Regulation of Bovine Papillomavirus Replicative Helicase E1 by the Ubiquitin-Proteasome Pathway

Proteins and DNA plasmids.BPV1 E1 protein, Xenopus cyclin E, and Xenopus Cdk2 were prepared by translation of mRNA-encoding constructs in RNase-treated translation extracts from Xenopus eggs in the presence of [³⁵S]methionine as described previously (13). The E1 mutant in which the three Cdk phosphorylation sites were changed to alanines (E1-AAA) was obtained by using a transformer site-directed mutagenesis kit from Clontech and then confirmed by dideoxy sequencing. BPV1 E1 protein was purified from recombinant baculovirus-infected Sf9 cells (7). ³⁵S-labeled BPV1 E2 was prepared by coupled transcription-translation reactions in a rabbit reticulocyte lysate (Promega).

Glutathione S-transferase (GST)-tagged human Cdk2, GST-tagged human Cdk2K33R (47), GST-ubiquitin (49), 6His-tagged ubiquitin, and 6His-tagged K48R ubiquitin (1, 53) were expressed in Escherichia coli BL21 and purified by affinity chromatography on glutathione-Sepharose or nickel-agarose. The GST fusion protein of ubiquitin contains a protein kinase A site between the GST and the ubiquitin portion of the fusion protein to allow specific labeling (49). Methylated ubiquitin was prepared by the method of Hershko and Heller (20). The green fluorescent protein (GFP)-E1 construct was generated by inserting wild-type E1 into the BamHI site of pEGFP-C2 (Clontech, Palo Alto, Calif.). The BPV1 E1 expression vectors pCGEag, pCGE2, and pSKori⁺, containing a 160-bp origin-bearing BPV1 DNA fragment (nucleotides 7855 to 81) in the pBluescript SK+ vector, have been described previously (8, 57). pCDNA₃-6HisUb and pCDNA₃-HAUb were provided by M. Piechaczyk, Institut de Genetique Moleculaire de Montpellier, Montpellier, France.

Preparation of soluble extracts and in vitro DNA replication assays.Crude Xenopus interphase egg extracts for translation assays and membrane-free interphase egg cytosol used for E1-dependent replication assays were prepared essentially as described previously (13). Replication assays were carried out by first mixing 18 μl of membrane-free egg cytosol supplemented with an ATP-regenerating system and with 2 μl of radiolabeled E1 protein for 30 min at 25°C before the addition of 300 ng of either pSKori⁺ or pSKori⁻. Reaction mixtures were incubated at 25°C, and samples were taken for both protein and DNA analysis. Samples to be analyzed for DNA synthesis were supplemented with 2 μCi of [³²P]dCTP.

The 293 cell extracts for cell-free replication were prepared as described previously (32). Cells were synchronized in early S phase by a double block in culture medium containing 2.5 mM thymidine. The conditions for cell-free replication in the presence of 2 μCi of [³²P]dCTP have been described (7). Reaction mixtures (50 μl) containing 300 ng of pSKori DNA, 100 ng of E1 protein, and 200 μg of 293 cell cytoplasmic extract were incubated at 37°C for 60 min. Aphidicolin was added at a final concentration of 40 μg/ml in both cell-free systems. Samples for DNA analysis were deproteinized with proteinase K (500 μg/ml) and 0.5% sodium dodecyl sulfate (SDS) for 60 min at 37°C, followed by two phenol-chloroform extractions, and analyzed on agarose gels.

In vitro E1 degradation and ubiquitination assays.For the degradation assays, Xenopus egg extracts were depleted with anti-Cdk2 antibody as previously described and supplemented with an ATP-regenerating system (13). NLVS (4-hydroxy-5-iodo-3-nitrophenylacetyl-Leu-Leu-Leu-vinylsulfone) (Calbiochem), an irreversible inhibitor of the three proteolytic activities of the proteasome 26S (5), was added to extracts before addition of 1 to 2 μl of radiolabeled E1 protein. Ubiquitination assays in crude Xenopus egg extracts were carried out by supplementing egg extracts with ubiquitin aldehyde (4 μM; Sigma) (21) and ATPγS [adenosine 5′-O-(3-thiotriphosphate); 4 mM] in the presence or absence of either hexahistidine-ubiquitin (6His-ubiquitin), methylated ubiquitin, or K48R ubiquitin at a final concentration of 1.25 mg/ml, before addition of 2 μl of radiolabeled E1 protein.

