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Introduction of the herpes simplex virus thymidine kinase gene into mouse cells using virus DNA or transformed cell DNA
Cell, Vol.
13, 581-587,
March
1978,
Copyright
0 1978 by MIT
Introduction of the Herpes Simplex Virus Thymidine Kinase Gene into Mouse Cells Using Virus DNA or Transformed Cell DNA A. C. Minson,* P. Wildy,* Darby *Division of Virology Department of Pathology University of Cambridge Cambridge, England t Department of Medical Medical School University of Birmingham Birmingham, England
A. Buchant
and G.
Microbiology
Summary Cells lacking the enzyme thymidine kinase (LMTK- cells) have been transformed to a kinasepositive phenotype using sheared herpes simplex virus (HSV) DNA, and the enzyme found in these transformed cells is HSV-specific. One of the cell lines is able to complement the functional defect found in two temperature-sensitive mutants of HSV 1, and reversion of the cells to a thymidine kinase-negative phenotype results in the loss of this capability. The HSV thymidine kinase gene can also be introduced into LMTK- cells using DNA extracted from transformed cells, and the high efficiency of this procedure suggests that the state of the virus DNA in transformed cells is different from that of DNA in virus particles. Introduction It is well established that herpes simplex viruses contain a gene for the enzyme thymidine kinase (Kitt and Dubbs, 1963, 1965; Klemperer et al., 1967; Buchan et al., 1970). This gene can be introduced into the genotype of mammalian cells which lack this enzyme either by infecting ceils with virus inactivated with ultraviolet light (Munyon et al., 1971) or by inoculating cells with fragments of virus DNA (Bacchetti and Graham, 1977; Maitland and McDougall, 1977; Wigler et al., 1977). Following these treatments, cells which have acquired a thymidine kinase enzyme can be simply selected (Littlefield, 1964), and the enzyme can be shown to be virus-specific. Cells ‘transformed” in this way have been shown to contain a small number of copies (l-5) of only part of HSV genome (Kraiselburd, Gage and Weissbach, 1975; Davis and Kingsbury, 1976). Little is known of the state of the herpes DNA sequences in these transformed lines, and studies of the stability of the kinase-positive phenotype has thrown little light on this problem because the stability varies considerably from one cell line to another (Davidson, Adelstein and Oxman, 1973; Bacchetti and Graham, 1977) and also because reversion to the kinase-negative pheno-
type may be due to the lack of expression of the virus gene rather than its loss (Davidson et al., 1973; Kaufman and Davidson, 1975). In this report, we describe experiments analogous to those of Bacchetti and Graham (1977) in which we have transformed LMTK- cells to a kinase-positive phenotype using HSV type 1 and type 2 DNA, and we describe some of the properties of the transformed cells. In addition, we have extended the usefulness of this system by showing that DNA extracted from transformed cells can also be used to transfer the kinase gene, and the high efficiency of this gene transfer implies that the chemical or physical state of the kinase gene in transformed cells is different from that of the gene in virus particles. Results Transformation with Sheared HSV DNA LMTK- cells seeded at 5 x lo5 cells per 60 mm dish were inoculated with HSV DNA sheared to a molecular weight of lo-20 x lo6 daltons. After growth for 48 h in nonselective medium, the cells were incubated in selective medium. Colonies appeared after 14 days and continued to appear until 25 days, after which no colonies appeared in dishes which had previously contained none. The minimum colony size at this stage was about 30 cells. At later times (~35 days), positive dishes contained numerous colonies, most of which were presumably secondaries. The data given therefore always relate to the twenty fifth day after inoculation. Table 1 shows the influence of the dose of HSV2 DNA on the frequency of transformation. Where more than one colony appears on a dish, we are unable to distinguish between primary and secondary colonies, so the number of colonies need not reflect the number of transformation events. Nevertheless, it is apparent from Table 1 that at high DNA concentrations, the number of events is independent of dose, since a dose of 2.5 pg gave a maximum of 14 events, while a dose of 0.1 pg gave a minimum of 9 or 8 events in two experiments. At lower doses, the number of events appears to become dosedependent, although the data are inadequate to determine whether a “single hit” is required for transformation. Properties of the Transformed Cells Four clonally unrelated transformed cell lines were established from experiments using sheared type 1 DNA and nine from experiments using type 2 DNA. All the cell lines were found to contain thymidine kinase activity, and this activity could be neutralized by antisera raised in rabbits against herpesinfected cells (Thouless and Wildy, 1975).
Cell 502
Table 1. Influence Frequency
of HSV DNA Dose
on Transformation
Experiment
1
Experiment
Dose of HSV-2 DNA per Dish (/.a)
Colonies in IO Dishes
Proportion of Dishes with Colonies
Colonies in IO Dishes
2.5
14
9/10
1 .o
20
IO/IO
0.25
28
IO/IO
0.10
16
Proportion of Dishes with Colonies
lO/lO 8110
0.03
8
4110
0.01
0
O/IO
0.003
0
0110
0
O/IO
0
O/IO
2. Inactivation
Kinase
Kinase
Source
by Specific
Sera
% Kinase Activity Remaining
Anti-Type 2 Serum
Anti-Type 1 Serum
Din
10,270
76
6
Dls
6,230
81
9
Dlr
3,180
88
13
‘321
2,900
15
33
D23
4,850
30
74
D25
9,990
8
25
D26
3,695
33
55
BHK
3,250
102
105
L-M
9,390
102
104
Type
Table 2 shows the results of an experiment with three lines transformed with type 1 DNA (DIP, D13, D14) and four lines transformed with type 2 DNA (D2,, D23, D2,. D2,). Antiserum raised in rabbits against type 1 or type 2 HSV-infected RK 13 cells had no effect on the thymidine kinase in BHK or L cells, but neutralized the kinase from transformed cells. Furthermore, serum raised against type 1 virus-infected cells neutralized the kinase from type 1 transformed cells more efficiently than the kinase from type 2 transformed cdls. Antiserum against type 2 virus behaved in a converse way. The results given in Table 2 are not entirely satisfactory, since there is a considerable difference between the neutralization of enzyme from lytically infected ceils and from transformed cell lines. In our hands, this serological test is very specific in that the antiHSV sera used never showed neutralizing activity against the enzyme from L cells or BHK cells, and each berum always neutralized the homologous enzyme more than the same concentration of the heterologous enzyme. Similarly, each enzyme was always neutralized more by the homologous serum than by the heterologous serum. Nevertheless, the actual level of neutralization produced by a particular enzyme-serum combination was very variable in different experiments. This was particularly noticeable in experiments with the type 2 specific enzyme. Different levels of thymidine kinase were found in different ceil lines, but the level in any particular cell line was characteristic and was independent of passage number in selective medium, at least until passage 20. The results of simultaneous estimations of the enzyme activity in nine type 2 transformed lines are given in Figure 1. The enzyme levels are distributed over an approximately 4-fold range, and these levels may be clustered in groups. As yet, insufficient numbers of cell lines have been
of Thymidine
Activity after Incubation with Preimmune Serum (w-V
2
13
0
9/l 0
24
Table
1 Infected
BHK
22,700
27
4
Type 2 Infected
BHK
10,700
5
79
a The data given are the means of duplicate samples 20 min of reaction. Samples were also taken after reactions were linear up to 20 min.
