Mouse cells surviving polyoma virus infection generally retain the whole viral genome

Virus Research, Elsevier

1

4 (1985) 1-18

VRR 00221

Mouse cells surviving polyoma virus infection generally retain the whole viral genome Elizabeth

Herring-Gillam, Louis Delbecchi, CCcile Royer, Daniel Gendron, Danielle Bourgaux-Ramoisy and P. Bourgaux *

D&partement de Microbiologic, FacultC de Mkdecine, Umoersitt! de Sherbrooke, Sherbrooke, Qukbec, Canada Jl H 5N4 (Accepted

for publication

16 July 1985)

Summary Permissive mouse 3T6 cells were exposed to polyoma virus - either wild-type or early mutant - at high multiplicities of infection. From colonies arising from surviving cells, so-called lines and clones were derived under conditions precluding superinfection. These lines and clones were examined for the presence of viral genetic information, using a variety of techniques. Two salient findings were made: (1) most lines or clones analyzed had retained viral genetic material; (2) generally, this material was nondefective, as evidenced by the production of virus and/or viral DNA molecules of genomic size. These findings indicate that mouse cells can survive for many generations while carrying a complete, infectious, and potentially cytocidal polyoma virus genome. polyoma

virus, permissive

mouse cells, survivors

Introduction Unlike their SV40 counterparts, polyoma virus-transformed cells have rarely been found to carry a viral genome that could be rescued (Koprowski et al., 1967; Watkins and Dulbecco, 1967; Dulbecco, 1968). For a long time, the most notable exceptions to this rule were cells which, being transformed by the ts-a mutant of polyoma virus were continuously propagated at the temperature restricting expres-

* To whom reprint

016%1702/85/$03.30

requests

should

be sent.

0 1985 Elsevier Science Publishers

B.V. (Biomedical

Division)

2 sion of the viral A function, and thus viral DNA replication: from such cells, virus could be readily recovered following transfer to the nonrestrictive temperature (Vogt, 1970; Summers and Vogt, 1970; Folk, 1973). To account for these findings, it was suggested that continuous expression of the viral A function precludes the growth of cells, or that of transformants (Summers and Vogt, 1970). Thus, transformants would be most likely to retain a viral genome which, while being active in transformation, would be unable to replicate. More recently, this interpretation received support from observations indicating that transformed cells often do not carry the entire sequence coding for large T antigen, the product of the viral A gene (Hutchinson et al., 1978; Birg et al., 1979; Lania et al., 1980). If indeed a constitutive block to virus replication was a condition favoring the growth of transformed cells, then mouse transformants, being permissive for polyoma virus replication, would be expected to all carry a defective viral genome (Tooze, 1980). We were therefore surprised to observe that, when shifted from 39 to 33°C mouse cells transformed by the early mutant ts-P155 of polyoma virus gave rise to survivors (R cells) which had retained an infectious viral genome, and were synthesizing a full-size large T antigen (Herring et al., 1980a, 1980b). Because these R cells had remained fully permissive, it appeared that only a paucity of cellular factors required for the initiation of the vegetative cycle, that is for instance the excision of integrated viral DNA, could account for their continuous growth at 33°C (Herring et al., 1980a). If indeed valid, this interpretation implied the existence of a yet unrecognized type of interaction between polyoma virus and its natural host cell. In order to assess the frequency of moderate infections of in vitro cultured cells by polyoma virus, we have exposed mouse 3T6 cells of the kind used to make viral DNA stocks, to large doses of various wild-type and mutant viruses. Survivors were isolated and cloned, then analyzed for the presence of viral genetic material. Our data indicate that most cells retained not only viral genetic material but also the ability to produce virus, at least through the 50 generations that followed the initial infection. Therefore, it would appear that defectiveness of the viral genome is not essential to the survival of mouse cells exposed to polyoma virus.

Materials and Methods Cells and viruses

Mouse cells used in the experiments described below were from the established Swiss 3T6 line (kindly provided by Dr. Walter Eckhart). Viruses were propagated at low multiplicity of infection on secondary cultures of mouse embryo (ME) cells, as previously described (Bourgaux, 1964; Bourgaux et al., 1971). Culture medium was Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated calf serum. Polyoma virus early ts mutants ts-a (Fried, 1965), ts-P155 and ts-52 (Eckhart, 1969, 1974), were obtained from Dr. Walter Eckhart, and plaquepurified on ME cells at 33°C. hr-t mutants NG-18 and NG-59 (Benjamin, 1970) were provided by Dr. Thomas Benjamin.

