Rescue of simian virus 40 (SV40) from SV40-transformed cells by fusion with anucleate monkey cells and variation in the yield of virus rescued by fusion with replicating or nonreplicating monkey cells

VIROLOQY

60,

Rescue

s-95

(1974)

of Simian

Fusion

with

Virus

Anucleate

of Virus

40 (SV40) Monkey

Rescued

from Cells

by Fusion

Nonreplicating G. POSTE, Department of Experimental

SV40-Transformed and

with

Monkey

Variation Replicating

Cells

by

in the Yield or

Cells

B. SCHAEFFER

AND

Pathology, Roswell Park Memorial Institute, Buffalo, New York 14808 AND

P. REEVE,’

AND

D. J. ALEXANDER2

Department of Virology, Royal Postgraduate Medical School, Du Cane Road, London W.19, England Accepted February 28, 1974 The formation of heterokeryons by fusion of anucleate African green monkey kidney cells with simian virus 40 (SV40)-transformed mouse 3T3 (SV3T3) cells resulted in the rescue of infective SV40. The yield of SV40 rescued from the heterokaryons was influenced by the physiological state of the monkey cells. Fusion of SV3T3 cells with monkey cells from actively dividing subconfluent log phase cultures resulted in the rescue of significantly lower yields of SV40 than from heterokaryons produced between SV3T3 cells and monkey cells from nondividing confluent stationary phase cultures. This effect was found with both anucleate and nucleated

monkey cells. INTRODUCTION

pccts of this problem we have investigated the rescue of infective SV40 from SV40transformed mouse 3T3 (SV3T3) cells after fusion with African green monkey kidney cells enucleated by cytochalasin B (Carter, 1967; Poste, 1972, 1973). The results reported here show that anucleate permissive cells can achieve rescue of infective SV40 from SV3T3 cells, indicating that the nucleus of the permissive cell is not required for the initiation or maintenance of SV40 replication and the release of infective virions from heterokaryons. When the work reported here was being prepared for publication a short communication by Croce and Koprowski (1973) appeared describing similar rescue of SV40 from SV40-transformed mouse mKS BUlOO cells on fusion with anucleate CV-1 cells. The present work confirms their observations but also introduces the new finding that

The recovery of simian virus 40 (SV40) from SV40-transformed cells by cocultivation or fusion with permissive cell lines to form heterokaryons has been described extensively (Gerber, 1966; Koprowski el al., 1967; Watkins and Dulbecco, 1967; Knowles et al., 1968; Kit and Brown, 1969; Jensen and Koprowski, 1969; Uchida and Watanabe, 1969; Kit et al., 1970; Watkins, 1970; Suarez et al., 1972). The mechanism of SV40 “rescue” by cell fusion and the respective contribution of the nucleus and the cytoplasm of the permissive cell in enabling SV40 replication to take place in heterokaryons are still unclear. In an effort to clarify as1 Present address: Department of Bacteriology, University College Hospital Medical School, London, England. *Present address: Central Veterinary Laboratory, Weybridge, England. 85 Copyright All rights

@ 1974 by Academic Press, of repmduction in any form

Inc. r-ed.

86

POSTE

the rate of division of the pcrmissivc cells at the time of fusion exerts a significant effect on the yield of SV40 rescued from transformed cells. Significant variation in the yield of rescued SV40 has been detected between subconfluent actively dividing cells and confluent nondividing monkey cells when fused with virus-transformed cells. This difference was found with both nucleated and anuclcatc monkey ccl1 populations. MATERIALS

AND

METHODS

Cells. Primary and secondary African green monkey kidney (AGMK) cells were cultivated in Eagle’s Basal Medium supplcmented with 10 % fetal bovine serum. A continuous line of African green monkey kidney cells, BS-C-1 (Hopps et al., 1963), and SV40transformed mouse 3T3 (SV3T3) cells were grown in Dulbecco’s modification of Eagle’s Medium supplemented with 10% fetal bovine serum (Poste et al., 1973). Enucleation of cell cultures. AGMK and BS-C-1 cells were enucleated by high speed centrifugation in culture medium supplcmentcd with 10 pg/ml cytochalasin B by the methods described previously (Poste, 1973). The properties of anucleate cells have been described in detail elsewhere (Post?, 1972, 1973; Goldman et al., 1973). The yield of anucleate cells was assessed from covcrslippreparations stained with May-Grtinwald and Giemsa. Sendai virus. Tho M34996 strain of Srndai virus obtained originally from Dr. H. Pereira, Medical Research Council, London, was propagated in 11-day-old fertile hen’s eggs and collected by methods described elsewhere (Harris et al., 1966). The concentration of virus was expressed in terms of the number of hemagglutinating units (HAU) per milliliter of harvested chorioallantoic fluid as determined by the standard WHO plate hemagglutination test. Virus prcparations used in cell fusion experiments were inactivated by /3-propiolactone as described previously (Postc, 1973). Cell fusion and detection of heterokaryons. The methods described previously for the fusion of anucleate and nucleated cells by inactivated Sendai virus were used (Poste, 1973; Poste and Reeve, 1972) with the minor

