Human Papillomavirus Type 16 E7 Oncoprotein Associates with E2F6

ABSTRACT The papillomavirus life cycle is intimately coupled to the differentiation state of the infected epithelium. Since papillomaviruses lack most of the rate-limiting enzymes required for genome synthesis, they need to uncouple keratinocyte differentiation from cell cycle arrest and maintain or reestablish a replication-competent state within terminally differentiated keratinocytes. The human papillomavirus (HPV) E7 protein appears to be a major determinant for this activity and induces aberrant S-phase entry through the inactivation of the retinoblastoma tumor suppressor and related pocket proteins. In addition, E7 can abrogate p21 and p27. Together, this leads to the activation of E2F1 to E2F5, enhanced expression of E2F-responsive genes, and increased cdk2 activity. E2F6 is a pRB-independent, noncanonical member of the E2F transcription factor family that acts as a transcriptional repressor. E2F6 expression is activated in S phase through an E2F-dependent mechanism and thus may provide a negative-feedback mechanism that slows down S-phase progression and/or exit in response to the activation of the other E2F transcription factors. Here, we show that low- and high-risk HPV E7 proteins, as well as simian virus 40 T antigen and adenovirus E1A, can associate with and inactivate the transcriptional repression activity of E2F6, thereby subverting a critical cellular defense mechanism. This may result in the extended S-phase competence of HPV-infected cells. E2F6 is a component of polycomb group complexes, which bind to silenced chromatin and are critical for the maintenance of cell fate. We show that E7-expressing cells show decreased staining for E2F6/polycomb complexes and that this is at least in part dependent on the association with E2F6.

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Association of the human papillomavirus type 16 E7 protein with the S-phase-specific E2F-cyclin A complex

Molecular and Cellular Biology ◽

10.1128/mcb.13.10.6537-6546.1993 ◽

1993 ◽

Vol 13 (10) ◽

pp. 6537-6546 ◽

Cited By ~ 1

Author(s):

M Arroyo ◽

S Bagchi ◽

P Raychaudhuri

Keyword(s):

Cell Cycle ◽

Human Papillomavirus ◽

Simian Virus 40 ◽

Cyclin A ◽

T Antigen ◽

S Phase ◽

Cellular Proteins ◽

Hpv 16 ◽

Adenovirus E1a ◽

Cell Extracts

The transcription factor E2F has been shown to be involved in the expression of several cell cycle-regulated genes, and the activity of this factor is controlled by cellular proteins such as pRB and p107. E2F is also a target of the DNA virus oncoproteins (adenovirus E1A, simian virus 40 T antigen, and human papillomavirus [HPV] E7) (see the review by J. R. Nevins [Science 258: 424-429, 1992]). These viral oncoproteins dissociate an inactive complex between E2F and the retinoblastoma tumor suppressor protein (pRB), and this dissociation of the E2F-pRB complex correlates with a stimulation of the E2F-dependent transcription. In the S phase of the cell cycle, E2F forms a complex with p107, cyclin A, and the cdk2 kinase (E2F-cyclin A complex). The cellular function of this S-phase-specific complex is unclear. The adenovirus E1A protein dissociates the E2F-cyclin A complex. The HPV type 16 (HPV-16) E7 protein, which possesses significant sequence homology with E1A, does not dissociate the E2F-cyclin A complex. We find that the HPV-16 E7 protein associates very efficiently with the E2F-cyclin A complex. This association is dependent on the sequences that are also necessary for the transforming activity of E7. Moreover, the E7 protein of a low-risk HPV (type 6b) is much less efficient in binding to the E2F-cyclin A complex compared with that of the high-risk type. We also find that the E2F-cyclin A complex remains endogenously associated with the E7 protein in extracts of Caski cells, which express high levels of HPV-16 E7 protein. Finally, we have extensively purified the E2F-cyclin A complex from mouse L-cell extracts and show that, in cell extracts, the E2F-cyclin A complex remains associated with other cellular proteins.