taken after 10 min. All
examined to encourage confidence in this observation. Since DNA fragments of molecular weight lo-20 x lo6 dalton have been used to produce these transformed lines, it is reasonable to suppose that in addition to the thymidine kinase gene, other nonselected herpes-specific functions may be acquired by the transformed cells. One approach toward detecting such functions is to test the ability of the transformed cell to support the growth of temperature-sensitive mutants of HSV at the nonpermissive temperature (McNab and Timbury, 1976). LMTKcells and transformed cells were therefore infected with ts- mutants at a multiplicity of 3 pfu per cell, and after 20 hr at the nonpermissive temperature, the yield of virus was compared with the yield of wild-type virus grown under identical conditions. Table 3 shows the results of two such experiments involving ts- mutants N102, N103, 85 and Bl, together with the parental type 1 isolate HFEM. The yield of wild-type virus varied from experiment to experiment and from cell line to cell line in individual experiments, but the combined results of five experiments show that no transformed cell line differs from any others or from LMTK- cells in its ability to support the growth of wild-type virus. None of the cell lines examined gave elevated yields of Bl or B5, but mutants N102 and N103 were complemented efficiently by cell line 02,. Mutants N102 and N103 do not complement each other, but both complement efficiently with Bl and B5, and we therefore suppose that N102 and
HSV Thymidine 583
Kinase
Gene
Transfer
Table 3. Complementation of HSV. Type Transformed with HSV Type 2 DNA 6
Experiment Virus Cell Line
TMP p moles L
1
Yield (pfu
Wild-Type
N103
LMTK-
60
0.06
D21
25
6.0
D22 D&
40
D2r
x 10-5) N102
by Cell Lines
Experiment
2
Virus
(pfu
Yield
Wild-Type
Bt
x IO-3 85
260
0.10
0.08
35.0
260
0.05
0.15
0.19
0.8
250
0.04
0.10
180
0.27
1 .2
ia0
0.08
0.12
200
0.20
1.7
240
‘0.04
0.05
D25
120
0.28
1 .4
380
0.12
0.10
D2e
180
0.22
1.6
220
0.05
0.08
D2r
20
0.05
D&
27
0.05
130
0.18
D2s
I .2
1. Mutants
1.0 ND 1.3
ND
ND
ND
270
0.10
0.08
ND
ND
ND 1.
10
20
Time (mlnutes ) Figure
1, Thymidine
Kinase
Levels
in Transformed
Cell Lines
The cell lines were grown to confluence in selective medium, harvested by trypsinization and suspended at 4 x IO’ cells per ml. Extracts were prepared by ultrasonic disintegration and assayed for kinase activity. The data shown are thymidine-phosphorylated by extracts of lo5 cells. All cell lines were at passage 5 in selective medium.
N103 are defective in the same function and that the absence of this function is compensated by D21 ceils. We could find no evidence of rescue of the virus gene resident in D2, cells since the progeny virus resulting from infection with N102 and N103 was all of mutant phenotype. Mutants N102 and N103 were also used in complementation experiments with type 1 transformed cell lines Dl,, Dl,, Dls and Dl,. None of these cell lines complemented either mutant. Thus we have been able to demonstrate intertypic complementation of these mutants by type 2 transformed cells, but not intratypic complementation, presumably because of the small number of type 1 transformed cell lines tested. It is known that type 2 virus function will compensate the defect in mutant N102, because this mutant has been used to generate intertypic recombinants of I-JSV (Morse et al., 1977). It appears that the introduction of the HSV kinase gene into LMTK- cells is rarely accompanied by the acquisition of a function which will complement mutants N102 and N103 regardless of whether type 1 DNA or type 2 DNA is used as a source of the kinase gene.
