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Genome Replication and Regulation of Viral Gene Expression

Identification and Functional Characterization of Phosphorylation Sites of the Human Papillomavirus 31 E8^E2 Protein

Saskia van de Poel, Marcel Dreer, Ana Velic, Boris Macek, Praveen Baskaran, Thomas Iftner, Frank Stubenrauch
Lawrence Banks, Editor
Saskia van de Poel
aUniversity Hospital Tuebingen, Institute for Medical Virology and Epidemiology of Viral Diseases, Division of Experimental Virology, Tuebingen, Germany
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Marcel Dreer
aUniversity Hospital Tuebingen, Institute for Medical Virology and Epidemiology of Viral Diseases, Division of Experimental Virology, Tuebingen, Germany
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Ana Velic
bProteome Center Tuebingen, University of Tuebingen, Tuebingen, Germany
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Boris Macek
bProteome Center Tuebingen, University of Tuebingen, Tuebingen, Germany
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Praveen Baskaran
cQuantitative Biology Center, University of Tuebingen, Tuebingen, Germany
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Thomas Iftner
aUniversity Hospital Tuebingen, Institute for Medical Virology and Epidemiology of Viral Diseases, Division of Experimental Virology, Tuebingen, Germany
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Frank Stubenrauch
aUniversity Hospital Tuebingen, Institute for Medical Virology and Epidemiology of Viral Diseases, Division of Experimental Virology, Tuebingen, Germany
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Lawrence Banks
International Centre for Genetic Engineering and Biotechnology
Roles: Editor
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DOI: 10.1128/JVI.01743-17
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ABSTRACT

The papillomavirus E2 protein regulates transcription, replication, and nuclear retention of viral genomes. Phosphorylation of E2 in the hinge region has been suggested to modulate protein stability, DNA-binding activity, and chromosomal attachment. The papillomavirus E8^E2 protein shares the hinge domain with E2 and acts as a repressor of viral replication. Mass spectrometry analyses of human papillomavirus 31 (HPV31) E8^E2 and E2 proteins identify phosphorylated S78, S81, and S100 in E8^E2 and S266 and S269 in E2 in their hinge regions. Phos-tag analyses of wild-type and mutant proteins indicate that S78 is a major phosphorylation site in E8^E2, but the corresponding S266 in E2 is not. Phosphorylation at S78 regulates E8^E2's repression activity of reporter constructs, whereas the corresponding E2 mutants do not display a phenotype. Phosphorylation at S78 does not alter E8^E2's protein stability, nuclear localization, or binding to DNA or to cellular NCoR/SMRT complexes. Surprisingly, in the context of HPV31 genomes, mutation of E8^E2 S78 does not modulate viral replication or transcription in undifferentiated or differentiated cells. However, comparative transcriptome analyses of differentiated HPV31 E8^E2 S78A and S78E cell lines reveal that the expression of a small number of cellular genes is changed. Validation experiments suggest that the transcription of the cellular LYPD2 gene is altered in a phospho-S78 E8^E2-dependent manner. In summary, our data suggest that phosphorylation of S78 in E8^E2 regulates its repression activity by a novel mechanism, and this seems to be important for the modulation of host cell gene expression but not viral replication.

IMPORTANCE Posttranslational modification of viral proteins is a common feature to modulate their activities. Phosphorylation of serine residues S298 and S301 in the hinge region of the bovine papillomavirus type 1 E2 protein has been shown to restrict viral replication. The papillomavirus E8^E2 protein shares the hinge domain with E2 and acts as a repressor of viral replication. A large fraction of HPV31 E8^E2 is phosphorylated at S78 in the hinge region, and this is important for E8^E2's repression activity. Surprisingly, phosphorylation at S78 in E8^E2 has no impact on viral replication in tissue culture but rather seems to modulate the expression of a small number of cellular genes. This may indicate that phosphorylation of viral transcription factors serves to broaden their target gene specificity.

INTRODUCTION

Infections with high-risk human papillomaviruses (HPVs) such as HPV16, -18, and -31 can result in anal, cervical, oropharyngeal, penile, and vaginal cancer (1). HPVs have a double-stranded covalently closed DNA genome of ∼8,000 bp. Upon infection of undifferentiated keratinocytes, HPV genomes undergo a first round of amplification that increases copy number to ∼100 viral genomes per cell. Upon differentiation of the infected cell, a second amplification step results in several thousands of viral genomes and the transcription and translation of the viral L1 and L2 capsid genes. The first round of genome amplification depends upon the viral E1 and E2 proteins, which form a complex that recognizes the viral origin of replication (2, 3). E1 then acts as a replicative helicase and recruits cellular replication enzymes to initiate replication (2). E2 not only activates replication via interaction with E1 but also can modulate viral transcription and tethers viral genomes to the host chromosomes in mitosis to facilitate their retention, maintenance, and partitioning (3). E2 is composed of an N-terminal domain of ∼200 amino acids (aa), which mediates the interaction with E1 and cellular proteins, and a C-terminal DNA-binding and dimerization domain of ∼100 aa (3). Both domains are connected by a flexible hinge domain that, in contrast to the N- and C-terminal domains, is not highly conserved among papillomaviruses (PVs).

The initial amplification of HPV genomes is limited by the HPV E8^E2 protein, which acts as an inhibitor of both E1/E2-dependent replication and viral transcription (4–11). E8^E2 shares the hinge region and the DNA-binding/dimerization domain with E2 but differs in its N-terminal domain, which is required for E8^E2's repressive activities and recruits cellular NCoR/SMRT corepressor complexes (4, 9, 11–14). HPV16 and -31 E8^E2 knockout genomes replicate to ∼10- and ∼30-fold-higher copy numbers, respectively, in short-term assays than wild-type (WT) genomes (7, 10, 11). In long-term assays, only HPV16 genomes are maintained as extrachromosomal elements at higher copy numbers, whereas HPV31 genomes integrate into the host chromosome (7, 9, 10).

The E2 proteins of different PV types have been shown to be modified by phosphorylation, acetylation, and sumoylation (15–22). S298 and S301, which are located in the hinge region, are the major phosphorylation sites of bovine papillomavirus 1 (BPV1) E2 (19). Mutation of these residues in the context of complete BPV1 genomes resulted in an increased genome replication (23, 24). This has been linked to an increased proteasomal degradation of phospho-S301 (p-S301) E2, and recent studies also indicated that phosphorylation of S298 and S301 decreases the DNA binding affinity of BPV1 E2 (24, 25). HPV16 E2 is phosphorylated at S243 and T286, which are also located within the hinge region (15, 18). Phosphorylation of HPV16 E2 S243 has been described to be important for the binding of E2 to mitotic chromosomes and to increase the E2 protein's half-life, whereas the functional consequences of the phosphorylation of T286 in HPV16 E2 remain unclear (15). HPV8 E2 is a highly phosphorylated protein (26). Phosphorylation of S253 in the hinge region stabilizes E2 and also increases binding to cellular chromosomes (21). Taken together, these findings indicate that the hinge of E2 proteins is a common target for serine/threonine kinases. Interestingly, the functional consequences of E2 phosphorylation differ among PVs: phosphorylation destabilizes the BPV1 E2 protein and weakens its DNA binding affinity, whereas phosphorylated HPV8 and -16 E2 proteins are stabilized and display an increased binding to host chromosomes.

