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Molecular and Cellular Biology, February 2005, p. 1013-1024, Vol. 25, No. 3
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.3.1013-1024.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Human Papillomavirus Oncoprotein E7 Targets the Promyelocytic Leukemia Protein and Circumvents Cellular Senescence via the Rb and p53 Tumor Suppressor Pathways

Oliver Bischof, Karim Nacerddine, and Anne Dejean*

Nuclear Organisation and Oncogenesis Unit, INSERM U 579, Institut Pasteur, Paris, France

Received 20 February 2004/ Returned for modification 27 April 2004/ Accepted 29 October 2004


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ABSTRACT
 
Cellular senescence can be triggered by a variety of signals, including loss of telomeric integrity or intense oncogenic signaling, and is considered a potent, natural tumor suppressor mechanism. Previously, it was shown that the promyelocytic leukemia protein (PML) induces cellular senescence when overexpressed in primary human fibroblasts. The mechanism by which the PML IV isoform elicits this irreversible growth arrest is believed to involve activation of the tumor suppressor pathways p21/p53 and p16/Rb; however, a requirement for either pathway has not been demonstrated unequivocally. To investigate the individual contributions of p53 and Rb to PML-induced senescence, we used oncoproteins E6 and E7 from human papillomaviruses (HPVs), which predominantly target p53 and Rb. We show that E7, but not E6, circumvents PML-induced senescence. Using different E7 mutant proteins, dominant negative cyclin-dependent kinase 4, and p16 RNA interference, we demonstrate that Rb-related and Rb-independent mechanisms of E7 are necessary for subversion of PML-induced senescence and we identify PML as a novel target for E7. Interaction between E7 and a functional prosenescence complex composed of PML, p53, and CBP perturbs transcriptional activation of p53, thus highlighting a significant effect also on the p53 tumor suppressor pathway. Given the importance of HPV in the pathogenesis of cervical cancer, our results warrant a more detailed analyses of PML in HPV infections.


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INTRODUCTION
 
Cellular senescence was first recognized by Hayflick and Moorhead (26) as a mechanism that limits the life span of primary human fibroblasts in culture. We now know that this mechanism, referred to as replicative senescence, is linked to the integrity of telomeres (51). Stimuli that do not affect telomeric integrity were also shown to lead to a permanent cell cycle arrest showing features of cellular senescence (49). These stimuli irreversibly arrest growth after only a few cell divisions, and the phenomenon therefore is referred to as premature senescence. Stimuli leading to premature senescence include DNA damage (55) and intense mitogenic signaling, as, for example, by oncogenic Ras, Raf1, or MEK (35, 50, 63).

The commonality among factors inducing premature senescence is that all have the potential to cause or contribute to cancer. Thus, cellular senescence appears to be a mechanism for irreversibly arresting the growth of cells at risk for tumorigenesis (9). The most compelling link between cellular senescence and tumor suppression is their mutual dependence on tumor suppressor genes such as these p16, p21, p53, and Rb genes (8).

Recent results indicate that another candidate tumor suppressor, promyelocytic leukemia protein (PML), is involved in controlling cellular senescence (5, 22, 41). The PML gene was initially identified in patients with acute promyelocytic leukemia, in whom it is fused to the retinoic acid receptor {alpha} gene as a result of the t(15;17) chromosomal translocation. The expression of the PML-retinoic acid receptor {alpha} fusion protein is sufficient to induce leukemia (37), and complete loss of PML function results in increased cell proliferation and tumor susceptibility (57). PML exists in at least seven isoforms, designated PML I to VII, which are all generated by differential splicing (31). The specific functions of the splice variants most likely rely on their C-terminal sequences. Ectopic expression of only one PML isoform, PML IV, elicits cellular senescence in primary human fibroblasts, whereas other PML isoforms fail to do so (5). PML IV-induced senescence is characterized by an increase in the levels of p21/p53 and p16/Rb tumor suppressor proteins (5, 22, 41). A hallmark for PML IV-induced senescence is the formation of a functional complex between p53, CBP, and PML (41). Complex formation results in acetylation and transcriptional activation of p53 followed by induction of p53 response genes involved in cell cycle arrest and senescence. Moreover, the senescence response is independent of intact PML-associated nuclear bodies (NBs) (5). The integrity of these subnuclear structures is compromised in viral infections, hinting at a role for PML in antiviral response mechanisms (47). Consistent with this idea is the finding that PML gene expression is upregulated by interferons (45). Other reports provided evidence for a connection between NBs and DNA virus replication, and from these studies it appears to be a general feature of nuclear-replicating DNA viruses that they associate with NBs (21).

Human papillomaviruses (HPV) are among those DNA viruses that appear to utilize NBs as their replication centers (11, 53). The E6 and E7 early gene products of high-risk HPV type 16 (HPV-16) stimulate cellular progression through the G1/S transition despite the presence of various G1 arrest signals in their host cells. This suggests that both viral proteins have evolved to interact with key factors of the cell cycle machinery (54). The best-described target for HPV-16 E6 is the p53 tumor suppressor protein. Binding of E6 to p53 promotes degradation through a ubiquitin-dependent mechanism (46). E7 is best known for its interaction and degradation of the Rb family of proteins (Rb, p107, and p130) (6, 18). The E7 protein can be divided into three domains: conserved region 1 (CR1, residues 1 to 15); conserved region 2 (CR2, residues 16 to 38), which contains the Rb-binding motif LXCXE; and conserved region 3 (CR3, residues 39 to 98), forming a Zn finger structure. E7 is the major transforming protein of HPV. Recently, however, the one-dimensional perception of E7 based solely on Rb function has been called into question. Mutational analyses of E7 have provided evidence that high-affinity binding to Rb is not sufficient for transformation. Several E7 mutants, particularly those with mutations in the CR3 region, retain the ability to bind to Rb, yet they are transformation deficient. These results led to the conclusion that E7 targets multiple regulators of the G1/S transition for bypassing cell cycle arrest induced by DNA damage, differentiation, or senescence (28, 38, 65).

