Episomal maintenance of plasmids with hybrid origins in mouse cells

Abstract

Bovine papillomavirus type 1 (BPV1), Epstein-Barr virus (EBV), and human herpesvirus 8 genomes are stably maintained as episomes in dividing host cells during latent infection. The mitotic segregation/partitioning function of these episomes is dependent on single viral protein with specific DNA-binding activity and its multimeric binding sites in the viral genome. In this study we show that, in the presence of all essential viral trans factors, the segregation/partitioning elements from both BPV1 and EBV can provide the stable maintenance function to the mouse polyomavirus (PyV) core origin plasmids but fail to do so in the case of complete PyV origin. Our study is the first which follows BPV1 E2- and minichromosome maintenance element (MME)-dependent stable maintenance 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 minimal origin together with the MME or family of repeats (FR) element can be monitored easily for 1 month under nonselective conditions. Our data demonstrate clearly that the PyV LT-dependent replication function and the segregation/partitioning function of the BPV1 or EBV are compatible in certain, but not all, configurations. The quantitative 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 maintenance functions from different viruses are principally interexchangeable and can provide a segregation/partitioning function to different heterologous origins in a variety of cells.

FIG. 1.

Schematic representation of PyV hybrid origin constructs. (A) Schematic representation of the PyV wt genetic origin of replication comprising 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 partly overlapping tandem repeats at the opposite strands of the ori. All the plasmids were constructed using pUC19 as backbone as described in Materials and Methods. (B) Plasmids which share the PyV wt origin (enhancer element represented as open oval ring, core origin represented as filled rectangle) and in addition 1, 5, or 10 E2 BS (indicated as shadowed square; the numbers of E2 BS are indicated). (C) Constructs where the wt enhancer element is removed or replaced by E2 BS. (D) Reporter constructs which carry a eukaryotic selection cassette, such as the Geneticin resistance gene (indicated as open rectangles), which makes it possible to screen transfected cells in stable maintenance assays.

FIG. 2.

(A) Schematic representation of designed E2 point mutations and their properties in the BPV1 life cycle, which are described in more detail by Abroi et al. (1). (B) Western blot analysis of the expression of wt E2 protein (lanes 2 and 3) in constructed COP5 derivate cell lines COP5E2/Neo and COP5E2/Puro, respectively. Cells from semiconfluent 60-mm-diameter dishes were lysed in 100 μl of Laemmli sample buffer and one-third of the cell lysate was loaded in each lane. Negative-control lysate was prepared from COP5 cells (lane 4). The purified E2 protein expressed in bacteria was used as a positive control (lane 1). E2 protein-specific 3F12 antibody was used (23). (C) Comparison of the expression levels of wt and mutated E2 proteins from cell lines harvested after 2 months. E2 proteins were immunoprecipitated from lysates by E2-specific antibodies and protein G as described in Materials and Methods. According to Bradford assay results, 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 22 copies of the episomally replicating BPV1 genome per haploid genome was used as a reference (lane 5). Five and 10 ng of purified E2 protein were used as a positive control (lanes 6 and 7, respectively). Horseradish peroxidase-conjugated 5E11 monoclonal antibody, which recognizes E2 proteins, was used. Arrows indicate full-length E2 proteins and transcription repressor E2C.

FIG. 3.

Southern blot analysis. BPV1 E2 protein and its BS are required for stable maintenance of PyV origin plasmids. (A) Transient- and stable-replication properties of PyV chimeric plasmids in wt E2 protein-expressing cell line COP5E2/Neo. Episomal or total DNA was extracted from cells 4, 11, 21, and 34 days after transfection. For selecting the PyV origin containing cells from the total population, cotransfection with linearized vector pBabePuro and puromycin selection were used. Purified DNA was digested with restriction endonucleases HindIII and DpnI. Filters were probed with a radiolabeled PyV core origin and a plasmid containing 10 E2 BS. Three to 300 picograms of linear plasmid containing PyV core origin and 10 E2 BS was used as a marker. Transfected constructs are schematically represented at the top of the panel (lines 1 to 9, see also Fig. 1 for explanation). (B) In the cell line COP5 LT protein alone is not sufficient to provide the maintenance function to PyV origin-containing plasmids. Four and 19 days after transfection low-molecular-weight DNA was extracted and digested with restriction endonucleases HindIII and DpnI. Filters were probed with a radiolabeled probe corresponding to the PyV core origin and the plasmid containing 10 E2 BS. One to 300 picograms of linear plasmid containing PyV core origin and 10 E2 BS was used as a marker. Transfected constructs are schematically represented at the top of the panel (lines 1 to 9; see also the Fig. 1 legend for an explanation). For Southern blot analysis either material from approximately 500,000 cells (in the case of episomal DNA) or 2 μg of total DNA was analyzed.