The E8^E2 protein shares the hinge region with E2 and thus might also be a target of serine/threonine kinases. Therefore, we investigated the phosphorylation status of the HPV31 E8^E2 and E2 proteins and analyzed the functional consequences.

RESULTS

Identification and validation of HPV31 E2 and E8^E2 phosphorylation sites.To investigate the phosphorylation status of the HPV31 E8^E2 protein, we generated a HeLa cell line using the lentiviral pInducer vector system to express a hemagglutinin (HA)-tagged HPV31 E8^E2 protein in a doxycycline-inducible manner (27). After induction, E8^E2 was immunoprecipitated with anti-HA magnetic beads. The immunoprecipitate was subjected to mass spectrometry and analyzed for phosphorylated E8^E2 peptides. In the first experiment, peptides were generated by trypsin digest, which resulted in a sequence coverage of 68% and the detection of phosphoserines 78 and 81 (Fig. 1). In the second experiment, peptides were obtained after combined trypsin and LysC digestion, which increased the coverage rate to 94% and detected phospho-S100 in addition to S78 and S81. Furthermore, HPV31 E2-HA was immunoprecipitated from transiently transfected HeLa cells, and peptides were obtained by trypsin (experiments 1 and 2) or trypsin-LysC (experiment 3) digestion and also subjected to mass spectrometry (MS). Coverage rates varied from 35 to 61%, and only in one experiment was phosphorylation of S266 and S269 detected, which corresponded to S78 and S81 in E8^E2 (Fig. 1A and B), although peptides containing these serines could be detected in all experiments regardless of whether E8^E2 or E2 was expressed. An alignment of all available alpha-HPV E8^E2 sequences showed that S78 and the downstream residues SV/LD are present in HPV11, -16, -26, -31, -39, -42, -43, -44, -51, -52, -59, -67, -68, -74, -82, -85, -87, and -125 (Fig. 2). S81 is present in HPV11, -16, -31, -35, -43, -44, -52, -74, and -87. S100 is only present in HPV26, -31, -35, -39, -51, and -82. This suggests that S78 is not specific for high-risk HPV and is moderately conserved among alpha-HPVs.

FIG 1
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FIG 1

Mass spectrometric analysis identifies serines 78, 81, and 100 of HPV31 E8^E2 and serines 266 and 269 of HPV31 E2 as potential phosphorylation sites. (A) Phosphorylated serines on the HPV31 E8^E2 and E2 proteins identified by nano-LC-MS/MS analysis from anti-HA immunoprecipitates from HeLa pInducer HPV31 E8^E2-HA cells or HeLa cells transfected with plasmids expressing HA-tagged HPV31 E2 proteins. (B) Schematic drawing of the HPV31 E2 and E8^E2 protein. Potential phosphorylated serines are underlined. (C) Nano-LC-MS/MS spectra of phosphopeptides containing S78 (GDpSVDSVNCGVISAAACTNQTR).

FIG 2
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FIG 2

Partial sequence alignment of alpha-HPV E8^E2 proteins. The alignment was obtained from pave.niaid.nih.gov. Alpha9-HPV types are indicated in boldface.

To confirm phosphorylation of E8^E2 and E2 proteins by an independent method, HeLa cells were transiently transfected with expression vectors for E2-HA or E8^E2-HA. Whole-cell extracts were prepared, subjected to SDS-PAGE in the absence or presence of Phos-tag reagent, and then analyzed by immunoblotting with an anti-HA antibody. Phos-tag reagent binds to phosphorylated ions and thereby retards the migration of phosphorylated proteins in SDS-PAGE (28). As can be seen in Fig. 3A, E8^E2 gave rise to six species with different migration patterns. In order to gain insight into whether some of these species are related to phosphorylation at S78, S81, or S100, serine-to-alanine mutations were generated in E8^E2 and analyzed. E8^E2 S78A displayed only one major fast-migrating band and two slower-migrating bands. In contrast, the E8^E2 S81A or S100A mutant proteins showed patterns more similar to the WT protein. This indicated that S78 is the major phosphorylation site in E8^E2. We also performed these experiments using a keratinocyte cell line that maintains extrachromosomal HPV31 genomes. WT E8^E2 gave rise to two major and several minor bands (Fig. 3B). Similar to HeLa cells, only the S78A mutant protein but not the S81A or S100A mutant proteins showed a different migration pattern with one major band that might represent the nonphosphorylated form. This suggested that S78 is the major phosphorylation site of E8^E2 in different keratinocyte-derived cell lines. E2 displayed only one major band and two minor slower-migrating bands in both HeLa and HPV31 WT cells. E2 S266A, S269A, and S288A mutant proteins showed a pattern similar to the WT protein (Fig. 3). This indicates that only a minor fraction of HPV31 E2 is phosphorylated, which is consistent with the difficulties of reproducibly detecting phosphorylation sites in E2 by mass spectrometry.

FIG 3
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FIG 3

Phos-tag SDS-PAGE analysis confirms phosphorylation of HPV31 E8^E2 and E2. HeLa cells (A) and NHK-HPV31 WT cells (B) were transfected with the empty expression vector (control [con]), expression vectors for HPV31 E8^E2-HA or E2-HA proteins (wild type or serine-to-alanine mutants). Whole-cell extracts were analyzed 48 h after transfection by immunoblotting with anti-HA antibody after Phos-tag SDS-PAGE. Arrows indicate the different migrating species of the proteins.

Phosphorylation of HPV31 E8^E2 regulates its repression activity.To investigate the functional consequences of E8^E2 and E2 phosphorylation, the wild-type and mutant proteins were tested in transcription reporter assays in HeLa cells. In addition to S-to-A mutants, which can no longer be phosphorylated, S-to-E mutations, which mimic constitutive phosphorylation, were introduced into E8^E2 and E2 expression vectors. All E2 mutants were able to activate transcription from the reporter plasmid comparable to the WT protein (Fig. 4A). Since the identification of phosphorylated serines used HA-tagged E8^E2, we compared native and HA-tagged E8^E2 WT and mutant proteins in HeLa cells and found no functional differences (data not shown). The analysis of E8^E2 mutants in HeLa cells revealed that the S78A and the S100E mutants significantly lost the ability to repress the reporter plasmid (Fig. 4A). In addition, S81E and S100A mutants showed a reduced repression activity, but this did not reach statistical significance. The S81A mutant behaved essentially like the WT E8^E2 protein. Consistent with phosphorylation of S78 being important for the repression activity of E8^E2, the S78E mutant reversed the transcriptional phenotype and repressed as efficiently as WT E8^E2 (Fig. 4A). These experiments were additionally carried out in keratinocytes that stably maintain episomal HPV31 WT genomes, which confirmed that the S78A mutant significantly lost repression activity, whereas the S78E mutant repressed the reporter similar to the WT (Fig. 4B). The S100E mutant also lost repression activity, whereas the S100A mutant did not, but this did not reach statistical significance. The differential behavior of the S100A mutant in HeLa versus NHK-HPV31 WT cells indicates cell-type-specific differences. This, however, was not further analyzed since no phenotype was observed in the physiologically more relevant NHK-HPV31 WT cell line and the HPV-negative keratinocyte RTS3b cell line (Fig. 5). In summary, these data suggest that phosphorylation of S78 is important for transcriptional repression by E8^E2 in different keratinocyte cell lines.