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Association of the human papillomavirus type 16 E7 protein with the S-phase-specific E2F-cyclin A complex.

Molecular and Cellular Biology ◽

10.1128/mcb.13.10.6537 ◽

1993 ◽

Vol 13 (10) ◽

pp. 6537-6546 ◽

Cited By ~ 73

Author(s):

M Arroyo ◽

S Bagchi ◽

P Raychaudhuri

Keyword(s):

Cell Cycle ◽

Human Papillomavirus ◽

Simian Virus 40 ◽

Cyclin A ◽

T Antigen ◽

S Phase ◽

Cellular Proteins ◽

Hpv 16 ◽

Adenovirus E1a ◽

Cell Extracts

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Intestinal Hyperplasia Induced by Simian Virus 40 Large Tumor Antigen Requires E2F2

Journal of Virology ◽

10.1128/jvi.01658-07 ◽

2007 ◽

Vol 81 (23) ◽

pp. 13191-13199 ◽

Cited By ~ 20

Author(s):

M. Teresa Sáenz-Robles ◽

Jennifer A. Markovics ◽

Jean-Leon Chong ◽

Rene Opavsky ◽

Robert H. Whitehead ◽

...

Keyword(s):

Transcriptional Repression ◽

Cultured Cells ◽

Simian Virus 40 ◽

Normal Growth ◽

Simian Virus ◽

T Antigen ◽

S Phase ◽

Large Tumor ◽

Intestinal Hyperplasia

ABSTRACT The simian virus 40 large T antigen contributes to neoplastic transformation, in part, by targeting the Rb family of tumor suppressors. There are three known Rb proteins, pRb, p130, and p107, all of which block the cell cycle by preventing the transcription of genes regulated by the E2F family of transcription factors. T antigen interacts directly with Rb proteins and disrupts Rb-E2F complexes both in vitro and in cultured cells. Consequently, T antigen is thought to inhibit transcriptional repression by the Rb family proteins by disrupting their interaction with E2F proteins, thus allowing E2F-dependent transcription and the expression of cellular genes needed for entry into S phase. This model predicts that active E2F-dependent transcription is required for T-antigen-induced transformation. To test this hypothesis, we have examined the status of Rb-E2F complexes in murine enterocytes. Previous studies have shown that T antigen drives enterocytes into S phase, resulting in intestinal hyperplasia, and that the induction of enterocyte proliferation requires T-antigen binding to Rb proteins. In this paper, we show that normal growth-arrested enterocytes contain p130-E2F4 complexes and that T-antigen expression destroys these complexes, most likely by stimulating p130 degradation. Furthermore, unlike their normal counterparts, enterocytes expressing T antigen contain abundant levels of E2F2 and E2F3a. Concomitantly, T-antigen-induced intestinal proliferation is reduced in mice lacking either E2F2 alone or both E2F2 and E2F3a, but not in mice lacking E2F1. These studies support a model in which T antigen eliminates Rb-E2F repressive complexes so that specific activator E2Fs can drive S-phase entry.

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C/EBPα Inhibits Cell Growth via Direct Repression of E2F-DP-Mediated Transcription

Molecular and Cellular Biology ◽

10.1128/mcb.20.16.5986-5997.2000 ◽

2000 ◽

Vol 20 (16) ◽

pp. 5986-5997 ◽

Cited By ~ 114

Author(s):

Beatrix A. Slomiany ◽

Kenneth L. D'Arigo ◽

Margaret M. Kelly ◽

David T. Kurtz

Keyword(s):

