Multi-PDZ Domain Protein MUPP1 Is a Cellular Target for both Adenovirus E4-ORF1 and High-Risk Papillomavirus Type 18 E6 Oncoproteins

ABSTRACT A general theme that has emerged from studies of DNA tumor viruses is that otherwise unrelated oncoproteins encoded by these viruses often target the same important cellular factors. Major oncogenic determinants for human adenovirus type 9 (Ad9) and high-risk human papillomaviruses (HPV) are the E4-ORF1 and E6 oncoproteins, respectively, and although otherwise unrelated, both of these viral proteins possess a functional PDZ domain-binding motif that is essential for their transforming activity and for binding to the PDZ domain-containing and putative tumor suppressor protein DLG. We report here that the PDZ domain-binding motifs of Ad9 E4-ORF1 and high-risk HPV-18 E6 also mediate binding to the widely expressed cellular factor MUPP1, a large multi-PDZ domain protein predicted to function as an adapter in signal transduction. With regard to the consequences of these interactions in cells, we showed that Ad9 E4-ORF1 aberrantly sequesters MUPP1 within the cytoplasm of cells whereas HPV-18 E6 targets this cellular protein for degradation. These effects were specific because mutant viral proteins unable to bind MUPP1 lack these activities. From these results, we propose that the multi-PDZ domain protein MUPP1 is involved in negatively regulating cellular proliferation and that the transforming activities of two different viral oncoproteins depend, in part, on their ability to inactivate this cellular factor.

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The Human Papillomavirus (HPV) E6* Proteins from High-Risk, Mucosal HPVs Can Direct Degradation of Cellular Proteins in the Absence of Full-Length E6 Protein

Journal of Virology ◽

10.1128/jvi.00539-09 ◽

2009 ◽

Vol 83 (19) ◽

pp. 9863-9874 ◽

Cited By ~ 30

Author(s):

David Pim ◽

Vjekoslav Tomaić ◽

Lawrence Banks

Keyword(s):

High Risk ◽

Tumor Suppressor ◽

Pdz Domain ◽

Direct Interaction ◽

Human Papillomaviruses ◽

Full Length ◽

Cellular Proteins ◽

Hpv E6 ◽

E6 Protein ◽

Hpv 18

ABSTRACT The E6 oncoproteins from high-risk mucosotrophic human papillomaviruses (HPVs) target a range of cellular proteins for proteasome-mediated degradation. Apart from the tumor suppressor p53 and proapoptotic Bcl-2 family member Bak, many targets contain class 1 PDZ domains and are involved in cell junction stability and signaling. The targeting mechanism is considered to function by the E6 protein acting as an adaptor molecule linking a cellular ubiquitin ligase to the target protein. In each case, whether the target is the p53 tumor suppressor or a member of the group of PDZ domain-containing targets, this mechanism relies on a direct interaction between E6 and its cellular target. This study focuses on the impact of the HPV type 18 (HPV-18) E6*I protein on the stability of Akt, Dlg, MAGI-1, MAGI-2, and Scribble. We show that HPV-18 E6* expression can downregulate the expression levels of Akt, Dlg, and Scribble in the absence of full-length HPV-18 E6 protein. The reduction in Dlg levels by E6* is independent of transcription and does not require a direct interaction between the two proteins although the proteasome pathway is involved. Further, we provide evidence that activation of certain signal transduction pathways has a profound effect on the targeting of Dlg by E6* and suggest that high-risk HPV E6 oncoproteins can target certain substrates both directly and indirectly through the E6* proteins and may cooperate in their degradation.

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Recognition of high-risk HPV E6 oncoproteins by 14-3-3 proteins studied by interactomics and crystallography

10.1101/2020.07.24.220376 ◽

2020 ◽

Author(s):

Gergo Gogl ◽

Kristina V. Tugaeva ◽

Pascal Eberling ◽

Camille Kostmann ◽

Gilles Trave ◽

...

