JOURNAL OF VIROLOGY, June 1991, p. 3029-3043

Vol. 65, No. 6

0022-538X/91/063029-15$02.00/0 Copyright C 1991, American Society for Microbiology

Host Range Specificity of Polyomavirus EC Mutants in Mouse Embryonal Carcinoma and Embryonal Stem Cells and Preimplantation Embryos FRANQOISE MELIN,1 ROLF KEMLER,2 CHANTAL KRESS,1t HUBERT PINON,lt

AND

DANIEL BLANGYl*

Laboratoire Virus et DiffJrenciation de l'Universite Pierre et Marie Curie, UPR 272, Centre National de la Recherche Scientifique, Institut de Recherches Scientifiques sur le Cancer, BP 8, 94801 Villejuif Cedex, France,' and Arbeitsgruppe Molekulare Embryologie, Max-Planck-Institut fur Immunologie, D-7800 Freiburg im Breisgau, Germany2 Received 14 September 1990/Accepted 22 February 1991

New polyomavirus mutants (PyEC-C) selected on LT1 cells and exhibiting a strong cytopathic effect in all embryonal carcinoma (EC) cell lines tested have been isolated. They were derived by a sequence duplication event from a new multiadapted mutant isolated in PCC4 cells. A quantitative analysis of viral DNA replication and transcription in 3T6 and EC cell lines was performed to compare PyEC-C mutants and PyEC mutants previously isolated on F9 or PCC4 cell lines. Analysis of the results indicated that PyEC-C mutants were more efficient in all EC cell lines tested than all other PyEC mutants; on the contrary, they were less adapted to 3T6 cells than wild-type polyomavirus. In both 3T6 and EC cells, uncoupling between early transcription and viral DNA replication was observed; different viruses were shown to replicate with the same efficiency, while their levels of early transcripts differed by two orders of magnitude. Attempts to correlate the genome structure of the mutants with their biological properties indicate that duplication of protein-binding sequences is not the only event responsible for their phenotype. PyEC mutants were also analyzed with respect to their interactions with early mouse embryos and embryonal stem (ES) cell lines derived from the inner cell mass of blastocysts. They showed different degrees of expression in ES cells and preimplantation embryos. ES cells were most efficiently infected and lysed by mutants which exhibit both a multiadapted and a lytic phenotype in EC cells. Preimplantation embryos were not permissive to any PyEC mutants. However, EC-multiadapted mutants were infectious in blastocysts after two days of in vitro culture. tions suggest that control of PyV early gene expression is dependent on a complex interplay between positive and negative regulatory factors and on the availability of these factors in each EC cell line. This suggestion is further substantiated by the fact that mutants selected on PCC4 cells all share similar genomic rearrangements which are different from those of mutants selected on F9 cells (28). We have reported that a mutant isolated on PCC4 cells, PyEC-M206, had acquired a multiadapted phenotype and expressed T antigen in all EC cell lines tested, including the F9 cell line; still, it did not harbor the characteristic PyEC-F9 rearrangements (27). Although it displayed an extended host range with regard to early viral gene expression, this mutant was unable to produce a visible lytic effect in EC cells; this result indicated that additional or different mutations might be required to obtain viruses which would be as efficient in EC cells as wt PyV in differentiated mouse fibroblasts. On the other hand, our attempts to isolate PyEC mutants on LT1 cells infected with wt A2 or evl001 PyV (22), from which all previous mutants were derived, were always unsuccessful. We inferred that more extensive rearrangements would be necessary to generate such mutants, and we undertook their selection according to the previously described procedure (27) with two major modifications. (i) LT1 cells were infected with PyEC multiadapted mutants isolated from evl001-infected PCC4 cells. (ii) Viruses and viral DNAs were initially propagated exclusively in LT1 cells in order to prevent a possible counterselection of mutants upon infection of secondary mouse embryo cells which were used previously to prepare viral stocks and viral DNA.

The inability of polyomavirus (PyV) to develop in embryonal carcinoma (EC) cells has been shown to result from a block at the levels of early transcription (2) and DNA replication (7). The viral noncoding regulatory region is composed of a mosaic of cis-acting sequences which can interact with several DNA binding proteins (for a review, see reference 15). Whether these proteins are involved in the control of viral replication and/or transcription is largely unknown, since most of them were characterized only by gel retardation assays, DNase I footprinting, or methylation interference experiments. In F9 and PCC4 cells, two of these factors, PEAl (AP-1/Jun) and PEA2, which interact with the A regulatory domain, are not detectable, but they are induced upon differentiation as these cells become permissive to wild-type (wt) PyV (20), and a third factor interacting with the same A domain, PEA3, is detected in both F9 (42) and differentiated (24) cell extracts. On the other hand, a single point mutation in the B enhancing element (mutant PyEC-F9-1 in Fig. 1) relieves transcriptional inhibition mediated by an adenovirus type 5 ElA-like activity present in F9 cells (11) and creates a binding site for another factor (19), first called GTII-C (46) and then, after isolation from HeLa cells, called TEF-1 (3). However, viral adaptation to F9 cells can also result from a deletion affecting the A enhancer domain, as in mutant PyEC-F9-5000 (39). These observa* Corresponding author. t Present address: URA 1148, Centre National de la Recherche Scientifique, Institut Pasteur, 75724 Paris Cddex 15, France. t Present address: URA 360, Centre National de la Recherche Scientifique, Universite Clermont-Ferrand 2, Les Cezeaux, 63177 Aubiere Cedex, France.

We report here the isolation and genome structure of 3029

a

3030

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MtLIN ET AL.

