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Journal of Virology, September 2008, p. 9056-9064, Vol. 82, No. 18
0022-538X/08/$08.00+0     doi:10.1128/JVI.01080-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Identification of an Arginine-Rich Motif in Human Papillomavirus Type 1 E1^E4 Protein Necessary for E4-Mediated Inhibition of Cellular DNA Synthesis In Vitro and in Cells

Sally Roberts,^1,* Sarah R. Kingsbury,^2, Kai Stoeber,² Gillian L. Knight,¹ Phillip H. Gallimore,¹ and Gareth H. Williams^2*

Cancer Research UK Institute for Cancer Studies, University of Birmingham, Birmingham, B15 2TT, United Kingdom,¹ Wolfson Institute for Biomedical Research and Research Department of Pathology, University College London, Gower Street, London, WC1E 6BT, United Kingdom²

Received 22 May 2008/ Accepted 3 July 2008

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Productive infections by human papillomaviruses (HPVs) are restricted to nondividing, differentiated keratinocytes. HPV early proteins E6 and E7 deregulate cell cycle progression and activate the host cell DNA replication machinery in these cells, changes essential for virus synthesis. Productive virus replication is accompanied by abundant expression of the HPV E4 protein. Expression of HPV1 E4 in cells is known to activate cell cycle checkpoints, inhibiting G₂-to-M transition of the cell cycle and also suppressing entry of cells into S phase. We report here that the HPV1 E4 protein, in the presence of a soluble form of the replication-licensing factor (RLF) Cdc6, inhibits initiation of cellular DNA replication in a mammalian cell-free DNA replication system. Chromatin-binding studies show that E4 blocks replication initiation in vitro by preventing loading of the RLFs Mcm2 and Mcm7 onto chromatin. HPV1 E4-mediated replication inhibition in vitro and suppression of entry of HPV1 E4-expressing cells into S phase are both abrogated upon alanine replacement of arginine 45 in the full-length E4 protein (E1^E4), implying that these two HPV1 E4 functions are linked. We hypothesize that HPV1 E4 inhibits competing host cell DNA synthesis in replication-activated suprabasal keratinocytes by suppressing licensing of cellular replication origins, thus modifying the phenotype of the infected cell in favor of viral genome amplification.

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Human papillomaviruses (HPVs) are a large group (>100 types) of small DNA viruses that replicate in keratinocytes of squamous epithelia. HPV infections produce hyperproliferative warts that are in most instances benign. A small subset of HPV types, however, form lesions on the skin, and on the oropharyngeal- and anogenital-tract mucosae, that have a significant risk of malignant transformation. The most common cancer attributable to infection with the high-risk HPV types is cancer of the uterine cervix (35). Despite the differences in pathogenesis between virus types, their life cycles are similar (10), beginning with infection of keratinocytes within the basal cell compartment of squamous epithelia. Here the HPV genome is replicated as a low-copy-number (50 to 100 copies per cell) episome in synchrony with the replication of the host cell genome, a process that requires HPV E1 and E2 functions. HPV early proteins E6 and E7 act to expand the population of HPV-infected keratinocytes once they migrate up from the basal layer, by stimulating cell cycle entry and cell survival. The virus then utilizes the host cell's replication machinery, which has been activated in these cells, to amplify the HPV DNA to many thousands of copies per cell during the vegetative stage of the life cycle. Finally, the capsid proteins L1 and L2 are produced, and new progeny are assembled in the most superficial cells prior to their release from the highly differentiated squames.

A major protein produced during the HPV life cycle is the E4 protein. It is expressed as an E1^E4 fusion protein from spliced transcripts formed between the N terminus of the E1 open reading frame and almost the complete open reading frame of E4 (21). The precise function of E4 has not been defined, but loss of expression of the full-length E1^E4 polypeptide has a severe adverse effect on viral genome amplification of HPV type 16 (HPV16), HPV18, and HPV31 following introduction of mutant genomes unable to support E1^E4 expression into keratinocytes and subsequent induction of cellular differentiation (20, 38, 39). Failure to complete the vegetative stage of the virus life cycle is also the outcome of loss of E1^E4 expression in rabbit papillomas induced by a mutant cottontail rabbit papillomavirus genome (22). These studies suggest that E4 function is necessary for efficient vegetative replication of the virus, a hypothesis supported by coincidence between onset of viral genome amplification and induction of high-level E4 production in natural papillomavirus infections (23).