Keyword(s):

High Risk ◽

Fluorescence Polarization ◽

Pdz Domain ◽

Human Papillomaviruses ◽

Binding Motif ◽

Hpv E6 ◽

Sequence Variations ◽

Hpv18 E6 ◽

Isoform Specificity ◽

High Risk Hpv

AbstractIn tumors induced by high-risk mucosal human papillomaviruses (hrm-HPVs), HPV E6 oncoproteins inhibit apoptotic processes and sustain cell proliferation. E6 from all hrm-HPVs harbor a C-terminal short PDZ domain-binding motif (PBM), whose phosphorylation down-regulates PDZ binding but triggers E6 binding to 14-3-3 proteins. Here we classify PBMs of E6 proteins depending on their principle ability to be phosphorylated and subsequently acquire a 14-3-3-binding motif III consensus, (pS/pT)XX-COOH. Systematic competitive fluorescence polarization measurements show that the PBMs from four selected E6 oncoproteins bind all seven human 14-3-3 isoforms with distinct, wide-ranging affinities, obeying remarkable trends assigned to 14-3-3 isoform specificity and small E6 sequence variations. We crystallized the hrm-HPV18 E6 PBM bound to 14-3-3ζ, revealing a 14-3-3-motif III complex at 1.9 Å resolution. Using fluorescence polarization and crystallography, we also demonstrate that fusicoccin, a molecule that reinforces many known 14-3-3 complexes, destabilizes the 14-3-3-E6 interaction, indicating the druggability of that complex.

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Protein tyrosine phosphatase H1 is a target of the E6 oncoprotein of high-risk genital human papillomaviruses

Journal of General Virology ◽

10.1099/vir.0.83123-0 ◽

2007 ◽

Vol 88 (11) ◽

pp. 2956-2965 ◽

Cited By ~ 21

Author(s):

Stephanie Töpffer ◽

Andreas Müller-Schiffmann ◽

Konstantin Matentzoglu ◽

Martin Scheffner ◽

Gertrud Steger

Keyword(s):

High Risk ◽

Cervical Carcinoma ◽

Protein Tyrosine Phosphatase ◽

Carcinoma Cell ◽

Tyrosine Phosphatase ◽

Pdz Domain ◽

Human Papillomaviruses ◽

Binding Motif ◽

Protein Tyrosine ◽

Pdz Domain Proteins

The E6 proteins of high-risk genital human papillomaviruses (HPV), such as HPV types 16 and 18, possess a conserved C-terminal PDZ-binding motif, which mediates interaction with some cellular PDZ domain proteins. The binding of E6 usually results in their ubiquitin-mediated degradation. The ability of E6 to bind to PDZ domain proteins correlates with the oncogenic potential. Using a yeast two-hybrid system, GST pull-down experiments and coimmunoprecipitations, we identified the protein tyrosine phosphatase H1 (PTPH1/PTPN3) as a novel target of the PDZ-binding motif of E6 of HPV16 and 18. PTPH1 has been suggested to function as tumour suppressor protein, since mutational analysis revealed somatic mutations in PTPH1 in a minor fraction of various human tumours. We show here that HPV16 E6 accelerated the proteasome-mediated degradation of PTPH1, which required the binding of E6 to the cellular ubiquitin ligase E6-AP and to PTPH1. The endogenous levels of PTPH1 were particularly low in HPV-positive cervical carcinoma cell lines. The reintroduction of the E2 protein into the HPV16-positive cervical carcinoma cell line SiHa, known to lead to a sharp repression of E6 expression and to induce growth suppression, resulted in an increase of the amount of PTPH1. Our data suggest that reducing the level of PTPH1 may contribute to the oncogenic activity of high-risk genital E6 proteins.