Cell Growth ◽

Ectopic Expression ◽

Simian Virus 40 ◽

Normal Liver ◽

Simian Virus ◽

Binding Activity ◽

T Antigen ◽

S Phase ◽

3T3 Cells ◽

L Cells

ABSTRACT Using an inducible transcription system which allows the regulated expression of C/EBP isoforms in tissue culture cells, we have found that the ectopic expression of C/EBPα, at a level comparable to that found in normal liver tissue, has a pronounced antimitogenic effect in mouse L cells and NIH 3T3 cells. The inhibition of cell division by C/EBPα in mouse cells cannot be reversed by simian virus 40 T antigen, by oncogenic ras, or by adenovirus E1a protein. When expressed in thymidine kinase-deficient L cells or 3T3 cells, C/EBPα is detected in a protein complex which binds to the E2F binding sites found in the promoters of the genes for E2F-1 and dihydrofolate reductase (DHFR). Bacterially expressed C/EBPα has no affinity for these E2F sites, but when recombinant C/EBPα is added to nuclear extracts from mouse fibroblasts, a new E2F binding activity appears, which contains the C/EBPα protein. Using an E2F-DP1-responsive promoter linked to a reporter gene, it can be shown that C/EBPα directly inhibits the induction of this promoter by E2F-DP1 in transient-transfection assays. Furthermore, C/EBPα can be shown to inhibit the S-phase induction of the E2F and DHFR promoters in permanent cell lines. These findings delineate a straightforward mechanism for C/EBPα-mediated cell growth arrest through repression of E2F-DP-mediated S-phase transcription.

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PTPN14 degradation by high-risk human papillomavirus E7 limits keratinocyte differentiation and contributes to HPV-mediated oncogenesis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1819534116 ◽

2019 ◽

Vol 116 (14) ◽

pp. 7033-7042 ◽

Cited By ~ 20

Author(s):

Joshua Hatterschide ◽

Amelia E. Bohidar ◽

Miranda Grace ◽

Tara J. Nulton ◽

Hee Won Kim ◽

...

Keyword(s):

Gene Expression ◽

Human Papillomavirus ◽

High Risk ◽

Gene Expression Signature ◽

Human Keratinocytes ◽

Keratinocyte Differentiation ◽

Oncogenic Transformation ◽

Hpv E7 ◽

Oncogenic Activity ◽

High Risk Hpv

High-risk human papillomavirus (HPV) E7 proteins enable oncogenic transformation of HPV-infected cells by inactivating host cellular proteins. High-risk but not low-risk HPV E7 target PTPN14 for proteolytic degradation, suggesting that PTPN14 degradation may be related to their oncogenic activity. HPV infects human keratinocytes but the role of PTPN14 in keratinocytes and the consequences of PTPN14 degradation are unknown. Using an HPV16 E7 variant that can inactivate retinoblastoma tumor suppressor (RB1) but cannot degrade PTPN14, we found that high-risk HPV E7-mediated PTPN14 degradation impairs keratinocyte differentiation. Deletion ofPTPN14from primary human keratinocytes decreased keratinocyte differentiation gene expression. Related to oncogenic transformation, both HPV16 E7-mediated PTPN14 degradation andPTPN14deletion promoted keratinocyte survival following detachment from a substrate. PTPN14 degradation contributed to high-risk HPV E6/E7-mediated immortalization of primary keratinocytes and HPV+but not HPV−cancers exhibit a gene-expression signature consistent with PTPN14 inactivation. We find that PTPN14 degradation impairs keratinocyte differentiation and propose that this contributes to high-risk HPV E7-mediated oncogenic activity independent of RB1 inactivation.

