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Characterization of defective SV40 isolated from SV40-transformed cells
VIROLOGY
63, 512-522 (1975)
Characterization
of Defective
SV40
Isolated
from SV40-Transformed
Cells l K. HUEBNER, D. SANTOLI, The Wistar Institute
of Anatomy
and Biology,
C. M. CROCE,
AND
H. KOPROWSKI
36th Street at Spruce, Philadelphia,
Accepted
Pennsylvania
19104
October 4, 1974
Defective SV40 viruses were isolated from SV40-transformed monkey, human and hamster cells after Sendai virus-mediated fusion of the transformed cells with TC7 cells, a stable line of African green monkey kidney cells. Viral isolates were concentrated and purified and the defective viruses examined by electron microscopy. The buoyant densities in CsCl of the defective viruses ranged’ between 1.32 and 1.33 g/cc. DNA isolated from defective viruses was characterized by dye-buoyant density centrifugation and by velocity sedimentation in neutral CsCl. The DNA was heterogeneous in size and contained some covalently closed double-stranded circular molecules. INTRODUCTION
The Sendai virus-mediated fusion of SV40-transformed nonpermissive (mouse) cells with African green monkey kidney (AGMK) cells, which are permissive to SV40 lytic infection, allows replication of the SV40 genome resident in the transformed cells (Koprowski et al., 1967; Watkins and Dulbecco, 1967). However, cells of many SV40-transformed permissive (monkey) and semipermissive (human and hamster) lines do not yield infectious virus after fusion with permissive cells (Knowles et al., 1968; Kit et al., 1970), and it has been proposed that these “nonrescuable” transformed cells contain defective virai genomes (Knowles et al., 1968). Recently we have reported formation of microplaques in AGMK cultures exposed to heterokaryocytes formed by fusion of nonrescuable human and hamster cells with AGMK cells (Huebner et al., 1974). Heterokaryocytes produced by fusion of AGMK cells with SV40-transformed human-mouse hybrid cells also formed mi‘This work Training Grant Nos. CA 10815 RR 05540 from and funds from
was supported in part by USPHS No. CA 05163 and Research Grant from the National Cancer Institute, the Division of Research Resources, the Commonwealth of Pennsylvania.
Copyright 0 1975 by Academic Press. Inc. All rights of reproduction in any term reserved.
croplaques on AGMK monolayers (Croce et al., 1974). Preliminary investigations led us to propose that these microplaques were formed by a defective SV40 rescued from the transformed cells (Huebner et al., 1974; Croce et al., 1974). This paper presents biological and physical characterizations of defective SV40 virions isolated from SV40-transformed monkey, human and hamster cell lines that had failed to yield wild type virus after fusion with permissive AGMK cells. MATERIALS
AND
METHODS
Cells. TC7, a line of AGMK cells (Robb and Huebner, 1973), was used for production of all SV40 pools and as the permissive cell in all rescue experiments. A summary of the properties of the SV40-transformed cell lines used in this study is presented in Table 1. H5 and T22 cells lines were kindly supplied by V. Defendi; A-12-1, 2A-1, WI26VA4, and H50 cell lines were obtained from A. Girardi. The 52-62-l-5 cells are LN-SV x Cl-1D hybrid cells containing the entire complement of Cl-1D mouse chromosomes plus human chromosome 7. Cells were subcultured in minimum essential Eagle’s medium supplemented with 10% fetal bovine serum (Flow Laboratories, Rockville, MD).
DEFECTIVE
SV40 FROM
TRANSFORMED
TABLE
513
CELLS
1
SV‘%TRANSFORMED CELL LINES Cell line”
mKS-BUlOO (Dubbs et al., 1967) 2A-1 (Koprowski et al., 1967) A-12-1 (Jensen and Defendi, 1968) H5 (Shiroki and Shimojo, 1971) T22 (Shiroki and Shimojo, 1971) W18VA2W (Weiss et al., 1968) WI26VA4 (Girardi et al., 1965) LN-SV (Croce et al., 1973) F5-1 (Girardi et al., 1963) H50 (Ashkenazi and Melnick, 1963) 52-62-l-5 (Croce et al., 1973)
Species
Transforming agent
Rescuable* ?
Mouse
sv40
65-70
Yes (Dubbs and Kit, 1968)
Monkey
sv40
64-70
No (Koprowski
Monkey
Human
Irradiated adeno 7-SV40 hybrid Irradiated adeno 7-SV40 hybrid Irradiated defective SV40 sv40
400-410
No (Jensen and Defendi, 1968) No (Shiroki and Shimojo, 1971) No (Shiroki and Shimojo, 1971) No. (Huebner et al., 1974)
Human
sv40
200-205
No (Knowles
Human Hamster Hamster
sv40 sv40 sv40
45-50 150-155 90-95
No (Huebner et al., 1974) No (Koprowski et al., 1967) No (Melnick et al., 1964)
LN-SV x Cl-1D hybrid
sv40
15-20
Monkey Monkey
210-215 a-90 80-85
et al., 1967)
et al., 1968)
No (Croce et al., 1974)
n References in this column are for transformation of these cell lines. b Rescuable means that standard plaque forming SV40 virus is obtained after fusion of the indicated with monkey kidney cells; references in this column are for reports on rescuability of the cell line. c Cl-1D cells are derived from mouse L-cells (Dubbs and Kit, 1964).
Sendai virus. Sendai virus was prepared as previously described (Steplewski and Koprowski, 1970) and inactivated in 0.025% B-propiolactone. Fusion. For rescue of SV40 from the SV40-transformed cells, 1 x 10’ TC7 cells were fused with 1 x 10’ transformed cells in the presence of Sendai virus according to techniques described previously (Croce et al., 1972). The fused cultures were seeded in roller bottles in minimum essential Eagle’s medium containing 10% fetal bovine serum. After lo-14 days cultures were frozen for later isolation of virus. For detection of plaque formation by heterokaryocytes, 5 x lo8 TC7 cells were fused with an equal number of transformed cells. Fused cultures were serially diluted and plated on TC7 monolayers in petri dishes as described previously (Huebner et al., 1974). Twenty hours after seeding, the medium was removed from the petri dishes and 8 ml of overlay medium containing 1%
ceil lines
agar in minimum essential Eagle’s medium with 8% fetal bovine serum was added. An additional 3 ml of overlay medium was added after 10 days of incubation at 37”. Ten days later, 3 ml of overlay medium containing l:lO,OOO neutral red was added and the number of plaques recorded one day later. mKS-BUlOO cells, which yielded large plaques after fusion with TC7 cells, were used as a positive control in all rescue experiments whether the method of detection was by SV40 particle isolation or production of plaques. SV40 T and V antigens. Coverslip cultures of TC7 cells that had been infected with wild type or defective SV40 were fixed at 2, 3, 4, 7 and 10 days after infection and tested for presence of T or V antigen by the indirect immunofluorescence method (Pope and Rowe, 1964). Labeling and purification of rescued SV40. Roller bottles of fused cell cultures to
be tested for presence of rescued virus were
514
HUEBNERETAL.
frozen at ten days after fusion as described above. After three cycles of freezing and thawing, the culture fluid was clarified by low speed centrifugation and the supernatant was centrifuged at 60,000 g to sediment virus and/or remaining debris. The pellets obtained after high speed sedimentation were resuspended in minimum essential medium containing 10% fetal calf serum and the concentrated suspensions adsorbed to TC7 monolayers. To label the DNA moiety of the virus, the monolayer cultures were supplemented at 18 hr after infection with 5 &i/ml of [aH]thymidine (specific activity 52 Ci/mmole, New England Nuclear, Boston, MA) or 10 &i/ml of [YP]phosphate (carrier-free, New England Nuclear). To label the protein moiety of the virus, cultures were supplemented with 5 &i/ml of [aH]leucine (specific activity 32 Ci/mmole, New England Nuclear). Ten to 14 days later, cultures were frozen-thawed three times and virus was purified by a standard procedure (Swetly et al., 1969) consisting of sedimentation by high speed centrifugation, treatment with sodium desoxycholate, banding by centrifugation in a steep KBr gradient, pooling of all virus bands and freeing them of KBr by dialysis. If no virus bands were visible in the KBr gradient, the KBr fractions corresponding to those fractions in which the virus was present in positive control samples were collected and dialyzed. After dialysis the virus pools, which will be referred to as the KBr purified pools, were processed in several ways: a sample was taken for electron microscopic examination; a sample was inoculated into TC7 cell cultures and coverslips were later examined for presence of SV40-induced T or V antigens; an aliquot was centrifuged to equilibrium in CsCl solution (j = 1.33) and fractions assayed for density and total radioactivity; and finally DNA was extracted from an aliquot and analyzed for the presence of closed circular double-stranded DNA. Electron microscopy. Purified virus was spread on carbon-coated copper grids, stained with potassium phosphotungstate (PTA), pH 7.0, and observed in an electron microscope (Hitachi HS-8) at a magnification of; 24,000 and 33,000 x .