Revertant Cell Lines Thymidine kinase-negative revertants of cell line D2, were obtained by plating IO4 cells per dish in medium containing 50 pg/ml BUDR. A few colonies appeared in each dish, and three such colonies from different dishes were established as cell lines in nonselective medium (D2,Rl, D2,R2, D2,R3). These cell lines contained no detectable thymidine kinase and failed to plate in medium containing methotrexate (plating efficiency ~10-~). The revertant lines were tested for their ability to support the growth of ts N103 at the nonpermissive temperature, and as shown in Table 4, the revertants have lost this function, while their ability to support the growth of wild-type virus is unimpaired. Selection against the herpes-specified thymidine kinase thus simultaneously selects against the function which complements ts N103. Transformation with DNA from Transformed Cells DNA was extracted from cell lines D2, and D2,, and used to transform LMTK- cells to a kinase-positive phenotype (Table 5). In four independent experiments, inoculation of DNA from transformed cells resulted in the appearance of colonies in a majority of dishes, while no transformants appeared with L cell DNA or with DNA extracted from a thymidine kinase-negative revertant of cell line D21. Four cell lines were established from clonally unrelated colonies induced by D2, DNA in experiment 1 (D2, Tl , D2, T2, D2, T3,02, T5). These will subsequently be referred to as transfected lines. Serological neutralization of the thymidine kinase from these cell lines (Table 6) showed that the enzyme was HSV2-specific. Furthermore, each of the four lines showed very similar levels of kinase activity. Figure 2 shows four independent estimations of the en-
Cell 584
Table
4. Growth
of Mutant
N103 in TKVirus
Revertants
Yield (pfu
Cell Line
Wild-Type
LMTK-
300
of D2,
x lo-“)
Table DNA
5. Transformation
of LMTK-
Cells with Transformed
N103 Dose per Dish
0.1
D21
390
D2,Rl
200
190 0.15
D2,R2
450
0.08
D2,R3
440
0.18
zyme from cell line D2, and simultaneous estimations of the enzyme from the four transfected cell lines. The transfected ceils contain about half the enzyme level found in D2, cells, and it appears that while transformation with virus DNA results in cell lines with widely different enzyme levels (see Figure l), transformation with D2, DNA yields cell lines which are rather uniform in this character. Each of the transfected lines was examined for its ability to complement ts N103, and the results (Table 7) showed that none of the lines had acquired this character. It appears that transformation with D2, DNA, like transformation with virus DNA, does not normally result in the transfer of the N103 complementing function. To determine whether co-transfer of the kinase gene and the N103 complementing gene could be achieved using transfecting DNA of higher molecular weight, D2, DNA of molecular weight 20-25 x lo6 daltons was used to transfect LMTKcells (Table 5, experiment 6). Three clonally unrelated cell lines were established from this experiment, and these also failed to complement mutant N103. Discussion We have shown that the thymidine kinase gene of herpes simplex virus can be introduced into the genotype of LMTK- cells using either virus DNA or transformed cell DNA as a gene source. The cell lines derived from experiments using HSV2 DNA as a transforming agent contain a thymidine kinase which is HSV2-specific, and one of these lines, D2,, is capable of complementing the DNA-negative ts mutants N102 and N103 of HSVl. Selection of kinase-negative revertants from this cell line results in the loss of this capability. The simplest interpretation of this result is that the kinase gene and the N103 complementing gene are introduced on the same fragment of virus DNA, and that selection against the kinase gene results in the loss of the entire virus sequence or of its expression. Reversion to the kinase-negative phenotype in the instance of cell line D2,R, presumably results from gene loss, since DNA derived from these cells
of DNA
Cell
Proportion of Dishes with Colonies
Experiment
Source
1
HSV-2 virions D2, cells Salmon sperm
0.5 10.0 10.0
WI0 9/l 0 O/l 0
2
HSV-2 virions D23 cells Salmon sperm
0.5 10.0 10.0
4/a 5/6 O/l 0
3
HSV-2 virions D21 cells Salmon sperm
0.5 10.0 10.0
lO/lO 619 o/10
4
HSV-2 virions L-cells
0.5 10.0
E/10 o/10
5
HSV-2 virions HSV-2 virions D2,Rl cells
0.5 0.05 10.0
B/10 2/10 O/l 0
6
HSV-2 virions D2, cells Salmon sperm
0.5 10.0 10.0
IO/10 9/l 0 o/10
All DNA was sheared to a molecular daltons, except in experiment 6, where molecular weight of 20-25 x lo6 daltons.
(CLS)
weight of lo-20 x lo6 D2, DNA was used at a
cannot be used to introduce the kinase gene into LMTK- cells. Since only one of nine cell lines examined is capable of complementing N103, it is probable that the two genes are usually separated when the HSV DNA is sheared. This interpretation is supported by recent evidence concerning the relative positions of the thymidine kinase gene and the ts N102 complementing gene on the HSV2 genome. The kinase gene is believed to be located in the L region between 55 and 65% from the left end of the genome (Maitland and McDougall, 1977). The position of the ts N102 complementing function is unknown, but restriction enzyme analysis of DNA from ts+ intertypic recombinants generated using ts N102 as the type 1 parent (Morse et al., 1977) suggests that this function is separated from the site of the kinase gene by at least 15% of the genome (that is, by a sequence >15 x lo6 dalton molecular weight). This probably explains why co-transfer has proved difficult to achieve even when DNA of molecular weight 20-25 x IO6 daltons has been used. The transfer of the thymidine kinase gene to LMTK- cells using transformed cell DNA as a transforming agent (transfection) is surprisingly efficient. Reference to Tables 1 and 5 shows that IO pg D2, DNA transforms at least as efficiently as 0.1 pg HSV DNA, and since we have not used smaller amounts of D2, DNA, this could be an underesti-
HSV Thymidine 585
Kinase
Gene
Transfer
Table 6. Serological Neutralization Transfected Cell Lines
Source of Thymidine Kinase D2,-Tl
of Thymidine
Activity after Incubation with Preimmune Serum (cpm)
9,500
D2 ,-T3 D2 ,-T5
Anti-Type 1 Serum
Anti-Type 2 Serum
9.6
22
7.5
a Neutralization tests with the enzyme from this cell line were performed on a different occasion from tests with the other three, and an unclarified ceil sonicate was used.
6 TMP p moles
10 Time Figure fected
2. Thymidine Lines
Kinase
Levels
20 (minutes) in Cell
7. Growth
Line
D2, and Trans-
Conditions were as described in the legend to Figure 1. The for D2, cell extracts were obtained on different occasions cells at different passage numbers. Data for the transfected were obtained from parallel assays of extracts prepared from at passage 4.
