View larger version (44K): [in this window] [in a new window] [Download PPT slide]   Figure 2. EVERs and ZnT-1 do not influence intracellular zinc accumulation and cell resistance to zinc. HaCaT cells (A and B) were transiently transfected with plasmid encoding ZnT-1, EVER1, EVER2, or control empty plasmid. Keratinocytes (C and D) were isolated from a patient suffering from EV (EVER2–/–) and from a healthy subject (EVER2+ /+). The cells were incubated for 24 h in a standard culture medium (5 µM basal zinc) or in the medium supplemented with indicated concentrations of zinc. The total intracellular zinc content was determined by flame absorption spectrometry (A and C) (29) and the cell number was determined by measurement of DNA (B) or protein (D) content in cell lysates. All the experiments were performed in triplicate. Error bars represent ± the SD.

View larger version (19K): [in this window] [in a new window] [Download PPT slide]   Figure 3. EVER2 influences intracellular distribution of free zinc and cell growth. (A) Keratinocytes with wild-type (EVER2+ /+) or mutated (EVER2–/–) gene were loaded with free zinc-specific indicator (Zinquin), incubated for 15 min in 140 µM zinc, and analyzed by phase-contrast microscopy (left) or fluorescence (right). A strong fluorescence is visible in nucleoli in both cell lines (arrows). Bars, 10 µm. (B) The fluorescence intensity in nucleoli was measured in 90 cells and presented as a percentage of fluorescence of EVER2+/+ cells. (C) EVER2–/– and EVER2+/+ keratinocytes were grown in triplicate in standard culture medium, and cell numbers were determined at different times as indicated. P < 0.01, as determined by Students' t test. Error bars represent ± the SD.

View larger version (17K): [in this window] [in a new window] [Download PPT slide]   Figure 4. EVERs and ZnT-1 down-regulate transcription factor activity. (A–D) EVER and ZnT-1 inhibit zinc-induced transcription factor MTF-1. HaCaT cells were transiently (A and B) or stably (C) transfected with control plasmid (pCINeo) or plasmids expressing full-length ZnT-1, EVER1, and EVER2 proteins, mutant EVER1* (R94X) (13) and EVER2* (T150fsX3) proteins or TMC and 3'COOH regions of EVER1 and EVER2. HaCaT cells, as well as keratinocytes with a wild-type (+/+) or mutant (–/–) EVER2 gene (D), were transfected with a luciferase reporter plasmid (pMT1/luc) under the control of transcription factor MTF-1 and grown for 24 h at different ZnSO4 concentrations (A, C, and D) or at 120 µM ZnSO4 (B). (E) HaCaT cells stably expressing the control pCiNeo plasmid (mock) or ZnT-1 were transfected with pMT1/luc and Renilla luciferase plasmids in the presence of control negative siRNA or two different siRNAs specific for ZnT-1 (siRNA#1 and siRNA#2). After 24 h, cells were harvested for luciferase activities. The results were scored as the ratio of firefly luciferase activity normalized to Renilla luciferase activity. (F–J) EVER and ZnT-1 inhibit transcription factors. HaCaT cells expressing the indicated plasmids were transfected with a mixture of plasmids encoding the transactivation domain of c-Jun (F and G) and Elk-1 (H) fused to the Gal4 DNA binding domain and a reporter luciferase plasmid containing binding sites for Gal4 (Gal4/luc). HaCaT cells (I) and keratinocytes with a wild-type (+/+) or mutant (–/–) EVER2 gene (J) were also transfected with a luciferase reporter gene under the control of AP-1 transcription factors (pAP-1/luc). The cells were grown without fetal calf serum and supplemented with 50 ng/ml EGF (F and H–J) or 10 ng/ml TGF (G). Luciferase activities and cell number were determined as described in the Materials and methods. Values ± the SD obtained with c-Jun, Elk, and AP-1 expression plasmids in the presence of control pCiNeo plasmid were taken as 100%. *, P < 0.05; #, P < 0.01.

