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Journal of Virology, September 2008, p. 8695-8705, Vol. 82, No. 17
0022-538X/08/$08.00+0     doi:10.1128/JVI.00579-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Human Papillomavirus Type 16 E7 Oncoprotein Associates with E2F6{triangledown}

Margaret E. McLaughlin-Drubin, Kyung-Won Huh,{dagger} and Karl Münger*

Infectious Diseases Division, The Channing Laboratory, Brigham and Women's Hospital, and Department of Medicine, Harvard Medical School, Boston, Massachusetts

Received 14 March 2008/ Accepted 19 June 2008


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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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INTRODUCTION
 
Cervical cancer is the second most common cancer among women worldwide and is the most common female cancer in developing countries (46, 55). The most significant risk factor for cervical cancer is infection with high-risk human papillomavirus (HPV) types (35); in fact, over 99% of all cervical cancers examined have been associated with high-risk HPVs (79). During carcinogenic progression, the HPV genome frequently integrates into the host chromosome, and as a result, E6 and E7 are the only viral proteins consistently expressed in HPV-positive cervical cancers. Therefore, carcinogenic progression is an accidental event and not part of the HPV life cycle. Instead, HPV-induced carcinogenesis is a result of the deregulated expression of HPV E6 and E7 (reviewed in reference 87). HPVs do not encode rate-limiting enzymes for DNA replication; thus, viral genome synthesis is linked to cellular DNA replication. During the normal life cycle, E6 and E7 function to establish and/or maintain a cellular milieu that supports viral genome replication (reviewed in reference 69). Because HPV genome replication and virion production occur in the most differentiated layers of the epithelium, E6 and E7 subvert pathways that signal growth arrest during differentiation; for instance, high-risk HPV E6 and E7 compromise the p53 and pRB tumor suppressors, respectively (24, 81). Aberrant cell proliferation in the absence of external stimuli is a key feature of many tumors, suggesting that the normal control of the cell cycle has been disturbed; indeed, the p53 and pRB tumor suppressor pathways are rendered dysfunctional in almost all human solid tumors (reviewed in reference 30). Therefore, a detailed mechanistic understanding of the aberrations in cell cycle regulation that are caused by HPV oncoproteins during cervical cancer development may yield insights that are generally relevant for the genesis of human solid tumors.

Part of the strategy developed by papillomaviruses to aberrantly activate cellular DNA synthesis machinery involves the functional abrogation of cellular inhibitory factors. The ability of HPV E7 to bind to and destabilize pRB and, consequently, inactivate pRB-E2F repressor complexes, allowing for uncontrolled cell cycle progression, is instrumental for this strategy (reviewed in reference 50). E2F proteins regulate genes that play key roles in cell cycle progression, nucleotide synthesis, DNA replication, DNA repair, and apoptosis (reviewed in reference 21). In addition to pRB, E7 also interacts with and inactivates the other RB family members, p107 and p130 (17, 23).

The ability of E7 to interact with pRB family members and other cell cycle regulators may thus be related to the capacity of E7 to uncouple differentiation from cell cycle progression by the modulation of the transcription of different subsets of genes, thereby establishing an environment that is more conducive to viral replication (13). In addition to disrupting pRB family member/E2F complexes by targeting pRB family members, E7 has also been reported to directly interact with E2F1 to activate E2F1-driven transcription (34). E7 from high- risk HPV types can also abrogate the inhibitory activities of the cyclin-dependent kinase inhibitors p21CIP1 (26, 36) and p27KIP1 (86).

Using tandem affinity purification (TAP) of HPV type 16 (HPV16) E7-associated host cellular protein complexes, we have discovered that the HPV16 E7 oncoprotein associates with E2F6. E2F6 lacks both the N-terminal homology domain found in E2F1 to E2F3 and the C-terminal domain, which contains the binding domain for pRB family members and a transactivation domain, that is common to E2F1 to E2F5 (11, 18, 22, 27, 48, 73). E2F6 functions as a repressor of transcription (11, 27, 48, 73); repression involves the association of E2F6 with polycomb group (PcG) transcriptional repressive complexes and histone methyltransferases (5, 54, 74). PcG complexes were first identified in Drosophila melanogaster as being regulators of Hox gene expression and have since been shown to be involved in a number of key cellular processes including cell fate determination and maintenance, stem cell maintenance, cell cycle control, genetic stability, and epigenetic heritability of transcriptional programs (reviewed in reference 63). E2F6 null mice display homeotic transformations of the axial skeleton (68) that are similar to those seen in PcG-deficient mice (77), indicating that E2F6 contributes to the biological activities of PcG proteins in vivo. Intriguingly, our TAP analyses showed that HPV16 E7 also associates with several PcG proteins including Bmi1, Mel18, Ring1, hpc2, and lethal 3 malignant brain tumor-like 2 (L3MBT-like 2) protein.

While it is known that E2F6 functions as a transcriptional repressor through its association with PcG complexes, the exact biological role(s) of E2F6 is not fully understood. Numerous studies have implicated E2F6 in the regulation of cell growth and proliferation, including one study that demonstrated that E2F6 overexpression in U2OS cells led to an arrest of cells in S phase (11). In contrast, asynchronously growing NIH 3T3 cells were unaffected by E2F6 overexpression, while exogenous E2F6 inhibited S-phase entry of quiescent NIH 3T3 cells stimulated to exit G0 (28). Additionally, an E2F6-PcG complex, E2F6-G0, has been shown to be associated with E2F target promoters in G0, suggesting an involvement of this complex in gene repression in quiescent cells and, thus, G0 maintenance (54). However, E2F6 is expressed at all cell cycle stages, suggesting a functional role beyond that in G0 or S phase. Experiments implicating E2F6 in the pRB-independent repression of transcription of G1/S-regulated genes as cells progress through S phase clearly support this notion (28). Therefore, it appears that E2F6 may be a component of a feedback mechanism that dampens the expression of E2F-responsive genes that are induced by pRB-regulated E2F family members during S-phase entry and progression and thus represents an attractive target for viral oncoproteins, including HPV E7. In this study, we report the association of HPV16 E7 with E2F6 and provide evidence for the deregulation of E2F6 function by the HPV16 E7 oncoprotein.