Plaque purification of wild type and defective SV40 viruses. Plaque isolates of
rescued from mKS-BUlOO, virus W18VA2W and H50 cells were prepared by picking up material from plaques formed after seeding of heterokaryocytes on TC7 cell monolayers. After addition of neutral red to the petri dishes at 20 days after seeding of the heterokaryocytes, the wild type plaques (rescued from mKS-BUlOO cells) or microplaques (rescued from W18VA2W and H50 cells) (Huebner et al., 1974) could be visualized as light areas of dead cells that did not take up neutral red. Virus was picked up from the plaques by putting a sterile needle through the agar and scraping the plaque area. The material in the needle was then resuspended in 0.5 ml of MEM containing 10% fetal calf serum; 0.2 ml of this suspension was inoculated into a culture of TC7 cells and after 2 weeks the culture and medium were frozen-thawed three times and this crude virus stock was stored at -20”. SV40-B, SV40-P and SV40-K are virus stocks prepared in this way from plaque isolates obtained after rescue of mKS-BUlOO, H50, and W18VA2W respectively. “Transformation”
by defective
viruses.
ICR mouse embryo fibroblasts were inoculated with SV40-B, SV40-P or with a crude cellular extract prepared from mockinfected TC7 cells. The cells will subsequently be referred to as ICRB (infected with SV40-B plaque isolate rescued from mKS-BUlOO cells), ICRP (infected with SV40-P plaque isolate rescued from H50 cells) and ICREF (mock infected). Human diploid fibroblasts that were inoculated with SV40-B, SV40-K or were mock infected will be referred to as HJB (infected with SV40-B), HJK (infected with SV40-K plaque isolate from W18VA2W cells) and HJ (mock infected). After infection or mock infection these cell lines were subcultured and tested at each passage for appearance of SV40-induced T or V antigen by indirect immunofluorescence. Cloning. ICRP cells at passage 30 were cloned by seeding 200 cells per 70-mm petri dish. Fourteen days after seeding, cloning cylinders were placed over visible colonies using sterile vacuum grease, and the colo-
DEFECTIVE
SV40 FROM
nies were trypsinized and transferred to flasks. Cell cultures derived from individual colonies were tested for SV40 T antigen by indirect immunofluoresence. Three clones were obtained in which 100% of the cells were T antigen-positive and fifteen clones were obtained in which 100% of the cells were T antigen-negative. Agar suspension culture for detection of transformed cells. The ICRB, ICRP and
ICREF cells as well as a T antigen-positive and a T antigen-negative clone of ICRP cells were tested for ability to form colonies in semisolid medium (MacPherson, 1969). Five-tenths per cent agar medium was prepared by mixing one part distilled water containing 1.0% agar (Difco bacto agar) with one part prewarmed double-strength minimum essential medium (containing twice the normal concentration of all components and 20% fetal bovine serum). Base layers were prepared by distributing 7 ml of 0.5% agar medium into petri dishes. Cell suspensions were prepared by mixing one volume of cells in minimum essential medium with 2 volumes of 0.5% agar medium. When the base layers were set, 3 ml of the agar cell suspensions were layered into each petri dish. For each cell line tested, lo’, 10s, and lo2 cells per petri dish were plated. Cultures were incubated at 37” in a humidified atmosphere and at 12 days the petri dishes were examined for appearance of colonies. Isolation of SV40 DNA. DNA was isolated from KBr purified virus pools by a phenol extraction procedure (Swetly et al., 1969). After removal of residual phenol from the aqueous phase by extraction with ether, the DNA was precipitated by addition of ethanol and dissolved in NTE buffer (0.05 MTris, 0.13 A4 NaCl, 0.001 MEDTA, pH 7.8). Extraction of cellular DNA. Cellular DNA was extracted from SV40-transformed cells by the Hirt procedure (Hirt, 1967). The pellet obtained after the Hirt extraction was resuspended in 1 x SSC (0.15 M NaCl, 0.015 M sodium citrate) and dialyzed for 24 hr against 1 x SSC. The resuspended pellet was treated with 100 pg/ml of Pronase for 30 min at 37”. The
TRANSFORMED
CELLS
515
DNA was deproteinized by mixture with two volumes of redistilled phenol, and the aqueous phase was dialyzed against 1 x SSC for 24 hr. The concentration of the DNA was estimated spectrophotometricallY. Passage of transformed
cell DNA on TC7
cells. A preparation of transformed cell DNA, 0.3 ml, containing 100 pg of DNA was mixed with 0.3 ml of a 1 mg /ml solution of DEAE-dextran and the mixture was inoculated on 2 x lo6 TC7 ceils. After 15 min at room temperature the inoculum was removed and the monolayer washed three times with PBS. Medium was added and the culture incubated for 14 days. Cells were then disrupted by three cycles of freezing and thawing and the crude preparation was tested for infectious SV40 by plaque assay and by ability to induce SV40 T and V antigen in TC7 cells. SV40pZaque assay. Two-tenths milliliter of serial tenfold dilutions of virus stock was seeded on duplicate TC7 monolayers. After adsorption an 8-ml overlay of 1.0% agar in double strength Eagle’s medium was added. At 10 days a 3-ml overlay was added and at 20 days a 3-ml overlay containing l:lO,OOOneutral red was added and plaques were counted the next day. RESULTS
Electron
Microscopy
Electron microscopic examination of the KBr purified virus preparations revealed the presence of papova particles in preparations obtained by rescue of SV40-transformed monkey, human, hamster and mouse cells. Virions rescued from the SV40-transformed mouse cell line, mKS-BUlOO, from which wild type virus is routinely rescued, are shown in Fig. If. These virions have an average diameter of 43 nm, and most are complete virions. In comparison with the wild type virus preparation, very few particles were observed in each of the defective virus preparations, and most of the defective virions were coreless with an average diameter of 46 nm.
516
HUEBNER
ET AL.
FIG. 1. Electron micrographs of SV40 particles (x 160,800) rescued from: a) SV40-transformed human cell line, LN-SV; b) human-mouse hybrid cell line, 52-62-l-5; c) SV40-transformed human cell line, WI26VA4; d) SV40-transformed hamster cell line, F5-1; e) SV40transformed monkey cell line, 2A-1; f) SV40-transformed mouse cell line, mKSBU100.