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High-Risk Human Papillomavirus E6 Oncoproteins Interact with 14-3-3ζ in a PDZ Binding Motif-Dependent Manner

Journal of Virology ◽

10.1128/jvi.02074-12 ◽

2012 ◽

Vol 87 (3) ◽

pp. 1586-1595 ◽

Cited By ~ 45

Author(s):

Siaw Shi Boon ◽

Lawrence Banks

Keyword(s):

Cervical Cancer ◽

Human Papillomavirus ◽

High Risk ◽

Pdz Domain ◽

Binding Motif ◽

Dependent Manner ◽

Hpv E6 ◽

Hpv Types ◽

High Risk Hpv ◽

Hpv 18

ABSTRACTCervical cancer develops through the combined activities of the human papillomavirus (HPV) E6 and E7 oncoproteins. A defining characteristic of E6 oncoproteins derived from cancer-causing HPV types is the presence of a PDZ binding motif (PBM) at the extreme carboxy terminus of the protein which is absent from E6 proteins derived from the so-called low-risk HPV types. Within this PBM is also a protein kinase A (PKA) phospho-acceptor site, which is thought to negatively regulate the association of E6 with its PDZ domain-containing substrates. We can now show that phosphorylation of E6 by PKA and/or AKT confers the ability to interact with 14-3-3ζ. The interaction is direct and specific for the high-risk HPV E6 oncoproteins, although there are significant differences in the efficiencies with which HPV-16, HPV-18, and HPV-31 E6 oncoproteins can associate with 14-3-3ζ; this correlates directly with their respective susceptibilities to phosphorylation by PKA and/or AKT. We demonstrate here that the interaction between E6 and 14-3-3ζ also requires integrity of the E6 PBM, and downregulation of 14-3-3ζ results in a marked reduction in the levels of HPV-18 E6 expression in HeLa cells. Using phospho-specific anti-E6 antibodies, we also demonstrate significant levels of E6 phosphorylationin vivo. These studies redefine the potential relevance of the E6 PBM in the development of cervical cancer, suggesting that interaction with 14-3-3ζ, as well as the more well-established interactions with PDZ domain-containing substrates, is likely to be responsible for the biological activities attributed to this region of the high-risk HPV E6 oncoproteins.

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A unique insert in the genomes of high-risk human papillomaviruses with a predicted dual role in conferring oncogenic risk

F1000Research ◽

10.12688/f1000research.19590.1 ◽

2019 ◽

Vol 8 ◽

pp. 1000 ◽

Cited By ~ 6

Author(s):

Noam Auslander ◽

Yuri I. Wolf ◽

Svetlana A. Shabalina ◽

Eugene V. Koonin

Keyword(s):

High Risk ◽

Pdz Domain ◽

Human Papillomaviruses ◽

Machine Learning Techniques ◽

Genome Comparison ◽

Binding Motif ◽

E6 Oncoprotein ◽

C Terminus ◽

Tumorigenic Potential ◽

E6 And E7

The differences between high risk and low risk human papillomaviruses (HR-HPV and LR-HPV, respectively) that contribute to the tumorigenic potential of HR-HPV are not well understood but can be expected to involve the HPV oncoproteins, E6 and E7. We combine genome comparison and machine learning techniques to identify a previously unnoticed insert near the 3’-end of the E6 oncoprotein gene that is unique to HR-HPV. Analysis of the insert sequence suggests that it exerts a dual effect, by creating a PDZ domain-binding motif at the C-terminus of E6 as well as eliminating the overlap between the E6 and E7 coding regions in HR-HPV. We show that as a result, the insert might enable coupled termination-reinitiation of the E6 and E7 genes, supported by motifs complementary to the human 18S rRNA. We hypothesize that the added functionality of E6 and positive regulation of E7 expression jointly account for the tumorigenic potential of HR-HPV.