Reaction mixtures were incubated at 28°C for 30 to 60 min, and 3-μl aliquots were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) followed by autoradiography. The conjugation of ubiquitin to E1 for replication in 293 cell extracts was carried out by supplementing reaction mixtures with 1 μg of ³²P-labeled GST-ubiquitin and ubiquitin aldehyde (4 μM). After 30 min at 37°C, E1 was immunoprecipitated with polyclonal anti-C-terminal E1 antibody. Immunoprecipitates were subjected to polyacrylamide gel electrophoresis, and radioactively labeled bands were visualized by autoradiography.

Cell culture and DNA transfection.COS-7 and 293 cells were grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. To inhibit protein synthesis, cells were cultured in the presence of 10 μg of cycloheximide per ml for 4 h. After this, the cells started to die. Inhibition of the proteasome was performed by culturing cells for up to 18 h in the presence of 5 μM MG132 (Calbiochem). A stock solution of MG132 was made in dimethyl sulfoxide and used at a 1:2,000 dilution.

Cells were 80% confluent when trypsinized for electroporation (56). Each electroporation mixture contained 10 μg of pEGFP-E1, 5 μg of pCGE2, 3 μg of supercoiled pSKori, and 50 μg of carrier DNA. Electroporation was performed with 6.6 million cells in 250 μl of growth medium containing 10% fetal bovine serum and 5 mM BES buffer (N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid, pH 7.2) at 960 μF and 170 V for 293 cells and 180 V for COS-7 cells, with a Bio-Rad Gene Pulser with a capacitance extender.

Western blotting and antibodies.Transfected cells were harvested from a 35-mm plate in 100 μl of RIPA buffer (0.5% NP-40, 0.5% Tween 20, 0.5% deoxycholic acid, 0.15 M NaCl, 10 mM KCl, 20 mM Tris-HCl [pH 7.5], and 1 mM EDTA) and boiled with 1% SDS, 48 h after electroporation. Then 50 μg of protein was loaded onto SDS-PAGE gels. Western blotting was performed by standard procedures with 5,000-fold dilutions of anti-GFP (Torrey Pines Biolabs) or monoclonal antihemagglutinin (HA) antibody (generous gift of A. Debant, Centre de Recherches de Biochimie Macromoleculaire, Montpellier, France).

Transient-replication assays in transfected cells.Transfected 293 cells used in transient-replication assays were plated onto 100-mm-diameter dishes, and samples were taken at 24-h intervals on days 2 to 4. DNA plasmids were isolated as described before (31), and DNA samples were digested with a mix of SmaI and DpnI. The samples were run on 1% agarose gels and blotted to nitrocellulose under standard conditions for Southern blot analysis. High-specific-activity probes were generated by random priming of pSKori⁺ DNA with a kit from Amersham (Rediprime), and the blots were visualized and quantitated with a PhosphorImager.

In vivo ubiquitination assays.Asynchronously growing COS-7 cells were transfected with 10 μg of pEGFP-E1, 5 μg of pCDNA₃-6HisUb, or 5 μg of pCDNA₃-HAUb and with or without 5 μg of pCGE2 and 3 μg of pSKori. Control transfections used the same amount of empty vectors. Cells were cultured in the presence of 5 μM MG132 overnight and lysed after 48 h. For the ubiquitination assay with HA-ubiquitin, cells were trypsinized, pelleted, and lysed in 50 μl of RIPA buffer containing MG132 and ubiquitin aldehyde to block isopeptidase activity. For the ubiquitination assay with 6His-ubiquitin, cells from three 100-mm-diameter dishes were lysed in 5 ml of 6 M guanidine-HCl-0.1 M Na₂HPO₄/NaH₂PO₄-0.01 M Tris-HCl (pH 8.0). 6His-ubiquitin conjugates in the extracts were purified on nickel-agarose.

