Infection of rat cells by avian sarcoma virus: Factors affecting transformation and subsequent reversion

VIROLOGY

106,217-233 (1980)

Infection of Rat Cells by Avian Sarcoma Virus: Factors Affecting Transformation and Subsequent Reversion JOHN A. WYKE AND KRISTINA Imperial

Cancer Research Fund,

P.O. Box 125, Liacoln’e

Inn

QUADE

Fields,

London

WC2A SPX, England

Accepted May 25, 1980

We have investigated factors that influence (1) the initial incidence of transformation in rat cells infected by avian sarcoma virus B7’7 and (2) the transformed phenotype of the cell and its stability. Transformation is not limited to a genetically highly competent subpopulation of either viruses or cells but can potentially involve any of the viruses or hosts. The kinetics of transformation are one-hit, even at input virus multiplicities of infection (m.o.i.) of 1 to 10, suggesting that a single virus particle is sufficient to induce transformation and that there is not a physiologically highly susceptible cell population whose response reaches a plateau at high m.o.i. However, as judged by levels of virus recovery and morphological reversion, transformed clones obtained after infection at high m.o.i. show a more uniform virus expression than those obtained at low m.o.i. Moreover, analysis of integrated proviral DNA in cloned transformed cells indicated that seven clones transformed at low (0.01) m.o.i. contain only single proviruses whereas five clones derived from high (1 or ‘7.5) m.o.i. infections each bear a minimum of three integrated proviruses. These findings suggest that, in the high m.o.i. transformants, multiple viral genome copies contribute to the transformed cell phenotype, making it unlikely that transformation is due simply to randomly occurring virus-cell interactions. Studies on transformants obtained at low m.o.i. show that heterogeneity in virus recovery and morphological reversion can be stable but is not, in most cases, due to virus mutants. Since levels of virus recovery and morphological reversion in these clones did not show a uniform inverse relationship, it is also unlikely that their phenotypic variability is due simply to modulations in overall levels of virus expression. Most revertants isolated from clones transformed at low m.o.i. show decreased but detectable rescuability of transforming virus, suggesting that virus expression is reduced, but reversion by other mechanisms has also been detected in these clones (Varmus et al., 1980).

Levinson et al., 1978), full virus expression and production of progeny ASV very rarely The structure and function of the trans- occur (Eisenman et al., 1974; Brugge et al., forming src gene of avian sarcoma viruses 1977; Deng et al., 19’77; Krzyzek et al., (ASV) is being elucidated and it is now im- 1979). Thus, virus spread is minimized and, portant to ask whether and how expression since mammalian cells are readily cloned, it of this gene and the action of its product is is feasible to study the outcome of single modulated in the host cell by both viral and virus-cell interactions. (3) The endogenous cellular factors. ASV-transformed mam- viruses of mammalian hosts are not closely malian cells seem promising tools for such related genetically to ASV and so are unstudies for the following reasons: (1) likely to confuse experimental analyses. Transformation of mammalian cells is main- (4) ASV-transformed mammalian cells octained by the action of the viral arc gene casionally revert to a nontransformed (Graf and Friis, 1973). (2) Although trans- phenotype (Macpherson, 1965; Boettiger, formed mammalian cells contain the STC 1974b). This reversion could occur by sevgene product pp60”‘” and other virus- eral different mechanisms involving the specific proteins (Brugge et al., 1978; functioning of the src gene and its product INTRODUCTION

217

00426822/80/140217-17$02.00/O Copyright All rights

0 1980 by Academic Press. Inc. of reproduction in any form reserved.

218

WYKE

AND QUADE

(for review see Wyke et al., 1979) and identifying these mechanisms should help in understanding viral transformation. Transformation of mammalian cells by ASV is, however, a relatively rare event. Some strains of ASV, such as B77 and viruses of envelope subgroup D, efficiently penetrate mammalian cells (Boettigeret al., 1975) and synthesize and integrate proviral DNA in a large proportion of infected cells (Varmus et al., 1973, 1974), but even these mammal-tropic viruses transform permissive chicken cells at least lo2 times as efficiently as they transform mammalian hosts (Altaner and Temin, 1970; Boettiger, 1974a). There is, however, evidence that many, perhaps even a majority, of mammalian cells exposed to high multiplicities of B77 harbor proviruses without showing an altered phenotype (Boettiger, 1974a; Varmus et al., 1974). These “silently” infected cells may, on prolonged culture, segregate transformed variants (Boettiger, 1974a; L. Turek, H. Oppermann, and P. K. Vogt, personal communication). The studies outlined above have involved various virus strains and mammalian host species, so they are not all strictly comparable. Hence, we wished to investigate the transformed phenotype and, in particular, the mechanism of phenotypic reversion in a single line of rat cells infected with a cloned B77 strain ASV. However, since only a small minority of ASV-infected mammalian cells permit expression of the src gene soon after infection, cells that are destined to become transformed are not necessarily representative of the majority of uninfected ceils which are the usual controls for work on transformation. For this reason we have prefaced our studies on reversion by investigating factors which affect the outcome of ASV infection, either by altering the incidence of cell transformation or by modifying the phenotype of the transformed cell. Our most important conclusions from these initial investigations are that cells transformed by infections at low multiplicity show a hitherto unreported variability of phenotype not observed in transformants obtained at high virus multiplicity, presumably because the latter transformants have a phenotype determined by more than

one proviral genome. A preliminary report of these studies was presented at the 44th Cold Spring Harbor Symposium on Quantitative Biology (Wyke et al., 1979). MATERIALS