In this study, we set out to gain insights into the individual contributions of p53 and Rb to PML IV-induced cellular senescence in primary human fibroblasts. Utilizing HPV oncoproteins E6 and E7 and different E7 mutant proteins as well as dominant negative cyclin-dependent kinase (Cdk) 4 and p16 RNA interference, we showed that PML IV simultaneously activates the p53 and Rb tumor suppressor pathways to ensure complete dropout from the cell cycle. Interestingly, we found that E7 alone is able to inhibit PML IV-induced senescence due to its ability to simultaneously disrupt Rb, p53/CBP, and PML functionality through direct interaction.


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MATERIALS AND METHODS
 
Vectors, viruses, and cell culture. Retroviral vectors pBABE-puro and pLXSN-neo were as reported previously (5). pLXSN-E6, -E7, and -E6E7 were a gift of D. Galloway; C-terminal E7-FLAG, CR2, and CR3 mutant E7 constructs were kindly provided by T. Kouzarides and J. Choe (7, 34); pWZLHy-Cdk 4R24C was a gift of S. Lowe (50); and small interfering RNA (siRNA)-p16 vector retro-pRSHyg-16 was a gift of R. Agami (56). Wild-type cDNAs for PML III and IV (new nomenclature) were as described previously (31); pBABE-RasV12, pBABE-PML IV, and pBABE-PML III were as reported previously (5). Genes were cloned by standard procedures into retroviral vectors pBABE-puro and pLXSN-neo, pcDNA3 (Invitrogen), or pSG-5 (Stratagene). Infections of primary fibroblasts by retrovirus-mediated gene transfer were performed with Phoenix packaging cells as previously described (5). At 24 to 48 h postinfection, cells were selected with 3 µg of puromycin per ml (pBABE), 400 µg of G418 per ml (pLXSN), or 100 µg of hygromycin B per ml. Day 0 is defined as the time when all noninfected cells were dead after pharmaceutical selection. WI38 fibroblasts (population doubling 13) and U2OS cells were obtained and cultured as previously described (5).

Senescence analysis. Senescence was assessed with several assays. For growth curves, cells were plated in triplicates at 2.0 x 104 per well in 12-well plates. Relative cell numbers were estimated at various time points by using a crystal violet incorporation assay, and population doublings (PDs) were calculated by using the equation n = (log10F – log10I) x 3.32, where n is the PD, F is the number of cells at the end of one passage, and I is the number of cells that seeded at the beginning of one passage. For life span studies, cells were subcultured when 70 to 80% confluent at 2 x104/cm2. Proliferative capacity was assessed by labeling cells for 72 h with [methyl-3H]thymidine (10 µCi/ml) followed by autoradiography to determine the percent radiolabeled nuclei (5). These cells were also costained for senescence-associated ß-galactosidase (SA-ß-Gal), as described previously (16).

Immunoprecipitation, immunoblotting, and antibodies. Cells were extracted either in buffer (20 mM Tris-HCl [pH 7.6], 200 mM NaCl, 1 mM EDTA, 0.5% NP-40, 1 mM dithiothreitol) supplemented with 5 µM trichostatin A (Calbiochem), 1 mM nicotinamide (Sigma), and protease inhibitors (Boehringer) or as described by Dignam et al. (15) with minor modifications. For immunoprecipitations, equal amounts of lysate (containing 5 to 10 mg of total cellular protein or 50 mg of nuclear lysates in the case of Caski cells) were incubated with 2 µg of either mouse anti-p53 antibody (DO1), mouse anti-E7 antibody (ED17), mouse anti-FLAG antibody (M2), rabbit anti-PML (no. 83) (58), or rabbit anti-CBP antibody (A22) plus protein A/G beads (Pierce) overnight at 4°C. Precipitates were washed extensively in extraction buffer, and bound complexes were eluted with 2x sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer with or without ß-mercaptoethanol and separated by SDS-4 to 15% PAGE. Immunoblotting was performed according to standard procedures. When immunoprecipitation was not performed, total protein lysates were prepared in 2x SDS-PAGE sample buffer, and 50 µg of protein was separated by SDS-4 to 15% PAGE. Antibodies were detected by chemiluminescence with Galactostar (Tropix). The following primary antibodies were used: rabbit polyclonal anti-p16 (H156) and anti-p21 (C19) and mouse monoclonal anti-p53 (DO-1), anti-CBP (C1), and anti PML (PG-M3) (Santa Cruz); rabbit polyclonal anti-acetylated lysine and anti-phospho-Rb Ser780, 795, and 807/11 (Cell Signaling); mouse monoclonal antitubulin (Ab1) (Calbiochem); mouse monoclonal anti-Rb (G3-245) (PharMingen); and rabbit-polyclonal anti-FLAG (Sigma).