ET .4L.

modification that fusion of the cell mixtures was done in medium buffered to pH 8.0 which has been shown to incrcasc> th(> frcquency of cell fusion by Scndai virus (Crocc et al., 1972). Bicarbonate-free culture mpdium was buffrrcd by the addition of I.5 rnfi1 N - 2 - hydroxycthylpiperazino -S - cthancsulfonic acid (HEPES) and then adjusted to pH 8.0. The same methods were also used to product both hctrrokaryons bct,wcacn nuclratrd monkey and mouse cells and SV3T3, AGMK, and BS-C-1 homokaryons. A series of replicate covcrslip cultures wcrc prepared from the same fused cell population for mcasurrmrnt of the number of hctcrokaryons, and the pcrccntage of hctcrokaryons yielding SV40 using an identical protocol to that drscribed by Watkins (1970). Heterokaryon formation between anucleate monkey cells and nucleated SV3T3 cells was assessed on covcrslip cultures by the demonstration of monkey-specific antigens on the surface of nucleated or multinucleated SV3T3 cells by the mixed hemadsorption (HAD) technique as described previously (Watkins and Grace, 1967; Poste and Reeve, 1972). The extent of heterokaryon formation was calculated by expressing the number of HAD-positive cells containing one or more SV3T3 nuclei as a percentage of the total number of multinucleatc cells present in the same culture. Although a small number of nucleated monkey cells remained in the culture after (nucleation (see results) and reacted positively in the mixed HAD test these could be easily distinguished from SV3T3 cells and SV3T3-anucleate monkey cell hrtcrokaryons by the different morphology of their nuclei. In coverslip cultures stained by haematoxylin and rosin or Giemsa the nuclei of AGMK and BS-C-1 monkey cells contain one or two spherical nucleoli and very little or no heterochromatin whereas the nuclei of 3T3 and SV3T3 cells contain five or six irregularly shaped nucleoli and numerous small darkly staining areas of hetcrochromatin (see Watkins and Dulbccco, 1967). Additional proof of fusion between anucleate AGMK or BS-C-1 cells and SV3T3 cells was provided by microscopic counts on stained coverslip preparations of the mixed

SV40 RESCUE cell populations before and after addition of Sendai virus, which revealed a significant decrease in the number of anucleate cells after treatment with virus. Anucleate cells are more susceptible to lysis by Sendai virus than nucleated cells (Poste, 1973). However, the decrease in the number of anucleate cells after treatment with Sendai virus in the presence of SV3T3 cells was 60-80 % greater than the decrease of between 9 and 17 % of the total cell population in similar anucleate cell populations treated with virus in the absence of SV3T3 cells, indicating that this proportion of the decrease in anucleate cell members was due to fusion with SV3T3 cells to form heterokaryons. Heterokaryons formed by fusion of SV3T3 cells and nucleated AGMK or BS-C-1 cells were identified by the presence of morphologically distinct nuclei from both cell types within the same multinucleate cell. The extent of heterokaryon formation between nucleated cells was estimated from microscopic counts on coverslip cultures of mixed cell cultures after treatment with Sendai virus. A minimum of 5000 cells were counted on four separate replicate coverslips and the number of multinucleate cells containing nuclei from both parent cell types (heterokaryons) was expressed as a percentage of the total number of multinucleate cells counted in the same microscope fields. Further proof of mouse-monkey heterokaryon formation between nucleated mouse and monkey cells was obtained by the identification of mouse-specific antigens by mixed HAD on the surface of multinucleatc cells which contained monkey cell nuclei. Antisera against SV3T3 and BS-C-l cells for use in mixed HAD tests were prepared in rabbits by intravenous inoculation of 1 X 10’ cells at weekly intervals for 4 weeks and serum was separated from whole blood samples obtained 1 week after the last inoculation of cells. The antisera showed marked species specificity and the SV3T3 antiserum did not react with the BS-C-1 cells or vice versa. Detection of infective SV40 in cell-free homogenates. Cell cultures were suspended in 3.0 ml of culture medium without serum, frozen and thawed twice (-60” to 37”) and