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MATERIALS AND METHODS
 
Plasmids. HPV and control plasmids used in this study include pOZ-C (control) (52), pOZ-C18E7 (32), pOZ-C6bE7, pOZ-C11E7, pOZ-C16E7, pOZ-C16E7 delP6-E10, pOZ-C16E7 delD21-C24, pOZ-C16E7 CVQ68-70AAA, pOZ-C16E7 delL79-L83, pOZ-C16E7 C91S (33), p1435 (human β-actin HPV16 E7 expression plasmid), p1318 (human β-actin control plasmid) (51), pBABE, pBABE-16E7 (57), pBABE-16E7 delP6-E10, pBABE-E7 delD21-C24, pBABE-E7 CVQL79-L83, and pBABE-E7 C91S. Other plasmids used in this study include pcDNA3.1 (Invitrogen); pGL36XE2F (49), a generous gift from Kristian Helin, Biotech Research and Innovation Centre, Copenhagen, Denmark; pAdE2luc (4), a generous gift from Ann Roman, Indiana University School of Medicine, Indianapolis, IN; E-box luciferase (64), a generous gift from William Walker, University of Pittsburgh, Pittsburgh, PA; 13S-SVE (31), a generous gift of Philip Hinds, Tufts University School of Medicine, Boston, MA; pCMV-TAg (9), a generous gift of James DeCaprio, Harvard Medical School, Boston, MA; and full-length and truncation mutants of E2F6 (74): pCMV-neo-Bam E2F6, pCMV-neo-bam E2F6 62-281, pCMV-neo-Bam E2F6 128-281, pCMV-neo-Bam E2F6 1-240, pCMV-neo-Bam E2F6 1-178, and pCMV-neo-Bam E2F6 179-281 (generous gifts from Jacqueline Lees, MIT, Cambridge, MA).

Cell lines and culture. HeLa S3 suspension cells stably expressing C-terminally FLAG- and hemagglutinin (HA)-tagged HPV6b E7, HPV11 E7, HPV16 E7, HPV18 E7, HPV16 E7 delP6-E10, HPV16 E7 delD21-C24, HPV16 E7 CVQ68-70AAA, HPV16 E7 delL79-L83, and HPV16 E7 C91S (32, 33), C33A (ATCC), 293 (ATCC), and 293T (ATCC) cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) supplemented with 10% newborn calf serum (NCS), 50 U/ml penicillin, and 50 µg/ml streptomycin. C33A cells stably maintaining C-terminally FLAG- and HA-tagged HPV16 E7 (7) were maintained in DMEM supplemented with 10% NCS, 50 U/ml penicillin, 50 µg/ml streptomycin, and 1 mg/ml G418. RKO cells (ATCC) were maintained in modified McCoy's medium (Invitrogen) supplemented with 10% NCS, 50 U/ml penicillin, and 50 µg/ml streptomycin. RKO 7.6 (E7-expressing) cells (66), a generous gift from Kathy Cho, Johns Hopkins University, Baltimore, MD, were maintained in modified McCoy's medium (Gibco Invitrogen) supplemented with 10% NCS, 50 U/ml penicillin, 50 µg/ml streptomycin, and 500 µg/ml G418.

Primary human foreskin keratinocytes (HFKs) were isolated from anonymous newborn circumcisions. Briefly, two to five human foreskins per culture were washed with phosphate-buffered saline (PBS), cut into small pieces, and incubated in 25 mg dispase/ml PBS overnight at 4°C. The epidermis was separated from the dermis, minced, and trypsinized into a single-cell suspension. HFKs were maintained in keratinocyte-serum-free media supplemented with human recombinant epidermal growth factor 1-53, bovine pituitary extract (Gibco Invitrogen), 50 U/ml penicillin, 50 µg/ml streptomycin, 10 µg/ml gentamicin, and 0.75 µg/ml amphotericin B. HFK cells stably expressing HPV16 E7 were created by transfecting primary HFK populations with p1435, a human β-actin HPV16 E7 expression plasmid, or p1318, the parental plasmid (51), as a control, together with pcDNA3.1 (Invitrogen), using the Amaxa human keratinocyte Nucleofector kit (Amaxa Biosystems) according to the manufacturer's instructions. Following selection with G418, the cells were maintained in keratinocyte-serum-free media supplemented with human recombinant epidermal growth factor 1-53, bovine pituitary extract (Gibco), 50 U/ml penicillin, 50 µg/ml streptomycin, 10 µg/ml gentamicin, and 0.75 µg/ml amphotericin B.

Primary human foreskin fibroblasts (HFFs) were isolated from anonymous newborn circumcisions. Dermis that was separated from the epidermis during HFK preparation was minced and incubated in a 2-mg/ml collagenase solution in DMEM and trypsin (4:1) for 2 to 4 h at 37°C. HFFs were maintained in DMEM supplemented with 10% calf serum, 50 U/ml penicillin, and 50 µg/ml streptomycin. HFF cells stably expressing wild-type or mutant HPV16 E7 were generated from primary HFF populations infected with the following pBABE-puromycin-based retroviral constructs: pBABE, pBABE-16E7, pBABE-16E7 delP6-E10, pBABE-16E7 delD21-C24, pBABE-16E7 CVQL79-L83, and pBABE-16E7 C91S. Recombinant retroviruses were produced as previously described (57). Infections of 50% confluent HFFs were performed with a mixture of 2 ml viral supernatant, 8 µg/ml Polybrene, and 2 ml DMEM for 6 h. The cells were maintained in DMEM supplemented with 10% NCS, 50 U/ml penicillin, and 50 µg/ml streptomycin following selection with 1 µg/ml puromycin.

