in both Epstein-Barr virus (EBV)-induced DNA polymerase activity and early ... EBV DNA polymerase, but also, to a lesser degree, the cellular polymerase a.
Vol. 29, No. 1
JOURNAL OF VIROLOGY, Jan. 1979, p. 1-10 0022-538X/79/01-0001/10$02.00/0
Characterization of an Epstein-Barr Virus-Induced DNA Polymerase T. OOKA,l* G. LENOIR,2 AND J. DAILLIE' Departement de Biologie Generale et Appliquee, Universite Claude Bernard (Lyon I), 69621 Villeurbanne,' and International Agency for Research on Cancer, 150 cours Albert Thomas, 69372 Lyon,2 France Received for publication 7 July 1978
The addition of iododeoxyuridine to P3HR-I cell cultures led to a large increase in both Epstein-Barr virus (EBV)-induced DNA polymerase activity and early antigen-positive cells. This EBV-induced DNA polymerase was separated from the cellular a- and ,B-polymerases by sequential column chromatography on Sepharose 6B, DEAE-cellulose, and phosphocellulose, resulting in partial purification of about 320-fold. The partially purified-EBV DNA polymerase could be distinguished from the cellular DNA polymerases by its activation by salts, its catalytic properties, and its degree of sensitivity to N-ethylmaleimide, phosphonoacetic acid, araATP, and araCTP. The viral polymerase showed properties similar to those reported for other herpesvirus DNA polymerases. The enzyme exhibited optimal activity for copying activated calf DNA in the presence of 50 mM (NH4)2SO4 and was resistant to 150 mM (NH4)2SO4. It utilized with high efficiency template-primer poly(dC)-oligo(dG)12 18 or poly(dA)-oligo(dT)12-18, but failed to copy poly(rA)-oligo(dT)1o and oligo(dT)IO, indicating that this enzyme has characters distinct from DNA polymerase y, reverse transcriptase, and terminal deoxynucleotidyl transferase. Phosphonoacetic acid inhibited not only EBV DNA polymerase, but also, to a lesser degree, the cellular polymerase a. AraATP did not severely inhibit viral activity, whereas the polymerase a was inhibited most effectively. Both EBV polymerase and polymerase a were inhibited at a comparable level by araCTP.
Herpesviruses such as herpes simplex virus (HSV) (11, 22, 30), human cytomegalovirus (9), Marek's disease virus (2), equine herpes virus (1, 5, 12), and varicella-zoster virus (18) induced a new DNA polymerase activity in infected cells that differs from the corresponding cellular enzymes with respect to physical properties and some biological activities such as template specificity and salt and chemical-inhibitor sensitivity. In the case of Epstein-Barr virus (EBV), the existence of such a type of DNA polymerase was strongly suspected, since the appearance of late viral gene expression, such as viral capsid antigens, was inhibited by DNA synthesis inhibitors such as phosphonoacetic acid (PAA), which have little effect on host DNA synthesis (27). The partial purification and characterization of EBV DNA polymerase is a necessary prerequisite for the understanding of its role, not only in the replication process of the virus during the productive cycle, but also in the viral DNA replication during the latent phase induced after immortalization of B lymphocytes by this virus. The absence of a fully permissive cell system
for EBV replication renders the study of such a putative enzyme difficult. However, our previous studies (T. Ooka, J. Daillie, 0. Costa, and G. Lenoir, in G. de The, W. Henle, and F. Rapp, ed., Oncogenesis and Herpesviruses, vol. Im, in press) have revealed that when some human lymphoblastoid cell lines carrying the EBV genome and expressing the EB nuclear antigen were treated with 5-iododeoxyuridine (IUdR), the derepression of the viral genome led to the induction of early antigens (EA) and of a new DNA polymerase activity resembling the herpes-induced enzymes. Similar results have recently been reported by Miller et al. (17) in an IUdR-treated epithelial/Burkitt hybrid cell line. Based on these findings, we decided to purify partially the DNA polymerase induced after IUdR treatment of P3HR-I cells, to compare its biological properties with those of the cellular DNA polymerases and to examine its relationship with EA. The EBV-induced DNA polymerase was purified 320-fold and the salt sensitivity, template specificity, and chemical inhibitor sensitivity were studied.
