For generation of the Syntenin evolutionary trees, representative Syntenin sequences were obtained from The UniProt Consortium (2017), aligned by the L-Ins-I algorithm of the MAFFT package (Katoh et al., 2002), and subjected to tree calculation by the T-Rex server (Boc et al., 2012) using the RAxML method (Stamatakis, 2006). Uniprot accession numbers for sequences analysis: human Syntenin-1 (SDCB1_HUMAN), human Syntenin-2 (SDCB2_HUMAN), murine Syntenin-1 (SDCB1_MOUSE), murine Syntenin-2 (SDCB2_MOUSE), zebrafish Syntenin-1 (Q6PUR1_DANRE), zebrafish Syntenin-2 (Q803J6_DANRE), xenopus Syntenin-1 (Q66JA3_XENLA), xenopus Syntenin-2 (Q801P2_XENLA), anolis Syntenin-1 (G1KE37_ANOCA), anolis Syntenin-2 (H9GDS9_ANOCA), chicken Syntenin-1 (Q5ZHM8_CHICK), chicken Syntenin-2 (R4GGF6_CHICK), mosquito Syntenin (Q7PK99_ANOGA), tick Syntenin (B7PNL5_IXOSC), nematostella Syntenin (A7S2D9_NEMVE), sponge Syntenin (I1FJB9_AMPQE), capsaspora Syntenin (A0A0D2VN74_CAPO3), monosiga Syntenin (A9UPR7_MONBE).

We have previously described the generation of the pLXSN-based retroviral vectors coding for HPV8-E6 (Akgül et al., 2010). PLXSN-HPV8-E6 based mutants were generated by site-directed mutagenesis using specific primers. For retroviral infection, 3 × 10⁵ cells were seeded into 6 cm dishes and treated as described in Hufbauer et al. (2013). Selection of infected cells was started 2 days later using 250 μg/ml G418. Positive clones were pooled and expanded.

RTS3b (Purdie et al., 1993) and PM1 keratinocytes (Proby et al., 2000) were maintained in RM+ media [consisting of a 3:1 ratio of DMEM:F12 with 10% fetal bovine serum (FBS), 1% glutamine, 0.4 μg hydrocortisone, 10^-10M cholera toxin, 5 μg/ml transferrin, 2 × 10^-11M liothyronine, 5 μg/ml insulin, 10 ng/ml epidermal growth factor, 1× Penicillin/Streptomycin mixture] (Akgül et al., 2003). HaCaT cells (Boukamp et al., 1988) were grown in DMEM with 10% FBS. N/TERT cells (Dickson et al., 2000) were either cultivated in KGM-Gold (which contains low concentrations of calcium to prevent differentiation) or in RM+ media. All cell lines were cultivated at 37°C and 6% CO₂. For global demethylation of DNA, N/TERT cells were treated with 5-aza-2′-deoxycytidine (5-Aza) (Sigma–Aldrich, Steinheim, Germany) diluted in dimethyl sulfoxide (DMSO) with different final concentrations of 0 or 10 μM for 72 h with media renewal every 24 h. Differentiation of N/TERT cells grown in KGM-Gold media was accomplished by treatment with 2 mM CaCl₂ for up to 8 days with 24 h media renewal.

Silencing of PI(4,5)P₂ generating kinase gene expression was achieved through transfection of siRNA-SMARTpools (Dharmacon, GE Healthcare, Freiburg, Germany) with the RNAiMAX kit (Thermo Scientific, Waltham, MA, United States) according to the manufacturer’s protocol (siPIP4KIIα: M-006778-01; siPIP4KIIβ: M-006779-03; siPIP5KIα: M-004780-02; siPIP5KIγ: M-004782-00). These siRNAs have previously been successfully used to decrease kinase expression (Bultsma et al., 2010; Chao et al., 2010). The siRNA against Syntenin-2 had the following sequence: siSyn-2: GCAACGGGCTCCTCACCAATT. Cells transfected with a scramble siRNA (Cat. Nr.: 98129_A, Biospring, Frankfurt, Germany) were used as controls.