TABLE 1. Origin and differentiation potential of the EC and ES cell lines used Differentiation potential

Cell line

Origin

PCC4 F9 LT1 ES-D3-5

Embryoid body OTT 6050 from mouse strain 129/Sv Embryoid body OTT 6050 from mouse strain 129/Sv Ovarian cyst 72484 from mouse strain LT Blastocysts from mouse strain 129/Sv

new multiadapted mutant selected on PCC4 cells and of two mutants derived from it but selected on LT1 cells. A

quantitative analysis of viral DNA replication, early and late expression, and viral production in 3T6 and EC cells reveals that these two mutants selected on LT1 cells are more efficient than all other PyEC mutants in all EC lines tested. In the present study, we have also extended the analysis of PyEC host range specificity. We have taken advantage of the recently described embryonal stem (ES) cells, which are derived from the inner cell mass (ICM) of blastocysts (4, 5, 23). These cells keep their embryonal phenotype as long as they are cocultured on an embryonal fibroblastic feeder layer, although their in vitro culture in cell-conditioned medium has abolished the need for feeder cells (34). ES cells are thought to be closer than EC cells to early embryos for two main reasons. First, they differentiate spontaneously in highly organized structures, e.g., the myocardium and yolk sacs with blood islands (4). Second, ES cells colonize the germ line with high frequency when reinjected into blastocysts (10). We have analyzed the ability of different PyEC mutants to infect ES cells with the aim of selecting mutants able to infect preimplantation embryos. We show here that only lytic multiadapted PyEC-C mutants are efficiently infectious and express the whole repertoire of viral functions in ES cells. This result suggests that identical or closely related regulatory mechanisms control mutant PyV expression in both EC and ES cells. In contrast, preimplantation embryos are not permissive to any PyEC mutants. However, multiadapted mutants are infectious for the ICM of blastocysts after 2 days of in vitro culture.

gene

MATERIALS AND METHODS Preimplantation embryos. 129/Sv female mice were hormonally stimulated and mated with C57BL/6 males. The appearance of a vaginal plug was taken as day one of embryonal development. Preimplantation embryos were removed from the oviduct, freed of their zona pellucida, and cultured in Whitten's medium as described previously (18). Cell lines and viruses. PCC4, F9, LT1 EC cells, and 3T6 cells were cultured as previously described (27). The origin

In vivo

In vitro

Multipotent Nullipotent Nullipotent Multipotent

Multipotent Multipotent Nullipotent Multipotent

Reference

30 14 35 4

and differentiation potential of the EC cell lines are indicated in Table 1. The NIH 3T3 cell line, obtained from the American Type Culture Collection, was seeded at 5 x 105 cells per 90-mm-diameter plate and split every 3 days. The cells were grown in Dulbecco modified Eagle medium (DMEM) supplemented with 10% calf serum. The ES-D3-5 cell line was established from 129/Sv blastocysts. The culture conditions and differentiation procedure were essentially as described previously (4). Briefly, ES cells were cultured on a mitomycin-treated primary embryonal fibroblast feeder layer obtained from 15-day-old BALB/c embryos. The cells were replated every other day by preparing a single-cell suspension with a 0.01% trypsin solution (Sigma; type XI). ES cell differentiation was induced in suspension culture, where ES cells form aggregates, after the feeder cells were removed by preplating. ES cells were also cultured without a feeder layer by using medium conditioned by the C5637 cell line as previously reported (34). wt A2 PyV was obtained from M. Fried (Imperial Cancer Research Fund, London, England). Strain evl001 was obtained from cloned DNA of evl001 in a pBR322 plasmid lacking all BglI sites (gift from G. Magnusson, Uppsala University Biochemical Center, Uppsala, Sweden). The origin, properties, and genome structure of the various PyEC mutants are described in Table 2 and Fig. 1. All viral stocks were propagated on secondary mouse embryo cells as described previously (27). To quantitate viral production, 2 x 105 3T6 or EC cells per 60-mm-diameter dish were either infected at 1 PFU per cell 18 h after being seeded or transfected with 4 ,ug of viral DNA 24 h after being seeded. Virus was recovered 72 h later and titrated on secondary mouse embryo cells. For viral infection, single ES cell suspensions (1 x 106 to 2 x 106) in 300 Wl of DMEM were incubated with virus (200 to 300 PFU per cell) for 2 h at 37°C. Subsequently, 10 ml of DMEM and 15% fetal calf serum were added, and the cells were cultured in non-tissue culture-treated bacterial dishes, where ES cells do not adhere and grow in aggregates. In a second series of experiments, ES cells grown on feeder cells on cover slips were washed three times in phosphatebuffered saline (PBS) without Ca2" and Mg2" and infected with virus for 2 h as described above. In a third series of

TABLE 2. Origin and biological properties of PyEC mutants studied in this article PyEC strain

F9-1 P5000 M206 M112 C201 C306 wt-C306 M112-ev

Origin

wt wt

ev1001 evlO01 M112 M112 wt, C306 evlO01, M112

Cell line or means of selection

Phenotype in EC cells

Reference

F9 PCC4 PCC4 PCC4 LT1 LT1 In vitro reconstruction In vitro reconstruction

Adapted to F9 Adapted to PCC4 Multiadapted Multiadapted Lytic, multiadapted Lytic, multiadapted Multiadapted Multiadapted