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 HindIII and with DpnI, which digests bacterially methylated unreplicated input DNA, and analyzed by Southern blotting (lanes 1 to 3). Transfected plasmids are schematically represented at the top of the panel (see also Fig. 1D). Marker lanes contain 125, 250, or 500 pg of linearized plasmid, which contains the PyV core origin, 10 E2 BS, and the neomycin selection cassette. (B) E2 chromatin attachment function is required to provide the stable maintenance for the PyV core origin in conjunction with MME. Cell lines expressing LT, wt E2, or one of the mutant E2 proteins, R68A or E39A, were transfected with constructs which are schematically indicated at the top of the panel. After transfection 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 DpnI and analyzed by Southern blotting. Five hundred picograms of a linearized plasmid which contains the PyV core origin, 10 E2 BS, and Geneticin selection cassette was used as a marker.

FIG. 5.

The PyV core origin in conjunction with MME is stably maintained as the episome. (A) LT- and wt E2-expressing cells were transfected with plasmids indicated schematically at the top of the panel. After 2 months of growing cells without Geneticin, episomal DNA was extracted and analyzed using linearizing enzyme HindIII (lanes 1 and 4), with noncutter NdeI (lanes 2 and 5) and plasmid nicking enzyme Nb.Bpu10I together with noncutter enzyme NdeI (lanes 3 and 6). The plasmid containing the PyV core origin, 10 E2 BS, and a Geneticin selection cassette was used as 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 HindIII and 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 nicking enzyme Nb.Bpu10I (lane 12). Arrows indicate CCC, linear (Lin.), OC, and oligomerized forms of DNA. All restriction reaction mixtures contained 2 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, the PyV core origin, and a Geneticin selection cassette into the COPE2/Puro cell line, total DNA was extracted. Two micrograms of uncut total DNA was processed for a plasmid rescue assay as described in Materials and Methods. (B) In lanes 1 to 12, the uncut rescued plasmids are represented from 12 separate colonies. Lane M contains marker LambdaDNA/HindIII (Fermentas). (C) Analysis of rescued plasmids with endonuclease BglI.(lanes 1 to 12). Lane 13 represents BglI digestion of DNA extracted from the colony on the control plate (plasmid rescue assay with uncut total DNA from cells which carry the reporter plasmid with the wt PyV origin and Geneticin selection cassette). (D) BglI digestion fragments of rescued plasmids analyzed by Southern blot with an MME-specific probe (lanes 1 to 13). (E) BglI digestion fragments of rescued plasmids analyzed by 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.

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 were COP5E2/PuroMMEG (B), COP5E2/PuroMMEG* (C), and COP5EBNA1/PuroFRG* (D).

FIG. 7.

Southern blot analysis of the COP5E2/PuroMMEG* (A) and COP5EBNA/PuroFRG* (B) cell lines after removal of Geneticin selection. At the indicated times after removal of selection total DNA was extracted from cells and double-digested with DpnI and MluI (linearizes pMMEG* and pFRG* plasmids). A. Lanes 1 to 5 (from the left), 10 μg of total DNA from the COP5E2/PuroMMEG* cell line (24- to 336-h time point); lanes 6 to 10, marker plasmid pMMEG* (100 to 500 pg) linearized with MluI. B. Lanes 1 to 4, 3 μg of total DNA from the COP5EBNA/PuroFRG* cell line (24- to 228-h time point); lanes 5 to 7, marker plasmid pFRG* (50 to 150 pg) linearized with MluI. At the same time the decrease in the percentage of d1EGFP-expressing cells was monitored with fluorescence-activated cell sorter (presented under Southern blot analysis).