2
OOKA, LENOIR, AND DAILLIE
J. VIROL.
MATERIALS AND METHODS
of DNA at 37°C for 30 min. After incubation at 77°C for 5 min, the DNA was precipitated by alcohol and dissolved in 10 mM Tris-hydrochloride (pH 7.5) buffer. About 20% of DNA treated in this manner was rendered acid soluble. Heat-denatured DNA was prepared by heating a DNA solution (500 Mg/ml) at 100°C for 10 min, followed by immediate chilling in ice. For the reaction mixture for the activated DNA-directed DNA synthesis, a total volume of 125 [l was comprised of: 100 mM Tris-hydrochloride buffer (pH 8.5), 4 mM MgCl2, 34 jiM activated DNA, 50 MM each of dTTP, dGTP, dCTP, and dATP containing 0.5 jiCi of [3H]dTTP (specific activity of 20 Ci/mmol), 50 Mg of bovine serum albumin per ml, and DNA polymerase. Enzyme assays were carried out in the absence or presence of ammonium sulfate at a concentration of 150 mM. At this concentration, the cellular DNA polymerases were completely inhibited whereas viral DNA polymerase was partially resistant, and at a concentration of 50 mM the viral enzyme was slightly stimulated (Ooka et al., in press; see Table 2). For the polymerization of [3H]dTTP or [3H]dGTP, directed by the synthetic polynucleotides-oligonucleotides or oligonucleotides, a total volume of 125 Ml was composed of: 100 mM Tris-hydrochloride buffer (pH 8.5), 4 mM MgCl2, 17MuM synthetic template-primer, 50 /M of dTTP or dGTP containing 0.5 MCi of [3H]dTTP (specific activity 20 Ci/mmol) or [3H]dGTP (specific activity 10.1 Ci/mmol), 1 mM EDTA, 50 Mg of bovine serum albumin per ml, and DNA polymerase. The reaction mixture was incubated at 37°C for 30 or 60 min. The reaction was then stopped in ice, and the nucleotides incorporated in acid-insoluble form were processed as already described (20). Briefly, the reaction mixtures were transferred on Whatman GF/c glass fiber disks, DNA was precipitated by a 5% cold trichloroacetic acid solution, and, after repeated washing with the 5% trichloroacetic acid solution, the disks were dried with ethanol and ether and counted. Purification of EBV-induced DNA polymerase. (i) Sepharose 6B chromatography. The combined supernatant fluids (8 ml), adjusted to 20% (wt/vol) glycerol, were loaded onto a column of Sepharose 6B (type K26/70, Pharmacia), which was equilibrated with TKMD buffer (pH 7.5) containing 20% glycerol. (ii) DEAE-cellulose chromatography. The peak fractions of 150 mM (NH4)2SO4-resistant DNA polymerase obtained from Sepharose 6B chromatography (indicated in Fig. 2) were pooled and applied directly, without dialysis, to a 5-ml column of DEAE-cellulose equilibrated with TKMD buffer (pH 7.5) containing 20% glycerol. After washing with 7 bed volumes of the same buffer, elution was performed with 12 bed volumes of a linear gradient ranging from 0 to 0.3 M NaCl (pH 7.5) in the same buffer. After addition of 500Mg of bovine serum albumin per fraction, all fractions (1 ml) were dialyzed overnight against TKMD buffer (pH 8.0) containing 0.1 M KCl. (iii) Phosphocellulose chromatography. The peak fractions obtained from DEAE-cellulose chromatography and the corresponding EBV-induced DNA polymerase activity were loaded onto a 1.5-ml column of acid- and base-washed phosphocellulose (P11), which was equilibrated overnight with TKMD buffer (pH 8.0) containing 0.1 M KCl. After washing
Reagents. [3H]dTTP and [3H]dGTP were purchased from Amersham (the Radiochemical Centre, England). The synthetic template-primers were obtained from Collaborative Research Inc. (Waltham, Mass.), Miles Laboratories (Lausanne, Switzerland), and P-L Biochemicals (Milwaukee, Wis.). PAA was the kind gift of Abbott Laboratories (North Chicago, Ill.). AraATP and araCTP were obtained from P-L Biochemicals. DEAE-cellulose (DE 52) and phosphocellulose (P 11) were supplied by Whatman Ltd. (Kent, England). Sepharose 6B was obtained from Pharmacia Fine Chemicals (Uppsala, Sweden). The unlabeled deoxyribonucleoside triphosphates were purchased from Boehringer Mannheim (Mannheim, West Germany), and deoxyribonuclease I came from Worthington Biochemicals (Freehold, N.J.). Growth of cells. Cells from EBV producer (P3HR1; 8) and nonproducer (Raji; 23) cell lines were seeded at 5 x 105 cells per ml and grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, 100 U of penicillin per ml, and 250 jig of streptomycin per ml at 37°C. Induction with IUdR. P3HR1 cells were seeded at 106 cells per ml in culture medium. After 24 h of cultivation at 37°C, IUdR was added in culture medium at a final concentration of 50 1ig/ml for 48 h. In these conditions the final percentage of EA-positive cell ranged between 20 and 30%. The evaluation of the percentage of EA-positive cells was carried out on smears by the indirect immunofluorescence test as described by Henle and Henle (7). For this purpose, samples of cells were removed and centrifuged at 