Stabilization of HPV16 E6 protein by PDZ proteins, and potential implications for genome maintenance

Cancer-associated human papillomaviruses (HPVs) express E6 oncoproteins that target the degradation of p53 and have a carboxy-terminal PDZ ligand that is required for stable episomal maintenance of the HPV genome. We find that the E6 PDZ ligand can be deleted and the HPV genome stably maintained if cellular p53 is inactivated. This indicates that the E6-PDZ interaction promotes HPV genome maintenance at least in part by neutralization of an activity that can arise from residual undegraded p53. P apillomaviruses induce benign squamous epithelial neoplasms (papillomas) in vertebrates, and the viral DNA genome is maintained as a plasmid within the papilloma. Some virally induced papillomas may evolve over time to produce malignancies (reviewed in reference 1); this typically occurs after many months of chronic infection. Thus, human papillomavirus (HPV) genome maintenance is important to the development of malignancy.Cancer-associated HPV types are termed "high-risk" HPV. This group expresses virally encoded E7 and E6 oncoproteins that target the degradation of cellular retinoblastoma family proteins and the p53 tumor suppressor, respectively (reviewed in references 2 and 3). The high-risk E7 oncoprotein associates with and targets the degradation of all three retinoblastoma protein (RB) family members, thereby inducing the stabilization of p53 (4) and sensitization of infected keratinocytes to apoptosis and autophagy (5, 6). Although p53 is stabilized, it remains transcriptionally inactive in E7 transduced cells (7).The alpha genus HPV E6 proteins, both high and low risk, bind to an LXXLL motif (LQELL) on the cellular E3 ubiquitin ligase E6AP (8, 9). The high-risk type E6-E6AP complex then recruits and ubiquitinates p53, leading to its proteasome-mediated degradation (10,11).In addition to the targeted degradation of p53, high-risk E6 oncoproteins contain a short peptide sequence at the carboxy terminus that associates with a subset of cellular proteins that contain PDZ domains. The proteins bound by the E6 PDZ binding motif (termed PBM) are diverse and include the human homologues of Drosophila tumor suppressor scaffold proteins DLG (12) and SCRIB (13), tyrosine phosphatases PTPN3 (14) and PTPN13 (15), and multiple other scaffolding proteins implicated in epithelial polarity, membrane trafficking, and cell signaling (16)(17)(18)(19)(20). The E6 PBM is subject to phosphorylation and then cytoplasmic tethering to 14-3-3 proteins (21). High-risk E6 can target the degradation of bound cellular PDZ proteins in vitro and in vivo, which has been associated with many phenotypes, including altered epithelial differentiation in transgenic mice (22,23), reduced growth factor dependence in human keratinocytes (14), promotion of the epithelial to mesenchymal transition (24), and cooperation with ras to induce anchorage-independent colony formation (15). In the context of the entire HPV genome, deletion of the E6 PBM causes loss of the viral plasmid upon cell passaging (25). The capacity of high-risk E6 to target the degradati...

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confidence: 85%

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confidence: 99%

Papillomavirus E6 PDZ Interactions Can Be Replaced by Repression of p53 To Promote Episomal Human Papillomavirus Genome Maintenance

Brimer

Pol

2014

“…The E6 oncoproteins of high-risk HPV types have a C-terminal PDZ-binding motif (PBM) that is important for a normal viral life cycle and progeny production (for a review, see reference 3). PBM deletion causes viral genome integration, loss of replicative competence, and an increased potential for oncogenic transformation (4)(5)(6)(7)(8).…”

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confidence: 99%

PDZRN3/LNX3 Is a Novel Target of Human Papillomavirus Type 16 (HPV-16) and HPV-18 E6

Thomas

Banks

2015

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High-risk human papillomavirus (HPV) E6 proteins have a C-terminal PDZ binding motif through which they bind, and target for proteasome-mediated degradation, a number of PDZ-containing cellular targets. Recent studies have suggested that the RING-containing ubiquitin-protein ligase PDZRN3 might also be an HPV E6 target. In this analysis, we show that HPV-16 and HPV-18 E6 can target PDZRN3 in a PDZ-and proteasome-dependent manner and provide a connection between the HPV life cycle and differentiation-related STAT signaling. High-risk human papillomaviruses (HPVs) are the causative agents of cervical cancer, other anogenital cancers, and some head and neck cancers (1, 2). The E6 oncoproteins of high-risk HPV types have a C-terminal PDZ-binding motif (PBM) that is important for a normal viral life cycle and progeny production (for a review, see reference 3). PBM deletion causes viral genome integration, loss of replicative competence, and an increased potential for oncogenic transformation (4-8).E6 targets several PDZ-containing proteins (9-12), and recently, a proteomic screen of DNA tumor virus interactions (13) and an HPV-16 E6 C-terminal peptide screen of the human PDZome (14) Extracts of 293 cells transfected with pCDNA3.1 (lanes C) or pCDNA-FLAG-PDZRN3A (lanes P) were incubated with GST, GST-HPV18E6, or GST-HPV18E6T156E, as indicated.Afterwashing,theboundproteinswereanalyzedbyaWesternblotassayprobedwithanti-PDZRN3andanti-GSTantibodiestoconfirmequalloadingofGSTproteins. (C) HPV-18 E6-induced degradation of PDZRN3 is proteasome dependent. A plasmid expressing FLAG-tagged PDZRN3A was transfected into 293 cells alone or with an HPV-18 E6-expressing plasmid. After overnight incubation, the cells were treated with the proteasome inhibitors carbobenzoxy-Leu-Leu-leucinal (CBZ) and N-acetyl-L-leucyl-L-leucyl-L-norleucinal for 3 h before harvesting. Cell extracts were analyzed by Western blotting with anti-FLAG antibody; ␤-galactosidase was included as a control for transfection efficiency.

