C33A (A) or U2OS (B) cells were cotransfected with the Notch-responsive firefly luciferase plasmid (300 ng), a control Renilla luciferase plasmid (6 ng), a plasmid encoding activated Notch1 ICN1 (50 ng), and plasmids encoding the following proteins: BPV-1 or β-HPV E6 (300 ng or 500 ng); Zer1 or HPV16 E6 (500 ng); or DN MAML1, a specific inhibitor of the Notch transcription complex (50 ng). The luciferase activity is reported as relative luciferase units (RLUs), as calculated by normalizing the firefly luciferase reading with its corresponding internal Renilla luciferase control. The luciferase activity is expressed as fold activation relative to cells transfected only with empty control plasmid. (C) BPV-1 and β-HPV (i.e., HPVs 8, 17a, and 38) E6 proteins repress Jagged2-mediated transcriptional activation of a Notch reporter gene. U2OS cells were cotransfected with a Notch-responsive firefly luciferase reporter plasmid and a control Renilla luciferase plasmid, and 300 ng of the indicated plasmids (except for 50 ng in the case of DN MAML1). Transfected U2OS cells were then plated onto control 3T3 or Notch-activating 3T3-Jagged2 cells 24 h after transfection. A GSI, compound E, was used at a 1-μM concentration as an additional Notch pathway inhibitor control in the indicated cocultures. Dual-luciferase assays were then performed on the lysates of cocultured cells 48 h after transfection. The relative luciferase activity was calculated as in A. (D) β-HPV (HPV8 and HPV17a) E6 repression of MAML1-mediated Notch transcriptional activation is dependent on interaction with MAML1. U2OS cells were cotransfected with luciferase reporter plasmids as in C, and 16 ng of full-length MAML1 (FL), 50 ng of the non–E6-binding mutant MAML1(ADDAA), or 300 ng of HPV8 or HPV17a E6 were cotransfected into the U2OS cells as indicated. Dual-luciferase assays were then performed on the lysates of cocultured cells 48 h after transfection. The luciferase activity was calculated as in A.

Fig. 5.

Fig. 5.

β-HPV E6 represses expression of endogenous Notch-responsive genes. (A) Endogenous basal Notch-responsive gene expression is repressed by β-HPV E6s. qRT-PCR analysis of Notch-responsive gene expression in the indicated stable N/Tert-1 cell lines. Reverse transcription was performed, followed by qRT-PCR. Transcript levels were normalized to the housekeeping gene Rad21 and then presented relative to levels in the control N/Tert-1 sample. (B) Notch-responsive gene expression is repressed by HPV17a E6 even in the presence of ligand stimulation of Notch signaling. The N/Tert-1 cells were cocultured with control 3T3 cells or 3T3-Jagged2 cells for 48 h before harvesting for RNA extraction. qRT-PCR analysis of Notch-responsive gene expression was performed and analyzed as in A.

Fig. P1.

Fig. P1.

Inhibition of Notch transcriptional activation by β-HPV E6 proteins. Upon activation of Notch signaling, the intracellular domain of Notch (ICN) migrates to the nucleus and forms a core transcription complex with the transcription factor RBPJ and the transcriptional coactivator MAML1, leading to the expression of Notch target genes. Other coactivators may also be recruited to this ternary complex during Notch activation. During β-HPV infection of squamous epithelial cells, β-HPV E6 proteins interact with the LDDLL motif of MAML1 and repress the expression of Notch target genes.

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