Western blotting and immunoprecipitation. For cross-linking experiments, cells were treated with 1 mM dithio-bis-succinimidyl-propionate (DSP; Sigma) for 30 min prior to lysis. Cross-linking was quenched by the addition of 10 mM glycine. Cell lysates for immunoblots, glutathione S-transferase (GST) pull-downs, and immunoprecipitation analyses were prepared by incubating the cells in ML buffer (300 mM NaCl, 0.5% Nonidet P-40 [NP-40], 20 mM Tris-HCl [pH 8.0], 1 mM EDTA) supplemented with one Complete EDTA-free protease inhibitor cocktail tablet (Roche) per 50 ml lysis buffer. The cells were then scraped, and the extracts were cleared by centrifugation at 16,000 x g for 20 min. Protein concentrations were determined by the Bradford method (Bio-Rad). Samples containing 200 µg of protein were boiled in sodium dodecyl sulfate (SDS)-containing sample buffer, separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto polyvinylidene difluoride membranes (Immobilon-P; Millipore). The membranes were blocked for 2 h in 5% nonfat dry milk in TNET buffer (200 mM Tris-HCl, 1 M NaCl, 50 mM EDTA, 0.1% Tween 20 [pH 7.5]) and subsequently probed with the appropriate antibody. Primary antibodies were used at the following dilutions: a mixture of 8C9 at a 1:150 dilution (Zymed/Invitrogen) and ED17 at a 1:200 dilution (Santa Cruz Biotechnology) for HPV16 E7, sc-53273 at a 1:500 dilution (Santa Cruz) and sc-8366 at a 1:100 dilution (Santa Cruz) for E2F6, AB-5 at a 1:100 dilution (Oncogene Research) for pRB, 3818-1 at a 1:2,000 dilution (Clontech) for GST, Ab16848 (Abcam) for simian virus 40 (SV40) large T antigen (TAg), M73 Ab28305 (Abcam) for adenovirus E1A (Ad E1A), M2 (Sigma) for FLAG, and MAB1501 at a 1:1,000 dilution (Chemicon) for actin. A secondary anti-mouse horseradish peroxidase-conjugated antibody (Amersham) was used at a 1:10,000 dilution, and a secondary anti-goat horseradish peroxidase-conjugated antibody (Amersham) was used at a 1:5,000 dilution. Proteins were visualized by enhanced chemiluminescence (Western Lightning Chemiluminescence Reagent Plus; Perkin-Elmer Life Sciences, Inc.) and exposed on Kodak BioMax XAR film or electronically acquired with a Kodak 4000R image station (Kodak) equipped with Kodak Imaging software, version 4.0. Equal loading was controlled using β-actin immunoblots.

For immunoprecipitation analyses, cells were lysed as described above. Five micrograms of either E7 antibody (ED19) or E2F6 antibody (sc-53273) was mixed with 50 µl ImmunoPure Immobilized Protein G Plus (Pierce) for 30 min at 4°C with constant rotation, followed by incubation with samples for 4 h at 4°C with constant rotation. Anti-FLAG M2 agarose (Sigma) was used for FLAG immunoprecipitations. Immunocomplexes were washed three times with ML buffer and subjected to SDS-PAGE and immunoblot analysis.

GST pull-down assay. For GST pull-downs, RKO whole-cell lysates were prepared as described above. GST control, GST-wild-type HPV16 E7, and GST-HPV16 E7 C91S mutant fusion proteins were expressed in BL21(DE3)pLysS cells (Invitrogen). The cells were induced with 100 µM isopropyl-β-D-thiogalactopyranoside (IPTG) overnight at 30°C once the optical density at 600 nm reached ~0.7. For each pull-down, 1 ml of induced bacterial culture was pelleted, lysed using ML buffer, and cleared by centrifugation. The supernatant was mixed with 50 µl glutathione-Sepharose 4B slurry (GE Healthcare) and incubated for 1 h at 4°C. The Sepharose beads were then washed several times with ML buffer and incubated with RKO whole-cell lysates. After 4 h at 4°C, beads were washed three times with ML buffer, and samples were analyzed via Western blotting as described above.

Immunofluorescence. For immunofluorescence analysis, cells were plated onto glass coverslips, fixed with 4% paraformaldehyde in PBS, washed with PBS, and permeabilized with 1% Triton X-100 in PBS for 6 min at room temperature. Following permeabilization, the cells were washed with wash buffer (PBS containing 0.02% [wt/vol] saponin, 0.05% [wt/vol] sodium azide, and 1% [wt/vol] bovine serum albumin), blocked with 10% normal donkey serum (Jackson Immunoresearch) and 0.1% cold water fish skin gelatin (Sigma), and incubated overnight with anti-E2F6 (sc-8366; Santa Cruz) at 4°C. Blocking solution and antibodies were diluted in wash buffer. Secondary antibody was donkey anti-goat Alexa Fluor 568 (Invitrogen Molecular Probes) and was used at a 1:1,000 dilution. Nuclei were counterstained with Hoechst 33258 stain. All images were acquired using a Nikon Eclipse TE2000-E apparatus equipped with a 60x, 1.4-numerical-aperture, Plan Apo objective (Nikon) and with Metamorph 6.3r7 (Molecular Devices) software. The X-Cite 120 PC apparatus (Expo) was used as the illumination source, and images were acquired using an Orca 285 charge-coupled-device camera (Hamamatsu). Fluorescence was excited and detected using the HcRed filter set (part number 41043; Chroma).

Transfections and luciferase assays. C33A/CE7 and 293T cells were transfected in 60-mm plates for immunoprecipitation mapping studies, and C33A cells were transfected in six-well plates for luciferase assays using FuGene6 reagent (Roche) according to the manufacturer's instructions. Immunoprecipitations for mapping studies were performed as described previously. For luciferase assays, dose-response curves were performed to determine the optimal concentrations of E2F6 and HPV E7 constructs to be used (data not shown). Five hundred nanograms of pGL3 6XE2F, pAdE2, or E-box luciferase was cotransfected with 1 µg pOZ-C6bE7, pOZ-C11E7, pOZ-C16E7, or pOZ-C18E7 (32, 33) and 25 ng of pCMV neo Bam E2F6. The cells were lysed 72 h posttransfection in 500 µl passive lysis buffer (dual-luciferase reporter kit; Promega) per well. The supernatants were subjected to the dual-luciferase assay. As there is variability in the ability of wild-type HPV16 E7 to relieve E2F6-mediated transcriptional repression, possibly due to the density of the plate upon transfection, each transfection and luciferase assay was performed at least three times.