E1 is protected from degradation within cyclin E/Cdk2 complexes.Coincubation of E1, cyclin E, and Cdk2 translated separately in egg extracts depleted of Cdk2 led to the generation of a tight complex that was coimmunoprecipitated by either anti-Cdk2 or anti-E1 antibodies, as previously described (13). The relative level of E1 was similar in both immunoprecipitates, suggesting that radiolabeled E1 molecules are detected only when associated with cyclin E-Cdk2 (Fig. 1A). To determine whether cyclin E/Cdk2 protects E1 from degradation, E1 was first mixed with increasing amounts of Xenopus cyclin E and Cdk2 and then added to Xenopus Cdk2-depleted interphase egg extract (5). While E1 alone was mostly degraded (Fig. 1B, lane 2), increasing E1 levels were detectable upon addition of cyclin E/Cdk2 (19 and 28% protection in lanes 3 and 4, respectively).

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FIG. 1.

E1 is unstable in Xenopus egg extract in the absence of cyclin E/Cdk2. (A) BPV1 E1 (E1), Xenopus cyclin E (CycE), and Xenopus Cdk2 (Cdk2), translated separately in Cdk2-depleted Xenopus egg extracts in the presence of [³⁵S]methionine, were mixed together, and immunoprecipitations (IP) were performed with anti-Cdk2 antibodies (K2) or with an anti-C-terminal E1 polyclonal antibody (E1). Immunoprecipitates were analyzed by SDS-PAGE and autoradiographed. (B) ³⁵S-labeled E1 translated in Xenopus Cdk2-depleted extracts was incubated in the absence (lane 2) or presence of increasing amounts of labeled Xenopus cyclin E and Xenopus Cdk2 (lanes 3 and 4) obtained by translation in Xenopus Cdk2-depleted extracts. After 30 min of incubation at 25°C, equal amounts of each mixture were added to fresh Cdk2-depleted egg cytosol and incubated for an additional 30 min. E1 was recovered by immunoprecipitation with an anti-C-terminal E1 polyclonal antibody and analyzed by SDS-PAGE and autoradiography. Input E1, lane 1. (C) Purified complexes reconstituted with ³⁵S-labeled E1, ³⁵S-labeled Xenopus cyclin E, and GST-Cdk2 (Cdk2) or GST-Cdk2K33R (Cdk2 K33R) were added to egg extract and incubated for the indicated times at 25°C. Samples were analyzed by SDS-PAGE and autoradiography. (D) ³⁵S-labeled E1 translated in undepleted Xenopus egg extracts was added to a fresh membrane-free interphase egg extract and incubated for the indicated times. After 60 min of incubation, a sample of extract was incubated with beads coupled to the cyclin/Cdk-associated protein Suc1 (suc1). The behavior of radiolabeled E1 was analyzed by SDS-PAGE and quantitated as a percentage of that at 0 min.

E1 is phosphorylated within E1/cyclin E/Cdk2 complexes but is not released from the kinase after phosphorylation (13, 36). To test whether the protective effect of cyclin E-Cdk2 is a direct consequence of binding or phosphorylation of E1, we examined the stability of E1 within complexes reconstituted with cyclin E and either catalytically inactive or active recombinant GST-Cdk2. In interphase egg extracts, E1 levels remained constant in complexes formed with both forms of Cdk2, indicating that the kinase activity had no effect on the stability of E1. The presence of cyclin E-Cdk2 in the E1 immunoprecipitates, as well as the protective effect observed with catalytically inactive kinase complexes, strongly suggests that E1 is stabilized as a consequence of its interaction with cyclin E-Cdk2.

In undepleted egg extracts, a significant proportion of E1 molecules were degraded within 30 min (Fig. 1D). The E1 molecules that remained stable over the incubation time bound to a Cdk affinity matrix, suc1-agarose, indicating that this fraction of E1 molecules was associated with endogenous cyclin E/Cdk2 complexes. These results indicate that all the E1 molecules in stoichiometric excess relative to the available cyclin E/Cdk2 complexes are potentially unstable, while those bound to endogenous cyclin E/Cdk2 complexes become protected from degradation.

Protection of E1 bound to cyclin E/Cdk2 is reversed as a consequence of replication.The effect of BPV DNA replication on the stability of E1 was then monitored. Membrane-free interphase egg extracts supplemented with E1 translated in egg extracts catalyzed the efficient replication of plasmid DNA molecules containing the BPV origin of replication (Fig. 2A, lane 1). The reaction was almost completely inhibited by aphidicolin, which induces the accumulation of early-replicating intermediates by blocking DNA polymerase α activity (Fig. 2A, lane 2). As shown previously, the replication was dependent on the presence of a BPV origin of replication, since very little DNA synthesis was observed with a template lacking BPV sequences (Fig. 2A, lane 3).