3 Isolation of cell lines from survivors Subconfluent 3T6 cells were infected at 39°C and at high m.o.i. (50-100 pfu per cell for the ts mutants, 25 pfu per cell for the hr-t mutants). In all cases, cytopathic effect (CPE) reached a maximum within 1 week after infection, while colonies of surviving cells were picked after 2-3 weeks. One colony was picked from each 60 mm Petri dish, and cultured at 39°C. From this moment, 0.1% mouse antipolyoma antiserum (Microbiological Associates) was added to the culture medium. Lines derived from the colonies were named A, B, C . . or 1, 2, 3.. . depending on the experiment . Cloning of lines Two passages after the picking of colonies, cells from each line were frozen, while others were used for cloning. In the latter instance, 60 mm petri dishes were seeded each with lo-100 cells. After incubation for lo-14 days at 39’C, colonies were again picked (one colony per petri dish) and subcultured, yielding so-called clones. These cells were frozen after 2 or 3 passages, i.e. 4 or 5 passages after line isolation. Clones were designated using a number or letter following that indicating the line (for example, clones 5-l and 5-3 both originate from the line 5). With few exceptions, characterization of lines and clones was carried out within three passages of thawing. Subcloning of (ts-P155) C-a and (ts-P155) 5-3 Cells of these 2 clones were thawed and, after three passages at 39°C recloned twice (C-a) or three times (5-3). Three clones were picked during the first round of cloning, for each of C-a and 5-3, while a total of 10 subclones (numbered from 1 to 10) derived from these three clones, were isolated from the last cloning. Total cellular DNA was extracted from confluent cultures at the 3rd and 13th passage since the last round of cloning. Virus extraction and hemagglutination assay Before cells were harvested for virus extraction, all cultures were incubated at 33°C for 48 h in medium containing no antipolyoma antiserum. Virus was extracted from cell debris with receptor-destroying enzyme and quantified as hemagglutinating units (HAU), as described elsewhere (Bourgaux et al., 1978). Extraction of low molecular weight DNA This DNA was extracted by the method of Hirt (1967) from cells that had been either maintained at 39°C or transferred to 33°C for 48 h. The Hirt supernatants were extracted twice with saturated phenol and once with chloroform/isoamyl alcohol (24 : 1) before the DNA was ethanol precipitated. Pellets were resuspended in TE buffer (10 mM Tris-HCl/l mM EDTA, pH 8.0; 0.2 ml per 100 mm petri dish). Marker DNAs were prepared essentially in the same way, but further purified by banding in cesium chloride/ethidium bromide (CsCl-EtBr) gradients (Bourgaux et al., 1971).

4 Extraction of total DNA Total cellular DNA was extracted from cells kept at 39°C according to either Gross-Bellard et al. (1973) or Perucho et al. (1981), with essentially identical results. In the first instance, the DNA was purified by two cycles of a treatment that included proteinase K (Merck Laboratories) digestion, extraction with saturated phenol followed by chloroform/isoamyl alcohol, and finally dialysis against TE buffer; pancreatic RNAse (Sigma) digestion was performed between the two cycles. In the second instance, the DNA was spooled on a glass rod during ethanol precipitation, after only one cycle of deproteinization (see above). It was then washed with ethanol at increasing concentrations, dried on the glass rod at 37°C and resuspended in TE buffer (0.05-0.1 ml per 100 mm petri dish). DNA was quantified following electrophoresis through 1% agarose vertical slab gels (20 x 10 cm) in E buffer (40 mM Tris/5 mM sodium acetate/l mM EDTA, pH 7.9) using lambda DNA (Bethesda Research Laboratories: BRL) as a concentration marker. Gels were stained in ethidium bromide solution (EtBr) and photographed under UV light (254 nm) with a Polaroid camera. DOT test Cell suspensions were spotted onto nitrocellulose filters, denatured with 0.2 N NaOH and neutralized (Brandsma and Miller, 1980). Filters were dried, baked at 80°C under vacuum, hybridized with a 32P-labeled viral probe and subjected to autoradiography. Restriction enzyme digestion, electrophoresis and transfer onto nitrocellulose Restriction enzymes were supplied by BRL or The Amersham Corporation, and used as recommended by the suppliers. After digestion, samples were electrophoresed through l-1.2% agarose horizontal slab gels (15 x 20 or 30 X 20 cm) in E buffer. After electrophoresis and staining with EtBr, DNA was blotted onto nitrocellulose paper according to Southern (1975). Nitrocellulose filters were dried, baked for 2 h at 80°C under vacuum and subjected to hybridization with a 32P-labeled viral probe. Nick-translation, hybridization and autoradiography 32P-labeled probes were prepared by the procedure of Maniatis et al. (1975) using polyoma virus DNA cloned in plasmid pBR322 as a template. The denatured probe was annealed with the blotted DNA as described by van der Ploeg and Flavell (1980) with minor modifications (Chartrand et al., 1981). About 5 X lo6 cpm of probe were used per nitrocellulose filter. Dried blots were autoradiographed at - 70°C using intensifying screens (Cronex Par Speed, DuPont). Immunofluorescence Cells were seeded onto microscope slides, immediately after infection (or mockinfection). 48 h later, the monolayers were fixed in acetone/methanol (2 : 1) for 15 min at 4’C, dried, and covered with mouse antipolyoma antiserum (Microbiological Associates). After 30 min of incubation at 37’C and washing three times with

5 PBS(A), FITC-conjugated was added, and incubation

sheep antiserum raised against mouse IgG (Amersham) was continued for another 30 min period at 37°C.

