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Journal of Virology, April 2009, p. 2907-2916, Vol. 83, No. 7
0022-538X/09/$08.00+0     doi:10.1128/JVI.02490-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

E7 Oncoprotein of Novel Human Papillomavirus Type 108 Lacking the E6 Gene Induces Dysplasia in Organotypic Keratinocyte Cultures {triangledown}

Rui Jorge Nobre,1,§ Elsa Herráez-Hernández,1 Jian-Wei Fei,1 Lutz Langbein,2 Sylvia Kaden,3 Hermann-Josef Gröne,3 and Ethel-Michele de Villiers1*

Division for the Characterization of Tumorviruses, Deutsches Krebsforschungszentrum, Heidelberg, Germany,1 Division of Skin Carcinogenesis, Deutsches Krebsforschungszentrum, Heidelberg, Germany,2 Department of Cellular and Molecular Pathology, Deutsches Krebsforschungszentrum, Heidelberg, Germany3

Received 4 December 2008/ Accepted 12 January 2009


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ABSTRACT
 
The genome organization of the novel human papillomavirus type 108 (HPV108), isolated from a low-grade cervical lesion, deviates from those of other HPVs in lacking an E6 gene. The three related HPV types HPV103, HPV108, and HPV101 were isolated from cervicovaginal cells taken from normal genital mucosa (HPV103) and low-grade (HPV108) and high-grade cervical (HPV101) intraepithelial neoplasia (Z. Chen, M. Schiffman, R. Herrero, R. DeSalle, and R. D. Burk, Virology 360:447-453, 2007, and this report). Their unusual genome organization, against the background of considerable phylogenetic distance from the other HPV types usually associated with lesions of the genital tract, prompted us to investigate whether HPV108 E7 per se is sufficient to induce the above-mentioned clinical lesions. Expression of HPV108 E7 in organotypic keratinocyte cultures increases proliferation and apoptosis, focal nuclear polymorphism, and polychromasia. This is associated with irregular intra- and extracellular lipid accumulation and loss of the epithelial barrier. These alterations are linked to HPV108 E7 binding to pRb and inducing its decrease, an increase in PCNA expression, and BrdU incorporation, as well as increased p53 and p21CIP1 protein levels. A delay in keratin K10 expression, increased expression of keratins K14 and K16, and loss of the corneal proteins involucrin and loricrin have also been noted. These modifications are suggestive of infection by a high-risk papillomavirus.


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INTRODUCTION
 
A large number of human papillomavirus (HPV) types have been associated with benign and malignant lesions of the genital tract. High-risk papillomaviruses encode two oncoproteins, E6 and E7, which independently immortalize human keratinocytes, although their combined actions have complementary and synergistic effects (5, 22, 23, 35, 43). High-risk E7 protein binds to a multitude of cellular proteins in vitro. Probably its most important action is targeting the pocket protein pRb for degradation and thereby allowing uncontrolled cell cycle progression (30, 34). More recent studies, however, have indicated that the pRb-independent functions of E7 are sufficient to induce disruption of terminal differentiation and mild hyperplasia (3). The key role of high-risk E6, on the other hand, is to inactivate the oncosuppressive protein p53 functionally by inducing its degradation through the ubiquitin-proteosome pathway (31). Expression of high-risk E7 alone stabilizes the p53 protein, leading to increased levels (12), although its transcriptional activity is disturbed (32).

The genome organizations of the majority of human-pathogenic papillomaviruses are characterized by an early region containing five genes (E1, E2, E4, E6, and E7) and two genes (L1 and L2) in the late region. The HPV types of the genus Alphapapillomavirus usually harbor an E5 gene in the region between the early and late genes, whereas this gene is absent in the HPV types commonly associated with cutaneous lesions and grouped in the genera Betapapillomavirus, Gammapapillomavirus, Deltapapillomavirus, Mupapillomavirus, and Nupapillomavirus (13). The E6 gene, known to play a very important role in the pathogenesis of malignant disease of mucosal origin in humans (43), is present in almost all HPV types, except for the recently described HPV101 and HPV103 (9). A typical E6 gene can be present or absent in the genera comprising animal papillomaviruses (13).

The three related HPV types HPV101, HPV103, and HPV108 were isolated from cervicovaginal cells taken from high-grade cervical intraepithelial neoplasia (HPV101), normal genital mucosa (HPV103), and low-grade cervical intraepithelial neoplasia (HPV108) (reference 9 and this report). Their unusual genome organization, against the background of considerable phylogenetic distance from the other HPV types usually associated with lesions of the genital tract, prompted us to investigate whether HPV108 E7 per se is sufficient to induce the above-mentioned clinical lesions. The present study describes the isolation and characterization of the full-length novel HPV108 genome lacking both E6 and E5 genes. We further demonstrate that expression of HPV108 E7 in organotypic keratinocyte (NIKS) cultures induces epithelial dysplasia with loss of terminal epidermal differentiation and with nuclear pleomorphisms and increased proliferation, as well as abnormal apoptosis and an accumulation of lipids. These alterations are characterized by an increased expression of keratin K14, a delay in expression of keratin K10, the induction of keratin K16, and an altered expression of keratins K4 and K13. Expression of involucrin and loricrin is greatly diminished. HPV108 E7 binds to and decreases pRb, which is accompanied by an increase in PCNA, p53, and p21CIP1 protein levels. Tight junctions are disrupted, and electron-microscopic examination revealed an increase in intercellular spaces with an abundance of prominent microvillus-like structures. No consistent changes in desmosomes are observed.