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A unique insert in the genomes of high-risk human papillomaviruses with a predicted dual role in conferring oncogenic risk

F1000Research ◽

10.12688/f1000research.19590.2 ◽

2019 ◽

Vol 8 ◽

pp. 1000 ◽

Cited By ~ 1

Author(s):

Noam Auslander ◽

Yuri I. Wolf ◽

Svetlana A. Shabalina ◽

Eugene V. Koonin

Keyword(s):

High Risk ◽

Pdz Domain ◽

Human Papillomaviruses ◽

Machine Learning Techniques ◽

Genome Comparison ◽

Binding Motif ◽

E6 Oncoprotein ◽

C Terminus ◽

Tumorigenic Potential ◽

E6 And E7

The differences between high risk and low risk human papillomaviruses (HR-HPV and LR-HPV, respectively) that contribute to the tumorigenic potential of HR-HPV are not well understood but can be expected to involve the HPV oncoproteins, E6 and E7. We combine genome comparison and machine learning techniques to identify a previously unnoticed insert near the 3’-end of the E6 oncoprotein gene that is unique to HR-HPV. Analysis of the insert sequence suggests that it exerts a dual effect, by creating a PDZ domain-binding motif at the C-terminus of E6, as well as eliminating the overlap between the E6 and E7 coding regions in HR-HPV. We show that, as a result, the insert might enable coupled termination-reinitiation of the E6 and E7 genes, supported by motifs complementary to the human 18S rRNA. We hypothesize that the added functionality of E6 and positive regulation of E7 expression jointly account for the tumorigenic potential of HR-HPV.

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Recognition of high-risk HPV E6 oncoproteins by 14-3-3 proteins studied by interactomics and crystallography

10.21203/rs.3.rs-48671/v1 ◽

2020 ◽

Author(s):

Gergő Gógl ◽

Kristina Tugaeva ◽

Pascal Eberling ◽

Camille Kostmann ◽

Gilles Trave ◽

...

Keyword(s):

High Risk ◽

Fluorescence Polarization ◽

Pdz Domain ◽

Human Papillomaviruses ◽

Binding Motif ◽

Hpv E6 ◽

Sequence Variations ◽

Hpv18 E6 ◽

Isoform Specificity ◽

High Risk Hpv

Abstract In tumors induced by high-risk mucosal human papillomaviruses (hrm-HPVs), HPV E6 oncoproteins inhibit apoptotic processes and sustain cell proliferation. E6 from all hrm-HPVs harbor a C-terminal short PDZ domain-binding motif (PBM), whose phosphorylation down-regulates PDZ binding but triggers E6 binding to 14-3-3 proteins. Here we classify PBMs of E6 proteins depending on their principle ability to be phosphorylated and subsequently acquire a 14-3-3-binding motif III consensus, (pS/pT)XX-COOH. Systematic competitive fluorescence polarization measurements show that the PBMs from four selected E6 oncoproteins bind all seven human 14-3-3 isoforms with distinct, wide-ranging affinities, obeying remarkable trends assigned to 14-3-3 isoform specificity and small E6 sequence variations. We crystallized the hrm-HPV18 E6 PBM bound to 14-3-3ζ, revealing a 14-3-3-motif III complex at 1.9 Å resolution. Using fluorescence polarization and crystallography, we also demonstrate that fusicoccin, a molecule that reinforces many known 14-3-3 complexes, destabilizes the 14-3-3-E6 interaction, indicating the druggability of that complex.

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Role of the PDZ Domain-Binding Motif of the Oncoprotein E6 in the Pathogenesis of Human Papillomavirus Type 31

Journal of Virology ◽

10.1128/jvi.78.22.12366-12377.2004 ◽

2004 ◽

Vol 78 (22) ◽

pp. 12366-12377 ◽

Cited By ~ 97

Author(s):

Choongho Lee ◽

Laimonis A. Laimins

Keyword(s):