AND METHODS

Cells and viruses. The F2408 line of Fischer rat cells (Mishra and Ryan, 1973), referred to here as Rat-l, was obtained from Dr. A. M. C. Fried, (I.C.R.F., London) and subcloned. A single subclone, E5, was used for most studies but some experiments also utilized a second subclone, 208F (Quade, 1979). Rat-l and its derivatives were routinely cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum (Gibco Europe, Glasgow, Scotland). Cloning of Rat-l cells, transformed Rat-l, and the revertant derivatives of transformed clones was achieved by aspirating isolated colonies of appropriate morphology, growing a small cell population, and cloning from this population by micromanipulation (Macpherson, 1969a). B77 strain ASV was originally obtained from Dr. P. K. Vogt and subcloned in this laboratory. A clone showing good tropism for mammalian cells was isolated in 1973 and studies on this clone were reported by Boettiger (1974a) who called it B77,. The work described here mainly used a subclone, B77, (v), and its derivatives. Since we obtained the B77 stock it has been grown and assayed in chick helper factor positive (chf+) Brown leghorn x White leghorn chick embryo cells (Wickham Laboratories, Wickham, Hampshire, England) cultured by standard techniques (Vogt, 1969). Virus was cloned by aspirating single, well-spaced foci of transformed chick cells from under agar. Infection and transformation of Rat-l by BY?‘. To infect monolayer cultures 2 x lo5 Rat-l cells were plated in 35mm plastic tissue culture dishes (Nunc A/S, Roskilde, Denmark) and after attachment were exposed at 37” to a 2 ml virus inoculum. For infection in suspension a l-ml aliquot of cells and virus was kept in a plastic tube for 2 hr at room temperature, shaking occasionally. Foci of transformed cells ap-

TRANSFORMATION

OF RAT CELLS

peared in monolayer culture and were counted 1 to 2 weeks after infection, expressing titers as focus forming units (FFU)/ml (where accurate quantitation was needed cultures were sometimes overlaid 1 day postinfection with medium containing 0.5% agar). Transformation was also assayed by suspending cells in 0.33% agar medium (Macpherson, 1969b) and counting colonies after 2 to 3 weeks. Infections were sometimes performed in the presence of 1 a dexamethasone (Sigma, Poole, England) and/or 2 pg/ml polybrene (hexadimethrine bromide, Aldrich, Gillingham, England). All infections were performed with virus which was freshly cloned to minimize the levels of defective virus in the stock. Rescue of infectious virus from Rat-l cells. The method of Steimer and Boettiger (1977) was used. Briefly, Rat-l cells were killed with mitomycin C (Sigma) and known numbers were seeded on monolayers on chf+ chick embryo cells. The next day the cells were fused using polyethylene glycol (PEG 6000; Koch-Light, Colnbrook, England) and were overlaid with agar medium a day later as in a standard focus assay (Vogt, 1969). Preparation and analysis of Rat-l cellular DNA. Confluent monolayer cultures were washed once, then l-3 ml of DNA buffer (Varmus et al., 1980), containing 0.5% sodium dodecyl sulfate (SDS) and 100 pg/ml proteinase K (BDH Chemicals, Poole, England) was added and the dishes were incubated a further 12-16 hr at 37”. The cell extracts were deproteinized by sequential exposure to phenol (once), phenol:chloroform:isoamyl alcohol (25:24:1; PCI) (at least twice, or until no material was present at the interface), and lastly once with chloroform:isoamyl alcohol (24:l; CI). The DNA was dialyzed in 3-4 changes of 10 m.ilf Tris-HCl, pH 8:l mM EDTA. Contaminating RNA was degraded by addition of RNase T, (Sigma) to 20 units/ml and RNase T, (Sigma) to 0.5 units/ml for at least 4 hr at 37”. NaCl was added to 100 mM and the mixture was extracted once with PC1 and once with CI. The DNA was precipitated with ethanol, spooled, washed three times with 70% ethanol, air-dried,

BY ASV

219

and finally resuspended in 5 mM Tris, pH 7.5:0.5 n&f EDTA. DNA concentrations were estimated by the method of Burton (1956) and purity was judged both by the ratio and by testing for conObdOD,,, taminating nuclease in the presence of 10 nil4 MgCl,. For digestion of DNA with restriction enzymes 10 pg of DNA plus enzyme and buffer were incubated in 30 ~1 at 37” for at least 3x the necessary time calculated from the enzyme/substrate ratio. Digests with BglII (Bethesda Research Laboratories, Rockville, Md.), Hind111 (New England Biolabs, Beverly, Mass.), and KpnI (New England Biolabs) were performed in accordance with the manufacturers’ instructions, and digests with EcoRI (Miles Laboratories, Slough, England) as described by Arrand et al. (1974). The completeness of digestion was monitored by incubating 10% of the final reaction mixture with either 250 ng polyoma DNA (kindly provided by Dr. J. R. Arrand I.C.R.F., London) for EcoRI, HindIII, and KpnI, or 500 ng of lambda DNA (New England Biolabs) for BglII. Molecular weight markers of 14.5, 5.9, 4.1, 2.7, 1.4, and 1.2 x lo6 M, (Daniels et al., 1980) were prepared from digests of lambda DNA with Hind111 labeled as described by Arrand and Roberts (1977). DNA digests were electrophoresed in 0.8% SeaKem agarose (Uniscience, Cambridge, England) gels, essentially as described by Humphries et al. (1979), and transferred to 0. l-pm Sartorius membrane filters (V. A. Howe and Co., London, England) by a modification of the procedures of Southern (1975). Viral 70s RNA was prepared from the supernatants of Prague A strain ASVinfected chick embryo cultures essentially as described by Smith and Quade (1976) except that the RNA was first poly(A) selected by chromatography on poly(U)sepharose (Pharmacia, Uppsala, Sweden) (Eiden and Nichols, 1973) prior to isolation of the 70 S RNA complex by sucrose gradient sedimentation. 32P-labeled DNA complementary to the viral genome (cDNA) was prepared by incubation of 1 pg RNA with 1 mg calf thymus DNA primers (Taylor

220

WYKE AND QUADE

VIRUS

INOCULUM

FFU-2~10~

2x105

2x104 VIRUS

ld06-FFU

INOCULUM

FIG. 1. Transformation of 2 x lo5 Rat-l cells in monolayer at different multiplicities of infection (m.0.i.) of B77&). (A) Rat-l clone E5. (B) Rat-l clone 208F; note the lower level of transformation of this clone. 0, No additives; A, 1 M dexamethasone added at time of infection; n , 2 pg/ml of polybrene added at time of infection; V, both dexamethasone and polybrene added at time of infection. Closed and open symbols in A represent two separate experiments performed at an 18 month interval. Each point is the mean of two to six determinations. To aid clarity lines are fitted, and ranges and number of determinations for each point (in parentheses) are given only for the symbols 0 and ‘1.

