Journal of General Virology (2003), 84, 2099–2104
Short Communication
DOI 10.1099/vir.0.18961-0
Preferential nuclear localization of the human papillomavirus type 16 E6 oncoprotein in cervical carcinoma cells Murielle Masson,1 Colette Hindelang,2 Annie-Paule Sibler,1 Georges Schwalbach,1 Gilles Trave´1 and Etienne Weiss1 1
UMR 7100, Ecole Supe´rieure de Biotechnologie de Strasbourg, Boulevard Se´bastien Brant, BP 10413, 67412 Illkirch cedex, France
Correspondence Murielle Masson
2
[email protected]
Institut de Ge´ne´tique et de Biologie Mole´culaire et Cellulaire, Service de Microscopie Electronique, 1 rue Laurent Fries, 67400 Illkirch cedex, France
Received 7 November 2002 Accepted 4 April 2003
The E6 protein of the high-risk human papillomavirus type 16 (HPV-16) is involved in the tumorigenesis of human cervical cells by targeting numerous cellular proteins. We characterized new anti-E6 monoclonal antibodies and used them for precise localization of the E6 oncoprotein within carcinoma cells. Overexpressed E6 protein was predominantly detected in the nucleus of transiently transfected HaCaT cells. While mostly localized at the periphery of condensed chromatin, E6 was also associated with nuclear ribonucleoproteic ultrastructures and with some ribosomal areas in the cytoplasm of SiHa and CaSki cells. The chimeric b-galactosidase–E6 protein expressed in transfected HeLa cells was essentially localized in the nuclear compartment. Together, these data indicate that the E6 sequence of HPV-16 may encode a nuclear localization signal. The preferential nuclear distribution of this viral oncoprotein in HPV-transformed cells correlates with its activities at the transcriptional level.
High-risk strains of human papillomaviruses (HPVs) such as HPV-16 are associated with lesions that can progress to cervical cancer (zur Hausen, 1999). The genome of these viruses encodes an oncoprotein, E6, which mediates degradation of the cellular p53 tumour suppressor protein in vitro (Scheffner et al., 1992) and in vivo (Lechner et al., 1992). Although this particular property of E6 remains the main explanation of its transforming activity, recent findings indicate that E6 is a multifunctional protein with p53-independent activities. It has been shown that E6 activates transcription of the telomerase gene (hTERT; Klingelhutz et al., 1996) and acts as a DNA-binding protein with high affinity for four-way DNA junctions (Ristriani et al., 2000, 2001). In addition to p53, E6 interacts with a variety of cellular proteins, of which a significant number act at the transcriptional level (Mantovani & Banks, 2001). Previous studies on the localization of HPV-16 E6 within human carcinoma cells have led to contradictory results, most probably due to the low level of endogenous E6 protein and the poor reactivity of the available anti-E6 antibodies. E6 protein was localized in the cytoplasmic perinuclear region of SiHa cells (Daniels et al., 1998) and co-localized with p53 in the cytoplasm of CaSki cells (Liang et al., 1993). In transfected COS1 cells, overexpressed HPV-16 E6 protein is essentially found in the nuclear compartment (Kanda et al., 1991; Schwalbach et al., 2000; Sherman & Schlegel, 1996), while in transfected human cells, E6 of HPV-18, 0001-8961 G 2003 SGM
Printed in Great Britain
another malignant strain, is evenly distributed in the cytoplasm and the nucleus (Guccione et al., 2002). We generated monoclonal antibodies that specifically bind to the recombinant E6 protein of HPV-16. The aim of this study was to determine the precise intracellular localization of the E6 protein in transformed cells in order to establish whether the cellular distribution of this oncoprotein correlates with its recently described p53-independent biological functions. Here, we have shown by immunomicroscopy that HPV-16 E6 is predominantly localized in the nuclei of either transfected or transformed cells of established human cell lines. Analysis of the cellular localization of a b-galactosidase–E6 fusion protein with the same reagents supported the finding that E6 is essentially a nuclear protein. This correlates with its implied role in the transcriptional regulation of several cellular genes (Kumar et al., 2002, and references therein). The monoclonal antibodies were obtained by injecting purified GST–HPV-16 E6 fusion protein into mice. Two antibodies, named 6F4 (described previously by Giovane et al., 1999) and 4C6, were cloned. While 6F4 and 4C6 contained different CDR3-H and CDR3-L coding sequences, they reacted equally with SDS-denatured maltosebinding protein (MBP)–HPV-16 E6 fusion protein and did not recognize MBP–HPV-6 E6 or MBP–HPV-11 E6 fusion proteins, processed in parallel (not shown). The epitopes of these antibodies were mapped by peptide phage 2099
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display (Fig. 1a). The consensus amino acid sequence recognized by 6F4 (F/YQSPF/YXR) is nearly identical to the N-terminal region of HPV-16 E6 encompassing residues 9–15 (Choulier et al., 2002). The same approach was used to identify the epitope of the 4C6 antibody. A library of 12-mer peptides inserted at the N terminus of the protein III of M13 phage (Ph.D.-12 Phage Display Peptide Library, New England Biolabs) was screened for binding to 4C6 immobilized on protein A beads. After three rounds of panning,