Results Isolation of 3T6 cells survived infection A desirable strategy for the isolation of such cells was one that could produce at least some cells having retained the whole viral genome, while allowing for relatively straightforward comparisons between all cells isolated. Therefore, we decided to challenge mouse 3T6 cells with several viruses (wild-type or mutant), but under only one set of conditions: a high m.o.i. and an incubation temperature of 39°C restrictive for the replication of the ts mutants. Three weeks after this initial exposure, colonies originating from survivors were isolated and subcultured in medium containing anti-polyoma virus antiserum. This eventually gave rise to lines, clones and subclones (see Materials and Methods), all cells deriving from the same initial colony constituting one lineage. Viral genetic information in lines and clones As soon as possible after isolation, lines and clones were tested for (1) the production of viral hemagglutinin at 33°C indicative of the persistence of a viral genome that could replicate autonomously at this temperature; and (2) the presence of viral DNA at 39°C detectable by the DOT test (Brandsma and Miller, 1980). Then, for each positive result in either test, all lines and clones belonging to the same lineage were examined for (3) the presence of viral DNA by the Southern (1975) technique (see next section). The lines and clones scoring positively in either test 1 or test 3 - which incidentally included all those positive in test 2 - were considered as having retained viral genetic information. The main conclusion from this analysis was that such ‘positive’ lines and clones were the majority (Table 1). As to the 36 lines and clones that scored negatively in both tests 1 and 2, only 8, representing a total of 5 lineages, were examined for the presence of viral sequences by the Southern technique, all with negative results. Finally, some lines and clones were found that scored negatively in one test and positively in another; these we will further consider below. Next, we focussed our attention on the positive lineages, those to which belonged at least one positive line or clone. This time, viral DNA detectable at 39°C and viral hemagglutinin produced at 33°C were quantified (Table 2). Thus, another striking observation was made: variable but often large amounts of free viral DNA were extracted from most cultures, even though they were derived from cells that had been routinely propagated in the presence of anti-polyoma virus antiserum since the picking of the individual colonies. However, some lines or clones which had been producing virus at one point in time were found at a later time to be completely free of viral DNA (see, for instance, clone B-b). Considering the tests upon which we relied, false positives were clearly improbable. The mere fact that the lines and clones in question belonged to otherwise positive lineages strongly suggested that

TABLE

1

PROPORTION TION Infection with

ts-P155 ts-52 ts-a NG-18 NG-59 P16 = Total

OF LINES

AND

Number

CLONES

HAVING

RETAINED

VIRAL

GENETIC

INFORMA-

Ratio 1 : 2

of lines or clones

Positive

Analyzed

(1)

(2)

17 12 3 5 9 14 60

25 21 9 5 9 27 96

0.68 0.57 0.33 1 1 0.52 0.63

Cell cultures were tested for the production of viral hemagglutinin at 33°C and for the presence of viral DNA at 39’C. In the latter instance, total cell DNA was digested with BglII, electrophoresed, blotted and annealed with radioactive viral DNA. When scoring positively in either one of these tests, the line or clone was compiled in column 1. a These survivors were not further characterized.

these positive results were indeed genuine (compare B-a with B-b). Furthermore, some lines and clones yielded DNA preparations that contained no free viral genomes, but a defective viral genome in an integrated form (see C and C-a), whereas for other clones, free viral genomes were found in some preparations, and only integrated viral sequences in others (5-1, 5-3, 3-a). Quite striking was the observation that in these instances, the fraction of viral DNA retained in an integrated form was not the same in all cells from the same lineage (compare C with C-a, and 5-1 with 5-3). In only three positive clones were we unable to demonstrate the presence of integrated viral sequences (B-b, C-l and l-4). Finally, in one clone (7-l) free viral DNA could not be detected in cells grown at 39°C while several copies of defective viral DNA were found covalently linked to host cell DNA (see below). We believe that these findings shed light on the mechanism whereby the whole viral genome persisted in cells of the majority of lines and clones, through many cell generations (see Discussion).