et al., 1976; kindly provided by Dr. C. Dickson, I.C.R.F., London) and 20 units avian myeloblastosis virus reverse transcriptase (provided by Dr. J. Beard, Life Sciences, St. Petersburg, Fla.) in 50 mM Tris-HCl, pH 8.38 m&f MgCl,:lOO mM KC1:20 mM dithiothreitol:200 fl each dATP, dGTP, and dCTP, and 100 &i [ a-32P]dTTP (~350 Wmmol, The Radiochemical Centre, Amersham, England) for 2 hr at 37”. Unincorporated dTTP was removed by chromatography on G-100 Sephadex (Pharmacia) and the peak fractions were pooled, made 0.5% in SDS, and extracted once with PCI. The cDNA probe was made 100 mfl4 in NaOH and after 1624 hr was neutralized to approximately pH 7 and made 1 mM in EDTA. Hybridization conditions were essentially as described by Shank et al. (1978) except that only 10 @g/ml yeast RNA was used, supplemented with 10 pg/ml of ribosomal RNA prepared from Rat-l E5 cells. Un-

annealed cDNA was removed as described by Hughes et al. (19’79). After drying, the filters were exposed to Fuji Rx Medical X-ray film at -70” in the presence of a DuPont Cronex Lighting plus intensifying screen (Swanstrom and Shank, 1978). RESULTS

Transformation of Rat-l cells by B77, (v) is linearly related to input virus multiplicity with a slope of 1 (Fig. 1). This agrees with the results of Altaner and Temin (1970) and shows that a single virus particle is able to transform a cell. However, from Fig. 1 it can be calculated that transformation of chick cells by B77,(v) is in the order of lo3 to lo4 times more efficient than transformation of rat cells. There are several possible reasons for this observed low incidence of Rat-l transformation. (1) Transformation is a property of

TRANSFORMATION

221

OF RAT CELLS BY ASV

genetically variant subpopulations of viruses or cells. (2) Transformation occurs only in rare cells which are physiologically altered (for example in showing an unusual pattern of transcription) or through the agency of physiologically abnormal viruses. (3) Transformation results from a random event of low probability which can occur in any virus-cell interaction. For example, though B77 proviruses can integrate at many different sites in mammalian cells (Hughes et al., 1978) there may be a limited number of sites at which integration results in stable cell transformation and if integration is random the “target size” for transformation may thus be far smaller than the whole cell. Such random probabilities may of course be superimposed on a background of genetic or physiological heterogeneity. (4) Transformation occurs more frequently than we observe but our methods of detecting it in this system may be too insensitive. A number of experiments have been performed in attempts to distinguish these possibilities. Genetic Variation The possibility that transformation is due to virus mutants that are very efficient at transforming rat cells or to cell mutants that are very susceptible to B77 transformation is one with greatest implications for subsequent studies on the transformed cells. Certain ASV strains clearly have a genetically determined ability to transform mammalian cells which is due in part (Boettiger et al., 1975), though probably not entirely (Altaner and Temin, 1970), to their envelope properties. Within the mammaltropic ASV strain B77,, five different subclones all transformed Rat-l cells with an efficiency lop5 to 2 x lop4 times their efficiency on chick cells (Table 1, part A), suggesting that all B77, have the ability to transform rat cells with low probability. These observations, together with the finding of Boettiger (1974a) that B77, rescued from “silent” infections of NRK rat cells can nonetheless transform fresh NRK, do not rule out the possibility that certain

TABLE 1 RELATIVE TRANSFORMINGCAPACITY OF B77 CLONESON CHICK AND RAT-~ CELLS

A. Subclones of B77,

Clone B77,(i) B77,Jii) B77,(iii) B77,(iv) B77,(v)

Relative eot” 9.3 6.8 1.3 5.2 2.0

x x x x x

10-S 10-j 10-4 lo-” 10-J

B. B77,Jv) rescued from transformed Rat-l, clone E5, cells Clone” A21 (1) A21 (5) A31 (1) A31 (6) A + 11 (1) A + 11 (6)

Relative eot 6.8 1.4 4.2 1.3 2.3 4.6

x x x x x x

lo-” 10-s lo-” 10-q lo-” lo-”

(1Relative efficiency of transformation (cot) = FFU on Rat-l, clone E5/FFU on chick. * A21, A31, and A + 11 are clones of B77-transformed Rat-l, E5 cells obtained from the experiments shown in Figs. 2 and 3. Their characteristics are described in Table 4. Virus was reseued from these clones by PEG-mediated fusion to chick cells. For each cell clone, two rescued virus clones (identified by numbers in parentheses) were tested for their ability to transform Rat-l cells.

virus mutants transform Rat-l very efficiently and arise regularly in subclones of B77,. Indeed, the 20-fold fluctuation in efficiency of transformation among subclones of B77, (Table 1, part A) suggests that this might be the case. However, this explanation seems unlikely, since viruses rescued from transformed Rat-l or NRK cells are no more mammal-tropic than the viruses used for the original infection (Table 1, part B and Boettiger, 1974a). Applying similar arguments to the host cells, Rat-l 208F, Rat-l E5, and eight subclones of Rat-l E5 show comparable susceptibilities to transformation by B77, (Table 2, part A). These results, together with the observations that (1) NRK cells carrying unexpressed ASV genomes are susceptible to retransformation (Boettiger, 1974a) and (2) many originally nontransformed cells may later segregate transformed variants (L. Turek, H. Oppermann, and P. K. Vogt, personal communication), all suggest that there is not a population of rat cells that is highly susceptible to B7i’. It remains possible that such susceptible

222

WYKE AND QUADE TABLE 2

of Rat-l cells is not due to a highly competent subpopulation of either viruses or cells.