“…The presence of a PBM on the carboxy termini of cancer-causing E6 oncoproteins is essential for the viral life cycle (17)(18)(19)(20)(21) and contributes toward cell transformation (8,13) and the induction of malignancy (15,16). While it has been known for some time that this region of E6 could be phosphorylated, which, in turn, would be expected to inhibit E6-PDZ interactions (23,25), it has only recently become clear that this phosphorylation also confers upon E6 the ability to interact with 14-3-3 family members (23).…”

Section: Discussionmentioning

confidence: 99%

“…Mutations in this region of E6 in the context of the whole viral genome result in marked defects to the viral life cycle, with reduced rates of viral DNA replication and a reduction in the expansion of replicationcompetent cells in the basal layers of the organotypic cultures (17,18). Furthermore, such viral genomes appear unstable over time, with rapid loss of viral episomes upon continued passaging of the cells (17)(18)(19)(20)(21).…”

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confidence: 99%

Cancer-Causing Human Papillomavirus E6 Proteins Display Major Differences in the Phospho-Regulation of Their PDZ Interactions

Boon

Tomaić

Thomas

et al. 2015

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Previous studies have shown that the cancer-causing high-risk human papillomavirus (HPV) E6 oncoproteins have PDZ binding potential, an activity which is important for their ability to support the viral life cycle and to cooperate in the induction of malignancy. However, PDZ interactions are not constitutive, and they can be negatively regulated by phosphorylation within the E6 PDZ binding motif (PBM). In this study, we have investigated the differential regulation of the HPV E6 PBMs from diverse highrisk HPV types. We show that, depending on the HPV type, PDZ binding activity can be regulated by phosphorylation with protein kinase A (PKA) or AKT, which, in turn, inhibits PDZ recognition. Such regulation is highly conserved between E6 proteins derived from HPV-16, HPV-18, and HPV-58 while being somewhat weaker or absent from other types such as HPV-31, HPV-33, and HPV-51. In the case of HPV31, PKA phosphorylation occurs within the core of the E6 protein and has no effect on PDZ interactions, and this demonstrates a surprising degree of heterogeneity among the different high-risk HPV E6 oncoproteins in how they are regulated by different cellular signaling pathways. IMPORTANCEThis study demonstrated that the cancer-causing HPV E6 oncoproteins are all subject to posttranslational modification of their extreme C-terminal PDZ binding motifs through phosphorylation. However, the identities of the kinase are not the same for all HPV types. This demonstrates a very important divergence between these HPVs, and it suggests that changes in cell signaling pathways have different consequences for different high-risk virus infections and their associated malignancies.H uman papillomaviruses (HPVs) are the causative agents of cervical cancer, which remains a leading cause of death in women throughout the world. Over 120 different HPV types have been identified, 12 of which are defined as cancer causing (1, 2). Of these, HPV-16 and HPV-18 are the most important, accounting for approximately 70% of cervical cancers. The remaining cancers are caused by other high-risk (HR) HPV types, which include 2). HPVinduced carcinogenesis arises from the combined activity of the two major viral oncoproteins E6 and E7, which, by deregulating multiple cellular pathways, including cell cycle control and apoptosis, ultimately induce cell immortalization and, eventually, malignancy (3, 4).A unique characteristic of the HR HPV E6 oncoproteins is the presence of a class I PDZ (PSD-95/Dlg/ZO-1) binding motif (PBM) at the extreme carboxy terminus, which is absent in the low-risk (LR) non-cancer-causing HPV E6 proteins (5, 6). This region of E6 allows it to interact with a number of cellular PDZ domain-containing proteins, many of which are involved in the regulation of cell junctional integrity and cell signaling pathways (reviewed in reference 7). The first such targets to be identified were the cell polarity regulators Discs Large (hDlg1) (5,7,8) and Scribble (hScrib) (9), which were shown to be degraded by HPV-16 and HPV-18 E6 in a protea...