For Western blot analysis, cells were trypsinized, pelleted by centrifugation and lysed on ice in LSDB buffer [50 mM Tris/HCl (pH 8.0), 20% Glycerol, 100 mM KCl, 0,1% NP40, 1 mM DTT, 50 mM NaF, 1 mM orthovanadate, 1 mM PMSF] supplemented with 1× Cocktail Protease Inhibitors (Roche Diagnostics, Mannheim, Germany). The resulting extracts were sonicated, and protein concentration was determined using the Biorad Protein Assay (Biorad, Munich, Germany). Cell extracts were resolved by SDS–PAGE and transferred to a nitrocellulose membrane. After the membrane had been blocked with 5% skimmed milk in TBST [10 mM Tris/HCl (pH 8.0), 150 mM NaCl, 0.05% Tween 20] for 1 h, the blots were probed with antibodies against Syntenin-2 (rabbit polyclonal, Cat. no. 10407-1-AP, diluted 1:1500; Proteintech, Chicago, IL, United States), Loricrin (rabbit polyclonal, Cat. no. ab85679, diluted: 1:1000, Abcam, Cambridge, United Kingdom), PIP5K1α (rabbit polyclonal, Cat. no. PA5-29405, diluted 1:500, Thermo Scientific) and Tubulin (rat monoclonal, Cat. no. YL1/2, diluted 1:10,000; Abcam), which was used as loading control. Immunoreactive proteins were visualized using horseradish peroxidase-coupled IgG secondary antibodies (Dako, Hamburg, Germany) and the BM Chemiluminescence Blotting Substrate Detection System (Roche Diagnostics). The blots were exposed to autoradiographic films and signals were quantified using the ImageJ software.

Anonymized EV skin lesions were obtained from routine surgical excisions. The use of the EV tissue for “studies on the mechanisms of HPV oncoprotein action in cutaneous oncogenesis” was approved by the local ethics-committee at the Medical University of Warsaw. HPV typing of these lesions was performed as described in Heuser et al. (2016). Four-micrometer formalin-fixed, paraffin-embedded sections were deparaffinized with xylene. Samples were hydrated through a descending alcohol series (100, 90, and 70%; 5 min each) and endogenous peroxidases were inactivated by incubation in 3% H₂O₂ in Methanol for 20 min. Antigen unmasking was performed by boiling the tissue sections in 10 mM citric acid buffer for 3 min in a beaker in a microwave followed by 15 min resting at RT. Further incubation steps were performed in a humid chamber to prevent drying-out. Blocking of unspecific antigen sites was achieved with 50% goat serum (Thermo Scientific) in PBS for 1 h at RT. Incubation with primary antibody against rabbit-anti-Syntenin-2 (ProteinTech) was done in a dilution of 1:250 in 2% goat serum over night at 4°C, followed by three washes with PBS. Sections of EV lesions were stained with rabbit anti-HPV8 E4 antibodies raised against a glutathione S-transferase (GST)-E1^∧E4 fusion protein (Borgogna et al., 2012). Detection of primary Syntenin-2 antibody was achieved by incubating the sections with a HRP-conjugated goat-anti-rabbit-IgG antibody (Dako, Santa Clara, CA, United States) diluted 1:2000 in 2% goat serum for 1 h at RT, followed by three washes with PBS. The fluorescence staining was performed with the “TSA^TM-Plus Fluorescein System” (PerkinElmer, Waltham, MA, United States). For E4 detection, sections were incubated with anti-rabbit secondary antibody conjugated with Alexa Fluor 488 (1:500 dilution in 2% FCS; Invitrogen) for 1 h at room temperature. The sections were counterstained with DAPI and embedded with Immumount (Thermo Fisher Scientific, St. Leon-Rot, Germany). Specific signals were visualized on the Leica DMI 6000B fluorescence microscope.