of Mutant
N103 in Cells Transformed
Virus
24
25
8,200
Table DNA
10
43
8,000
from
% Kinase Activity Remaining
22
6,700
D2,-TZa
Kinase
data using lines cells
mate of its efficiency. Since 10 pg is the DNA content of lo6 cells, and since 0.1 pg HSV DNA represents lo9 genomes, it follows that to account for the efficiency of transformation by D2, DNA in terms of the thymidine kinase gene concentration, we would require each D2, cell to contain lo3 copies of the kinase gene. This seems improbable in view of the very small amounts of virus DNA which have been found in herpes-transformed cells (Kraiselburd et al., 1975; Davis and Kingsbury, 1976; Frenkel et al., 1976; Minson et al., 1976). An alternative proposal is that the kinase gene in transformed cells is in a different physical or chemical state from the gene extracted from virus particles and transforms more efficiently in consequence. For example, the virus DNA in transformed cells could be integrated so that the virus sequence
Yield
Cell Line
Wild-Type
LMTK-
300
(pfu
with
D2,
x lo-“) N103 0.10
D21
390
D2,-Tl
760
0.05
D2,-T2
820
0.08
D2 ,-T3
750
0.33
D2,-T5
510
0.30
190.0
derived from such cells is flanked by host sequences. If these flanking sequences were highly reiterated in the recipient cell chromosomes, then an increased rate of integration of virus sequences might result from recombination of homologous sequences. We can eliminate the possibility that the virus sequences must be flanked on both sides by host sequences in transfecting DNA, since this model would predict that the entire virus sequence in the donor cell should be acquired by the recipient cell. In fact, cells transformed using D2, DNA as a gene donor do not carry the ts N103 complementing function. The idea that transformation using DNA from transformed cells as a gene donor is qualitatively different from transformation with virus DNA is supported by the properties of transfected cells. The transformed lines show a wide range of kinase levels, while the transfected lines are uniform in this character. Only four lines have been examined, so this could be a fortuitous observation, but it is worth noting that of the nine transformed lines examined, only one, D2!,, has the same enzyme level as if found in all four transfected lines. It is obviously important to determine which herpes DNA sequences are present in our transformed lines and to find the number of sequences in each cell. We have not excluded the possibility that each cell contains thousands of copies of the HSV thymidine kinase gene, but it seems more probable that each cell contains only a few copies and that these sequences have unusual properties which render them very efficient in gene transfer experiments. There have been a number of reports of gene transfer from cell to cell, including transfer of thymidine kinase genes, mediated by chromosome uptake (McBride and Ozer, 1973; Willecke et al., 1976; Wullens van der Horst and Bootsma 1977), but the frequency of transformation is lower than reported here, and as yet there have been no confirmed reports of gene transfer from mammalian cell to mammalian cell using direct DNA uptake.
Cell 586
Experimental
Procedures
Cells and Tissue Culture LMTK- cells were obtained from Dr. M. Thouless. BHK/Pi cells were used for virus assays and growth of virus stocks. Hep-2 cells were used for large-scale virus propagation for virus DNA preparation. All cells were grown in Glasgow modified Eagle’s medium supplemented with 10% tryptose phosphate broth and 10% newborn calf serum (Macpherson and Stoker, 1962). Selection of cells containing thymidine kinase was achieved by supplementing with thymidine, adenosine, guanosine, glycine and methotrexate as described by Munyon et al. (1971), and unless otherwise stated, all transformed lines were maintained in this medium. Selection against cells containing thymidine kinase was achieved by supplementing with 50 +g/ml BUDR. Viruses HSV type 1 strain HFEM and type 2 strain 25766 (obtained from Professor K. R. Dumbell, Wright Fleming Institute, London, England) were used throughout. Mutants Bl, 85. N102 and N103 were independently isolated from strain HFEM using either BUDR (prefix 6) or nitrous acid (prefix N) as mutagens. All mutants have a relative plaquing efficiency of <1O-3 at 38.5”C compared with 33°C. Mutant 85 is DNA-positive at the restrictive temperature. The remainder are DNA-negative. (These mutants were isolated by Dr. A. Buchan; details of their isolation and characterisation will appear elsewhere.) Hep-2 cells were infected at a multiplicity of 1 pfu per cell, and cell-released virus was purified from the medium 48 hr after infection. Cells infected with type 1 virus were incubated at 37”C, and cells infected with type 2 were incubated at 33°C. Virus DNA was extracted from purified particles as described by Wilkie (1973). DNA was extracted from cells by the method of Varmus, Vogt and Bishop (1973). Transformation 5 x lo5 cells were seeded in 60 mm dishes, and after 24 hr, the medium was removed and the cells inoculated with DNA as described by Bacchetti and Graham (1977). The DNA dose per dish was always made to 10 pg using salmon sperm DNA as carrier. The DNA was first sheared by passing a solution at 100 pglml3 times through a 21 gauge x 1.5 inch syringe needle, and the fragment size was then estimated by electrophoresis in agarose gels using Eco RI-restricted HSVP DNA as a source of size markers. After 1 hr at room temperature, the inoculum was removed and replaced with nonselective medium. The dishes were changed to selective medium after a further 48 hr, and the medium was then changed every third day. Complementation of ts Mutants Preformed monolayers of 3-6 x lo5 cells were infected at a multiplicity of 3 pfu per cell, and the virus was allowed to absorb for 1 hr at 37°C. The monolayers were then washed 3 times with warm medium and sealed in plastic bags. All procedures to this point were carried out in a 37°C warm room. The monolayers were then incubated under water at 38.5”C, and after 30 hr, they were harvested and sonicated, and the virus yield was assayed at 33°C. In all experiments, monolayers were infected in parallel with mutant and wild-type virus. Where evidence of complementation was obtained, the progeny virus was assayed again at 38.5”C. Thymldine Klnase Assays Cells were trypsinized and suspended in 0.01 M Tris-Cl(pH 7.5) at 4 x 10’ cells per ml. The cells were disrupted by ultrasonic vibration and immediately assayed for thymidine kinase activity by the method of Klemperer et al. (1967). For serological neutralization tests, cell extracts were clarified by centrifuging for 30 min at 100,000 x g at 4°C. The supernatants were mixed with an equal volume of preimmune or immune serum, and the thymidine kinase activity was determined after 2
hr at 4%. The enzyme in high-speed supernatants was neutralized more efficiently than the enzyme in crude cell sonicates. The observed enzyme levels found in supernatants, however, were very variable, presumably because of the instability of the enzymes. Anti-type 1 serum (192) and anti-type 2 serum (409) were gifts from Dr. Margaret Thouless and were prepared by immunization of rabbits with infected RK13 cells (Thouless and Wildy, 1975; Thouless, Chadha and Munyon, 1975). Acknowledgments We are grateful to Lynne Jeffrey and Susanne Bell for excellent technical assistance, and to Silvia Bacchetti and Peter Sheldrick for helpful comments and discussion. This work was supported by the Cancer Research Campaign. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received
September
28,1977;
revised
December
13,1977
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Willecke, K., Lange, Pi., Kruger, A. and Reber, fer of two linked human genes into cultured Nat. Acad. Sci. USA 73,1274-1276. Wullens, G. J., van der Horst, J. and Bootsma, of human genes coding for thymidine kinase to Chinese hamster cells and human-Chinese brids. Somatic Cell Genet. 3, 261-293.