16 28 27 This article This article This article This article This article

VOL. 65, 1991

experiments, ES cells grown in conditioned medium without a feeder layer were infected as described above. Preimplantation embryos at different stages, as indicated in Results, were infected with virus for 2 h and subsequently cultured in Whitten's medium. Indirect immunofluorescence. ES cells grown on coverslips were washed three times in PBS without Ca2' and Mg2' and fixed with cold (-20°C) methanol-acetone (60:40, vol/vol) for 15 min at room temperature. ES cell aggregates washed in PBS were fixed with freshly prepared 4% formaldehyde in PBS (pH 7.2) for 15 min, washed in PBS, and frozen in Tissue-Tek 11 (18) on a cryostat (Reichert-Jung). Cryostat sections (8 ,um) were fixed with cold methanol-acetone as described above and washed in PBS. Preimplantation embryos were attached to glass slides by a series of ethanol steps (20 to 96% ethanol in water), fixed with cold methanolacetone, and washed in PBS. Indirect immunofluorescence tests were carried out in a humid chamber at room temperature; each antibody incubation (30 min) was followed by several 20-min washes in PBS. The following antibodies were used: rat anti-T (PyV T antigens) serum; sheep anti-V (PyV late structural proteins) serum; ECMA-7, a mouse immunoglobulin M (IgM) monoclonal antibody which reacts with ES cells (17); and TROMA-1, a rat monoclonal antibody which reacts with cytokeratin-like polypeptides of intermediate filaments (18). Bound antibodies were detected with the respective fluorescein isothiocyanate-labeled second antibodies: goat F(ab')2 anti-rat IgG (Cappel), rabbit anti-sheep IgG (Nordic), and rabbit anti-mouse IgM (Miles). Cells were examined under a Leitz Dialux 20 EB microscope, and photographs were taken with Ilford HP5 film. Replication assay. 3T6 and EC cells were seeded at 4 x 105 cells per 60-mm-diameter dish and infected 18 h later at a multiplicity of infection of 10 PFU per cell. The efficiency of infection was monitored by infecting (at 100 PFU per cell) coverslips in 35-mm-diameter dishes seeded the day before with 2 x 105 cells and by measuring T-antigen immunofluorescence. Viral DNA was extracted by the Hirt procedure (13) 24, 48, and 72 h postinfection (p.i.). One-fourth of the DNA extracted at each time point was digested with BamHI and run on a 1.4% agarose gel. The gels were stained with ethidium bromide, transferred to nitrocellulose filters, and hybridized with an in vitro 32P-labeled PyV DNA probe as described previously (1). PyV early, late, and a-actin probes. The AvaI-to-EcoRI (nucleotides [nt] 1010 to 1562) fragment overlapping the early coding region and the HinclI fragment (nt 2964 to 3468) overlapping the late coding region were cloned at the unique EcoRI and HincII sites, respectively, of the Bluescript SK(+) plasmid (Stratagene). The 930-bp a-actin-encoding fragment from PAL 41 plasmid (29) was cloned at the unique PstI site of Bluescript SK(+). After digestion with BamHI, 32P-labeled RNA transcripts complementary to the cellular RNA transcripts of PyV and a-actin were synthesized with the T7 polymerase promoter. Early and late viral RNA quantification. 3T6 and EC cells were seeded at 5 x 105 cells per 90-mm-diameter dish and infected 18 h later at 50 PFU per 3T6 cell and 200 PFU per EC cell. Total RNA was isolated 30 h p.i. for 3T6 cells and 48 h p.i. for EC cells by the guanidinium-cesium chloride centrifugation technique (21). RNAs were denatured by incubation with 1 M glyoxal (44) without dimethyl sulfoxide. Samples (5, 10, and 15 ,ug) of RNAs isolated from each virus-infected EC line were transferred in duplicate onto a reinforced nitrocellulose mem-

PyEC MUTANTS AND MOUSE EMBRYONAL CELLS

3031

brane by using a Minifold II apparatus (Schleicher & Schuell). The membranes were baked for 2 h at 80°C and hybridized according to the Stratagene protocol in 50% deionized formamide at 65°C. Because of a higher background and a lower level of viral RNA expression in EC cells than in 3T6 cells, the washing temperature in 0.1 x SSC (1 x SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-0.1% sodium dodecyl sulfate was gradually increased from 72 to 82°C. One set of blots was hybridized to the early-coding PyV probe, and the other set was hybridized to the latecoding PyV probe. Each set was then dehybridized in 2 mM Tris-1 mM EDTA (pH 8) for 1 h at 100°C and rehybridized to the a-actin probe. Different exposures of each blot on Fuji X-ray films were scanned with a Chromoscan 3 scanner (Joyce-Loebl, Ltd.), and the amounts of early or late PyV transcripts were normalized with respect to the a-actin transcripts. Plasmids, transfections, and luciferase and 0-galactosidase assays. pPyLuc plasmids are derivatives of ptkLuc (25) in which the noncoding region of PyV, between the BclI and the BstXI sites (nt 5022 to 167 according to the numbering scheme of Tyndall et al. [38]), is substituted, in the early orientation, for the BglII-SacI fragment of ptkLuc, thereby placing the luciferase gene under the control of the PyV early promoter/enhancer sequences. Plasmid pPyC306LacZ, used as an internal standard in transfections, is derived from pCH110 (Pharmacia) and contains the lacZ gene under the control of the early promoter/enhancer sequences of the PyEC-C306 mutant. Four hours before transfection, 2 x 105 EC or 3T3 cells per dish were seeded in 60-mm-diameter dishes. Cells were cotransfected with 10 ,Ig of pPyLuc and 2 pkg of pPyC306LacZ plasmid by the calcium phosphate procedure (43). The medium was changed 18 h after transfection, and cell extracts were prepared 24 h later as described below. The transfected cells were washed three times in cold PBS, scraped in 1 ml of cold extraction buffer (100 mM potassium phosphate [pH 7.8], 1 mM dithiothreitol), centrifuged, resuspended in 100 [lI of extraction buffer, and subjected to three cycles of freeze-thawing (-70 to 37°C). The extracts were centrifuged for 15 min at 12,000 x g and stored at -20°C. The protein concentration was determined by the Bio-Rad protein assay. Luciferase activity was measured as described elsewhere (25) with an Analytical Luminescence Monolight 2010 luminometer, and P-galactosidase activity was measured by using 4-methylumbelliferyl-p-Dgalactoside as the substrate (36) with a Hoefer Scientific Instruments DNA fluorimeter. RESULTS

Isolation, cloning, and genome structure of PyEC mutants selected on LT1 cells. Two viral mutants, PyEC-M206 and PyEC-M112, isolated on PCC4 cells were used to infect LT1 cells. They were derived from the evl001 PyV strain, a spontaneous variant of wt A2 PyV harboring a duplication including both the A and B enhancer domains and the origin of replication (22). Mutant PyEC-M206 has been previously described (27), whereas PyEC-M112 has not. The latter has a genome structure very similar to that of the former, except that the deletion in the duplicated region extends from nt 5119 to 5242 instead of nt 5123 to 5226 (Fig. 1). They both behaved as if multiadapted to EC cell lines with regard to T-antigen expression as measured by immunofluorescence. LT1 cells, infected at a multiplicity of infection of 200 PFU per cell with PyEC-M112 or PyEC-M206 or with a 1:1

MtLIN ET AL.