RNA was isolated from cells using the RNeasy kit, and DNase digestion was performed on-column using RNase-free DNase according to the manufacturer’s instructions (Qiagen, Hilden, Germany) (Lazic et al., 2011; Hufbauer et al., 2015). One microgram of total RNA was reverse transcribed using the Omniscript RT kit (Qiagen, Hilden, Germany) with 10 μM random nonamers (TIB MOLBIOL, Berlin, Germany) and 1 μM oligo(dT₂₃) primer (Sigma), as well as 10 units of RNase inhibitor (Thermo Fisher Scientific). Quantitative PCR (qPCR) was performed using the Light-Cycler system (Roche Diagnostics). The total copy number of target genes was normalized to the total copy number of the housekeeping gene encoding hypoxanthine phosphoribosyltransferase 1 (HPRT1). One PCR mixture contained 2 μl of 1:10 diluted cDNA in a total volume of 20 μl, 1.25 units Platinum Taq polymerase and the buffer provided in the kit (Invitrogen, Karlsruhe, Germany), 4 mM MgCl₂, 1.6 μl of a 1:1000 dilution of SYBR^® Green (Sigma), 5% DMSO, 0.5 μM each forward and backward primer, 500 ng/μl non-acetylated bovine serum albumin (Thermo Fisher Scientific), and 0.2 mM deoxynucleotide triphosphate. To generate absolute standards, PCR fragments, amplified with primers also used for subsequent qPCR analysis, were cloned into pJET1.2 (Thermo Fisher Scientific). Samples were analyzed in duplicates together with a 10-fold dilution series of standard plasmid. The cycling protocol conditions were 60 s at 95°C, followed by 40 cycles of 1 s at 95°C (20°C/s), 5 s at annealing temperature (melting temperature of primer minus 5°C) (20°C/s), and 15 s at 72°C (20°C/s). The primers used in this study had the following 5′-to-3′ sequences:

Cells were seeded on coverslips and fixed in 4% paraformaldehyde for 10 min at RT and further permeabilized with 0.1% Triton X-100 for 5 min at RT. After blocking in 10% goat serum (diluted in PBS) for 30 min at RT, cells were incubated with primary antibodies against Syntenin-2 (ProteinTech) diluted 1:250 in 2% goat serum over night at 4°C. On the following day cells were incubated with corresponding fluorescently labeled secondary antibodies, counterstained with DAPI and embedded using Immumount (Thermo Fisher Scientific). Fluorescent signals were visualized on a Leica DMI 6000B fluorescence microscope.

All qRT-PCR experiments were repeated a minimum of three times in duplicates. The results are expressed as mean ± SEM. Western blots were repeated in n = 3 independent experiments. The data presented as immunoblots or images of immunofluorescence analysis are from a representative experiment, which was qualitatively similar in the replicate experiments. Statistical significance was determined with unpaired 2-tailed Student’s t-test. The asterisks shown in the figures indicate significant differences of experimental groups (^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001). Non-significant changes are labeled as “ns.”

The PDZ protein family of Syntenins is comprised of two family members, Syntenin-1 and Syntenin-2. Retrieving Syntenin sequences from UniProt and constructing phylogenetic trees, we identified that both genes are the result of a gene duplication event that occurred during vertebrate evolution from a single ancestral Syntenin gene. Since the host range of papillomaviruses seems to be restricted to vertebrates (Rector and Van Ranst, 2013; Lopez-Bueno et al., 2016), the proposed timing of Syntenin gene duplication correlates with the evolution of the first papillomaviruses (Figure 1A). In addition, Syntenin-1 and Syntenin-2 show extensive structural similarity within the different vertebrate lineages (Figure 1B). Thus, Syntenin gene duplication may have contributed to both divergent and convergent evolution of Syntenin-1 and Syntenin-2 during vertebrate evolution.