It must be stressed that all the experiments were performed using artificial synthetic transcription factors. To investigate the influence of ZnT-1 and EVERs on the natural transcription factor present in human keratinocytes, we studied transactivation of a luciferase reporter plasmid with a minimal responsive promoter for AP-1 transcription factors (pAP-1/luc). ZnT-1 and EVER inhibited luciferase expression (Fig. 4 I), whereas a significantly increased luciferase activity was observed in EVER2^–/– keratinocytes (Fig. 4 J).

These data indicate that ZnT-1 and EVER are negative regulators of AP-1. Because AP-1 is a key transcription factor for HPV (2), a mutation in either EVER gene should facilitate the transcription of the viral genome, particularly the expression of E6 and E7 genes.

HPV16 E5 protein inhibits EVER and ZnT-1 activities
Although EV HPV are responsible for asymptomatic infections that are widespread in the general population (6, 7), they induce lesions only in EV patients where high amount of virions are produced. It must be stressed that EV patients have an abnormal susceptibility to EV HPV, but are not prone to infection with other cutaneous or genital HPV, such as the oncogenic genital HPV16 and HPV18 (4, 5). It can be speculated that EVER and ZnT-1 proteins are involved in the control of EV HPV expression. This prompted us to use a coimmunoprecipitation assay to search for a possible interaction between EVER1, EVER2, or ZnT-1 and early proteins specific for cutaneous and genital HPVs. This study was further justified by the recent study showing an interaction between HPV16 E5 and ZnT-1 in a two-hybrid assay (27). Importantly, the EV HPV genome lacks an E5 open reading frame (16).

No interaction was detected between EVER or ZnT-1 proteins and E6 or E7 of HPV5, HPV9, a nononcogenic EV HPV, and HPV16 (unpublished data). In contrast, it was found that the E5 protein of HPV16 (16E5) binds to EVER1 and EVER2 and, as expected with ZnT-1, also binds with an EVER1–EVER2 complex (Fig. 5 A). In addition, EVER and ZnT-1 colocalized with E5 in transiently transfected HaCaT cells (Fig. 5 B). Moreover, in keratinocytes transfected with ZnT-1 or EVER, 16E5 prevented ZnT-1/EVER-mediated inhibition of MTF-1 transcriptional activity (Fig. 5 C). Similarly, 16E5 blocked EVER2-mediated down-regulation of c-Jun transactivation domain activity in HaCaT cells with constitutive expression of EVER2 (Fig. 5 D). More importantly, 16 E5 increased luciferase activity by a factor of 1.6 also in EVER2^+/+ keratinocytes (Fig. 5 E). Interestingly, no effect of 16 E5 protein was observed in EVER2^–/– keratinocytes, which further suggests EVER complex as an important target for 16 E5 in keratinocytes. On the other hand, EVER2 deficiency seems to compensate the lack of functional 16 E5 protein, as shown with truncated 16 E5 corresponding to the N- or C-terminal half of the protein (Fig. 5 E). This led to MTF-1 transcriptional activity comparable to one induced by 16 E5 in wild-type keratinocytes. All these data indicate that 16 E5 is able to interact with endogenous EVER and ZnT-1 and to counteract their down-regulation.