FIG 4
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FIG 4

Phosphorylation of E8^E2 S78 is required for transcriptional repression of reporter plasmids. (A) HeLa cells were transfected with 100 ng of the pC18-Sp1-luc firefly luciferase reporter, 10 ng of the empty expression vector (pSG5), or the expression vectors for wild-type or mutant HPV31 E8^E2 (left graph) or HPV31 E2 (right graph) and 0.5 ng pCMV-Gluc as an internal control. (B) NHK-HPV31 wild-type cells were transfected with 300 ng pC18-Sp1-luc, 30 ng of the empty vector, or the indicated HPV31 E8^E2 expression vectors (wild type or mutants) and 0.5 ng pCMV-Gluc. Values are presented as the ratio of firefly luciferase (Fluc) to Gaussia luciferase (Gluc) activities. Error bars indicate the standard error of the mean (SEM) from at least seven independent experiments (HeLa) or three independent experiments (NHK-HPV31 WT) performed in duplicate. Statistical significance was determined with a one-way ANOVA and Dunnett's multiple-comparison test: *, P < 0.05; ***, P < 0.001.

FIG 5
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FIG 5

Phosphorylation of E8^E2 S78 is required for repression of replication in a reporter-based replication assay. RTS3b cells were transfected with 0.5 ng of pCMV-Gluc, 50 ng of a reporter plasmid containing the HPV31 URR and the viral early promoter driving the firefly luciferase gene (pGL31URR), and expression vectors for HPV31 E1 (100 ng), E2 (10 ng), and wild-type or mutant E8^E2 (10 ng). Differences in the amounts of DNA were adjusted with the empty expression vectors (pSG5). Values are presented as the ratio of firefly luciferase (Fluc) to Gaussia luciferase (Gluc) activities. Error bars indicate the SEM from five independent experiments performed in duplicate. Statistical significance was determined with a one-way ANOVA and Dunnett's multiple-comparison test: *, P < 0.05; ***, P < 0.001.

To address the effects of these mutations on the modulation of E1/E2-dependent replication, an HPV31 URR luciferase construct was cotransfected with expression vectors for wild-type E1, E2, and E8^E2 or the respective serine mutants into the HPV-negative RTS3b keratinocyte cell line as described previously (4, 29). The E1/E2-induced replication of the reporter leads to an increase in activity of the viral major early promoter that drives firefly luciferase expression. WT E8^E2 repressed E1/E2-induced luciferase activity 10-fold (Fig. 5). In contrast, E8^E2 S78A and S100E displayed significantly reduced repression activities of 2.5- and 2.1-fold, respectively. Reduced repression was not observed with the S78E and S100A mutants. E8^E2 S81A did not show an effect, and the S81E mutant displayed a reduced repression activity that was not statistically significant. In summary, the activities of the E8^E2 serine mutants in replication assays reflected their behavior in transcription assays. However, in contrast to the complete loss of transcriptional repression observed with the pC18-Sp1-luc reporter construct, only a partial loss of replication repression activity of the S78A and S100E mutants in the presence of E1 and E2 on the HPV31 URR construct could be seen.

Phosphorylation of the hinge region of BPV1, HPV16, and HPV8 E2 has been reported to influence protein stability (15, 18, 21, 25). We therefore determined steady-state levels of HA-tagged E8^E2 WT and mutant proteins in HeLa cells after transient transfection (Fig. 6A and B). Quantification of several independent experiments revealed that S78A, S81A, and S100A protein levels were similar to WT levels, whereas S78E and S100E protein levels were slightly increased and S81E levels slightly decreased. We then analyzed the subcellular localization of E8^E2 S-to-A and S-to-E mutants after transient transfection of HeLa cells by immunofluorescence microscopy (Fig. 6C). All mutants showed a nuclear distribution similar to the WT protein. This indicated that the impaired repression by E8^E2 S78A and S100E is neither due to decreased protein levels nor due to its localization.

FIG 6
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FIG 6

Phosphorylation of E8^E2 does not affect its stability and cellular localization. HeLa cells were transfected with the indicated expression vectors for HPV31 E8^E2-HA (wild type or mutant) or the empty vector (control [con]). (A) Cells were harvested 48 h after transfection, and whole-cell extracts were analyzed by immunoblotting using an anti-HA antibody and anti-α-tubulin as a reference. (B) Immunoblots were quantified using Image Studio software, and the relative E8^E2 protein levels were normalized to α-tubulin levels. Error bars indicate the SEM from at least three independent experiments. (C) Cells were stained with an anti-HA antibody to detect E8^E2 proteins and analyzed by immunofluorescence microscopy 48 h after transfection. DNA was stained with DAPI (blue).

Recent studies indicated that phosphorylation of BPV1 E2 of S298 and S301 influences not only protein stability but also DNA-binding activity (24). We therefore performed electrophoretic mobility shift assays (EMSAs) using nuclear extracts from transfected HeLa cells and an oligonucleotide representing the E2 binding site (E2BS) of the pC18-Sp1-luc reporter plasmid. This showed that the S78A mutant bound the DNA to a similar extent to the WT and S78E mutant proteins (Fig. 7A). In addition, chromatin immunoprecipitation (ChIP) analyses were carried after cotransfection of the pC18-Sp1-luc reporter plasmid with expression vectors encoding HA-tagged WT E8^E2 or the S78A, S78E, and S100E mutant versions to address binding to E2BS in vivo. Quantitative PCR (qPCR) analyses using an amplicon covering the E2BS of pC18-Sp1-luc revealed that WT E8^E2 was 7.6-fold enriched compared to the empty vector control, and comparable binding was observed for both S78A and S78E mutants (Fig. 7B). In contrast, binding of the S100E mutant was slightly decreased to 52% of WT levels. This indicated that phosphorylation of S78 in the hinge region does not influence DNA binding of E8^E2 in vitro or in vivo, whereas phosphorylation of S100 may slightly decrease DNA binding in vivo.

FIG 7
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FIG 7

Phosphorylation of HPV31 E8^E2 S78 does not affect DNA binding activity. (A) EMSA analysis of nuclear proteins binding to the E2BS of the pC18-SP1-luc reporter plasmid. Nuclear extracts of HeLa cells transfected with expression vectors for HPV31 E8^E2, HPV31 E8^E2 S78A, or HPV31 E8^E2 S78E were incubated with the DY681-labeled oligonucleotides containing E2BS. (B) HeLa cells were cotransfected with the pC18-Sp1-luc reporter plasmid and the empty expression vector (pSG5) or expression vectors for HA-tagged HPV31 E8^E2, HPV31 E8^E2 S78A, HPV31 E8^E2 S78E, or HPV31 E8^E2 S100E. ChIP assays were performed with anti-HA antibody and anti-histone 3 (H3) antibodies as a control. ChIP-enriched DNA was analyzed by quantitative reverse transcription-PCR (qRT-PCR) using specific primers spanning the E2BS of the pC18-Sp1-luc reporter plasmid. Values are presented as relative HA enrichment levels normalized to the H3 enrichment levels and expressed as fold increase over the empty vector controls. Error bars indicate the SEM from at least three independent experiments.