87

finally sonicated at 10 kc for 5 min at 4”. The cell debris was then removed by lowspeed centrifugation and the supernatant assayed for the presence of infective SV40 by plaque titration as described below (0.1 ml undiluted sample per 60-mm dish; 10 dishes per sample). SV40 plaque assay. The presence of SV40 in cellular extracts was assayed by the plaque technique in confluent AGMK or BS-C-1 cell monolayers in 60 mm X 15 mm plastic petri dishes (Falcon Plastics, Oxnard, California). Aliquots of 0.1 ml of undiluted sonicated cell-free extract were added to the cells in dishes from which the culture medium had been removed and replaced with 0.3 ml of 0.01 M (pH 7.4) tris(hydroxymethyl)aminomethane (Tris) buffer as a diluent. The extracts were allowed to adsorb for 2 hr at 37” and then 4.0 ml of overlay was added to each dish. The overlay consisted of Eagle’s Basal h’ledium supplemented with 10 r/;l fetal bovine serum and 0.8 % agar per ml. The dishes were then incubated at 37” in a 95 % air: 5 % COz environment for 8 days at which time a second overlay plus a 1: 20,000 dilution of neutral red was added, and a final plaque count was made 14 days after inoculation and the yield of SV40 expressed as the number of plaque-forming units (PFU) per ml of cellular extract. Quantitation of virus rescue from heterokaryons. The number of heterokaryons that yielded infective SV40 was determined by measuring the number of positive SV40 foci present 11-14 days after fusion in coverslip cultures of fused cell populations using the methods described by Watkins (1970). Parallel measurements of the yield of infective SV40 rescued from heterokaryons derived from the same fused cell population was measured by plaque assay as described above. Demonstration of “V” and “T” antigens. Coverslip cultures were fixed in methanol: acetic acid (7:3, v/v) for detection of “V” antigen, or in acetone for “T” antigen. Rabbit antisera agianst “V” antigen was obtained from the Grand Island Biological Co., Grand Island, New York. When tested against several lines of transformed cells no cross-reaction with SV40 “T” antigen was

88

POSTE ET AL.

found. Hamster antiserum against SV40 “T” antigen was obtained from Flow Laboratories, Irvine, Scotland. Fluorescein-labeled goat antirabbit-globulin and fluorescein-labeled rabbit antihamster-globulin were obtained from Miles Laboratorirs, Kankakee, Illinois and Cappel Laboratories, Downington, Philadelphia. Antisera against the respective antigens was added to the fixed cultures for 30 min at 37” followed by fluorescein-conjugated antirabbit or antihamster antiserum for 30 min at 37”. The coverslips were then washed in distilled water and mounted in 50% (v/v) glycerol saline, and examined for specific fluorescence under ultraviolet illumination using a Leitz Ortholux microscope with UG-1 excitation and K-530 barrier filters. Radioautography. The synthesis of DNA in cells grown on coverslips was detected by the incorporation of 10 &i/ml 3H-thymidine. Radioautographs were prepared by the methods described previously (Poste, 1972). B (Imperial Reagents. Cytochalasin Chemical Industries, Macclesfield, Cheshire, England and Aldrich Chemical Co., Milwaukee, Wisconsin) was dissolved as a 0.1% (w/v) stock solution in dimethyl sulphoxide and stored at -20”. For experiments, the drug was diluted in the cell culture medium at a final concentration of 10 pg/ml. RESULTS Absence of Infective SVdO in SVSTS Cells

To establish that the SV3T3 cells did not contain infective SV40, samples of 1 X 10’ cells were taken at several passagelcvols and a cell-free extract prepared and inoculated onto fresh AGMK cell monolayers and assayed for infective SV40. Infective virus was not detected in SV3T3 cells at any of the passage levels used in the experiments reported below. Similarly, cocultivation of SV3T3 cells with AGMK cells for up to 2 weeks did not result in the recovery of detectable amounts of SV40. Also, infective SV49 was not detectable in any of the Sendai virus preparations used in the cell fusion experiments. Rescue of Infective SV.$O from SVSTS Cells by Fusion with Anucleate Monkey Cells