Examination of E4 activity in epithelial cell cultures has revealed diverse biological actions that perhaps imply a multifunctional role for this viral protein in the virus life cycle. These include disruption of ND10 body organization, which might be required for viral DNA replication, either by organization of viral replication centers or by inactivation of a host antiviral response mediated through the nuclear ND10 body (8, 29). A potent G₂ arrest function is a conserved function of E4 proteins between HPV types with dissimilar tropism, and it is thought that division arrest of infected cells might be necessary to support efficient viral DNA amplification (5, 13, 19). E4 inclusion bodies found in the cytoplasm of cells of HPV1 skin warts contain the kinase SRPK1, a binding partner of E1^E4 proteins, which is associated with regulating the function of splicing factors (1). Sequestration of SRPK1 by E4 could be an HPV mechanism to regulate expression of viral late transcripts at the late stages of the replication cycle (1). Late in the infectious cycle, the E4 protein may also act to diminish the integrity of the keratinocyte by disrupting the keratin cytoskeleton and cornified envelope formation and inducing apoptosis through alteration of mitochondrial function to facilitate egress of the newly formed HPV virions (3, 6, 24, 26).

Execution of multiple functions might be assisted by conversion of the E4 protein into multiple forms, brought about by a combination of sequential N-terminal proteolysis of the E1^E4 polypeptide (7, 25) and by phosphorylation (9). Indeed, a study of the interaction between E4 and cell growth revealed an interesting relationship between modification of the HPV1 E4 protein and dysregulation of the cell cycle (13). During the HPV1 infectious cycle, N-terminal sequences are removed from the full-length 17-kDa E1^E4 polypeptide to produce smaller E4 species of 16, 11, and 10 kDa that progressively replace the full-length protein as the replication cycle proceeds (7, 25). Expression of the 17-kDa E1^E4 protein in the presence of a protein mimicking the 16-kDa polypeptide in keratinocyte cells inhibits G₂-to-M transition of the cell cycle, and in a population of cells, prohibition of entry into S phase is also observed (13). The negative effect on S-phase entry, however, is not apparent in cells expressing the individual forms of E4, although expression of the truncated 16-kDa protein alone was sufficient to block cell division (13). Further analysis revealed that HPV1 E4 employs two distinct mechanisms to inhibit G₂-to-M transition: the first, mediated by the combined expression of 17- and 16-kDa proteins, was found to be dependent on maintenance of high levels of the Wee1 kinase to inhibit Cdk1 activity, and the second, mediated by the 16-kDa protein, is associated with insufficient production of cyclin B1 to enable the cells to transverse G₂ to M (13, 14). Employment of two distinct mechanisms to inhibit cell division suggests that the G₂ arrest function of HPV1 E4 is important in the HPV life cycle. In this study, we investigated how HPV1 E4 inhibits progression of cells into S phase and showed that HPV1 E4 affects a key step in the cellular DNA replication process.

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HPV1 E4 expression plasmids. Construction of plasmids based on pcDNA3.1 that deliver expression of the 17-kDa full-length HPV1 E1^E4 (E4-17K) and an N-terminal truncation (E4-16K) equivalent to the 16-kDa E4 polypeptide was described previously (29). A set of previously described deletions within the full-length E1^E4 coding sequence (25) were excised and inserted into the BamHI restriction site of pcDNA3.1 (Invitrogen, Carlsbad, CA). The sequence integrity of plasmid inserts was verified by bidirectional DNA sequencing. The substitution-containing proteins E4R45A, E4R47A, and E4R48A are described elsewhere (1).

Expression of recombinant proteins. HPV1 E4 and Xenopus laevis Cdc6 (His₆-XeCdc6) proteins were purified from Sf9 insect cells following infection with appropriate recombinant baculoviruses, as previously described (28, 31). Histidine-tagged human geminin (His₆-HsGeminin) was expressed in Escherichia coli and purified as described previously (36). For expression of histidine-tagged HPV1 E4 protein in bacteria, the E1^E4 wild-type and mutant coding sequences were inserted into the BamHI-EcoRI cloning site of the expression vector pRSET-C (Invitrogen) and expressed in E. coli strain BL21(DE3) pLysS (Novagen, Madison, WI). The recombinant protein was purified by immobilized metal affinity chromatography using NiCl₂-charged HiTrap chelating columns (GE Healthcare Europe GmbH, Munich, Germany). Nonspecifically bound proteins were removed with 4 column volumes (c.v.) of 10% elution buffer (30 mM Tris-Cl [pH 8], 300 mM imidazole, 30 mM NaCl, 0.1 mM phenylmethylsulfonyl fluoride) and 8 c.v. of 20% elution buffer. The E4 proteins were eluted with 2 c.v. of 100% elution buffer and desalted into 20 mM Tris-Cl (pH 8), 50 mM NaCl.