To test if the reduced repression activity of S78A is due to a decreased binding to NCoR/SMRT corepressor complexes (4, 13), coimmunoprecipitation experiments were carried out. The repression-deficient E8^E2 KWK mutant was included as a negative control as it does not bind to NCoR/SMRT complexes (4). Both WT E8^E2-HA and the S78A mutant bound to similar amounts of the NCoR/SMRT complex components histone deacetylase 3 (HDAC3) and TBLR1, whereas the E8^E2 KWK mutant did not (Fig. 8). This indicated that the reduced repression activity of the E8^E2 S78A mutant is also not due to a diminished binding to NCoR/SMRT complexes.

FIG 8
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FIG 8

Phosphorylation of HPV31 E8^E2 S78 does not affect the interaction with NCoR/SMRT complex components. Whole-cell extracts from HeLa cells transfected with expression vectors for the HPV31 E8^E2 WT or HPV31 E8^E2 S78A or HPV31 E8^E2 KWK mutant (mt) were directly analyzed (input) or precipitated with anti-HA antibody (IP) and subjected to immunoblot analysis with the indicated antibodies.

Phosphorylation of E8^E2 at S78, S81, or S100 has no impact on viral replication in undifferentiated and differentiated keratinocytes.To address the role of E8^E2 serine phosphorylation during the viral life cycle, we generated E8^E2 S78A, S78E, S81A, S100A, and S100E mutations in the context of the HPV31 genome. The hinge domain of E8^E2 and E2 overlaps with the E4 gene. The S78A mutation is silent in E4, whereas the S78E, S81A, S100A, and S100E mutations also introduce mutations in E4. Stable human keratinocyte lines were established by transfection of recircularized DNA and drug selection using cells from different donors. Total cellular DNA was isolated from cells maintained in monolayer culture, the physical state of the viral genomes was analyzed by Southern blotting, and viral copy number was quantified by qPCR. All mutants were present as extrachromosomal elements at similar copy numbers (Fig. 9A). The S100E cell line used in this experiment showed partial integration, but this was not seen when using cells from a different donor. Quantification of viral copy numbers by qPCR indicated on the average 288 virus copies/cell (c/c) in the WT and 372 c/c in the S78A line, 257 c/c in the S78E line, 256 c/c in the S81A line, 541 c/c in the S100A line, and 242 c/c in the S100E line (Fig. 9B). We also quantified the amounts of E1 and E2 transcripts in WT and mutant cell lines by qPCR. This revealed that neither E1 nor E2 transcript levels in HPV31 mutant cell lines differed significantly from the HPV31 WT cell lines (Fig. 9C). In summary, these data surprisingly indicated that mutation of S78 or S100 does not dramatically change copy number, influence extrachromosomal maintenance, or alter the levels of viral transcripts in a phosphorylation-dependent manner.

FIG 9
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FIG 9

Phosphorylation of E8^E2 does not affect genome copy numbers in undifferentiated keratinocytes. (A) Southern blot analysis of total DNA isolated from low-passage-number HPV31 cell lines (wild type or mutant) grown in monolayer culture. NotI- or HindIII-digested DNA was transferred to a membrane and detected by hybridization with a 32P-labeled HPV31 genomic probe. As a marker (M), 100 pg of linearized HPV31 WT genome was used. (B) HPV31 copy numbers were quantified by qPCR. Total genomic DNA (50 ng) from the different HPV31 cell lines was analyzed by multiplex qPCR. Values are presented as HPV31 E2 copies per 2 β-actin copies. Data are derived from 2 to 3 independent DNA preparations. Error bars indicate the standard errors of the means. (C) Total RNA from the different HPV31 cell lines was analyzed by qPCR for the expression of E1 and E2 transcripts. The PGK1 gene was used as a reference gene. Data are derived from four independent RNA preparations and are presented relative to the HPV31 WT. Error bars indicate the SEM.

The HPV31 E8^E2 S78A mutant genome also expresses E2 S266A. Since the repression activity of the E8^E2 S78A mutant is less pronounced in the presence of WT E1 and E2 (Fig. 5), we investigated the possibility that E8^E2 S78A regains its repression activity in the presence of E2 S266A. The pGL31URR reporter plasmid was cotransfected with expression vectors for WT E1, E2, and E8^E2 or WT E1, E2 S266A, and E8^E2 S78A into RTS3b cells. This revealed that E2 S266A induced replication together with E1 to slightly higher levels than WT E2, but these were reduced to a similar extent in the presence of WT E8^E2 (Fig. 10). Comparable to the data shown in Fig. 4 E8^E2 S78A showed a 3-fold-reduced repression in the combination with WT E2 and a 6.5-fold-reduced repression in combination with S266A, but this difference was not significant (Fig. 10). These data demonstrate that E2 S266A does not counteract the reduced repression by E8^E2 S78A.

FIG 10
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FIG 10

E2 S266A does not restore repression activity of E8^E2 S78A. RTS3b cells were transfected with 0.5 ng of pCMV-Gluc, 50 ng of pGL31URR reporter plasmid, expression vectors for HPV31 E1 (100 ng), and the indicated expression vectors for HPV31 E2 (10 ng) and E8^E2 (10 ng). Differences in the amounts of DNA were adjusted with the empty expression vectors (pSG5). Values are presented as the ratio of firefly luciferase (Fluc) to Gaussia luciferase (Gluc) activities. Error bars indicate the SEM from four independent experiments performed in duplicate.

To explore whether E8^E2 S78 or S100 displays a phosphorylation-dependent phenotype in the complete HPV31 life cycle, the respective cell lines were grown as organotypic cultures for 12 days, and then RNA and DNA were isolated and analyzed by qPCR (Fig. 11). The analysis of keratin 10 RNA levels revealed a differentiation-dependent 4.4-fold induction in WT cells (Fig. 11A). Mutant cell lines expressed similar amounts of keratin 10 RNA in organotypic cultures. Immunofluorescence analysis also confirmed suprabasal keratin 10 staining in both WT and mutant cell lines, indicating that the mutants do not influence differentiation (Fig. 11A). Generally, viral copy numbers in organotypic cultures were similar to the ones determined for monolayer cultures, consistent with the idea that only a small fraction of differentiated cells initiates genome amplification (30, 31). DNA copy numbers of S78A genomes were slightly increased compared to the WT (280 versus 212), but the S78E cell lines displayed even higher levels (431 c/c), indicating that these small changes do not follow the phosphorylation-dependent phenotype of E8^E2's repression activity (Fig. 11B). The S100A cell lines displayed the highest copy numbers (575 c/c), whereas the S100E cell lines only had 65 c/c. However, all differences were not statistically significant (one-way analysis of variance [ANOVA] and Dunnett′s multiple-comparison test). The analysis of viral transcripts in WT cell lines revealed differentiation-dependent increases for E6*(2.8-fold), E1 (3.6-fold), E2 (3.5-fold), E8^E2 (1.8-fold), E1^E4 (3.3-fold), and L1 (28.1-fold), indicating a successful induction of viral late functions (Fig. 11C). A slight decline for E6* transcripts in S78A cell lines compared to the WT and S78E was observed (Fig. 11C). E1, E2, E8^E2, and E1^E4 transcripts were present at similar levels in the WT, S78A, and S78E cell lines. In contrast, L1 transcript levels were increased 4.6-fold in the S78A line and 5.3-fold in the S78E line compared to the WT, but these differences were not statistically significant (one-way ANOVA and Dunnett′s multiple-comparison test). Compared to the WT, S100A cell lines showed slightly reduced E6* levels, slightly increased E8^E2 and E1^E4 levels, and greatly increased L1 levels (29-fold compared to the WT). In S100E cell lines, all transcripts were slightly decreased compared to S100A cell lines. None of these changes were statistically significant (one-way ANOVA and Dunnett′s multiple-comparison test). In summary, these data suggest that phosphorylation of E8^E2 at S78 or S100 does not regulate viral replication in undifferentiated or differentiated keratinocytes.

