episomal maintenance of plasmids with hybrid origins in mouse cells

JOURNAL OF VIROLOGY, Dec. 2005, p. 15277–15288 Vol. 79, No. 240022-538X/05/$08.00�0 doi:10.1128/JVI.79.24.15277–15288.2005Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Episomal Maintenance of Plasmids with Hybrid Origins in Mouse CellsToomas Silla,1 Ingrid Haal,1 Jelizaveta Geimanen,1 Kadri Janikson,1 Aare Abroi,2

Ene Ustav,3 and Mart Ustav1,3*Department of Microbiology and Virology, Institute of Molecular and Cell Biology, Tartu University, 23 Riia St., Tartu 51010,

Estonia1; Estonian Biocentre, 23 Riia St., Tartu 51010, Estonia2; and Department of Biomedical Technology,Institute of Technology, Tartu University, 21 Vanemuise St., Tartu 51010, Estonia3

Received 22 June 2005/Accepted 29 September 2005

Bovine papillomavirus type 1 (BPV1), Epstein-Barr virus (EBV), and human herpesvirus 8 genomes are stablymaintained as episomes in dividing host cells during latent infection. The mitotic segregation/partitioning functionof these episomes is dependent on single viral protein with specific DNA-binding activity and its multimeric bindingsites in the viral genome. In this study we show that, in the presence of all essential viral trans factors, thesegregation/partitioning elements from both BPV1 and EBV can provide the stable maintenance function to themouse polyomavirus (PyV) core origin plasmids but fail to do so in the case of complete PyV origin. Our study isthe first which follows BPV1 E2- and minichromosome maintenance element (MME)-dependent stable mainte-nance function with heterologous replication origins. In mouse fibroblast cell lines expressing PyV large T antigen(LT) and either BPV1 E2 or EBV EBNA1, the long-term episomal replication of plasmids carrying the PyV minimalorigin together with the MME or family of repeats (FR) element can be monitored easily for 1 month undernonselective conditions. Our data demonstrate clearly that the PyV LT-dependent replication function and thesegregation/partitioning function of the BPV1 or EBV are compatible in certain, but not all, configurations. Thequantitative analysis indicates a loss rate of 6% per cell, doubling in the case of MME-dependent plasmids, and 13%in the case of FR-dependent plasmids in nonselective conditions. Our data clearly indicate that maintenancefunctions from different viruses are principally interexchangeable and can provide a segregation/partitioningfunction to different heterologous origins in a variety of cells.

Several eukaryotic DNA viruses maintain their genomes asextrachromosomal multicopy nuclear episomes in proliferatinghost cells. Such episomal maintenance is characteristic of latentinfection of bovine papillomavirus type 1 (BPV1), Epstein-Barrvirus (EBV), and Kaposi’s sarcoma-associated human herpesvi-rus 8 (HHV8). Two functions of the viral genome are abso-lutely critical for extrachromosomal maintenance in dividingcells: viral genome replication during the S phase and propersegregation and partitioning of the replicated genomes intodaughter cells during host cell mitosis. For BPV1 and twomembers of the gammaherpesvirus family, EBV and HHV8,effective segregation of viral genomes into daughter cells andnuclear retention during mitosis are mediated through a singleviral protein serving as a molecular linker, which attaches viralgenomes to the host mitotic chromosomes (4, 11, 12, 19, 24,35). This linker protein is viral regulatory protein E2 for BPV1(18, 24, 35), viral transactivator EBNA1 for EBV (19), andviral transcription repressor LANA1 for HHV8 (4, 5).

For the initiation of the DNA replication of a BPV1-basedreplicon in vivo, the minimal origin region in cis and two viralproteins, E1 and E2, in trans, are absolutely essential (39, 40).However, the minimal origin is not sufficient for stable extra-chromosomal replication in dividing cells (30). An additionalelement, the minichromosome maintenance element (MME),ensures the long-term episomal persistence of the genome inthe presence of viral E1 and the E2 proteins in the dividingcells (30). In the BPV1 genome, in total 17 E2 protein binding

sites (BS) with different affinities for E2 can be identified; 12 ofthese are located in the noncoding upstream regulatory region(URR) (26). We have shown that, for efficient partitioning/segregation of the episomal plasmid, MME activity is providedby a sufficient number of high-affinity E2 BS (30). The functionof multimeric E2 BS in the stable maintenance of the BPV1genomes is to provide the anchoring function for the E2 pro-tein, which therefore tethers MME-containing plasmids to mi-totic chromosomes (18, 24). This linkage between the BPV1genome and host chromatin ensures also that the viral genomeis maintained in the nucleus when the nuclear membrane isreassembled during mitosis. In the case of EBV, the stablemaintenance of replicated genomes is achieved due to theEBNA1 protein and family of repeats (FR) element, which iscomposed of multimeric EBNA1 protein binding sites (19, 27).

We have shown that both the BPV1 E2 protein-dependentMME (1) and EBV EBNA1-dependent FR (A. Mannik, K.Janikson, and M. Ustav, unpublished data) segregation/parti-tioning and chromatin attachment activities function indepen-dently from replication of the plasmids (18). The stable-mainte-nance function of EBNA1/FR has been used to ensure long-timeepisomal maintenance for non-OriP origins, usually the cellularreplication origins (22, 41). In the case of OriP, the enzymaticactivity required for initiation of replication is the same as incellular origins (14, 37). The E2/MME-dependent stable-main-tenance function has not been tested with heterologous repli-cation origins. In the present study, we have further examinedthe compatibility of viral segregation/partitioning elementswith heterologous replication origins. For this purpose, differ-ent reporter plasmids were constructed that combine theE2/MME- and EBNA1/FR-based stable-maintenance function

* Corresponding author. Mailing address: Department of Microbi-ology and Virology, Institute of Molecular and Cell Biology, TartuUniversity, Riia 23 St., Tartu 51010, Estonia. Phone: 372-7-375047.Fax: 372-7-420286. E-mail: [email protected].

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and different variants of the mouse polyomavirus (PyV) repli-cation origin. The mouse polyomavirus is a lytic virus, whichreplicates its DNA very fast during productive infection. In-fected cells contain up to 200,000 molecules of viral DNA, andthe maximal copy number is reached about 50 h postinfection(10). The replication origin of PyV contains the transcription/replication enhancer responsible for the high level of replica-tion (13). We tested the stable maintenance of plasmids thatcontained E2/MME in conjunction with the wild-type (wt) PyVor core (enhancerless) origin of replication in cell lines ex-pressing PyV large T antigen (LT) and E2 or its mutants. Alsothe stable maintenance of a plasmid containing the FR and thePyV core origin was tested in cell lines expressing LT andEBNA1. The results from these experiments show convincinglythat the segregation/partitioning functions of BPV1 and EBVcan effectively be used for stable episomal maintenance of thePyV core origin. In addition, efficient chromatin attachmentrather than a high level of activation of replication is requiredfor stable episomal maintenance.

MATERIALS AND METHODS

Plasmids. For constructing hybrid replicons (Fig. 1B and C) containing thePyV origin (wild type or core origin), we used vector pUC19 as the basic backbone,where we cloned 1, 5, or 10 head-to-tail copies of high-affinity E2 BS 9. PyV wt andthe core origin were amplified by PCR from vectors pmu1046/CAT andpmu1047/CAT (29) using primers Py4963 (5�-AGGGAGCTACTCCTGATG-3�)and Py174 (5�-CTACCACCACTCCGACTT-3�). Amplified PyV origin frag-ments were digested with enzymes EheI and BclI and inserted betweenBamHI and HincII sites of the pUC19 vector containing different numbers ofBPV1 E2 BS.