Free viral DNA in positive lineages: nondefective genomes When total cell DNA from routinely grown cultures was digested with a restriction enzyme that does not cleave polyoma virus DNA, such as BglII, and analyzed for the presence of viral DNA by the Southern (1975) procedure, free viral DNA was generally detected, and often found in large amounts (see Table 2). In all instances but one, this DNA co-migrated with marker polyoma DNA, as if consisting almost exclusively of cyclic non-defective viral genomes. This conclusion was verified by digesting the same DNA preparations with enzymes which cut polyoma virus DNA once, like EcoRI or BamHI, or several times, such as MboI or HpaII: the fragments thus generated migrated as those from similarly cleaved marker DNA (Fig. 1). Under our experimental conditions, however, a deletion or insertion smaller than 50

7 TABLE

2

VIRAL

GENETIC

Clone

ts-P155 B-a B-b C” C-a 4-3 4-4 5-1 5-2 5-3

INFORMATION Virus at 33°C (HAU per 100 mm petri dish) a

IN CELLS

FROM

POSITIVE

LINEAGES

Viral DNA at 39’C Free viral genomes (copies per cell)

6 400 6400 10 12800 5120 10 10 12800 800

2000-2500 0 0 0 50 800-l 000 5-10 200-250 800-l 000

ts-52 7-1 C-l c-4

100 10 1600

0 1-2 2000-2 500

ts-a l-4 2-l 2-5

10 12800 12800

5-10 1000-5000 50000-1OOooc

NG-18 l-a l-b 2-b

NT” NT NT

NG-59 2-a 3-a 3-b 4-c 5-b

NT NT NT NT NT

20-40 400 4000

200 200 100000 30000 30000

Integrated viral genomes (copies per cell) b

? 0

0.4 0.7 ? ? 0.7 ? 0.9

s-10 0 ?

0 ? ‘7

? ? 0

0.4 ? ?

Results shown in the different columns were obtained on separate occasions for a given clone or line, but always with cells between the 7th and the 9th passage after picking the initial colony. On average, the cultures analyzed for virus production and for the presence of viral DNA had gone through respectively 45-50 and 50-55 generations since exposure to virus. All results compiled here apply to clones rather than lines, with only one exception (C line). a Since 1 HAU represents about 10’ physical particles, one would expect to harvest 1 HAU of virus per 100 mm petri dish (ca. 10’ cells) if each cell produced one virus particle. As one infected mouse cell yields about lo5 virus particles, one would extract about lo5 HAU of virus from one petri dish if all cells in it were completing a lytic cycle. b The question marks in this column indicate an abundance of free genomes making detection of integrated viral sequences impossible. Otherwise, the number of integrated copies was assessed from the intensity of bands on autoradiograms of Southern transfers and, especially for those cells carrying less than a complete viral genome, from the mapping of the integrated sequences (see below). ’ C-a is a clone from line C. However, when each could be analyzed for virus production, both had gone through approximately the same number of generations. d The clones not tested (NT) for virus pr~uction had already been found positive in the DOT test.

8 a.

123456789

b.

123 MboI 4A

4B

Fig. 1. Viral DNA in clones derived from surviving 3T6 cells. (a) 5 pg of total DNA from cells grown at 39’C were treated with BarnHI, electrophoresed, blotted onto nitrocellulose paper, hybridized to 32P-labe1ed viral DNA and subjected to autoradiography (see Materials and Methods). Tracks 2 through 9: DNA from clones (ts-P155) 5-2, 5-3, B-a, 4-4, (ts-52) C-4, (ts-a) 1-4, 2-l and 2-5, respectively. Track 1 contains 10S4 pg of ts-P155 DNA digested with EcoRI. L: Linear form of ts-P155 DNA. Time of exposure was longer for tracks 1 and 7. (b) DNA from some of the preparations already analyzed in (a) was treated with Mbol and processed as before. Track 1: 2.5 x 10m5 pg of ts-P155 DNA: track 2: 5 pg of DNA from clone (ts-P155) 4-4; track 3: 5 pg of DNA from clone (ts-P155) 5-3. Fragment MboI-G (2.5 m.u. in size) was not detected on the autoradiogram.

base pairs (bp), equivalent to about 1 map unit (m.u.) on the physical map of polyoma DNA (see below), would have remained undetected. Judging from these results, therefore, the viral genetic information most frequently retained by cells surviving infection consisted in or was inclusive of the whole viral genome. Free viral DNA in positive lineages: defective genomes Clone (ts-P155) 5-l: Total DNA isolated from routinely grown cultures was found to contain on a per cell basis, 5-10 copies of a defective species, and about one copy of full-length polyoma DNA (Fig. 2a). The defective species, designated fg5-1(90%), was found to have a deletion of about 11.5 m.u., the deleted segment encompassing the BamHI site at 58.1 m.u. and the SstI site at 52.1 m.u. (Tooze, 1980). From these results and others not shown, we concluded that the deletion in fg5-1(90%) mapped entirely between 46.5 and 64 m.u. on the viral genome (Fig. 2b). With the hope of further characterizing fg5-1(90%), low molecular weight DNA was extracted from 5-l cells that had been transferred to 33°C. Indeed, fg5-1(90%) having a deletion in the late coding region (Tooze, 1980), one might have expected such transfer to the nonrestrictive temperature to activate the viral A gene, thereby triggering viral DNA excision and/or replication (Bourgaux et al., 1978). Somewhat surprisingly, the species of viral DNA that appeared to accumulate under these conditions was full-length polyoma DNA. However, the amount of DNA detected was still small, the equivalent of 150-200 copies per cell. If one assumes that these copies had been produced in lytic cycles triggered by the temperature shift-down,

9

such cycles would have involved less than 1% of the cells in the cultures. Incidentally, the integrated viral sequences mapped subsequently in 5-l cells represented only 70% of the polyoma genome (see below). *

*

56

1234

*

789

oc, :11-A

cc,

HindIll-A

b

-A

Hind 111-8 -B

II-

a

90

Fig. 2. See legend on p. 10.