To address the mechanisms through which HPV8-E6 regulates gene expression in a cell line model, Syntenin-2 protein levels were determined in different keratinocyte cell lines. Total cell extracts of the HPV-negative PM1, HaCaT and RTS3b cells were analyzed by Western blotting in addition to N/TERT cells grown either in RM+ (N/TERT^RM+) or in KGM-Gold (N/TERT^KGM) media. As shown in Figure 2A, PM1, HaCaT and RTS3b cells showed expression of Syntenin-2. However, compared to isogenic N/TERT^RM+ cells, Syntenin-2 was found to be downregulated in N/TERT^KGM. These data indicated that Syntenin-2 expression is culture media and differentiation dependent. To further confirm differentiation dependence, N/TERT^KGM were exposed to high extracellular calcium concentrations (2 mM) for up to 8 days. Parallel to the induction of the differentiation marker loricrin, confirming the differentiation-inducing cell culture conditions, Syntenin-2 was found to be upregulated in a calcium- and time-dependent manner (Figure 2B).

To further evaluate the expression pattern of Syntenin-2 in skin tumors induced by betaPV infection, we stained HPV8 positive skin tumors of EV patients. Staining against the viral E4 protein, which can have both a nuclear and cytoplasmic staining pattern in betaPV-induced tumors was used as surrogate marker for betaPV infection. To visualize E4, a broadly cross-reactive polyclonal antibody raised against the E4 protein of HPV5 and HPV8 was used (Borgogna et al., 2012). As demonstrated in Figure 2C, Syntenin-2 is expressed in the cytoplasm and nuclei of suprabasal keratinocytes in E4 negative EV skin and is absent in E4 positive lesional skin. Taken together, these findings implicated that Syntenin-2 expression is dependent on cell differentiation and is downregulated in skin with productive betaPV infection.

Given that HPV8-E6 mediated Syntenin-2 repression was previously identified in primary human keratinocytes (Lazic et al., 2012), we next tested whether E6 may also interfere with Syntenin-2 regulation in keratinocyte cell lines. As shown in Figure 3A, E6 mediated repression of Syntenin-2 transcription was only found to be significant in N/TERT^KGM and was not observed in HPV8-E6 positive N/TERT^RM+, RTS3b, HaCaT and PM1 cells. Immunocytochemical analysis revealed nuclear and cytoplasmic localization of Syntenin-2 in empty vector transduced N/TERT^KGM keratinocytes and a marked reduction of mainly cytoplasmic Syntenin-2 in HPV8-E6 positive cells (Figure 3B). To further elucidate the role of conserved amino acids in HPV8-E6 involved in regulation of Syntenin-2 expression, seven mutants of E6 were generated. To test whether repression of Syntenin-2 is a direct consequence of the previously described function of E6 to inhibit Mastermind-like protein 1 (MAML1) dependent differentiation processes (Brimer et al., 2012; Tan et al., 2012; Meyers et al., 2013), the mutants L61A, W63A and L61A/W63A were used. We could show in another study, that the E6 mutants L61A and W63A are still able to bind to MAML1 whereas the double mutant L61A/W63A is deficient in MAML1 binding (own unpublished data). Based on previously published data on HPV5-E6 mutants, the HPV8-E6 mutants V68A, D96A and D126A represent functional mutants, which are still able to inhibit UV-induced apoptosis but are incapable to induce proteasomal degradation of the pro-apoptotic protein Bak (Simmonds and Storey, 2008). The HPV8-E6-K136N mutant is, in contrast to wild type E6, deficient in inhibiting UV-induced DNA damage repair (Hufbauer et al., 2015). In line with wild type E6, the mutants L61A, W63A and the double mutant L61A/W63A (all positions are located within the first zinc-finger) were able to repress Syntenin-2 expression, whereas mutations located near the C-terminus (V68A, D96A, D126A, K136N) failed to inhibit Syntenin-2 expression (Figures 3C,D). This data provided evidence that E6 mediated downregulation of Syntenin-2, is a mechanism that is independent of E6 mediated blockade of MAML1 dependent differentiation processes.