Fusion of anucleate AGMK or BS-C-1 cells obtained from confluent monolaycrs

with SV3T3 cells resulted in the rcscuc of infective SV40 (Table 1; experiments 1 and 3). However, the yield of SV40 rescued by fusion of anucleate monkey cells with SV3T3 cells was significantly lower than in control experiments in which conventional hctcrokaryons were formed by fusion of SV3T3 cells with similar numbers of nucleated monkey cells (Table 1). This difference in yield appears to reflect the higher efficiency of heterokaryon formation in the control cultures, since the yield of SV40 rescued per heterokaryon was similar in both the control and the anucleate cultures (Table 1). The plaque-forming agent rescued from the heterokaryons was identified as SV40 by neutralization of its ability to induce plaques by incubation with SV40 antiserum prior to inoculation onto fresh AGMK cell cultures. No infective virus was dctccted in cell-free extracts of homokaryons produced by fusion of SV3T3 cells or in homokaryons of AGMK or BS-C-1 cells. Since the anucleate monkey cell populations used in the rescue experiments shown in Table 1 contained a small number of cells that had retained their nuclei, it was necessary to establish that the rescued virus was not derived solely from heterokaryons formed between SV3T3 cells and these remaining nucleated permissive cells. Microscopic counts indicated that the anucleate AGMK cell populations used in the four expcriments to provide the mean values given in Table 1 contained 2.7 ‘;;, 1.6 %, 2.3 ‘/; , and 3.1 ‘/I nucleated cells, respectively. Similarly, the three separate anuclcate BS-C-1 cell populations used to provide the mean values given in Table 1 w’crc contaminated with 3.4 5, 2.s (2, and 3.7 5; nucleated cells, rcspcctively. To determine the contribution of this number of nucleated cells to virus rescue, the same number of nucleated AGMK or BS-C-1 cells were fused with SV3T3 cells and the yield of SV40 rescued compared with that obtained by fusion of SV3T3 cells and anuclcatc monkey cells. The results (Table 1; experiments 2 and 4) revealed that the yield of infective SV40 rescued by the nucleated cells present as contaminants in the original anucleate cell populations was significantly lower than that obtained when anuclcatc cells wcrc also present. This suggcsts that a significant proportion (50-SO?I>

89

SV40 RESCUE

TABLE 1 Rsscux OF SV40 FROMSV3T3 CELLS (1 X lOa) BY FUSION WITH ANUCLEATE AGMK CKLLS (1 X 1Oe)nou ANUCLEATE BS-C-1 CIGLLS (1 X 100)” OR LIMITED NUMBERS OF NUCLEATED AGMK OR B&C-l CELL&~ Expt. No. Cell type and number of monkey cells

Mean virus yield (PFU/ml)

Mean yield Mean % number of Positive as % maximum infectious heteroyield in centers (= karyons control number of rey;ze

Virus yield per positive heterokaryon

Virus yield per positive heterokaryon as yo control for same cell types

karyons) Control 1” % Control 3c 4c

SV3T3 + AGMK SV3T3 + AGMK SV3T3 + AGMK SV3T3 + BS-C-1 SV3T3 + BS-C-1 SV3T3 + BS-C-P

a Mean value * The number cells remaining c Mean value d The number cells remaining

nucleated (1 x 10”) anucleate (1 x 10”) nucleated* nucleated (1 x 10’) anucleate (1 x 10”) nucleated

1.9 x 10’

109

3.2 x lOa

16.8

8.5 x lo*

4.4

9.2 x 10’

106

1.6 X 104

17.4

4 x 108

4.6

69

2.3

2.7 X lO*

106

14.2

2.1

2.3 X lo2

85

0.9

2.2 x 10%

81

Gl

3.1

1.5 x 103

106

10

2.3

1.6 x 10a

106

1.7

1.3 x lo*

86

3.76

3.3

derived from four separate experiments. of nucleated AGMK cells used corresponds to the number of residual nucleated AGMK in the anucleate cell populations in experiment 1 (see text). derived from three separate experiments. of nucleated BS-C-1 cells used corresponds to the number of residual nucleated BS-C-1 in the anucleate cell populations used in experiment 3.