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MATERIALS AND METHODS
 
Specimens and HPV108 detection. HPV108 was detected in a cervicovaginal sample from a 41-year-old female with a low-grade cervical lesion and was collected and diagnosed at the Portuguese Institute for Oncology at Coimbra. HPV DNA was initially identified by PCR amplification using FAP primers (FAP59 and FAP64) located in the L1 open reading frame (ORF), and amplification was performed as previously described (18). PCR products (480 bp) were purified from agarose gel using a PCR Clean-Up Gel Extraction Kit (Genomed, Germany) and cloned using the TA Cloning Kit (Invitrogen, Carlsbad, CA).

Isolation of the full-length genome of HPV108. Total cellular DNA extracted from cervical cells was subjected to rolling-circle amplification using a Templiphi Amplification Kit (Amersham Biosciences, Little Chalfont, Buckinghamshire, England) according to the manufacturer's instructions. The full-length genome was amplified by long PCR, using the Expand Long Template PCR Kit (Roche, Mannheim, Germany) and two HPV108-specific primers (HPV108 forward, 5'-TAAAGGACACTGAAAATCCTAACGGGTA-3', and HPV108 reverse, 5'-GCTTGTTAAATAAAGGATGCCCTGTAGA-3'). These back-to-back primers were designed on the partial L1 sequence. The amplicon with the size of a full-length genome was purified and cloned into the pCR2.1 vector (Invitrogen, Carlsbad, CA). This full-length HPV108 DNA genome was sequenced on both strands on an ABI model sequencer with Big Dye terminator chemistry (Perkin-Elmer Applied Biosystems Division).

Sequence analyses. The sequence of the full-length L1 ORF was compared to those of all other papillomaviruses. Alignment was performed with the ClustalW program by using a gap creation penalty of 10 and a gap extension penalty of 5. The phylogenetic tree (with neighbor-joining analysis) was constructed using the Growtree program and based on distance matrix calculation with Kimura distance correction (all GCG Wisconsin package, version 10.2; Accelrys). The tree was displayed by using the Treeview program from the University of Glasgow.

Plasmid constructs. HPV108 E7 was obtained by PCR amplification on the HPV108 genome as a template, using primers HPV108E7-forward (5'-TTAGCTAGCACCATGAGAGGCAAAGCACCTACAATTAAGG-3') and HPV108E7-reverse (5'-TTAGGATCCTTAGTGTCTTTGTCCTCCATTTCGTTCT-3'). (Italics denote the restriction sites for the enzymes NheI and BamHI, respectively, used for cloning). Flag-tagged HPV108 E7 was PCR amplified using the flag108E7-forward (5'-TTAGCTAGCACCATGGACTACAAGGACGACGACGACAAGAGAGGC-3') and HPV108E7-reverse primers. Both amplicons were confirmed by DNA sequencing and cloned into the vector pcDNA3.1 (Invitrogen), resulting in the constructs pcDNA3.1(+)HPV108E7 and pcDNA3.1(+)flagHPV108E7. The above-mentioned primers were also used to construct pLXSN-flagHPV108E7 and pLXSN-HPV108E7, except that the NheI restriction site was replaced with an EcoRI site in the forward primer. These amplicons were confirmed by sequencing and cloned into the vector pLXSN (BD Biosciences Clontech, Palo Alto, CA). pLXSN-HPV16E6/E7 containing both E6 and E7 genes of HPV16, used as a positive control, was a gift from A. Alonso. Full-length glutathione S-transferase (GST) fusion proteins of HPV108 E7 and HPV16 E7 were generated by PCR amplification with type-specific primers and HotStar HiFidelity DNA polymerase. Both HPVE7s were subcloned into the expression vector pGEX-5x-3 (Amersham Biosciences AB, Uppsala, Sweden) at the EcoRI and XhoI restriction sites. The DNA sequences and orientations of the DNA inserts were verified by sequencing of the constructs.

Cell lines and transfection. NIKS cells, a spontaneously immortalized human foreskin keratinocyte cell line (2), were maintained as subconfluent cultures on a mitomycin C-treated J23T3 fibroblast feeder layer in NIKS medium (3:1 Ham's F-12 medium and Dulbecco's modified Eagle's medium [DMEM] supplemented with 5% fetal bovine serum [FBS], adenine [24 µg/ml], cholera toxin [8.4 ng/ml], epidermal growth factor [EGF] [10 ng/ml], hydrocortisone [0.4 µg/ml], insulin [5 µg/ml], and 1% penicillin/streptomycin). The cells were grown at 37°C in a humidified chamber with 5% CO2. The small-cell lung carcinoma cell line H1299 was grown in DMEM, 10% FBS, and 1% penicillin/streptomycin. H1299 cells were transiently transfected using Lipofectamine reagent (Invitrogen, Karlsruhe, Germany) according to the manufacturer's instructions. Transfection was performed with the vectors pcDNA3.1(+)HPV108E7 and pcDNA3.1(+)flagHPV108E7. The empty vector pcDNA3.1(+) was used to equalize the total amount of transfected DNA. The transfected cells were harvested 24 h later. Transfection efficiency was confirmed by coexpression of β-galactosidase (pCMV-β-gal) (15), and experiments were performed at least three times for each construct.