FIG 11
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FIG 11

Genome copy numbers are not influenced by phosphorylation of E8^E2 in differentiated keratinocytes. (A) Total RNA from the different HPV31 cell lines grown in monolayer culture or for 12 days in organotypic cultures was analyzed by qPCR for the expression of K10 using PGK1 as a reference gene. Data are derived from at least four different RNA preparations for each cell line and are presented relative to the HPV31 WT (Monolayer). Error bars indicate the SEM. Frozen sections of organotypic cultures were stained with antibodies against keratin 10 (K10) and DAPI to detect DNA. (B) Total cellular DNA from the HPV31 WT or mutated organotypic cultures was isolated and analyzed by multiplex qPCR. Values are presented as HPV31 E2 copies per 2 β-actin copies. Data are derived from at least four independent organotypic raft cultures. Error bars indicate the SEM. (C) Total RNA from the different HPV31 cell lines grown in organotypic cultures and from HPV31 WT cell lines grown in monolayer culture (WT M) was analyzed by qPCR for the expression of E6*, E1, E2, E8^E2, E1^E4, and L1 using the PGK1 gene as a reference gene. Data are derived from at least four independent organotypic cultures and are presented relative to the HPV31 WT. Error bars indicate the SEM.

E8^E2 S78 phosphorylation regulates transcription of cellular genes.Phosphorylation of E8^E2 at S78 might be involved in the regulation of the expression of cellular genes. We therefore compared the transcriptome of cell lines with HPV31 E8^E2 S78A genomes with the phosphomimetic E8^E2 S78E mutant genomes instead of WT genomes to maximize differences. We subjected RNA isolated from the HPV31 E8^E2 S78A and S78E cell lines grown in organotypic cultures (n = 3 with different donors) to transcriptome sequencing (RNA-seq) analysis. Using a false-discovery rate-adjusted P value cutoff of 0.05 did not yield significantly differentially regulated genes. Without false-discovery rate adjustment, a log fold change cutoff of 1, and a nominal P value cutoff of 0.05, 92 differentially regulated genes were detected (Fig. 12A). The expression of 43 genes was decreased and that of 49 genes increased in S78A cell lines. Of those, we validated the expression of CALB1 (P = 0.0008), IFI27 (P = 0.03), INSR1 (P = 0.02), LYPD2 (P = 0.000004), and UCA1 (P = 0.005) by qPCR in the samples used for RNA-seq and three additional RNA samples. This revealed that only LYPD2 (Ly6/PLAUR domain-containing protein 2) was significantly differentially expressed in differentiated S78A and S78E cell lines (Fig. 12B). The comparison with differentiated WT cell lines indicated that LYPD2 RNA levels are similar in the WT and S78E cell lines, providing further evidence for a phosphorylation-specific regulation of this gene by E8^E2. The analysis of RNA from undifferentiated WT, S78A, and S78E cell lines showed that LYPD2 expression is 28-fold increased by differentiation in WT cell lines and similarly in mutant cell lines (Fig. 12B). Interestingly, LYPD2 levels are also ∼2-fold decreased in undifferentiated S78A cell lines compared to the WT and S78E cell lines. Taken together, these data suggest that phosphorylation of E8^E2 at S78 regulates the transcription of LYPD2 in undifferentiated and differentiated HPV31-positive cell lines.

FIG 12
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FIG 12

Phosphorylation of E8^E2 at S78 regulates the transcription of LYPD2. (A) Volcano plot showing the distribution of upregulated genes (red dots) or downregulated genes (blue dots) in S78E versus S78A cells. (B) Total RNA from the HPV31 E8^E2 S78A and HPV31 E8^E2 S78E cell lines grown in organotypic raft cultures was analyzed by qPCR for the expression of CALB1, IFI27, INSR1, and UCA1 using the PGK1 gene as a reference gene. Data are derived from six independent organotypic raft cultures and are presented relative to HPV31 E8^E2 S78A. Error bars indicate the SEM. (C) Total RNA from the HPV31 WT and HPV31 E8^E2 S78A and HPV31 E8^E2 S78E cell lines grown in monolayer culture (M) or organotypic raft cultures (R) was analyzed by qPCR for the expression of LYPD2 using the PGK1 gene as a reference gene. Data are derived from four different RNA preparations for each cell line grown in monolayer culture and six independent organotypic raft cultures. Data are presented relative to HPV31 E8^E2 S78A organotypic raft culture. Error bars indicate the SEM. Statistical significance was determined with a paired t test (*, P < 0.05).

DISCUSSION

Phosphorylation of serines in the hinge region of the papillomavirus E2 protein has been linked to protein stability, modulation of specific DNA binding, and the interaction with host chromatin (15, 18, 21, 24–26).

We have now determined for the first time that the HPV31 E8^E2 protein is also phosphorylated at S78, S81, and S100 in the hinge region. The major phosphorylation site of E8^E2 is S78, and, surprisingly, both mass spectrometry and Phos-tag labeling experiments indicate that only a small fraction of the corresponding serine, S266, in E2 is phosphorylated. This suggests that the extent of phosphorylation of the hinge region is modulated by the different N termini of E8^E2 and E2. In line with this, it has been reported that the N-terminal domain of HPV16 E2 recruits the calcineurin phosphatase, which results in a dephosphorylation of E2 (32). Whether a similar mechanism contributes to the different phosphorylation levels of S78 and S266 in the HPV31 E8^E2 and E2 proteins remains to be determined. Alternatively, the different sizes or structures of the N termini (12 residues in E8^E2 versus 200 residues in E2) may influence the structure of the hinge and result in a differential exposure of phosphorylation sites.