T. (1976). Cotransmouse cells. Proc. D. (1977). Transfer and galactbkinase hamster cell hy-
13, 581-587,
March
1978,
Copyright
0 1978 by MIT
Introduction of the Herpes Simplex Virus Thymidine Kinase Gene into Mouse Cells Using Virus DNA or Transformed Cell DNA A. C. Minson,* P. Wildy,* Darby *Division of Virology Department of Pathology University of Cambridge Cambridge, England t Department of Medical Medical School University of Birmingham Birmingham, England
A. Buchant
and G.
Microbiology
Summary Cells lacking the enzyme thymidine kinase (LMTK- cells) have been transformed to a kinasepositive phenotype using sheared herpes simplex virus (HSV) DNA, and the enzyme found in these transformed cells is HSV-specific. One of the cell lines is able to complement the functional defect found in two temperature-sensitive mutants of HSV 1, and reversion of the cells to a thymidine kinase-negative phenotype results in the loss of this capability. The HSV thymidine kinase gene can also be introduced into LMTK- cells using DNA extracted from transformed cells, and the high efficiency of this procedure suggests that the state of the virus DNA in transformed cells is different from that of DNA in virus particles. Introduction It is well established that herpes simplex viruses contain a gene for the enzyme thymidine kinase (Kitt and Dubbs, 1963, 1965; Klemperer et al., 1967; Buchan et al., 1970). This gene can be introduced into the genotype of mammalian cells which lack this enzyme either by infecting ceils with virus inactivated with ultraviolet light (Munyon et al., 1971) or by inoculating cells with fragments of virus DNA (Bacchetti and Graham, 1977; Maitland and McDougall, 1977; Wigler et al., 1977). Following these treatments, cells which have acquired a thymidine kinase enzyme can be simply selected (Littlefield, 1964), and the enzyme can be shown to be virus-specific. Cells ‘transformed” in this way have been shown to contain a small number of copies (l-5) of only part of HSV genome (Kraiselburd, Gage and Weissbach, 1975; Davis and Kingsbury, 1976). Little is known of the state of the herpes DNA sequences in these transformed lines, and studies of the stability of the kinase-positive phenotype has thrown little light on this problem because the stability varies considerably from one cell line to another (Davidson, Adelstein and Oxman, 1973; Bacchetti and Graham, 1977) and also because reversion to the kinase-negative pheno-
type may be due to the lack of expression of the virus gene rather than its loss (Davidson et al., 1973; Kaufman and Davidson, 1975). In this report, we describe experiments analogous to those of Bacchetti and Graham (1977) in which we have transformed LMTK- cells to a kinase-positive phenotype using HSV type 1 and type 2 DNA, and we describe some of the properties of the transformed cells. In addition, we have extended the usefulness of this system by showing that DNA extracted from transformed cells can also be used to transfer the kinase gene, and the high efficiency of this gene transfer implies that the chemical or physical state of the kinase gene in transformed cells is different from that of the gene in virus particles. Results Transformation with Sheared HSV DNA LMTK- cells seeded at 5 x lo5 cells per 60 mm dish were inoculated with HSV DNA sheared to a molecular weight of lo-20 x lo6 daltons. After growth for 48 h in nonselective medium, the cells were incubated in selective medium. Colonies appeared after 14 days and continued to appear until 25 days, after which no colonies appeared in dishes which had previously contained none. The minimum colony size at this stage was about 30 cells. At later times (~35 days), positive dishes contained numerous colonies, most of which were presumably secondaries. The data given therefore always relate to the twenty fifth day after inoculation. Table 1 shows the influence of the dose of HSV2 DNA on the frequency of transformation. Where more than one colony appears on a dish, we are unable to distinguish between primary and secondary colonies, so the number of colonies need not reflect the number of transformation events. Nevertheless, it is apparent from Table 1 that at high DNA concentrations, the number of events is independent of dose, since a dose of 2.5 pg gave a maximum of 14 events, while a dose of 0.1 pg gave a minimum of 9 or 8 events in two experiments. At lower doses, the number of events appears to become dosedependent, although the data are inadequate to determine whether a “single hit” is required for transformation. Properties of the Transformed Cells Four clonally unrelated transformed cell lines were established from experiments using sheared type 1 DNA and nine from experiments using type 2 DNA. All the cell lines were found to contain thymidine kinase activity, and this activity could be neutralized by antisera raised in rabbits against herpesinfected cells (Thouless and Wildy, 1975).
Cell 502
Table 1. Influence Frequency
of HSV DNA Dose
on Transformation
Experiment
1
Experiment
Dose of HSV-2 DNA per Dish (/.a)
Colonies in IO Dishes
Proportion of Dishes with Colonies
Colonies in IO Dishes
2.5
14
9/10
1 .o
20
IO/IO
0.25
28
IO/IO
0.10
16
Proportion of Dishes with Colonies
lO/lO 8110
0.03
8
4110
0.01
0
O/IO
0.003
0
0110
0
O/IO
0
O/IO
2. Inactivation
Kinase
Kinase
Source
by Specific
Sera
% Kinase Activity Remaining
Anti-Type 2 Serum
Anti-Type 1 Serum
Din
10,270
76
6
Dls
6,230
81
9
Dlr
3,180
88
13
‘321
2,900
15
33
D23
4,850
30
74
D25
9,990
8
25
D26
3,695
33
55
BHK
3,250
102
105
L-M
9,390
102
104
Type
Table 2 shows the results of an experiment with three lines transformed with type 1 DNA (DIP, D13, D14) and four lines transformed with type 2 DNA (D2,, D23, D2,. D2,). Antiserum raised in rabbits against type 1 or type 2 HSV-infected RK 13 cells had no effect on the thymidine kinase in BHK or L cells, but neutralized the kinase from transformed cells. Furthermore, serum raised against type 1 virus-infected cells neutralized the kinase from type 1 transformed cells more efficiently than the kinase from type 2 transformed cdls. Antiserum against type 2 virus behaved in a converse way. The results given in Table 2 are not entirely satisfactory, since there is a considerable difference between the neutralization of enzyme from lytically infected ceils and from transformed cell lines. In our hands, this serological test is very specific in that the antiHSV sera used never showed neutralizing activity against the enzyme from L cells or BHK cells, and each berum always neutralized the homologous enzyme more than the same concentration of the heterologous enzyme. Similarly, each enzyme was always neutralized more by the homologous serum than by the heterologous serum. Nevertheless, the actual level of neutralization produced by a particular enzyme-serum combination was very variable in different experiments. This was particularly noticeable in experiments with the type 2 specific enzyme. Different levels of thymidine kinase were found in different ceil lines, but the level in any particular cell line was characteristic and was independent of passage number in selective medium, at least until passage 20. The results of simultaneous estimations of the enzyme activity in nine type 2 transformed lines are given in Figure 1. The enzyme levels are distributed over an approximately 4-fold range, and these levels may be clustered in groups. As yet, insufficient numbers of cell lines have been
of Thymidine
Activity after Incubation with Preimmune Serum (w-V
2
13
0
9/l 0
24
Table
1 Infected
BHK
22,700
27
4
Type 2 Infected
BHK
10,700
5
79
a The data given are the means of duplicate samples 20 min of reaction. Samples were also taken after reactions were linear up to 20 min.