Hybrid replicons containing a Geneticin resistance gene (Fig. 1D) were estab-lished by replacing the URR in plasmid pNeoBgl40 (30) with PyV wt origin, coreorigin, or core origin with 10 E2 BS, which were amplified by PCR and digestedwith enzymes BamHI and Ecl136II and cloned into BamHI and HindIII sites inpNeoBgl40.

Three types of the enhanced green fluorescent protein (EGFP) marker containingplasmids were designed. First, a fragment comprising the PyV minimal origin and 10E2 BS was added to a plasmid containing a Geneticin resistance marker (expressedfrom the simian virus 40 promoter). Then either an EGFP or destabilized greenfluorescent protein (d1EGFP) marker was added (named either pMMEG orpMMEG* plasmid; see Fig. 6A). EGFP expression cassettes, which are under thecontrol of the cytomegalovirus promoter, were taken either from pEGFP-C1 orpd1EGFP-N1 plasmids (Clontech). For the third plasmid, the first EBV FR elementwas added to the pUC19 plasmid containing the PyV core origin. Then the fragmentcontaining the PyV minimal replication origin and 10 copies of E2 BS 9 fromplasmid pMMEG* was replaced by the fragment containing the PyV minimal originand EBV FR element (plasmid pFRG*; see Fig. 6A).

Construction of cell lines. For construction of cell lines which express BPV1 wtE2 protein and its mutant forms E39A and R68A, the vector pBabePuro (28) waslinearized using enzyme SalI and was ligated with an equal amount of E2expression vectors (pCGE2, pCGE2/R68, pCGE2/E39 [2, 39]), which were lin-earized with XhoI endonuclease. One microgram of ligated hybrid plasmids waselectroporated into the COP5 cell line (38). Electroporation experiments werepreformed with a Bio-Rad Gene Pulser with capacitance and voltage settings of975 �F and 220 V. For selection puromycin (2 �g/ml) was added. The expressionof the proteins was analyzed by Western blotting.

A cell line expressing wt E2 and carrying a Geneticin selection cassette wasconstructed by the same protocol described above using vector pBabeNeo (28)instead of pBabePuro.

A cell line expressing PyV T antigens and EBV EBNA1 protein was generatedas a result of transfection of the NotI-linearized plasmid pBabePuro/EBNA1(EBNA1 coding sequence inserted into EcoRI/SalI sites in the pBabePuro vec-tor) into the COP5 cell line and selection for puromycin (2 �g/ml). The expres-sion of the proteins was analyzed by Western blotting. The cell line was namedCOP5EBNA1/Puro.

Cells and transfection. COP5 cells (38) and their derivatives COP5E2/Puro,COP5E2/Neo, COP5R68/Puro, COP5E39/Puro, and COP5EBNA1/Puro ex-pressing PyV T antigens and BPV1 wt E2 or its mutant forms or EBNA1 weregrown in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with10% fetal calf serum. For selection Geneticin (500 �g/ml) or puromycin (2�g/ml) was added, depending on the selection marker. Electroporation experi-ments were performed with a Bio-Rad Gene Pulser with capacitance and voltagesettings of 975 �F and 220 V, respectively.

COP5E2/Puro cells transfected with neomycin constructs were selected withGeneticin at 500 �g/ml. COP5E2/Neo cells cotransfected with pBabePuro (28)

FIG. 1. Schematic representation of PyV hybrid origin constructs. (A) Schematic representation of the PyV wt genetic origin of replicationcomprising the enhancer sequence as a combination of binding sites for transcription activators, the physical origin for initiation of replication (ori),and element A, which contains three tandem large T-antigen binding sites. The ori contains four large T-antigen binding sites built as partlyoverlapping tandem repeats at the opposite strands of the ori. All the plasmids were constructed using pUC19 as backbone as described inMaterials and Methods. (B) Plasmids which share the PyV wt origin (enhancer element represented as open oval ring, core origin represented asfilled rectangle) and in addition 1, 5, or 10 E2 BS (indicated as shadowed square; the numbers of E2 BS are indicated). (C) Constructs where thewt enhancer element is removed or replaced by E2 BS. (D) Reporter constructs which carry a eukaryotic selection cassette, such as the Geneticinresistance gene (indicated as open rectangles), which makes it possible to screen transfected cells in stable maintenance assays.

15278 SILLA ET AL. J. VIROL.

were selected with puromycin at 2 �g/ml. After transfection with 500 ng ofplasmids carrying the Geneticin resistance marker and EGFP coding sequence,the COP5EBNA1/Puro cell line was grown in IMDM containing 500 �g/mlGeneticin (medium contained no puromycin).

Southern blot analysis. Total DNA was extracted from cells by following astandard protocol (3). Extraction of low-molecular-weight DNA from cells andanalysis of origin construct levels in both low-molecular-weight- and total-DNApreparations were performed as described previously (30, 39). All restriction reac-tions included DpnI to eliminate bacterially methylated input DNA. In the case ofthe nicking reaction 0.2 units of nicking enzyme Nb.Bpu10I (Fermentas, Vilnius,Lithuania) was added. During episomal- or total-DNA studies always equal numbersof cells or equal amounts of DNA were loaded onto each lane. Specific probes werelabeled with [32P]dCTP by random-decamer-primed synthesis using the DecaLabelkit (Fermentas). PyV origin- and MME-specific probes were made by PCR with[32P]dCTP. Hybridizing species were visualized by autoradiography. Radioactivesignals on the blots were quantified on PhosphorImagerSI using ImageQuantsoftware (Molecular Dynamics, Amersham Biosciences, Little Chalfont, UnitedKingdom).

Immunoprecipitation. Cells (1.5 � 107) were lysed with ice-cold 1% sodiumdodecyl sulfate (SDS)–phosphate-buffered saline on ice, collected in a 15-mltissue culture tube, and sonicated. From this step an aliquot for the Bradfordassay was taken. SDS was diluted to 0.1% by adding ice-cold radioimmunopre-cipitation assay (RIPA) buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1%NP-40, 0.5% deoxycholate, 0.1 mM dithiothreitol [DTT], 0.5 mM phenylmeth-ylsulfonyl fluoride, protease inhibitors). The insoluble fraction was sedimentedby centrifugation at 5,000 � g for 15 min. The soluble fraction was transferred toa new tube and incubated with 5H4, 3E8, 1E4, and 3F12 antibodies (22) over-night at 4°C. Then protein G-Sepharose (Amersham Biosciences) was added andincubated for 1 h. Sepharose beads were washed three times with RIPA bufferand resuspended in SDS loading buffer and subjected to immunoblotting analysiswith horseradish peroxidase-conjugated 5E11 (subclone of MAb 3F12) antibody(Quattromed AS, Tartu, Estonia).

Immunoblotting. Total protein from the same number of cells lysed in stan-dard loading buffer supplemented with 100 mM DTT was separated by electro-phoresis on an 8% polyacrylamide-SDS gel and transferred to an Immobilon-Pmembrane (Millipore). Antibody 1E4 (23) was used to detect E2 proteins.Antibodies BM3167 and BM1083 (DPC Biermann) were used to detect EBNA1protein. Peroxidase-conjugated goat anti-mouse antibody and the enhancedchemiluminescence detection kit (ECL Western blotting reagents; AmershamBiosciences) were used for subsequent development of the blot, using a standardprotocol provided by the supplier.