In vitro cellular DNA replication assays. Nuclei and cytosolic extracts were prepared from synchronized NIH 3T3 and HeLa S3 cells and supplemented as described previously (11, 15, 31, 32). In vitro DNA replication assays were performed as described previously (15, 31) (Fig. 1a). Briefly, reaction mixtures contained 30 µl of cytosolic extracts (250 to 300 µg of protein), 10 µl of premix buffer (160 mM K-HEPES [pH 7.8], 28 mM MgCl_2, 12 mM ATP, 0.4 mM of GTP, CTP, UTP, dATP, dGTP, and dCTP, 1 µM biotin-16-dUTP, 2 mM dithiothreitol, 160 mM creatine phosphate, 20 µg phosphocreatine kinase), 1 x 10⁵ nuclei, and, where indicated, up to 10 µl of recombinant protein(s). His₆-XeCdc6 protein in 20 mM HEPES (pH 7.4), 150 mM NaCl, 5% glycerol, 1 mM dithiothreitol was added to in vitro replication reaction mixtures at a final concentration of 0.65 µM, baculovirus-expressed HPV1 E4 proteins in 10 mM phosphate buffer (pH 7.4), 0.1 mM dithiothreitol at 3 µM, bacterially expressed His₆-E4 proteins in 40 mM Tris-HCl (pH 7.6), 30 mM NaCl, and His₆-HsGeminin protein in 50 mM sodium phosphate at 4 µM. Equal volumes of appropriate buffers were added to control reaction mixtures. All components of the replication reaction mixtures were incubated together on ice for 15 min prior to the addition of S-phase cytosol and incubation for 3 h at 37°C. For analysis of in vitro DNA synthesis reactions by confocal microscopy, reactions were stopped by diluting with 500 µl of phosphate-buffered saline (PBS), and nuclei were fixed for 5 min in 4% paraformaldehyde. After fixation, nuclei were spun through a 30% sucrose-PBS cushion onto poly-L-lysine-coated coverslips. All subsequent washing and staining steps were carried out in PBS, 0.2% Triton X-100, 0.04% sodium dodecyl sulfate. Coverslips were washed, stained for incorporated biotin-16-dUTP with fluorescein-linked streptavidin (1:100 dilution; Amersham) and for DNA with propidium iodide-RNase A (both at 50 ng/ml), washed again, and mounted in Vectashield (Vector Laboratories, Ltd., Peterborough, United Kingdom). Confocal fluorescence microscopy of random fields of nuclei was performed on a Leica TCS DMRE confocal microscope, and the numbers of nuclei incorporating biotin-16-dUTP in vitro and nonreplicating nuclei were counted. Routinely, 800 to 1,000 nuclei were scored blind by a single individual for each reaction and quantitated as percentages of the total number of nuclei that synthesized DNA in vitro. More than one preparation of nuclei was assayed, in triplicate, for each set of experiments, and analysis was performed by two individuals. Statistical analysis of data from multiple independent experiments was performed by single-factor analysis of variance.

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FIG. 1. HPV1 E4 inhibits initiation of cellular DNA synthesis in an in vitro replication assay. (a) Cell-free cellular DNA replication system. Nuclei (N) prepared from G1-phase NIH 3T3 fibroblasts, synchronized by release from quiescence (G0), initiate a single round of semiconservative DNA replication in cytosolic extracts (SC) from S-phase HeLa cells following incubation in buffer A (BA) (which supports elongation), nucleotides (dNTPs, NTPs), and an ATP regeneration system (CP, CK). Nuclei were stained with propidium iodide (red) to reveal DNA and with fluorescein-streptavidin (green) to detect biotin-16-dUTP incorporation resulting from in vitro DNA synthesis. (b) Coomassie-stained sodium dodecyl sulfate-polyacrylamide gel of baculovirus recombinant HPV1 E4 proteins. Lane 1, molecular mass standards (12.3, 17.2, 30, 42.7, 66, and 76 kDa); lanes 2 and 3, purified HPV1 E4 proteins WT38 and WT43 containing variable levels of full-length E1^E4 (17-kDa) and processed (16- and 11-kDa) species. (c) NIH 3T3 G1 nuclei were incubated in cytosolic extracts from S-phase HeLa cells, which induces initiation in competent nuclei, or in elongation buffer (buffer A), which supports elongation DNA synthesis only in nuclei that are already in S phase. Addition of E4 (but not E4) and Cdc6 to coincubation mixtures inhibits DNA synthesis at a level comparable to that achieved with geminin. Results are expressed as the percentage of nuclei replicating (mean ± standard deviation), and asterisks indicate a significance of >99.99% in the decrease of replicating nuclei compared to control replication assays. (d) Addition of E4 and Cdc6 to coincubation mixtures of NIH 3T3 S-phase nuclei and HeLa S-phase cytosol had no effect on replication potential. Similarly, addition of geminin did not affect ongoing DNA synthesis. Data were analyzed as described for panel c.

Chromatin-binding assay. In vitro DNA replication assays were set up as described above. After 3 h incubation, nuclei were pelleted by low-speed (1,300 x g) centrifugation and used in chromatin-binding reactions performed as described previously (12). Samples were immunoblotted with antibodies against Mcm2 (BD Biosciences; 610701), Mcm7 (Neomarkers; Lab Vision, Suffolk, United Kingdom; MS-862-P), Cdc6 (Santa Cruz, Santa Cruz, CA; 9964), HPV1 E4 (MAb 4.37) (7) and histone H1 (Santa Cruz; 10806). Analysis of protein bands using densitometry was determined using ImageJ (http://rsb.info.nih.gov/ij). Chromatin-binding reactions were repeated at least twice in separate in vitro DNA replication assays.