taken after 10 min. All
examined to encourage confidence in this observation. Since DNA fragments of molecular weight lo-20 x lo6 dalton have been used to produce these transformed lines, it is reasonable to suppose that in addition to the thymidine kinase gene, other nonselected herpes-specific functions may be acquired by the transformed cells. One approach toward detecting such functions is to test the ability of the transformed cell to support the growth of temperature-sensitive mutants of HSV at the nonpermissive temperature (McNab and Timbury, 1976). LMTKcells and transformed cells were therefore infected with ts- mutants at a multiplicity of 3 pfu per cell, and after 20 hr at the nonpermissive temperature, the yield of virus was compared with the yield of wild-type virus grown under identical conditions. Table 3 shows the results of two such experiments involving ts- mutants N102, N103, 85 and Bl, together with the parental type 1 isolate HFEM. The yield of wild-type virus varied from experiment to experiment and from cell line to cell line in individual experiments, but the combined results of five experiments show that no transformed cell line differs from any others or from LMTK- cells in its ability to support the growth of wild-type virus. None of the cell lines examined gave elevated yields of Bl or B5, but mutants N102 and N103 were complemented efficiently by cell line 02,. Mutants N102 and N103 do not complement each other, but both complement efficiently with Bl and B5, and we therefore suppose that N102 and
HSV Thymidine 583
Kinase
Gene
Transfer
Table 3. Complementation of HSV. Type Transformed with HSV Type 2 DNA 6
Experiment Virus Cell Line
TMP p moles L
1
Yield (pfu
Wild-Type
N103
LMTK-
60
0.06
D21
25
6.0
D22 D&
40
D2r
x 10-5) N102
by Cell Lines
Experiment
2
Virus
(pfu
Yield
Wild-Type
Bt
x IO-3 85
260
0.10
0.08
35.0
260
0.05
0.15
0.19
0.8
250
0.04
0.10
180
0.27
1 .2
ia0
0.08
0.12
200
0.20
1.7
240
‘0.04
0.05
D25
120
0.28
1 .4
380
0.12
0.10
D2e
180
0.22
1.6
220
0.05
0.08
D2r
20
0.05
D&
27
0.05
130
0.18
D2s
I .2
1. Mutants
1.0 ND 1.3
ND
ND
ND
270
0.10
0.08
ND
ND
ND 1.
10
20
Time (mlnutes ) Figure
1, Thymidine
Kinase
Levels
in Transformed
Cell Lines
The cell lines were grown to confluence in selective medium, harvested by trypsinization and suspended at 4 x IO’ cells per ml. Extracts were prepared by ultrasonic disintegration and assayed for kinase activity. The data shown are thymidine-phosphorylated by extracts of lo5 cells. All cell lines were at passage 5 in selective medium.
N103 are defective in the same function and that the absence of this function is compensated by D21 ceils. We could find no evidence of rescue of the virus gene resident in D2, cells since the progeny virus resulting from infection with N102 and N103 was all of mutant phenotype. Mutants N102 and N103 were also used in complementation experiments with type 1 transformed cell lines Dl,, Dl,, Dls and Dl,. None of these cell lines complemented either mutant. Thus we have been able to demonstrate intertypic complementation of these mutants by type 2 transformed cells, but not intratypic complementation, presumably because of the small number of type 1 transformed cell lines tested. It is known that type 2 virus function will compensate the defect in mutant N102, because this mutant has been used to generate intertypic recombinants of I-JSV (Morse et al., 1977). It appears that the introduction of the HSV kinase gene into LMTK- cells is rarely accompanied by the acquisition of a function which will complement mutants N102 and N103 regardless of whether type 1 DNA or type 2 DNA is used as a source of the kinase gene.