Retrovirus production and infection of NIKS cells. Phoenix amphotropic packaging cells (Orbigen Inc., San Diego, CA) and J23T3 cells were maintained in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. Retroviruses were produced by transfecting subconfluent Phoenix amphopackaging cells with the empty vector pLXSN, pLXSN-flagHPV108E7, pLXSN-HPV108E7, or pLXSN-HPV16E6/E7. NIKS cells were kept in low-calcium (0.66 mM Ca2+) NIKS medium without a feeder layer and were exposed for 48 h to medium containing recombinant retrovirus or parental virus. Polybrene (6 µg/ml; Sigma-Aldrich catalog no. 10768-9) was added to increase the retroviral gene transfer (10). Transduced NIKS cells were trypsinized and seeded onto a mitomycin C-treated J23T3 feeder layer in complete NIKS medium prior to selection for stable transduced cells with Geneticin sulfate (G418) (100 µg/ml) for 4 days. Stable clones were pooled and subsequently kept on fresh mitomycin C-treated feeder layer in NIKS complete medium, expanded (four to eight passages), and used for organotypic raft cultures.

Organotypic raft culture. Collagen dermal equivalents composed of collagen mixture (8 parts collagen type 1, 1 part reconstitution buffer, and 1 part 10x DMEM) and embedded live J23T3 fibroblasts (41) were maintained in incomplete NIKS medium (without EGF) in six-well deep plates (Biocoat; Becton Dickinson, San Jose, CA) for 4 days. Stable transduced pLXSN-, pLXSN-HPV108E7-, pLXSN-flagHPV108E7-, or pLXSN-HPV16E6/E7-NIKS cells (1 x 106 cells/plate) were seeded onto dermal equivalents, the medium was changed to complete NIKS medium (with EGF) 24 h later, and the cells were cultured for 5 days. After that time, the medium was carefully removed from the inner well, leaving the cells exposed to air, and fed through the collagen matrix with incomplete NIKS medium. The medium was changed every second day before the rafts were harvested on day 14. Bromodeoxyuridine (BrdU) (50 µg/ml) was added 12 h prior to harvest. After being harvested, the rafts were either fixed overnight in 4% formalin and embedded in paraffin (for immunohistochemistry and immunofluorescence), embedded in Tissue-Tek (Sakura Finetechnical, Tokyo, Japan) and frozen for barrier function assays, or fixed in Karnovsky's fixative and embedded in Araldite for electron microscopy analysis. Organotypic raft cultures were repeated at least three times for each stable cell line.

Generation and expression of the GST fusion proteins and pull-down assays with GST-HPV108 E7 and GST-HPV16 E7. The recombinant fusion proteins GST-HPV108 E7 and GST-HPV16 E7 were expressed in the Escherichia coli BL21 strain (Promega, Madison, WI) and purified using the MicroSpin GST purification Module kit (Amersham, Freiberg, Germany). Nuclear extracts from NIKS cells were isolated as described previously (15). Precleaned nuclear extract was incubated at 4°C overnight with GST or the respective GST-fused proteins, and the GST pull-down assays were performed as previously described (16). The isolation and characterization of interacting proteins were analyzed by immunoblotting.

Western blot analyses. Proteins were extracted from transfected H1299 cells as previously described (15). Organotypic raft cultures were carefully collected from the collagen matrix and lysed manually in a chilled glass tissue grinder (Dounce) in buffer containing 50 mM Tris-HCl, pH 8, 10 mM NaCl, 5 mM EDTA, 10 mM NaF, 0.5% NP-40, 2 mM dithiothreitol, 1 mM Na3VO4, and 1% protease inhibitor cocktail (Sigma P8340) (6). Western blot analyses were performed as described previously (15). For Flag tag detection, membranes were blocked in 3% nonfat dry milk in 1x Tris-buffered saline, and for other antibodies, in 5% nonfat dry milk in 1x Tris-buffered saline, 0.1% Tween 20. The primary antibodies used were anti-Flag (Stratagene, CA), anti-GST (Amersham, Freiburg, Germany), anti-pRb (OP-136) and anti-p21 (OP-64) (both from Calbiochem), anti-p53 (sc-126) and anti-PCNA (sc-56) (both from Santa Cruz Biotechnology, CA), anti-β-galactosidase (Promega, Madison, WI), anti-β-actin (ICN, Aurora, OH), and anti-keratin K10 and anti-keratin K14 (both from Progen, Heidelberg, Germany). The secondary antibody used was either anti-mouse (W402B; Promega, Madison, WI) or anti-guinea pig (Southern Biotech, Birmingham, AL), coupled to horseradish peroxidase and detected by chemiluminescence (enhanced chemiluminescence system). β-Actin was measured as a loading control.

Immunohistochemical analyses. Serial sections (3 µm) of paraffin-embedded raft cultures were stained with hematoxylin-eosin or used for immunohistochemistry. Antigen retrieval was achieved by heating them at 98°C three times for 5 min each time in sodium citrate buffer (10 mM, pH 6.0). Slides were cooled at 4°C for 30 min and washed in phosphate-buffered saline (PBS) for 5 min. Samples were trypsinized (0.001% trypsin in 0.05 M Tris-HCl) for 15 min at 37°C.