Cell transfection and cell cycle analysis. Cos-1 cells were transfected with the appropriate combinations of HPV1 E4 expression plasmids, or the pcDNA3.1 empty vector as a control plasmid, as described previously (13). At 48 h posttransfection, the cells were incubated with 5-bromodeoxyuridine (BrdU) at a final concentration of 33 µM for 2 h. Cells were then fixed, incubated with an anti-BrdU antibody conjugated to fluorescein isothiocyanate, labeled with propidium iodide, and analyzed by dual-parameter flow cytometry as described previously (13). Statistical analysis of data derived from multiple independent experiments was performed by analysis of variance. Expression of E4 proteins was confirmed by immunoblot analysis using monoclonal antibody MAb 4.37.

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HPV1 E4 inhibits cellular DNA replication in vitro. We sought to establish whether the negative effect of HPV1 E4 upon S-phase entry reflected an interaction between the viral protein and the process of cellular DNA synthesis itself. To do this, we took advantage of an established cell-free DNA replication system that supports efficient cellular DNA synthesis under somatic cell cycle control (31) (Fig. 1a). Previous characterization of this system had revealed that nuclei prepared from quiescent (G₀) NIH 3T3 fibroblasts cannot initiate DNA synthesis in S-phase cytosolic extracts of HeLa cells, while G₁ nuclei become competent to initiate DNA replication in the S-phase extracts when prepared 16 to 18 h after release from G₀ (31).

NIH 3T3 G₁ nuclei were combined with HeLa S-phase cytosol and incubated for 3 h in the presence of an ATP generation system and nucleotides (nucleoside triphosphates [NTPs] and deoxynucleoside triphosphates [dNTPs]), including biotin-labeled dUTP as a marker to enable detection of DNA synthesis by confocal microscopy (Fig. 1a). We observed that 19.3% of G₁ nuclei were capable of DNA synthesis in the presence of S-phase cytosol, in comparison to only 2.2% following incubation of the G₁ nuclei in a physiological buffer (buffer A) that supports elongation but not initiation of DNA replication (Fig. 1c). The small proportion of replication-competent nuclei observed in buffer A represent contaminating S-phase nuclei present in the G₁ nuclear preparation that continue DNA synthesis at replication forks established in vivo prior to their isolation (31). Thus, 17.1% of G₁ nuclei undergo true replication initiation in the presence of S-phase cytosol. To confirm that our preparations of G₁ nuclei and S-phase cytosol respond to exogenous factors, we first tested the response to recombinant preparations of the replication-licensing factor (RLF) Cdc6 (His₆-XeCdc6) and geminin (His₆-HsGeminin), a known cellular repressor of replication licensing (18).

The percentage of replicating nuclei increased from 19.3% to 23% in reaction mixtures containing His₆-XeCdc6 (Fig. 1c). Since Cdc6 is known to be rate limiting for replication competence after release from G₀ (31), the small but consistent increase in replicating nuclei in the presence of His₆-XeCdc6 indicates that a low number of G₁ nuclei are responsive to this RLF. In contrast, in the presence of His₆-HsGeminin there was a marked and significant decrease (7.2%) in the percentage of replication-competent nuclei (Fig. 1c).

To investigate if HPV1 E4 might interfere with cellular DNA synthesis, the viral protein was expressed in Sf9 insect cells using a recombinant baculovirus and the purified protein (Fig. 1b, WT38) titrated into in vitro replication reaction mixtures (Fig. 1c). While we observed no significant effect of E4 on the percentage of nuclei synthesizing DNA in coincubations of G₁ nuclei and S-phase cytosol (19.7%), when E4 was added to reaction mixtures that also contained exogenous Cdc6 (His₆-XeCdc6), the percentage of replicating nuclei decreased significantly from 23% to 10.6%, indicating that 54% of the replication-competent nuclei failed to initiate DNA synthesis (Fig. 1c). Notably, the scale of E4-induced replication inhibition was comparable to the inhibitory effect (67%) of His₆-HsGeminin (Fig. 1c).

The E4 protein added to the in vitro replication assays contained the full-length E1^E4 protein (17 kDa) plus small quantities of truncated polypeptides (16, 11, and 10 kDa) (Fig. 1b, WT38). Interestingly, addition of recombinant E4 protein that contained the truncated proteins but no full-length E1^E4 polypeptide (Fig. 1b, WT43) to the cell-free replication assay did not inhibit DNA synthesis in the G₁ nuclei in either the absence (data not shown) or presence (Fig. 1c, E4) of exogenous Cdc6. This observation suggests that E4-induced inhibition of cellular DNA synthesis in vitro requires the presence of the full-length E1^E4 protein.

To validate the specificity of our findings, identical replication reactions to those described above but using a separate preparation of baculovirus-expressed E4 protein containing a similar profile of E4 species to WT38 and G₁ nuclei prepared from human WI38 diploid fibroblasts achieved a similar level of replication inhibition (55%; data not shown).

