1 UMR 7100, Ecole Supérieure de Biotechnologie de Strasbourg, Boulevard Sébastien Brant, BP 10413, 67412 Illkirch cedex, France
2 Institut de Génétique et de Biologie Moléculaire et Cellulaire, Service de Microscopie Electronique, 1 rue Laurent Fries, 67400 Illkirch cedex, France
Correspondence
Murielle Masson
masson{at}esbs.u-strasbg.fr
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ABSTRACT |
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MAIN TEXT |
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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, 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
-galactosidaseE6 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 GSTHPV-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 maltose-binding protein (MBP)HPV-16 E6 fusion protein and did not recognize MBPHPV-6 E6 or MBPHPV-11 E6 fusion proteins, processed in parallel (not shown). The epitopes of these antibodies were mapped by peptide phage display (Fig. 1
a). 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 915 (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 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 HPV-negative C33A cells were lysed by sonication and the soluble material of these lysates was incubated with 6F4- or 4C6-treated protein ASepharose 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).
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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 AralditeEpon 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 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 MBPE6 (118) 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 4060 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
-galactosidase. The pHM829 plasmid encodes a
-galactosidaseGFP 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-E6-encoded 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,
-galactosidaseE6 was observed in the nuclei of the transfected cells (Fig. 3
b), whereas
-galactosidaseGFP was essentially localized in the cytoplasm (Fig. 3a
). To show definitively that E6 is capable of carrying the
-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 (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.
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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.
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ACKNOWLEDGEMENTS |
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Received 7 November 2002;
accepted 4 April 2003.