3032

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FIG. 1. Genome structure of wt A2, evlOOl, and PyEC mutants isolated in vivo and reconstructed in vitro as described in this article. The numbering is that used by Tyndall et al. (38). ORI designates the wt PyV origin of replication. The thin lines indicate sequences deleted, the medium-thick lines indicate sequences present in the genome, and the thick lines indicate the duplicated copies of the A domain. The CT in PyEC-M206 indicates two additional nucleotides of unknown origin. C is the point mutation at nt 5204 present in the duplicated copy of PyEC-C201 and PyECC306. The sequence of the evlOOl strain differs from that of evlOOlh previously described by M,lin et al. (27): the point mutation observed by Magnusson and Nilsson (22) at nt 5228 in the second copy of the evlOO1 B enhancer was not found. Therefore, evlOOl contains a perfect duplication of the wt PyV genome from nt 5100 to 48.

mixture of both viruses, were subcultured and checked for T-antigen expression at intervals. The proportion of T-antigen-positive cells increased from less than 0.1% at 72 h p.i. to about 30% at 6 weeks p.i. in PyEC-M112 or doubly infected cells. At the same time, a significant degree of cell death was observed. No increase of T-antigen-expressing cells and no cytopathic effect were evidenced in PyECM206-infected cells. Virus was recovered from the cells and the culture medium and concentrated by centrifugation. This

viral ministock was used to infect a series of LT1 plates from which a larger stock was prepared. Viral DNA was extracted, purified, and cloned after digestion with BamHI in the unique BamHI site of plasmid pAT153. Cloned DNA was used to transform Escherichia coli HB101; plasmid DNA was prepared from transformed bacterial clones, cut with BamHI, and tested for its ability to produce virus upon transfection in LT1 cells. Only one type of infectious DNA could be recovered from each experiment. These DNAs were designated PyEC-C201 and PyEC-C306. In vitro reconstruction experiments indicated that the PyEC-C phenotype could be obtained by substituting the BcII-BgIl fragment (nt 5021 to 87) of wt DNA with the equivalent fragment from the mutants. The nucleotide sequences between the BclI and BglI sites of both mutants, determined by the method of Maxam and Gilbert (26), are mapped in Fig. 1. Both were clearly derived from PyECM112 through a duplication event leading to the presence of three copies of the adenovirus type 5 ElA-like core sequence (nt 5107 to 5117), two copies of the simian virus 40-like core sequence (nt 5189 to 5198), and two origins of replication. Nucleotide 5203 in the second duplicated sequence is followed by a C instead of a G in the wt PyV genome. Although isolated from two independent infections, both mutants displayed very similar genome structures and identical biological properties. Therefore only one, PyECC306, is discussed further in this article. Viral stocks were prepared by transfecting either secondary mouse embryo or EC cells in case mutants were counterselected in differentiated cells. Analysis of the structure of viral DNA from Hirt supernatants and of the infectious properties of the viral stocks showed that no bias was introduced by the cell lines used. To define more precisely the structural features of the PyEC-C mutant genomes responsible for their phenotype (i.e., the roles of the duplication of the PyEC-M112 enhancer region and the duplication of the wt origin of replication), two hybrid DNAs were constructed in vitro (Fig. 2). PyECM112-ev was identical to PyEC-C201, PyEC-C306, and PyEC-M112 up to the first origin of replication and to evlO01 between the first origin and the BglI site. Conversely, PyEC-wt-C306 was identical to wt and evlO01 up to the first origin and to PyEC-C306 from this origin to the BglI site. Expression of viral early and late RNAs. Early and late viral RNAs were quantified by slot blot hybridization of total RNA extracted 30 h p.i. from 3T6 cells and 48 h p.i. from EC cells. The results are shown in Fig. 3. No viral RNA could be detected by this method on wt PyV-infected EC cells, and very large differences between the various mutants in the levels of both early and late RNAs were observed. The amount of early transcripts was correlated with the proportion of T-antigen-positive cells detected by immunofluorescence (data not shown). PyEC-M112, a deleted version of PyEC-M206, exhibited strongly enhanced expression compared with the latter. PyEC-M112 and PyEC-C306, which was derived from it, were the most efficient viruses on all EC cells, while both in vitro-reconstructed hybrids, PyEC-wtC306 and PyEC-M112-ev, were severely impaired in PCC4 and LT1, but this effect was less pronounced in F9 cells. The implications of these results are discussed below. It is also apparent from Fig. 3 that good adaptation to EC cells was deleterious to viral RNA expression in differentiated cells, since PyEC-M112 and PyEC-C306 were much less efficient on 3T6 cells than was evlO01 PyV, from which they derived. Still, the level of viral early RNA in 3T6 cells 30 h after infection at 50 PFU per cell with wt or evlO01 was

3033

PyEC MUTANTS AND MOUSE EMBRYONAL CELLS

VOL. 65, 1991

EV1 001 ORI

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10- to 20-fold higher than in PCC4 cells 48 h after infection at 200 PFU per cell with PyEC-M112 or PyEC-C306. Furthermore, this level dropped 5- to 10-fold in F9 cells and 10- to 20-fold in LT1 cells compared with PCC4 cells. Nevertheless, all PyEC-C306-infected EC cells could be kept for only , 3T6

5.

two or three passages in culture; by then, a strong cytopathic effect killed all the cells. Differences in the relative amounts of late RNAs, which reflect the control of late transcription by large T antigen, the number of templates generated through replication, and the rLL'B