HPV1 E4 does not arrest ongoing cellular DNA synthesis in vitro. Unlike G₁ nuclei, nuclei isolated from cells in the S phase of the cell cycle contain active replication forks and are thus competent for DNA synthesis in the absence of cytosolic S-phase extracts (31). Addition of geminin, an inhibitor of origin licensing, to in vitro replication reaction mixtures containing NIH 3T3 S-phase nuclei as the source of the template failed to affect ongoing DNA synthesis (Fig. 1d), consistent with previous reports (18, 33, 40). Significantly, cellular DNA elongation was also not affected by the addition of recombinant E4 protein (WT38) to S-phase nuclei, in either the absence or presence of His₆-XeCdc6 (Fig. 1d). The data from the in vitro replication assay (Fig. 1) indicate that in the presence of exogenous Cdc6, HPV1 E4 inhibits initiation of DNA synthesis but fails to arrest ongoing DNA synthesis.

HPV1 E4 blocks recruitment of replication licensing proteins onto chromatin in vitro. Initiation of cellular DNA replication is achieved by the ordered assembly of prereplicative complexes (pre-RCs) at origins of replication (34). During late mitosis (M) and early G₁ phase, the RLFs Cdc6 and Cdt1, by interacting with the origin recognition complex, load the putative DNA replicative helicase Mcm2-7 onto chromatin to form pre-RCs. In the subsequent S phase, DNA replication is initiated at these "licensed" origins by the concerted action of cyclin-dependent kinases and Cdc7-Dbf4. To investigate whether HPV1 E4 might inhibit replication initiation by blocking assembly of pre-RCs onto chromatin, we resolved chromatin-bound protein fractions prepared from G₁- and S-phase nuclei subjected to in vitro replication reactions by gel electrophoresis and probed for the RLFs Cdc6, Mcm2, and Mcm7 (Fig. 2). Histone H1 levels were used as a loading control. Chromatin prepared from G₁ nuclei incubated with S-phase cytosol showed a twofold increase in the binding of endogenous Cdc6 and MCM factors compared to the elongation control reaction (buffer A) (Fig. 2a). Addition of recombinant Cdc6 to replication reaction mixtures led to a 1.5-fold increase in the total amount of chromatin-bound Cdc6 and further increased levels of chromatin-bound MCMs (Fig. 2a), correlating with the observed small increase in the percentage of replication-competent nuclei (Fig. 1c). In contrast, geminin inhibited origin licensing by blocking loading of Mcm2 onto chromatin (Fig. 2a), indicating that increased levels of chromatin-bound RLFs in nuclei subjected to the replication assay are a result of genuine pre-RC assembly in vitro. Addition of HPV1 E4 protein (WT38) with His₆-XeCdc6 to the replication reaction mixtures was associated with a 3.5-fold and 2-fold decrease in chromatin-bound Mcm2 and Mcm7 proteins, respectively, compared to reaction mixtures containing His₆-XeCdc6 alone (Fig. 2a). Notably, the reduced levels of chromatin-bound MCM proteins were close to base levels measured in G₁ nuclei incubated with buffer A (Fig. 2a). The reduction of chromatin-bound MCM proteins by the HPV1 protein correlated with its ability to inhibit replication, as Mcm2 and Mcm7 levels were not affected by addition of E4 in the absence of exogenous Cdc6 or by the addition of E4 protein lacking full-length E1^E4 protein (E4) to reaction mixtures containing His₆-XeCdc6 (Fig. 2a). There was no evidence that E4 became bound to chromatin, in either the presence or absence of exogenous Cdc6 (see Fig. 4b).

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FIG. 2. HPV1 E4 suppresses recruitment of MCM proteins onto chromatin in vitro. Immunoblots of chromatin-bound protein fractions prepared from NIH 3T3 G1-phase (a) and S-phase (b) nuclei subjected to in vitro replication assays are shown at the top. The densities of protein bands relative to those measured in nuclei incubated in S-phase cytosol (SC), after normalization against histone H1 loading, are shown in the histograms at the bottom.

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FIG. 4. The arginine-rich motif in E1^E4 is necessary for inhibition of cellular DNA synthesis initiation in vitro. (a) Addition of bacterial recombinant wild-type HPV1 E4 protein (WTE4) and Cdc6 to coincubations of NIH 3T3 G1 nuclei and HeLa S-phase cytosol inhibits cellular DNA synthesis in replication-competent nuclei. E4-mediated inhibition is relieved following addition of mutant E4 proteins containing either a deletion of residues 44 to 48 (E444-48) or a single alanine replacement of arginine 45 (E4R45A), together with Cdc6. Data from three independent experiments are given as means ± standard deviations, and the asterisk indicates a >99.99% significance in the decrease of replicating nuclei compared to a control replication assay containing exogenous Cdc6. (b) Immunoblot of chromatin-bound protein fractions prepared from NIH 3T3 G1 nuclei subjected to in vitro replication reactions in mixtures containing wild-type and mutant E4 proteins. The histogram shows the densities of the protein bands, after normalization against histone H1 loading, relative to those in nuclei incubated in S-phase cytosol (SC).