Protein expression and in vitro binding assays. Glutathione S-transferase (GST), GST-E7, or GST-E7 mutant proteins were expressed in BL21DE Escherichia coli cells and purified according to standard procedures. Untagged recombinant E7 proteins were produced by introducing a TEV protease site into pGEXT1 and releasing GST-free E7 by treatment with TEV protease (17). [35S]methionine-labeled in vitro-translated proteins were prepared with the TNT-coupled reticulocyte lysate kit (Promega, Madison, Wis.). GST pull-down assays were performed on in vitro-translated, [35S]methionine-labeled proteins as described previously (48). Bound complexes were washed under stringent conditions, eluted, resolved by SDS-PAGE, and analyzed by autoradiography.

In vivo GST pull-down assays. Nuclear lysates were prepared as described by Dignam et al. (15) from WI38 fibroblasts expressing PML IV and pretreated with DNase I (160 U/ml) for 30 min at 37°C, after which ethidium bromide was added to 250 ng/ml. Pretreated nuclear lysates were incubated with either GST or GST-E7 glutathione-Sepharose for 4 h at 4°C. The resin was washed with 10 volumes each of phosphate-buffered saline (PBS) plus 0.5% NP-40, PBS plus 0.2% NP-40, and PBS. Proteins were eluted by boiling in 2x SDS-PAGE sample buffer. Eluted proteins were analyzed by SDS-PAGE and Western blotting for PML.

Immunofluorescence. Cells were seeded onto coverslips in six-well dishes at a density of 3 x 105 cells/plate and either transfected 24 h later with the indicated plasmids by using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions or left untreated. Cells were cultured for 1 to 2 days, fixed, and stained with primary and secondary antibodies as described previously (5). For each colocalization experiment, parallel single labelings were performed to guard against the possibility of immunological or optical cross talk. The primary antibodies used were mouse monoclonal anti-E7 (ED17) (Santa Cruz), mouse monoclonal anti-E7 (Zymed), and rabbit polyclonal anti-PML (no. 83) (58). Coverslips were mounted in VectaShield containing DAPI (4',6'-diamidino-2-phenylindole) (Vector Laboratories) at 0.4 µg/ml to visualize nuclear DNA and were viewed by single-laser confocal microscopy. Images were captured with a charge-coupled-device camera and merged by using Canvas (Deneba).

Reporter assays. Cells (5 x 103 to 8 x 103/cm2 on 35-mm-diameter dishes) were transfected with 0.6 µg of p21-luciferase (p21-Luc) (20) and 0.1 µg of pCMV-ß-Gal reporters by using Lipofectamine Plus (Invitrogen). ß-Galactosidase and luciferase activities were measured with the Galacto-Star (Tropix) and Luciferase Assay Systems (Promega) luminescence assay kits according to the suppliers' instructions. Luciferase activities were normalized to ß-galactosidase activities.


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RESULTS
 
E7 overcomes PML IV-induced cellular senescence. To selectively distinguish between p53 and Rb pathways in eliciting PML IV-induced cellular senescence, we used the HPV oncoproteins E7 and E6. The main oncogenic activity of E6 has been attributed to its ability to promote degradation of p53 via the proteasome (46), while E7's main inhibitory function relies on its interaction with the Rb family of proteins (6). Normal human primary lung WI38 fibroblasts were transduced first with the retroviral vector pLXSN (LX) or its derivatives encoding HPV-16 E7 or E6. Infected cells were kept under neomycin selection for 10 days and subsequently superinfected either with pBABE expressing PML IV, RasV12, or empty vector (B0), followed by puromycin selection. At this point, (day 0, PD = 0), we started monitoring the proliferative properties of all infected cell populations by growth curves and incorporation of [methyl-3H]thymidine into DNA.

Cells constitutively expressing PML IV, E6/PML IV, Ras, E6/Ras, or E7/Ras rapidly arrested growth (Fig. 1A). The fraction of cells that synthesized DNA over a 3-day interval dropped from >75 to <10% by 10 to 15 days postinfection in all cases (Fig. 1B). At the same time, >70% of all cell populations expressed SA-ß-Gal (Fig. 1C). In contrast, cells expressing PML IV in combination with E7 proliferated in a manner that was indistinguishable from that of E7 control cells and produced no SA-ß-Gal (Fig. 1A to C). Interestingly, expression of E7 together with PML IV in fibroblasts led to alterations in cell morphology that were similar to those seen in the presence of PML IV alone, indicating that some features of PML signaling are not perturbed by the presence of E7 (Fig. 1D).



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  		FIG. 1. Effects of E6 and E7 on cell proliferation, DNA synthesis, and induction of cellular senescence by PML IV and Ras. (A) Growth curves of normal WI38 human fibroblasts expressing combinations of PML IV, oncogenic RasV12, or empty vector (B0) plus E6, E7, or empty vector (LX). After drug selection, the number of PDs over the indicated period of time was determined. Day 0 is the first day after selection. PDs foreach time point are the mean values from triplicates. (B) DNA synthesis and (C) SA-ß-Gal expression in normal human cells. WI38 cells were infected and selected as described in (A). After selection, [3H]thymidine was added at the indicated time points and left for 3 days (B), and cells were subsequently histochemically stained for SA-ß-Gal expression (C) followed by autoradiography as described in Materials and Methods. A minimum of 200 cells were counted to determine the percentages of positive SA-ß-Gal expression and radiolabeled nuclei (LN). A cell was considered SA-ß-Gal positive only when it was not radiolabeled. (D) Morphology of cells expressing E7/PML IV or LX/PML IV. Cells were photographed under phase-contrast optics at day 10 postselection. Note the similar alterations in cell morphology in LX/PML IV- and E7/PML IV-expressing cells. (E) Inhibition of the Rb tumor suppressor pathway does not rescue PML-induced senescence. Growth curves of WI38 fibroblasts expressing Cdk 4R24C or siRNA-p16 plus empty vector B0 or PML IV are shown. After drug selection, the number of PDs was determined as in (A). Shown are also the protein levels for Cdk 4 and p16 in the respective cell populations.