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efficiency of the late promoter, are more difficult to interpret. However, the abundance of late RNAs was paralleled by the abundance of early RNAs and was proportional in all cell lines to virus production, as measured by a plaque assay using secondary mouse embryo cells (data not shown). Replication of PyEC mutants. 3T6, PCC4, F9, and LT1 cells were infected with the various PyV strains described in Fig. 1, and the kinetics of viral DNA synthesis were measured as described in Materials and Methods. The results are shown in Fig. 4. In differentiated 3T6 fibroblasts, the efficiency of replication measured 72 h p.i. ranged from 50 to 150% of the wt virus efficiency for all strains tested. Therefore, no major influence of genome rearrangements on DNA replication was evident in these cells. In contrast, a strong hierarchy between the different EC mutants was observed in EC cells. The patterns of PyEC replication were very similar in PCC4 and LT1 cells, although the extent of replication, as also observed for viral transcription, was lower in LT1 cells. In both cell lines, PyEC-C306 was clearly the most efficiently replicated and PyEC-M112 was two- to threefold more efficient than PyEC-M206. A different situation prevailed in F9 cells, in which F9adapted (PyEC-F9-1), PCC4-adapted (PyEC-M112), and LT1-adapted (PyEC-C306) mutants exhibited similarly high replication levels and PyEC-M112 was about 20 times more efficient than PyEC-M206. Furthermore, compared with PyEC-C306, hybrid viruses PyEC-wt-C306 and PyEC-

M112-ev were less impaired than in PCC4 and LT1 cells, as was already shown to be the case for RNA expression. Finally, we observed an uncoupling between early RNA expression and DNA replication in all the cell lines. For example, the level of early RNAs in PyEC-M112-infected PCC4 cells was at least 100 times higher than in evlOO1 PyVor PyEC-F9-1-infected cells, while all three viruses replicated to a similar extent. The same observation held when PyEC-P5000 and PyEC-C306 viral RNA and DNA replication levels in 3T6 cells were compared. Transfection of EC cells with pPyLuc plasmids. In order to quantify the efficiency of the early promoter/enhancer region of the different PyEC mutants, their noncoding sequences were cloned in front of the luciferase gene. These various pPyLuc constructs were cotransfected in EC and 3T3 cells with pPyC306LacZ. A promoterless plasmid (pLuc) was used as a control. Luciferase activities determined 48 h after transfection were normalized for transfection efficiency by measuring ,-galactosidase activities and protein concentrations (Fig. 5). In transfected LT1 cells, the relative levels of luciferase expression driven by the different PyV early promoter/ enhancer sequences were very similar to those of early RNAs in infected cells (Fig. 3 and 5). This comparison did not hold for the other cell lines, in which the pattern of luciferase activity was quite different, both quantitatively and qualitatively, from the results of early RNA expression.

PyEC MUTANTS AND MOUSE EMBRYONAL CELLS

VOL. 65, 1991

3035

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For example PyEC-C306 expressed luciferase at only a 2.5-fold-higher level than evlOO1 PyV in PCC4 cells, whereas the level of early RNAs was at least 200-fold higher in infected cells. In F9 transfected cells, PyEC-M206 appeared to be almost as efficient as PyEC-M112, while in infected cells it was 10-fold less active. Infection of ES cells. In an initial series of experiments, ES cells grown on a feeder layer were infected with PyV. Under these culture conditions, ES cells form aggregates of around 10 to 100 cells attached to the fibroblasts. This made an unambiguous examination of large T antigen expression rather difficult, mainly for three reasons. First, it was not certain that all cells were equally well infected within one aggregate. Second, the localization of positive cells within aggregates was difficult to achieve. Third, feeder cells are susceptible to PyV infection. However, it could be noted that wt PyV was negative on ES cells, PyEC-P5000 and PyEC-M206 (occasionally) and PyEC-F9-1 yielded around 5 to 8% positive cells, and a higher percentage of cells expressing large T was obtained with the PyEC-M112 and PyEC-C306 mutants. To ensure that all ES cells were equally infected and to unambiguously characterize large-T-positive cells, ES cells were infected as single-cell suspensions. For this purpose, the embryonal fibroblast feeder cells were removed by two cycles of preplating and ES cells were infected in suspension. The cells were kept for 45 h in suspension, where they formed aggregates. Under these culture conditions, the first differentiated cells appear in the outer cell layer and are of endodermal phenotype (17). This was monitored with monoclonal antibodies which recognize cell-type-specific markers (Fig. 6). TROMA-1 recognizes intermediate filament proteins specific for endodermal cells appearing gradually at the surface of the aggregate. In most cases, only few endodermal

cells were detected (Fig. 6a and b), and occasionally a ring of endodermal cells had been formed (Fig. 6c and d). The inner cells of the aggregates were composed of embryonal cells, as monitored with monoclonal antibody ECMA-7 (Fig. 6e). Aggregates of wt PyV-infected cells were always negative for large T (Fig. 7a). After PyEC-F9-1, PyEC-P5000, and PyEC-M206 infection, few large-T-positive cells were found exclusively in the outer cell layer of the aggregates (Fig. 7b). After PyEC-M112 infection, more outer cells and a few cells localized beneath the outer layer or inside the aggregates expressed T antigen (Fig. 7c). The percentage of large-Tpositive cells was greatly increased after PyEC-C306 infection (Fig. 7d). Positive cells were distributed over the whole aggregates, in which anti-large T antibodies stained equally well the outer cells and the embryonal cells of the aggregates. However, within aggregates, not all cells were found to be positive, even at a high multiplicity of infection. The expression of late structural PyV proteins (V antigens) was studied 72 h after viral infection. No V-antigenpositive cells were observed after wt PyV, PyEC-F9-1, PyEC-P5000, or PyEC-M206 infection (not shown). However after PyEC-M112 and PyEC-C306 infection, V-antigenpositive cells were found (Fig. 7e). As observed for large T antigen, cells expressing V antigens were distributed within the aggregates, although not all cells in a given aggregate were found to be positive and PyEC-M112 yielded many fewer V-antigen-positive cells than PyEC-C306. Infection of ES cells grown in conditioned medium led to the same general pattern of PyEC mutant expression as was found in ES cell aggregates. To investigate whether the different viruses could undertake efficient lysis of ES cells, aggregates were grown in suspension culture for 6 days after viral infection. Aggregates of wt PyV-, PyEC-F9-1-, PyEC-P5000-, or PyEC-

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FIG. 6. Immunofluorescence tests on cryostat sections of ES-D3-5 cell aggregates. Sections were stained with TROMA-1 (a through d) for cytokeratin intermediate filament proteins and with ECMA-7 (e) as a marker of ES cells. After 40 h of suspension culture, the differentiated cells appear at the periphery of the aggregates, as monitored with TROMA-1. (a and c) Phase contrast; (b, d, and e) immunofluorescence. Magnification, ca. x 155 (a through d) or x232 (e).