High Risk ◽

Pdz Domain ◽

Immunohistochemical Analysis ◽

Human Papillomaviruses ◽

Binding Motif ◽

Wild Type ◽

Pdz Proteins ◽

Viral Copy Number ◽

Viral Copy

ABSTRACT A number of PDZ domain-containing proteins have been identified as binding partners for the oncoprotein E6 of the high-risk type human papillomaviruses (HPVs). These include hDlg, hScrib, MAGI-1, MAGI-2, MAGI-3, and MUPP1. The PDZ domain-binding motif (-X-T-X-V) at the carboxy terminus of E6 is essential for targeting PDZ proteins for proteasomal degradation. The presence of this motif only in the high-risk HPVs suggests its possible role in HPV-induced oncogenesis. To investigate the role of the PDZ domain-binding motif of E6 in the HPV life cycle, two mutant HPV31 genomes were constructed: E6ValΔ, with a deletion of the last amino acid residue of E6 (valine), and E6ETQVΔ, with a deletion of the entire PDZ domain-binding motif of E6 (ETQV). Three human foreskin keratinocyte (HFK) cell lines were established which maintained transfected wild-type HPV31 or either of two mutant genomes. Cells containing either of two mutant genomes were significantly retarded in their growth rates and reduced in their viral copy numbers compared to those transfected with wild-type genomes. Western analysis did not reveal any significant changes in the levels of PDZ proteins following stable transfection of any HPV31 genomes into HFKs. Although the E6ETQVΔ-transfected HFKs exhibited a pattern of morphological differentiation that appeared different from the HPV31 wild-type-transfected HFKs in organotypic raft cultures, immunohistochemical analysis failed to identify substantial changes in the differentiation-dependent membrane localization of hDlg proteins. These results suggest that binding of E6 to PDZ proteins modulates the early viral functions such as proliferation and maintenance of the viral copy number in undifferentiated cells.

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E6AP-Dependent Degradation of DLG4/PSD95 by High-Risk Human Papillomavirus Type 18 E6 Protein

Journal of Virology ◽

10.1128/jvi.01712-06 ◽

2006 ◽

Vol 81 (3) ◽

pp. 1379-1389 ◽

Cited By ~ 48

Author(s):

Keisuke Handa ◽

Takashi Yugawa ◽

Mako Narisawa-Saito ◽

Shin-ichi Ohno ◽

Masatoshi Fujita ◽

...

Keyword(s):

High Risk ◽

Tumor Suppressor ◽

Pdz Domain ◽

Mouse Skin ◽

Human Papillomaviruses ◽

Binding Motif ◽

Human Homologue ◽

Large Tumor ◽

Hpv16 E6 ◽

Carboxy Terminal

ABSTRACT In most cervical cancers, DNAs of high-risk mucosotropic human papillomaviruses (HPVs), such as types 16 and 18, are maintained so as to express two viral proteins, E6 and E7, suggesting that they play important roles in carcinogenesis. The carboxy-terminal PDZ domain-binding motif of the E6 proteins is in fact essential for transformation of rodent cells and induction of hyperplasia in E6-transgenic mouse skin. To date, seven PDZ domain-containing proteins, including DLG1/hDLG, which is a human homologue of the Drosophila discs large tumor suppressor (Dlg), have been identified as targets of high-risk HPV E6 proteins. Here, we describe DLG4/PSD95, another human homologue of Dlg, as a novel E6 target. DLG4 was found to be expressed in normal human cells, including cervical keratinocytes, but only to a limited extent in both HPV-positive and HPV-negative cervical cancer cell lines. Expression of HPV18 E6 in HCK1T decreased DLG4 levels more strongly than did HPV16 E6, the carboxy-terminal motif of the proteins being critical for binding and degradation of DLG4 in vitro. DLG4 levels were restored by expression of either E6AP-specific short hairpin RNA or bovine papillomavirus type 1 E2 in HeLa but not CaSki or SiHa cells, reflecting downregulation of DLG4 mRNA as opposed to protein by an HPV-independent mechanism in HPV16-positive cancer lines. The tumorigenicity of CaSki cells was strongly inhibited by forced expression of DLG4, while growth in culture was not inhibited at all. These results suggest that DLG4 may function as a tumor suppressor in the development of HPV-associated cancers.

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