RELATIVE TRANSFORMING CAPACITY OF B77,W ON DIFFERENT CLONES OF RAT-~ B”

A

Clone Rat-l, E5 Subclone 3 Subclone 4 Subclone 5 Subclone 6 Subclone 7 Subclone 8 Subclone 9 Subclone 10 Rat-l, 208F

cot*

2.5 0.9 0.7 1.1 1.5 0.7 1.7 0.8 0.2

CloneC B31 Revertant Revertant Revertant Revertant Revertant Revertant Revertant Revertant Revertant Revertant

clone B clone C clone E clone F clone H clone I clone J clone M clone N clone 0

eotb

1.2 0.1 1.0 4.5 0.3 2.1 0.8 6.6 1.2 0.9

a The data in this column are also shown in Varmus et al. (1980). * Transformation efficiency relative to that on Rat-l, clone E5 (=l.O). c B31 is a clone of B77-transformed Rat-l, E5 obtained from the experiment shown in Figs. 2 and 3. This clone has yielded revertants, morphologically very similar to uninfected Rat-l, either by complete loss of the single integrated B77 provirus (revertants H and 0) or by mutations resulting in a loss of function in the B77 src gene (all other revertants). There are no apparent host cell factors involved in this reversion to a normal phenotype (Varmus et GE., 1980).

cells arise regularly in all cell clones, but the results in Table 2, part B make this unlikely, for they show that revertants of cells which had been transformed are no more susceptible to transformation than the original Rat-l cells. The revertants in Table 2, part B have become normal by loss of the B77 provirus or by mutation in the proviral src gene, not by suppression of viral functions (Varmus et al., 1980). Thus, their failure to show high susceptibility to B77 transformation is probably not a result of the reversion mechanism. It is noteworthy that rat cells transformed by ASV with a temperature-sensitive src gene show a susceptibility to wild-type virus transformation at restrictive temperature equivalent to that shown by uninfected cells (Chen et al., 1977; J. A. Wyke, J. A. Beamand, and S. Evans, in preparation). Thus available evidence suggests that B77 transformation

Physiological Events

Variation

and Random

Since genetic heterogeneity does not explain the low transformation efficiency of Rat-l cells by B77 we presume it is due either to a small proportion of physiological variants among the viruses or cells or to the random, low probability occurrence of appropriate, and perhaps specific, virus-cell interactions. As discussed below, our experimental results make it impossible to distinguish these two explanations. To some extent this failure is not important, for both possibilities have similar implications: (1) any transformed cells which may be the subject of subsequent studies contain viral and cellular genomes which are representative of the genomes of the original virus and cell populations and are not rare variants and (2) since transformation is not a property of only a small proportion of the viruses and cells it may be possible to alter transformation frequencies by experimental manipulations. Some investigations of this latter point are shown in Fig. 1. The response of transformation of input virus multiplicity remains linear and shows no tendency to reach a plateau even when the multiplicity of infection is 5 to 10 FFU per cell. At these multiplicities almost every cell should be exposed to virus and the lack of a plateau is evidence against a physiologically highly susceptible subpopulation of cells. Moreover, transformation can be increased several-fold if cells are infected in the presence of either the polycation polybrene (2 pg/ml) or the synthetic steroid dexamethasone (1 a). The effects of polybrene and dexamethasone are seen when B77 infects Rat-l E5 (Fig. lA), 208F (Fig. lB), and secondary Lewis rat embryo fibroblasts (P. Vesely and J. A. Wyke, unpublished data) so they do not seem a peculiarity of individual cell clones. In the presence of these additives the transformation frequency still shows a linear relationship to virus multiplicity with no evidence of

TRANSFORMATION

OF RAT CELLS

saturating the population of susceptible cells. Polybrene probably increases transformation by enhancing virus adsorption to cells (Toyoshima and Vogt, 1969). Dexamethasone probably acts by a different mechanism for its effect is additive to that of polybrene when both are present (Fig. 1). The Effect of Virus Multiplicity on the Phenotype of B77 Transformed Rat-l The results described above suggest that there is not a physiologically variant subpopulation of Rat-l cells which is highly susceptible to transformation by B77. The findings are, however, consistent with the concept that transformation results from a rare and randomly occurring virus-cell interaction in which the kinetics are one-hit but the target is smaller than the individual rat cell. Such a situation would pertain, for example, if proviruses integrate randomly but are expressed only at a limited number of sites. A corollary of this model is that when a cell carries more than one integrated provirus it would be very rare for more than one of these proviruses to be expressed and to contribute to the transformed cell phenotype. To test this concept a number of transformed clones were isolated by micromanipulation after infecting Rat-l E5 cells with B77,(v) at multiplicities of 0.01, 0.1, 1.0, and 7.5 FFU per cell in the presence or absence of 1 fl dexamethasone. We reasoned that transformants obtained at the higher multiplicity might contain multiple proviruses, while this was very unlikely in cells transformed at m.o.i. 0.1 or less. The transformed clones were tested for their efficiency of plating (cop) on a plastic substrate and in 0.33% agar suspension, and morphological peculiarities were noted. The eop of different clones on plastic varied from 19 to 85% when lo3 cells were seeded in a 60-mm dish and the eop in suspension showed even greater variation. However, there was no correlation between eop and the original multiplicity of infection; for example, transformants derived at m.o.i. 7.5 showed the same range of eop as other transformants (25% to 85%). The morphology of the clones was also variable;

BY ASV

223

transformed cells showed a rounded, fusiform, or “cobblestone” appearance, but here again there was no correlation with the original m.o.i. and indeed the morphology within a single clone could show great variation. A correlation with m.o.i. emerged, however, when we studied the ease of virus rescue from these cells and the incidence of phenotypic reversion to normal morphology. It is perhaps significant that these two parameters of the transformed cell may reflect directly the level of expression of the virus genome, for previous workers have shown reversion to be associated with both decreased levels of virus recovery and decreased levels of virus-specific RNA (Boettiger, 1974b; Deng et al., 1974; 1977). It cannot be assumed in these studies that phenotypic reversion and virus rescuability necessarily correlate with expression of the virus genome. The levels of and species of virus-specific RNAs and proteins have not yet been determined in these clones, nor is the extent to which host cell factors modulate both reversion and virus rescue in the absence of alterations in viral gene products known. However with this caveat, these two parameters are useful indicators of functional virus expression which permit large-scale screening of transformed clones. All transformants obtained at high (7.5) m.o.i. yielded virus readily upon fusion with chick cells; between 1 and 10% of the rat cells gave rise to a focus of transformed chick cells after fusion (Fig. 2). In some transformants obtained at m.o.i. 0.1 and 0.01 virus was also readily rescued but in other clones rescue was low or undetectable (range
WYKE AND QUADE

levels of both virus rescue and reversion in the transformed cell and this effect masks the phenotypic variability seen in transformed clones obtained after low m.o.i. infection. This explanation contradicts the concept that transformation results simply from a rare and randomly occurring viruscell interaction.