the peptide-coding regions of the retained phages were sequenced. In this case, no clear consensus was found, but most peptides contained an aromatic residue (F/Y) and an arginine interspaced by five residues (Fig. 1a), reminiscent of the epitope of 6F4. However, these peptides did not contain the Q residue (position 10 of the E6 sequence) which is systematically retained in all clones interacting with 6F4, suggesting that the two antibodies may have different binding properties. Both antibodies were tested for their
Fig. 1. Analysis of the binding properties of monoclonal antibodies 6F4 and 4C6. (a) Results of the epitope consensus sequence determination by peptide phage display. (b) HeLa (lanes 3 and 7), SiHa (lanes 4 and 8), CaSki (lanes 5 and 9) and C33A (lanes 6 and 10) cells were lysed by sonication and cleared by centrifugation. The corresponding extracts (5 mg of total proteins) were submitted to immunoprecipitation with either 6F4 (lanes 1–6) or 4C6 (lanes 7–10) antibodies immobilized on protein A beads. COS1 cells (66105) transfected with the empty vector pcDNAI/Amp (lane 1) or the vector pcDNA-HPV16E6/K2 (lane 2) were treated in parallel. The precipitated proteins were analysed by SDS-PAGE and blotted to nitrocellulose. The blot was incubated with 6F4 antibody and then developed with peroxidase-conjugated secondary antibodies (Sigma) and Enhanced Chemiluminescence Substrate (Amersham). The presence of E6 is shown in the lower panel and the light chains of the 6F4 and 4C6 antibodies are shown in the upper panel. (c) Detection of overexpressed HPV-16 E6 protein in transfected HaCaT cells by immunofluorescence microscopy. HaCaT cells were transiently transfected with the expression vector pcDNA-HPV16-E6/K2 (lower panels) or empty vector pcDNAI/Amp (upper panels) and fixed 48 h post-transfection. The expressed E6 polypeptides were detected with 6F4 antibody and FITC-labelled rabbit anti-mouse immunoglobulins (left panels). The nuclei of cells were stained with Hoechst dye (right panels). 2100
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ability to immunoprecipitate the E6 protein present in cervical carcinoma cell extracts (Fig. 1b). HPV-16-positive CaSki and SiHa cells, HPV-18-positive HeLa cells and HPVnegative C33A cells were lysed by sonication and the soluble material of these lysates was incubated with 6F4- or 4C6treated protein A–Sepharose beads, as described previously (Giovane et al., 1999). COS1 cells transiently transfected with pcDNA HPV-16 E6/K2 (Giovane et al., 1999) were treated in parallel. The proteins bound to the beads were then analysed by Western blotting. After incubation with 6F4 antibody, the E6 protein present in the CaSki, SiHa and transfected COS1 cell extracts was detected as a faint band migrating at approximately 19 kDa, in agreement with its calculated molecular mass (Fig. 1b). This band, migrating as a single species, was not obtained with C33A, HeLa and non-transfected COS1 cell extracts, which indicates that the selected anti-E6 antibodies 6F4 and 4C6 specifically recognize native HPV-16 E6 protein. Interestingly, in COS1 cells transfected with pcDNA HPV-16 E6/K2, the 6F4 antibody also detected a band that co-migrates with the light-chain of the 6F4 antibodies (molecular mass approximately 28 kDa). The intracellular localization of HPV-16 E6 was first evaluated in a transiently transfected human keratinocyte cell line (HaCaT cells) by indirect immunofluorescence microscopy. Forty-eight hours after transfection with the expression vector pcDNA-HPV16-E6/K2, the cells were fixed with 4 % paraformaldehyde and permeabilized with PBS containing 0?1 % Triton X-100. After incubation with 6F4 antibody or 4C6 antibody (data not shown) and subsequent treatment with FITC-conjugated rabbit antimouse immunoglobulins (Dako), they were examined with a Zeiss Axioplan fluorescence microscope. Overexpressed E6 was predominantly localized in the nuclei of the treated HaCaT cells and no reaction was obtained in HaCaT cells transfected with an empty vector (Fig. 1c). Interestingly, the overexpressed HPV-16 E6 was also detected preferentially in the nucleus of transiently transfected HeLa cells (data not shown). Using the same approach, we were unable to detect with confidence the endogenous E6 protein in cells from both the CaSki and SiHa cell lines. This indicates that the expression level of E6 in these transformed cells is far below that observed in transfected HaCaT and HeLa cells. To improve the sensitivity of E6 detection in transformed cells, we used electron microscopic immunogold staining, which is also believed to be of higher resolution while preserving the cellular ultrastructures, as compared with conventional immunofluorescence. Approximately 105 cells from the CaSki, SiHa and C33A cell lines, cultured in parallel, were fixed with PBS containing 4 % paraformaldehyde and 0?1 % glutaraldehyde. After dehydratation in graded ethanol, the cells were embedded in Araldite–Epon resin (Stoeckel et al., 1985). Ultra-thin sections of these preparations were collected on Maxtaform grids and processed essentially as for immunofluorescence microscopy, except that the secondary antibodies used were goat anti-mouse immunoglobulins conjugated to colloidal gold http://vir.sgmjournals.org