Quantitative real-time RT-PCR. Total RNA was extracted from HFFs and HFFs with the stable expression of HPV16 E7 using the Total RNA Isolation Mini kit (Agilent) according to the manufacturer's instructions. Quantitative reverse-transcription PCR (RT)-PCR analysis was performed by use of a 7300 real-time PCR system (Applied Biosystems) with Sybr green fluorescence. Primers for RPIA, UXT, and ZCCHC7 were purchased from SuperArray. For cDNA synthesis and quantitative RT-PCR, we used the QuantiTect Sybr green RT-PCR kit (Qiagen) according to the manufacturer's instructions. Cycling parameters were as follows: 30 min at 50°C for cDNA synthesis and 15 min at 95°C for DNA strand denaturation, followed by 40 cycles for 15 s each at 94°C for denaturation, 30 s at 55°C for annealing, and 30 s at 72°C for primer extension. Dissociation curve analysis (95°C for 15 s, 60°C for 15 s, and 95°C for 15 s) was performed at the end of 40 cycles to verify PCR product identity. Each RNA sample was tested in triplicate. Data were analyzed using the 2–{Delta}{Delta}CT method (41).

Interactome analyses. The HPV16 E7 interactome was analyzed using the STRING database of known and predicted protein-protein interactions (78).

Statistical methods. A Student's t test was used to evaluate the results of at least three independent experiments and at least 100 cells per experiment.


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RESULTS
 
HPV16 E7 associates with E2F6 complexes. In order to identify cellular protein complexes that associate with HPV16 E7 in cervical cancer cells, we stably expressed carboxy-terminally FLAG/HA epitope-tagged HPV16 E7 in HeLa cervical carcinoma cells and performed TAP, followed by mass spectrometric identification of individual E7-interacting partners (33). HeLa cells were selected for these studies because they are derived from a cervical cancer and contain an inherently intact retinoblastoma tumor suppressor pathway (58, 82), and ectopic expression of HPV16 E7 can rescue the biological and biochemical defects that are caused by the loss of expression of endogenous HPV18 E7 (6, 80). We isolated known HPV16 E7-interacting partners, such as RB family members, E2F family members, cdk2, cyclin A, and cyclin E, as well as novel E7 targets, such as p600 (33), steroid coactivator 1 (7), and cullin-2 (32). In addition, we identified E2F6 and the related PcG proteins Bmi1, Mel18, Ring1, hpc2, and L3MBT-like 2 protein as a putative E7-interacting complex(s) (Fig. 1A). The power of the TAP assay is that it allows the identification of directly, as well as indirectly, associated proteins and thus allows the identification of entire complexes of proteins, as evidenced by the E2F6-PcG complex(s) that we identified in the HPV16 E7 TAP assay (Fig. 1B). The fact that E2F6 does not contain a pRB-binding site and that its functions are not regulated by association with pRB family members strongly suggested that E2F6 is a pRB-independent cellular E7 target and was thus of interest to us to study. The association of untagged HPV16 E7 with E2F6 was confirmed by coprecipitation experiments with HPV16 E7-expressing RKO colon cancer cells (Fig. 2A). These cells were chosen because they express relatively high levels of E2F6.


Figure 1
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  		FIG. 1. HPV16 E7 associates with E2F6 and related polycomb group proteins. (A) TAP of cellular protein complexes associated with C-E7 expressed in HeLa cells. Representative HPV16 E7-associated cellular proteins isolated by this procedure are indicated (33). Vector-transfected HeLa cells (lane C) were used as a control. (B) Schematic of the partial E7-E2F6-PcG interactome. The schematic is based on the STRING database of known and predicted protein-protein interactions (78) and results described previously by Ogawa et al. (54).


Figure 2
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  		FIG. 2. HPV16 E7 associates with endogenous E2F6 and relieves E2F6-mediated transcriptional repression. (A) Coprecipitation of E2F6 with E7 antibodies in HPV16 E7-expressing RKO colon carcinoma cells. Parental RKO cells were used as a control. IP, immunoprecipitation. (B) Effect of HPV16 E7 on E2F6-mediated transcriptional repression. C33A cells were transiently transfected in triplicate with 500 ng of the reporter construct indicated at the top. pGL36xE2F (6XE2F) contains the luciferase gene driven by six consensus E2F sites upstream of a TATA box, pAdE2luc (AdE2) contains the luciferase gene driven by the Ad E2 promoter, and E-box-luciferase (E-box) contains the luciferase gene driven by two consensus E-box motifs upstream of a minimal thymidine kinase promoter. These reporters were either (i) transfected alone to determine baseline luciferase values, (ii) cotransfected with 25 ng of pCMV neo Bam E2F6 or 1 µg of pOZ-C16E7 to determine the effect of E2F6 or HPV16 E7 on the reporter construct, or (iii) cotransfected with a combination of 25 ng of pCMV neo Bam E2F6 and 1 µg of pOZ-C16E7 to determine the ability of E2F6 to repress transcription in the presence of HPV16 E7. The amount of expression plasmid was kept constant in all assays by the addition of empty pOZC vector. Luciferase activity was determined 72 h after transfection. The data are presented as the repression of E2F6 alone [reporter construct/(reporter construct and E2F6)] versus the repression of E2F6 in the presence of HPV16 E7 [(reporter construct and HPV16 E7)/(reporter construct, HPV16E7, and E2F6)]. The values shown are averages of triplicate transfections for representative experiments (experiments were repeated at least three times). Raw luciferase data for the 6XE2F reporter in the absence and presence of E2F6 were 39.38 and 7.98 relative light units (RLU), respectively. Raw luciferase data for the Ad E2 reporter in the absence and presence of E2F6 were 229.69 and 23.54 RLU, respectively. Raw luciferase data for the E-box reporter in the absence and presence of E2F6 were 574.59 and 108.11 RLU, respectively. (C) Effect of HPV16 E7 on endogenous E2F6-responsive genes. The expression of endogenous RPIA, UXT, and ZCCHC7 mRNA was analyzed by quantitative real-time RT-PCR using total RNA extracted from HFF cells and HFF cells with the stable expression of HPV16 E7. Data are represented as changes in mRNA levels in the presence of HPV16 E7.

HPV16 E7 relieves E2F6-mediated transcriptional repression. E2F6 has been shown to repress the transcription of E2F-responsive genes (11, 27, 73). In addition, it was previously reported that E2F6 can bind to and repress E-box-containing promoters, although the physiological relevance of this has not yet been verified (53). In order to determine if the association of E7 with E2F6 altered the ability of E2F6 to function as a transcriptional repressor of E2F or E-box-responsive genes, we performed luciferase assays utilizing three reporter constructs, two that contain E2F elements and one that contains an E-box element (Fig. 2B). C33A cells were utilized for these assays, as they are HPV-negative cervical carcinoma cells that have mutated pRB, thus relieving repression by canonical E2F family members. As expected, E2F6 repressed transcription from all three reporters used. Interestingly, the ability of E2F6 to repress transcription was reduced 1.7- to 7-fold when cotransfected with HPV16 E7 (Fig. 2B).