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Cell-Specific Modulation of Papovavirus Replication by Tumor Suppressor Protein p53

Journal of Virology ◽

10.1128/jvi.74.10.4688-4697.2000 ◽

2000 ◽

Vol 74 (10) ◽

pp. 4688-4697 ◽

Cited By ~ 3

Author(s):

Dina Lepik ◽

Mart Ustav

Keyword(s):

Tumor Suppressor ◽

Cell Lines ◽

P53 Protein ◽

Chinese Hamster ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

S Phase ◽

Human Papillomaviruses ◽

Mouse Cells

ABSTRACT Small DNA tumor viruses like human papillomaviruses, simian virus 40, and adenoviruses modulate the activity of cellular tumor suppressor proteins p53 and/or pRB. These viruses replicate as nuclear multicopy extrachromosomal elements during the S phase of the cell cycle, and it has been suggested that inactivation of p53 and pRb is necessary for directing the cells to the S phase. Mouse polyomavirus (Py), however, modulates only the pRB protein activity without any obvious interference with the action of p53. We show here that Py replication was not suppressed by the p53 protein indeed in all tested different mouse cell lines. In addition, E1- and E2-dependent papillomavirus origin replication was insensitive to the action of p53 in mouse cells. We show that in hamster (Chinese hamster ovary) or human (osteosarcoma 143) cell lines the replication of both Py and papillomavirus origins was efficiently blocked by p53. The block of Py replication in human and hamster cells is not caused by the downregulation of large T-antigen expression. The deletion analysis of the p53 protein shows that the RPA binding, proline-rich regulatory, DNA-binding, and oligomerization domains are necessary for p53 action in both replication systems. These results indicate that in mouse cells the p53 protein could be inactive for the suppression of papovavirus replication.

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The kinetics of simian virus 40-induced progression of quiescent cells into S phase depend on four independent functions of large T antigen.

Journal of Virology ◽

10.1128/jvi.68.9.5496-5508.1994 ◽

1994 ◽

Vol 68 (9) ◽

pp. 5496-5508 ◽

Cited By ~ 36

Author(s):

A Dickmanns ◽

A Zeitvogel ◽

F Simmersbach ◽

R Weber ◽

A K Arthur ◽

...

Keyword(s):

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

S Phase ◽

Large T Antigen ◽

Quiescent Cells ◽

Independent Functions ◽

Kinetics Of

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WAF1 retards S-phase progression primarily by inhibition of cyclin-dependent kinases.

Molecular and Cellular Biology ◽

10.1128/mcb.17.8.4877 ◽

1997 ◽

Vol 17 (8) ◽

pp. 4877-4882 ◽

Cited By ~ 131

Author(s):

V V Ogryzko ◽

P Wong ◽

B H Howard

Keyword(s):

Mammalian Cells ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

S Phase ◽

In Vivo Studies ◽

Nuclear Antigen ◽

Phase Progression

The p21(WAF1/CIP1/sdi1) gene product (WAF1) inhibits DNA replication in vitro (J. Chen, P. Jackson, M. Kirschner, and A. Dutta, Nature 374:386-388, 1995; S. Waga, G. Hannon, D. Beach, and B. Stillman, Nature 369:574-578, 1994), but in vivo studies on the antiproliferative activity of WAF1 have not resolved G1-phase arrest from potential inhibition of S-phase progression. Here, we demonstrate that elevated WAF1 expression can retard replicative DNA synthesis in vivo. The WAF1-mediated inhibitory effect could be antagonized by cyclin A, cyclin E, or the simian virus 40 small-t antigen with no decrease in the levels of WAF1 protein in transfected cells. Proliferating-cell nuclear antigen (PCNA) overexpression was neither necessary nor sufficient to antagonize WAF1 action. Expression of the N-terminal domain of WAF1, responsible for cyclin-dependent kinase (CDK) interaction, had the same effect as full-length WAF1, while the PCNA binding C terminus exhibited modest activity. We conclude that S-phase progression in mammalian cells is dependent on continuing cyclin and CDK activity and that WAF1 affects S phase primarily through cyclin- and CDK-dependent pathways.