. 0

8 .

x

.

:

.

. 0

Analysis of Integrated Proviral DNA

8” .

Analysis of integrated proviral DNA in both high and low m.o.i. transformants is in accord with the hypothesis that multiple proviruses contribute to the cell phenotype. Figure 4 shows the analysis of DNA from a selection of representative trans-

. .

10

8 I

R 011

Dkll Multiplicity

of

lkl

7%

infection

I

- I

.

0

FIG. 2. Correlation between the level of virus rescue from B7Ptransformed Rat-l clones and the m.o.i. of virus infection. Rat-l E5 was infected with B77,(v) at multiplicities of 0.01 to 7.5 FFU per cell. At each multiplicity three separate dishes were infected in the presence (0) of 1 /.&f dexamethasone and three in its absence (0). Two transformed clones were isolated by micromanipulation from each dish. Virus rescue by PEG-mediated fusion to chick cells was quantitated by the method of Steimer and Boettiger (1977). The two clones on the abscissa at m.o.i. 0.1 showed no rescue after fusing a total of lo6 transformed rat cells to recipient chick embryo cells.

of revertants may simply mean that reversion occurred very early in the clone’s history. However the high level of reversion in several clones of the low multiplicity transformants does demonstrate that this group shows a higher reversion rate than the clones transformed at high m.o.i. These results demonstrate a close correlation between high input virus multiplicity and both high levels of virus rescue and low levels of phenotypic reversion to normal in the transformed clones. The simplest explanation of this association is that after high m.o.i. infection multiple proviruses have a role in determining the

Multlpllcity

of

Infrction

FIG. 3. Correlation between the incidence of morphological reversion in B77-transformed Rat-l clones and the m.o.i. of virus infection. The origin of the clones is described in the legend to Fig. 2. Reversion incidence was assessed for 44 of the 48 clones by seeding 10 petri dishes (60 mm) each with 103 cells, Giemsa-staining cell colonies 8 days later, and scoring the number of revertants by microscopic examination. The eop of the clones varied from 19 to 85%; thus the minimum level of revertants detectable in this way varied (depending on the clone) from 0.01 to 0.5%. The downward pointing arrows at m.o.i. 7.5 and m.o.i. 0.1 indicate clones that showed no detectable reversion in this screening.

TRANSFORMATION

OF RAT CELLS BY ASV

formed cells with the restriction endonucleases KpnI and HindHI. Both these enzymes cleave the B77 provirus into two major pieces (Shank et ccl., 1978; Taylor et al., 1978) so that each integrated provirus should yield two fragments which anneal with cDNA, their sizes varying with differences in the distance between the point of provirus integration and the nearest cleavage site in the flanking cellular DNA. Four clones transformed at m.o.i. 7.5 and one clone transformed at m.o.i. 1.0 all show complex patterns with both enzymes, demonstrating that they all contain multiple proviruses. Counting the bands annealing to cDNA the numbers of integrated proviruses can be estimated as 6 for C + 22, 5 for Dll, 3 for D41, 5 for D + 12, and 6 for D + 22. These are, however, minimum estimates, first because crowding of the fragments may obscure some bands and second because some proviruses may be defective and could have deleted the cleavage site for one or the other enzyme. In contrast, all seven of the Rat-l clones transformed at m.o.i. 0.01 show only two virus-specific bands for both enzymes and thus clearly contain proviruses singly integrated at a single site. These clones were obtained from six different infected dishes and display six completely different patterns of cleavage, showing that the single integration sites are different in the genomes of the various cell clones, in agreement with the findings of Hughes et al. (1978). Two clones, A + 11 and A + 12, were obtained from the same infected dish, and, since they show cleavage patterns indistinguishable from one another they are probably sister clones descended from the same transformed parent. It is formally possible that, although there is only a single provirus integration site in these low m.o.i. clones, both the provirus and its cellular site may be present at several locations in the cell genome. This seems unlikely first because in every case such amplification must involve the enzyme cleavage sites in the flanking cellular DNA as well as the provirus and second because the intensity of the labeled bands in the low m.o.i. clones is far less than in the high m.o.i. transformants, suggesting that they contain far

A. c+ D 22 II

D 41

225

A II

D+ D+ Rat-l 12 22 Es

A A 23 31

A+ 11

A+ 12

I";

:;

A+ 22

A+ 32

-211-

6. c+ 110 22

D 41

D+ n+ Rat-1 12 22 ES

I?

e-2.7

-

-1.4

-

A

3:

2*;

37

FIG. 4. Analysis (performed as described in Materials and Methods) of DNA from B’7’7-transformed Rat-l clones with the restriction endonucleases KpnI (Panel A) and Hind111 (Panel B). The nomenclature of the transformed clones is as follows: The letter represents the B77 m.o.i. (A, 0.01; B, 0.1; C, 1.0; D, 7.5). This is followed by a + if infection occurred in the presence of dexamethasone. The first number represents individual dishes in any one series of infections and the second number designates single cell clones isolated by micromanipulation. If clones differ in their letter, presence of + symbol, or first number then they must be derived from different transforming events, e.g., All and A + I1 derive from transforming events in different dishes and so must be different clones. If only the second number differs then the clones could be derived from the same transforming event, e.g., A + 11 and A + 12 were obtained from the same infected dish and so could be sister clones; however, this is very unlikely in high m.o.i. infections where many transformed colonies arise in each dish. The reversion incidence and levels of virus rescue in these clones are listed in Table 3.

fewer copies of the provirus. However, we cannot yet rigorously exclude this possibility and it must be considered in interpreting these data. The findings with KpnI and Hind111 were strengthened by the results of digestion with BgZII (Fig. 5, Panel A). This enzyme

226

WYKE AND QUADE

11

B. D D+ D+R&-1 W-D 22 11 11*2 22 I?5

A 23

A 31

A+ A+ A+ 11 12 22

* 32

A A A Ai A+A+A+ 11 23 31 11 12 22 32

FIG. 5. Digestion of DNA from B77-transformed Rat-l clones with restriction endonucleases BglII (Panel A) and EcoRI (Panel B). The analyses were performed as described in Materials and Methods. For nomeclature of clones see the legend to Fig. 4.