To rule out the possibility that inhibition of PML IV-induced senescence by E7 is merely the consequence of destabilizing the Rb tumor suppressor pathway, we inactivated this pathway by two other ways. We double infected cells with a combination of either pWZLHy-Cdk 4R24C and PML IV or a retroviral siRNA-p16 construct (pRS-Hyg-p16) and PML IV. Cdk 4R24C is a mutant Cdk4 protein that is insensitive to p16 (61) and that cooperates with cyclin D1 to prevent cell cycle arrest induced by p16 overexpression (3). Cyclin D1 and Cdk 4R24C inactivate the p16/Rb pathway by phosphorylating Rb in a p16-insensitive manner. In contrast to E7/PML IV-expressing cells, cells coexpressing Cdk 4R24C/PML IV or siRNA-p16/PML IV ceased growth as quickly as cells expressing PML IV alone (Fig. 1E).

Together, these results demonstrate that E7 alone, but not E6, bypasses PML IV-induced senescence. For this process to occur, E7 must simultaneously target the p16/Rb and p53 pathways, as independent disruption of either pathway alone by either dominant negative Cdk 4, siRNA-p16, or E6 is not sufficient to override the senescence response elicited by PML IV. Furthermore, neither E6 nor E7 can inhibit Ras-induced cellular senescence, which therefore must differ mechanistically from senescence mediated by PML IV.

Rb-related and Rb-independent activities of E7 are necessary to block senescence. A number of studies have demonstrated the importance of the E7-CR2 region for destabilization of Rb and transformation of a number of cell types. However, there is evidence that high-affinity binding of E7 to Rb is not sufficient for extension of the cell life span and effective transformation. In particular, regions located in the Zn finger-containing C terminus of E7 (CR3) have also been shown to contribute to the transforming potential of E7 (13, 19, 27, 32, 43).

To determine the involvement of the CR2 and CR3 regions of the E7 protein for impeding PML IV-induced senescence, we transduced WI38 fibroblasts with different well-characterized E7 mutant retroviral constructs. These mutant proteins included three with mutations located in the CR3 region that target Rb for degradation (LX-E7C91G, LX-E7L82.83R, LX-E7L67R) (hereafter called CR3 mutants) and one with a mutation situated in the CR2 region that is deficient for Rb binding (LX-E7C24G) (Fig. 2A). Following primary infection with E7 or E7 mutant constructs, cells were superinfected with pBABE-PML IV or empty vector. After drug selection, the effect on proliferation was determined as described above, and the functionality of E7 and E7 mutant proteins was assessed by quantitating the overall level as well as the phosphorylation status of Rb by Western blot analysis (Fig. 2B). Expression of E7 alone or in combination with PML IV significantly prolonged the life span of fibroblasts, by 10 to 13 population doublings compared to empty vector controls. In contrast, fibroblasts expressing E7 mutant proteins alone showed a lower, yet significant, life span extension of three to six PDs. While E7/PML IV-expressing fibroblasts proliferated unfettered, cells expressing E7 mutants in combination with PML IV ceased proliferation as rapidly as cells expressing PML IV alone. These data confirm that E7 relies on an intact CR2 and CR3 domain for full functionality and that both Rb-related and Rb-independent activities of E7 contribute to inhibition of PML IV-induced senescence.



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  		FIG. 2. Rb-related and Rb-independent mechanisms of E7 are necessary to block PML IV-induced senescence and extend life span. (A) Diagram of E7, showing the locations of substitution mutations. (B) Growth curves of WI38 fibroblasts expressing either empty vector LX or its derivatives expressing E7 or mutant E7 genes superinfected with empty vector pBABE (B0) or its derivative expressing PML IV. After drug selection, the number of PDs over the indicated period of time was determined (upper panel). Also shown is the overall level and phosphorylation status of Rb in these cell populations as determined by Western blotting (lower panel). (C) Protein levels of p16, p21, and p53 in cells infected with the indicated viruses at 10 days postselection. Tub, tubulin.

Cyclin-dependent kinase inhibitors (CKIs) p16 and p21 inhibit Cdks 2, 4, and 6, thus keeping Rb in its functional, hypophosphorylated state. Progressive Rb phosphorylation during G1 phase forces cells to enter S phase and complete the cell cycle. Additionally, p21 is a direct reporter for transcriptionally active p53. Given the importance of p16, p21, and p53 in cell cycle regulation and senescence, we determined the levels of these proteins in cells expressing PML IV, E7, or E7/PML IV by Western blotting (Fig. 2C). Expression either of PML IV or E7 alone led to similar increases in p16, p21, and p53 levels (compare lanes 2 and 4 to lane 1), an effect that was not further enhanced upon coexpression of PML IV together with E7 (lane 3). Since increased expression of these proteins normally correlates with the onset of senescence, this observation implies that p16, p21, and p53 should be kept in an inactive state in E7-expressing cells. Indeed, we found that Cdk 2 and Cdk 4 activities were maintained in E7/PML IV-expressing cells irrespective of the high levels of p16 and p21, whereas cells expressing PML IV alone (data are available upon request) or together with the E7 mutant genes (data not shown) exhibited a dramatic decrease in Cdk 2 and Cdk 4 activities. It has been proposed that the sustained Cdk 2 activity in E7-expressing cells is a consequence either of a direct inhibition of p21 by E7 (24, 33) or of a delocalization of p21 to the cytoplasm (60). Our findings suggest that in human fibroblasts, E7 renders p21 dysfunctional through direct interaction rather than cytoplasmic delocalization (data are available upon request). Why Cdk 4 activity is not reduced in E7-expressing cells is an open question and is currently under investigation.