Figure la is a photomicrograph of virions rescued from the SV40-transformed human cell line, LN-SV, and Figure lb is a photomicrograph of virions rescued from the LN-SV x Cl-1D hybrid cell line, 52-62-l-5,
in which the only human chromosome is chromosome number 7, which is the site of SV40 integration (Croce et al., 1973; Croce et al., 1974). Virus rescued from LN-SV (Fig. la), 52-62-l-5 (Fig. lb) and the
DEFECTIVE
SV40 FROM
SV40-transformed hamster cell line, F5-1 (Fig. Id) were the only preparations in which partially degraded but complete virions were observed by electron microscopy. In virus preparations rescued from the SV40-transformed human cell line, WI26VA4 (Fig. lc), and from the SV40-transformed monkey cell line, 2A-1 (Fig. le), only coreless virions were observed. The SV40-transformed human and hamster cell lines and the SV40-transformed monkey cell line, 2A-1, were all transformed after exposure to wild type sv40. In addition to these cell lines, several monkey cell lines transformed after exposure to defective SV40 pools were investigated. The T22 cell line was derived from transformed foci of AGMK cells after exposure to a defective virion fraction of SV40 which had been inactivated by ultraviolet irradiation (Shiroki and Shimojo, 1971). No virus particles were observed in KBr purified preparations after fusion of T22 cells with TC7 cells. The A-12-l. (Jensen and Defendi, 1968) and the H5 (Shiroki and Shimojo, 1971) cell lines were derived from AGMK cells transformed by irradiated adenovirus 7-SV40 hybrid virus strain LL-E46, which contains 60% of the SV40 genome (Lebowitz et al., 1974) and 90% of the adenovirus 7 genome (Kelly and Rose, 1971). Particles were observed in virus preparations rescued from the A-12-1 (40-50 nm) and H5 (45-90 nm) cell lines. Characterization of the antigenic properties of these particles is under investigation. Biological Properties Viruses
of Rescued Defective
The initial observation that led US to investigate the rescue of defective viruses was that heterokaryocytes formed by fusion of nonrescuable SV40-transformed human and hamster cells with TC7 cells formed microplaques three weeks after seeding on TC7 monolayers (Huebner et al., 1974). We have subsequently observed the formation of microplaques on TC7 monolayers after seeding heterokaryocytes formed by fusion of SV40-transformed
TRANSFORMED
CELLS
517
monkey cells (2A-1) with TC7 cells. Mateformed by rial from microplaques WlSVA2W-TC7 heterokaryocytes and H50-TC7 heterokaryocytes was picked up and virus stocks prepared as described in Materials and Methods. The virus stocks prepared from a W18VA2W-TC7 microplaque (SV40-K), a mKS-BUlOO-TC7 large plaque (SV40-B) and an H50-TC7 microplaque (SV40-P) were tested for ability to induce SV40 T and V antigens in TC7 cells and for ability to transform human or mouse cells. Aliquots of each virus stock were inoculated into TC7 cultures and coverslips were later removed and tested for SV40 T and V antigen. By ten days after infection neither T nor V antigen was detectable in cultures infected with SV40-P or SV40-K. Cultures infected with SV40-B were 99% T antigenpositive and 97% V antigen-positive by 60 hr after infection. Human diploid fibroblasts (HJ cells) were infected with SV40-B (HJB cells) or SV40-K (HJK cells); ICR mouse embryo fibroblasts (ICREF cells) were infected with SV40-B (ICRB cells) or SVCO-P (ICRP cells). The infected human and mouse fibroblasts were subcultured in parallel with mock-infected sister cultures and tested at each passage level for SV40 T antigen. After subculturing six times, the HJB cell line and the ICRB cell line were 99% T antigen-positive, noncontact inhibited, and appeared morphologically transformed, while the HJK and ICRP cell lines contained some SV40 T antigen-positive cells but the percentage varied between 10 and 50% in subsequent passages and never exceeded 50% of the cells. Cells of the mock-infected ICREF and HJ cell lines were always T antigen-negative. The HJK cell line is now in the 20th passage in tissue culture and grows very slowly; there are very few dividing cells and the cells can be subcultured only once monthly. Thus, these cells have not been further characterized. The ICRP mouse cells, like the mockinfected ICREF cells have become a stable line but resemble the ICREF cells rather than the wild type SV40-transformed ICRB cells. Three T antigen-positive clones (100% of cell exhibited T antigen)
518
HUEBNER ET AL.
and 15 T antigen-negative clones were established from the ICRP cell line. The T antigen-positive and T antigen-negative cell clones grew to saturation densities similar to that of ICREF cells (1.1-1.5 x lo6 cells/cml). The ICRB cells grew to a saturation density of 4.8 x lo6 cells/cm2. The ICRB, ICRP, and ICREF cells and cells of a T antigen-positive and a T antigen-negative clone of ICRP cells were tested for production of colonies in soft agar (MacPherson, 1969). Only ICRB cells produced visible colonies in soft agar. In order to determine if purification and concentration of defective viruses might influence the ability of these viruses to induce viral antigens early after infection of TC7 cells, an aliquot of each of the concentrated, KBr purified virus pools was inoculated into TC7 cultures, and coverslips removed at 2, 3, 4, 7 and 10 days after infection were tested for presence of SV40induced T and V antigens by indirect immunofluorescence. Neither T nor V antigen was detected in cultures infected with the defective virus isolates. It has been reported that stocks of a large plaque variant of SV40 contain defective virions with a density of 1.33 g/cc which are biologically inert as far as antigen formation is concerned (Yoshiike, 1968). Isopycnic Centrifugation Gradients
in CsCl Density
We have previously reported (Croce et al., 1974) that defective SV40 virus isolated from LN-SV and a LN-SV x Cl-1D hybrid subclone had a buoyant density in CsCl of 1.325 g/cc. The results obtained after isopycnic centrifugation in CsCl of defective virus pools isolated from several other nonrescuable transformed cell lines are shown in Figs. 2 and 3. The virus rescued from mKS-BUlOO cells (Fig. 2, panel a) has a buoyant density in CsCl of 1.34 g/cc which is the expected density for infectious SV40 virus. The viruses rescued from W18VA2W (Fig. 2, panel b), 2A-1 (Fig. 2, panel c), and WI26VA4 (Fig. 2, panel d) have densities between 1.32 and 1.33 g/cc. The virus rescued from H5 cells has a density of 1.34 g/cc (Fig. 3, panel a). T22
l.’ : i6 80.a . L iiIL I’i 40 . %’ :
: P
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if ” ,I R I 0
*i
.
i
,-
,~
i
l*
.
t....
LO- 4 -7 05-
?’ i
. ‘..*.Q..
:
..#.... .. .. .... .. .. .
4.0-
i
!!
2,0uJ I 2
3
VOLUME
(ml)
BOTTOM
4
5 TOP
FIG. 2. Isopycnic centrifugation in CsCl solution of KBr purified [S*P]phosphate- and [SH]leucinelabeled SV46 preparations. An aliquot of the KBr purified virus pool was centrifuged to equilibrium (40,000 rpm, 24 hr, 4” in CsCl (~5= 1.33) in the SW 50.1 rotor of a Spinco centrifuge. Fractions were collected from the bottom of the tube and IO ~1 aliquots were assayed for density (A-A) and 56 rl aliquots were assayed for counts per minute, ‘?p (O---O) and W (O-O). a) Virus rescued after fusion of mKS-BU106 cells with TC7 cells; b) virus rescued after fusion of WI8VA2W cells with TC7 cells; c) virus rescued after fusion of 2A-1 cells with TC7 cells; d) virus rescued after fusion of WI26VA4 cells with TC7 cells.
cells did not yield defective virus which was detectable in a CsCl gradient (Fig. 3, panel b). Several attempts were made to obtain defective virus from SV40-transformed human cells without fusion with TC7 cells and.without further passage on TC7 cells. Ten days after addition of [sH]thymidine, 10” W18VA2W cells were frozen and the medium and cell debris subjected to the procedure for SV40 purification. W18VA2W cells appeared to be synthesizing defective virions that banded at a density of 1.325; however, the amount of
DEFECTIVE
SV40 FROM
TRANSFORMED
CELLS
519
the virus peak after CsCl gradient purification was too small to allow further processing, DNA was extracted from aliquots of the KBr purified virus pools. There is very little, if any, nonvirion DNA in the KBr purified virus pools since, as can be seen in panel b of Fig. 3, the CsCl fractions obtained after centrifugation of the KBr purified preparation isolated from T22, Characteristics of DNA Isolated from the virus-negative sample, do not contain Defective SV40 Viruses significant amounts of labeled DNA. In order to determine whether the DNA The isolated virions obtained from the nonrescuable cell lines appeared to be extracted from the KBr purified virus pools unstable as evidenced by the large amount contained closed circular double-stranded of DNA found in the lower fractions of the DNA and/or DNA of the size of SV40CsCl gradients (Figs. 2 and 3). This DNA DNA, the DNA extracted from the virus appeared to derive from virions since if the pools was analyzed by ethidium bromidevirus containing fractions from the first CsCl centrifugation (Radloff et al., 1967) CsCl centrifugation were pooled and cen- and by velocity sedimentation in neutral trifuged to equilibrium a second time in CsCl (Yoshiike and Furuno, 1969). As can be seen in Fig. 4, the DNA CsCl almost all the DNA again appeared in the lower fractions of the gradients with a extracted from virus rescued from WIcorresponding decrease in the number of 26VA4 contained some closed circular double-stranded DNA (p = 1.59 g/cc) which counts found at the density of the virus. Since the amount of DNA remaining in migrated with the closed circular doublestranded DNA (component I) extracted from virus rescued from mKS-BUlOO cells. In addition, virus rescued from LN-SV, 3.0 2A-1 and 52-62-l-5 contained varying amounts of closed circular DNA (not shown in figure). g 2.0 The results of velocity sedimentation of z viral DNA in neutral CsCl is presented in 2 IL Fig. 5 and Table 2. The DNA extracted 5 I .o from virus rescued from LN-SV cells con0 tained DNA that sedimented slightly more I slowly than component I (21 S) DNA from z virus rescued from mKS-BUlOO cells, indi* I.0 n cating that the closed circular DNA from 4 LN-SV is slightly shorter than wild type SV40 component I DNA (Yoshiike and 0.5 Furuno, 1969). There was also DNA present that sedimented faster (29 S) than SV40 component I DNA. DNA extracted from virus rescued from 52-62-l-5 also I 2 3 4 5 BOTTOM contained heterogeneous DNA that sediTOP VOLUME (ml) mented at 29 S, 21 S, and 16 S (Table 2). FIG. 3. Isopycnic centrifugation in CsCl solution of These data indicate that defective virion [8H]thymidine-labeled virus preparations as depreparations rescued from these nonrescuscribed in the legend for Fig. 2. a) Virus rescued after able transformed cell lines contain DNA of fusion of H5 cells with TC7 cells; b) KBr purified the size and conformation of SV40 compopreparation obtained after fusion of T22 cells with TC7 cells. nent I DNA.
virus obtained (as determined by the amount of isotopic label in the virus peak in CsCl) was too low to perform further analysis. Thus all further experiments were performed using rescued virus; that is, virus obtained after fusion of the transformed cells with TC7 cells using Sendai virus.