We next wished to determine if the studies of transfected promoter constructs reflected the activity of endogenous E2F6-responsive promoters. Generally, it is challenging to determine the effects of a particular E2F on gene expression because of the fact that multiple E2Fs bind to the same promoter. However, a set of promoters that are preferentially bound by E2F6 and not E2F1 or E2F4 has been identified (84). We chose three genes from this list, RPIA, UXT, and ZCCHC7, and performed quantitative real-time RT-PCR to determine if the mRNA levels of these genes changed in the presence of HPV16 E7. The levels of RPIA, UXT, and ZCCHC7 mRNA were each increased in the presence of HPV16 E7 (Fig. 2C).

The integrity of the carboxy-terminal zinc-binding domain of HPV16 E7 is necessary for association with the repression domain of E2F6. In order to gain insight into the association of HPV16 E7 and E2F6, we mapped the domain of HPV16 E7 that is necessary for the association with E2F6. HPV16 E7 is composed of three domains: the amino terminus, which has homology to a portion of conserved region 1 (CR1) of Ad E1A; a region that is homologous to Ad E1A CR2; and the carboxy-terminal domain, which contains a cysteine-rich zinc-binding domain (25, 40, 56) (Fig. 3A). HeLa cells stably expressing various carboxy-terminally FLAG/HA epitope-tagged HPV16 E7 mutants were generated (33), E7-associated cellular proteins were isolated by immunoprecipitation with FLAG antibody, and coprecipitation of E2F6 was assessed by Western blotting (Fig. 3B). These experiments showed that the HPV16 E7 carboxyl-terminal mutant C91S is defective for association with E2F6 (Fig. 3B), while the pRB-binding-deficient HPV16 E7 delD21-C24 associated with E2F6 at levels similar to those of wild-type HPV16 E7 (Fig. 3B). Since these experiments were performed in HeLa cells that contain endogenous HPV18 E7, we wished to rule out the possibility that dimers of wild-type HPV18 E7 and mutant HPV16 E7 mediate binding to E2F6. This is significant since the C91S mutant was previously reported to be incompetent for dimer formation (15). Hence, we performed GST pull-down experiments using HPV-negative RKO cell extracts. Consistent with the coimmunoprecipitation experiments with HeLa cells, the HPV16 E7 C91S mutant was defective for E2F6 association, whereas it retained pRB-binding activity as expected (Fig. 3C) (15). Therefore, the integrity of the carboxy-terminal zinc-binding domain of HPV16 E7 is necessary for the association with E2F6 and is independent of E7's ability to interact with pRB family members.


Figure 3
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  		FIG. 3. Cysteine 91 in the C terminus of HPV16 E7 and the marked box of E2F6 are critical for the functional association of E7 and E2F6. (A) Schematic representation of HPV16 E7 functional regions. (B) Association of E2F6 with different HPV16 E7 mutants. HeLa cells with the stable expression of HPV16 E7 (wild type [WT] or the indicated mutants) with FLAG/HA epitopes at their C termini were cross-linked, and lysates were immunoprecipitated with FLAG antibody, followed by Western blotting with E2F6 antibody; E7 (using FLAG antibody) and actin blots are shown as controls. A longer exposure of the E7 blot from lysates is shown to document the expression of the HPV16 E7 del6-10 mutant. IP, immunoprecipitation. (C) Lack of association of E2F6 with the HPV16 E7 C91S mutant. Shown is a Western blot analysis of GST and GST-16E7 pull-downs in RKO cells. pRB is shown as a positive control, as it is known to associate with HPV16 E7. The blot was also probed for E2F6 and GST. (D) Effect of the wild type and HPV16 E7 mutants on E2F6-mediated transcriptional repression. C33A cells were transiently transfected in triplicate with 500 ng of pGL36xE2F. The reporter was either (i) transfected alone to determine baseline luciferase values, (ii) cotransfected with 25 ng of pCMV neo Bam E2F6 or 1 µg of pOZC16E7 (wild type or the indicated mutant) to determine the effect of E2F6 or HPV16 E7 on the reporter construct, or (iii) transfected with a combination of 25 ng of pCMV neo Bam E2F6 and 1 µg of pOZC16E7 (wild type or the indicated mutant) to determine the ability of E2F6 to repress transcription in the presence of HPV16 E7 (wild type or the indicated mutant). The amount of expression plasmid was kept constant in all assays by the addition of empty pOZC vector. Luciferase activity was determined 72 h after transfection. The data are presented as the repression of E2F6 alone [reporter construct/(reporter construct and E2F6)] versus the repression of E2F6 in the presence of HPV16 E7 [(reporter construct and HPV16 E7)/(reporter construct, HPV16E7, and E2F6)]. The values shown are averages of triplicate transfections for representative experiments (experiments were repeated at least three times). Raw luciferase data in the absence and presence of E2F6 are 274.81 and 18.24 RLU, respectively. (E) Schematic representation of E2F6 functional regions. (F) Association of E7 with different E2F6 functional regions. Lysates of C33A cells with the stable expression of HPV16 C-E7 and transient expression of E2F6 (wild type and the indicated mutant) were immunoprecipitated with FLAG antibody, followed by Western blotting with E2F6 antibodies: the top panel was probed with an E2F6 antibody (sc-53273) that recognizes E2F6 residues 1 to 281, 62 to 281, 128 to 281, and 179 to 281, and the second E2F6 panel was probed with an E2F6 antibody (sc-8366) that recognizes E2F6 residues 1 to 281, 1 to 240, and 1 to 178. HPV16 C-E7 (using FLAG antibody) and actin blots are shown as controls. Arrows indicate E2F6 proteins, and asterisks indicate the immunoglobulin G light chain.

Further characterization of the HPV16 E7 mutants in the luciferase assay of E2F6-mediated transcriptional repression revealed that the HPV16 E7 C91S mutant was defective for abrogating E2F6-mediated transcriptional repression (Fig. 3D).