After being unmasked, the slides were washed in H2O and PBS. Endogenous peroxidase activity was inactivated by treatment with 3% hydrogen peroxide/methanol for 15 min at room temperature, followed by a wash in PBS. Tissue sections were blocked in blocking serum (1% bovine serum albumin in PBS) for 60 min and incubated with the respective first antibody overnight at 4°C in a humid chamber. The first antibodies used were mouse monoclonal antibodies against pRb (NCL-RB; Novocastra Laboratories Ltd., United Kingdom), and p53 (clone B3-11) and p21 (clone WA-1) (both from Progen Biotechnic, Heidelberg, Germany). The slides were then washed in PBS and incubated with biotin-labeled anti-mouse secondary antibody for 30 min, and the labeling signals were visualized using Vectastain ABC reagent (Vector Laboratories, Peterborough, United Kingdom) and 3,3'-diaminobenzidine (BD Bioscience) as a substrate for peroxidase. The sections were counterstained with Mayer's hemalaun, dehydrated, and mounted.

For immunofluorescence staining, antigen retrieval was performed as described above. Sections were blocked for 60 min with 5% bovine serum albumin in PBS and subsequently incubated overnight at 4°C with the respective primary antibodies: mouse anti-PCNA (sc-56; Santa Cruz Biotechnology, CA), anti-involucrin (clone SY5; catalog no. I9018; Sigma-Aldrich), rabbit anti-loricrin (Covance, CA) and guinea-pig anti-keratin K2, anti-keratin K4, anti-keratin K10, anti-keratin K13, anti-keratin K14, anti-keratin K16, and anti-cingulin (all from Progen, Heidelberg, Germany). Primary antibodies were detected using Cy3-conjugated goat anti-mouse and Cy3-conjugated goat anti-guinea pig (Jackson Immuno Research Laboratories, Suffolk, United Kingdom) or goat anti-rabbit coupled to Alexa Fluor 488 (Molecular Probes Inc.). Secondary-antibody incubations were supplemented with Hoechst dye 33258 (Sigma) for nuclear counterstaining. Incorporated BrdU was detected using streptavidin-Alexa 488 (S-32354; Invitrogen), and nuclei were counterstained with Hoechst 33258 (Sigma).

All slides were examined using a Leitz DM RBF microscope (Leitz Instruments, Wetzlar, Germany). Immunohistochemical staining was documented with a digital color camera (Colorview II; Olympus, Hamburg, Germany) and fluorescent staining with an F-View II firewire fluorescence camera (Soft Imaging System GmbH, Münster, Germany).

Barrier functions assays. Lucifer Yellow (1 mM) in PBS (pH 7.4) was applied to the apical surfaces of epidermal organotypic (raft) cultures for 1 h. The raft cultures were subsequently embedded in Tissue-Tek (Sakura Finetechnical, Tokyo, Japan), frozen, and cryosectioned at a thickness of ca. 5 µm. The cryosections were counterstained with Hoechst dye 33258 (Sigma), and penetration of the dye was assessed by immunofluorescence microscopy.

Electron microscopy. Electron microscopy was performed as described previously (19). Ultrathin sections were viewed in a Zeiss EM 900 transmission electron microscope.


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RESULTS
 
Identification, cloning, and characterization of HPV108. HPV108 was identified as a partial fragment of the L1 ORF amplified from a low-grade cervical intraepithelial neoplasia (CIN1). Cellular DNA was initially subjected to PCR amplification using CP, GP, and FAP primers to enable detection of all known, as well as putative new, HPV types (14, 18). An amplicon with the expected size for a partial HPV fragment was obtained only with the FAP primers. This amplicon was cloned and sequenced. Sequence analyses of six clones indicated a single infection of HPV108 in this biopsy. This isolate was most closely related to HPV103 (75%) and HPV101 (70%) (9), defining it as a member of a putative new HPV type (13). The complete genome was amplified from the cervicovaginal sample, cloned, and sequenced. Sequence analysis of the full-length genome (7,149 bp) (accession no. FM212639) revealed an atypical organization for HPVs in that both the E5 and E6 genes were lacking. It constituted only five large ORFs, potentially representing three early genes (E7, E1, and E2) and two late genes (L2 and L1). The full-length HPV108 L1 ORF shared 75% nucleotide identity with HPV103 L1 and 63% identity with the HPV101 L1 ORF. Both these HPV types also lack the E5 and E6 genes (9). The genomes of a number of animal papillomaviruses deviate from the generally accepted five early genes and two late genes. Several of them, e.g., bovine papillomavirus type 3, also lacks a distinct E6 gene (13). Phylogenetic analyses of the L1 ORF sequences group these three HPV types into a new genus clustering with the genera Gammapapillomavirus and Pipapillomavirus (Fig. 1A). The L1 nucleotide sequence of HPV108 shared 60 to 61% homology with HPV4 (genus Betapapillomavirus) and hamster oral papillomaviruses (genus Pipapillomavirus), analogous to what was described for the HPV101 and HPV103 L1 ORFs (9). The amino acid similarities, however, were below 60%. This degree of nucleotide homology points to a discrepancy with the present nomenclature (13) and underlines the necessity for a revision. Similarities to all other ORFs (both nucleotide and amino acid sequences) indicate a much closer similarity to HPV101 and HPV103 than to the above-mentioned papillomavirus types. The nucleotide positions of the respective ORFs and the amino acid numbers of their putative encoded proteins, as well as nucleotide homologies and amino acid similarities to other papillomaviruses, are presented in Fig. 1B and C.