10

B

10

Clone (ts-52) 7-l: No free viral DNA could be found in DNA preparations from cells grown continuously at 39°C. However, free viral DNA was regularly produced after a shift to 33°C. This DNA was extensively characterized and found to consist of two distinct species: one species with a deletion of 1.5 m.u. mapping on the viral genome between 67.4 and 70 m.u. (fragment PvuII-D), and a second species with the same deletion plus another of 15-16 m.u. located between 70 and 92.2 m.u. (fragment PvuII-B). Tandem arrays of both types of deleted genomes were found in an integrated form (data not shown). Integrated viral genomes in positive lineages Relatively few preparations of total cellular DNA were obtained that contained sufficiently low amounts of free viral DNA to allow for the detection of integrated viral sequences (see Table 2). In three of these rare instances, no integrated viral DNA could be found, even though the clones considered, (ts-P155) B-b, (ts-52) C-l and (ts-a) l-4, had been producing virus at an earlier passage. However, integrated sequences were found in DNA preparations, devoid of free viral genomes, from five clones and one line: (ts-P155) 5-1, 5-3, C, C-a, (ts-52) 7-l and (NG-59) 3-a. Physical mapping of the integrated viral sequences in line C and in clones C-a, 5-l and 5-3 was rather straightforward. Southern analysis of total cell DNA digested with an enzyme like BglII, which does not cleave polyoma DNA, clearly indicated the presence of a single insertion of viral DNA (not shown). After digestion of the same DNA preparations with enzymes which cut polyoma DNA once or more, of which PstI and especially MboI proved the most useful (Fig. 3) rather accurate maps of these insertions could be drawn (Fig. 4). Such maps were consistent with the presence of a single copy of a defective viral genome, different and flanked by different cellular sequences in each case. A common feature of the integrated genomes was the deletion of a portion of DNA coding exclusively for large T antigen (see Fig. 4). This is not surprising since the

Fig. 2. Free viral DNA in (ts-P155) 5-l cells. (a) Southern analysis of total DNA extracted from cells grown at 39°C. The DNA was analyzed as outlined in Fig. 1. Marker ts-P155 DNA was run in the three tracks marked by a star. Track 1: 5 X 1V5 pg of untreated DNA, in the covalently-closed (CC) and open circular (OC) forms; track 5: 10K5 pg of EcoRI-digested DNA. migrating as linear (L) Py DNA, and 10U5 pg of HindIII-digested DNA, migrating as fragments HindIII-A and HindIII-B; track 7: 5 X10-s ng of each of HincII-digested DNA and WI-digested DNA. The other tracks are run with 5 pg of total DNA from (ts-P155) 5-l cells. Digestion was with: track 2: BglII (no-cut enzyme); track 3: EcoRI (single-cut enzyme) - notice the strong band produced by fg5-1(90%) and the weak band produced by full-length molecules; track 4: BamHI (single-cut) - unlike full-length molecules (L), fg5-1(90%) remains uncut; track 6: Hind111 (two-cut) - the shortened HindIII-A from fg5-1(90%) co-migrates with HindIII-B; track 8: HincII (two-cut) - fg5-1(90%) produces a fragment shorter than HincII-A; track 9: SstI (multi-cut) - the strong band originates from a fragment of fg5-1(90%) which consists of sequences from both the viral A and B fragments. (b) Map of the fg5-1(90%) molecule. The cleavage site for EcoRI (R,) defines the O-100 m.u. position. The fragments identified by letters between the two concentric circumferences are those produced by MboI. The hatched segment stands for sequences which in genomic viral DNA are part of MboI-B or MboI-F and are joined here at some point between mu. 46.5 and 64.0, the 11.5 m.u. deletion having removed the MboI/BamHI site at 58.1 mu.