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FIG. 2.

E1 protection is reversed by replication. (A) ³⁵S-labeled E1 was added to a membrane-free interphase egg extract supplemented or not with aphidicolin (Aphi) before addition of pSKori⁺ or pSKori⁻. Samples to be analyzed for DNA synthesis were supplemented with [³²P]dCTP. After a 30-min incubation at 25°C, DNA products from pSKori⁺ (lanes 1), pSKori⁺ and aphidicolin (lanes 2), and pSKori⁻ (lanes 3) extracts were purified, subjected to agarose gel electrophoresis, and detected by incorporation of [³²P]dCTP. FI and RI designate the migration positions of supercoiled monomer circles of pSKori⁺ and pSKori⁻ markers and replicative intermediates, respectively. (B) Samples from the same reactions were taken at intervals to monitor E1 behavior and analyzed by SDS-PAGE and autoradiography. (C) Complementation with BPV1 E2. ³⁵S-labeled E1 was incubated in a membrane-free interphase egg extract in the absence (lane 1) or presence (lane 2) of ³⁵S-labeled E2 before addition of pSKori⁺ and [³²P]dCTP. The replication was allowed to proceed for 10 min, and the DNA products were analyzed by agarose gel electrophoresis. (D) The levels of E1 and E2 before addition of pSKori⁺ (lane 1) and after 30 min of incubation with pSKori⁺ (lane 2) were analyzed by SDS-PAGE and autoradiography.

As shown in Fig. 1D, E1 molecules not bound to cyclin E/Cdk2 were rapidly degraded after addition to membrane-free interphase egg cytosol. To specifically follow the behavior of E1 molecules stabilized by association with cyclin E/Cdk2 complexes, E1 was therefore incubated for 30 min in membrane-free interphase egg extract prior to the addition of DNA template. As shown in Fig. 2B, the fraction of E1 molecules stably bound to cyclin E/Cdk2 was degraded 20 to 30 min after addition of the BPV origin-containing plasmids. This was not observed in the reaction carried out in the presence of aphidicolin or after addition of a plasmid without a BPV origin of replication (Fig. 2B), demonstrating that E1 protection is reversed by replication.

While BPV E2 is dispensable in this cell-free system, we examined whether it could affect E1 stability during the in vitro replication process. In vitro-translated and -labeled E2 protein stimulated E1-dependent replication, as demonstrated by the enhanced level of late replicative intermediates at an early time point (Fig. 2C). While E2 levels remained unchanged, the E1 levels were diminished at the end of the reaction (Fig. 2D). Thus, the successful execution of E1-directed replication events causes E1 degradation, a phenomenon that is unaffected by the presence of E2.

E1 degradation in egg extract occurs by the ubiquitin-proteasome pathway.Analysis of E1 for the in vitro replication process revealed the transient appearance of high-molecular-weight E1 species in parallel with the gradual loss of E1 monomers (Fig. 3A). This behavior is characteristic of proteins that are modified by polyubiquitination for degradation by the proteasome. To test this in the case of E1, we identified E1-ubiquitin conjugates by supplementing the egg extracts with different modified forms of ubiquitin as well as by inhibiting the activity of cellular isopeptidases, which cleave ubiquitin molecules from protein substrates. As shown in Fig. 3B, addition of hexahistidine-tagged ubiquitin led to a notable increase in very large E1 conjugates (Fig. 3B, lane 3). This ubiquitin chain elongation was inhibited by incorporation of methylated ubiquitin, which terminates the elongation of ubiquitin chains and led to the appearance of apparently monoubiquitinated E1 (Fig. 3B, lane 4). The addition of K48R ubiquitin decreased the overall level of ubiquitin conjugates (Fig. 3B, lane 5).

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FIG. 3.