of the yield of rescued SV40 in the experiments with anucleate AGMK and BS-C-1 cell populations shown in Table 1 was rescued from heterokaryons formed by direct fusion of SV3T3 cells and anuclcate monkey cells. Comparison of the yields of rescued virus from the various fusod cell populations does not, however, provide unequivocal evidence for the rescue of virus by heterokaryon formation between anucleatc permissive cells and transformed cells. For example, the above results indicate that fusion of SV3T3 cells with anucleate monkey cells rescued up to three times more virus than would be cxpetted from the number of residual nucleated monkey cells. This larger yield could be due, however, not to the formation of heterokaryons between the anucleate cells and transformed cells, but to the formation of complex heterokaryons involving fusion of SV3T3 cells with at least one residual nucleated monkey cell together with one or more anucleate monkey cells; the additional cyto-

plasm contributed by the anucleatc cells being responsible for the larger yield of rescued virus. This interpretation is considered unlikely, however, since the avcragc yield of SV40 rescued from individual positive hcterokaryons does not differ significantly between heterokaryons in cultures containing anuclcatc cells and those in cultures containing only nucleated cells (Table 1). Mom direct evidence that rescue of SV40 occurred in hctcrokaryons between transformed cells and anucleatc permissive cells was provided by observations on the synthosis of SV40 capsid (“V”) antigen by such hctcrokaryons. Since “V” antigen is synthcsized only in hcterokaryons in which virus rcscuc has occurred, the presence of this antigcn in cells containing only SV3T3 nuclei would effectively demonstrate that rescue can occur following fusion of anucleate monkey cells with SV3T3 cells Covcrslip cultures of fused mixtures of SV3T3 cells and anuclcatc monkey cells were cxamincd 4 days after fusion for the prcscncc

90

ET AL.

POSTE

of cells containing “V” antigen. Simultancously, replicate coverslip cultures of the same fused cell population wcrc fixed and stained with haematoxylin and cosin and the number of cells containing nuclei showing nuclear alterations characteristic for SV40 activation were counted. These alterations involve cnlargement of the affected nuclei, loss of the normal granular appearance of the nucleoplasm and its replaccmcnt by small intranuclear inclusions and the appearance of a poorly stained halo between the nuclcoplasm and the nuclear mcmbranc (see, Watkins and Dulbccco, 1967). Further replicate cultures derived from the same fused ccl1 population vzrc prepared and the number of heterokaryons yielding SV40 measured by infectious ccntcr assay. The results, shown in Table 2, rcvcaled that a number of cells containing only SV3T3 nuclei showed a positive immunofluorcsccncc staining reaction for SV40 “V” antigen. These were interpreted as heterokaryons formed by fusion of SV3T3 cells and anuclcate monkey cells in which SV40 rescue had occurred. A reasonable agrccmcnt was found between the number of thcsc hetcrokyarons and the number of similar hetcrokaryons containing only SV3T3 nuclei which showed SV4O-specific nuclear alterations (Table 2). Importantly, although cvidcncc of SV40 rescue was also obscrvcd in certain hctcrokaryons containing both mouse and monkey

nuclei (Table 2), thesct rctprcscntcbd only a minor proportion of the total number of ccblls in the culturcb that showed clvid(lnccl of SV40 replication. Thesca results thcrc>forcl providr clear cvidonco that anucloatcx ~~~11s arc able to induce r(‘scu(’ of SV40 from transformcld ~~~11s. Finally, the total number of cells showing cvidcncct of SV40 replication by cytological criteria was in rcasonablr agreement with the number of SV40-positive hctcrokaryons in the culture dctcrmincd by infcctivo ccxntcr assay (Table 2). Variation in the Yield of SV40 Rescued from SVSTS Cells after Fusion. with Mon.key Cells from Subconfluent Log Phase Populations or Confluent Stationary Phase Populations Bocausc of initial technical difficulties in the cnuclcation of large numbers of monkry cells in confluent monolaycrs due to the drtachment of large sheets of the monolayer during ccntrifugation (15,000 y for 26 min) our early cxpcrimcnts on SV40 rescue from SV3T3 cells by fusion with anucleate monkey cells wcrc done with anuclcatc cells harvested from subconflucnt cell populations which wcrc more resistant to detachment from the glass during centrifugation (SPC Postc, 1973). However, when suitable mcthods were dcvelopcd for the cficicnt cnuclcation of confluent cell populations using a lower centrifugal force (6000 y for 50 min)

TABLE 2 THE NUMH~R OF HI;TI
ANTIGEN 011~V~O-~PI~:CIFICNUCLLW ALTIDL~TION AND THE Nu~u~m OF HISTI~;BOK.\RYONS YIELDING INFIXTIVK VIRUS IN FUSED MIXTURES OF SV3T3 CISLLS (1 x 106) AND ANUCLUTR BS-C-1 CELLS (1 X 10’)

Expt. No.”