600 x g for 2 min. The cells were then washed twice in phosphate-buffered saline, seeded on glass cover slips, dried, and fixed in cold acetone for 10 min. Preparation of cell extracts. All manipulations during the preparation were carried out at 0 to 40C. The cells from the suspension culture were centrifuged at 600 x g for 6 min and washed twice with TKMD buffer (50 mM Tris-hydrochloride [pH 7.5]-10 mM KCl-1 mM MgCl2-1 mM dithiothreitol). After resuspension at a concentration of 2 x 108 to 3 x 108 cells per ml in TKMD buffer, the cells were sonically disrupted for four 30-s periods at a high (4) setting with an MSE Sonicator. The sonically disrupted suspension was centrifuged at 105,000 x g for 60 min. After centrifugation the supernatant fluid was collected (S1), and the pellets were resuspended in 1/10 of initial volume of TKMD buffer. This suspension was sonically treated and centrifuged as before. The second supernatant (S2) was combined with the first (S1), and these pooled supernatants were used as a crude extract preparation. No significant EBV-induced DNA polymerase activity was detected in the pellets obtained after the last centrifugation, showing that almost all viral enzyme activity could be found in the supernatant as prepared by the above extraction procedure. Enzyme assays. Most of the DNA polymerase assays have been described previously by Ooka and Daillie (20). In brief, activated calf thymus DNA was prepared by digestion with 0.024 U of DNase I per mg
VOL. 29, 1979 with 20 ml of column buffer, the EBV-induced DNA polymerase activity was eluted with a 20-ml linear gradient of 0.1 to 1.0 M 'KCl in column buffer. The EBV-induced DNA polymerase fractions obtained from this column were dialyzed against TKMD buffer (pH 8.0) and stored at -70°C. Preparation of DNA polymerases a and /8. To compare the EBV-induced DNA polymerase with the cellular a- and fi-polymerases, partially purified P3HR-1 cellular polymerases were prepared as follows. DNA polymerase a came from the DEAE-cellulose fraction which was eluted at 0.12 M NaCl (see Fig. 3). For P3HR-1 cell DNA polymerase fi, nuclei were obtained by Dounce homogenization and detergent treatment, as previously described (20). To extract the ,B polymerase activity, the nuclear preparations were suspended in TKMD buffer and treated with an equal volume of 4 M NaCl at 0 to 40C for 2 h, followed by centrifugation at 40,000 rpm for 4 h. The supernatant fluid from NaCl extraction was dialyzed for 24 h against TKMD buffer. The nuclear extracts were then applied directly on a phosphocellulose chromatography column and eluted with a linear gradient of KCI from 0.1 to 1.0 M. The fl-polymerase activity was eluted at 0.4 M.
RESULTS Induction of the EBV-induced DNA polymerase activity after addition of IUdR. Our previous reports have indicated the presence of the EBV-induced DNA polymerase activity, distinguishable from cellular DNA polymerases by its high salt resistance [150 mM (NH4)2SO4 in Burkitt's lymphoma-derived cell lines which express the EA (Ooka et al., in press). Using 150 mM ammonium sulfate in DNA polymerase assays, the kinetics of the appearance of EBV-induced DNA polymerase activity in total cell extracts of IUdR-treated or nontreated P3HR1 were studied. Induction of EBVspecific EA-positive cells was also monitored by assaying in the indirect immunofluorescence test. The addition of IUdR in P3HR1 cell cultures led to a large increase in both viral DNA polymerase activity and EA-positive cells (Fig. 1). The viral DNA polymerase activity increased in a linear fashion and after 48 h reached a rate about four to five times higher than that at 0 h. A percentage of 29.7% of EA-positive cells was obtained after 48 h, the time at which the maximum induction is generally obtained in this system. In contrast, both the EBV-induced polymerase activity and the percentage of EA-positive cells in P3HR1 cell cultures not treated by IUdR showed a negligible variation with time. The observed increase in the activity of EBV-induced DNA polymerase, which paralleled the appearance of EA-positive cells, suggests that this enzyme is related to EA synthesis. Therefore, to
EBV-INDUCED DNA POLYMERASE
0
24
3
48
Hours after addition of lUdR
FIG. 1. Kinetics ofinduction ofEBV-induced DNA polymerase activity and EA-positive cells in P3HR-1 cells treated with IUdR. At the indicated times after addition of IUdR (50 pg/ml) at zero time, 4 x 108 IUdR-treated or untreated P3HR-1 cells were harvested for assaying either EBV-specific EA by the indirect immunofluorescence or DNA polymerase activity in the presence of 150 mM (NHj2SO4, which completely inhibited the activity of cellular DNA polymerases. DNA polymerase activity in IUdR-treated cells (-@*) and in untreated cells (0. .....0). The percentage ofEA-positive cells is indicated in parentheses.