11 **

*

6

12345

*

789

10 11

Mbo A

b

B

b

4

C+D)

c

Fig. 3. Analysis of integrated viral sequences. Preparations of total cellular DNA and marker viral DNA (stars) were treated with PstI (left of figure) or MboI (right of figure) and analyzed as indicated in legend of Fig. 1. PstI digests: 5 gg of DNA from line C (track I), clone C-a (track 2) and clone 5-3 (track 3); 10V5 pg of ts-P155 DNA (track 4) and 5 x 10m5 pg of the same (track 5). MboI digests: 5 x lo-' pg of ts-P155 DNA (tracks 6 and 10); 5 Fg of DNA from clone 5-3 (track 7), clone C-a (track 8), line C (track 9) and clone 5-1 (track 11). Longer exposures of the same blot indicated quite clearly the existence of a fragment migrating as MboI-F in track 11.

absence of these sequences rendered the integrated viral DNA unable to excise and replicate, thus making its successful mapping much easier. One of the integrated viral structures, that of clone 5-1, displayed features worth stressing. Firstly, the early region was nearly - if not completely - preserved, its terminus mapping within a broadly defined segment of DNA that included the ‘right’ viral-cellular junction (see Fig. 4). As mentioned above, 5-1 cells had been found to carry two species of free viral DNA at 39°C one of which was amplifiable at 33°C. Secondly, the viral DNA ended at the left viral-cellular junction with sequences which bordered the deletion in fg5-1(90%). Even though fg5-1(90%) includes some viral sequences absent from the integrated viral structure, it is tempting to speculate that the two structures are related (see Discussion). A relationship of this nature was more obvious in the case of (ts-52) ‘7-l (see above). fn this clone, two species of free viral DNA were found, one carrying a 1.5 m.u. deletion, another carrying the same deletion plus a second one of 15-16 mu. Tandem arrays of both kinds of deleted genomes were found in an integrated form. However, the data appeared too complex to allow for drawing a map of the integrated viral DNA, or even decide about the number of separate viral insertions. Viral DNA in subclones

Curiously,

of(ts-P155)

the maps shown

C-a and 5-3

in Fig. 4 were difficult

to match

with what else we

2;

-..

3%

10

50

--a;-;b-;;o

6 oiloo-2

2.

-z-s

Fig. 4. Physical maps of viral sequences integrated in one line and three clones. In all four instances, the virus used in the selection was ts-P155. When clearly present, viral sequences are shown as empty boxes. Cellular DNA is shown by filled boxes. Hatched boxes define the limits within which were mapped the viral-cellular junctions. The physical map of Py DNA shown at the bottom is divided into 100 m.u., with the letters A to I identifying the fragments produced by Mbol (Tooze, 1980). The ‘enhancer region’ is positioned according to de Villiers and Schaffner (1981) and Tyndall et al. (1981). In the case of clone 5-1, it is unclear whether the BamHI/MboI site at the left of the viral insertion is viral or cellular: no viral site could be unambiguously mapped at the left of it; yet, digestion of DNA from 5-I cells with MboI seems to generate viral fragment F (see Fig. 3).

knew about the viral genetic information associated with the clones. ‘1he integrated viral genomes mapped were all defective, even though they were those of cells whose ancestors had proven able to produce whole virus (see Table 2). One way to explain such an apparent paradox was to assume that all populations studied had been initially heterogeneous, but that mapping of integrated viral sequences had always been carried out when a dominant class of cells had outgrown all others. We decided to explore this possibility using two of the so-called clones for which it appeared most likely. Frozen cells from the first stocks made after isolation of clones (ts-P155) C-a and 5-3 were thawed, put into culture and re-cloned two or three times consecutively as described in Materials and Methods. Three and thirteen passages after such re-cloning, total cell DNA was isolated from each of the ten resulting subclones and examined for the presence of free or integrated viral sequences by the Southern procedure (1975) as described above. Only integrated viral sequences were found in this manner. These sequences were mapped after digesting the DNA either with two enzymes known to cleave the viral insertion in the parental clone, each within a short distance from one of the viral-cellular joints (EcoRI and Sal1 for 5-3, BamHI and XbaI for C-a, see Fig. 4); or with one enzyme making several cuts

13

1 2 3 4

5 6

7 8 9 10 11 12 13 14 15 16 17 18 19

Fig. 5. Integrated viral sequences in derivatives of clone (ts-P155) 5-3. Samples of 5 pg of total DNA from various subclones were treated with MboI and analyzed as indicated in legend of Fig. 1. Each pair of tracks from 1 through 18 corresponds to a different subclone, from 5-3-l to 5-3-9, analyzed 3 (odd-numbers tracks) and 13 (even-numbered tracks) passages after isolation. Track 19 was run with 5 x lo-’ pg of Mbol-digested ts-P155 DNA. Notice that the fragments including the viral-cellular joints, indicated by the empty circles on the left of the panel, migrated as if they were the same in all digests of cellular DNA.

the viral insertion, MboI in both instances. Hence, blots such as that shown in Fig. 5 were obtained. These blots indicated that, regardless of passage number, the integrated viral sequences in the subclones were neither different from those already found in the parental clones, nor linked to the adjacent cellular DNA in a different manner. These data demonstrated that the populations of 5-3 and C-a cells that were subjected to subcloning were not grossly heterogeneous. Failure to find subclones carrying a complete viral genome may indicate that cells possessing them were either too few or too quickly disappearing in the population at the time of subcloning to be detectable in this fashion. Incidentally, the complete disappearance of free viral DNA is consistent with the expected inability of the ts viral genome to replicate autonomously at 39°C.

