Induction of ribonucleotide reductase activity in cells infected with African swine fever virus

VIROLOGY

187, 73-83 (1992)

Induction

of Ribonucleotide Reductase Activity in Cells Infected with African Swine Fever Virus CELSO V. CUNHA AND Joi@

Gulbenkian

Institute of Science, Apartado

v.

COSTA’

14, P-278 1 Oeiras Codex, Portugal

Received January 25, 199 1; accepted

November

6, 193 1

Infection of Vero cells with African swine fever virus (ASFV) resulted in a marked increase in ribonucleotide reductase activity. The induction of ribonucleotide reductase was detected early after infection and was proportional to the multiplicity of infection. Inhibition of viral DNA replication did not affect the induction of the enzyme. Several characteristics could distinguish the virus-induced from the normal cell enzyme. ASFV-induced ribonucleotide reductase was inhibited by magnesium, was more strongly inhibited by hydroxyurea, and had a fourfold lower K,. The virus-induced enzyme was inhibited by deoxyribonucleoside triphosphates and by ATP. The isolation of hydroxyurea-resistant ASFV mutants provided genetic evidence for the viral origin of the induced ribonucleotide reductase. The resistance to hydroxyurea was due to a threefold overproduction of ribonucleotide reductase. as compared to enzyme induction by wild-type ASFV. Hydroxyurea had similar effect in vitro on ribonucleotide reductases induced by wild-type or mutant virus. The gene for the small subunit of the viral enzyme was mapped within a 2.3-kb fragment by hybridization with an oligonucleotide probe designed from a conserved aminoacid sequence of eukaryotic and viral ribonucleotide reduc0 1992 Academic Press, Inc. tases.

that catalyzes the reduction of all four ribonucleoside diphosphates to their corresponding deoxyribonucleoside diphosphates. Escherichia co/i and mammalian ribonucleotide reductases have been purified and extensively characterized. They are composed of two subunits, a regulatory subunit containing sites for allosteric effecters and a catalytic subunit containing a tyrosyl radical that interacts with ferric cations (for reviews see Thelander and Reichard, 1979; Reichard and Ehrenberg, 1983; Reichard, 1988; Eriksson and Sjoberg, 1989). New ribonucleotide reductase activities are induced in cells infected with herpes simplex viruses (Cohen, 1972) Epstein-Barr virus (Henry et al., 1978), pseudorabies virus (Lankinen et a/., 1982), and vaccinia virus (Slabaugh eta/., 1984). In this paperwe reportthe identification and characterization of a ribonucleotide reductase activity induced in cells infected with ASFV. We also describe the isolation and characterization of ASFV mutants that are resistant to hydroxyurea.

INTRODUCTION African swine fever virus (ASFV), the causative agent of a devastating disease of swine, is an icosahedral enveloped DNA virus that grows in the cytoplasm of infected cells. It is the only representative of a new unnamed family (for reviews see Vif’iuela, 1985, 1987; Costa, 1990). The replication of cytoplasmic DNA viruses, like ASFV and poxviruses, requires enzymatic activities that are not normally present in the cytoplasm of animal cells. ASFV must therefore encode or recruit from the nucleus the enzymes that catalyze DNA replication and transcription. A number of ASFV-specific enzymes has been identified: DNA polymerase (Polatnick and Hess, 1972) thymidine kinase (Polatnick and Hess, 1970) RNA polymerase (Kuznar er a/., 1980) poly-A polymerase and capping enzymes (Salas et a/., 1981), protein kinase (Polatnick et al., 1974), nucleoside triphosphatase (Kuznar et a/., 1981), and a singlestranded DNA nuclease (Barros et a/., 1986). The enzymes needed fortranscription and for mRNA processing are packaged in the viral particles and allow the transcription of early genes, including those encoding the enzymes involved in DNA synthesis. Viral DNA synthesis is dependent on the supply of deoxyribonucleoside triphosphates. The only route for de nova synthesis of these precursors is provided by ribonucleotide reductase (EC 1.17.4. l), an enzyme

MATERIALS

AND METHODS

Virus and cells Monkey Vero cells were grown in Dulbecco’s modified Eagle’s medium (DME) with 8% newborn calf serum and 50 pug/ml gentamycin. The cells were routinely tested for mycoplasma contamination. ASFV, Lisbon-60 isolate adapted to grow in monkey cells, was produced in Vero cells and titrated as described