520
HUEBNER
I
2
3
4
BOTTOM
5 TOP
VOLUME
Imll
FIG. 4. [‘Hlthymidine-labeled DNA extracted from virus rescued from mKSBU100 cells (O--O) was mixed with [‘2p]phosphate-labeled (O--O) DNA extracted from virus rescued from WI26VA4 and centrifuged at 40,000 rpm for 48 hr at 20” in CsCl solution containing 100 pglml of ethidium bromide. Fractions were assayed for total radioactivity and density. The band at 1.59 g/cc corresponds to closed circular double-stranded DNA (component I), whereas that at 1.56 g/cc corresponds to nicked circular or linear DNA.
ET AL.
used in this study contain the entire SV40 genome, cellular DNA was extracted from 2 x 10’ cells of each of the SV40-transformed cell lines listed in Table 1. Triplicate cultures of TC7 cells were inoculated with 100 kg per culture of DNA from each of the transformed lines as described in Materials and Methods. After 14 days the DNA-infected cultures were frozen and thawed three times and the crude preparations were tested for the ability to induce SV40 T and V antigen in TC 7 cultures and for the ability to produce plaques on TC7 cells. Infectious SV40 was not obtained from any of the SV40-transformed cell lines tested, including mKS-BUlOO, which is known to contain the entire SV4O genome. Thus, this test, under our conditions, cannot be used to determine which SV40-transformed cell lines contain the entire SV40 genome. DISCUSSION
FIG. 5. Velocity sedimentation in neutral CsCl. [“Hlthymidine-labeled DNA extracted from virus rescued from mKS-BUlOO cells (O---O) was mixed with [3aP]phosphate-labeled DNA (O---O) from virus rescued from LN-SV cells and layered on 3.0 ml of neutral CsCl solution (5 = 1.50) and centrifuged in a SW 50.1 rotor of a Spinco centrifuge at 35,000 rpm for 4.5 hr. Fractions were collected from the bottom and assayed for total radioactivity.
Infection of TC7 Cells with SV40-Transformed cells
DNA
from
It has been reported that DNA extracted from nonrescuable SV40-transformed cells produced SV40 virus if transferred to permissive cells, thus demonstrating that some nonrescuable SV40-transformed cells contain the entire SV4Ogenome (Boyd and Butel, 1972). In order to determine if the DNA from the SV40-transformed cells
In summary, defective viruses isolated from nonrescuable transformed monkey, human and hamster cells are capable of replication to a limited extent as evidenced by the formation of microplaques on TC7 monolayers and by the incorporation of radioactive precursors into virion DNA and protein. However, the defective viruses have a reduced ability to induce detectable levels of virar antigens in permissive, semipermissive and nonpermissive cells. T antigen-positive mouse cell clones, established after infection with a defective virus stock rescued from H50 cells, do not have the characteristics of wild type SV40-transformed cells (except that they are T antigen-positive) and are presumably nontransformed cells. Recovery of defective viruses from these transformed cell lines is consistent with the proposal that cells which are permissive or semipermissive to SV4Q lytic infection and which survive SV40 infection to become transformed contain defective viral genomes. The instability of the defective viruses and their low yields have thus far hampered complete analysis of the protein and DNA moieties of the viruses. We are currently preparing defective viruses in larger
DEFECTIVE
SV40 FROM
TRANSFORMED
TABLE
521
CELLS
2
SUMMARYOF CHARACTERISTICS OF RESCUEDDEFECTIVEVIRUSES Visible band in KBr ?
Particles observed byEM?
Density WCC) of virus in CsCl
Dens$;2/;c) of
2A-1 A-12-1 H5 T22
Yes No Yes No
Yes Yes Yes No
WI26VA4 W18VA2W
Yes Yes
Yes Yes
LN-SV F5-1 52-62-l-5
Yes No Yes
Yes Yes Yes
1.33 -a 1.34 1.33 1.325 1.325 1.325
1.59, 1.56 1.56 1.59, 1.56 1.56 1.59, 1.56 1.59, 1.56
Cell line of origin
S values of DNA in neutral CsCl
EB-CsCl
<16 S <16 S 16 S 16s 29 s, 20 s, 14 s 29S,21S, 16s
n This symbol means that there was no viral peak (no counts) in CsCl, thus these samples were not analyzed for DNA content.
quantities in order to determine if the DUBEI~,D. R., KIT, S., DE TORRES,R., and ANKEN,M. (1967). Virogenic properties of bromodeoxyuridinevirions contain cellular DNA sequences, sensitive and bromodeoxyuridine-resistant simian and, if so, if these sequences are unique.
virus 40-transformed mouse kidney cells. J. Viral. 1,
ACKNOWLEDGMENTS
The authors thank Anthony Girardi for helpful discussion throughout the course of this study and Emma de Jesus for excellent technical assistance. REFERENCES
ASHKENAZI,A., and MELNICK,J. L. (1963). Tumorige. nicity of simian papovavirus SV40 and of virustransformed cells. J. Nat. Cancer Inst. 30, 1227-1243. BOYD, V., and BUTEL, J. (1972). Demonstration of infectious deoxyribonucleic acid in transformed cells. I. Recovery of simian virus 40 from yielder and nonyielder transformed cells. J. Viral. 10, 399-409. CROCE,C. M., KOPROWSKI,H., and EAGLE,H. (1972). Effect of environmental pH on the efficiency of cellular hybridization. Proc. Nat. Acad. Sci. USA 69, 1953-1956. CROCE,C. M., GIRARDI, A. J., and KOPROWSKI,H. (1973). Assignment of the T-antigen gene of simian virus 40 to human chromosome C-7. Proc. Nat. Acad. Sci. USA 70, 3617-3620. CROCE,C. M., HUEBNER, K., GIRARDI, A. J., and KOPROWSKI,H. (1974). Rescue of defective SV40 from mouse-human hybrid cells containing human chromosome 7. Virology 60, 276-281. DUBBS,D. R., and KIT, S. (1964). Effect of halogenated pyrimidine and thymidine on growth of L-cells and a subline lacking thymidine kinase. Exp. Cell Res. 33, 19-28. DUBB~,D. R., and KIT, S. (1968). Isolation of defective lysogens from simian virus 40-transformed mouse kidney cultures. J. Viral. 2, 1272-1202.
968-979.
GIRARDI,A. J., SWEET,B., and HILL.EMAN,M. (1963). Factors influencing tumor induction in hamsters by vacuolating virus, SV40. Proc. Sot. Exp. Biol. Med. 112, 662-667.
GIRARDI,A. J., JENSEN,F., and KOPROWSKI, H. (1965). SV40-induced transformation of human diploid cells: crisis and recovery. J. Cell. Comp. Physiol. 65, 69-83. HIRT, B. (1967). Selective extraction of polyoma DNA from infected mouse cell cultures. J. Mol. Biol. 26, 365-369. HUEBNER,K., CROCE, C. M., and KOPROWSKI,H. (1974). Isolation of defective viruses from SV40-transformed human and hamster cells. Virology 59, 570-573. JENSEN, F., and DEFENDI,V. (1968). Transformation of African green monkey kidney cells by irradiated adenovirus ‘I-simian virus 40 hybrid. J. Viral, 2, 173-177. KELLY, T., JR., and ROSE,J. (1971). Simian virus 40 integration site in an adenovirus ‘I-simian virus 40 hybrid DNA molecule. Proc. Nat. Acad. Sci. USA 68, 1037-1041. Krr, S., KURIMURA, T., BROWN,M., and DUBBS, D. R. (1970). Identification of the simian virus 40 which replicates when simian virus 40-transformed human cells are fused with simian virus IO-transformed mouse cells or superinfected with simian virus 40 deoxyribonucleic acid. J. Viral. 6, 69-77. KNOWLES,
B. B., JENSEN,
F.