Revertant Cell Lines Thymidine kinase-negative revertants of cell line D2, were obtained by plating IO4 cells per dish in medium containing 50 pg/ml BUDR. A few colonies appeared in each dish, and three such colonies from different dishes were established as cell lines in nonselective medium (D2,Rl, D2,R2, D2,R3). These cell lines contained no detectable thymidine kinase and failed to plate in medium containing methotrexate (plating efficiency ~10-~). The revertant lines were tested for their ability to support the growth of ts N103 at the nonpermissive temperature, and as shown in Table 4, the revertants have lost this function, while their ability to support the growth of wild-type virus is unimpaired. Selection against the herpes-specified thymidine kinase thus simultaneously selects against the function which complements ts N103. Transformation with DNA from Transformed Cells DNA was extracted from cell lines D2, and D2,, and used to transform LMTK- cells to a kinase-positive phenotype (Table 5). In four independent experiments, inoculation of DNA from transformed cells resulted in the appearance of colonies in a majority of dishes, while no transformants appeared with L cell DNA or with DNA extracted from a thymidine kinase-negative revertant of cell line D21. Four cell lines were established from clonally unrelated colonies induced by D2, DNA in experiment 1 (D2, Tl , D2, T2, D2, T3,02, T5). These will subsequently be referred to as transfected lines. Serological neutralization of the thymidine kinase from these cell lines (Table 6) showed that the enzyme was HSV2-specific. Furthermore, each of the four lines showed very similar levels of kinase activity. Figure 2 shows four independent estimations of the en-
Cell 584
Table
4. Growth
of Mutant
N103 in TKVirus
Revertants
Yield (pfu
Cell Line
Wild-Type
LMTK-
300
of D2,
x lo-“)
Table DNA
5. Transformation
of LMTK-
Cells with Transformed
N103 Dose per Dish
0.1
D21
390
D2,Rl
200
190 0.15
D2,R2
450
0.08
D2,R3
440
0.18
zyme from cell line D2, and simultaneous estimations of the enzyme from the four transfected cell lines. The transfected ceils contain about half the enzyme level found in D2, cells, and it appears that while transformation with virus DNA results in cell lines with widely different enzyme levels (see Figure l), transformation with D2, DNA yields cell lines which are rather uniform in this character. Each of the transfected lines was examined for its ability to complement ts N103, and the results (Table 7) showed that none of the lines had acquired this character. It appears that transformation with D2, DNA, like transformation with virus DNA, does not normally result in the transfer of the N103 complementing function. To determine whether co-transfer of the kinase gene and the N103 complementing gene could be achieved using transfecting DNA of higher molecular weight, D2, DNA of molecular weight 20-25 x lo6 daltons was used to transfect LMTKcells (Table 5, experiment 6). Three clonally unrelated cell lines were established from this experiment, and these also failed to complement mutant N103. Discussion We have shown that the thymidine kinase gene of herpes simplex virus can be introduced into the genotype of LMTK- cells using either virus DNA or transformed cell DNA as a gene source. The cell lines derived from experiments using HSV2 DNA as a transforming agent contain a thymidine kinase which is HSV2-specific, and one of these lines, D2,, is capable of complementing the DNA-negative ts mutants N102 and N103 of HSVl. Selection of kinase-negative revertants from this cell line results in the loss of this capability. The simplest interpretation of this result is that the kinase gene and the N103 complementing gene are introduced on the same fragment of virus DNA, and that selection against the kinase gene results in the loss of the entire virus sequence or of its expression. Reversion to the kinase-negative phenotype in the instance of cell line D2,R, presumably results from gene loss, since DNA derived from these cells
of DNA
Cell
Proportion of Dishes with Colonies
Experiment
Source
1
HSV-2 virions D2, cells Salmon sperm
0.5 10.0 10.0
WI0 9/l 0 O/l 0
2
HSV-2 virions D23 cells Salmon sperm
0.5 10.0 10.0
4/a 5/6 O/l 0
3
HSV-2 virions D21 cells Salmon sperm
0.5 10.0 10.0
lO/lO 619 o/10
4
HSV-2 virions L-cells
0.5 10.0
E/10 o/10
5
HSV-2 virions HSV-2 virions D2,Rl cells
0.5 0.05 10.0
B/10 2/10 O/l 0
6
HSV-2 virions D2, cells Salmon sperm
0.5 10.0 10.0
IO/10 9/l 0 o/10
All DNA was sheared to a molecular daltons, except in experiment 6, where molecular weight of 20-25 x lo6 daltons.
(CLS)
weight of lo-20 x lo6 D2, DNA was used at a
cannot be used to introduce the kinase gene into LMTK- cells. Since only one of nine cell lines examined is capable of complementing N103, it is probable that the two genes are usually separated when the HSV DNA is sheared. This interpretation is supported by recent evidence concerning the relative positions of the thymidine kinase gene and the ts N102 complementing gene on the HSV2 genome. The kinase gene is believed to be located in the L region between 55 and 65% from the left end of the genome (Maitland and McDougall, 1977). The position of the ts N102 complementing function is unknown, but restriction enzyme analysis of DNA from ts+ intertypic recombinants generated using ts N102 as the type 1 parent (Morse et al., 1977) suggests that this function is separated from the site of the kinase gene by at least 15% of the genome (that is, by a sequence >15 x lo6 dalton molecular weight). This probably explains why co-transfer has proved difficult to achieve even when DNA of molecular weight 20-25 x IO6 daltons has been used. The transfer of the thymidine kinase gene to LMTK- cells using transformed cell DNA as a transforming agent (transfection) is surprisingly efficient. Reference to Tables 1 and 5 shows that IO pg D2, DNA transforms at least as efficiently as 0.1 pg HSV DNA, and since we have not used smaller amounts of D2, DNA, this could be an underesti-
HSV Thymidine 585
Kinase
Gene
Transfer
Table 6. Serological Neutralization Transfected Cell Lines
Source of Thymidine Kinase D2,-Tl
of Thymidine
Activity after Incubation with Preimmune Serum (cpm)
9,500
D2 ,-T3 D2 ,-T5
Anti-Type 1 Serum
Anti-Type 2 Serum
9.6
22
7.5
a Neutralization tests with the enzyme from this cell line were performed on a different occasion from tests with the other three, and an unclarified ceil sonicate was used.
6 TMP p moles
10 Time Figure fected
2. Thymidine Lines
Kinase
Levels
20 (minutes) in Cell
7. Growth
Line
D2, and Trans-
Conditions were as described in the legend to Figure 1. The for D2, cell extracts were obtained on different occasions cells at different passage numbers. Data for the transfected were obtained from parallel assays of extracts prepared from at passage 4.