Taken together, our results show that only fully functional E7 is able to create and maintain a cell state conducive for proliferation and for the subversion of PML IV-induced premature senescence.

E7 interferes with PML IV-promoted acetylation of p53. One hallmark of PML IV-induced senescence is CBP-mediated acetylation of p53 through formation of a PML IV/p53/CBP ternary complex, thus turning p53 into a transcriptional activator (5, 41). Consequently, we asked whether E7 influences these processes. To address this question, we coimmunoprecipitated CBP and p53 in the presence or absence of E7 in PML IV-expressing fibroblasts. As a negative control, we used cell lysates from PML III-infected cells. In contrast to PML IV, PML III does not elicit premature senescence upon overexpression in primary fibroblasts (5). As shown in Fig. 3A, the amount of p53 precipitated by CBP is strongly reduced in E7/PML IV and PML III cell lysates compared to PML IV lysates. Moreover, acetylated p53 is completely absent in E7/PML IV- and PML III-expressing cells. Performance of this experiment with cells coexpressing PML IV and E7C24G or E7L82.83R showed that neither E7 mutant protein affected p53 acetylation (Fig. 3B). To demonstrate whether E7 also disturbs formation of PML/CBP or PML/p53 complexes, we coimmunoprecipitated these proteins in the presence or absence of E7 and mutant derivatives thereof. As can be seen in Fig. 3C, whereas E7 slightly disrupts the interaction between PML and CBP, the binding between PML and p53 seems to be somewhat enhanced. To analyze this phenomenon more precisely, we performed in vitro pull-down assays with GST-p53 and in vitro-translated PML IV protein in the presence of increasing amounts of recombinant E7 protein. Indeed, we could confirm that E7 is able to tighten the complex between p53 and PML (Fig. 3D). The functional consequence of this increased stability remains to be determined.



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  		FIG.3. E7 perturbs a functional complex between PML, p53, and CBP. (A) Nuclear lysates were prepared from WI38 fibroblasts infected with either PML III/LX, PML IV/LX, or PML IV/E7 and immunoprecipitated (IP) with either anti-CBP or anti-p53 antibody. Immunoprecipitated complexes were collected on protein A/G beads and analyzed by Western blotting (WB) with antibodies specific for CBP, p53, or acetylated lysine. IgG-H+L, immunoglobulin G heavy plus light chains. (B) Nuclear lysates were prepared from WI38 fibroblasts infected with either PML IV/E7, PML IV/E7C24G, or PML IV/L82.83R and immunoprecipitated with anti-p53 antibody. Immune complexes were collected on protein A/G beads and analyzed by Western blotting with an antibody specific for p53 or acetylated lysine. (C) Nuclear lysates were prepared from WI38 cells infected either with PML IV/LX, PML IV/E7, PML IV/E7C24G, or PML IV/L82.83R and immunoprecipitated with anti-PML antibody. Immune complexes were collected on protein A/G beads and analyzed by Western blotting with an antibody specific for PML, p53, or CBP. Expression of PML, p53, and E7 was evaluated by immunoblotting of total nuclear lysates. (D) Radiolabeled, in vitro-translated PML IV was incubated with GST-p53 and increasing amounts of recombinant wild-type E7 or E7 L82.83R mutant (mt) proteins (1, 10, and 100 ng). (E) WI38 normal human fibroblasts were infected with the indicated retroviral combinations, selected, and transiently cotransfected when growth arrested with a p21-Luc reporter vector together with a pCMV-ß-galactosidase normalization vector. Cells were assayed for ß-galactosidase and luciferase reporters. Normalized p21 reporter activity is luciferase/ß-galactosidase activity, with the activity of p21-Luc in proliferating LX/B0 cells set at 1. Shown are the averages and standard deviations from three independent experiments. Within each experiment, transfections were done in triplicate. (F) WI38 normal human fibroblasts were infected with the indicated retroviral combinations and selected, and the number of PDs over the indicated period of time was determined.

Next, we investigated whether the observed impairment of p53 acetylation in E7/PML IV cells affected transcriptional activation of p53. To this end, we transiently transfected PML IV-arrested cells and matched E7/PML IV- or mutant E7/PML IV-infected cells with a p53 reporter gene. PML IV alone or in conjunction with the E7 mutant genes activated the promoter ~5-fold compared to control cells; however, this activation was absent in E7/PML IV-expressing cells (Fig. 3E).

Given the effect of E7 on the transcriptional activation of p53 by PML IV, we then asked whether E7 mutants that are able only to inactivate Rb and not prevent p53 activation would regain the ability to overcome PML-induced senescence if p53 was simultaneously removed. To this end, we triple infected normal human fibroblasts with viruses expressing a p53 dominant negative mutant, p53V175A (p53dn), thereby deleting p53 function; E7L82.83R, thus inactivating Rb; and PML IV. As illustrated in Fig. 3F, the combination of the p53dn and E7 mutant proteins rendered cells refractory to PML IV-induced senescence, thus confirming that E7, in addition to blocking the Rb pathway, also interferes with activation of the p53 tumor suppressor pathway.