1 2 3

Number of positive heterokaryons determined by infectious center assay”

95 78 103

Number of heterokaryons

containing

Nuclear alteration

“V” antigen Mouse nuclei onlyc

Mouse and monkey nucleid

Mouse nuclei onlyc

Mouse and monkey nucleid

39 40 59

11 9 14

54 49 63

12 8 13

a The anucleate BS-C-l populations in experiments respectively . * Derived from counts on six coverslip cultures. ’ Heterokaryons

containing

SV3T3 nuclei

only

l-3 contained

and no monkey

2.8, 3.3, and 3.6O/o nucleated

cells,

nuclei.

d Heterokaryons containing both SV3T3 and BS-C-1 nuclei but scored as positive even if only the SV3T3 nuclei were altered.

91

SV40 RESCUE

and confluent cell populations at the same passage level. The results (Table 3) confirmed the observation that the yield of SV40 rescued from SV3T3 cells was significantly greater when the anucleate cells were harvested from confluent rather than subconfluent populations. To establish that the variation in the yield of virus rescued using replicating log phase cells compared with nondividing stationary cells was not peculiar to the anucleate cell state, the experiments were repeated using equivalent numbers of nucleated monkey cells. The results revealed a similar significant difference in the yield of virus rescued using subconfluent and conflucnt monkey cells. Infectious center assays on the fused cell populations revealed that the

the rescue experiments were repeated using anucleatc monkey cells from confluent stationary-phase cell populations. Coincidental with this change in the method of obtaining the anucleate cells it was found that the fusion of anucleato AGMK or BS-C-1 cells from confluent populations with SV3T3 cells rcsultcd in the rescue of significantly higher yields of SV40 than in earlier experiments using anucleate monkey cells harvested from subconfluent log phase cultures. To establish that this difference in the yield of rescued virus was not due to an undctectcd change in the properties of the cells during passage in the laboratory, identical rescue experiments were done using anucleate monkey cells obtained from subconfluent TABLE

3

RISSCU~ OF SV40 FROM SV3T3 CELLS (1 X 106) BY FUSION WITH NUCLICATED (1 X 106) OR ANUCLEATE (1 X 106) BS-C-1 CELLS HARVKSTKD FROM SUBCONFLUI
Control

1

2

3

4

5

6

7

Cell type

Confluent SV3T3 + confluent nucleated BS-C-1 Confluent SV3T3 + confluent anucleate BS-C-1 Confluent SV3T3 + subconfluent anucleate BS-C-1 Subconfluent SV3T3 + confluent anucleate BS-C-1 Subconfluent SV3T3 + subconfluent anucleate BS-C-1 Confluent SV3T3 + subconfluent nucleated BS-C-1 Subconfluent SV3T3 + confluent nucleated BS-C-1 Subconfluent SV3T3 + subconfluentnucleated BS-C-1

Virus yield @W-J/ml)

2.1 x

106

4.9

x

104

8.0 x

‘% Yield as Number of Positive % maximum infectious heteroyield in centers (= karyons number of control positive heterokaryons)

Virus yield per positive heterokaryon

Yield per positive heterokaryon as y0 control

86

1.9

2.5 X 1Oa

106

23.5

18

3.2

2.7 X lo3

106

10”

3.8

14

2.6

5.7 x

4.0 x

10’

19.1

17

1.3

2.3 X lo3

1.5 x

10”

7.3

15

2.5

1.9 x

10’

9.4

73

2.3 X lo6

3.1 x

104

100

108

12.5

a Subconfluent cells harvested from 50-mm petri dish cultures * Confluent cells harvested from similar petri dish cultures

1 x

102

23

91

103

40

2.7

2.7 X lo2

11

70

4.1

3.2 X lo3

129

69

3.4

4.5

x 102

with a density of 8 X 10’ cells/cm*. at a density of 6 X 104 cells/cm*.

18

92

POSTE ET AL.