Plasmid rescue assay. Two micrograms of uncut genomic DNA was electro-transformed into Escherichia coli strain DH10B. The electrocompetent cells wereprepared as described previously (34), and the transformations were performedusing a Pulser apparatus and 2-mm electroporation cuvette (Bio-Rad Laborato-ries, Hercules, CA) according to the manufacturer�s instructions. The cells wererecovered by centrifugation and were grown on medium containing ampicillin at100 �g/ml. Plasmid DNA from single colonies was purified and analyzed usingrestriction endonucleases.

Flow cytometry analysis. EGFP expression was analyzed by flow cytometryusing a Becton Dickinson FACSCalibur flow cytometer with associatedCellQuest software. One hundred thousand to 200,000 signals were analyzedfrom each sample. The threshold for autofluorescence was set to 99% of thesignals from the mock-transfected control cells. All the signals above the thresh-old were considered to correspond to EGFP-positive cells. For calculating theepisomal rates of loss in Table 1, EGFP expression data were analyzed on days0 and 12 (pEGFP-C1 and pd1EGFP-N1), on days 0 and 55 for pMMEG, on days0 and 37 for pMMEG*, and on days 0 and 30 for pFRG* (day 0 is the time pointwhen selection was removed). For this calculation a first-order rate-of-loss modelwas used: rate of loss (�) � (�1/t)(ln Nt/N0) (41), where N0 is the percentage ofgreen cells at the beginning of the experiment at nonselective conditions and Nt

is the percentage of green cells after t generations.

RESULTS

BPV1 E2 protein and its multimeric binding sites activatethe replication of PyV core origin and provide the segregation/partitioning function to the origin plasmid. The BPV1 E2protein is a multifunctional protein which is involved in tran-scriptional regulation and viral DNA replication and segrega-tion. It has been shown that, for stable episomal replication of

BPV1, E1 and E2 proteins and the MME, which consists ofmultimeric E2 BS, are required (30). E2 protein can also ac-tivate the transient replication of the PyV core origin in vivo inE2 multimeric BS-dependent fashion (29). We decided tostudy if the BPV1 E2 BS in the hybrid PyV origin can, inaddition to the activation of the initiation of replication, alsoprovide the long-term maintenance function to the PyV-derivedreplicator in cells expressing LT and E2 protein.

Replication of the PyV origin requires LT as the only viralreplication factor; all other components are derived from thehost cell (9, 15). LT is an origin recognition factor and DNAhelicase, thus directly participating in initiation and elongationof the replication of the viral origin (43). We constructedmouse cell lines expressing PyV LT and the BPV1 E2 proteinusing the cell line COP5, which constitutively produces LTfrom the integrated replication-defective PyV genome (38).Individual colonies were allowed to expand in the presence ofselection (puromycin or Geneticin), and PyV LT- and BPV1E2-positive double-expression cell lines were identified andcharacterized. The cell lines expressing E2 protein at the high-est level were used in further assays (referred to as COP5/E2/Puro or COP5/E2/Neo, selected for puromycin or Geneticinselection markers, respectively). The same approach was usedfor construction of cell lines which express mutant forms of theE2 proteins, E39A and R68A (referred to as COP5/E39/Puroand COP5/R68/Puro). As described earlier, both these mu-tants are at least partially functional in E2 BS-dependent tran-scriptional activation and initiation of the BPV1 origin repli-cation as well as in activation of initiation of PyV core originreplication; however, they fail to attach to the host cell chro-mosomes and do not support segregation/partitioning of theMME plasmids (1, 2; A. Abroi et al., submitted for publica-tion). Expression of the wt E2 protein in the cell linesCOP5E2/Neo and COP5E2/Puro was verified using Westernblot analysis (Fig. 2B). In both cell lines the expression of thewt E2 protein was maintained at a detectable level for a pro-longed period without selection, which is essential for the studyof the maintenance of the episomal plasmids. We determinedthe expression level of E2 proteins in the constructed COP5E2/Puro, COP5E39/Puro, and COP5R68/Puro cell lines, as well asthe E2 expression level from C127 cells stably maintaining theBPV1 genome as an episome. Immunoprecipitation with E2-specific antibodies and following normalized immunoblottingshowed that the expression level of E2 proteins in constructed celllines is higher than the full-length-E2 expression level in theBPV1-transformed cell line (Fig. 2C). Thus, the E2 protein ex-pression level in our constructed cell lines is not limiting in stable-maintenance experiments.

The constructed cell lines were used in further experimentsto study the effect of the BPV1 E2 protein and E2 BS oninitiation of replication and maintenance of the constructedplasmids. At first, we examined the chimeric origins, compris-ing the PyV wt origin (Fig. 1B) or the core origin (Fig. 1C)linked to different numbers of E2 BS in cell lines COP5/E2/Neo (expressing constitutively PyV LT and BPV1 E2) andCOP5 (expressing constitutively PyV LT), respectively. Ninety-six hours after transfection strong replication signals of wtorigin plasmids were detected in both cell lines (Fig. 3A and B,4-day time points, lanes 1 to 4). Additional E2 BS had ratheran inhibitory effect on the replication of the wt PyV origin.

VOL. 79, 2005 MME AND FR ARE FUNCTIONAL IN HETEROLOGOUS SYSTEMS 15279

However, the E2 protein-dependent activation of replicationwas clearly detected in the cases when the PyV enhancerlesscore origin was linked to different numbers of E2 BS. Additionof one E2 BS had no effect on the initiation of replication ofthe core origin; however, the addition 5 or 10 E2 BS activatedcore origin replication to almost the wt origin replication levelin an E2 protein-dependent fashion (compare Fig. 3A and B,lanes 5 to 9). In the COP5 cell line lacking E2 protein, thereplication enhancer function of the E2 BS to the core origincannot be detected (Fig. 3B). These results are in principalagreement with the data previously published by Nilsson et al.(29), showing that the replication of the PyV enhancerlessorigin can be activated by BPV1 E2 and its BS.

We further studied the stable episomal maintenance of dif-ferent PyV origin-containing constructs in two experimentalsettings—first, without any selective pressure, and second, un-der puromycin selection—after cotransfection of the origin

FIG. 2. (A) Schematic representation of designed E2 point muta-tions and their properties in the BPV1 life cycle, which are describedin more detail by Abroi et al. (1). (B) Western blot analysis of theexpression of wt E2 protein (lanes 2 and 3) in constructed COP5derivate cell lines COP5E2/Neo and COP5E2/Puro, respectively. Cellsfrom semiconfluent 60-mm-diameter dishes were lysed in 100 �l ofLaemmli sample buffer and one-third of the cell lysate was loaded ineach lane. Negative-control lysate was prepared from COP5 cells (lane4). The purified E2 protein expressed in bacteria was used as a positivecontrol (lane 1). E2 protein-specific 3F12 antibody was used (23).(C) Comparison of the expression levels of wt and mutated E2 proteinsfrom cell lines harvested after 2 months. E2 proteins were immuno-precipitated from lysates by E2-specific antibodies and protein G asdescribed in Materials and Methods. According to Bradford assayresults, samples were normalized and analyzed by Western blotting.Lanes 1 to 3 represent signals of wt E2 and mutants E39A and R68A,respectively. Negative control was prepared from C127 cells (lane 4).To estimate E2 expression levels, the C127 cell line containing 22copies of the episomally replicating BPV1 genome per haploid genomewas used as a reference (lane 5). Five and 10 ng of purified E2 proteinwere used as a positive control (lanes 6 and 7, respectively). Horse-radish peroxidase-conjugated 5E11 monoclonal antibody, which rec-ognizes E2 proteins, was used. Arrows indicate full-length E2 proteinsand transcription repressor E2C.