E1 is degraded by the ubiquitin-proteasome pathway. (A) ³⁵S-labeled E1 was added to a membrane-free interphase egg extract 30 min before addition of pSKori⁺. Aliquots were taken at intervals and analyzed by SDS-PAGE and autoradiography. The autoradiogram shows the appearance of high-molecular-weight labeled E1 species (lanes 2 and 3). (B) E1 is ubiquitinated in egg cytosol. Radiolabeled E1 was incubated in a membrane-free interphase egg treated with ubiquitin aldehyde and supplemented with either 6His-ubiquitin (lane 3), methylated ubiquitin (lane 4), or K48R ubiquitin (lane 5). No supplement was used in lane 2, and input was used in lane 1. The formation of large radiolabeled ubiquitin-E1 (Ub-E1) conjugates (lane 3) was blocked by addition of methylated ubiquitin (lane 4) and decreased in the presence of K48R ubiquitin (lane 5). (C) Radiolabeled E1 protein was added to Cdk2-depleted membrane-free interphase egg supplemented with NLVS at a final concentration of 50, 100, or 200 μM (lanes 3 to 5, respectively) or dimethyl sulfoxide (DMSO) as a control (lane 2). TO, input, lane 1. Quantitation of the fluctuation of E1 levels is shown on the lower panel. (D) Radiolabeled wild-type E1 (lanes 1 to 3) or E1-AAA (lanes 4 to 6) was added in a membrane-free interphase egg supplemented with ubiquitin aldehyde and either 6His-ubiquitin (lanes 2 and 5), methylated ubiquitin (lanes 3 and 6), or no ubiquitin complement (lanes 1 and 4). Aliquots of reaction mixtures were subjected to SDS-PAGE, and radiolabeled E1 species were visualized by autoradiography (upper panel). A shorter exposure is shown on the bottom panel to emphasize the accumulation of monoubiquitinated E1 species in the presence of methylated ubiquitin, indicated by an asterisk on the right.

We then tested the effects of a potent proteasome inhibitor (NLVS) on E1 degradation in membrane-free interphase egg extracts. As shown in Fig. 3C, NLVS stabilized radiolabeled E1 molecules translated in the absence of cyclin E-Cdk2 in egg extracts. This is consistent with the fact that the covalent attachment of ubiquitin to E1 targets the protein for degradation by the 26S proteasome (12). In addition, a mutant in which the three cyclin/Cdk phosphorylation sites were mutated to alanines (E1-AAA) was ubiquitinated with the same efficiency as the wild-type E1 protein (Fig. 3D). Restriction to monoubiquitination by the addition of methylated ubiquitin yielded similar amounts of monoubiquitinated E1 molecules in both the wild-type E1 and E1-AAA reactions, suggesting that the targeted degradation of E1 is independent of its phosphorylation status.

E1 ubiquitination in S-phase extracts from human 293 cells depends on replication.Given that replication caused E1 degradation in Xenopus egg extracts, we tested for a similar process in mammalian cell extracts with cytoplasmic fractions from human 293 cells. These extracts replicated the BPV origin-containing plasmid in the presence of E1 protein produced from baculovirus vectors (Fig. 4A, lane 2). Interestingly, the purified protein was phosphorylated and tightly associated with a kinase activity from insect cells (5). Replication and cell cycle factors are much less concentrated in this replication system than the Xenopus extracts, necessitating 100-fold more E1. We were unable to detect ubiquitinated E1 species on immunoblots following in vitro replication into the cytosol from asynchronously growing 293 cells.

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FIG. 4.

E1 is ubiquitinated in a replication-dependent manner in a cell-free system from S-phase 293 cell extracts. (A) A total of 100 ng of E1 produced from baculovirus vector in Sf9 cells was added to reaction mixtures containing a cytoplasmic extract prepared from 293 cells synchronized in S phase by a double block with thymidine, followed by release into the culture medium for 1 h. Reaction mixtures were supplemented with aphidicolin (lane 1) or dimethyl sulfoxide (lane 2) before addition of pSKori⁺. Half of each reaction mixture was supplemented with [³²P]dCTP and used in the BPV1 origin replication assay. Radiolabeled DNA products were purified after 60 min of incubation at 37°C and subjected to agarose gel electrophoresis. The fastest-migrating supercoiled form I (FI) and early and late replication intermediates (RI) are marked. (B) Half of the reaction mixtures described in A containing aphidicolin (lane 2) or dimethyl sulfoxide (lane 3) were used to monitor E1 ubiquitination (Ub-E1) for the replication assay. As a control, a reaction mixture lacking pSKori⁺ was used (lane 1). Reactions were supplemented with ³²P-radiolabeled GST-ubiquitin and ubiquitin aldehyde and incubated for 30 min at 37°C. E1 was recovered by immunoprecipitation (IP), and radioactively labeled bands were visualized by autoradiography.