difference in the yield of SV40 rescued from SV3T3 cells by fusion with confluent permissive cells compared with subconfluent permissive cells was not due simply to differences in cell fusion capacity and the number of heterokaryons present in the cultures (Table 3). These results indicate that the differences in the yield of SV40 rescued from heterokaryons using confluent or subconfluent permissive cells reflected true variation in the physiological state of the permissive cells. Significantly greater yields of SV40 were rescued from heterokaryons between SV3T3 cells and monkey cells obtained from confluent monolaycrs irrespective of whether the SV3T3 cells were harvested from confluent or subconfluent cultures (Table 3). The effect of the rate of replication of monkey cells on their ability to rescue SV40 from SV3T3 cells was examined in more detail. Since the variation in yield of rescued virus between subconfluent and confluent cells was exhibited by both nucleated and anucleate cells, the remaining experiments were done for technical convenience with nucleated BS-C-1 cells To examine further the influence of the growth rate of the permissive cell component on virus rescue from heterokaryons, confluent BS-C-1 cell monolayers were trypsinized and reseeded in plastic petri dishes at 1 X lo5 cells per dish and harvested and fused with SV3T3 cells 2, 6, 12, 18, and 24 hr later. The results (Table 4) show that

heterokaryons produced by fusion of SV3T3 cells and BS-C-l cells obtained 6, 12, 18, and 24 hr after trypsinization gave a significantly lower yield of infect’ive SV40 than heterokaryons formed by fusion of SV3T3 cells and BS-C-1 cells obtained 2 hr after trypsinization or control heterokaryons between SV3T3 cells and BS-C-1 cells harvested from monolayers that had been confluent for 2 days (Table 4). Autoradiographic observations of DNA synthesis and measurement of 3H-thymidine incorporation in the BS-C-1 cells revealed resumption of cellular DNA synthesis beginning from 5-12 hr after trypsinization which further suggests that the lower yield of SV40 rescued from heterokaryons using subconfluent monkey cells may be related to an alteration in the physiology of these cells associated with cellular replication, DNA synthesis or both. The major question posed by these results is whether the observed differences in the yield of virus rescued by monkey cells under different growth conditions reflect variation in the ability of these cells to initiate and/or support lytic SV40 replication at different times in the cell cycle. Experiments on the rescue of SV40 using synchronized monkey cell populations should provide insight into this problem. DISCUSSION

The rescue of infective SV40 from SV40transformed 3T3 cells by fusion with anucle-

TABLE

4

RESCUE OF SV40 FROM SV3T3 CI~;LLS (1 X 106) BY FUSION WITH BBC-1 CKLLS (1 X 106) HAWIE+T~D INTERVALS AFTER TRYPSINIZATION OF CONFLUENT BS-C-l MONOLAYERS Expt. NO.

Hours after trypsinization

Untreated control” 1

2 3 4 5

Virus yield (PFU/ml) from fused cultures

experiments.

Yield per \:g:: kwynw,“,“l %

100

66

1.8 x

103

100

1.3 8.5 1.7 7.6 2.1

111.2

76 62 80 58 70

1.7 1.4 2.2 1.3 3.0

103 101 10” 10” 10’

95 76 12 7 16

a Control BS-C-1 monolayer 8138 in the fusion

Virus yield per positive heterokaryon

1.2 x 106

0

2 6 12 18 24

Yield as yo max- Number of infectious imum yield in centers (= control number of positive heterokaryons)

x x x X

106 10’ 10’ lo3

x 104 was ace&d

70.4 14.6 6.3 17.3 originally

at 1 X 10’ cells/ml

x x x x x

and cultured

AT

for 4 days before

i

,

SV40RESCUE ate AGMK or BS-C-1 cells indicates that the rescue process does not require the participation of a functional permissive cell nucleus in the heterokaryon. These results support previous observations on the rescue of SV40 from hcterokaryons between nucleated transformed and pcrmissivc cells in which SV40 replication was first detected in the transformed cell nuclei, and infective virions were rescued before SV40 replication was dctcctable in the permissive ccl1 nuclei (Steplcwski et al., 1968; Wever et al., 1970). That the nucleus of the permissive cell does not contribute to the initiation of the virus rescue process from transformed cell-permissive cell heterokaryons is also supported by the rcccnt findings of Koprowski and his colleagues which show that in addition to the rescue of SV40 from SV40-transformed mouse cells by fusion with anucleate CV-1 monkey cells (Croce and Koprowski, 1973), virus rescue can also be achieved by fusion of the transformed cells with nucleated CV-1 cells lethally irradiated with X-rays or pretreated with actinomycin D and mitomycin C (Huebner et al., 1973). The effect of the rate of division of the permissive cells on the yield of SV40 rescued from transformed cell-permissive cell heterokaryons described here with both anucleate and nucleated monkey cells has not been reported previously. The present results indicate that fusion of SV3T3 cells with pcrmissive cells obtained from nondividing confluent stationary phase cell populations results in rescue of a significantly higher titer of SV40 than from heterokaryons between similar SV3T3 cells and permissive cells harvested from replicating subconfluent log phase cultures. Importantly, this variation in the yield of virus rescued using replicating or stationary phase cells does not appear to be due to differences in the susceptibility of the cells to fusion and the extent of heterokaryon formation. Instead, the results indicate that the physiological state of the permissive cells exerts a direct effect on the amount of virus rescued from the heterokaryons. Unfortunately, information on the condition of the permissive cells used in previous reports of SV40 rescue by heterokaryon formation has not been documented. The general similarity of the yield of SV40 res-