C., STJZPLEWSKI,
2..
and KOPROWSKI,H. (1968). Rescue of infectious SV40 after fusion between different SV40-transformed cells. Proc. Nat. Acad. Sci. USA 61,42-45.
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KOPROWSKI,H., JENSEN,F. C., and STEPLEWSKI,Z. (1967). Activation of production of infectious tumor virus SV40 in heterokaryon cultures. Proc. Nat. Acad. Sci. USA 58, 127-133. Lrmowrrz, P., KELLY, T. J., JR., NATHANS,D., LEE, T. N. H., and LEWIS,A. M., JR. (1974). A colinear map relating the simian virus 40 (SV40) DNA segments of six adenoviru-SV40 hybrids to the DNA fragments produced by restriction endonuclease cleavage of SV40 DNA. Proc. Nat. Acad. Sci. USA 71, 441-445. MACPHERSON,I. (1969). Agar suspension culture for quantitation of transformed cells. In “Fundamental Techniques in Virology,” (K. Habel and N. P. Salzman, eds.), pp. 214-228. Academic Press, New York. MELNICK, J. L., KHERA, K. S., and RIP, F. (1964). Papova virus SV40: Failure to isolate infectious virus from transformed hamster cells synthesizing SV40-induced antigens. Virology 23, 430-432. POPE,J. H., and ROWE,W. P. (1964). Detection of specific antigen in SV40 transformed cells by immunofluorescence. J. Exp. Med. 120, 121-127. RADLOFF,R., BAUER,W., and V~OGRAD,J. (1967). A dye-buoyant density method for the detection and isolation of closed circular duplex DNA: the closed circular DNA in HeLa cells. Proc. Nat. Acad. Sci. USA 57, 1514-1521. ROBB,J. A., and HUEBNER,K. (1973). Effect of cell
chromosome number on simian virus 40 replication. Exp. Cell Res. 81, 120-126. SHIROKI,K., and SHIMCJJO, H. (1971). Transformation of green monkey kidney cells by SV40 genome: The establishment of transformed cell lines and the replication of human adenoviruses and SV40 in transformed cells. Virology 45, 162-171. STEPLEWSKI,Z., and KOPROWSKI,H. (1970). Somatic cell fusion and hybridization. Methods Cancer Res. 5, 155-191. S WETLY,P., BARBANTI-BRODANO, G., KNOWLES,B., and KOPROWSKI, H. (1969). Response of simian virus IO-transformed cell lines and cell hybrids to superinfection with simian virus 40 and its deoxyribonucleic acid. J. Virol. 4, 348-355. WATKINS,J. F., and DULBECCO,R. (1967). Production of SV40 virus in heterokaryons of transformed and susceptible cells. hoc. Nat. Acad. Sci. USA 58, 1396-1403. WEISS, M. C., EPHRUSSI,B., and SCALET~A,L. J. (1968). Loss of T-antigen from somatic hybrids between mouse cells and SV40-transformed human cells. Proc. Nat. Acad. Sci. USA 59, 1132-1135. YOSHIIKE, K. (1968). Studies on DNA from low-density particles of SV40. II. Noninfectious virions associated with a large-plaque variant. Virology 34, 402-409. YOSHIIKE,K., and FURUNO,A. (19601. Heterogeneous DNA of simian virus 40. Fed. hoc. 28, 1899-1903.
63, 512-522 (1975)
Characterization
of Defective
SV40
Isolated
from SV40-Transformed
Cells l K. HUEBNER, D. SANTOLI, The Wistar Institute
of Anatomy
and Biology,
C. M. CROCE,
AND
H. KOPROWSKI
36th Street at Spruce, Philadelphia,
Accepted
Pennsylvania
19104
October 4, 1974
Defective SV40 viruses were isolated from SV40-transformed monkey, human and hamster cells after Sendai virus-mediated fusion of the transformed cells with TC7 cells, a stable line of African green monkey kidney cells. Viral isolates were concentrated and purified and the defective viruses examined by electron microscopy. The buoyant densities in CsCl of the defective viruses ranged’ between 1.32 and 1.33 g/cc. DNA isolated from defective viruses was characterized by dye-buoyant density centrifugation and by velocity sedimentation in neutral CsCl. The DNA was heterogeneous in size and contained some covalently closed double-stranded circular molecules. INTRODUCTION
The Sendai virus-mediated fusion of SV40-transformed nonpermissive (mouse) cells with African green monkey kidney (AGMK) cells, which are permissive to SV40 lytic infection, allows replication of the SV40 genome resident in the transformed cells (Koprowski et al., 1967; Watkins and Dulbecco, 1967). However, cells of many SV40-transformed permissive (monkey) and semipermissive (human and hamster) lines do not yield infectious virus after fusion with permissive cells (Knowles et al., 1968; Kit et al., 1970), and it has been proposed that these “nonrescuable” transformed cells contain defective virai genomes (Knowles et al., 1968). Recently we have reported formation of microplaques in AGMK cultures exposed to heterokaryocytes formed by fusion of nonrescuable human and hamster cells with AGMK cells (Huebner et al., 1974). Heterokaryocytes produced by fusion of AGMK cells with SV40-transformed human-mouse hybrid cells also formed mi‘This work Training Grant Nos. CA 10815 RR 05540 from and funds from
was supported in part by USPHS No. CA 05163 and Research Grant from the National Cancer Institute, the Division of Research Resources, the Commonwealth of Pennsylvania.
Copyright 0 1975 by Academic Press. Inc. All rights of reproduction in any term reserved.
croplaques on AGMK monolayers (Croce et al., 1974). Preliminary investigations led us to propose that these microplaques were formed by a defective SV40 rescued from the transformed cells (Huebner et al., 1974; Croce et al., 1974). This paper presents biological and physical characterizations of defective SV40 virions isolated from SV40-transformed monkey, human and hamster cell lines that had failed to yield wild type virus after fusion with permissive AGMK cells. MATERIALS
AND
METHODS
Cells. TC7, a line of AGMK cells (Robb and Huebner, 1973), was used for production of all SV40 pools and as the permissive cell in all rescue experiments. A summary of the properties of the SV40-transformed cell lines used in this study is presented in Table 1. H5 and T22 cells lines were kindly supplied by V. Defendi; A-12-1, 2A-1, WI26VA4, and H50 cell lines were obtained from A. Girardi. The 52-62-l-5 cells are LN-SV x Cl-1D hybrid cells containing the entire complement of Cl-1D mouse chromosomes plus human chromosome 7. Cells were subcultured in minimum essential Eagle’s medium supplemented with 10% fetal bovine serum (Flow Laboratories, Rockville, MD).
DEFECTIVE
SV40 FROM
TRANSFORMED
TABLE
513
CELLS
1
SV‘%TRANSFORMED CELL LINES Cell line”
mKS-BUlOO (Dubbs et al., 1967) 2A-1 (Koprowski et al., 1967) A-12-1 (Jensen and Defendi, 1968) H5 (Shiroki and Shimojo, 1971) T22 (Shiroki and Shimojo, 1971) W18VA2W (Weiss et al., 1968) WI26VA4 (Girardi et al., 1965) LN-SV (Croce et al., 1973) F5-1 (Girardi et al., 1963) H50 (Ashkenazi and Melnick, 1963) 52-62-l-5 (Croce et al., 1973)
Species
Transforming agent
Rescuable* ?
Mouse
sv40
65-70
Yes (Dubbs and Kit, 1968)
Monkey
sv40
64-70
No (Koprowski
Monkey
Human
Irradiated adeno 7-SV40 hybrid Irradiated adeno 7-SV40 hybrid Irradiated defective SV40 sv40
400-410
No (Jensen and Defendi, 1968) No (Shiroki and Shimojo, 1971) No (Shiroki and Shimojo, 1971) No. (Huebner et al., 1974)
Human
sv40
200-205
No (Knowles
Human Hamster Hamster
sv40 sv40 sv40
45-50 150-155 90-95
No (Huebner et al., 1974) No (Koprowski et al., 1967) No (Melnick et al., 1964)
LN-SV x Cl-1D hybrid
sv40
15-20
Monkey Monkey
210-215 a-90 80-85
et al., 1967)
et al., 1968)
No (Croce et al., 1974)
n References in this column are for transformation of these cell lines. b Rescuable means that standard plaque forming SV40 virus is obtained after fusion of the indicated with monkey kidney cells; references in this column are for reports on rescuability of the cell line. c Cl-1D cells are derived from mouse L-cells (Dubbs and Kit, 1964).