We next performed a similar series of experiments to map the sequences of E2F6 that are necessary for the association with HPV16 E7. A series of E2F6 truncation mutants (Fig. 3E) was tested by transient transfection and coimmunoprecipitation assays in 293 cells for the ability to interact with HPV16 E7 (Fig. 3F). Deletion of the unique N-terminal domain of E2F6 had no detectable effect on the interaction between E2F6 and E7; moreover, an additional amino-terminal deletion of residues 62 to 128 revealed that the DNA-binding domain is also dispensable for the association with HPV16 E7. However, the deletion of residues 179 to 281 abolished the association between E2F6 and E7, indicating that the E7 binding domain is located within the repression domain of E2F6.

Low- and high-risk HPV E7 proteins associate with E2F6. We next wished to determine if the association with E2F6 was unique to HPV16 E7 or if it is a feature shared with other high- and low-risk HPVs. To address this issue, lysates of HeLa cells expressing carboxy-terminal FLAG/HA-tagged E7 proteins from HPV6b, HPV11, HPV16, and HPV18 (32, 33) were immunoprecipitated with anti-FLAG resin, resolved by SDS-PAGE, and immunoblotted with E2F6 antibody. These experiments revealed that low-risk as well as high-risk HPV E7 proteins can associate with E2F6 (Fig. 4A). When we tested the abilities of low-risk HPV6b and HPV11 E7 and high-risk HPV18 E7 in the E2F6 transcriptional repression reporter assays, we found that, similar to HPV16 E7, HPV6b, HPV11, and HPV18 E7 could also relieve E2F6-mediated transcriptional repression (Fig. 4B).


Figure 4
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  		FIG. 4. HPV18 E7 and low-risk HPV E7 proteins associate with E2F6. (A) HeLa cells with the stable expression of HPV6b, HPV11, HPV16, or HPV18 E7 tagged with FLAG/HA epitopes at their C termini were cross-linked and subjected to FLAG immunoprecipitation (IP), followed by immunoblot analysis for E2F6; E7 (using FLAG antibody) and actin blots are shown as controls. Arrows indicate E7 proteins. (B) Effect of HPV6b, HPV11, HPV16, and HPV18 E7 on E2F6-mediated transcriptional repression. C33A cells were transiently transfected in triplicate with 500 ng of pGL36xE2F. The reporter was either (i) transfected alone to determine baseline luciferase values, (ii) cotransfected with 25 ng of pCMV neo Bam E2F6 or 1 µg of pOZCE7 (HPV6b, HPV11, HPV16, or HPV18 E7) to determine the effect of E2F6 or HPV E7 on the reporter construct, or (iii) transfected with a combination of 25 ng of pCMV neo Bam E2F6 and 1 µg of pOZCE7 (HPV6b, HPV11, HPV16, or HPV18 E7) to determine the ability of E2F6 to repress transcription in the presence of HPV E7 (HPV6b, HPV11, HPV16, or HPV18 E7). The amount of expression plasmid was kept constant in all assays by the addition of empty pOZC vector. Luciferase activity was determined 72 h after transfection. The data are presented as the repression of E2F6 alone [reporter construct/(reporter construct and E2F6)] versus the repression of E2F6 in the presence of HPV E7 [(reporter construct and HPV E7)/(reporter construct, HPV E7, and E2F6)]. The values shown are averages of triplicate transfections for representative experiments (experiments were repeated at least three times). Raw luciferase data in the absence and presence of E2F6 were 569.33 and 1,182.73 RLU, respectively.

SV40 TAg and Ad E1A can also associate with E2F6. Given that Ad E1A and SV40 TAg associate with E2F1 to E2F5, we next wished to determine if the association with E2F6 is unique to HPVs or if it is a feature shared with other small DNA tumor viruses. To address this issue, lysates of 293 and 293T cells were immunoprecipitated with anti-E2F6 antibody, resolved by SDS-PAGE, and immunoblotted with E2F6, SV40 TAg, and Ad E1A antibodies. From these experiments, we conclude that E2F6 associates with SV40 TAg and Ad E1A (Fig. 5A). When we tested the ability of SV40 TAg and Ad E1A in the E2F6 transcriptional repression luciferase assays, we found that, similar to HPV16 E7, SV40 TAg and Ad E1A could also relieve E2F6-mediated transcriptional repression (Fig. 5B). We next performed transient transfection and coimmunoprecipitation assays to determine the sequences of E2F6 that are necessary for the association with SV40 TAg. These experiments revealed that the SV40 TAg-binding domain is located within the "marked-box" domain of E2F6, a domain that is included in the repression domain, the region necessary for HPV E7 binding.


Figure 5
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  		FIG. 5. Ad E1A and SV40 TAg associate with E2F6. (A) 293 and 293T cells were subjected to E2F6 immunoprecipitation (IP), followed by immunoblot analysis for TAg and E1A; E2F6 and actin blots are shown as controls. (B) Effect of HPV16 E7, Ad E1A, and SV40 TAg on E2F6-mediated transcriptional repression. C33A cells were transiently transfected in triplicate with 500 ng of pGL36xE2F. The reporter was either (i) transfected alone to determine baseline luciferase values; (ii) cotransfected with 25 ng of pCMV neo Bam E2F6, 1 µg of pOZC16E7, 1 µg 13S-SVE, or 1 µg pCMV-TAg to determine the effect of E2F6, HPV16 E7, Ad E1a, or SV40 TAg on the reporter construct; or (iii) transfected with a combination of 25 ng of pCMV neo Bam E2F6 and 1 µg of pOZCE7, 1 µg 13S-SVE, or 1 µg pCMV-TAg to determine the ability of E2F6 to repress transcription in the presence of HPV16 E7, Ad E1A, or SV40 TAg. The amount of expression plasmid was kept constant in all assays by the addition of empty pOZC vector. Luciferase activity was determined 72 h after transfection. The data are presented as the repression of E2F6 alone [reporter construct/(reporter construct and E2F6)] versus the repression of E2F6 in the presence of HPV16 E7, Ad E1a, or SV40 TAg [(reporter construct and HPV16 E7/Ad E1a/SV40 TAg)/(reporter construct, HPV16 E7/Ad E1a/SV40 TAg, and E2F6)]. The values shown are averages of triplicate transfections for representative experiments (experiments were repeated at least three times). Raw luciferase data in the absence and presence of E2F6 are 28.35 and 1.34 RLU, respectively. (C) Association of SV40 TAg with different E2F6 functional regions. Lysates of 293T cells with transient expression of E2F6 (wild type and the indicated mutant) were immunoprecipitated with TAg antibody, followed by Western blotting with E2F6 antibodies: the top panel was probed with an E2F6 antibody (sc-53273) that recognizes E2F6 residues 1 to 281, 62 to 281, 128 to 281, and 179 to 281, and the second E2F6 panel was probed with an E2F6 antibody (sc-8366) that recognizes E2F6 residues 1 to 281, 1 to 240, and 1 to 178. E7 (using FLAG antibody) and actin blots are shown as controls. SV40 TAg and actin blots are shown as controls. Arrows indicate E2F6 proteins, and asterisks indicate the immunoglobulin G light chain.