Cytosols were then prepared from 293 cells synchronized in S phase, and ³²P-radiolabeled GST-ubiquitin was added together with ubiquitin aldehyde, which blocks isopeptidases and enhances the detection of ubiquitin conjugates. In the absence of DNA, E1 conjugates were not detected (Fig. 4B, lane 1). However, under conditions in which DNA synthesis was initiated by adding BPV plasmids, high-molecular-weight radiolabeled E1 conjugates were present in E1 immunoprecipitates after 30 min (Fig. 4B, lane 3) that were reduced in aphidicolin-treated cytosol (Fig. 4B, lane 2). These data suggest that some E1 molecules are also ubiquitinated in a replication-dependent manner in extracts of mammalian cells synchronized in S phase.

E1 is degraded by the proteasome in living cells.These results indicated that E1 is an unstable protein in cell-free systems. To test this conclusion in vivo, we used an expression vector encoding wild-type E1 with the green fluorescent protein attached to the N terminus. This vector allows us to force E1 expression and readily detect it, since we were unable to detect E1 in a BPV-transformed C127 line. The intrinsic replication capacity of the GFP-E1 recombinant protein was first tested in vitro. As shown in Fig. 5A, the GFP-E1 protein generated similar amounts of early and late replicative intermediates in the cell-free system and thus displayed an activity equivalent to that of the nontagged E1 protein. We also did not observe any significant effect of the fusion to GFP on E1 interactions with other proteins or its stability in the cell-free system (data not shown).

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FIG. 5.

Functional GFP-E1 proteins are degraded by the proteasome in vivo. (A) In vitro-translated and ³⁵S-radiolabeled GFP, E1, and GFP-E1 were analyzed by SDS-PAGE and autoradiography (left panel). Membrane-free interphase egg extracts were supplemented with either GFP (lane 1), E1 (lane 2), or GFP-E1 (lane 3) before addition of pSKori⁺ and [³²P]dCTP. After a 30-min incubation at 25°C, DNA products were purified, subjected to agarose gel electrophoresis, and detected by incorporation of [³²P]dCTP. FI and RI designate the migration positions of supercoiled monomer circles of pSKori⁺ and replicative intermediates, respectively (right panel). (B) Transient replication of pSKori⁺ in the presence of E1 and E2 (lanes 1), GFP and E2 (lanes 2), and GFP-E1 and E2 (lanes 3) expression vectors. Low-molecular-weight DNA was extracted from 293 cells at 48, 72, and 96 h after transfection, digested with SmaI and DpnI, and analyzed by Southern blotting. Filters were probed with radiolabeled pSKori⁺ plasmid. pOri indicates the band generated after digestion of pSKori⁺ with SmaI. (C) 293 cells were transfected with GFP-E1 expression vector, and proteins from total cell lysate were resolved on an SDS gel and analyzed by Western blotting with an antibody directed against the GFP (lane 2). Cell lysate from untransfected cells is shown in lane 1 as a control. The presence of GFP-E1 is indicated (left panel). GFP-E1 expression plasmid was transfected into asynchronously growing 293 cells. Cells were treated with cycloheximide (CHX), and the level of GFP-E1 was evaluated at the indicated time (hours) by Western blotting with the GFP antibody (right panel). Only relevant parts of the blot are shown. 293 cells transfected with GFP-E1 and E2 expression vectors and pSKori⁺ plasmid were treated with cycloheximide as above in the presence of either MG132 (+) or dimethyl sulfoxide (−). Cells were harvested, and lysates were prepared for Western blotting with an antibody to GFP.

We then tested the ability of GFP-E1 to support BPV origin-dependent replication in vivo in the presence of BPV E2. In transiently transfected 293 cells, GFP-E1 yielded a level of replication comparable to that of the wild-type nontagged E1 protein at different times after cotransfection (Fig. 5B, compare lanes 3 with lanes 1). In the presence of GFP and E2, no replication was observed (Fig. 5B, lanes 2). Thus, E1 monomers carrying a GFP tag at the amino terminus are able to form a functional hexameric helicase on the BPV origin without affecting replication levels either in vitro or in vivo.