of Mutant
N103 in Cells Transformed
Virus
24
25
8,200
Table DNA
10
43
8,000
from
% Kinase Activity Remaining
22
6,700
D2,-TZa
Kinase
data using lines cells
mate of its efficiency. Since 10 pg is the DNA content of lo6 cells, and since 0.1 pg HSV DNA represents lo9 genomes, it follows that to account for the efficiency of transformation by D2, DNA in terms of the thymidine kinase gene concentration, we would require each D2, cell to contain lo3 copies of the kinase gene. This seems improbable in view of the very small amounts of virus DNA which have been found in herpes-transformed cells (Kraiselburd et al., 1975; Davis and Kingsbury, 1976; Frenkel et al., 1976; Minson et al., 1976). An alternative proposal is that the kinase gene in transformed cells is in a different physical or chemical state from the gene extracted from virus particles and transforms more efficiently in consequence. For example, the virus DNA in transformed cells could be integrated so that the virus sequence
Yield
Cell Line
Wild-Type
LMTK-
300
(pfu
with
D2,
x lo-“) N103 0.10
D21
390
D2,-Tl
760
0.05
D2,-T2
820
0.08
D2 ,-T3
750
0.33
D2,-T5
510
0.30
190.0
derived from such cells is flanked by host sequences. If these flanking sequences were highly reiterated in the recipient cell chromosomes, then an increased rate of integration of virus sequences might result from recombination of homologous sequences. We can eliminate the possibility that the virus sequences must be flanked on both sides by host sequences in transfecting DNA, since this model would predict that the entire virus sequence in the donor cell should be acquired by the recipient cell. In fact, cells transformed using D2, DNA as a gene donor do not carry the ts N103 complementing function. The idea that transformation using DNA from transformed cells as a gene donor is qualitatively different from transformation with virus DNA is supported by the properties of transfected cells. The transformed lines show a wide range of kinase levels, while the transfected lines are uniform in this character. Only four lines have been examined, so this could be a fortuitous observation, but it is worth noting that of the nine transformed lines examined, only one, D2!,, has the same enzyme level as if found in all four transfected lines. It is obviously important to determine which herpes DNA sequences are present in our transformed lines and to find the number of sequences in each cell. We have not excluded the possibility that each cell contains thousands of copies of the HSV thymidine kinase gene, but it seems more probable that each cell contains only a few copies and that these sequences have unusual properties which render them very efficient in gene transfer experiments. There have been a number of reports of gene transfer from cell to cell, including transfer of thymidine kinase genes, mediated by chromosome uptake (McBride and Ozer, 1973; Willecke et al., 1976; Wullens van der Horst and Bootsma 1977), but the frequency of transformation is lower than reported here, and as yet there have been no confirmed reports of gene transfer from mammalian cell to mammalian cell using direct DNA uptake.
Cell 586
Experimental
Procedures
Cells and Tissue Culture LMTK- cells were obtained from Dr. M. Thouless. BHK/Pi cells were used for virus assays and growth of virus stocks. Hep-2 cells were used for large-scale virus propagation for virus DNA preparation. All cells were grown in Glasgow modified Eagle’s medium supplemented with 10% tryptose phosphate broth and 10% newborn calf serum (Macpherson and Stoker, 1962). Selection of cells containing thymidine kinase was achieved by supplementing with thymidine, adenosine, guanosine, glycine and methotrexate as described by Munyon et al. (1971), and unless otherwise stated, all transformed lines were maintained in this medium. Selection against cells containing thymidine kinase was achieved by supplementing with 50 +g/ml BUDR. Viruses HSV type 1 strain HFEM and type 2 strain 25766 (obtained from Professor K. R. Dumbell, Wright Fleming Institute, London, England) were used throughout. Mutants Bl, 85. N102 and N103 were independently isolated from strain HFEM using either BUDR (prefix 6) or nitrous acid (prefix N) as mutagens. All mutants have a relative plaquing efficiency of <1O-3 at 38.5”C compared with 33°C. Mutant 85 is DNA-positive at the restrictive temperature. The remainder are DNA-negative. (These mutants were isolated by Dr. A. Buchan; details of their isolation and characterisation will appear elsewhere.) Hep-2 cells were infected at a multiplicity of 1 pfu per cell, and cell-released virus was purified from the medium 48 hr after infection. Cells infected with type 1 virus were incubated at 37”C, and cells infected with type 2 were incubated at 33°C. Virus DNA was extracted from purified particles as described by Wilkie (1973). DNA was extracted from cells by the method of Varmus, Vogt and Bishop (1973). Transformation 5 x lo5 cells were seeded in 60 mm dishes, and after 24 hr, the medium was removed and the cells inoculated with DNA as described by Bacchetti and Graham (1977). The DNA dose per dish was always made to 10 pg using salmon sperm DNA as carrier. The DNA was first sheared by passing a solution at 100 pglml3 times through a 21 gauge x 1.5 inch syringe needle, and the fragment size was then estimated by electrophoresis in agarose gels using Eco RI-restricted HSVP DNA as a source of size markers. After 1 hr at room temperature, the inoculum was removed and replaced with nonselective medium. The dishes were changed to selective medium after a further 48 hr, and the medium was then changed every third day. Complementation of ts Mutants Preformed monolayers of 3-6 x lo5 cells were infected at a multiplicity of 3 pfu per cell, and the virus was allowed to absorb for 1 hr at 37°C. The monolayers were then washed 3 times with warm medium and sealed in plastic bags. All procedures to this point were carried out in a 37°C warm room. The monolayers were then incubated under water at 38.5”C, and after 30 hr, they were harvested and sonicated, and the virus yield was assayed at 33°C. In all experiments, monolayers were infected in parallel with mutant and wild-type virus. Where evidence of complementation was obtained, the progeny virus was assayed again at 38.5”C. Thymldine Klnase Assays Cells were trypsinized and suspended in 0.01 M Tris-Cl(pH 7.5) at 4 x 10’ cells per ml. The cells were disrupted by ultrasonic vibration and immediately assayed for thymidine kinase activity by the method of Klemperer et al. (1967). For serological neutralization tests, cell extracts were clarified by centrifuging for 30 min at 100,000 x g at 4°C. The supernatants were mixed with an equal volume of preimmune or immune serum, and the thymidine kinase activity was determined after 2
hr at 4%. The enzyme in high-speed supernatants was neutralized more efficiently than the enzyme in crude cell sonicates. The observed enzyme levels found in supernatants, however, were very variable, presumably because of the instability of the enzymes. Anti-type 1 serum (192) and anti-type 2 serum (409) were gifts from Dr. Margaret Thouless and were prepared by immunization of rabbits with infected RK13 cells (Thouless and Wildy, 1975; Thouless, Chadha and Munyon, 1975). Acknowledgments We are grateful to Lynne Jeffrey and Susanne Bell for excellent technical assistance, and to Silvia Bacchetti and Peter Sheldrick for helpful comments and discussion. This work was supported by the Cancer Research Campaign. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received
September
28,1977;
revised
December
13,1977
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