Reduced detection of E2F6 polycomb bodies in HPV16 E7-expressing cells. E2F6 has been shown to function through its ability to interact with members of the PcG family of transcriptional repressors (5, 54, 74). PcG proteins form discrete heterochromatin-associated nuclear foci termed polycomb bodies (60). Given that the ability of E2F6 to function as a transcriptional repressor is altered in the presence of HPV16 E7, we next wished to determine if E2F6-associated polycomb bodies are altered as well. In order to address this issue, we performed immunofluorescence experiments for E2F6 polycomb bodies in HPV16 E7-expressing HFFs and HFKs (Fig. 6A) compared to vector-expressing control cells. Compared to control, non-E7-expressing cells, we detected fewer E2F6 polycomb bodies in E7-expressing cells (Fig. 6B). We next performed immunofluorescence experiments for E2F6 polycomb bodies in HFFs expressing a series of HPV16 E7 mutants. HFFs were used for these experiments because they exhibit more easily quantifiable E2F6 nuclear dots (Fig. 6C). These experiments showed that in addition to the carboxy-terminal zinc-binding motif (cysteine 91), which is critical for E2F6 association and the abrogation of transcriptional repression in reporter assays, amino acids 6 to 10 of HPV16 E7 are also necessary for the reduction of detectable E2F6-PcG bodies (Fig. 6D). The HPV16 E7 del6-10 mutant is expressed at levels similar to those of wild-type E7 in these assays; moreover, the levels of E2F6 are also similar in these cells (data not shown).


Figure 6
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  		FIG. 6. E2F6-polycomb body number is reduced in the presence of HPV16 E7. (A) Immunofluorescence analysis of endogenous E2F6 expression in HFFs and HFKs with stable expression of wild-type and mutant HPV16 E7. (B) Quantification of E2F6-PcG bodies/nuclei in HFFs and HFKs with empty vector or HPV16 E7. Bar graphs show the means of counts done in triplicate where >100 cells were analyzed per experiment. Error bars indicate standard deviations. (C) Immunofluorescence analysis of endogenous E2F6 expression in HFFs with stable expression of wild-type and mutant HPV16 E7. (D) Quantification of E2F6-PcG bodies/nuclei in HFFs with empty pBabe puro vector or HPV16 E7 (wild type or mutant). Bar graphs show the means of counts done in triplicate where >100 cells were analyzed per experiment. Error bars indicate standard deviations.


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DISCUSSION
 
In this study, we provide evidence that HPV E7, as well as SV40 TAg and Ad E1A, associates with and functionally deregulates the noncanonical E2F family member E2F6. While E2F6 has the capacity to act as a transcriptional repressor on promoters with E2F sites as well as E boxes, the exact biological role(s) of E2F6 has not yet been fully elucidated. Data from several studies are consistent with a role for E2F6 in the regulation of cell growth and proliferation; in particular, some studies indicated that E2F6 may be an S-phase regulator (11, 27). The notion of E2F6 as an S-phase regulator is further supported by experiments implicating E2F6 in pRB-independent repression of transcription of E2F-dependent G1/S-phase genes as cells progress through S phase: such experiments indicate that E2F6 establishes part of the mechanism that dampens the expression of E2F targets that are activated through a pRB-dependent mechanism upon S-phase entry and progression and thus contributes to the temporal control of gene expression during the cell cycle (28). Additionally, the association of an E2F6-PcG complex, E2F6-G0, with E2F target promoters in G0 suggests an involvement of this complex in gene repression in quiescent cells and, therefore, G0 maintenance (54). Surprisingly, studies with mouse embryo fibroblasts from E2F6-deficient mice indicated that E2F6 is dispensable for proliferation and quiescence but suggested that additional E2Fs, in particular, E2F4, may compensate for E2F6 in E2F6-deficient cells (28, 68). Hence, E2F6 may play multiple functional roles within cell-cycle-regulatory transcription factor complexes and represents an attractive cellular target for viral oncoproteins that subvert key regulatory pathways that control host cell replication, such as HPV E7, Ad E1A, and SV40 TAg.

The HPV life cycle is intimately associated with the differentiation state of the infected epithelial cell. With the noted exception of the E1/E2 origin binding complex, these viruses lack essential enzymes that are necessary for viral genome replication; consequently, HPVs need to retain or reestablish a replication-competent state in differentiated keratinocytes, which normally have withdrawn from the cell division cycle (reviewed in reference 69). As such, the HPV E6 and E7 proteins subvert pathways that signal growth arrest during differentiation. One of HPV E7's best-characterized functions is its ability to bind to and destabilize the retinoblastoma tumor suppressor protein (pRB) and thus inactivate the pRB-E2F repressor complex, thereby allowing uncontrolled cell cycle progression. However, E2F6 is not regulated by pRB family members; thus, the destabilization of pRB by E7 would not directly affect E2F6 activities. Intriguingly, the E2Fs, most notably E2F1, play a role in mediating the transcriptional control of the E2F6 gene. The E2F6 promoter contains two E2F consensus-binding sites, and E2F6 mRNA expression and E2F6 promoter activity are enhanced by the ectopic expression of E2F1 (43). E2F6 is upregulated at the G1/S-phase transition to exert an opposing effect on the activities of E2F-responsive promoters, thereby ultimately directing appropriate cell cycle exit and differentiation (43). Therefore, we envision that the functional deregulation of E2F6 by HPV E7 is needed to counterbalance the upregulation of E2F6 as a consequence of the activation of E2F1 to E2F5 by E7, thus ensuring that the cells remain in an S-phase-competent state that is necessary for the viral life cycle. Moreover, given E2F6's possible role in maintaining quiescence, it is conceivable that the deregulation of E2F6 by HPV E7 aids in allowing HPV to bypass negative growth signals, thus allowing cells to progress into S phase. While our experiments document the dysregulation of endogenous bona fide E2F6-dependent genes, it is not clear which genes may be responsible for this effect. Moreover, because E2F4 is known to compensate for E2F6 (28), E7 must also target E2F4 in order for the deregulation of E2F6 to have a functional consequence. As evidenced by our TAP results (Fig. 1), HPV16 E7 can target E2F4-containing complexes; moreover, a normally cytoplasmic E2F4-DP1 heterodimer accumulates in the nucleus in HPV16 E7-expressing cells (61).