M206-infected cells were indistinguishable from control aggregates after a 6-day culture (Fig. 8a). In contrast, aggregates of PyEC-C201-infected cells became necrotic and were composed of many lysed and few healthy cells (Fig. 8b), while the extent of lysis was less important after PyEC-M112 infection. These experiments indicate that, after PyEC-C infection, most ES cells express large T antigen and late structural proteins and that the virus can undergo a complete lytic cycle in these cells. Infection of preimplantation embryos. About 60 to 80 embryos in stages ranging from the two-cell to the morula stage were infected with PyEC-C viruses after removal of the zona pellucida, cultivated for 44 h, and scored for large T expression. Two-thirds of the infected two-cell embryos developed into eight-cell embryos and compact morulae, while the rest of the embryos ceased dividing. This effect was most likely due to the toxicity of the viral suspension and not to the expression of the virus, since no T antigen was detected in any of the infected embryos. Virtually all of the infected eight-cell embryos developed into blastocysts, and the infected compact morulae gave rise to expanded blastocysts. Of a total of nearly 200 infected embryos, all were negative for large T with one exception. One embryo infected at the two-cell stage, which had not divided during the subsequent culture period, was positive for T antigen (Fig. 9). Both nuclei of this embryo were rather unusually enlarged compared with those of control embryos. A possible explanation of this peculiar case is discussed below. In order to determine whether stem cells at later stages of development would become permissive, fully expanded blastocysts (day 4.5 of embryonal development) were transferred to tissue culture dishes, where they attached by trophoblast outgrowth while the ICM was exposed to the tissue culture environment. After 48 h of in vitro development, attached blastocysts were infected with PyEC mutants, and expression of T antigen was determined 48 h p.i. As for the ES cell aggregates cultured on feeder cells, unambiguous observation was difficult to achieve, especially for the ICM, in which it was difficult to distinguish between endodermal cells and ES cells. However, the general outcome of these experiments was that PyEC mutants but not wt PyV expressed T antigen in cells of the ICM, while only some trophoblastic cells showed fluorescence staining (Fig. 10). As already observed for ES cells, PyEC-C mutants were more efficiently expressed than the other PyEC mutants. DISCUSSION

PyV in EC cells. Acquisition by wt PyV of the ability to express its genetic program in EC cells requires DNA sequence modifications involving the promoter/enhancer domains of the viral genome. In this article, we describe three new mutant viruses which exhibit better adaptation to EC cell lines than all previously described mutants. PyEC-M112 has a genome structure very similar to that of the previously described mutant PyEC-M206 (27) but is replicated and transcribed more efficiently in all EC cell lines. The difference between the two viruses is most striking in F9 cells. Although it has been selected on PCC4 cells and

has a typical PyEC-PCC4 genome structure, PyEC-M112 is even more adapted to F9 cells than PyEC-F9-1 is. It should be pointed out that the only difference between PyEC-M206 and PyEC-M112, as shown in Fig. 1, concerns the boundaries of the deletion of the B domain. This deletion extends from nt 5118 to 5243 in PyEC-M112, as opposed to nt 5122 to 5227 in PyEC-M206. This extended deletion removes two key sequences for PyEC-F9 mutants. One sequence includes nucleotide 5233, which is the site of the PyEC-F9-1 point mutation responsible for most PyEC-F9 mutant phenotypes. Although DNA-protein interactions have not been detected in this region of wt PyV DNA by using F9 or PCC4 cell extracts but have been detected in PyEC-F9-1 DNA by using F9 cell extracts (19), it is still not clear whether this point mutation is only a prerequisite for the binding of a transcriptional activator or simultaneously prevents the binding of an inhibitor. The other key sequence deleted in PyEC-M112 compared with PyEC-M206 is located in the wt PyV A domain. One F9-adapted mutant, PyEC-F9-5000 (39) harbors a deletion from nt 5118 to 5142 but lacks the typical A -) G transition at nucleotide 5233. This suggests that a potential silencer overlaps the PEA2-binding site (nt 5122 to 5129), as shown by Wasylyk et al. (40). Analysis of the effects of in vitro-generated deletions (26a) also supports this hypothesis. Therefore, it is not currently possible to decide which of the deleted nucleotides are responsible for the higher level of expression of PyEC-M112 compared with PyEC-M206. In addition, the possibility that the different junctions in both mutants create a potential binding site, either for an inhibitory transcription factor in PyEC-M206 or for an activating factor in PyEC-M112, cannot be excluded. Experiments are in progress to distinguish between the different possibilities. Mutants PyEC-C201 and PyEC-C306, isolated on LT1 cells, are derived from PyEC-M112. The duplication event which gave rise to these mutants increased their expression in all three EC cell lines and their replication in PCC4 and LT1 cells. Replacement of one of the duplicated enhancer sequences, either proximal (in PyEC-M112-ev) or distal (in PyEC-wt-C306) to the early coding sequences, by its wt counterpart, although it preserves the duplicated origin of replication, severely impairs adaptation to LT1 and PCC4 cells at both the transcription and replication levels. This implies that acquisition of the PyEC-C phenotype results neither from the mere duplication of the wt PyV origin of replication nor from the mere duplication of binding sites for positive regulatory factors which interact with the wt PyV genome. This is probably also true for the acquisition of the PyEC-PCC4 phenotype, which is associated with a duplication of the A core. The minimal duplication contains the consensus binding sequences for two identified transcription factors, PEAl and PEA3 (24). In differentiated cells, PEA3, which acts synergistically with PEAl to stimulate transcription (41), is indispensable for a productive viral cycle (26a). PEA3 has also been detected by gel shift experiments in F9 cells, and although it has not been looked for in PCC4 cells, the duplication of its binding site observed in all PyEC-PCC4 mutants points to a crucial role of this factor for their