particles (12 nm; Chemicon). After extensive washing, the grids were post-fixed with 4 % paraformaldehyde and finally stained with 5 % uranyl acetate. Typical micrographs of these treated sections examined under a CM12 Philips electron microscope are shown in Fig. 2. For both SiHa and CaSki cells, the majority of the gold particles (about 70 %) were found to be localized in the interphasic nuclei (Fig. 2a, b). In particular, the periphery of perinuclear chromatin and that of intranuclear condensed chromatin were preferentially labelled (Fig. 2b). After treatment of the sections with EDTA regressive stain (Bernhard, 1969), gold particles were present at the periphery of the bleached chromatin and associated with perichromatin fibrils and interchromatin granules as well (Fig. 2d). Whilst a faint homogeneous labelling of the nucleolus was also observed (Fig. 2e), a minor part of the gold particles co-localized with ribosomes in the cytoplasm (Fig. 2c). In contrast, no specific gold particle labelling was observed in HPV-16-negative C33A cells treated in parallel (Fig. 2f). Furthermore, preincubation of either 6F4 or 4C6 antibody with an excess of purified MBP–E6 (1–18) fusion protein, which harbours the residues of the recognized E6 region (Fig. 1a), resulted in the absence of gold particles in both the nucleic and cytoplasmic areas of treated SiHa cells (Fig. 2g). Together, these results indicate that the E6 protein of HPV-16-transformed cells is preferentially localized in the nucleus, as observed after transfection of HaCaT cells. In eukaryotic cells, active passage of macromolecules larger than 40–60 kDa from the cytoplasm to the nucleus is mediated by specific amino acid sequences, referred to as nuclear localization signals (Yoneda, 2000). Since the molecular mass of E6 (19 kDa) would allow its passive diffusion through nuclear pore complexes, we tested whether the accumulation of E6 in the nuclei of human cells is regulated. To distinguish clearly between active transport and passive diffusion, we constructed a chimeric protein consisting of HPV-16 E6 protein fused to the C terminus of the bacterial enzyme b-galactosidase. The pHM829 plasmid encodes a bgalactosidase–GFP fusion protein, for which expression in HeLa cells has been reported to be exclusively cytoplasmic (Sorg & Stamminger, 1999). This plasmid was modified by replacing the GFP coding sequence with that of HPV-16 E6, thus generating pHM829-E6. The size of the pHM829-E6encoded chimeric protein (molecular mass of the monomer 123 kDa) is sufficiently large to prevent its passive diffusion into the nucleus. Forty-eight hours after transfection with pHM829 or pHM829-E6 vectors, HeLa cells were fixed with paraformaldehyde and stained with X-Gal (Weiss et al., 1997). By visualizing under the microscope the redistribution of the stain within the coloured cells, b-galactosidase– E6 was observed in the nuclei of the transfected cells (Fig. 3b), whereas b-galactosidase–GFP was essentially localized in the cytoplasm (Fig. 3a). To show definitively that E6 is capable of carrying the b-galactosidase enzyme to the nucleus, pHM829-E6-transfected cells were incubated with 6F4 antibody and FITC-labelled anti-mouse immunoglobulins and observed under the fluorescence microscope. The fluorescence emitted corresponded to the nuclei 2101
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Fig. 2. Subcellular localization of HPV-16 E6 in cervical carcinoma cells. CaSki, SiHa and C33A cell sections were incubated with either 6F4 or 4C6 antibodies and subsequently with goat anti-mouse immunoglobulins conjugated to colloidal gold particles. After washing, the sections were processed for electron microscopic analysis. The picture represents typical electron micrographs of CaSki (a, c), SiHa (b, d, e, g) and C33A (f) cell sections incubated with 6F4 (a, b, d, f, g) or 4C6 antibody (c, e). In (b), the arrows indicate areas at the periphery of the perichromatin (pc). The nuclear region shown in (d) was observed after EDTA regressive staining. The gold particles in the interchromatin granule (ig) are indicated. Those present at the periphery of bleached chromatin (c) and on perichromatin fibrils (pf) are indicated with white or black arrows, respectively. In (g), 6F4 antibody was preincubated with an excess of the purified MBP–E6 (1–18) fusion protein. C, cytoplasm; N, nucleus; nu: nucleolus. Bars, 0?2 mm. 2102
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proteasome inhibitors treatment (Guccione et al., 2002). The reason for these differences is not clear but it is possible that the level of expression of E6 protein in transfected cells may influence its intracellular localization.