Together, these results suggest that E7 perturbs the trimeric complex between PML, CBP, and p53 by modulating the parameters of binding between the different complex components. Perturbation of the PML/p53/CBP complex leads to the loss of transcriptional activation of p53 and of the consequent induction of p53-responsive genes, such as that for p21, which are pivotal for the establishment of senescence. For this to occur, both intact CR2 and CR3 domains of E7 seem to be necessary.

PML and E7 colocalize and interact in vitro as well as in vivo. In light of the results described above, we next investigated whether PML and E7 localize within the same cellular compartment. To this end, U2-OS cells were cotransfected with C-terminally FLAG-tagged E7 (E7-FLAG) and PML IV. In agreement with previous studies (4, 25), E7 shows a predominantly nuclear, diffuse distribution with a number of dot-like structures that we could show coincided with the PML-associated NBs (Fig. 4A). Similar results were obtained with fibroblasts infected with retroviruses expressing an untagged E7 protein and PML IV (Fig. 4B). The colocalization of PML and E7 in the NBs was also evident at the level of endogenous proteins in Caski cervical cancer cells, which naturally express the E7 protein (Fig. 4C). However, in this case, association of E7 with the PML NBs was observed in only a subset of cells. Colocalization could be enhanced when Caski cells were expressing PML IV exogenously (Fig. 4D).



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  		FIG. 4. E7 and PML colocalize. (A) U2-OS cells were cotransfected with pcDNA3.1-PML IV and pSG5-E7-FLAG and costained with anti-PML (green) and anti-E7 (red) antibodies. (B) WI38 fibroblasts were retrovirally infected with PML IV and untagged E7 and costained with anti-PML (green) and anti-E7 (red) antibodies. (C) Caski cells were costained with anti-PML (red) and anti-E7 (green) antibodies. (D) Caski cells were transfected with pcDNA3.1-PML IV and costained with anti-PML (green) and anti-E7 (red) antibodies. All samples were analyzed by laser scanning microscopy.

Next, we examined whether the two proteins physically interact in vivo. Primary fibroblasts were transduced sequentially with PML IV together with E7-FLAG or FLAG-green fluorescent protein (GFP) as a negative control. Coimmunoprecipitation assay with an anti-FLAG antibody followed by Western blot analysis with an anti-PML antibody revealed that a significant amount of PML coimmunoprecipitated with E7 but not with GFP (Fig. 5A). To further verify the interaction, we performed a GST-E7 pull-down assay with PML IV-expressing cell lysates. The GST-E7 fusion protein efficiently pulled down PML, while the GST protein alone did not (Fig. 5B). To enhance the physiological relevance of the observed interaction between PML and E7, coimmunoprecipitation experiments were carried out with Caski cells. As depicted in Fig. 5C, endogenous PML and E7 could be reciprocally brought down by each other, demonstrating the physical association of the two proteins under physiological conditions.



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  		FIG. 5. E7 and PML physically interact. (A) Nuclear lysates were prepared from WI38 fibroblasts infected with either LX-E7-FLAG/pBABE-PML IV or LX-FLAG-GFP/pBABE-PML IV and immunoprecipitated (IP) with anti-FLAG antibody. Immunoprecipitated complexes were collected on protein A/G beads and were analyzed by Western blotting (WB) with antibodies specific for FLAG and PML. (B) In vivo GST-pull down assay. Nuclear lysates were prepared from WI38 fibroblasts expressing PML IV and affinity purified on GST-E7 or GST beads. Bound protein complexes were analyzed by Western blotting with using an anti-PML antibody. (C) Nuclear lysates were prepared from Caski cells and immunoprecipitated with either anti-E7, anti-PML, or control immunoglobulin G (IgG) antibodies. Immunoprecipitated complexes were analyzed by Western blotting with antibodies specific for E7 and PML. Asterisks mark IgG chains. (D) Radiolabeled, in vitro-translated PML IV or PML IV{Delta}CC was incubated with either recombinant GST-E7 or -E7 mutant fusion protein products. Bound complexes were analyzed by autoradiography. (E) Growth curves of WI38 fibroblasts expressing the indicated retroviral constructs. After drug selection, the number of PDs over the indicated period of time was determined. Also shown is the in vitro interaction between GST-HPV-16 E7, GST-HPV-11 E7, and GST-HPV-6 E7 fusion proteins and radiolabeled, in vitro-translated PML IV.

To map the regions required for complex formation between E7 and PML, we performed in vitro pull-down assays. GST fusion proteins of E7 and mutant protein C24G or L82.83R were incubated with in vitro-translated PML IV or PML IV{Delta}CC, which lacks the coiled-coil domain. This domain of PML provides a dimerization interface for protein-protein interactions (42). As shown in Fig. 5D, wild-type E7 and the C24G mutant proteins have similar affinities for PML IV, whereas the interaction with the CR3 mutant protein L82.83R (as well as with L67R and C91G [data not shown]) is decreased by 50%. Likewise, deletion of the coiled-coil domain in PML IV entirely abolished binding to E7. Of note, E7 displays affinity for all PML isoforms to various degrees, with PML I showing the strongest interaction (data not shown). Moreover, PML IV also interacts with E7 proteins from both medium-risk HPV-11 and low-risk HPV-6, and, interestingly, both proteins equally antagonize PML IV-induced senescence (Fig. 5E).

In conclusion, these results show that E7 is targeted to PML-associated NBs and that E7 and PML physically interact. This suggests that E7 can inhibit PML function through direct interaction. The Zn finger region of E7 and the coiled-coil domain of PML appear to be important domains involved in complex formation. Moreover, the interaction is not limited to high-risk HPV-16 E7 but also extends to the E7 proteins of medium- and low-risk HPV-11 and -6, respectively.