93

cued from SV3T3 cells in the present experiments after fusion with nucleated monkey cells from confluent cultures with the values reported in previous papers on SV40 rescue from hcterokaryons, suggests that these earlier experiments might also have been done with monkey cells from confluent cultures. This conclusion also seems likely on practical grounds in that confluent cultures would prove more convenient as the source of cells for fusion experiments than the USC of a larger number of subconfluent cell cultures to provide the same number of cells. The mechanism by which the rate of division of the permissive cells used in hctcrokaryon formation might influence the yield of rescued SV40 is not clear from the present experiments. Theoretically, several mechanisms can be advanced. The difference in yield might reflect simple variation in the rate of SV40 replication in heterokaryons produced from SV3T3 cells and monkey cells from subconfluent or confluent monolayers. Alternatively, the difference in yield might be due to variation in the capacity of subconfluent or confluent permissive cells to “activate” SV49 replication which once initiated would occur at a similar rate irrespective of whether subconfluent or confluent permissive cells were used in heterokaryon formation. Finally, both of these mechanisms might be involved. The identification of the mechanism responsible for this phenomenon would help to clarify our understanding of the nature of cellular permissiveness to infection by SV40. A particularly important question is whether the difference in the yield of SV40 rescued using replicating and nonreplicating permissive cells is due to variation in the properties of the cell at different stages of the cell division cycle or reflects a more general difference between replicating and nonreplieating cells. In view of the greater yield of SV40rescued from SV3T3 cells by fusion of permissive cells from confluent cultures compared with subconfluent cultures, it is of interest to note that the yield of infective SV40 in lytic infection of CV-1 (Hahn and Sauer, 1971) and BS-C-1 monkey cell lines (Carp and Gilden, 1966) and primary AGMK cells (Carp and Gilden, 1966; Fischer and Munk, 1970; Hahn and Sauer, 1971) has

94

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been shown to be significantly higher in confluent stationary phase monolayers than in actively replicating subconflucnt log phase cultures. Pages et al. (1973) have shown that the synthesis of SV40 DNA during productive infection of CV-1 monkey cells is postponed by as much as one cell cycle in cells which were in S-phase at the time’of infection and is suppressed entirely when cells infected during S phase wcrc prcvented from entering the next mitotic cycle. Thus, if the “factors” responsible for activation of SV40 DNA synthesis and completion of the virus replicativc cycle during virus rescue in heterokaryons resemble the same events in productive infection of permissive cells then the lower yields of SV40 obtained here from heterokaryons involving actively replicating permissive cells might be due simply to the higher proportion of cells in the replicating population that were in S or Gz at the time of fusion and which would not provide a suitable cnvironmcnt for induction of SV40 DNA synthesis and virus rescue. ACKNOWLEDGMENTS This work was supported by Grant lROlCA13393 from the National Institutes of Health. We thank Joe Sambrook for his helpful advice. The technical assistance of P. Newhouse, K. Willett, and A. MacKearnin is gratefully acknowledged. REFERENCES CARP, It. I., and GILDEN, Ii. V. (1966). A comparison of the replication cycles of simian virus 40 in human diploid and African green monkey kidney cells. Virology 28, 150-162. CARTER, S. B. (1967). Effects of cytochalasins on mammalian cells. Nature (London) 213,261-264. CEOCI~;, C. M., and KOPROWSKI, H. (1973). Enucleation of cells made simple and rescue of SV40 by enucleated cells made even simpler. Virology 51, 227-229. CROCIC, C. M., KOPROWSICI, H., and EAGLI.:, H. (1972). Effect of environmental pH on the efficiency of cellular hybridization. Proc. Nat. Acad. Sci. U.S. 69, 1953-1956. ~ULBECCO, R., HART\~I~zLL, L. H., and VOGT, M. (1965). Induction of cellular DNA synthesis by polyoma virus. Proc. Nat. Acad. Sci. U.S. 53, 403410. FISCHISR, H., and MUNK, K. (1970). The effect of actinomycin 1) on DNA synthesis in SV40 in-

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