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expression in PCC4 cells. However, except for PyEC-M112, these mutants are poorly expressed in F9 cells. Hence, duplication of the binding site for PEA3 cannot account by itself for the PyEC-PCC4 phenotype. Factor PEAl (32) is present at a much higher level in differentiated cells than in EC cells and stimulates both transcription and replication of wt PyV in differentiated cells (24). Since the A core is the major enhancer core in differentiated cells and a minor one in EC cells (12), it has been hypothesized that duplication of the PEAl-binding site in PCC4 mutants results in an increased affinity for this factor. This would also imply a higher expression level for all PyEC-PCC4 mutants compared with wt PyV in 3T6 cells. Figure 3 shows that this is not the case except for PyEC-P5000, in which the junction consisting of nt 5192 to 5092 creates a third potential PEAl site. PyEC-P5000 exhibits a very high level of RNA expression in 3T6 and 3T3 cells but not in PCC4 cells; in contrast, PyEC-M112, which has only two PEAl-binding sites, is expressed poorly in 3T6 cells but very efficiently in PCC4 cells. Therefore, mechanisms other than the mere duplication of the PEAl- and PEA3-binding sites must be invoked to explain the PyEC-PCC4 or PyEC-C phenotypes. The sequences deleted and the new junctions created must have removed silencers and/or generated new binding sites for other positive factors which are still unknown. In addition, the positioning of the different regulatory factors might be of great importance. We are currently investigating which elements of the PyEC-PCC4 rearranged genomes are responsible for their expression. It should also be noticed that PyEC-M112 is always expressed at a higher level than PyEC-M206 in all EC cell lines, implying the existence of common transcription factors in PCC4, F9, and LT1 cells. Still, the behavior of

mutants such as PyEC-F9-1 suggests that some transcription factors are either cell line specific or expressed at unequal levels in the different types of EC cells. It should be recognized that, up to now, no complete investigation of any transcription factor in EC cells which would allow an understanding of the mechanisms involved in PyEC mutant expression has been achieved. Comparison of the relative levels of transcription of the different virus strains in 3T6 and EC cells shows that, with the exception of PyEC-F9-1, the mutants which are most efficient in EC cells (i.e., PyEC-M112 and PyEC-C306) are poorly expressed compared with the wt in 3T6 cells. In contrast, mutant PyEC-P5000, which is the least adapted to EC cells, is more efficient in 3T6 cells than wt virus. This is a further indication that some important PyV regulatory factors are qualitatively and/or quantitatively different in 3T6 and EC cells. Another outcome of these experiments is the observed lack of relationship between the levels of viral DNA replication and of gene expression. In 3T6 cells, the replication efficiency of the different viral strains ranges from 50 to 150% compared with the wt; in contrast, early RNAs are in the range of 5 to 200%, late RNAs are in the range of 5 to 500%, and there is always a good correlation between early and late RNA levels. A similar situation prevails in EC cell lines. Therefore, although the sequences controlling viral DNA replication and gene expression are intermingled and overlapping, some DNA modifications strongly affect transcription without altering replication and vice versa. This observation is consistent with the absence of transcomplementation for viral DNA replication between PyEC-F9-1 and wt PyV in doubly infected F9 cells (7). It also indicates that the threshold amount of T antigen necessary to allow effi-

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cient replication can be achieved with very few mRNA templates. Except in LT1 cells, the relative luciferase activity levels in EC cells transfected with the different pPyLuc constructs are not comparable with the relative early RNA transcript levels measured after infection with the corresponding viruses. While transcription varies over two to three orders of magnitude, luciferase activity varies by a factor of up to 7. An effect of bacterial vector sequences on transcription cannot account for the data, since similar results were obtained by transfecting viral DNA and by measuring either T-antigen expression by immunofluorescence or virion production by plaque assay (data not shown). Titration of a transcription inhibitor(s) with an excess of viral sequences has been invoked to explain the efficient expression of simian virus 40 DNA in transfected cells but not in virus-

infected F9 cells (9). On the other hand, the wt PyV enhancer was shown to be only three to four times less efficient in PCC3 than in 3T3 cells transfected with chloramphenicol acetyltransferase expression vectors (12). We point out that transfection and infection data are very similar only for LT1 cells, which are the most easily transfectable and the least able to differentiate in vitro and in vivo. Because of the very large difference (at least 500-fold) between the level of wt PyV expression in fibroblasts and in EC cells, induction by transfection of a low percentage of differentiated cells could lead to the results obtained in F9 and PCC4 cells. Two sets of experiments support this hypothesis. We have observed that a detectable number of F9 and PCC4 but not LT1 cells express T antigen, as measured by immunofluorescence assay, upon wt PyV infection if they have been pretreated 18 h before with Ca phosphate. Gorman et al. (9) have shown

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MELIN ET AL.