makes three cuts in the B77 provirus (Shank et al., 19’78) so that each complete integrated provirus yields two internal fragments and two “junction” fragments joined to flanking cell sequences. The clones derived at high m.o.i. all show an array of bands in addition to the internal fragments of M, 2.4 x lo6 and 1.9 x 10”. Analysis of these patterns is complicated by the possible presence of defective genomes, which may or may not have deleted BgZII cleavage sites, and which thus might migrate anomalously. The cutting patterns of the low m.0.i. transformants are very simple, showing the two internal fragments and either one or two “junction” fragments. The absence of a second junction fragment may be because it comigrates with another band, or it may reflect inconsistencies in the cDNA probe and/or the transfer procedure. It is also possible that the provirus might be amplified together with only one of the flanking cellular cleavage sites, although, as

mentioned above, this is unlikely. Finally, it is conceivable that, although the provirus is not amplified, variation in the position of the flanking cellular BglII cleavage sites may be generated among cells within the clone. We are testing these possibilities by analyzing the enzyme cleavage patterns in both transformed and revertant subclones of the low m.o.i. transformants. Once again, with the exception of clones A + 11 and A + 12, all the proviruses are shown to be integrated at different sites. Figure 5, Panel B, shows the same clones digested with EcoRI, an enzyme that produces three internal fragments of M, 2.5 x 10s, 2.1 x 106, and 1.6 x lo6 but whose junction fragments contain too little ASV-specific DNA to be detected by most representative cDNA probes (Hughes et al., 1978). The high m.o.i. transformants cleaved with EcoRI show predominantly the expected internal fragments generated from complete proviruses but also, in some cases, additional fragments, both smaller and larger. These probably represent proviruses which have suffered deletions, the larger fragments probably resulting from the deletion of an EcoRI cutting site. The band (approximately 0.7 x lo6 daltons) found in all high m.o.i. transformants may be derived from a transformation-defective virus present in the original B77Jv) stock or generated upon infection. The low m.o.i. transformants contain the characteristic EcoRI fragments but some, notably A23 and the A + 11/A + 12 pair, contain additional bands. Since analysis with the other enzymes strongly suggests that these clones contain proviruses which are singly integrated at single sites, these additional bands are difficult to explain. One possibility is that the cDNA probe contains a large amount of “strong stop” DNA (Haseltine et al., 1977; Friedrich et al., 19771, overrepresenting the 5’ end of the genome, which might then detect one of the EcoRI junction fragments. Other possible explanations are that mutant viruses are generated either by amplification of the original provirus or by segregation among daughter cells in the clone. It is hard to reconcile either of these possibilities with the results of the KpnI and Hind111 digestions,

TRANSFORMATION

227

OF RAT CELLS BY ASV

but they are being tested by examination of subclones, digestion with additional enzymes, and analysis with cDNA probes containing a limited set of specific viral sequences.

.

0

Phenotypic Variability in Rat-l Cells Transformed at Low moi by BY7 Regardless of the factors which determine whether or not a rat cell becomes transformed by B’77, the data of Figs. 2 and 3 show that the transformed clones obtained at low m.o.i. are phenotypically heterogeneous. One possible basis for this heterogeneity is variability in the level of B77 expression in different clones. As shown above, high levels of virus rescue and low levels of reversion are correlated in cells transformed at high m.o.i. Moreover, in other ASV-transformed mammalian cells it is known that phenotypic reversion is accompanied by both a decrease in the level of virus rescue (Boettiger, 1974b) and a decrease in ASV-specific RNA (Denget al., 1974, 1977). In Fig. 6 the data from Figs. 2 and 3 are replotted so that levels of both virus rescue and reversion incidence can be directly compared for all transformed clones irrespective of their original m.o.i. A number of clones show both low reversion and high rescuability but most of these are clones obtained after high m.o.i. infection. Aside from this cluster there is no negative correlation between virus rescue and reversion and, in particular, there are no clones which show high reversion and low rescue. Indeed, clones with high levels of revertants contain readily rescuable virus whereas some which revert only infrequently contain no detectable virus (these latter could be due to mutation in viral replicative genes and we have not tested for this). It thus seems that phenotypic variability in low m.o.i. transformants does not involve a constant inverse relationship between rescuability and reversion and thus it cannot be explained solely by modulation in overall levels of viral expression. Heterogeneity of the low m.o.i. transformants may, however, reflect some inherent variability of the cell or its trans-

.

. 0

. .

I

03

1.0 Rcrarrim

10 freqrancy

IhO (%I

FIG. 6. Lack of correlation between the level of virus rescue and the frequency of morphological reversion in Rat-l clones transformed by BW. The data from Figs. 2 and 3 are replotted so that the virus rescue and reversion of individual transformed clones can be compared, irrespective of the multiplicity at which they were originally infected.

forming virus. The data in Tables 1 and 2 suggest that there are no cells or viruses which are particularly likely to be involved in a transforming event but it is possible that once transformation occurs the phenotype may be influenced by genetic differences in the cells or viruses. Variants may preexist in the population or they may arise as a result of particular virus-cell interactions. To test this, virus was rescued from three transformed clones in which the levels of virus rescue varied from 23 to 4100 foci/lo5 cells but in which the levels of reversion were similar, the incidence being between 2 and 17%. Cloned recovered virus (Table 1, column B) was used to infect fresh rat cells at low m.o.i., new transformed clones were obtained and their level of virus rescue tested. It can be seen (Table 4) that there is no correlation between the level of virus rescue in the original clones and that in