FIG. 3. Southern blot analysis. BPV1 E2 protein and its BS arerequired for stable maintenance of PyV origin plasmids. (A) Tran-sient- and stable-replication properties of PyV chimeric plasmids in wtE2 protein-expressing cell line COP5E2/Neo. Episomal or total DNAwas extracted from cells 4, 11, 21, and 34 days after transfection. Forselecting the PyV origin containing cells from the total population,cotransfection with linearized vector pBabePuro and puromycin selec-tion were used. Purified DNA was digested with restriction endonucle-ases HindIII and DpnI. Filters were probed with a radiolabeled PyVcore origin and a plasmid containing 10 E2 BS. Three to 300 picogramsof linear plasmid containing PyV core origin and 10 E2 BS was used asa marker. Transfected constructs are schematically represented at thetop of the panel (lines 1 to 9, see also Fig. 1 for explanation). (B) In thecell line COP5 LT protein alone is not sufficient to provide the main-tenance function to PyV origin-containing plasmids. Four and 19 daysafter transfection low-molecular-weight DNA was extracted and di-gested with restriction endonucleases HindIII and DpnI. Filters wereprobed with a radiolabeled probe corresponding to the PyV core originand the plasmid containing 10 E2 BS. One to 300 picograms of linearplasmid containing PyV core origin and 10 E2 BS was used as amarker. Transfected constructs are schematically represented at thetop of the panel (lines 1 to 9; see also the Fig. 1 legend for anexplanation). For Southern blot analysis either material from approx-imately 500,000 cells (in the case of episomal DNA) or 2 �g of totalDNA was analyzed.


plasmids together with plasmid pBabePuro, which encodes thepuromycin resistance marker, to select out transfected cells(see Materials and Methods for details). The episomal persis-tence of the PyV origin-containing plasmids was analyzed bySouthern blotting. wt origin plasmids were lost from the cellsunder selective and nonselective conditions very fast in COP5E2/Neo and COP5 cells (Fig. 3A and B). However, the hybrid originscomprising the core origin and 5 or 10 E2 BS were capable oflong-term persistence (11 and 34 days, at least 27 doublings) inthe COP5E2/Neo cells, as analyzed by episomal DNA extractionor analysis of total DNA from the transfected cells. After 21 and34 days without selective pressure, the only origin construct thatwas efficiently maintained as an episome was the hybrid of thecore origin with 10 E2 BS (Fig. 3A, 21- and 34-day time pointswithout selection, lane 5). For the PyV origin with five E2 BS, aweak replication signal was detected only at longer exposure (datanot shown). We estimated the average copy number of the epi-somal plasmids in the culture using total-DNA Southern blotting(Fig. 3A, total DNA time points). The plasmid with 5 and 10 E2BS had, on average, 5 and 17 copies per cell, respectively, after 34days (with the assumption that all cells contain a replicon). Thesedata indicate that E2 and its BS can provide the episomal main-tenance function for chimeric PyV origin constructs that areotherwise lost from the cell population during cell growth.

Somewhat unexpectedly, we found that when the BPV1 seg-regation/partitioning element is linked to the PyV wt origin(short-term replication signals on Fig. 3A, 4-day time points,lanes 1 to 4) these replicons are not able to survive despite oftheir high-level replication (Fig. 3A, lanes 1 to 4). This could bedue to the overreplication of the intact enhancer-containing

origin plasmid, which could lead to cell death. Inspection of thetransfected culture indicated that indeed the wt PyV originplasmids induced extensive cell death at the later time points(data not shown).

Replication of the origin plasmids carrying an episomalselection marker. COP5E2/Puro cells were transfected withthree different plasmids carrying, in addition to the origin, aGeneticin selection marker (Fig. 1D). The eukaryotic selectioncassette in the plasmid makes it possible to select for cellscarrying reporter plasmids in transfected cells in the presenceof Geneticin. Transient transfection of COP5E2/Puro withneomycin reporter plasmids resulted in a strong replicationsignal for the wt origin construct (Fig. 4A, time points at 48 and72 h, lane 1) compared to a much lower replication signal forthe core origin construct (Fig. 4A, 48- and 72-h time points,lane 2). As expected, addition of 10 E2 BS to the core originincreased the transient-replication signal (Fig. 4A, 48- and 72-htime points, lane 3). The transfected cells were then grown inselective medium containing Geneticin, followed by a series ofcell divisions comparatively with and without selection. After 2months of cultivation, these pooled cell lines were analyzed forstable maintenance of the reporter plasmids using Southernblotting with a radioactively labeled probe against the PyVorigin. Ten E2 BS containing the reporter plasmid could es-tablish the extrachromosomal maintenance of autonomousepisomes in E2-positive cells (Fig. 4B, lanes 3, analysis after 2months). Removal of the selection reduced the replicationsignal, but it was still detectable in the episomal fraction after2 months (Fig. 4B, lanes 3, 2-month time points), and evenafter 5 months (data not shown).

FIG. 4. Efficient partitioning/segregation rather than high-level activation of replication is required for stable episomal maintenance.(A) Transient replication of neomycin selection cassette containing plasmids in cell lines expressing LT and wt E2 or one of its mutant forms,E39A or R68A. Low-molecular-weight DNA was extracted 48 and 72 h after transfection and digested with the single-cutting enzyme HindIIIand with DpnI, which digests bacterially methylated unreplicated input DNA, and analyzed by Southern blotting (lanes 1 to 3). Transfectedplasmids are schematically represented at the top of the panel (see also Fig. 1D). Marker lanes contain 125, 250, or 500 pg of linearizedplasmid, which contains the PyV core origin, 10 E2 BS, and the neomycin selection cassette. (B) E2 chromatin attachment function isrequired to provide the stable maintenance for the PyV core origin in conjunction with MME. Cell lines expressing LT, wt E2, or one ofthe mutant E2 proteins, R68A or E39A, were transfected with constructs which are schematically indicated at the top of the panel. Aftertransfection cells were grown in the presence (�) or absence (�) of Geneticin and analyzed for stable replication (lanes 1 to 3).Low-molecular-weight DNA was extracted 2 months after transfection and digested with the single-cutting enzyme HindIII and with DpnIand analyzed by Southern blotting. Five hundred picograms of a linearized plasmid which contains the PyV core origin, 10 E2 BS, andGeneticin selection cassette was used as a marker.