View larger version (55K): [in this window] [in a new window] [Download PPT slide]   Figure 5. HPV16 E5 protein binds to EVER1, EVER2, and ZnT-1 and interferes with their activity. (A) Clear lysates (CL) from HaCaT cells expressing FLAG-EVER and/or FLAG-ZnT-1, together with GFP-16 E5 or GFP alone, were immunoprecipitated (IP) with anti-GFP antibody and subsequently immunoblotted (IB) with the indicated antibodies. Immunoprecipitation experiments with ZnT-1 and HPV16 E5 gave a band of unknown origin with a migration pattern similar to that of EVER2. (B) HaCaT cells were transiently transfected with plasmids encoding FLAG-EVERs or ZnT-1 and GFP-16 E5 and stained with anti-FLAG monoclonal antibodies, followed by incubation with CY-3–conjugated secondary antibodies. Colocalization of EVER/ZnT-1 (red) and 16 E5 (green) is presented on merged images (yellow). Bars, 10 µm. (C) HaCaT cells were transiently transfected with pMT1/luc and plasmids expressing EVER or ZnT-1 or control empty plasmid in the absence (E5-) or presence (E5 + ) of a plasmid encoding 16 E5. Cells were cultured for 24 h in medium with 140 µM zinc, and luciferase activity was measured. (D) HaCaT cells stably expressing control pCiNeo plasmid or EVER2 were starvated and transfected with a plasmid encoding 16 E5 (or control plasmid) and with a mixture of plasmids expressing the luciferase reporter gene (pGal4/luc) and chimeric Gal4/c-Jun protein. Cells were incubated for 24 h in a culture medium supplemented with 50 ng/ml EGF, and luciferase activity was determined. (E) Keratinocytes with a wild-type (+/+) or mutant (–/–) EVER2 gene were transiently transfected with pMT1/luc, together with the control empty plasmid (control) or plasmids expressing 16 E5 or truncated 16 E5 corresponding to the N-terminal (M1 to I51) and C-terminal (I51 to T83) parts of the viral protein. Cells were cultured for 24 h in medium with 40 µM zinc, and luciferase activity was measured. *, P < 0.05; #, P < 0.01.

However, the importance of the EVER–ZnT-1 complex is probably not limited to the control of virus life cycle in keratinocytes. It has been reported that most of EV patients have an impaired cell-mediated immunity and, as was hypothesized, that EV could represent a primary deficiency of extrinsic immunity against the β papillomaviruses (28). Thus, it can be assumed that in addition to inhibition of EV HPV expression, regulation of zinc homeostasis by EVER and ZnT-1 proteins play a role in immune response, through the secretion of antiviral effectors such as cytokines, chemokines, or growth factors. Indeed, our preliminary data indicate that the proinflammatory cytokine IL-6 is down-regulated in EVER2^–/– cells (unpublished data).

	MATERIALS AND METHODS

Top ABSTRACT RESULTS AND DISCUSSION MATERIALS AND METHODS REFERENCES

 
Cell lines and culture conditions.
Cell lines were cultured at 37°C with 5% CO₂ in MEM (human HaCaT keratinocyte line) or Dulbecco's minimum essential medium (hamster BHK21 cell line) supplemented with 10% fetal bovine serum, 2 mmol/liter glutamine, 1% penicillin-streptomycin-gentamicin antibiotics, and 0.5% fungizone. Keratinocytes were obtained from hair follicles (29) from a healthy subject and from a Polish EV patient. The samples were taken according to the approval of the Local Ethical Committee at the Medical University of Warsaw. The informed consent was obtained from EV family individuals.

Yeast two-hybrid analysis.
DNA fragments corresponding to the TMC region of EVER1 (aa L514 to W673) and EVER2 (aa E362 to S530) were obtained by PCR amplification. Fragments were cloned into plasmid pGBKT7 containing the GAL4 DNA-binding domain and used as bait to screen a human keratinocyte (HaCaT) cDNA library for proteins that were capable of interacting with EVER regions. Yeast transformation and two-hybrid screening assays were done as previously described (17).

Plasmids.
Full-length EVER1, EVER2, and ZnT-1 and DNA fragments corresponding to the conserved TMC and C-terminal regions of EVER1 (L514 to W673 and Q674 to A805) and EVER2 (E362 to S530 and A622 to L725) were obtained by PCR amplification. Fragments were cloned and tagged with FLAG epitope or fused to GST or GFP protein using Gateway recombination technology (Invitrogen) as described by Mendoza et al. (17).

Immunoprecipitation and GST-pull down assays.
BHK21 cells were plated in P6 culture plates and grown to 50–80% confluence. Cells were transfected by the PEI method with a combination of recombinant plasmids, and cell extracts were prepared for immunoprecipitation and GST pulldown as previously described (17).