Fig. 3. Intracellular location of the b-galactosidase–E6 fusion protein. Subconfluent HeLa cells were transiently transfected with pHM829 (a, c) or pHM829-E6 (b, d) plasmids and fixed 48 h post-transfection. (a, b) Cells were incubated with X-Gal for 30 min before visualization under the optical microscope. (c) Cells were directly visualized under the fluorescence microscope with the FITC filter set. The green staining corresponds to the fluorescence emitted by the GFP. (d) Cells were incubated with 6F4 antibody and with FITC-labelled rabbit antimouse antibody immunoglobulins before microscopic analysis, as in (c).
(Fig. 3d) while in pHM829-transfected cells, fluorescence emitted by GFP was exclusively restricted to cytoplasmic areas (Fig. 3c). Identical results were obtained by transfecting COS1 cells, suggesting that HPV-16 E6 protein contains a targeting signal that mediates its efficient nuclear uptake in mammalian cells. Compelling evidence indicates that sustained expression of the E6 oncoprotein, albeit at a low level, is required for maintenance of the transformed malignant phenotype (zur Hausen, 1999). By investigating the subcellular localization of HPV-16 E6 protein in both transiently transfected and transformed cells using two well-characterized anti-E6 monoclonal antibodies, we observed that most of the expressed E6 protein resides in the nucleus. This contrasts with previous reports showing by indirect immunofluorescence that E6 of HPV-16 is preferentially localized in the cytoplasmic perinuclear region (Liang et al., 1993; Daniels et al., 1998). It is possible that our monoclonal antibodies might be more specific than polyclonal antisera previously used and that the electron microscopic immunogold staining is more sensitive than conventional immunofluorescence microscopy. While we observed in agreement with Sherman & Schlegel (1996) that E6 is predominantly nuclear in transiently transfected cells, it was shown that overexpressed E6 of HPV-18 is diffusely expressed throughout the cell and is relocalized to the nuclear compartment after http://vir.sgmjournals.org
Electron microscopic immunogold staining showed that E6 protein was often localized at the periphery of the nuclear condensed chromatin of the transformed cells. These perichromatin regions are the most significant sites of RNA transcription (Cmarko et al., 1999). The distribution of E6 within the nucleus is consistent with E6 having transcription factors (Lechner et al., 1992; Gross-Mesilaty et al., 1998; Ronco et al., 1998) or transcriptional coactivators (Patel et al., 1999; Kumar et al., 2002) as cellular binding partners. Interestingly, E6 was associated with perichromatin fibrils and interchromatin granules. The former are in situ forms of nascent transcripts (Cmarko et al., 1999), whereas the latter correspond to nuclear ribonucleoprotein particles and accumulation sites of splicing factors and heterogeneous nuclear RNAs (Spector et al., 1991; Hendzel et al., 1998). Whether these observations are related to the E6-induced malignancy or are simply due to the nucleic acid binding (Ristriani et al., 2001), basic pI or nuclear matrix-associating (Daniels et al., 1998) properties of E6 remains to be investigated. The most striking aspect of this work is that overexpressed E6 efficiently targets an appended large cytoplasmic protein to the nucleus, indicating that it has an intrinsic capacity to be transported into this compartment. It strongly suggests that E6 contains a nuclear localization signal, which is most probably located in its C-terminal zinc-binding domain that contains several clusters of basic residues. Consistently, Le Roux & Moroianu (2003) demonstrated very recently that HPV-16 E6 protein interacts with several karyopherins that mediate nuclear import in digitonin-permeabilized HeLa cells. Because some E6 was also found in the cytoplasm of the cervical carcinoma cells and has been shown to interact with several cytoplasmic partners (Pim et al., 2000), it may be possible that HPV-16 E6 protein is a more sophisticated protein, shuttling to some extent between the cytoplasm and nucleus. In conclusion, these data suggest that the subcellular localization of E6 is a regulated mechanism that may be influenced by putative post-translational modifications.
ACKNOWLEDGEMENTS We are grateful to Professor T. Stamminger for the generous gift of the pHM829 plasmid. We warmly acknowledge Dr E. Lalli for advice and Dr R. A. Atkinson for the critical review of this manuscript. We thank Bernard Boulay for technical assistance. This work was supported by the University Louis Pasteur, the Centre National de la Recherche Scientifique and the Association de Recherche contre le Cancer (ARC).
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