within

Permissivity of mouse cells surviving infection The data reported above raised the question of the mechanism whereby the cells under study had insured their survival. Had the survivors remained permissive to polyoma virus replication or not? The procedure used for the selection of these survivors was not likely to produce resistant cetls; that is, cells that would be unable to take up virions (Herring et al., 1980a, 1980b). Therefore, the problem under consideration could be addressed readily in an experiment of superinfection. Cells from four representative clones and one line were examined for the synthesis of viral capsid antigen following infection with ts-P15.5 or mock-infection (Table 3). Thus,

14 TABLE

3

PRODUCTION EAGES Cells

OF POLYOMA

VIRUS

CAPSID

ANTIGEN

After mock-infection’

BY CELLS

After infection

FROM

POSITIVE

h

Expt. 1

Expt. 2

Expt. 3

Expt. 1

Expt. 2

Swiss 3T6

i 0.2%(O)

< 0.01 %(O)

i 0.01 %(O)

66%(338)

49.6%(248)

ts-P155 C C-a 5-l

i 0.2%(O) < 0.2%(O) < 0.1%(O)

< 0.01 S(0) < 0.01 %(O) i 0.01 %(O)

< 0.01%(0) < 0.01 %(O) < 0.01%(O)

12.4(62) 34%(171) 5%(26)

14.3%(71) 8.9%(45)

11.6%(116) 40.2%(402) 8.3%(830)

NG-18 I-a

0.06%(6)

i 0.01 %(O)

0.02%(Z)

0.16%(16)

1.0%(5)

0.7%(70)

NG-59 3-b

0.6%(3)

4.8%(481)

2.6%(13)

2.8%(14)

5.7%(570)

3.5%(349)

LIN-

Expt. 3 -

Non-confluent cultures from uninfected 3T6 cells, the C line and from the four clones specified in the table were either mock-infected or infected with ts-P155 (10 pfu/cell) and examined after 48 h of incubation at 33°C for the synthesis of viral capsid antigen by the immunofluorescence test (see Materials and Methods). Only those cells with a brightly fluorescent nucleus were scored as positive. ” The number of microscope fields scanned was such that about 500, 1000 or 10000 cells were examined. The proportion of positive cells, estimated in relation to these totals, is given here as a percentage. The figures in brackets represent the number of positive cells actually counted. h Both positive and negative cells were counted. before the percentage of positive cells was calculated. Positive cells counted are indicated in brackets.

three main observations were made. Firstly, only those cells stably associated with a nondefective viral genome, i.e. the cells from clones (NG-18) l-a and (NG-59) 3-b, produced detectable viral capsid antigen in the absence of superinfection: as expected from other results (see Table 2), (NG-59) 3-b proved then the most positive of these two clones. Secondly, l-a and 3-b were the two clones responding least to superinfection. Finally, (ts-P155) C, C-a and 5-1, were found to be quite permissive for the replication of superinfecting virus. The significance of the apparent difference in permissivity between 3T6 cells and, for example, the C or C-a cells, is unclear. Indeed, growth kinetics are not comparable as, unlike the 3T6 cells which multiply slowly and are markedly contact-inhibited, the C and C-a cells grow as transformants (E. Herring-Gillam, unpublished observations).

Discussion The main conclusion from these studies is that mouse 3T6 cells which multiply after infection by polyoma virus often retain nondefective viral genetic information for at least 50 generations. Given the permissive nature of mouse cells and also the ability of polyoma DNA not only to replicate, but also to integrate into and excise from cellular DNA, the finding that such information almost invariably exists in the form of free viral molecules is hardly surprising. However, the mere presence of