Sendai virus. Sendai virus was prepared as previously described (Steplewski and Koprowski, 1970) and inactivated in 0.025% B-propiolactone. Fusion. For rescue of SV40 from the SV40-transformed cells, 1 x 10’ TC7 cells were fused with 1 x 10’ transformed cells in the presence of Sendai virus according to techniques described previously (Croce et al., 1972). The fused cultures were seeded in roller bottles in minimum essential Eagle’s medium containing 10% fetal bovine serum. After lo-14 days cultures were frozen for later isolation of virus. For detection of plaque formation by heterokaryocytes, 5 x lo8 TC7 cells were fused with an equal number of transformed cells. Fused cultures were serially diluted and plated on TC7 monolayers in petri dishes as described previously (Huebner et al., 1974). Twenty hours after seeding, the medium was removed from the petri dishes and 8 ml of overlay medium containing 1%
ceil lines
agar in minimum essential Eagle’s medium with 8% fetal bovine serum was added. An additional 3 ml of overlay medium was added after 10 days of incubation at 37”. Ten days later, 3 ml of overlay medium containing l:lO,OOO neutral red was added and the number of plaques recorded one day later. mKS-BUlOO cells, which yielded large plaques after fusion with TC7 cells, were used as a positive control in all rescue experiments whether the method of detection was by SV40 particle isolation or production of plaques. SV40 T and V antigens. Coverslip cultures of TC7 cells that had been infected with wild type or defective SV40 were fixed at 2, 3, 4, 7 and 10 days after infection and tested for presence of T or V antigen by the indirect immunofluorescence method (Pope and Rowe, 1964). Labeling and purification of rescued SV40. Roller bottles of fused cell cultures to
be tested for presence of rescued virus were
514
HUEBNERETAL.
frozen at ten days after fusion as described above. After three cycles of freezing and thawing, the culture fluid was clarified by low speed centrifugation and the supernatant was centrifuged at 60,000 g to sediment virus and/or remaining debris. The pellets obtained after high speed sedimentation were resuspended in minimum essential medium containing 10% fetal calf serum and the concentrated suspensions adsorbed to TC7 monolayers. To label the DNA moiety of the virus, the monolayer cultures were supplemented at 18 hr after infection with 5 &i/ml of [aH]thymidine (specific activity 52 Ci/mmole, New England Nuclear, Boston, MA) or 10 &i/ml of [YP]phosphate (carrier-free, New England Nuclear). To label the protein moiety of the virus, cultures were supplemented with 5 &i/ml of [aH]leucine (specific activity 32 Ci/mmole, New England Nuclear). Ten to 14 days later, cultures were frozen-thawed three times and virus was purified by a standard procedure (Swetly et al., 1969) consisting of sedimentation by high speed centrifugation, treatment with sodium desoxycholate, banding by centrifugation in a steep KBr gradient, pooling of all virus bands and freeing them of KBr by dialysis. If no virus bands were visible in the KBr gradient, the KBr fractions corresponding to those fractions in which the virus was present in positive control samples were collected and dialyzed. After dialysis the virus pools, which will be referred to as the KBr purified pools, were processed in several ways: a sample was taken for electron microscopic examination; a sample was inoculated into TC7 cell cultures and coverslips were later examined for presence of SV40-induced T or V antigens; an aliquot was centrifuged to equilibrium in CsCl solution (j = 1.33) and fractions assayed for density and total radioactivity; and finally DNA was extracted from an aliquot and analyzed for the presence of closed circular double-stranded DNA. Electron microscopy. Purified virus was spread on carbon-coated copper grids, stained with potassium phosphotungstate (PTA), pH 7.0, and observed in an electron microscope (Hitachi HS-8) at a magnification of; 24,000 and 33,000 x .
Plaque purification of wild type and defective SV40 viruses. Plaque isolates of
rescued from mKS-BUlOO, virus W18VA2W and H50 cells were prepared by picking up material from plaques formed after seeding of heterokaryocytes on TC7 cell monolayers. After addition of neutral red to the petri dishes at 20 days after seeding of the heterokaryocytes, the wild type plaques (rescued from mKS-BUlOO cells) or microplaques (rescued from W18VA2W and H50 cells) (Huebner et al., 1974) could be visualized as light areas of dead cells that did not take up neutral red. Virus was picked up from the plaques by putting a sterile needle through the agar and scraping the plaque area. The material in the needle was then resuspended in 0.5 ml of MEM containing 10% fetal calf serum; 0.2 ml of this suspension was inoculated into a culture of TC7 cells and after 2 weeks the culture and medium were frozen-thawed three times and this crude virus stock was stored at -20”. SV40-B, SV40-P and SV40-K are virus stocks prepared in this way from plaque isolates obtained after rescue of mKS-BUlOO, H50, and W18VA2W respectively. “Transformation”
by defective
viruses.
ICR mouse embryo fibroblasts were inoculated with SV40-B, SV40-P or with a crude cellular extract prepared from mockinfected TC7 cells. The cells will subsequently be referred to as ICRB (infected with SV40-B plaque isolate rescued from mKS-BUlOO cells), ICRP (infected with SV40-P plaque isolate rescued from H50 cells) and ICREF (mock infected). Human diploid fibroblasts that were inoculated with SV40-B, SV40-K or were mock infected will be referred to as HJB (infected with SV40-B), HJK (infected with SV40-K plaque isolate from W18VA2W cells) and HJ (mock infected). After infection or mock infection these cell lines were subcultured and tested at each passage for appearance of SV40-induced T or V antigen by indirect immunofluorescence. Cloning. ICRP cells at passage 30 were cloned by seeding 200 cells per 70-mm petri dish. Fourteen days after seeding, cloning cylinders were placed over visible colonies using sterile vacuum grease, and the colo-
DEFECTIVE
SV40 FROM
nies were trypsinized and transferred to flasks. Cell cultures derived from individual colonies were tested for SV40 T antigen by indirect immunofluoresence. Three clones were obtained in which 100% of the cells were T antigen-positive and fifteen clones were obtained in which 100% of the cells were T antigen-negative. Agar suspension culture for detection of transformed cells. The ICRB, ICRP and
ICREF cells as well as a T antigen-positive and a T antigen-negative clone of ICRP cells were tested for ability to form colonies in semisolid medium (MacPherson, 1969). Five-tenths per cent agar medium was prepared by mixing one part distilled water containing 1.0% agar (Difco bacto agar) with one part prewarmed double-strength minimum essential medium (containing twice the normal concentration of all components and 20% fetal bovine serum). Base layers were prepared by distributing 7 ml of 0.5% agar medium into petri dishes. Cell suspensions were prepared by mixing one volume of cells in minimum essential medium with 2 volumes of 0.5% agar medium. When the base layers were set, 3 ml of the agar cell suspensions were layered into each petri dish. For each cell line tested, lo’, 10s, and lo2 cells per petri dish were plated. Cultures were incubated at 37” in a humidified atmosphere and at 12 days the petri dishes were examined for appearance of colonies. Isolation of SV40 DNA. DNA was isolated from KBr purified virus pools by a phenol extraction procedure (Swetly et al., 1969). After removal of residual phenol from the aqueous phase by extraction with ether, the DNA was precipitated by addition of ethanol and dissolved in NTE buffer (0.05 MTris, 0.13 A4 NaCl, 0.001 MEDTA, pH 7.8). Extraction of cellular DNA. Cellular DNA was extracted from SV40-transformed cells by the Hirt procedure (Hirt, 1967). The pellet obtained after the Hirt extraction was resuspended in 1 x SSC (0.15 M NaCl, 0.015 M sodium citrate) and dialyzed for 24 hr against 1 x SSC. The resuspended pellet was treated with 100 pg/ml of Pronase for 30 min at 37”. The
TRANSFORMED
CELLS
515
DNA was deproteinized by mixture with two volumes of redistilled phenol, and the aqueous phase was dialyzed against 1 x SSC for 24 hr. The concentration of the DNA was estimated spectrophotometricallY. Passage of transformed
cell DNA on TC7
cells. A preparation of transformed cell DNA, 0.3 ml, containing 100 pg of DNA was mixed with 0.3 ml of a 1 mg /ml solution of DEAE-dextran and the mixture was inoculated on 2 x lo6 TC7 ceils. After 15 min at room temperature the inoculum was removed and the monolayer washed three times with PBS. Medium was added and the culture incubated for 14 days. Cells were then disrupted by three cycles of freezing and thawing and the crude preparation was tested for infectious SV40 by plaque assay and by ability to induce SV40 T and V antigen in TC7 cells. SV40pZaque assay. Two-tenths milliliter of serial tenfold dilutions of virus stock was seeded on duplicate TC7 monolayers. After adsorption an 8-ml overlay of 1.0% agar in double strength Eagle’s medium was added. At 10 days a 3-ml overlay was added and at 20 days a 3-ml overlay containing l:lO,OOOneutral red was added and plaques were counted the next day. RESULTS
Electron
Microscopy
Electron microscopic examination of the KBr purified virus preparations revealed the presence of papova particles in preparations obtained by rescue of SV40-transformed monkey, human, hamster and mouse cells. Virions rescued from the SV40-transformed mouse cell line, mKS-BUlOO, from which wild type virus is routinely rescued, are shown in Fig. If. These virions have an average diameter of 43 nm, and most are complete virions. In comparison with the wild type virus preparation, very few particles were observed in each of the defective virus preparations, and most of the defective virions were coreless with an average diameter of 46 nm.