228

WYKE

AND QUADE

the clones transformed with recovered virus. Thus ease of rescuability does not seem to be a stably inherited trait of the transforming virus. We have not yet performed similar experiments to test a possible viral inheritance of reversion incidence nor have we yet investigated the more difficult question of whether the host cell genotype influences the levels of virus rescue. The level of virus recovery appears to be relatively stable in a number of clones transformed at both high and low m.o.i. for it shows no great fluctuation but only a moderate decrease after If weeks of cell passage (Table 5). However, this stability may obscure the genesis of subclones which express much higher or lower levels of virus rescue. This work is intended as a prelude to a study of the mechanisms of phenotypic reversion (Wyke et al., 1979) and the variable levels of reversion seen in the clones transformed at low m.o.i. (Figs 3 and 6) suggest that they will be useful tools for such investigations. Two types of revertants of ASV-transformed mammalian cells have been described previously, those in which virus rescue and levels of virus-specific RNA are reduced (Boettiger, 1974b; Deng et al., 1974, 1977) and those in which recovery of virus and viral RNA levels remain high (Krzyzek et al., 1977, 1978, 1979). Table 3 shows that revertants with both high and low levels of virus rescuability can be obtained from B77-transformed Rat-l cells. Revertants from which virus is recovered at a high level have so far been isolated from only one clone, C + 22, which itself yielded transforming virus at a very high level upon fusion with chick cells. The reversion incidence in this clone is fairly low (1.5%) and the two revertants obtained are unusual in that they spontaneously retransform at a low level. We cannot say whether the mechanism of reversion in these cases is comparable to that described by Krzyzek and co-workers (1977, 1978, 1979), particularly since C + 22 was originally transformed at the moderately high m.o.i. of 1.0 and it contains several proviruses (Figs. 4 and 5). The viral genome responsible for the transformed phenotype may not be

that which is rescued upon fusion with chick cells. Revertants obtained from five clones transformed at m.o.i. 0.01 all show levels of rescue of transforming virus reduced to 20 foci/lo5 cells or less, though the magnitude of this reduction is very variable since levels of rescue from the parental transformed clones differed widely. The most remarkable observations are those on clones All and its revertants. This clone shows fairly high virus rescuability (1040 foci/lo5 cells) yet consisted of more than 95% revertant cells. All four of its revertants studied so far have levels of virus rescue reduced to less than 10 foci/lo5 cells so we must conclude either that the small proportion of transformed cells in All show very high levels of virus rescue or that some revertants which have yet to be isolated yield appreciable levels of recovered virus. If such revertants exist they should be very useful in elucidating reversion mechanisms. DISCUSSION

These experiments address two main topics: (1) the reasons why B77 transforms Rat-l cells far less efficiently than it transforms chick cells and (2) the factors which influence the phenotype of the transformed cell and, in particular, its tendency to revert to normal morphology. We have postulated four explanations for the low level of Rat-l transformation by B77. The most trivial is that our methods of detecting transformation in this system are inefficient. This cannot be ruled out conclusively but there is ample documentation of cells in which failure to transform or reversion to normality is associated with reduced levels of virus expression (Boettiger, 1974a, b; Deng et al., 1974, 1977). It is thus likely that the low level of mammalian cell transformation by mammal-tropic ASV is due, at least in part, to the src gene product failing to reach a critical threshold in most cells. Refinements in cultural conditions, such as the presence of dexamethasone (Fig. l), may significantly alter the incidence of transformation, possibly by increasing virus expression or decreasing the threshold of cell response, but it seems

TRANSFORMATION

OF RAT CELLS

unlikely that such changes will result in massive increases in the efficiency of transformation. A second possibility is that transformation is an attribute only of genetically variant subpopulations of viruses or cells. The data in Tables 1 and 2 argue strongly that this is not the case and they confirm and extend the findings of Boettiger (19’74a). We conclude that any virus or any cell is potentially capable of involvement in transformation, either because they are in a physiologically favorable condition or because transformation is a low probability event whose occurrence is entirely random. We cannot at present distinguish these two postulates, our relevant evidence being paradoxical. Figure 1 shows that at high input virus multiplicity the relationship between virus dose and transformation remains linear as would be expected if transformation resulted from a random event. There is no evidence for a physiologically highly susceptible cell population which becomes saturated when the majority of cells are exposed to virus. It could be argued that the calculated m.o.i. (based on titer on chick cells) and the effective m.o.i. (after virus penetration of Rat-l, provirus synthesis, etc.) may be very different and even at 10 FFU per cell one might not be exposing the target in all the susceptible cells to virus. However this argument is untenable because the data in Figs. 2 and 3 show that at a m.o.i. of 7.5 the transformed cells show a uniformity in both the high level of virus recovery and low level of reversion that is not seen at lower multiplicity and which reflects the presence of multiple proviruses in the cell (Figs. 4 and 5). This qualitative difference between high and low m.o.i. transformants suggests that in cells transformed at high m.o.i. more than one provirus is functioning to maintain the stable cell phenotype, showing that the effective m.o.i. is indeed high. However, this reasoning is also inconsistent with the concept that transformation is a randomly occurring rare event, for if truly random it would be extremely unusual, even in a multiply infected cell, for more than one provirus to be expressing its transforming capacity.

BY ASV

229

We cannot at present reconcile these apparently conflicting arguments. It is possible that there may be a physiologically susceptible subpopulation of cells and the linear response of transformation to input virus at high m.o.i. might be spurious. For example, cells which are not highly susceptible might transform if they contain more than one provirus, each expressed at subthreshold levels but, in concert, producing sufficient src gene product to transform the cell. Such cells would form a negligible proportion of transformants at low m.o.i. but would increase greatly in number at high m.o.i. If their increase occurs at levels of infection at which the highly susceptible population shows a plateau in transformation response then the result might mimic a continued linear response to increasing virus input. On the other hand, the initial virus-cell interaction leading to transformation may be random with one-hit kinetics but, once a virus is expressed in a cell at a level sufficient to induce transformation, then this event in some way augments expression in that cell of other viral genomes which would otherwise remain silent. Such an explanation could account both for the data in Fig. 1 and for the stably expressed phenotype of high m.o.i. transformants. Whatever the factors governing cell transformation, the data in Figs. 2 to 6 have wide implications for subsequent studies on the transformed cells. First, cells transformed at high m.o.i. have a high chance of acquiring more than one functioning provirus, a situation which could obscure further studies on cell control of virus expression. For example, revertants of the clone C + 22 (Table 3) display high levels of virus rescue and retransform spontaneously but we cannot say whether this is due to modulation of expression of a single provirus or to a combination of alterations in expression of more than one provirus. Second, transformants obtained at high m.o.i. do not show the heterogeneity of phenotype exhibited by transformed cells obtained at m.o.i. 0.1 or 0.01. The basis of this heterogeneity is clearly of great interest, for Fig. 6 suggests that it is not simply due to a variation in overall virus

230

WYKE

AND QUADE TABLE

3

BIOLOGICAL CHARACTERISTICS (VIRUS RESCUE AND REVERSIONFREQUENCY)OF B’77-TRANSFORMED

RAT-~ CELLS AND THEIR MORPHOMGICALLYNORMAL DERIVATIVES Virus rescue (foci/lo5 cells) Transformed clonea