Residue S301 of bovine papillomavirus (BPV) E2 is phosphorylated by casein kinase 2, an acidophilic kinase with the minimal consensus sequence S/T-X-X-D/E (24, 25, 33, 34). HPV31 E8^E2 S78 does not display a negative charge at the +3 position, but has an aspartic acid (D80) at the +2 position, making it a potential atypical casein kinase 2 substrate (34). Interestingly, D80 is conserved as well as S78 (Fig. 2). The negative charge at the +3 position can also be substituted for by a phosphorylated residue and therefore prime phosphorylation in a hierarchical manner. However, this appears not to be the case for S78 as the S81A mutation does not show a reduced intensity of the shifted bands in Phos-tag experiments (Fig. 3). HPV8 E2 residue S253 is located within the common kinase motif R-X-X-S and has been shown to be phosphorylated by protein kinase A (PKA) (21). As residue S78 in HPV31 E8^E2 also lies within an R-X-X-S consensus sequence, it can be speculated that it might also be a PKA substrate. However, in contrast to D80, R75 is not well conserved among alpha-HPVs (Fig. 2). Future studies are required to determine the kinase responsible for phosphorylation of S78.

Mutation of E8^E2 S78 or the corresponding S266 in E2 to alanine only influenced the activity of E8^E2, and the mutation of S78 to E reverted the phenotype only of E8^E2 and had no influence on E2. This strongly suggests that phosphorylation of S78 specifically regulates repression activity by E8^E2. Mutation of S100 also showed a phosphorylation-dependent phenotype only for E8^E2 but not E2 in reporter assays, but in contrast to S78, S100A maintained whereas S100E lost E8^E2's repressive activities. Chromatin immunoprecipitation (ChIP) analyses indicated that E8^E2 S100E has slightly reduced binding to DNA in vivo, which may contribute to the partial loss of repression activity. This was not the case for E8^E2 S78A which bound to E2BS comparably to the WT. Furthermore, E8^E2 S78A was present at similar protein levels to the WT. In addition, E8^E2 S78A bound comparably to the WT to HDAC3 and TBLR1, which are components of the NCoR/SMRT complex. This is consistent with the finding that E8^E2 residues 1 to 37 are sufficient for the interaction with HDAC3 (12). Taken together, these data suggest that phospho-S78 contributes to the transcriptional repression activity of E8^E2 in a novel way.

Surprisingly, HPV31 E8^E2 S78A genomes displayed copy numbers and viral transcript levels in undifferentiated and differentiated cells indistinguishable from those of WT cells, indicating that the phospho-S78-mediated repression of E8^E2 is not involved in establishment, maintenance, or productive amplification of HPV31 genomes in tissue culture. Along these lines, we observed that the E8^E2 S78A phenotype is less pronounced in the presence of the HPV31 E1 and E2 proteins. Sequence alignments of alpha-HPV E8^E2 proteins reveal that S78 is only moderately conserved and is not specific for high-risk HPV. This suggests that phosphorylation of E8^E2 proteins may contribute to type-specific differences but not to carcinogenicity.

Transcriptome analyses of differentiated HPV31 E8^E2 S78A and S78E cell lines revealed only a small number of differentially expressed cellular genes. Validation experiments using qPCR demonstrated that only the LYPD2 gene out of five tested genes was significantly differentially expressed between S78E and S78A cells. LYPD2 (Ly6/PLAUR domain-containing protein 2) transcript levels were lower in S78A than in S78E cells and in WT cells were similar to those in S78E cells, which is consistent with a phosphorylation-dependent regulation of LYPD2 by E8^E2. However, the reduced LYPD2 levels in S78A versus S78E cells argue against a direct regulation of LYPD2 by E8^E2 (e.g., by direct binding to the gene and repressing its transcription) but suggest a more complex regulation. We propose that the main function of the phospho-S78 form of HPV31 E8^E2 is the modulation of the expression of a small number of cellular genes.

The function of LYPD2 is currently unknown. Sequence predictions indicate that LYPD2 is most likely a glycosylphosphatidylinositol (GPI)-anchored membrane protein and thus might be involved in signal transduction pathways. As we have been unable to identify changes in the extent of HPV31 replication caused by p-S78 E8^E2, we think it is unlikely that LYPD2 modulates viral replication. Future studies are required to determine the role of LYPD2 during papillomavirus replication in vitro and in vivo.

In summary our data indicate that the phosphorylation of HPV31 E8^E2 at S78 regulates its repression activity, and this seems not to be important for the regulation of viral replication but rather for regulation of a small number of specific cellular genes. The observation that S78 is only moderately conserved among alpha-HPV types may indicate that this activity may only be advantageous for the replication of certain HPV types.

MATERIALS AND METHODS

Recombinant plasmids.Firefly luciferase reporter plasmids pC18-Sp1-luc and pGL31URR have been previously described (11, 14). The Gaussia luciferase reporter pCMV-gluc was commercially obtained (NEB). Expression plasmids for HPV31 E1, E2, and E8^E2 and HA-tagged versions of E2 and E8^E2 have been described previously (10, 35–37). Mutations in E2 or E8^E2 expression vectors were generated by overlap extension PCR and subcloned into the respective expression plasmids. Plasmid pInducer 31 E8^E2-HA was generated by recombination of pENTR-31 E8^E2-HA with pInducer 20 (27). Plasmid pENTR-31 E8^E2-HA was generated by inserting an NcoI/BamHI fragment from pSG 31 E8^E2-HA into a modified pENTR4 plasmid (Invitrogen). HPV31 mutant genomes were constructed by replacing restriction fragments obtained by overlap extension PCR in the context of pBR322-HPV31 (30). The S78A mutant is silent in E4. The S78E mutation changes E4 residues T73 to R and P74 to S, S81A to E4 V77L, S100A to E4 V96L, and S100E to E4 V96N. All constructs were verified by DNA sequencing.

Cell culture.HeLa cells and RTS3b cells were maintained as previously described (4). To generate the HeLa-pInducer 31 E8^E2-HA cell line, HeLa cells were infected with recombinant lentiviruses and selected with G418. Pooled cultures were used for expression. Cells were incubated with doxycycline (1 μg/ml) for 24 h to induce HPV31 E8^E2-HA expression. HPV31-positive human keratinocytes were established by transfection with recircularized HPV31 wild-type or mutant genomes and a G418 resistance plasmid using the Fugene HD reagent (Promega). Cell lines were selected and maintained as described previously (9). Organotypic cultures were grown as previously described and were harvested after 12 days (9). Frozen sections were stained with an anti-keratin 10 antibody (RKSE60; Research Diagnostics) as previously described (9).

Coimmunoprecipitation experiments.For immunoblot analysis, approximately 4 × 107 transfected HeLa cells were harvested 48 h after transfection, and for nanoscale liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) analysis, approximately 2 × 109 HeLa pInducer 31 E8^E2-HA cells or 4 × 108 E2-HA-transfected HeLa cells were harvested 16 h after doxycycline treatment or 48 h after transfection. Cells were then lysed in immunoprecipitation (IP) buffer (50 mM HEPES [pH 7.9], 150 mM NaCl, 0.3% [vol/vol], Igepal 630, 1 mM dithiothreitol [DTT], and protease and phosphatase inhibitors for HeLa cells or 50 mM HEPES [pH 7.9], 500 mM NaCl, 0.3% [vol/vol] Igepal 630, 1 mM DTT, and protease and phosphatase inhibitors for HeLa pInducer 31 E8^E2-HA cells) and precipitated with magnetic anti-HA-conjugated beads (Miltenyi Biotech). Washing of the beads with IP buffer was carried out using μMACS columns and a μMACS separator (Miltenyi Biotech). Bound proteins were eluted in 4× SDS gel loading puffer (Carl Roth) and heated to 95°C. Efficiency of the immunoprecipitation was analyzed by HA immunoblotting of an aliquot of the eluates.