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DISCUSSION
 
We report here that E7 inhibits PML IV-induced senescence by targeting Rb for destruction and simultaneously interfering with PML-mediated transcriptional activation of p53 by CBP, thus emphasizing the importance for the two Rb and p53 tumor suppressor pathways in PML-induced senescence. These findings extend recent results implicating Rb in PML-dependent senescence (36). Moreover, we identify PML as a novel target for E7, thus adding a new layer to E7 function.

We and others have shown previously that ectopic expression of PML IV in primary fibroblasts engenders permanent growth arrest reminiscent of cellular senescence (5, 22, 41). Consistent with its role in tumor suppression, cellular senescence is regulated by a number of tumor suppressor proteins, the most important of which are the p53 and Rb proteins (9). Overexpression of PML IV in primary human fibroblasts leads to extensive posttranslational modifications of p53, including acetylation by CBP, transforming it into a potent transcriptional activator. Transcriptionally active p53 upregulates a number of genes involved in cell cycle arrest, such as that for CKI p21 (1). Upon simultaneous upregulation of CKI p16, Rb becomes hypophosphorylated (i.e., active) as a result of the concerted inhibition of Cdk activities by CKIs p16 and p21 (52). Active Rb, in turn, represses E2F-dependent transcription of genes involved in cell cycle progression (30). Direct evidence, however, for a strict requirement for either p53 or Rb in PML IV-induced senescence has not been attained to date. Therefore, to assess the individual contribution of each tumor suppressor pathway in PML senescence, we used the well-studied HPV-16 high-risk oncoproteins E6 and E7. The main mode of action of E7 is destabilization of pocket domain proteins Rb, p107, and p130, whereas E6 promotes p53 degradation (6, 40, 46). Given the previous evidence implicating p53 in PML IV-induced senescence, it was surprising to find that expression of E7 alone, but not that of E6 or of a p53 dominant negative mutant (data not shown), was sufficient to abolish PML IV-induced senescence. This suggested that, in primary human fibroblasts, the p53 tumor suppressor pathway might not be essential for execution of senescence elicited by PML IV. Subsequent experiments, however, using dominant negative Cdk 4 or siRNA-p16, which target only the p16/Rb pathway, revealed that under these conditions, PML IV was still able to trigger senescence. From these results, we concluded that E7 simultaneously affects several cell cycle regulators to bypass PML IV-induced senescence. Consequently, we decided to dissect the functions of E7 for blocking PML-induced senescence by utilizing several well-characterized mutant variants. The different E7 mutant proteins tested included those that target the Rb family of proteins (CR3 Zn finger mutants C91G, L82.83R, and L67R) and one (CR2 mutant C24G) that does not. Interestingly, we found that both types of mutants not only were unable to block the senescence elicited by PML IV but were also compromised in extending the life span of fibroblasts compared to wild-type E7. Moreover, medium-risk HPV-11 and low-risk HPV-6 E7 proteins, both of which have a largely diminished ability to bind and degrade Rb (39), antagonized PML IV-induced senescence as well as high-risk HPV-16 E7 does. Together, these findings clearly indicate that Rb-dependent as well as Rb-independent activities of E7 are essential for circumventing the senescence program triggered by PML IV. In addition, they provide evidence that both activities contribute to the extension of the fibroblast life span. Our results are in contrast to those of Mallette et al. (36), who suggested that only the Rb pathway is relevant for PML IV-induced senescence. To support the idea for a unique requirement for Rb in PML IV-induced senescence, those authors, however, employed a single Rb binding-deficient mutant and not others that still target Rb.

It is well established that the CR3 domain plays a pivotal role in the transactivation and transformation functions of E7 (7, 13, 19, 27, 32, 43). From those studies, it was inferred that destabilization of Rb is necessary, but not sufficient, for E7 to disrupt cell growth control and that therefore other cellular targets of E7 are required. We show here that one target of E7 is PML. Both proteins colocalize in NBs and are found in a complex in vivo as well as in vitro. Full complex formation between the two proteins requires an intact Zn finger domain of E7 and the helical coiled-coil domain of PML. Since the coiled-coil domain is shared by all PML isoforms, it is not surprising that the interaction between E7 and PML IV is not isoform specific but occurs also to various degrees with other PML isoforms. We therefore assume that the individual C-terminal extensions of the various isoforms modulate the affinity for E7, as has been shown also for the interaction between PML and p53 (23). Although only PML IV induces cellular senescence, we have shown previously that some or all isoforms must cooperate for senescence to occur, as PML–/– fibroblasts are resistant to PML IV-induced senescence (5). It is therefore possible that in targeting all PML isoforms simultaneously, E7 is potentially able to modulate several cell growth-suppressive signals emanating from PML.

Our results bear strong similarities to published results demonstrating that E7 alone is able to circumvent HPV E2-triggered senescence in HeLa cervical carcinoma cells by, at least in part, deactivating the Rb tumor suppressor pathway (12, 14, 29, 44, 59). Our data add a new layer to E7 function by showing that it also perturbs PML signaling, thereby potentially impinging on multiple other pathways involved in cell cycle control and amplifying the proproliferative potential of E7. In this respect, it would be interesting to determine in what way the E7 Zn finger mutants used in this study modulate the senescence response in the HPV E2-induced senescence system and how PML loss in this system affects the senescence outcome.