Immunofluorescence microscopy.
HaCaT cells plated on 10-mm glass coverslips (200,000 cells) were transfected with different recombinant plasmids using the PEI transfection methods (17). 24 h later, the transfected cells were washed thrice with cold PBS, fixed in 3% paraformaldehyde, and permeabilized with 0.1% Triton X-100. Cells were incubated with primary mouse monoclonal or rabbit polyclonal antibodies for 1 h at 37°C. After washings, cells were incubated for 60 min with CY3-conjugated goat anti–mouse antibodies or FITC-conjugated anti–rabbit antibodies. Images were obtained using a TCS4D confocal microscope (Leica) (13). As negative controls, replicate samples were incubated with protein-blocking solution instead of primary antibodies.

For the detection of free zinc by fluorescence (22), the keratinocytes were grown to subconfluence on 30-mm plastic/glass dishes (MatTek Corporation), washed twice with incubation buffer (10 mM Hepes, 120 mM NaCl, 5.4 mM KCl, 5 mM glucose, 1 mM CaCl₂, 1 mM MgCl₂, 1 mM NaH₂PO₄, and 3 g/liter BSA, pH 7.35), and loaded with 25 µM Zinquin-E (Ex/Em 368nm/490nm; Qbiogene) in incubation buffer for 30 min at 37°C. The cells were washed twice with incubation buffer without BSA, and ZnSO4 (140 µM) was added for 15 min. The cells were investigated by fluorescence microscopy before and after incubation with zinc and photographs were taken at 200-fold magnification on a microscope (Axioplan 2; Carl Zeiss, Inc.) using a 365/520-nm filter.

Atomic absorption.
For determination of the total cellular zinc content, 3 x 10⁶ cells were washed with PBS twice and collected in 1 ml of PBS. 50 µl were taken to determine the protein content of the samples using the Bio-Rad protein assay (Bio-Rad Laboratories). The remaining cell suspension was centrifuged, and the pellet was treated with 100 µl 33% H₂O₂ and 100 µl 65% HNO₃ at 60°C for 1 h, followed by an overnight incubation at 85°C. Samples were dissolved in 0.5 ml 0.2% HNO₃ and the zinc concentration was determined by flame atomic absorption (22), using an atomic absorption spectrometer (model 2380; PerkinElmer).

Luciferase assay.
HaCaT cells were plated in P24 culture plates, grown to 50–80% confluence, and transfected by the PEI method (18). In each cotransfection experiment, total DNA (0.5 µg) was adjusted with control pCiNeo DNA. To estimate transfection efficiency, the cells were also transfected with the TK promoter-driven Renilla-luciferase plasmid (0.1 µg). After 24 h, cells were washed with phosphate-buffered saline and overlaid with 100 µl of passive lysis buffer (Promega) for 15 min at room temperature. The extracts were centrifuged 1 min at 14,000 g, and firefly and Renilla luciferase activities were measured by using the Dual-Glo luciferase kit assay (Promega) and a Lumat LB 9507 luminometer (Berthold Technologies). All experiments were performed 6–12 times to ensure reproducibility. Because expression of EVER and ZnT-1 proteins were found to down-regulate both Firefly and Renilla luciferase activities, data were normalized to cell number as determined by DNA content by fluorescence of bis benzamidine H33258 (30) or protein content using Bio–Rad protein assay. The statistical significance of data were calculated by Student's t test. For all analyses, P < 0.05 was considered significant.

Small interfering RNA (siRNA)–mediated ZnT-1 silencing.
HaCaT cells constitutively expressing ZnT-1 were cotransfected with siRNA (20 pmol) oligonucleotides, pMT1/luc plasmid, and Renilla luciferase plasmid using Lipofectamine 2000 reagent (Invitrogen) according to the instructions of the manufacturer. Two different predesigned siRNAs specific for ZnT-1 (siRNA ID#117632 and siRNA ID#117633), as well as a negative control siRNA, were purchased from Ambion. Luciferase activities were tested 24 h after transfection. Experiments were performed in quadruplicate to ensure reproducibility.

Online supplemental material.
Fig. S1 shows the localization of EVER and ZnT-1 proteins by immunofluorescence using a confocal microscope (model TCS4D; Leica). The online version of this article is available at http://www.jem.org/cgi/content/full/jem.20071311/DC1.

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