Protein in-gel digestion.Protein samples were purified on the SDS-PAGE. Coomassie-stained gel pieces were digested as previously described (38) using trypsin. Extracted peptides were desalted using C18 Stage tips and subjected to LC-MS/MS analysis.

Mass spectrometry.LC-MS/MS analyses were performed on an EasyLC nanoscale high-performance liquid chromatography (nano-HPLC) device (Proxeon Biosystems) coupled to an LTQ Orbitrap Elite (Thermo). Separations of the peptide mixtures were done on a 15-cm fused silica emitter with a 75-μm inner diameter (Proxeon Biosystems), in-house packed with reverse-phase ReproSil-Pur C18-AQ 3-μm resin (Dr. Maisch GmbH). Peptides were injected with solvent A (0.5% acetic acid) at a flow rate of 500 nl/min and separated at 200 nl/min. Separation was performed using a linear 60-min gradient of 5 to 33% solvent B (80% acetonitrile [ACN] in 0.5% acetic acid). The LTQ Orbitrap Elite was operated in the positive-ion mode. Precursor ions were acquired in the mass range from m/z 300 to 2,000 in the Orbitrap mass analyzer at a resolution of 120,000 followed by MS/MS spectrum acquisition. The 15 or 20 most intense precursor ions from the full scan were sequentially fragmented. High-energy C-trap dissociation MS/MS spectra were acquired with a resolution of 15,000 and a target value of 40,000. The normalized collision energy was set to 35, activation time to 0.1 ms, and the first mass to 120 Th. Fragmented masses were excluded for 60 s after MS/MS. The target values were 1E6 charges for the MS scans in the Orbitrap and 5,000 charges for the MS/MS scans, with maximum fill times of 100 ms and 150 ms, respectively.

Proteomic data analysis.The MS data were processed with MaxQuant software suite v.1.2.2.9 (39). Database search was performed using the Andromeda search engine (40), which is integrated in MaxQuant. Database search was performed against a human database obtained from UniProt taxonomy ID 9606, containing 91,646 protein entries and 285 commonly occurring laboratory contaminants, together with the HPV database and small databases for each tagged sequence. Endoprotease trypsin was fixed as the protease with a maximum missed cleavage of 2. Oxidation of methionines and N-terminal acetylation, as well as acetyl (K) and phospho (STY) were specified as variable modifications. Initial maximum allowed mass tolerance was set to 6 ppm. Carbamidomethylation on cysteines was defined as a fixed modification. Requantify was enabled. A false-discovery rate of 1% was applied at the peptide and protein levels.

Immunofluorescence analysis.For immunofluorescence analyses, 1.5 × 105 HeLa cells were seeded on coverslips in 6-well culture dishes 1 day before transfection. Cells were transfected with wild-type or mutant expression vectors for HPV31 E8^E2-HA using the Fugene HD reagent (Promega) and Opti-MEM (Life Technologies), and 48 h after transfection, cells were fixed and permeabilized with methanol-acetone for 2 min and incubated with the primary antibody against the HA tag (C29F4 at 1:1,000; Cell Signaling catalog no. 3724) diluted in phosphate-buffered saline (PBS)–3% bovine serum albumin (BSA) in a humidified chamber for 1 h at room temperature. After being washed with PBS, diluted secondary Alexa Fluor 555-labeled donkey anti-mouse antibody (Life Technologies A-31570 at 1:2,000) was added and incubated for 1 h at room temperature. Samples were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) and mounted with SlowFade Gold antifade mountant (Life Technologies). Immunofluorescence signals were recorded with a Zeiss Axio Observer microscope and the appropriate filter sets in combination with a Zeiss ApoTome. Images were recorded using fixed exposure times.

Luciferase-based reporter assays.HeLa (3 × 104), RTS3b (3 × 104), or NHK-HPV31 WT (4 × 104) cells were seeded into 24-well culture dishes 1 day before transfection. Cells were transfected with reporter plasmids as indicated in the figure legends using Fugene HD (Promega) and Opti-MEM (Life Technologies). In addition, the pCMV-Gluc plasmid (New England BioLabs) was cotransfected as an internal control. Luciferase assays were carried out 48 h after transfection.

Southern blot analysis.Total cellular DNA from stable keratinocyte lines was digested with NotI (noncutter) or HindIII (single cutter) and separated in a 0.8% agarose gels. Blotting and hybridization to a 32P-labeled HPV31 probe were carried out as previously described (9). After exposure of the membrane to PhosphorImager screens, signals were visualized using the AIDA software package (Raytest).

qPCR.HPV31 copy numbers were quantified in total cellular DNA by qPCR using amplicons in the HPV31 E2 and the cellular ACTB genes and copy number standards. Isolation of RNA and cDNA synthesis was performed as described previously (9). RNA (1 μg) isolated from HPV31-positive keratinocytes maintained in monolayer or organotypic cultures was reverse transcribed using the QuantiTect reverse transcription kit (Qiagen), and 25 ng of cDNA was analyzed in duplicates by qPCR in a LightCycler 480 for viral and cellular transcripts using Light-Cycler 480 SYBR green I master mix (Roche Applied Science) and 0.3 μM the following gene-specific primer pairs: for HPV31 E1, HPV31 1519 F (5′-GTGTGTAGCTGCGTTTGGAG-3′) and HPV31 1739 R (5′-CGTAATTTGGGTGGCTGAAT-3′); for HPV31 E2, HPV31 E2N F (5′-CTGTTGTGGAAGGGCAAGTT-3′) and HPV31 E2N R (5′-TCCCAGCAAAGGATATTTCG-3′); for HPV31 E8^E2, HPV31 1242 F (5′-ACTTCCAGACAGCGGGTATG-3′) and HPV31 3461 R (5′-GGTGGGTGTTTCTGTGCTCT-3′); for HPV31 E1^E4, HPV31 804 F (5′-TGTTAATGGGCTCATTTGGAA-3′) and HPV31 3373 R (5′-GGTTTTGGAATTCGATGTGG-3′); for HPV31 E6*, HPV31 E6* F (5′-AATTGTGTCTACTGCAAAGGTGTA-3′) and HPV31 508 R (5′-CCAACATGCTATGCAACGTC-3′); for HPV31 L1, HPV31 6338 F (5′-AATAGATCAGGCACGGTTGG-3′) and HPV31 6534 R (5′-TTGCCCCAACAAATACCATT-3′); for CALB1, CALB1 Ex4 F (5′-AATTTCCTGCTGCTCTTCCG-3′) and CALB1 Ex5 R (5′-TCTATGAAGCCACTGTGGTCAG-3′); for IFI27, IFI27 Ex2 F (5′-TCTGGCTCTGCCGTAGTTTT-3′) and IFI27 Ex4 R (5′-ATCTTGGCTGCTATGGAGGA-3′); for INSR1, INSR1 Ex16_17 F (5′-TGCCAGTGATGTGTTTCCAT-3′) and INSR1 Ex17 R (5′-TGAGGAACTCAATCCGCTCT-3′); for keratin-10, KRT10 F (5′-CGCCTGGCTTCCTACTTGG-3′) and KRT10 R (5′-CTGGCGCAGAGCTACCTCA-3′); for LYPD2, LYPD2 Ex2 F (5′-TGTGCAAGACCACACTCTACTC-3′) and LYPD2 Ex3 R (5′-ACAGCTCAGTATTGCAGCAG-3′); and for UCA1, UCA1 F (5′-AAAACGCTTAGGCTGGCAAC-3′) and UCA1 R (5′-TTGTGTTGTCCTGGATGCTG-3′) or PGK1 (9).