Efficient partitioning/segregation rather than a high level ofactivation of replication is required for stable episomal main-tenance. We compared the stable episomal maintenance of thehybrid origins in the cell lines expressing wt E2 with that in celllines expressing mutant forms of E2 carrying alanine substitutionsof the conserved charged residues in the N-terminal domain.These mutants have been previously characterized in BPV1 rep-lication, transactivation, sequence-specific DNA binding, and par-titioning assays (1, 2). E2 mutants E39A and R68A (Fig. 2A) areinactive in the chromatin attachment functions and failed to me-diate the segregation/partitioning of the BPV1 URR reporterplasmids but were still active in initiation of transient replicationand in transcription, where their relative activity was comparableto wt E2 (1). We transfected the COP5 cells with expressionconstructs for the BPV1 E2 mutant forms R68A or E39A aswell as a puromycin resistance marker; the puromycin-resistantclones were picked, expanded, and characterized for expres-sion of the desired proteins (Fig. 2C). The cells with the bestexpression were selected for the subsequent assays. In thefollowing short- and long-term replication assays, we used thereporter constructs carrying the selection marker that confersresistance to Geneticin selection (Fig. 1D). Both E2 mutantforms R68A and E39A activated PyV core origin replication inan E2 BS-dependent fashion in established cell lines (Fig. 4A,lane 3, 48- and 72-h time points). This suggested that E2mutant forms R68A and E39A behave as efficiently in repli-cation activation as wt E2 protein. The transfected cells weregrown in the media with and without Geneticin selection fortime periods up to 2 months. By this time, only the replicationof reporter plasmid with 10 E2 BS added to PyV the coreorigin was detectable in cells (Fig. 4B, lanes 1 to 3). In wtE2-expressing cells, this signal was present in Geneticin-se-lected cells as well as in control cells without selection. On theother hand, in the case of E2 mutant forms E39A and R68A,only a very weak replication signal was observed in cells grownunder Geneticin selection (Fig. 4B, lane 3). It is important tonote that further cultivation up to 5 months resulted in thecomplete loss of the episomal signal in mutant E2 cell lines(data not shown). The same results as in cell lines expressingmutant E2 proteins R68A and E39A were obtained from theexperiments with cell lines which express hybrid proteinsVP16/E2 and p53/E2, where the whole transactivation domainof the E2 protein is replaced with the respective activationdomain from VP16 or p53 protein, respectively (data notshown). These transactivation domains have been shown toactivate PyV replication very efficiently (6, 16; A. Abroi un-published data) and at least the VP16 activation domain doesnot support the plasmid partitioning function (1). These resultsshowed that the chromatin attachment function of E2 proteinis required to ensure stable maintenance of the chimeric PyVorigin and that the replication activation function alone is notsufficient for stable episomal maintenance.

Episomal state of chimeric origins. A high mutation frequency,especially for recombination, is often associated with replicationfrom the papillomavirus and polyomavirus origin-based vectorsystems (8, 42). Therefore, we decided to check for this possibilityin our experimental model. Episomal DNA was extracted fromCOP5/E2/Puro-derived cell lines to analyze the presence and thestatus of episomally maintained hybrid origin plasmids (Fig. 5A).Hybridization analysis with a neomycin gene-specific probe of the

linearized DNA from the PyV MME reporter-carrying cell linerevealed mostly one very discrete band that migrated similarly tothe unit size marker on the agarose gel (Fig. 5A, compare lane 1to lane 9). The sample digested with a noncutter (enzyme with norestriction sites in plasmid DNA) gave a pattern where opencircular (OC) and covalently closed circular (CCC) forms can bedetected (Fig. 5A, compare lane 2 with marker lanes 10 and 11).However, the additional slower-moving reporter-specific bandswere observed (Fig. 5A, lane 2 and 3). We suggest that the signalscorrespond to the oligomerized episomes; both forms have beenshown to appear, for example, during the episomal maintenanceof papillomavirus full-length genomes in several cell lines (32)and URR-containing plasmids in an E1/E2-positive cell line (30).To confirm that hybridization signals are not from integratedmaterial, the samples were digested with a noncutter enzymetogether with a nicking enzyme Nb.Bpu10I (Fig. 5A, lane 3).Nb.Bpu10I is a site- and strand-specific endonuclease that cleavesonly one strand of DNA within its recognition sequence on adouble-stranded DNA substrate. Thus, by nicking enzyme CCCDNA transfers to the OC form, a DNA mobility shift on agarosegel is observed (Fig. 5A, lane 12). Nb.Bpu10I does not changelinear DNA mobility as can be observed in Fig. 5A, lane 8, whichrepresents circular DNA digestion with a linearizing enzyme to-gether with a nicking enzyme. However, no hybridization signalwas observed on lanes containing the wt PyV origin (Fig. 5A,lanes 4 to 6). The results of this experiment show that the ana-lyzed episomal DNA fraction contained a reporter plasmid whichwas sensitive to the nicking enzyme so hybridization signals werenot from integrated material.

The presence of episomal DNA was also confirmed by plas-mid rescue into E. coli, using the uncut total DNA from the E2-and LT-expressing cells that were carrying a reporter plasmidwith the PyV core origin and 10 E2 BS. Analysis of uncutrescued plasmids showed an oligomerized pattern compared toinput DNA (Fig. 5B). Restriction analysis of rescued plasmidsby endonuclease BglI showed that, compared to input DNAsome rearrangements in the plasmid backbone can be ob-served (Fig. 5C, lanes 1 to 3, 5, 6, 10, and 12). Thus, some cellscarried plasmids with rearrangements. In addition, intact un-arranged DNA forms were also detected (Fig. 5C, lanes 4, 7 to9, and 11). To confirm that rescued plasmids still contain thePyV origin and MME, we analyzed the BglI digestion patternby Southern blot analysis with an MME- or PyV-specific probe(Fig. 5D and E, respectively). Southern blot analysis showedthat all rescued reporter plasmids contained the MME andPyV origin fragment (Fig. 5D and E). A plasmid rescue assaywith total DNA (total DNA was extracted from cells whoseepisomal DNA is analyzed in Fig. 5A, lanes 4 to 6) from cellscarrying the reporter plasmid with the wt PyV origin revealedonly one colony, which we analyzed for the existence of theMME or PyV origin fragment (Fig. 5D and E, lanes 13).Southern blot analysis indicated that plasmid DNA from thiscolony did not contain MME or the PyV origin (Fig. 5D and E,compare lane 13 with lanes wt input and input). The results ofthese experiments strongly suggest that the cell lines we ana-lyzed carry the input vectors as an extrachromosomal element.

Measurement of the episomal plasmid loss using flow cyto-metry analysis. Maintenance of plasmids containing the PyVcore origin, MME, selection marker (Geneticin resistance),and green fluorescent protein marker (either long-half-life