516
HUEBNER
ET AL.
FIG. 1. Electron micrographs of SV40 particles (x 160,800) rescued from: a) SV40-transformed human cell line, LN-SV; b) human-mouse hybrid cell line, 52-62-l-5; c) SV40-transformed human cell line, WI26VA4; d) SV40-transformed hamster cell line, F5-1; e) SV40transformed monkey cell line, 2A-1; f) SV40-transformed mouse cell line, mKSBU100.
Figure la is a photomicrograph of virions rescued from the SV40-transformed human cell line, LN-SV, and Figure lb is a photomicrograph of virions rescued from the LN-SV x Cl-1D hybrid cell line, 52-62-l-5,
in which the only human chromosome is chromosome number 7, which is the site of SV40 integration (Croce et al., 1973; Croce et al., 1974). Virus rescued from LN-SV (Fig. la), 52-62-l-5 (Fig. lb) and the
DEFECTIVE
SV40 FROM
SV40-transformed hamster cell line, F5-1 (Fig. Id) were the only preparations in which partially degraded but complete virions were observed by electron microscopy. In virus preparations rescued from the SV40-transformed human cell line, WI26VA4 (Fig. lc), and from the SV40-transformed monkey cell line, 2A-1 (Fig. le), only coreless virions were observed. The SV40-transformed human and hamster cell lines and the SV40-transformed monkey cell line, 2A-1, were all transformed after exposure to wild type sv40. In addition to these cell lines, several monkey cell lines transformed after exposure to defective SV40 pools were investigated. The T22 cell line was derived from transformed foci of AGMK cells after exposure to a defective virion fraction of SV40 which had been inactivated by ultraviolet irradiation (Shiroki and Shimojo, 1971). No virus particles were observed in KBr purified preparations after fusion of T22 cells with TC7 cells. The A-12-l. (Jensen and Defendi, 1968) and the H5 (Shiroki and Shimojo, 1971) cell lines were derived from AGMK cells transformed by irradiated adenovirus 7-SV40 hybrid virus strain LL-E46, which contains 60% of the SV40 genome (Lebowitz et al., 1974) and 90% of the adenovirus 7 genome (Kelly and Rose, 1971). Particles were observed in virus preparations rescued from the A-12-1 (40-50 nm) and H5 (45-90 nm) cell lines. Characterization of the antigenic properties of these particles is under investigation. Biological Properties Viruses
of Rescued Defective
The initial observation that led US to investigate the rescue of defective viruses was that heterokaryocytes formed by fusion of nonrescuable SV40-transformed human and hamster cells with TC7 cells formed microplaques three weeks after seeding on TC7 monolayers (Huebner et al., 1974). We have subsequently observed the formation of microplaques on TC7 monolayers after seeding heterokaryocytes formed by fusion of SV40-transformed
TRANSFORMED
CELLS
517
monkey cells (2A-1) with TC7 cells. Mateformed by rial from microplaques WlSVA2W-TC7 heterokaryocytes and H50-TC7 heterokaryocytes was picked up and virus stocks prepared as described in Materials and Methods. The virus stocks prepared from a W18VA2W-TC7 microplaque (SV40-K), a mKS-BUlOO-TC7 large plaque (SV40-B) and an H50-TC7 microplaque (SV40-P) were tested for ability to induce SV40 T and V antigens in TC7 cells and for ability to transform human or mouse cells. Aliquots of each virus stock were inoculated into TC7 cultures and coverslips were later removed and tested for SV40 T and V antigen. By ten days after infection neither T nor V antigen was detectable in cultures infected with SV40-P or SV40-K. Cultures infected with SV40-B were 99% T antigenpositive and 97% V antigen-positive by 60 hr after infection. Human diploid fibroblasts (HJ cells) were infected with SV40-B (HJB cells) or SV40-K (HJK cells); ICR mouse embryo fibroblasts (ICREF cells) were infected with SV40-B (ICRB cells) or SVCO-P (ICRP cells). The infected human and mouse fibroblasts were subcultured in parallel with mock-infected sister cultures and tested at each passage level for SV40 T antigen. After subculturing six times, the HJB cell line and the ICRB cell line were 99% T antigen-positive, noncontact inhibited, and appeared morphologically transformed, while the HJK and ICRP cell lines contained some SV40 T antigen-positive cells but the percentage varied between 10 and 50% in subsequent passages and never exceeded 50% of the cells. Cells of the mock-infected ICREF and HJ cell lines were always T antigen-negative. The HJK cell line is now in the 20th passage in tissue culture and grows very slowly; there are very few dividing cells and the cells can be subcultured only once monthly. Thus, these cells have not been further characterized. The ICRP mouse cells, like the mockinfected ICREF cells have become a stable line but resemble the ICREF cells rather than the wild type SV40-transformed ICRB cells. Three T antigen-positive clones (100% of cell exhibited T antigen)
518
HUEBNER ET AL.
and 15 T antigen-negative clones were established from the ICRP cell line. The T antigen-positive and T antigen-negative cell clones grew to saturation densities similar to that of ICREF cells (1.1-1.5 x lo6 cells/cml). The ICRB cells grew to a saturation density of 4.8 x lo6 cells/cm2. The ICRB, ICRP, and ICREF cells and cells of a T antigen-positive and a T antigen-negative clone of ICRP cells were tested for production of colonies in soft agar (MacPherson, 1969). Only ICRB cells produced visible colonies in soft agar. In order to determine if purification and concentration of defective viruses might influence the ability of these viruses to induce viral antigens early after infection of TC7 cells, an aliquot of each of the concentrated, KBr purified virus pools was inoculated into TC7 cultures, and coverslips removed at 2, 3, 4, 7 and 10 days after infection were tested for presence of SV40induced T and V antigens by indirect immunofluorescence. Neither T nor V antigen was detected in cultures infected with the defective virus isolates. It has been reported that stocks of a large plaque variant of SV40 contain defective virions with a density of 1.33 g/cc which are biologically inert as far as antigen formation is concerned (Yoshiike, 1968). Isopycnic Centrifugation Gradients
in CsCl Density
We have previously reported (Croce et al., 1974) that defective SV40 virus isolated from LN-SV and a LN-SV x Cl-1D hybrid subclone had a buoyant density in CsCl of 1.325 g/cc. The results obtained after isopycnic centrifugation in CsCl of defective virus pools isolated from several other nonrescuable transformed cell lines are shown in Figs. 2 and 3. The virus rescued from mKS-BUlOO cells (Fig. 2, panel a) has a buoyant density in CsCl of 1.34 g/cc which is the expected density for infectious SV40 virus. The viruses rescued from W18VA2W (Fig. 2, panel b), 2A-1 (Fig. 2, panel c), and WI26VA4 (Fig. 2, panel d) have densities between 1.32 and 1.33 g/cc. The virus rescued from H5 cells has a density of 1.34 g/cc (Fig. 3, panel a). T22
l.’ : i6 80.a . L iiIL I’i 40 . %’ :
: P
--h
if ” ,I R I 0
*i
.
i
,-
,~
i
l*
.
t....
LO- 4 -7 05-
?’ i
. ‘..*.Q..
:
..#.... .. .. .... .. .. .
4.0-
i
!!