What is the consequence of E7 binding to PML? The simplest idea would be that by interacting with PML, E7 is able to alter the prosenescence and growth-suppressive properties of PML most effectively. E7 could achieve this by perturbing the function of PML-containing complexes directly and/or by recruiting these complexes, which harbor a plethora of different enzymatic activities (e.g., acetylases, deacetylases, and kinases), to new substrates, including other E7-binding proteins, thereby exerting its antisenescence properties. An example of the former idea is our finding concerning the perturbation of a trimeric prosenescence complex between PML, p53, and CBP. The formation of this complex was shown to be one outcome of ectopic expression of PML IV in human fibroblasts, as evidenced by coimmunoprecipitation and immunofluorescence experiments (22, 41). Accordingly, this complex could thus be seen as supporting CBP-mediated acetylation of p53 and the consequent transcriptional upregulation of p53 response genes that mediate cell cycle arrest (5, 41). We found that all of these events appear to be diminished in the presence of E7. Specifically, E7 is able to abolish PML IV-mediated acetylation and transcriptional activation of p53 by CBP. Moreover, coimmunoprecipitation experiments revealed that PML IV-stimulated interaction between CBP and p53 is strongly reduced by E7, as is the association between PML and CBP. By contrast, formation of the complex between PML and p53 seems to be enhanced in the presence of E7. Functional impairment of p53 is not achieved by either the E7C24G or the CR3 mutant protein. The result with the E7C24G mutant protein was somewhat unexpected, since this protein binds as strongly as wild-type E7 to PML. We believe, therefore, that this mutant may have distinct PML binding properties which still permit formation and functioning of the PML/p53/CBP complex. A similar mechanism has been proposed for the acetylase complex pCAF; it still binds to the Zn finger mutant protein E7L67R, yet its enzymatic activity is not affected by this binding (2). In conclusion, it appears that E7 alters the binding properties of the different complex components and that both the CR2 and -3 domains of the E7 protein seem to be essential for this to occur. Thus, the simplest explanation for the function of E7 in blocking PML IV-induced senescence is the destabilization of such a PML/p53/CBP complex. Given the physical interaction between E7 and PML as well as that between E7 and CBP (4), it is possible that E7 simultaneously targets PML and CBP to render the trimeric complex dysfunctional. Thus, whether E7 must target PML or CBP individually or together to exert its antisenescence properties remains an open question.

In Fig. 6, we propose a model, which incorporates the results from this study and alludes to potential functions for PML during HPV infections. In contrast to the model presented by Mallette et al. (36), we believe that PML activates the p53 and p16/Rb tumor suppressor pathways simultaneously and that depending on cellular context, either one or the other takes the lead. Our results suggest that E7 uses multiple mechanisms to interfere with cell cycle control. One mechanism is inactivation of the Rb tumor suppressor through the LXCXE motif-mediated interaction. The results presented here and elsewhere, however, indicate that E7 also targets molecules unrelated to Rb, including PML, p21/p27, and chromatin remodeling complexes, through its C-terminal Zn finger (2, 7, 24, 33, 34, 62). In sum, this enables E7 to alter expression of cellular genes that would otherwise be unfavorable for viral propagation. Several associations between PML and HPV have been reported (11, 25, 53). Those studies suggest that PML might play a positive role in the replication of HPV. On the other hand, it has been proposed that PML plays an active role in interfering with viral infection mechanisms (10). Thus, the mode of action of PML in influencing the course of the viral infection process is complex, and therefore more work is needed to elucidate the mechanisms underlying this process.



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  		FIG. 6. Schematic representation of the inhibitory mechanism of E7. E7 circumvents PML IV-induced cellular senescence by directly targeting Rb for degradation and simultaneously perturbing a prosenescence PML/p53/CBP complex, resulting in the loss of transcriptional activation of p53 and induction of p53 response genes necessary for the execution of senescence. By directly targeting PML, E7 may also compromise the host's antiviral defense response against HPV infection, thereby securing virus survival. Double arrows indicate protein-protein interactions, single arrows indicate processing, and blunt lines indicate inhibitory events.

Taking into account all of the available data on E7, it becomes clear that by targeting multiple cell cycle regulators, E7 is able to create a cell milieu that is conducive for virus replication and survival. HPV has been identified as a causative agent of at least 90% of cancers of the cervix (64). Given the prominent role that PML plays in tumor suppression and viral infections, it comes as no surprise that it represents a prime target for E7. Therefore, our findings point to the need for more-detailed analyses of the involvement of PML in cervical cancer.


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ACKNOWLEDGMENTS
 
We thank Denise Galloway, Joonho Choe, Tony Kouzarides, Scott Lowe, and Reuven Agami for kindly providing DNA constructs. We further thank Françoise Breitburd, Françoise Thierry, and members of our lab for sharing reagents and advice.

This work was supported by grants from the Association par la Recherche sur le Cancer, the Ligue Nationale Contre le Cancer, the Fondation de France, and the Pasteur-Negri-Weizmann Council. O. Bischof was supported by the European Economic Community (grant QLG1), and K. Nacerddine was supported by the Ministère de la Recherche et la Technologie.


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FOOTNOTES
 
* Corresponding author. Mailing address: Nuclear Organisation and Oncogenesis Unit, INSERM U579, Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France. Phone: 33 1 45 68 88 86. Fax: 33 1 45 68 89 43. E-mail: adejean{at}pasteur.fr. Back


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Molecular and Cellular Biology, February 2005, p. 1013-1024, Vol. 25, No. 3
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.3.1013-1024.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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