Chromatin-binding studies on S-phase nuclei subjected to replication elongation assays showed that neither geminin nor E4 added together with His₆-XeCdc6 affected the chromatin-binding status of Mcm2 and Mcm7 (Fig. 2b).

Together, the in vitro replication and chromatin-binding data indicate that inhibition of initiation of cellular DNA synthesis by HPV1 E4 in the presence of His₆-XeCdc6 correlates with reduced MCM loading onto chromatin.

Repression of S-phase entry by HPV1 E4 in epithelial cells is dependent on an arginine-rich motif in the E1^E4 protein. To determine whether there is a relationship between the negative effect of HPV1 E4 on cell proliferation (13) and E4's ability to inhibit cellular DNA replication in vitro, we first identified the HPV1 E1^E4 sequences required for suppression of S-phase entry in epithelial cells. Since the negative effect of HPV1 E1^E4 on S-phase entry was dependent on the presence of a truncated 16-kDa E4 protein (13), HPV1 E1^E4 (E4-17K) expression plasmids containing small deletions that cover the majority of the E1^E4 sequence were individually cotransfected with the plasmid expressing the truncated protein (E4-16K) into Cos-1 epithelial cells, and cellular DNA synthesis was monitored by BrdU incorporation. In keeping with our previous findings (13), transient expression of the full-length E1^E4 protein together with E4-16K reduced S-phase BrdU incorporation nearly twofold, in comparison to cells expressing the individual proteins or control cells (Table 1). Of the E1^E4 deletion plasmids tested, only one, containing a deletion of residues 44 to 48 (GRPRR), did not inhibit S-phase entry following cotransfection with E4-16K (Table 1). The G₂ arrest function of this mutant E1^E4 protein, however, remained intact (data not shown).

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TABLE 1. Percentage of BrdU-positive S-phase Cos-1 epithelial cells following transfection with wild-type and mutant HPV1 E4 expression plasmidsa

The contribution of individual amino acids within the ⁴⁴GRPRR⁴⁸ sequence to E1^E4 function was examined by replacing the arginine residues at positions 45, 47, and 48 with alanine residues (E4R45A, E4R47A, and E4R48A). Following cotransfection of these mutant E1^E4 plasmids with the E4-16K expression plasmid into Cos-1 cells, BrdU incorporation revealed that inhibition of entry into S phase was sensitive to alanine substitution of Arg45 but not mutation of Arg47 or Arg48 (Fig. 3a). Loss of inhibitory action was not due to any change in the stability of the E4R45A E1^E4 protein in epithelial cells (Fig. 3b), and all three mutants promoted G₂ arrest to levels comparable to those obtained with E4-17/16K-expressing cells (data not shown).

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FIG. 3. Inhibition of S-phase entry by HPV1 E4 in epithelial cells is dependent on an arginine residue within the E1^E4 protein. Cos-1 epithelial cells were transfected with expression plasmids and pulse-labeled with BrdU, and the percentage of BrdU-positive S-phase cells was determined. (a) Coexpression of full-length E1^E4 with the truncated E4 protein E4-16K inhibits S-phase entry compared to expression of the polypeptides alone. Alanine replacement of arginine 45 (R45A), but not of arginine 47 (R47A) or 48 (R48A), is sufficient to relieve the inhibitory effect upon S-phase progression. Data are means plus standard deviations. The double and single asterisks indicate significances of 99.99% and 99.98%, respectively, in the decrease in the percentage of BrdU-positive cells compared to that in Cos-1 cells transfected with empty vector. The data were collected from seven independent experiments. The two-dimensional BrdU-PI profiles of cells expressing E4-16K, E1^E4+E4-16K, R45A+E4-16K, and R47A+E4-16K are also shown. (b) Immunoblot of protein extracts showing E4 protein expression in Cos-1 cells.

The arginine-rich motif in E1^E4 is necessary for inhibition of cellular DNA synthesis initiation in vitro. Next, we wanted to determine whether the ⁴⁴GRPRR⁴⁸ sequence E1^E4 was also involved in inhibition of cellular origin licensing in the in vitro replication assay. In this instance, the recombinant HPV1 E4 proteins were expressed in and purified from bacteria. To verify that a bacterial form of the E4 protein has an inhibitory function, the wild-type protein was titrated into the in vitro replication reaction mixtures. Consistent with the data obtained by using baculovirus recombinant E4 protein, we observed no significant effect of E4 on the percentage of nuclei synthesizing DNA following addition of the bacterial E4 protein to coincubation mixtures of NIH 3T3 G₁ nuclei and HeLa S-phase cytosol (24%; data not shown). However, when E4 was added to reaction mixtures that also contained exogenous Cdc6 (His₆-XeCdc6), the percentage of replicating nuclei decreased significantly, from 29% to 13.8%, indicating that in the presence of a bacterial HPV1 E4 protein nearly 53% of the replication-competent nuclei failed to initiate DNA synthesis (Fig. 4a). Inhibition of cellular replication initiation was, however, abrogated upon the addition of the E4 protein containing a deletion of ⁴⁴GRPRR⁴⁸ to the in vitro reaction mixtures containing His₆-XeCdc6 (23.2% [Fig. 4a]), and alanine substitution of arginine 45 (E4R45A) within this motif was sufficient to relieve the inhibitory effect of the HPV protein (26.5% [Fig. 4a]).