Figure 1
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  		FIG. 1. HPV108, together with HPV101 and HPV103, constitute a new genus of the family Papillomaviridae. (A) Phylogenetic tree of representative papillomavirus types based on full-length L1 ORF sequences. The numbers indicate the HPV type, and the other abbreviations refer to animal papillomavirus types. HPV108, HPV103, and HPV101 form a new genus (marked with a star) clustering with the genera Gammapapillomavirus and Pipapillomavirus. McPV2, Mastomys coucha papillomavirus type 2; HaOPV, hamster oral papillomavirus; CaPV2, canine papillomavirus type 2; PsPV, Phocoena spinipinnis papillomavirus; bpv1, bovine papillomavirus type 1; EcPV, Equus caballus papillomavirus; PePV, Psittacus erithacus timneh papillomavirus; FcPV, Fringilla coelebs papillomavirus; MnPV, Mastomys natalensis papillomavirus; COPV, canine oral papillomavirus; ROPV, rabbit oral papillomavirus; EdPV1, Erethizon dorsatum papillomavirus; TmPV, Trichechus manatus papillomavirus. (B) Putative ORFs of HPV108 (7,149 bp) with nucleotide positions and sizes of their putative encoded proteins (amino acids [aa]). (C) Identities between HPV108 ORFs and other HPV types (aa and nucleotides [nt]).

The putative encoded HPV108 E7 protein comprises 99 amino acids and possesses all characteristics previously determined to be necessary for a fully functional E7 protein (34). These include conserved region 1 (CR1), a CR2 containing the pRb binding site (LLCGE) and the casein kinase II phosphorylation site (TFEKEEQE), as well as two Cys-X-X-Cys domains (CGVC and CPAC) separated by 29 amino acids in the metal binding domain. The CR1 conserved sequence in HPV108 E7 is MRGKAPTIKDVDLEL. Both CR1 and CR2 in the amino terminus of HPV16 E7 are critical for transformation and are required for the induction of epidermal hyperplasia and tumor formation in transgenic mice (7, 11, 21). The zinc-binding domain in the C-terminal region of high-risk E7 is necessary for full Rb-binding ability and is also required for transformation (11, 27). In silico comparison of the amino acid compositions of HPV16 E7 and HPV108 E7 with the program Polyphen did not reveal any significant amino acid variation. However, additional in vitro mutational analyses are necessary to confirm these results.

We sought to investigate the neoplastic potential of HPV108 E7 by monitoring its influence on proliferation (BrdU and PCNA), differentiation (keratins K2, K4, K10, K13, K14, and K16, involucrin, and loricrin), and other proliferation-associated targets (pRb, p53, and p21CIP1), as well as for the barrier function of the epidermal layer.

HPV108 E7 expression in organotypic keratinocyte cultures. The HPV108 E7 gene (with and without a Flag tag) was introduced into NIKS immortalized keratinocytes via retrovirus-mediated gene transfer. Neomycin selection was performed, and pooled clones were expanded for four to eight passages prior to cells being seeded onto dermal equivalents. Stable transduced cells were lifted onto the raft 4 days later and were allowed to differentiate under organotypic culture conditions before being harvested on day 14. No obvious differences were noted between cultures expressing either Flag-tagged or non-Flag-tagged HPV108 E7. Nevertheless, in addition, we expressed Flag-tagged and non-Flag-tagged E7 under in vitro conditions in H1299 cells to control for any alterations induced by the Flag tag per se. The HPV108 E7 influence on the expression of the intrinsic cellular proteins pRb, p21CIP1, and PCNA was independent of the Flag tag (data not shown).

HPV108 E7 expression induces abnormal morphology. Raft cultures of cells transduced with control virus (pLXSN) showed the typical epidermal stratification with respective stages of keratinocyte differentiation: a well-organized basal compartment (stratum basale) and typical suprabasal compartments, including spinous (stratum spinosum), thin granular (stratum granulosum), and parakeratotic and keratotic (stratum corneum) layers (Fig. 2A). The width of the keratinocyte layer increased in the HPV108 E7-transduced raft cultures. The basal layer of the keratinocytes was composed of densely packed cuboidal cells, followed by irregularly arranged, more elongated cells with a slight degree of pleomorphism and polychromatism of the nuclei. A distinct separation into stratum spinosum and stratum granulosum was not evident. A decrease of the parakeratotic and keratotic strata could be seen, but they were seldom completely absent. Apoptotic cells were more numerous in the upper layer (Fig. 2B). HPV16 E6/E7-transduced keratinocyte rafts grown under the same conditions resulted in a highly dysplastic morphology with numerous mitotic figures in the multiple suprabasal layers. A stratum corneum, however, was present (Fig. 2C).


Figure 2
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  		FIG. 2. HPV108 E7 induces abnormal morphology. Shown is histology of paraffin-embedded sections of NIKS cells expressing empty vector (A), HPV108 E7 (B), and HPV16 E6/E7 (C). (B) The epidermal layer displays disorganized morphology and altered differentiation induced by HPV108 E7 in organotypic raft culture. Magnification, x200.