Reversion incidenceb

Transformed clone

Revertant clones

67/4400 (1.5)

10,400

IR: 2700d IIR: 4500d

Dll

13/3720 (0.4)

D41 D + 12 D + 22

O/4980 (cO.02) lb3520 (0.01)

2,500 7,506 4,000 4,200 1,040

c + 22

All

25/3770 (0.7) 3300/3450 (96)

A23

145/3000 (4.8)

20

A31 A + 11 A + 12

322/1880 (17) 110/4750 (2.3) 1320/4050 (33)

4,100

A + 22 A + 32

5/2097 (0.3)

760 470 5 25

4/6530 (0.06)

NT’ NT NT NT IR: 4 IIR: 8 IIIR: 3 IVR: 4 IR: 5 IIR: 4 IIIR: 18 IIR: 2 IR: 10 IIR: 21 NT NT

DThe clones tabulated here are those whose integrated proviruses have been investigated (Figs. 4 and 5). For nomenclature of clones see legend to Fig. 4. b Number of morphological revertantakotal number of cell colonies screened. c Percentage figures in parentheses. d These revertants are unstable and seareaate transformed subclones. e NT, Not tested. I

-

expression. In some cases, for example explain why chick cells containing spleen where virus rescue is undetectable, virus necrosis proviruses integrated at different mutation might account for the observed sites display different biological behavior phenotype. Altered proviruses are known (Keshet and Temin, 19’78; Keshet et al., 1979). In the transformed clones described to occur frequently in ASV-transformed mammalian cells (Hughes et aZ., 1978) and here phenotypic variation may reflect mutations can explain the existence of qualitative and/or quantitative differences transformed nonproducer rat cells (Steimer in virus-specific RNA species. It is also and Boettiger, 1977, 19’79). However, in conceivable that a parameter such as the many cases genetic alterations in the virus level of virus rescue depends partially or or cell cannot be detected (Tables 4 and 5 wholly on the number of integrated proand Figs. 4 and 5) and variability in virus viruses in a cell rather than on their level rescue and morphological reversion may be of expression. This is possible since we candue to different, and perhaps specific, not yet rigorously exclude the existence of virus-cell interactions. Such variations in multiple proviruses in the clones transthe relationship between the virus and host formed at low m.o.i. and we do not yet have have been proposed by Quintrell et al. information on virus-specific RNA in (1980) in studies on the viral RNA species in these cells. Heterogeneity in the level of reversion ASV-transformed rat cells. They may also

TRANSFORMATION

231

OF RAT CELLS BY ASV TABLE 4

VIRUS RECOVEREDFROMTRANSFORMEDRAT-~ CELLS DOES NOT SHOWSIMILAR LEVELS OF RESCUE WHEN IT TRANSFORMS FRESH RAT CELLS

Original transformed Rat-l clone”

Virus rescue from

Clone of

original clone (foci/lo5 cells)

recovered virus”

23

A21 A31 A + 11

A21 (5)

Clone of Rat-l transformed by recovered virus

Virus rescue from new transformed Rat-l clone (foci/105 cells)

1 3

1100

790

4100

A31 (6)

2 4

1 120

760

A + ll(6)

1 21 22

650 140 1300

’ For nomenclature of clones see legend to Fig. 4. * The nomenclature of these clones and their eot on Rat-l, clone E5, cells is shown in Table 1, Part B.

is one of the most interesting features of the low m.o.i. transformants. One clone, B31, which shows a very low level of spontaneous reversion has already been studied in detail (Varmus et al., 1930). Revertants isolated from this clone after negative selection have either a complete deletion of the single integrated provirus or mutation in the proviral src gene, the latter providing useful agents for analyzing src and its product. The revertants listed in Table 5 are all derived from transformed clones which show higher TABLE 5 STABILITY OF LEVEL OF VIRUS RESCUE IN TRANSFORMED RAT-~ CLONES

Clone” All A + B21 B + c21 c + Dll D +

12 21 22 12

Original level of virus rescue (foci/105 cells)b 1,040 470 co.5 450 410 10,400 2,500 4,000

Later level of virus rescue (foci/lo5 cells) 360 110

co.5 300 53 3,000 1,500 800

’ For nomenclature of clones see legend to Fig. 4. ’ Virus rescue tested soon after cloning. ’ Virus rescue tested after a further 11 weeks of continuous culture.

levels of reversion than B31 and, with the exception of derivatives of C + ‘22 mentioned above, they all show reduced, but detectable levels of virus rescue. They are thus comparable to the revertants studied by Boettiger (1974b) and Deng et al. (1974, 1977). Revertants of this type, exhibiting reduced levels of virus expression, would thus seem to be the commonest form in ASV-transformed mammalian cells. However, this group of revertants show some variability in behavior, even when derived from a single transformed clone (U. Rovigatti and J. Wyke, unpublished data), so the presumed phenotypic suppression may occur by a variety of mechanisms. The transformed cell clones described here are derived from a single clone of virus and a single clone of Rat-l cells. The variability of clones transformed at low m.o.i. and in particular the heterogeneity of reversion mechanism that low m.o.i. clones display, makes then excellent tools for investigating the control of src gene activity. We are now using these clones in attempts to define the host cell factors involved in phenotypic reversion. ACKNOWLEDGMENTS We should like to thank Drs. D. Chiswell, P. Goodfellow, and M. Fried for their critical comments on this work and Ms. J. Newton for secretarial assistance. K.Q. was a Special Fellow of the Leukemia Society of America.

WYKEANDQUADE

232

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VARMUS, H. E., QUINTRELL, N., and WYKE, J. A. (1980). Revertants of an ASV-transformed rat cell line have lost the complete provirus or sustained mutations in src. Virology, in press. VOGT, P. K. (1969). Focus assay of Rous sarcoma virus. In “Fundamental Techniques in Virology” (K. Habel and N. P. Salzman, eds.), pp. 198-211. Academic Press, New York. WYKE, J. A., BEAMAND, J. A., and VARMUS, H. E. (1979). Factors affecting phenotypic reversion of rat cells transformed by avian sarcoma virus. Cold Spring Harbor Symp. Quant. Biol. 44, in press.