2,0uJ I 2
3
VOLUME
(ml)
BOTTOM
4
5 TOP
FIG. 2. Isopycnic centrifugation in CsCl solution of KBr purified [S*P]phosphate- and [SH]leucinelabeled SV46 preparations. An aliquot of the KBr purified virus pool was centrifuged to equilibrium (40,000 rpm, 24 hr, 4” in CsCl (~5= 1.33) in the SW 50.1 rotor of a Spinco centrifuge. Fractions were collected from the bottom of the tube and IO ~1 aliquots were assayed for density (A-A) and 56 rl aliquots were assayed for counts per minute, ‘?p (O---O) and W (O-O). a) Virus rescued after fusion of mKS-BU106 cells with TC7 cells; b) virus rescued after fusion of WI8VA2W cells with TC7 cells; c) virus rescued after fusion of 2A-1 cells with TC7 cells; d) virus rescued after fusion of WI26VA4 cells with TC7 cells.
cells did not yield defective virus which was detectable in a CsCl gradient (Fig. 3, panel b). Several attempts were made to obtain defective virus from SV40-transformed human cells without fusion with TC7 cells and.without further passage on TC7 cells. Ten days after addition of [sH]thymidine, 10” W18VA2W cells were frozen and the medium and cell debris subjected to the procedure for SV40 purification. W18VA2W cells appeared to be synthesizing defective virions that banded at a density of 1.325; however, the amount of
DEFECTIVE
SV40 FROM
TRANSFORMED
CELLS
519
the virus peak after CsCl gradient purification was too small to allow further processing, DNA was extracted from aliquots of the KBr purified virus pools. There is very little, if any, nonvirion DNA in the KBr purified virus pools since, as can be seen in panel b of Fig. 3, the CsCl fractions obtained after centrifugation of the KBr purified preparation isolated from T22, Characteristics of DNA Isolated from the virus-negative sample, do not contain Defective SV40 Viruses significant amounts of labeled DNA. In order to determine whether the DNA The isolated virions obtained from the nonrescuable cell lines appeared to be extracted from the KBr purified virus pools unstable as evidenced by the large amount contained closed circular double-stranded of DNA found in the lower fractions of the DNA and/or DNA of the size of SV40CsCl gradients (Figs. 2 and 3). This DNA DNA, the DNA extracted from the virus appeared to derive from virions since if the pools was analyzed by ethidium bromidevirus containing fractions from the first CsCl centrifugation (Radloff et al., 1967) CsCl centrifugation were pooled and cen- and by velocity sedimentation in neutral trifuged to equilibrium a second time in CsCl (Yoshiike and Furuno, 1969). As can be seen in Fig. 4, the DNA CsCl almost all the DNA again appeared in the lower fractions of the gradients with a extracted from virus rescued from WIcorresponding decrease in the number of 26VA4 contained some closed circular double-stranded DNA (p = 1.59 g/cc) which counts found at the density of the virus. Since the amount of DNA remaining in migrated with the closed circular doublestranded DNA (component I) extracted from virus rescued from mKS-BUlOO cells. In addition, virus rescued from LN-SV, 3.0 2A-1 and 52-62-l-5 contained varying amounts of closed circular DNA (not shown in figure). g 2.0 The results of velocity sedimentation of z viral DNA in neutral CsCl is presented in 2 IL Fig. 5 and Table 2. The DNA extracted 5 I .o from virus rescued from LN-SV cells con0 tained DNA that sedimented slightly more I slowly than component I (21 S) DNA from z virus rescued from mKS-BUlOO cells, indi* I.0 n cating that the closed circular DNA from 4 LN-SV is slightly shorter than wild type SV40 component I DNA (Yoshiike and 0.5 Furuno, 1969). There was also DNA present that sedimented faster (29 S) than SV40 component I DNA. DNA extracted from virus rescued from 52-62-l-5 also I 2 3 4 5 BOTTOM contained heterogeneous DNA that sediTOP VOLUME (ml) mented at 29 S, 21 S, and 16 S (Table 2). FIG. 3. Isopycnic centrifugation in CsCl solution of These data indicate that defective virion [8H]thymidine-labeled virus preparations as depreparations rescued from these nonrescuscribed in the legend for Fig. 2. a) Virus rescued after able transformed cell lines contain DNA of fusion of H5 cells with TC7 cells; b) KBr purified the size and conformation of SV40 compopreparation obtained after fusion of T22 cells with TC7 cells. nent I DNA.
virus obtained (as determined by the amount of isotopic label in the virus peak in CsCl) was too low to perform further analysis. Thus all further experiments were performed using rescued virus; that is, virus obtained after fusion of the transformed cells with TC7 cells using Sendai virus.
520
HUEBNER
I
2
3
4
BOTTOM
5 TOP
VOLUME
Imll
FIG. 4. [‘Hlthymidine-labeled DNA extracted from virus rescued from mKSBU100 cells (O--O) was mixed with [‘2p]phosphate-labeled (O--O) DNA extracted from virus rescued from WI26VA4 and centrifuged at 40,000 rpm for 48 hr at 20” in CsCl solution containing 100 pglml of ethidium bromide. Fractions were assayed for total radioactivity and density. The band at 1.59 g/cc corresponds to closed circular double-stranded DNA (component I), whereas that at 1.56 g/cc corresponds to nicked circular or linear DNA.
ET AL.
used in this study contain the entire SV40 genome, cellular DNA was extracted from 2 x 10’ cells of each of the SV40-transformed cell lines listed in Table 1. Triplicate cultures of TC7 cells were inoculated with 100 kg per culture of DNA from each of the transformed lines as described in Materials and Methods. After 14 days the DNA-infected cultures were frozen and thawed three times and the crude preparations were tested for the ability to induce SV40 T and V antigen in TC 7 cultures and for the ability to produce plaques on TC7 cells. Infectious SV40 was not obtained from any of the SV40-transformed cell lines tested, including mKS-BUlOO, which is known to contain the entire SV4O genome. Thus, this test, under our conditions, cannot be used to determine which SV40-transformed cell lines contain the entire SV40 genome. DISCUSSION
FIG. 5. Velocity sedimentation in neutral CsCl. [“Hlthymidine-labeled DNA extracted from virus rescued from mKS-BUlOO cells (O---O) was mixed with [3aP]phosphate-labeled DNA (O---O) from virus rescued from LN-SV cells and layered on 3.0 ml of neutral CsCl solution (5 = 1.50) and centrifuged in a SW 50.1 rotor of a Spinco centrifuge at 35,000 rpm for 4.5 hr. Fractions were collected from the bottom and assayed for total radioactivity.
Infection of TC7 Cells with SV40-Transformed cells
DNA
from
It has been reported that DNA extracted from nonrescuable SV40-transformed cells produced SV40 virus if transferred to permissive cells, thus demonstrating that some nonrescuable SV40-transformed cells contain the entire SV4Ogenome (Boyd and Butel, 1972). In order to determine if the DNA from the SV40-transformed cells
In summary, defective viruses isolated from nonrescuable transformed monkey, human and hamster cells are capable of replication to a limited extent as evidenced by the formation of microplaques on TC7 monolayers and by the incorporation of radioactive precursors into virion DNA and protein. However, the defective viruses have a reduced ability to induce detectable levels of virar antigens in permissive, semipermissive and nonpermissive cells. T antigen-positive mouse cell clones, established after infection with a defective virus stock rescued from H50 cells, do not have the characteristics of wild type SV40-transformed cells (except that they are T antigen-positive) and are presumably nontransformed cells. Recovery of defective viruses from these transformed cell lines is consistent with the proposal that cells which are permissive or semipermissive to SV4Q lytic infection and which survive SV40 infection to become transformed contain defective viral genomes. The instability of the defective viruses and their low yields have thus far hampered complete analysis of the protein and DNA moieties of the viruses. We are currently preparing defective viruses in larger
DEFECTIVE
SV40 FROM
TRANSFORMED
TABLE
521
CELLS
2
SUMMARYOF CHARACTERISTICS OF RESCUEDDEFECTIVEVIRUSES Visible band in KBr ?
Particles observed byEM?
Density WCC) of virus in CsCl
Dens$;2/;c) of
2A-1 A-12-1 H5 T22
Yes No Yes No
Yes Yes Yes No
WI26VA4 W18VA2W
Yes Yes
Yes Yes
LN-SV F5-1 52-62-l-5
Yes No Yes
Yes Yes Yes
1.33 -a 1.34 1.33 1.325 1.325 1.325
1.59, 1.56 1.56 1.59, 1.56 1.56 1.59, 1.56 1.59, 1.56
Cell line of origin
S values of DNA in neutral CsCl
EB-CsCl
<16 S <16 S 16 S 16s 29 s, 20 s, 14 s 29S,21S, 16s
n This symbol means that there was no viral peak (no counts) in CsCl, thus these samples were not analyzed for DNA content.
quantities in order to determine if the DUBEI~,D. R., KIT, S., DE TORRES,R., and ANKEN,M. (1967). Virogenic properties of bromodeoxyuridinevirions contain cellular DNA sequences, sensitive and bromodeoxyuridine-resistant simian and, if so, if these sequences are unique.
virus 40-transformed mouse kidney cells. J. Viral. 1,
ACKNOWLEDGMENTS
The authors thank Anthony Girardi for helpful discussion throughout the course of this study and Emma de Jesus for excellent technical assistance. REFERENCES
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