that after mock transfection, 0.1 to 0.5% of F9 cells react with the TROMA-1 monoclonal antibody, which specifically recognizes intermediate filament proteins of differentiated F9 cells (18). Furthermore, it has been shown (6, 33) that the regulation of some genes (including nuclear transcription factors such as c-fos) is greatly affected by Ca phosphate transfection. These observations stress the limits of the validity of transfections for the analysis and interpretation of biological phenomena. PyV in ES cells and preimplantation embryos. The fact that better adaptation of PyV to EC cells also leads to adaptation to ES cells suggests that transcriptional regulatory factors are shared by these two types of cells. Indeed, the same hierarchy of mutant efficiency is observed in EC and ES cells. On the other hand, although ES cells are thought to resemble, more closely than EC cells, the pluripotent stem cells of early cleaving embryos, ES-adapted PyEC mutants are not infectious on early preimplantation embryos. Therefore, major differences in transcriptional regulation between ICM and earlier pluripotent embryonic cells are expected to be observed. It should be noticed that all PyEC mutants, but not wt virus, are able to infect the primitive endodermal cells which appear at the surface of ES cell aggregates, although with variable efficiencies. In contrast, differentiated parietal endodermal cells induced by retinoic acid treatment of F9 cells become fully permissive to wt PyV (8). This indicates that although they express a common set of markers, these two endodermal derivatives are not equivalent with regard to wt PyV restriction. Preimplantation embryos are refractory to virus infection from the two-cell stage to the morula stage, and viral exposure does not seem to impair the early stages of development. In contrast, Wirak et al. (45) observed that a plasmid carrying a complete wt PyV genome did replicate when microinjected into one of the nuclei of a two-cell embryo and that replication resulted in abnormal multicellular embryos. However, in those experiments several thousand copies of DNA were introduced directly into the nucleus, allowing the synthesis of threshold amounts of T antigen required for viral DNA replication. We have shown (26b) that the wt PyV early promoter/enhancer exhibits a very low but significant level of activity in microinjected two-cell embryos. Therefore, the different results obtained upon infection and transfection probably rely on quantitative rather than qualitative aspects. Of all embryos tested, only a single two-cell embryo infected with PyEC-C mutant was shown to express T antigen (Fig. 9). This embryo contained abnormally enlarged nuclei, suggesting that it might have resulted from an uncoupling between chromosomal replication and cell division. It has been shown (31) that treatment of fertilized eggs with cytochalasin B prevents cellular division but not chromosomal replication. Under these conditions, the eggs express a repertoire of proteins which no longer resemble those of fertilized eggs but are similar to those of cleaving embryos. It might well be that in this particular case the viral suspension exhibited some toxicity which impaired cell division and generated abnormal replication, leading to permissivity for PyEC-C. ICM cells at the stage of blastocyst outgrowth express T antigen upon infection with PyEC-M and PyEC-C mutants. This is in agreement with the results obtained with ES cell lines which are established from attached blastocysts and can be considered equivalent to ICM cells with regard to PyV restriction.

J. VIROL.

Differentiated trophectodermal cells of attached blastocysts are still refractory to wt PyV or PyEC infection. This observation must be correlated with the fact that PyV mutants adapted to trophoblast cells (37) exhibit specific genetic rearrangements and are not infectious for EC cells. In conclusion, we have shown that complex and multiple DNA rearrangements are necessary to generate EC-multiadapted lytic mutants from wt PyV. These viruses are far more efficient in EC cells than all previous EC mutants but are less adapted to differentiated 3T6 cells than wt virus. We also show that different PyEC mutants have different host range specificity in ES cells. PyEC-C mutants are the most efficient and might be the appropriate viruses to generate vectors to study the control of transcription and the effect of gene expression during early mouse development. Still, they are not expressed in early preimplantation embryos. We have therefore undertaken the isolation of a novel class of mutants from ES cells and the analysis of their expression in early cleaving embryos. ACKNOWLEDGMENTS We thank Margot Katz and Nicole Montreau for excellent technical assistance, Colin Stewart for the C5637 cell line, and Melvin De Pamphilis for the ptkLuc plasmid. This work was supported by the Institut National de la Santd et de la Recherche Medicale, the Association pour la Recherche sur le Cancer, the Ligue Nationale contre le Cancer, the Dr. Mildred Scheel Stiftung fur Krebsforschung, and the Deutsche Forschungsgemeinschaft. REFERENCES 1. Dandolo, L., J. Aghion, and D. Blangy. 1984. T-antigen-independent replication of polyomavirus DNA in murine embryonal carcinoma cells. Mol. Cell. Biol. 4:317-323. 2. Dandolo, L., D. Blangy, and R. Kamen. 1983. Regulation of polyomavirus transcription in murine embryonal carcinoma cells. J. Virol. 47:55-64. 3. Davidson, I., J. H. Xiao, R. Rosales, A. Staub, and P. Chambon. 1988. The HeLa cell protein TEF-1 binds specifically and cooperatively to two SV40 enhancer motifs of unrelated sequence. Cell 54:931-942. 4. Doetschman, T. C., H. Eistetter, M. Katz, W. Schmidt, and R. Kemler. 1985. The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium. J. Embryol. Exp. Morphol. 87:27-45. 5. Evans, M. J., and M. H. Kaufman. 1981. Establishment in culture of pluripotential cells from mouse embryos. Nature

(London) 292:154-156. 6. Foster, R., P.-E. Olsson, and L. Gedamu. 1989. Calcium phosphate-mediated transfection alters metallothionein gene expression in response to Cd2' and Zn2+. Mol. Cell. Biol. 9:41054108. 7. Fujimura, F. K., and E. Linney. 1982. Polyoma mutants that productively infect F9 embryonal carcinoma cells do not rescue wild-type polyoma in F9 cells. Proc. Natl. Acad. Sci. USA 79:1479-1483. 8. Georges, E., M. Vasseur, and D. Blangy. 1982. Polyoma virus mutants as probes of variety among mouse embryonal carcinoma cell lines. Differentiation 22:62-65. 9. Gorman, C. M., P. W. J. Rigby, and D. P. Lane. 1985. Negative regulation of viral enhancers in undifferentiated embryonic stem cells. Cell 42:519-526. 10. Gossler, A., T. Doetschman, R. Korn, E. Serfling, and R. Kemler. 1986. Transgenesis by means of blastocyst-derived embryonic stem cell lines. Proc. Natl. Acad. Sci. USA 83:90659069. 11. Hen, R., E. Borrelli, C. Fromenthal, P. Sassone-Corsi, and P. Chambon. 1986. A mutated polyoma virus enhancer which is inactive in undifferentiated embryonal carcinoma cells is not repressed by adenovirus-2 ElA products. Nature (London)

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