purify the EBV-induced DNA polymerase, P3HR-1 cells were harvested at 48 h after addition of IUdR, at which time a maximal percentage of EA-positive cells was obtained without major decrease in cell viability. Purification ofEBV-induced DNA polymerase. The purification steps are indicated in Table 1. Cellular extracts were prepared from approximately 4 x 109 IUdR-treated P3HR-1 cells and passed through a Sepharose 6B gel filtration. The fractions were assayed using ammonium sulfate for EBV-induced DNA polymerase and without the salt for cellular DNA polymerases. Figure 2 shows that an EBV-induced DNA polymerase activity that was partly resistant to 150 mM ammonium sulfate was found. The fact that this viral activity eluted just after the cellular activity indicates that the EBV-induced DNA polymerase seems to have a molecular weight lower than that of the cellular DNA polymerases. It should be pointed out that a large amount of nucleic acids, proteins, and some apolymerase activity can be also removed with this step. The fractions from Sepharose 6B were loaded
4
J. VIROL.
OOKA, LENOIR, AND DAILLIE
TABLE 1. Purification of EBV-induced DNA polymerase from IUdR-treated P3HR-1 cells Total protein Total activity Recovery Sp act (u/mg of Purification Fraction (fold) (mg) (units)a (%) protein)a 210.0 273.8 100 Crude extract 1.29 1 32.2 16.1 520.2 189 Sepharose 6B 13 2.96 147.4 DEAE-cellulose 54 50.0 39 0.099 41.4 15 418.4 326 Phosphocellulose 'A unit is defined as 1 nmol of deoxynucleotide incorporated into an acid-insoluble form in 60 min at 370C. The EBV-DNA polymerase reaction mixture was supplemented with 150 mM (NH4)2S04, and activated calf thymus DNA was used as template-primer (see the text).
E
0
w
r-
rn '0 D
x
(L
C.)
L
Fraction Number
FIG. 2. Gel filtration chromatographic analysis of EBV-induced DNA polymerase and cellular DNA polymerases. IUdR-treated P3HR-1 ceU extracts (210 mg of protein) were applied to a column of Sepharose 6B in TKMD buffer (pH 7.5) containing 20% glycerol. Samples (30;d) ofeach column fraction were assayed for DNA polymerase activity without added salt (A....A) and after supplementing the polymerase reaction mixture with 150 mM (@-*) (NH4)2SO4. Absorbance at 280 nm ( ).