HPV108 E7 binds pRb and modifies the expression of pRb, p21CIP1, and p53. The influence of HPV108 E7 expression on the cytoplasmic proteins p21CIP1, pRb, and p53 was assessed on serial sections for immunohistochemical staining and confirmed in Western blot analyses by using the respective antibodies. These three proteins were conspicuously expressed in all suprabasal layers of the HPV108 E7-transduced raft culture, even in the uppermost, rather flat suprabasal cell layer. This pattern, notably seen for the p21 and pRb staining, contrasted to the NIKS-pLXSN control cultures, where all three proteins were prominent but mostly restricted to the basal compartment. Some positive cells were also seen in the strata spinosum and granulosum (Fig. 3A). p21CIP1 is activated by high-risk E7 and is increased in E7-expressing cells (40), probably due to the stabilization of the p21CIP1 protein itself rather than resulting from its transcriptional induction (28, 36). Immunohistochemistry, as well as Western blot analyses, of raft protein extracts indicated an increase of p21CIP1 protein induced by HPV108 E7 (Fig. 3A and B). Both high- and low-risk mucosal HPV E7 proteins bind pRb, but only high-risk E7 targets its degradation (34). HPV108 E7 binds to pRb similarly to HPV16 E7, as demonstrated in in vitro GST pull-down assays (Fig. 3C). pRb expression seemed to be weaker in HPV108 E7-expressing rafts than in the pLXSN control cells, an observation that became more evident in Western blot analyses performed on protein extracts of these raft cultures. pRb reduction was similar in HPV16 E6/E7-expressing rafts (Fig. 3B). Steady-state levels of p53 are increased in high-risk E7-expressing cells (12), whereas it is targeted for degradation in the presence of high-risk E6. Our results demonstrated an increased number of cells expressing p53 in both the basal and suprabasal layers under simultaneous HPV108 E7 expression (Fig. 3A). This was confirmed by Western blot analyses, in which the absence/degradation of p53 by HPV16 E6/E7 was also demonstrated in parallel cultures (Fig. 3B).


Figure 3
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  		FIG. 3. HPV108 E7 modifies the expression of cellular proteins p21CIP1, pRb, p53, and PCNA. (A) Representative immunohistochemical staining of p21CIP1, pRb, and p53 in sections of NIKS raft cultures transduced with the parental vector and HPV108 E7. (B) Western blot analyses of extracts from organotypic raft cultures expressing HPV108 E7 and HPV16 E6/E7 and an empty vector as a control. The expression of Flag-HPV108 E7, p21, pRb, p53, PCNA, and keratins K10 and K14 is demonstrated. Actin served as a loading control. (C) HPV108 E7 binds pRb in vitro. Nuclear extracts from NIKS cells were precipitated onto GST-tagged HPV108 E7 protein. The pRb and GST proteins were analyzed by Western blotting. GST-HPV16 E7 was used as a positive control and GST as a negative control.

HPV108 E7 enhances proliferation and reduces differentiation. DNA synthesis was measured through incorporation of BrdU, which was added to the culture medium 12 h prior to harvest. Only single basal cells proliferated in NIKS-pLXSN control cultures (Fig. 4A), whereas BrdU incorporation was increased in many basal, as well as in a number of suprabasal, cells of HPV108 E7-expressing cultures. In HPV16 E6/E7, this incorporation was drastically enhanced to include most suprabasal layers. This increase in proliferation induced by HPV108 E7 and HPV16 E6/E7 expression was also evidenced by PCNA induction in the suprabasal strata (Fig. 4B). Western blot analyses confirmed this increase in PCNA (Fig. 3B). Keratin K16 expression is induced in epithelia with enhanced proliferation, as well as in squamous cell carcinoma (33, 42). The K16 expression occurred in the suprabasal layers of HPV16 E6/E7 cultures and was strongly enhanced to include nearly all of the suprabasal layers in HPV108 E7 raft cultures, except for the uppermost apical cell layer, in contrast to the pLXSN controls, where it was evident in only a few basal cells (Fig. 4C).


Figure 4
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  		FIG. 4. HPV108 E7 enhances proliferation and alters differentiation in an epidermal layer of immortalized foreskin keratinocytes. Stable transduced NIKS cells harboring the parental retrovirus, HPV108 E7, and HPV16 E6/E7 were allowed to differentiate on organotypic cultures. (A) Immunofluorescence staining of cells after BrdU incorporation (green) and against nuclear staining (blue) of all cells (Merge). (B) PCNA expression (red) in separate and merged images. Immunofluorescent staining of keratin K16 (C), keratin K4 (D), keratin K14 (E), keratin K10 (F), and involucrin (Inv) (G) (each in red) and loricrin (Lor) (green) (H) against nuclear staining (blue) of all cell is shown. The dotted lines indicate the upper cell layer, and the dashed lines indicate the basement membrane barrier. Magnification, x200.