Our analysis of pre-RC assembly in the in vitro replication assay had indicated that HPV1 E4 protein inhibited loading of MCM onto chromatin (Fig. 2). Therefore, we investigated whether failure to inhibit replication initiation by the mutant E1^E4 proteins correlated with efficient assembly of pre-RCs onto chromatin. Chromatin-bound protein fractions prepared from G₁- and S-phase nuclei subjected to in vitro replication reactions were probed for the RLFs Cdc6, Mcm2, and Mcm7 (Fig. 4b). Chromatin prepared from G₁ nuclei incubated with S-phase cytosol in the presence of His₆-XeCdc6 and the wild-type protein derived from bacteria showed 51% and 41% decreases in chromatin-bound Mcm2 and Mcm7, respectively, compared to the levels of these proteins in the reaction mixture containing His₆-XeCdc6 alone (Fig. 4b). The reductions in the levels of chromatin-bound MCM proteins in the presence of the bacterial preparation of E4 were similar to the decrease observed with the baculovirus recombinant E4 protein (Fig. 2). However, in the presence of the mutant E1^E4 proteins E444-48 and E4R45A, both Mcm2 and Mcm7 were efficiently recruited to chromatin (Fig. 4b).

Together, our data suggest that HPV1 E4 inhibits initiation of cellular DNA replication in vitro by blocking MCM loading onto chromatin and that this is dependent on an arginine-rich motif within the full-length form of the viral protein.

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Using a cell-free cellular DNA replication assay, we have shown that HPV1 E4 protein is a potent inhibitor of cellular replication licensing, a novel function for this protein. Chromatin-binding studies indicate that E4 blocks replication initiation in vitro by preventing loading of the licensing factors Mcm2 and Mcm7 onto chromatin. The functional effect of E4 mimics the cellular repressor of replication licensing geminin (18), but while geminin interacts with the RLF Cdt1 to inhibit assembly of MCMs into pre-RCs, the mechanism by which E4 blocks MCM loading appears to be different from that of the cellular repressor. Unlike geminin, inhibition of licensing in vitro by E4 requires addition of an exogenous supply of soluble Cdc6. The requirement for exogenous Cdc6 for E4-mediated inhibition of DNA replication may be explained by the differential regulation of Cdc6 in normal proliferating cells, where Cdc6 is found in both the soluble and chromatin-bound fractions (30), and during the G₀-S transition, where Cdc6 is synthesized de novo and immediately recruited to replication origins (12). In the in vitro replication assay used in this study, NIH 3T3 G₁ nuclei are prepared during release from density-dependent growth arrest (G₀) and therefore contain only chromatin-bound Cdc6 (31). How HPV1 E4 and the soluble form of the licensing factor Cdc6 function together to inhibit replication is not yet understood, but they are sufficient to block MCM recruitment to origins even in the presence of chromatin-bound Cdc6. One possibility is that E4 is able to complex with MCM factors in a soluble-Cdc6-dependent manner. There is evidence of an association between a glutathione S-transferase-HPV1 E1^E4 fusion protein and an epitope-tagged form of Mcm7 expressed in cell lysates containing soluble Cdc6 (I. Bell and Sally Roberts, unpublished data). Association between E1^E4 and this MCM factor, however, might not be a complete description of the mechanism of E4-mediated replication inhibition, since E4, the form of HPV1 E4 defective in inhibiting cellular DNA synthesis, can form an association with Mcm7 (I. Ashmole and S. Roberts, unpublished data). It is feasible that further modification (for example, a phosphorylation event) is necessary to achieve an "active" inhibitory complex, and this might be dependent on N-terminal sequences specific to the full-length E1^E4 polypeptide. Whatever the underlying mechanism, inhibition of initiation of cellular DNA replication in vitro and suppression of entry of epithelial cells into S phase are functions that are both dependent on arginine 45 in the HPV1 E1^E4 polypeptide, suggesting that these two functions are linked and hence that E4 can block cellular DNA synthesis in the presence of endogenous soluble Cdc6.

Our studies have shown that E4-induced inhibition of in vitro replication initiation is dependent on a full-length E1^E4 molecule. It is possible that the smaller forms of E4 that exist in the purified preparations of HPV1 E4 protein used in this study may contribute to this E1^E4 function. Indeed, HPV1 E4 expression studies show that coexpression of full-length and truncated forms of HPV1 E4 act to repress cell proliferation, while expression of the full-length form alone do not (13). Complex formation between the different E4 polypeptides (14) might be one explanation: the complex might either inhibit S-phase entry directly or, upon formation, deplete free full-length protein to a level that it is then active with regard to blocking cell proliferation. This latter explanation might well explain why there is no block in cell proliferation in cells expressing the E1^E4 protein alone even though a small amount of the truncated E4 species does accumulate in these cells.