onto a DEAE-cellulose column and eluted with a 0 to 0.3 M NaCl salt gradient (Fig. 3). A peak of salt-resistant viral DNA polymerase activity, which can be clearly separated from a-polymerase activity, was eluted at a level of about 0.08 M sodium chloride, whereas the level was about 0.12 M for a-polymerase. When the EBV-induced polymerase obtained from DEAE-cellulose columns was further chromatographed on phosphocellulose, the activity was eluted as a single peak at a salt (KCI) concentration of 0.29 M (Fig. 4). The degree of purification achieved with each column step is summarized in Table 1. Since all viral DNA polymerase assays were carried out in the presence of 150 mM ammonium sulfate, to inhibit completely the contaminating cellular DNA polymerase activities, the units reported in Table 1 are about 10-fold inferior to the maximal values
obtainable with 50 mM (NH4)2SO4 (see the section on the effect of ammonium sulfate), but represent a real viral DNA polymerase activity, which in these conditions was purified 320-fold from IUdR-treated P3HR-I cell extracts. Characteristics of EBV-induced DNA polymerase. For this study, the biologically active fractions eluted from the phosphocellulose column after the three-step purification procedures (Table 1) were used as source of EBV-induced polymerase. The a- and fi-polymerases were also prepared as described in Materials and Methods. (i) Effect of salt concentrations. The effect of increasing the amounts of ammonium sulfate clearly distinguished the EBV-induced DNA polymerase from the P3HR1 cell a- and /-polymerases (Table 2). Only EBV-induced DNA polymerase was stimulated about 1.5-fold with 50 mM ammonium sulfate and expressed again a
VOL.VEBV-INDUCED DNA POLYMERASE 29, 1979
residual activity at 150 and 200 mM (about 20 and 5%, respectively), whereas a- and ,B-polymerases were both sensitive to the presence of salt and were inhibited 99 to 100% by concentrations of up to 150 mM. (ii) Effect of N-ethylmaleimide. Although B8-polymerase activity was relatively resistant to
5
N-ethylmaleimide (the remaining 40% at 4 mM), the EBV-induced and a-polymerase activities were both inhibited by the drug: concentrations of up to 0.5 mM resulted in more than 80% inhibition of these enzyme activities (data not indicated). (iii) Primer-template studies. The EBV-in-
E 0 c 4i
'0
cD a)
x
Uia
co .0L0 Lo
.0
Fraction Number
FIG. 3. DEAE-cellulose chromatograms of DNA polymerases. The active fractions (fractions of 31 to 36) from each Sepharose6B gel filtration were pooled and passed through a DEAE-cellulose column which had been equilibrated with TKMD buffer (pH 7.5) containing 20% glyceroL After washing with the equilibration buffer, a column was eluted with an NaCI linear gradient from 0 to 0.3 M. Thirty microliters of each fraction was used for DNA polymerase assay. DNA polymerase activity (A ..... A) without added salt; (-4) with 150 mM (NH42$04. Absorbance at 280 nm (-).
E
9
0 m Cu
8 7
CD a)
'0 6 X5
c
O4
CD
Li3
C0 U)
0
ci
n
10
50 40 30 20 Fraction Number
60
70
FIG. 4. Phosphocellulose chromatograms of DNA polymerases. The active fractions (fractions of 31 to 37) from DEAE-cellulose peak I were pooled and passed through a phosphocellulose column which had been equilibrated with TKMD buffer (pH &0) containing 20% glycerol. After washing with the equilibration buffer, a column was eluted with KCI linear gradient from 0.1 to I M. EBV-induced DNA polymerase activity ). ....A) without added salt; (-4*) with 150 mM (NH4)2SO4. Absorbance at 280 nm ( (A.*
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OOKA, LENOIR, AND DAILLIE
J. VIROL.
duced DNA polymerase was tested for its capacity to utilize diverse synthetic and natural DNA templates. Table 3 shows the results obtained with the EBV-induced DNA polymerase and with the cellular DNA polymerases usin1g these various template-primers. All DNA polymerases could effectively use activated calf thymus DNA as template-primer, but poorly accepted denatured and native calf thymus DNA as templates. Synthetic template-primers, such as poly(dA)oligo(dT)12-18 and particularly poly(dC)oligo(dG)1218, were shown to be better templates than the activated calf thymus DNA for the viral enzyme. However, the a-polymerase also used synthetic poly(dC)-oligo(dG)12 18more efficiently, when compared to activated DNA, but not poly(dA)-oligo(dT)12-j8. Both EBV-induced and a-polymerases failed to copy poly(rA)oligo(dT)1o, whereas DNA polymerase ,B was
able to use this template-primer, though somewhat less efficiently than activated calf thymus DNA. All DNA polymerases were unable to use oligo(dT)jO as template. These observations indicate that the EBV-induced DNA polymerase is an enzyme with a character distinct from ypolymerase, reverse transcriptase, and terminal deoxynucleotidyl transferase. Effect of PAA. Since HSV-induced DNA polymerase activity has been reported to be specifically inhibited by PAA (3, 13, 15), we have determined the effect of this inhibitor on the activity of DNA polymerases (Fig. 5). The EBV-
50
TABLE 2. Effect of salt concentration on the activity of EBV-induced, a, and ,B DNA polymerases (NH4)2SO4 concentration (mM) 0 50 100 150 200
DNA polymerase activity without salta (%) .. ,II,
EBV
a
100 150 96 20 5
100 61 20