EGFP or short-half-life d1EGFP) was analyzed by flow cytom-etry. Transfection of these plasmids (schematically presentedin Fig. 6A), into the COP5E2/Puro cell line resulted in efficienttransient replication of these plasmids, which could be de-tected by Southern blot analysis (data not shown) as well asfollowed indirectly by measuring the fluorescence of plasmid-encoded EGFP. Two different variants of the EGFP proteinmarker were used comparatively to avoid potential problemscoming from by-fluorescence of long-half-life EGFP in thecase of short-term experiments. Transfected cells were grownin continuous culture in the presence or absence of Geneticinfor up to 96 days. The cells were passaged every second day(every day when grown without selection), assuring active celldivision. During each passage 100,000 to 200,000 cells weretaken for analysis and the proportion of EGFP-positive cellswas measured by flow cytometry. The percentage of cells(COP5E2/Puro) expressing EGFP above background (the fluo-rescence signal in the EGFP detection channel is higher thanthe cellular autofluorescence) was calculated for each trans-fected cell culture at each time point. Without Geneticin se-lection the percentage of the EGFP-fluorescent cells decreasedquite rapidly. Eleven days after transfection without selectionfew EGFP-positive cells could be detected using fluorescence-activated cell sorter analysis compared to the initial approxi-mately 50% EGFP-positive cells (Fig. 6B and C). Selection of

the COP5E2/Puro cells transfected with the plasmid carryingthe Geneticin resistance marker resulted in a cell culture whichhad nearly 100% EGFP-positive cells in the case of the plasmidexpressing long-half-life EGFP and approximately 50% whenthe plasmid expressed short-half-life d1EGFP (Fig. 6B and C).The percentage of EGFP-positive cells stayed constant formore than 20 cell generations, indicating that these cells arecapable of long-term maintenance of episomal genetic ele-ments that contain the PyV core origin and MME. When theGeneticin selection was removed, the percentage of EGFP-positive cells decreased from 90% to approximately 1% in 55days (from 64% to 2.4% in the case of d1EGFP in 37 days). Inthe case of integration of the episome the percentage of theEGFP-fluorescent cells remains constant even when the selec-tion is removed (41). In order to characterize the kinetics ofloss of the episomes, the rate of loss for each episomal con-struct during nonselective conditions was calculated for twoindependent experiments (Table 1, series 1 and 2). Two con-trol plasmids, pEGFP-C1 and pd1EGFP-N1 (control plasmidsfrom Clontech lacking the replication origin and MME), wereused in the flow cytometry study to provide a comparison to thenormal rate of loss of the episomes in the COP5E2/Puro cellline. After COP5E2/PuroMMEG cells were grown for 55 daysand COP5E2/PuroMMEG* cells for 37 days without selection,1% of the cells still contained the episome, as indicated by the

FIG. 5. The PyV core origin in conjunction with MME is stably maintained as the episome. (A) LT- and wt E2-expressing cells were transfectedwith plasmids indicated schematically at the top of the panel. After 2 months of growing cells without Geneticin, episomal DNA was extracted andanalyzed using linearizing enzyme HindIII (lanes 1 and 4), with noncutter NdeI (lanes 2 and 5) and plasmid nicking enzyme Nb.Bpu10I togetherwith noncutter enzyme NdeI (lanes 3 and 6). The plasmid containing the PyV core origin, 10 E2 BS, and a Geneticin selection cassette was usedas the marker (100 pg in each of lanes 7 to 12) and is represented in linearized form (lane 7), circular form digested with linearizing enzyme HindIIIand nicking enzyme Nb.Bpu10I (lane 8), circular form digested with linearizing enzyme HindIII in the presence of COP5E2/Puro episomal DNA(lane 9), noncut forms (lane 10), circular forms digested with noncutter NdeI (lane 11), and circular form digested with noncutter NdeI and nickingenzyme Nb.Bpu10I (lane 12). Arrows indicate CCC, linear (Lin.), OC, and oligomerized forms of DNA. All restriction reaction mixtures contained2 units of DpnI. (B to E) Plasmid rescue analysis of the COP5E2/Puro cell line. Two months after transfecting a plasmid containing 10 E2 BS, thePyV core origin, and a Geneticin selection cassette into the COPE2/Puro cell line, total DNA was extracted. Two micrograms of uncut total DNAwas processed for a plasmid rescue assay as described in Materials and Methods. (B) In lanes 1 to 12, the uncut rescued plasmids are representedfrom 12 separate colonies. Lane M contains marker LambdaDNA/HindIII (Fermentas). (C) Analysis of rescued plasmids with endonucleaseBglI.(lanes 1 to 12). Lane 13 represents BglI digestion of DNA extracted from the colony on the control plate (plasmid rescue assay with uncuttotal DNA from cells which carry the reporter plasmid with the wt PyV origin and Geneticin selection cassette). (D) BglI digestion fragments ofrescued plasmids analyzed by Southern blot with an MME-specific probe (lanes 1 to 13). (E) BglI digestion fragments of rescued plasmids analyzedby Southern blot with a PyV origin-specific probe (lanes 1 to 13). Input or wt input lanes contain the plasmid with the PyV core origin, 10 E2 BS,and Geneticin selection cassette or the plasmid with the PyV wt origin and Geneticin selection cassette, respectively.


flow cytometry analysis. The reapplication of Geneticin selec-tion at this point soon restored the proportion of EGFP-ex-pressing cells to the initial level (Fig. 6B and C).

Comparison of the segregation/partitioning effects providedby the BPV1 MME and EBV FR elements to the PyV coreorigin plasmid. To compare the effects of BPV1 MME andEBV FR-based elements on the segregation/partitioning of thePyV replication origin construct, we constructed the EBNA1-expressing COP5 cell line and PyV core origin and FR-con-taining reporter plasmid that was similar to those used in theE2/MME analysis described above (Fig. 6A). EBNA1 activatedPyV core origin replication in an FR-dependent manner (datanot shown). The long-term maintenance of a transfected re-porter plasmid (pFRG*) containing the PyV core origin, FRelement, Geneticin selection marker, and expression cassette

for d1EGFP was monitored by flow cytometry. In this case, thereplication function of the plasmid is provided by the PyV coreorigin and LT protein and the segregation/partitioning func-tion is provided by the FR element and EBNA1 protein.Transfected cells were grown in continuous culture in the pres-ence or absence of Geneticin for up to 75 days. Selection of thetransfected COP5EBNA1/PuroFRG* for Geneticin resultedin a cell culture which had approximately 40% d1EGFP-posi-tive cells (Fig. 6D). When the Geneticin selection was re-moved, the percentage of d1EGFP-positive cells decreasedfrom 40% to 1% in 30 days. When Geneticin selection on theCOP5E2/PuropFRG* cell line was restored at this point, theproportion of EGFP-expressing cells increased back to the initiallevel (Fig. 6D). These results are, in principle, identical tothose obtained from the similar experiments with the E2/MME-dependent segregation/partitioning system described inthe previous section (Fig. 6B and C). Therefore, EBNA1/FRelements and E2/MMEs confer comparable segregation/parti-tioning functions on the PyV core origin reporter plasmids inthe analyzed cell model.

To exclude the possibility that the loss of EGFP fluorescenceis due to inactivation of the promoter of EGFP, we also ana-lyzed the DNA content in the cells. After removal of Geneticinselection total DNA was extracted from cells and digested withMluI (linearizes pMMEG* and pFRG* plasmids) and DpnI.Equal amounts of total DNA were then analyzed using South-ern blotting with a radioactively labeled probe against thepMMEG* or pFRG* plasmid. As presented in Fig. 7 the lossof the episomal plasmid DNA from the cells grown withoutGeneticin selection correlates with the flow cytometry analysis.On the other hand, these results indicate that EGFP fluores-cence was indeed measured from plasmids which exist in the

FIG. 6. (A) Schematic representation of PyV hybrid origin constructs used in flow cytometry analysis. (B to D) Time course of long-term EGFP(B) or short-term d1EGFP (C and D) expression in the presence or absence of Geneticin selection for various cell lines. Cell lines used wereCOP5E2/PuroMMEG (B), COP5E2/PuroMMEG* (C), and COP5EBNA1/PuroFRG* (D).