’ To whom reprint requests should be addressed. 73

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74

CUNHA

(Esteves et al., 1986). When necessary, virus stocks were concentrated by centrifugation at 6000 gfor 16 hr at 4”. ASFV was purified by centrifugation in Percoll gradients as described by Carrascosa et a/. (1985). Preparation

of cell extracts

Vero cells were infected with 1O-20 PFU per cell or mock-infected. After 1 hr adsorption at 37’, DME with 2% newborn calf serum was added. At the indicated times p.i. the dishes were placed on ice and the medium was aspirated. All the following steps were done on ice or at 4”. The cells were washed twice and scraped into ice-cold PBS, centrifuged, and resuspended in 100 mM Tris-HCI, pH 7.8, at 2-5 X lo7 cells/ml. For the preparation of cytoplasmic extracts, the cells were treated with 0.5% NP-40 for 15 min on ice. The intact nuclei were removed by centrifugation at 800 g for 5 min at 4”. Alternatively, in some experiments, total cell extracts were prepared by 1 min treatment with ultrasound (18.5 kHz) in a water bath. In some experiments, an insoluble virosomal fraction was used. Infected cell cytoplasmic extracts were prepared as described above and centrifuged at 100,000 g for 1 hr at 4” through a cushion of 20% (w/v) sucrose in 100 mM Tris-HCI, pH 7.8. The pellets were resuspended in 50 ~1 ribonucleotide reductase reaction mixture. Assay of ribonucleotide

reductase

activity

The enzymatic assay was performed essentially as described by Slabaugh et al. (1984). Reaction mixtures contained 25 ~1 cell extract, 100 mMTris-HCI, pH 7.8, 100 pM FeCI,, 10 mM NaF, 10 mM dithiothreitol, 10 pM CDP, and 1 &Ii [3H]CDP (Amersham, 21 Ci/mmol) in a total volume of 50 ~1. Reaction mixtures for the assay of uninfected cell extracts also included 2.5 mM MgCI, and 4 mM adenylylimido-diphosphate (AMPPNP). The mixture was incubated at 37” for 30-60 min. The tubes were then transferred to ice and the reaction was stopped by addition of 5.5 ~1 10 M perchloric acid. Macromolecules were precipitated for 10 min and removed by centrifugation in a microcentrifuge. The supernatants were transferred to fresh microcentrifuge tubes and boiled for 15 min to hydrolyze the phosphotylated nucleosides to monophosphates. The solutions were then neutralized with 5 M KOH and cooled on ice. The resulting potassium perchlorate precipitate was removed by centrifugation. Aliquots of the supernatants were applied to plastic-backed cellulose thin-layer chromatographic sheets. Deoxyribonucleotides were separated by ascending chromatography for 12-l 6 hr using a solvent composed of ethanol, sat-

AND COSTA

urated sodium tetraborate, 5 M ammonium acetate, pH 9.8, and 250 mM EDTA(220:80:20:1). The position of unlabeled markers was determined by uv illumination and the spots containing labeled deoxyribonucleotide were cut from the chromatograms and counted by liquid scintillation. One unit (U) is defined as the amount of ribonucleotide reductase that catalyzes the reduction of 1 nmol CDP per min (Engstrbm et a/., 1979). Partial purification

of ribonucleotide

reductase

Cytoplasmic extracts (1 ml) from infected or uninfected cells were precipitated with lo/o streptomycin sulfate at room temperature for 20 min with stirring. The pellets were removed by centrifugation at 20,000 g. Solid ammonium sulfate (209 mg) was slowly added to the supernatants with stirring to a final concentration of 35% saturation. After 30 min incubation with stirring, the precipitates were centrifuged as above and resuspended in 100 ~1 100 mM Tris-HCI, pH 7.8. These samples were then applied to a column of DEAE-cellulose (1 X 10 cm). After extensive washing with 100 mM Tris-HCI, pH 7.8, adsorbed proteins were eluted at 5 ml/hr with a 50 to 500 mM NaCl linear gradient in the same buffer. Fractions of 500 ~1 were collected and assayed for protein concentration and for ribonucleotide reductase activity. Fractions containing enzyme activity were pooled. Synthesis

of viral DNA

Vero cell monolayers in 35-mm plastic dishes were infected at 1O-20 PFU per cell or mock-infected. After adsorption for 90 min, the inoculum was replaced by DME containing 2% dialyzed newborn calf serum and different concentrations of hydroxyurea. At different times after infection, the cells were labeled for 30 min with 20 &i/ml methyl-[3H]thymidine (Amersham, 47 Ci/mmol) and cytoplasmic extracts were prepared as described above. Samples of the extracts were applied to glass fiber filters, precipitated with cold 10% trichloroacetic acid, washed with ethanol, and counted by liquid scintillation. Isolation

of hydroxyurea-resistant

viral mutants

Vero cells were infected with ASFV at 0.2 PFU per cell in the presence of 1 mM hydroxyurea. After 48 hr, the medium was collected, diluted fivefold, and added to fresh, uninfected cells. Five successive blind passages were done in the presence of 5 mM hydroxyurea. The virus was then titrated and expanded in the presence of the inhibitor. The variant virus stocks were plated under an agar overlay containing 10 mM hy-

ASFV-INDUCED

RIBONUCLEOTIDE

REDUCTASE

tions equal to or higher than 2.5 mM resulted in a decrease in the virus titer of at least 3.5 log. As expected, the effect of hydroxyurea was at the level of DNA replication. Acid-precipitable incorporation of labeled thymidine in the cytoplasm of cells infected in the presence of 5 mM hydroxyurea was eightfold less than that of cells infected in the absence of the inhibitor (Fig. 1).