Immunoblot analyses.HeLa cells (3 × 105) were transfected with wild-type or mutant expression vectors for HPV31 E8^E2-HA, lysed 48 h after transfection in 4× SDS gel loading buffer (Carl Roth), heated to 95°C, separated in a 12% SDS-PAGE gel, and transferred onto a nitrocellulose membrane.

Membranes were incubated with the following primary antibodies at the indicated dilutions: α-tubulin (Calbiochem CP06 at 1:1,000), HA tag (C29F4 at 1:1,000; Cell Signaling catalog no. 3724), HDAC3 (1:500; Cell Signaling catalog no. 2632), and TBLR1 (1:500; Santa Cruz BT catalog no. SC-100908). Bound antibodies were detected with the following fluorescence-labeled antibodies: anti-mouse IRDye 680RD (1:15,000; Li-Cor catalog no. 926-68070), anti-mouse TrueBlot DyLight 800 (1:10,000; Rockland Antibodies and Assays catalog no. 18-4517-32), anti-rabbit IRDye 680RD (1:15,000; Li-Cor catalog no. 926–68071), anti-rabbit IRDye 800CW (1:15,000; Li-Cor catalog no. 926–32211), and anti-rabbit TrueBlot IRDye 800 (1:10,000; Rockland Antibodies and Assays catalog no. 18-3216-32) and recorded with an OdysseyFC infrared imaging system (Li-Cor Biosciences).

Phos-tag SDS-PAGE.For Phos-tag experiments, HeLa cells were transfected with HA-tagged HPV31 E8^E2 (wild type or mutant), and 48 h after transfection, cells were lysed in 4× SDS gel loading buffer (Carl Roth) and heated to 95°C. Equal aliquots were separated in a 15% SDS-PAGE gel containing 50 μM Phos-tag (Wako Chemicals) and 100 μM MnCl2. After electrophoretic separation, the gels were incubated in transfer buffer containing 1 mM EDTA for 10 min and then in transfer buffer without EDTA for an additional 10 min. Proteins were transferred onto a nitrocellulose membrane and detected by immunoblotting as described above.

EMSAs.For electrophoretic mobility shift assays (EMSAs), nuclear extracts from transfected HeLa cells were incubated with double-stranded 5′-Dy681-labeled oligonucleotides (40 fmol) for 15 min and separated in 5% polyacrylamide–0.5× Tris-borate-EDTA gels at 100 V. Fluorescent signals were recorded with an OdysseyFC infrared imaging system (Li-Cor Biosciences).

ChIP.Chromatin immunoprecipitation (ChIP) experiments were performed using the SimpleChIP Enzymatic Chromatin IP kit (Cell Signaling catalog no. 9003) according to the manufacturer's instructions. Briefly, HeLa cells (5 × 106) were cotransfected with the pC18-Sp1-luc reporter plasmid and the expression vectors for HA-tagged HPV31 E8^E2 (wild type or mutant), and 48 h after transfection, cells were cross-linked with formaldehyde at a final concentration of 1% for 10 min at room temperature. Cells were then lysed, and chromatin was harvested, digested with nuclease, and subjected to sonication for fragmentation. Chromatin immunoprecipitation was performed with rabbit anti-histone H3 antibody (1:50; Cell Signaling catalog no. 4620) or anti-HA tag antibody (1:100; Abcam catalog no. ab9110). After reverse cross-linking and DNA purification, immunoprecipitated DNA was quantified by qPCR using primers covering the four E2BSs of pC18-Sp1-luc: 5′-CCCCCTGAACCTGAAACATA-3′ (sense) and 5′-CAACAGTACCGGAATGCCAAG-3′ (antisense).

RNA-seq.RNA-seq libraries were prepared according to the manufacturer's instructions using the TruSeq stranded mRNA library preparation kit (Illumina, Inc.). The quality and concentration of RNA in each library were measured using Qubit and Bioanalyzer (Agilent). All samples had RNA integrity number (RIN) values of >7. High-quality samples were sequenced using HiSeq2500 system (Illumina, Inc.) as 125-bp paired-end reads at the Medical Genetics Genome Sequencing Facility of the University of Tuebingen. The quality of raw sequenced reads was assessed using FastQC (version V0.11.4), and the Cutadapt software (version 1.8.3) was used to trim Illumina adapter sequences and low-quality bases (41). The quality controlled reads were aligned to the human reference genome (version hg19) using Tophat2 software (Version 2.1.1) with default options (42). Reads aligned to features such as genes or exons were counted using HTSeq (version 0.6.0) (43). More precisely, reads were counted using the “union” mode on the “gene_id” feature. Features on both strands were counted by setting the stranded option to “no.” To identify differentially expressed genes from read counts, the R package DESeq2 (version 1.10.1) was used (44). Apart from standard R packages (version 3.2.1), ggplot2 was used to generate figures.

ACKNOWLEDGMENTS

This work was supported by grants from the Else-Kröner-Fresenius-Stiftung (2013_A277) and the Deutsche Forschungsgemeinschaft (STU 218/4-2) to F.S. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

FOOTNOTES

    • Received 5 October 2017.
    • Accepted 15 November 2017.
    • Accepted manuscript posted online 22 November 2017.
  • Copyright © 2018 American Society for Microbiology.

All Rights Reserved.

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Identification and Functional Characterization of Phosphorylation Sites of the Human Papillomavirus 31 E8^E2 Protein
Saskia van de Poel, Marcel Dreer, Ana Velic, Boris Macek, Praveen Baskaran, Thomas Iftner, Frank Stubenrauch
Journal of Virology Jan 2018, 92 (4) e01743-17; DOI: 10.1128/JVI.01743-17

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Identification and Functional Characterization of Phosphorylation Sites of the Human Papillomavirus 31 E8^E2 Protein
Saskia van de Poel, Marcel Dreer, Ana Velic, Boris Macek, Praveen Baskaran, Thomas Iftner, Frank Stubenrauch
Journal of Virology Jan 2018, 92 (4) e01743-17; DOI: 10.1128/JVI.01743-17
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KEYWORDS

DNA replication
E8^E2
papillomavirus
protein phosphorylation
regulation of gene expression
transcription factors

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