15 these free molecules makes the search for integrated viral sequences particularly difficult. Among the possible long-term interactions between viral and cellular genomeswe must consider first the chronic infection - or carrier state - and the perpetuation of the viral genome as a freely replicating plasmid. It is however difficult to understand how chronic infections could proceed with such regularity through successive clonings performed in medium containing antipolyoma serum, a procedure which we have noted for its effectiveness in neutralizing virus progeny (Bourgaux et al., 1978). Persistence of viral DNA in a free plasmidic form appears as a more realistic possibility, even though it is not completely convincing. Indeed, in the majority of instances considered, infection had been carried out using a ts viral genome. Although one would expect transient replication of a ts genome after a high multiplicity infection at 39°C such replication would be unlikely to persist under the conditions used to propagate the survivors. Even in clones where many free copies of viral DNA were produced at 39°C such as (ts-a) 2-5 (see Table l), it appeared that the viral genome retained was still ts (E. Herring-Gillam, unpublished observations). Therefore, stabilization of the thermolabile viral protein by cellular factors, already believed to occur in the case of similar mutants of SV40 (Imbert et al., 1983), might be responsible for the presence of high amounts of free viral DNA in some clones. Other clones, like (ts-P155) 5-l and (ts-52) 7-1, clearly demonstrated that the restriction imposed on viral DNA replication by the ts mutation was still operative in some cloned cells. Considering the existence of such restriction, it would seem that the ts viral genome would have a greater probability of surviving in dividing cells if incorporated into the cellular DNA. Only under certain conditions would excision followed by autonomous replication occur, resulting eventually in cell death and the release of virus progeny. Even though this interpretation appears the most likely (see also Zouzias et al., 1980) we have no direct proof that it applies to the majority of clones studied. In a small number of cases, partial or total defectiveness of the resident viral genome with respect to autonomous replication has allowed us to demonstrate the presence of integrated viral sequences. However, it was not completely clear at first how the defective sequences were related to the nondefective ones which had invariably pre-existed in these already cloned populations. Were the integrated defective genomes the remnants of integrated nondefective genomes, or the result of direct integration of defective molecules which had been replicating freely? Some data incline us to favor the former explanation. Firstly, free viral DNA found in cells grown at 39°C was always nondefective, except in the case of clone (ts-P155) 5-1. Therefore, the viral genetic information which we found integrated is not representative of the bulk of the free viral DNA. Actually, this consideration even applies to clone (ts-P155) 5-1, as the integrated viral sequences mapped in this clone carry a larger deletion than that found in fg5-1(90%). Secondly, two of those clones in which integrated viral sequences could be demonstrated provide us with information suggesting that free molecules indeed originate from integrated sequences. For instance, in the case of (ts-52) 7-1, free viral DNA is not detected unless the cells are shifted to 33°C. Of the two defective species produced at this temperature, one

16 carries a large deletion which renders it unable to code for large T antigen. Such a species is not expected to replicate without assistance from another viral genome, an unlikely requirement for a molecule which would have perpetuated itself as a plasmid. In the case of clone (ts-P155) 5-1 also, two species of free viral DNA were found, but neither one could have originated from the integrated sequences which we succeeded in mapping. One species was genomic viral DNA, the other fg5-1(90%): one of the boundaries assigned to the deletion in the latter species was similar to one of the boundaries assigned to the integrated viral structure (see Figs. 2 and 4). However, the two other boundaries were different, as if the integrated viral structure mapped had evolved through one more deletion from that which had been the precursor of f@-1(90%). In view of these considerations, the most remarkable feature of our results is that. while highly characteristic integrated viral structures could be assigned to twice cloned populations, cells from these clones had been ~ or were - producing free viral DNA distinct from that found integrated. These findings may not be contradictory, as the populations derived from the survivors may have been composed initially of several types of cells with different growth potentials. Indeed, the maps shown in Fig. 4 show little resemblance between either 5-l and 5-3, or C and C-a, whether from the point of view of the viral sequences or from that of the flanking cellular DNA. This means, for instance, that passing cells from the C colony without further cloning must have selected against C-a cells. A striking observation made with respect to the integrated viral structures is that we failed to identify similar sister clones or dissimilar subclones. This observation suggests that the event responsible for the final arrangement of sequences at and around the integration site occurred before &FW isoiation, that is before the nondefective free genomes tended to vanish. Thus, it is only after that event had occurred that cells carrying only a defective viral genome could emerge and outgrow those with a nondefective viral genome. Our data are consistent with the idea that the survivors which were at the origin of the positive lineages generally took up within their DNA, an insertion of viral DNA containing at least one complete copy of the polyoma genome (Della Valle et al.. 1981). While compatible with cell multiplication, the presence of this insertion generally resulted in the production of free viral DNA and virus progeny in a fraction of the cell population. Deletions - affecting the insertion only or both the insertion and the flanking sequences - sometimes generated cells which outgrew their parents. As indicated by the recloning of (ts-P155) C-a and 5-3, integrated viral DNA carrying large deletions within the early coding sequences proved remarkably stable upon subsequent cell passages, in agreement with the concept that rearrangement of viral sequences is actuated by large T antigen (Basilic0 et al., 1979; Colantuoni et al., 1982). Such deletions may also impart a cellular growth advantage, as in vivo experiments suggest that cells carrying a complete viral early region are subjected to a negative selection (Lania et al., 1981). Finally, it may be that these deletions made it possible for the cells to remain permissive for virus replication (see Table 3). Interestingly, cells having survived infection by an hr-t mutant virus appeared to carry viral genetic information which had suffered no deletion, but whose replication was subjected to some restriction.

17 Many of the difficulties which we encountered in this study came from the permissive nature of the cells studied. Our main conclusion justifies the effort. Indeed, the fact that mouse cells can multiply while carrying a complete polyoma genome is likely to have some influence on the course of polyoma virus infection in nature.

Acknowledgements This work was supported by a grant awarded to P.B. and D.B. by the Medical Research Council of Canada. CR. is the recipient of a postdoctoral fellowship from M.R.C.C. We thank P. Chartrand for critical reading of the manuscript and W. Eckhart and T. Benjamin for the supply of virus mutants.

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8 April 1985)