TABLE 1. Rate of plasmid loss calculated from the data in Fig. 6 andthe rates observed for two control plasmids lacking replication

origins (pEGFP-C1 and pd1EGFP-N1; Clontech)a

Construct Series Rate of loss per cellgeneration (%)

pMMEG 1 5.5pMMEG 2 6.5pMMEG* 1 6.6pMMEG* 2 5.9pFRG* 1 13.1pFRG* 2 13.6

Control plasmidspEGFP-C1 21.7pdEGFP-N1 29.8

a The experiment was performed twice (series 1 and 2).


episomal state. In the case of plasmid integration the hybrid-ization signals remains constant.

DISCUSSION

The E2/MME works efficiently as a partitioning/segregationdeterminant also with a heterologous replicon. Prior to thisstudy the only episomal maintenance element tested togetherwith its nonnative replication origin was EBNA1/FR linked todifferent cellular replication origins. In these cases as well as inthe case of replication of EBV OriP all the enzymatic activitiesrequired for replication are provided by the host cell. We havedeveloped a system to study the mechanism of stable replica-tion of a plasmid containing different maintenance elements incombination with their nonnative origins. More specifically, wehave examined the functioning of E2/MME and EBNA1/FRmaintenance elements in conjunction with the PyV replicon inthe present study. As shown on Fig. 3A, the establishment ofstable maintenance in this model depends on the simultaneouspresence of E2 and MME. The plasmid containing 10 E2 BSand the PyV core origin is still present in the cell population 1month after the initial enrichment of the transfected-reporter-carrying population based on antibiotic resistance selection.The same plasmid is efficiently maintained in the cells even ifno selection is applied. Analysis of episomal DNA with nickingenzyme Nb.Bpu10I and rescue onto bacteria (Fig. 5) indicatethat at least a majority of the stably maintained plasmids arenot integrated and exist in an episomal state in the cells. South-ern blot analysis and plasmid rescue assay indicated that somecells carry an oligomerized reporter plasmid (Fig. 5). Analysisof rescued plasmids showed that, in roughly one-half of theanalyzed colonies, some plasmid rearrangement occurred(Fig. 5C). It has been shown that spontaneous sequence dele-tion by homologous recombination in the bacterial cell mayoccur (46). We have also noticed that highly palindromic se-quences, like those of E2 BS-containing plasmids, are unstableduring bacterial manipulations (A. Abroi, I. Ilves, and K. Jan-ikson, personal communications). Whether these rearrange-

ments in our present experiment originated in eukaryotic or inbacterial cells remains unclear; it is, however, important tonote that all rearranged plasmids still carried the PyV minimalorigin and BPV1 maintenance element (Fig. 5D and E). Inaddition, a control plasmid rescue assay with total DNA fromcells carrying reporter plasmids with the wt PyV origin re-vealed only one colony whose low-molecular-weight DNA didnot contain an MME or PyV origin (Fig. 5D and E). Thus, theE2/MME provides the stable episomal maintenance functionnot only to a BPV1-based replicon but also to a PyV-basedreplicon. Our study shows that mitotic chromatin attachmentdeterminants of both BPV1 and EBV can provide the stableepisomal maintenance function to heterologous viral repliconsin addition to their native ones.

The episomally maintained reporter plasmids analyzed inour study do not cause major growth disadvantages for trans-fected cells and have a relatively low loss rate (Fig. 3 andTable 1). Previous studies have shown that, in the establishedcell lines carrying OriP, the plasmid loss is 2 to 8% per cellgeneration. However, the average plasmid copy number ofOriP decreases more than 100-fold during the first 2 weeksafter transfection into the cells expressing EBNA1 (25). In oursystem, certainly some decrease in the average copy numbercan be observed, but definitely not as fast. The quantitativeaspects of the establishment of BPV1 stable episomal mainte-nance were not addressed in the present study. However, ourdata suggest that this process may be even more effective thanin the case of EBNA1/OriP (25).

Overreplication of the wt PyV origin disrupts the establish-ment of stable maintenance. On the other hand, E2/MMEcannot provide stable episomal maintenance to plasmids withthe PyV wt origin, even under selective pressure (Fig. 3 and4B). PyV exhibits a replication pattern that is uncoupled fromthe regulatory mechanisms of the host cell, so that each viralgenome replicates many times within each cell cycle. The com-plete PyV origin includes transcriptional and replicational en-hancer sequences, which dictate the origin activity and theefficiency of replication in specific cells by determining theavailability of the replication factors and nucleotides. Papillo-mavirus origin replication control is similar to PyV replicationin the first, amplificational phase of replication. However, inthe latent-replication phase the copy number control mecha-nism is applied, which assures the controlled initiation of rep-lication of the episomal viral genome in the latent-replicationphase. We show that replacement of the wt PyV enhancer with5 or 10 synthetic BS for the BPV1 E2 protein can replacereplication enhancer function and makes it dependent on E2protein. These results are in accordance with earlier reports byNilsson et al. (29) and Abroi et al. (submitted) that at least twoE2 BS are required to activate the PyV core origin. It is inter-esting to note that adding 5 or 10 E2 BS to the PyV wt origindid not cause additional replication activation. This fact sup-ports the idea that replication from the episomal viral replica-tion origins has a certain maximal threshold level in the hostcell, which can be achieved by the presence (or addition) ofstrong enhancer elements. Further enhancement of the repli-cation is not possible, even if more enhancer elements areadded. It could be explained by the limiting levels of cellularreplication factors or by the saturation of the nucleus withactive genetic elements in the form of replication intermedi-

FIG. 7. Southern blot analysis of the COP5E2/PuroMMEG* (A) andCOP5EBNA/PuroFRG* (B) cell lines after removal of Geneticin selec-tion. At the indicated times after removal of selection total DNA wasextracted from cells and double-digested with DpnI and MluI (linearizespMMEG* and pFRG* plasmids). A. Lanes 1 to 5 (from the left), 10 �gof total DNA from the COP5E2/PuroMMEG* cell line (24- to 336-h timepoint); lanes 6 to 10, marker plasmid pMMEG* (100 to 500 pg) linearizedwith MluI. B. Lanes 1 to 4, 3 �g of total DNA from the COP5EBNA/PuroFRG* cell line (24- to 228-h time point); lanes 5 to 7, markerplasmid pFRG* (50 to 150 pg) linearized with MluI. At the same timethe decrease in the percentage of d1EGFP-expressing cells was mon-itored with fluorescence-activated cell sorter (presented under South-ern blot analysis).


ates. This observation is also supported by the replication ki-netics data, showing that the maximal level of replication bythe wt PyV origin is achieved already �24 h posttransfectionand that there is no more increase later; rather some decreaseis observed (A. Abroi, unpublished data). At the same time,the replication signal of the PyV core origin or E2-dependentcore origin increases in time. The toxic effect of the overrep-lication of the episomes on the cell can be suggested, as weobserved many floating dead cells after transfection with PyVwt origin constructs. Thus, even though additional experimen-tal data are needed to clarify this point, the most likely expla-nation for the inability of E2/MME to provide stable episomalmaintenance to PyV wt origins is the cellular response againstthe high level of replication intermediates and/or the high levelof replication itself.