HPV108 E7 modified the differentiation pattern for normal epithelium considerably. Keratins K4 (Fig. 4D) and K13 (data not shown) are expressed throughout the suprabasal compartment of noncornified (mucosal-type) stratified squamous epithelia but absent in cornified (epidermal) squamous epithelia (33). The general suprabasal expression of keratin K4 in HPV108 E7-expressing raft cultures was similar to a pattern seen in noncornified squamous epithelium. The keratin K4 expression in the control pLXSN and HPV16 E6/E7 raft cultures was restricted to the uppermost stratified layers, exemplifying a cornified squamous cell epithelium (Fig. 4D).

Keratin K10 is characteristically expressed in the suprabasal compartment of stratified epithelia (33). Keratin K14 is expressed in squamous cell carcinoma of the cervix, whereas keratin K10 is frequently present in keratinizing, as well as nonkeratinizing, cervical carcinomas (37). Keratin K14 was predominantly expressed in the basal layer of pLXSN control rafts, whereas its expression was extended to several suprabasal layers in HPV108 E7 and slightly reduced in HPV16 E6/E7 cultures (Fig. 4E). Keratin K10 expression was delayed in HPV108 E7 organotypic cultures, and it was not expressed in the first suprabasal layers, a typical feature observed in squamous cell carcinomas of mucosal origin (Fig. 4F). This was similar in HPV16 E6/E7-expressing raft cultures, in contrast to pLXSN control cultures, in which keratin K10 expression started in the first suprabasal layer. This "delay" in the keratin K10 expression was compensated for by the filling of this "gap" by the broadened expression of keratin K14 (Fig. 4E and F).

Both loricrin and involucrin are important major components of the cornified cell envelope formation and part of the barrier structure of the epidermis (8). Involucrin expression was observed throughout the suprabasal compartments of control cultures but was highly decreased and often locally concentrated in both HPV108 E7- and HPV16 E6/E7-expressing cultures, a profile that strongly resembles the reduced status of keratinocyte differentiation (Fig. 4G). Loricrin expression is usually restricted to the upper terminally differentiated living cell layers. Its expression was restricted to the uppermost cell layers in the pLXSN control cultures and virtually absent in HPV108 E7- and HPV16 E6/E7-expressing cultures. This absence of loricrin expression is in agreement with the decrease in cornification in HPV108 E7-harboring cells (Fig. 4H). Keratin K2 was not detected in any of the cultures (data not shown).

HPV108 E7 expression disrupts the epithelial barrier function. Partial absence of the stratum corneum, as well as the remarkable decrease or absence of loricrin and involucrin expression and the absence of a cornified envelope, prompted us to investigate whether the barrier function of these epidermal cultures was disrupted. We controlled for permeability of the upper cell layer by uptake of a fluorescent dye. The Lucifer yellow dye moved through all layers into the dermal equivalent in rafts with HPV108 E7, in contrast to control NIKS-pLXSN cells, where it hardly penetrated the cornified cell layer (Fig. 5A).


Figure 5
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  		FIG. 5. HPV108 E7 disrupts the epithelial barrier function of organotypic NIKS cultures. (A and A') Fluorescent-dye penetration in organotypic raft cultures of (A) NIKS-pLXSN and (A') NIKS-pLXSN-HPV108 E7. ep, epidermis; de, dermal equivalent. Magnification, x200. (B and B') Tight junctions (red) are present (B) in NIKS-pLSXN control cultures and absent (B') in NIKS-pLSXN-HPV108 E7-expressing cultures. Enlarged views are shown in insets. Nuclei were counterstained with Hoechst 33258 (blue). The dashed lines indicate the positions of the basement membrane barriers. Magnification, x200. (C and C') Ultrastructural detail of intracellular spaces and desmosomes in NIKS-pLSXN control cultures (C) and NIKS-pLSXN-HPV108 E7-expressing cultures (C') with large intercellular spaces and many microvillus-like structures. Scale bar, 1 µm. (D and D') Ultrastructural overview of organotypic raft cultures of empty vector (D) and HPV108 E7 (D'). Scale bar, 10 µm.

Intercellular junctional complexes and lipids form the epithelial diffusion barrier. Adherens and tight junctions, as well as desmosomes, are barrier structures. Desmosomes play an important role in the tissue integrity of epithelia by linking cytoskeletal components, such as keratin filaments, to cellular membranes (25, 29, 39). A complex series of lipids, either covalently attached to proteins of the cornified envelope or as intercellular lamellae, constitute a major component of the epithelial barrier (8, 26). Immunofluorescent staining of cingulin revealed intact tight junctions in the control pLXSN keratinocyte cultures, whereas staining was completely absent in HPV108 E7-expressing rafts (Fig. 5B). By ultrastructure, HPV108 E7 rafts revealed pronounced alterations of cell-cell adhesion in all layers, with wide intercellular spaces with many microvillus-like structures protruding into the intercellular clefts (Fig. 5D). No consistent change in desmosome morphology was observed (Fig. 5C and D). The cellular protrusions were also abundant on the apical surface of the raft in the absence of a cornifying layer. Irregular lipid accumulation was an additional sign of a disturbed barrier function. Apoptotic cells were irregularly distributed in upper cell layers and were more numerous than in control keratinocyte layers.