The exact mechanism of repression by E2F6 has not yet been fully elucidated, but it is believed that the biological functions of E2F6 are mediated by its association with mammalian PcG proteins (74). Interestingly, we have also found that HPV16 E7 associates with several PcG proteins, including Bmi1, Mel18, Ring1, hpc2, and L3MBT-like 2 protein, and we detected fewer E2F6-PcG complexes in HPV16 E7-expressing cells. In addition to sequences in the carboxyl terminus of E7, sequences in the amino-terminal CR1 homology domain were also necessary for this activity of HPV16 E7. These results are consistent with our model that E2F6 binding is necessary but not sufficient for the reduction of E2F6-PcG complexes. Conceptually, this is similar to a previous study where we showed that pRB binding is not sufficient for the disruption of pRB/E2F complexes but that additional C-terminal sequences are also necessary (83). In addition, HPV16 E7 may associate with multiple components of E2F6-PcG complexes. It will be interesting to determine which components of the E2F6-PcG complex(s) that HPV16 E7 associates with directly. The fact that HPV16 E7 associates with E2F6's repression domain suggests that E7 expression does not generically inhibit the DNA-binding activity of E2F6. The reduced E2F6 PcG staining thus suggests that HPV16 E7 expression inhibits and/or disrupts the formation of discrete nuclear structures through multiple mechanisms.

PcG proteins were first discovered in Drosophila (37, 39) and are best known for their role in maintaining the stable transcriptional repression of Hox genes during development (29, 62). PcG functions are exerted by the formation of multimeric complexes that modify chromatin structures and regulate numerous posttranslational histone modifications (42, 59, 76). In PcG mutant mice, the expression boundaries of certain Hox genes are shifted anteriorly (1-3, 16, 19, 71). Similar to PcG mutant mice, mice lacking E2F6 display posterior homeotic transformations of the axial skeleton (68). In addition to their role in development (8, 45), Hox genes have also been implicated in the regulation of adult tissues, including skin (38, 47, 65). During keratinocyte differentiation, HOXA7 functions to silence differentiation-specific genes, and it is thought that a transient increase in HOXA7 expression may initially limit the rate at which differentiation progresses (38). Therefore, it will be interesting to determine whether promoter occupancy and/or the expression of certain Hox genes may be altered as a consequence of E7-mediated E2F6 deregulation, thus allowing differentiation to become uncoupled from cell cycle progression. Additionally, HPV16 E7-mediated disruption of E2F6-PcG complexes may have an impact on transformation, as growing evidence suggests that Hox genes may play an important role in the development of cancer (10, 12, 14, 20, 72).

It was suggested that papillomaviruses infect basal cells with stem-cell-like properties (44). High-risk mucosal HPVs infect reserve cells in the squamocolumnar transformation zone of the cervix (67, 75); alternatively, papillomaviruses may convert an infected cell to a more stem-cell-like state. Recently, it was reported that normal diploid cells can be converted to stem cells via the alteration of the expression of genes that are usually epigenetically silenced (70, 85). Hence, it is an exciting possibility that HPV E7 expression may modulate the "stemness" of the infected basal epithelial cell through alterations in E2F6-PcG complexes. The induction of a stem-cell-like state would present a clear advantage to the viral life cycle, as it would ensure long-term persistent infection of the squamous epithelia. In addition, once high-risk HPV integrates into the host genome, epigenetic alterations induced as a consequence of HPV E7's association with PcG complexes may also contribute to the formation of "cancer stem cells".

In summary, we have identified E2F6 and the related PcG proteins Bmi1, Mel18, Ring1, hpc2, and L3MBT-like 2 protein as novel associating partners of HPV16 E7 and have provided evidence that HPV16 E7 deregulates E2F6. In addition, we have shown that this association is not limited to high-risk HPV16 E7, as low-risk HPV6b and HPV11 E7 and high-risk HPV18 E7 also associate with E2F6. Moreover, SV40 TAg and Ad E1a also associate with a similar, but not identical, domain of E2F6. The integrity of the carboxyl-terminal zinc-binding domain of HPV16 E7 is necessary for associations with E2F6; interestingly, although these sequences are not conserved with SV40 TAg and Ad E1A, it appears that the subversion of E2F6 activity is in fact conserved among these DNA tumor viruses. It will be of interest to determine which sequences of SV40 TAg and Ad E1A mediate the association with E2F6.


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ACKNOWLEDGMENTS
 
This work was supported by PHS grants CA066980 and CA081135 (K.M.) and American Cancer Society fellowship PF-07-072-01-MBC (M.E.M.-D.).

We thank P. Silver for use of microscopy facilities, D. Drubin for microscopy assistance, and our colleagues for their gifts of reagents.


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FOOTNOTES
 
* Corresponding author. Mailing address: The Channing Laboratory 861, 181 Longwood Ave., Boston, MA 02115. Phone: (617) 525-4282. Fax: (617) 525-4283. E-mail: kmunger{at}rics.bwh.harvard.edu Back

{triangledown} Published ahead of print on 25 June 2008. Back

{dagger} Present address: Department of Biochemistry and Molecular Biology, School of Medicine, Louisiana State University Health Science Center, New Orleans, LA. Back


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Journal of Virology, September 2008, p. 8695-8705, Vol. 82, No. 17
0022-538X/08/$08.00+0     doi:10.1128/JVI.00579-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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