We do not know at this stage of our investigations whether this novel HPV1 E4 function is conserved between the different phylogenetic types. Arginine 45 lies in a region of HPV1 E4 that is rich in basic amino acids, and indeed, similar (but not identical) regions are to be found in E4 proteins of types with a tropism dissimilar to that of HPV1, such as HPV16 and -18, which have a preference for epithelia of the oral and anogenital tracts (1). The basic region of HPV16 E4 forms part of the G₂ arrest domain (5), but in HPV1, E4 is not a required element of the G₂ arrest function, nor do these regions contribute to the interaction with the keratin cytoskeleton (25, 27). An association between HPV1 E4 and the SR protein kinase SRPK1 is dependent on arginine 45 (1), although other sequences required to maintain this interaction do not contribute to inhibition of cell proliferation, suggesting that this E4 binding partner is unlikely to be involved in the underlying mechanism of replication inhibition by HPV1 E4. Therefore, arginine 45 either mediates an association to a novel E4-binding protein or dictates a specific cellular localization necessary for replication inhibition.

Host cell DNA synthesis is blocked during the Epstein-Barr virus (EBV) lytic infection cycle, during which there is high-level amplification of the EBV genome (17). EBV inactivates MCM helicase function by phosphorylation of MCM proteins, and this might be sufficient to block cellular DNA synthesis in lytic infected cells (16). Infection by another DNA virus, cytomegalovirus, also abrogates cellular replication licensing by inhibiting chromatin loading of MCM proteins (2, 37). Even though the underlying mechanisms of repression of cellular DNA replication by EBV and CMV were not identified, taken together with our study, this observation implies that unrelated DNA viruses may have evolved similar strategies to selectively inhibit host cell DNA synthesis. This function could prove advantageous to viruses that depend on the host cell for the supply of essential replication enzymes and nucleotides for viral DNA synthesis. Papillomaviruses have three phases of replication: establishment and maintenance of the genome in basal cells are followed by vegetative genome amplification in cells that have migrated up from the basal layer and differentiated (10). Because keratinocyte differentiation normally correlates with exit from the cell cycle, the virus induces S-phase gene activity in these cells, and eventually they initiate vegetative viral genome replication, whereby the viral genome is amplified to high copy number (4). Notably, the switch to genome amplification is associated with induction of E4 protein (23). Furthermore, more recently, it was shown that this switch also correlates with suppression of cellular DNA synthesis in replication-activated HPV16-containing keratinocytes (20). We therefore hypothesize that in these cells, E4 acts to preserve the supply of essential host replication factors by inhibiting licensing of cellular origins of replication and thus repress competing cellular DNA synthesis. Combined with action on G₂-to-M transition of the cell cycle (5, 13, 19), E4 could be a key player in ensuring successful replication of the virus. Indeed, loss of expression of the full-length E1^E4 protein is associated with an abrogation of efficient vegetative genome replication in systems that recapitulate the productive replication life cycles of HPV16, -18, and -31 and cottontail papillomavirus (20, 22, 38, 39).

Viral factors such as E4 could provide powerful molecular tools that can be utilized to dissect the molecular mechanisms regulating initiation of eukaryotic DNA replication. Furthermore, because the origin licensing machinery has been proposed as a novel attractive target for anticancer therapy, the design of E4-based mimetic compounds could provide novel nongenotoxic agents.

 
We thank Ron Laskey for insightful discussions during the early stages of the project. We are grateful to Renos Savva for help producing bacterial E1^E4 proteins and to Emma Yates for technical assistance.

This work was supported by Cancer Research UK Programme grants to S.R. (C427/A3919) and to G.H.W. and K.S. (C428/A2281). S.R.K. was supported by a Medical Research Council studentship.

 
* Corresponding author. Mailing address for Sally Roberts: CR-UK Institute for Cancer Studies, University of Birmingham, Vincent Drive, Birmingham B15 2TT, United Kingdom. Phone: 44(0)121-4147459. Fax: 44(0)121-4144486. E-mail: s.roberts{at}bham.ac.uk. Mailing address for Gareth Williams: Wolfson Institute for Biomedical Research and Department of Pathology, University College London, Gower Street, London WC1E 6BT, United Kingdom. Phone: 44(0)207-6796304. Fax: 44(0)207-3384408. E-mail: gareth.williams{at}ucl.ac.uk

Published ahead of print on 16 July 2008.

These authors contributed equally to this work.

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Journal of Virology, September 2008, p. 9056-9064, Vol. 82, No. 18
0022-538X/08/$08.00+0     doi:10.1128/JVI.01080-08
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