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DISCUSSION
 
The pathogenesis of cervical carcinoma has largely been elucidated through detailed in vitro and in vivo functional analyses of the high-risk HPV E6 and E7 oncoproteins. The HPV types analyzed were mainly HPV16 and HPV18 compared to low-risk HPV6 and HPV11. The prevailing present view is that both E6 and E7 oncoproteins of high-risk HPV types are required for full transformation of keratinocytes. They each interact with and then disrupt the functions of cellular proteins known to control normal growth and differentiation of the epithelium (31, 34, 43). We isolated and characterized the full-length genome of HPV108 from a patient with a low-grade cervical lesion. Similar to a number of animal papillomavirus types and to HPV101 and HPV103, HPV108 does not have an E6 gene. Interestingly, these HPV types are more closely related to HPV types usually associated with cutaneous lesions and a number of animal papillomaviruses than to the main group of mucosa-associated HPV types of the genus Alphapapillomavirus. The isolation of novel HPV types lacking an E6 oncoprotein from low- and high-grade genital lesions (reference 9 and this report) posed the question of whether the E7 proteins of these HPV types may, in addition, substitute for functions usually attributed to E6.

One of the characteristics of high-grade E6 oncoproteins is that they impair the terminal differentiation of epithelial cells, causing expansion of less differentiated compartments. This function has been shown to be p53 independent (31, 38). HPV108 E7 expression leads to a dysplastic morphology in which the original normal epidermal stratification and sequence of keratinocyte layers are widely disorganized. p53 protein was expressed in the basal, as well as suprabasal, cell layers, which differs from previous reports in which HPV16 E7-induced p53 expression was seen only in the suprabasal layers (17). The accumulation of p53 protein also indicates that HPV108 E7 may disturb p53 degradation and that its functional activity may be impaired (32). Inactivation of both pRb and p21CIP1 by high-risk E7 contributes to the subversion of cell cycle control (24). High-risk E7-induced inactivation of pRb is required for accumulation of the cyclin-dependent kinase inhibitor p21CIP1, a transcriptional target of p53 (4, 40). The p21CIP1 increase may, however, be attributed to protein stabilization and not to transcriptional induction (28). The in vitro binding of HPV108 E7 to pRb and reduction of pRb levels under HPV108 E7 expression indicate that HPV108 E7 may be responsible for the degradation of this cellular protein and thereby interfere with cell cycle control.

Phenotypes observed in transgenic mice expressing high-risk E7 include, besides basal and suprabasal proliferation and p21CIP1 induction, an expansion of both keratin K14- and K10-positive layers of the epidermis (4). Expression of HPV108 E7 in our organotypic cell cultures induced similar phenotypes. Proliferation was induced, as monitored by enhanced BrdU incorporation, induction of PCNA, and enhanced keratin K16 expression. This process was accompanied by an expansion of keratin K14 to the suprabasal layers and a delay in keratin K10 expression into the upper suprabasal layers. This decrease in keratinocyte differentiation was also confirmed by a loss of markers for late differentiation, involucrin and loricrin. These features all point to a decrease in differentiation of the epithelial layer. Keratinocytes in epithelial layers are held together by strong cell-cell adhesions, i.e., adherens junctions and desmosomes. A progressive reduction in adherens junctions and desmosomes occurs during the transition from normal keratinocytes to HPV16-transformed cells (20), and an altered expression of desmosomal proteins distinguishes low-grade cervical lesions from high-grade lesions (1). High-grade E6 has been shown to contribute more to the progression stage of carcinogenesis (38). PDZ domain proteins form the structural backbone of tight junctions. The transforming ability of high-risk E6 is linked to its interaction with these proteins, leading to disruption of cell junctions (31). This was also induced by HPV108 E7 expression, as evidenced by the disruption of tight junctions and the formation of large intercellular spaces with many prominent microvillus-like structures.

HPV108 E7 per se induces a phenotype qualitatively similar in quite a few morphological and molecular features to that described for the high-risk E7 oncoprotein. Although the degree of dysplasia is less in HPV108 E7 cultures than in HPV16 E6/E7-transformed keratinocytes, the current data point to the potential for HPV108 E7 to induce dysplasia and initiate a tumorigenic sequence. Further isolation and identification of this virus from clinical samples may clarify its role in the pathogenesis of genital and extragenital lesions.


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ACKNOWLEDGMENTS
 
We thank Andreas Hunziker for sequencing, Silke Prätzel-Wunder for excellent technical assistance, and Louise Chow for valuable advice. We thank Teresa Martins and Luis Almeida for their continuous support and the Portuguese Institute for Oncology at Coimbra for providing the original patient sample.

R.J.N. is the recipient of a Ph.D. fellowship (SFRH/BD/19165/2004) from the Portuguese Research Council. This study was supported in part by the Ministry of Health, Berlin, Germany, and by Wilhelm Sander-Stiftung, Munich, Germany (to L.L.).


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FOOTNOTES
 
* Corresponding author. Mailing address: Division for the Characterization of Tumorviruses, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 242, 69120 Heidelberg, Germany. Phone: 49-6221-424655. Fax: 49-6221-424822. E-mail: e.devilliers{at}dkfz.de Back

{triangledown} Published ahead of print on 19 January 2009. Back

§ Present address: Molecular Pathology Laboratory, Portuguese Institute for Oncology at Coimbra, EPE and Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal. Back


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Journal of Virology, April 2009, p. 2907-2916, Vol. 83, No. 7
0022-538X/09/$08.00+0     doi:10.1128/JVI.02490-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.




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