The stable maintenance element from an EBV replicon thatreplicates strictly once per cell cycle can confer stable episomalmaintenance properties to replication origin constructs derivedfrom lytically replicating virus. The replication origins of BPV1and PyV are fired several times during their amplificationalreplication in the single S phase of the host cell cycle, and therespective initiator proteins, E1 and LT, have many biochem-ical and structural similarities. However, these viruses havedifferent time courses of productive infection. PyV is a lyticvirus, and thus the viral DNA does not need to be stablymaintained. During the stable replication of the BPV1 genomeor URR, the origin is not restricted to precisely once replica-tion round in each cell cycle (30, 31, 36). At the same time theEBV latent origin OriP replicates strictly once per cell cycle,exactly the same way as chromosomal DNA. Thus, in theseterms, the replication modes of PyV and OriP are completelydifferent. As shown on Fig. 6, the BPV1 E2/MME and EBVEBNA1/FR element can provide a stable maintenance func-tion to the PyV core origin plasmids in the presence of viraltrans factors. Our data presented here suggest that stable main-tenance of the episomes provided by the function of MMEor the FR element is not connected to the mode of replicationof the episome. The FR element can provide a stable mainte-nance function to several types of origins, in its natural contextwithin the EBV latent origin OriP, in the plasmids where thechromosomal origin of replication from cellular DNA is linkedto the FR element, and in our hybrid replicon together with thePyV core origin (22, 41). These data also show that the repli-cation function is not connected to the stable-maintenancefunction of the virus; replication origins of different viruses canbe combined with heterologous stable-maintenance elementswithout the loss of either function. The cellular receptors ofBPV1 E2 protein and EBV EBNA1 protein, which link theepisomes to mitotic host chromatin and therefore provide thestable-maintenance function, are different (20, 21, 33, 44, 45,47). And, most likely, E2/MME- and EBNA1/FR-dependentplasmids are localized on chromosomes in different places.Our data presented here indicate that the different localiza-tions of the episome on mitotic chromosomes do not interferewith the replication of the PyV minimal replication origin.

The chromatin attachment/partitioning function, not activa-tion of replication, is responsible for stable episomal mainte-nance of a heterologous origin. Structural intactness of the E2protein is very important in order to provide the MME-depen-dent partitioning function. A recent study from our laboratory

showed that single point mutations might affect the chromatinattachment of E2 protein or its ability to mediate chromatintethering of URR reporters even if the effect on replicationinitiation or transcription activation is relatively modest (1). E2mutant proteins E39A and R68A were used to analyze the roleof the transcription activation properties of the E2 protein inits functioning as a trans factor for stable episomal mainte-nance of a PyV-derived replicon. These E2 mutants wereshown to be inactive in chromatin attachment, URR plasmidtethering, and segregation/partitioning assays in our studiesusing Chinese hamster ovary cells (1); they were, however,functional in Brd4 binding and chromatin binding assays usingCV1 cells derived from African green monkey kidney cells (7).These mutant E2 proteins though seemed to be nonfunctionalin supporting long-term episomal maintenance of the hybridMME/PyV core ori plasmids in C127 mouse cells, as shown inthis paper. The apparent contradiction in the features of theseE2 mutants may be attributed to the difference in the speciesof cells used to measure the functions of the E2 mutants. Theinteraction of these E2 mutants could be different with mouse,hamster, and monkey Brd4 proteins, which could be due to thedifference in the sequences between Brd4 proteins of the dif-ferent species. The cell lines used in our assays have beenshown to be functional in supporting BPV1 origin replication,segregation/partitioning, and long-term maintenance of episo-mal replication, which is not necessarily the case with CV1 cells.

In addition, we tested the stable replication of the E2/MME-dependent PyV replicon also in cell lines expressing LT andVP16-E2 or p53-E2 (containing the activation domains fromVP16 and p53, respectively). They both are very potent trans-activators and capable of activating the replication of the PyVcore origin in transient assays. However, none of these mutantor hybrid E2 variants was capable of ensuring effective stableepisomal maintenance, in the case of VP16-E2 and p53-E2 noteven under selective conditions (Fig. 4B and data not shown).Our results show clearly that chromatin attachment, but nottransactivation and the consequent replication initiation activ-ity of E2 protein, is essential to provide stable maintenance forchimeric constructs used in this study and that random parti-tioning of the episomal plasmids cannot provide a reliablemechanism for stable episomal maintenance of the plasmidseven in the presence of selection for the episomal selectionmarker. These results suggest that MME-mediated partition-ing in conjunction with the PyV origin or its natural BPV1origin is achieved by using the same strategy, i.e., throughchromatin attachment.

Rate of loss of episomal plasmids. In the present study wehave analyzed the episomal maintenance of plasmids contain-ing the PyV minimal replication origin and either BPV1 MMEor the EBV FR element (Fig. 6A) in cells where the appro-priate viral trans factors (either PyV LT and BPV1 E2 or PyVLT and EBV EBNA1 protein) were stably expressed. In thecase of plasmids containing the PyV minimal replication originand BPV1 MME, the rate of episomal loss was �6% per celldivision in the absence of selection. For plasmids containingthe PyV minimal origin and EBV FR element, the rate ofepisomal plasmid loss was higher (�13%), but it is still signif-icantly lower than 22 to 30%, which we observed in the case ofcontrol plasmids (pEGFP-C1 and pd1EGFP-N1) without aeukaryotic replication origin and segregation elements. The


rate of loss of plasmids containing the PyV minimal replicationorigin and FR element (pFRG*) is also different from thepreviously published results on the rate of loss of several rep-licating plasmids that contained the FR element as a stablemaintenance factor, where it was 2.1 to 7.8% (41); however, itis very similar to the 15% rate of loss previously estimated forOriP-containing plasmids (17). The difference in the rate ofloss may be due to the differences in the expression level ofEBNA1, the configuration of the test plasmids used, or thenature of the chromatin receptor for EBNA1, because in ourexperiments the mouse cell line COP5, not human cells, wasused. The difference between the d1EGFP-positive and EGFP-positive cells in cell culture grown under Geneticin selection isprobably due to the sensitivity of detection. As the half-life ofthe d1EGFP protein is 1 hour and as d1EGFP does not accu-mulate in the cells, the level of d1EGFP in cells with lowerreporter plasmid copy number may probably be insufficient forits detection from the autofluorescence background and thus acertain fraction of cells which in fact carry the reporter willprobably be regarded as “EGFP negative”. However, the factthat both long- and short-half-life EGFP reporters gave similarrates of loss indicates that our method for measuring the rateof plasmid loss is adequate.

In conclusion, all these data together indicate that the main-tenance elements from different DNA viruses are interchange-able with each other and can work in conjunction with differentreplicons, even with those from lytically replicating viruses.However, in heterologous systems as well as in native configu-rations, a certain loss of plasmid exists. In order to compensatethe loss of episomes, viruses have evolved systems to acceleratethe host cell proliferation compared to uninfected cells.

ACKNOWLEDGMENTS

We thank Anne Kalling for technical assistance, Ivar Ilves for care-ful reading of the manuscript, Kertu Runkorg for constructing theCOP5E2/Neo cell line, Michael Botchan for VP16-E2 expression con-struct, Mari Sepp for the BPV1-transfected C127 cell line, and GoranMagnusson for the COP5 cell line and PyV origin constructs.

This study was supported in part by grants 4475, 4476, 5999, and5998 from the Estonian Science Foundation, grant INTNL 55000339from the Howard Hughes Medical Institute, grant CT96-0918 from theEuropean Union, and target financial project 0182566s03.

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