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Non-specific elongation of cell cycle phases by cycloheximide in rat 3Y1 cells, and specific reduction of G1 phase elongation by simian virus 40 large T antigen

Journal of Cell Science ◽

10.1242/jcs.91.2.295 ◽

1988 ◽

Vol 91 (2) ◽

pp. 295-302

Author(s):

A. Okuda ◽

G. Kimura

Keyword(s):

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

S Phase ◽

G1 Phase ◽

Protein Accumulation ◽

Large T Antigen ◽

Coding Region ◽

Sv40 Large T Antigen ◽

In Cells

Partial inhibition of protein synthesis by cycloheximide caused prolongation of G1, S and G2 phases in rat 3Y1 fibroblasts. In cells expressing simian virus 40 (SV40) large T antigen, by infection with SV40 in the previous generation, the prolongation of G1 phase in the presence of cycloheximide was suppressed. However, the prolongation of S and G2 phases in the presence of cycloheximide was not suppressed in cells expressing large T antigen, by infection with SV40 in the current generation. Similarly, when density-arrested cells (cells in G0 phase) were infected with SV40 (either wild-type strain or a mutant deleted in the unique coding region for small t antigen) and reseeded sparsely in the presence of cycloheximide, the cycloheximide-induced delay of entry into S phase was suppressed. In this case, the reduction in [35S]methionine incorporation, that in protein accumulation and that in cell volume increase, were not surmounted by SV40 infection. In T-antigen-negative cells, all the regions in G1 phase seemed to be sensitive to cycloheximide, i.e. they suffered elongation. These results suggest that, in comparison with cells that enter S phase by the action of growth factors, cells expressing large T antigen can enter S phase more efficiently through a quite different process.

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p21cip1 Degradation in Differentiated Keratinocytes Is Abrogated by Costabilization with Cyclin E Induced by Human Papillomavirus E7

Journal of Virology ◽

10.1128/jvi.75.13.6121-6134.2001 ◽

2001 ◽

Vol 75 (13) ◽

pp. 6121-6134 ◽

Cited By ~ 37

Author(s):

Francisco Noya ◽

Wei-Ming Chien ◽

Thomas R. Broker ◽

Louise T. Chow

Keyword(s):

Human Papillomavirus ◽

Dna Replication ◽

Cyclin E ◽

Human Keratinocytes ◽

S Phase ◽

Brdu Incorporation ◽

Primary Human Keratinocytes ◽

Differentiated Cells ◽

Hpv E7 ◽

Cyclin E2

ABSTRACT The human papillomavirus (HPV) E7 protein promotes S-phase reentry in a fraction of postmitotic, differentiated keratinocytes. Here we report that these cells contain an inherent mechanism that opposes E7-induced DNA replication. In organotypic raft cultures of primary human keratinocytes, neither cyclin E nor p21cip1 is detectable in situ. However, E7-transduced differentiated cells not in S phase accumulate abundant cyclin E and p21cip1. We show that normally p21cip1 protein is rapidly degraded by proteasomes. In the presence of E7 or E6/E7, p21cip1, cyclin E, and cyclin E2 proteins were all up-regulated. The accumulation of p21cip1 protein is a posttranscriptional event, and ectopic cyclin E expression was sufficient to trigger it. In constract, cdk2 and p27kip1 were abundant in normal differentiated cells and were not significantly affected by E7. Cyclin E, cdk2, and p21cip1 or p27kip1 formed complexes, and relatively little kinase activity was found associated with cyclin E or cdk2. In patient papillomas and E7 raft cultures, all p27kip1-positive cells were negative for bromodeoxyuridine (BrdU) incorporation, but only some also contained cyclin E and p21cip1. In contrast, all cyclin E-positive cells also contained p27kip1. When the expression of p21cip1 was reduced by rottlerin, a PKC δ inhibitor, p27kip1- and BrdU-positive cells remained unchanged. These observations show that high levels of endogenous p27kip1 can prevent E7-induced S-phase reentry. This inhibition then leads to the stabilization of cyclin E and p21cip1. Since efficient initiation of viral DNA replication requires cyclin E and cdk2, its inhibition accounts for heterogeneous viral activities in productively infected lesions.

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