Since we do not have a suitable E1-specific antibody to analyze its levels by Western blotting, we used a GFP antibody that can detect low levels of GFP-E1 (Fig. 5C). To measure E1 steady-state levels in 293 cells transfected with the GFP-E1 expression vector, cells were treated with cycloheximide to block de novo protein synthesis, and extracts were prepared at various times thereafter. Anti-GFP immunoblots showed no apparent changes in E1 levels during the 4-h treatment.

Since degradation of E1 was only detectable in the presence of E2 and BPV plasmids, i.e., conditions that allow viral replication, 293 cells were cotransfected with the GFP-E1 and E2 expression vectors as well as the BPV origin-containing plasmid and then cultured in the presence of cycloheximide. One transfection was cultured in the presence of the proteasome inhibitor MG132, while the other was cultured with dimethyl sulfoxide, the solvent for MG132. In contrast to the apparent stability of E1 when expressed alone in 293 cells, significant degradation of E1 was observed in the presence of E2 and BPV plasmids, with an estimated half-life of less than 2 h. In addition, MG132 stabilized E1 over the 4 h of cycloheximide treatment, confirming the proteasome-dependent turnover of E1 under these conditions.

E1 is polyubiquitinated in vivo.To investigate whether E1 ubiquitination can be detected in vivo, we cotransfected COS-7 cells with the GFP-wild-type E1 expression vector and a hexahistidine-tagged ubiquitin (6His-ubiquitin) expression vector either with or without the E2 expression vector and the BPV origin-containing plasmid. Cell extracts were prepared and analyzed by immunoblotting with the antibody to GFP (Fig. 6A, lanes 1 to 3). An aliquot was also bound to nickel affinity resin prior to immunoblotting (Fig. 6A, lanes 4 to 6). A ladder of slower-migrating E1 species was specifically detected in samples from total cells as well as in the 6His-ubiquitin-conjugated protein purified fraction (Fig. 6A, lanes 1 and 4). However, cotransfection of the E2 and BPV plasmids with the GFP-E1 plasmid strongly increased the amount of polyubiquitinated E1 (Fig. 6A, lanes 2 and 5).

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FIG. 6.

In vivo polyubiquitination of E1. (A) COS-7 cells were transiently cotransfected with pEGFP-E1 and pCDNA3-6HisUb in the absence (lanes 1 and 4) or presence (lanes 2 and 5) of pCGE2 and pSKori⁺. COS-7 cells transfected with pCDNA3-6HisUb were used as a control (lanes 3 and 6). After 48 h, cells were harvested and lysed. An aliquot corresponding to 1% of each cell extract was kept for input analysis (lanes 1 to 3). The ubiquitinated proteins were purified from the extracts on nickel-agarose (lanes 4 to 6). Purified proteins were subjected to SDS-PAGE and analyzed by immunoblotting with anti-GFP antibody. Polyubiquitinated GFP-E1 proteins are detected as a smear of high-molecular-weight species (lanes 1, 2, 4, and 5). _6xHisUb, 6His-ubiquitin. (B) COS-7 cells were transfected with the indicated plasmids. After 48 h, cells were lysed in the presence of MG132 and ubiquitin aldehyde. Cell lysates were immunoprecipitated with anti-E1 C-terminal (Cter) antibody or with preimmune antiserum (α-PI), and the Western blot was probed with anti-HA. Ubiquitin-E1 (Ub-E1) conjugates were detected in cells transfected with pEGFP-E1, pCGE2, pSKori⁺, and pCDNA₃-HAUb. _6xHAUb, HA-ubiquitin. Sizes are shown in kilodaltons.

To further demonstrate that these high-molecular-weight species contain ubiquitin, an immunoprecipitation-Western blot assay was used in which HA-tagged ubiquitin was cotransfected with the GFP-E1 plasmid into COS-7 cells, once again with or without the E2 and BPV origin-containing plasmids. Extracts were immunoprecipitated with an anti-E1 or control antibody, and the ligation of HA-tagged ubiquitin to E1 was evaluated by Western blotting with an antibody to the HA tag. As shown in Fig. 6B, a robust signal of polyubiquitinated E1 species was detected only when E2 and BPV origin-containing plasmids were present in COS-7 cells. These ubiquitination experiments, together with our inability to detect subtle changes in E1 levels without E2 and BPV origin-containing plasmids, indicate that conditions allowing viral replication facilitate the detection of ubiquitin-mediated E1 degradation within living cells.