Hypermethylation of promoter regions can be involved in transcriptional regulation of cellular genes and E6 mediated promoter hypermethylation has previously been described for HPV16-E6 (D’Costa et al., 2012; Schütze et al., 2015; Yin et al., 2017). To test the hypothesis that Syntenin-2 promoter activity may also be methylation-dependent, the effect of 5-Aza-2′-deoxycytidine (5-Aza) mediated inhibition of DNA methylation on Syntenin-2 promoter regulation was measured in the presence and absence of HPV8-E6. E6 expression in cells used for these assays was confirmed by qRT-PCR (Supplementary Figure S1). Syntenin-2 mRNA levels were low in DMSO treated cells but significantly increased following treatment with 5-Aza in control cells. This increase in mRNA was also mirrored on the protein level (Figure 3E). Syntenin-2 mRNA levels were lower in HPV8-E6 expressing cells compared to empty vector positive and DMSO treated keratinocytes. Syntenin-2 transcription levels were increased about 30-fold following 5-Aza treatment in E6 positive cells, restoring them to the same level that was detected in 5-Aza treated control cells (Figure 3E). This complete restoration supports hyper-methylation dependent regulation of Syntenin-2 expression by HPV8-E6.

It has been shown by Mortier et al. (2005), that nuclear PI(4,5)P₂ is required for the sub-nuclear enrichment of Syntenin-2. PI(4,5)P₂ generating kinases are present in the nucleus and have an impact on gene expression by contributing to chromatin unfolding and transcriptional regulation (Delage et al., 2013). In principle, PI(4,5)P₂ is synthesized by either the phosphorylation of phosphatidylinositol-4-phosphate (PI4P) by the lipid kinase phosphatidylinositol-4-phosphate-5-kinase type I (PIP5KI) or by the phosphorylation of phosphatidylinositol-5-phosphate (PI5P) through phosphatidylinositol-5-phosphate-4-kinase type II (PIP4KII) (van den Bout and Divecha, 2009). Each of these kinases comprises three isoforms, given the designations α, β and γ. To evaluate the functional correlation between the PI(4,5)P₂ pathway and Syntenin-2 gene expression, we next analyzed the impact of PI(4,5)P₂ generating kinases on Syntenin-2 expression through silencing of the single kinase isoforms. The kinase isoform PIP4KIIγ is not a nuclear protein and was therefore not studied (Itoh et al., 1998; Clarke et al., 2008) in addition to PIP5KIβ, which is not expressed in N/TERT^KGM (data not shown). A strong knockdown of the kinase isoforms could be achieved for all four tested enzymes using kinase specific siRNA pools in E6 negative and positive cells (Figure 4A). The expression of HPV8-E6 in these cells was confirmed by qRT-PCR and was not significantly changed in all siRNA treated cells (Supplementary Figure S2). Knockdown of PIP4KIIβ and particularly PIP5KIα led to a significant upregulation of Syntenin-2 mRNA expression in wild type keratinocytes. Interestingly, HPV8-E6 was able to counteract siPIP4KIIβ and siPIP5KIα mediated re-activation of Syntenin-2 transcription (Figure 4B). On protein level, kinase silencing led to the induction of Syntenin-2 expression in siPIP4KIIα, siPIP4KIIβ, siPIP5KIα and siPIP5KIγ transfected cells with siPIP5KIα showing the strongest effect in empty vector positive N/TERT^KGM cells. HPV8-E6 blocked Syntenin-2 protein re-expression in siPIP4KIIα, siPIP4KIIβ and siPIP5KIγ treated cells and counteracted siPIP5KIα-mediated re-expression of Syntenin-2 by 50% (Figures 4C,D). To further determine whether, on the other hand, Syntenin-2 is a regulator of PIP5K1α expression, we also measured protein levels in keratinocytes treated with Syntenin-2 specific siRNA. Unexpectedly, knockdown of Syntenin-2 led to a strong downregulation of PIP5K1α protein expression (Figure 4E).