6

ASFV induces infected cells o-o 0

2.5

5

7.5

10

Hydroxyurea, mM FIG. 1. Effect of hydroxyurea on the replication of wild-type ASFV (solid circles) and hydroxyurea-resistant mutants HUR-4 (solid squares) and HUR-21 (solid diamonds). Vero cell monolayers were infected at 1 PFU per cell in the presence of different hydroxyurea concentrations. Extracellular virus was harvested at 48 hr p.i. and titrated. The effect of hydroxyurea on wild-type ASFV DNA replication was determined by counting the acid-insoluble radioactivity in the cytoplasmic fraction from infected cells labeled with [3H]thymidine (open circles).

droxyurea and 49 well-isolated and expanded. Hybridization

75

with oligonucleotide

plaques were picked

probes

Degenerate oligonucleotide probes corresponding to conserved regions of the two subunits of viral and eukaryotic ribonucleotide reductases were synthesized and 5’-end-labeled with [T-~‘P]ATP (Sambrook et al., 1989). The probe sequences for the large subunit gene (Ml) and for the small subunit gene (M2) were respectively (A/G)TA(AIG)TACAT(AK/G/T)CC(AKYG/ T)GT(C/T)TT and (Crr)TC(A/G)AA(A/G)AA(A/G)rr(A/C/ G/T)GT (Tengelsen eta/., 1988; Slabaugh et al., 1988). Viral DNA was extracted from purified virus as described by Caeiro et al. (1990). Genomic ASFV DNA or cloned fragments were digested with restriction enzymes, separated by agarose gel electrophoresis, and analyzed by Southern blot hybridization according to standard techniques (Sambrook et a/., 1989).

ribonucleotide

activity

in

Ribonucleotide reductase activity was considerably increased in Vero cells after infection with ASFV. In different experiments with total cell extracts, increases in enzyme activity ranged from 2.6- to 5.8-fold. The difference was more pronounced when cytoplasmic extracts were used. The activity in nuclear fractions from infected and uninfected cells was practically the same, representing one-third of the total enzyme activity in uninfected cells (average, 0.04 U/mg cell protein). Most of the induced activity in infected cells was thus present in the cytoplasm and was about 8- to IO-fold higher than in the cytoplasm of uninfected cells. The virosomal fraction was not clearly enriched in ribonucleotide reductase as only 16.3% of the cytoplasmic activity in infected cells sedimented with the virosomal fraction. The enzyme activity in purified virus was very low, about 0.2 pmol/min/mg, indicating that the enzyme is probably not encapsidated into the virions. As shown in Fig. 2, the induction of ribonucleotide reductase activity was detected after 2 hr of synchro-

0

RESULTS

reductase

4

0

12

hr p.i. ASFV replication reductase

is dependent

on ribonucleotide

As a first approach to study the role of ribonucleotide reductase in ASFV replication, we analyzed the effect of hydroxyurea on ASFV production. As shown in Fig. 1, hydroxyurea inhibited viral replication. Concentra-

FIG. 2. Time course of induction of ribonucleotide reductase activity by ASFV. Cells were infected at 20 PFU per cell. Cytoplasmic extracts were prepared and assayed for ribonucleotide reductase activity at the indicated times after infection (solid circles). Parallel experiments were performed with cells infected in the presence of 100 wg/ml cytosine arabinoside (open circles) or 200 pglml phosphonoacetic acid (open squares).

76

CUNHA

AND COSTA

TABLE 1 EFFECTOF VIRUS DOSE AND OF MODIFICATIONSOF THE REACTIONMIXTURE ON RIBONUCLEOTIDEREDUCTASEACTIVITY Infected Activity (UX 102/mg)a

Uninfected Activity %b

(U

x 10Ymg)

%b

Virus dose (PFU per cell) 0 1 5 20

21 .o 31 .o 38.1

Modifications pH: 6.5 7.0 7.5 7.8 8.0 8.5

8.0 22.8 25.7 38.3 34.6 23.2

21 60 67 100 91 61

0.7 3.2 4.5 5.8 5.1 5.1

12 55 78 100 88 88

Mg2+ OmM 1 mM 2.5 mM 5mM 10mM

40.2 26.2 25.0 18.4 18.0

100 65 62 46 45

3.7 5.1 5.7 3.7 2.4

65 89 100 65 42

AMP-PNP OmM 2.5 mM 5mM 10mM

36.1 21.1 16.7 12.0

100 58 46 33

2.3 3.6 3.4 2.8

63 100 94 78

Standard mixture -NaF -Fe& -dithiothreitol +l mM EDTA +l mM hydroxyurea + 10 mM hydroxyurea + 2 mM pyridoxal phosphatee

37.2 32.4 10.3 8.7 8.6 12.6 3.1 4.5

100 87 28 23 23 34 8 12

3.5 3.4 1.2 1.3 0.7 1.5 1.0 0.8

100 96 36 38 20 42 28 16

3.8

of the reaction mixtured

a One unit (U) is defined as the amount of ribonucleotide reductase that catalyzes the reduction of 1 nmol CDP per min. b Enzyme activities are expressed as the percentage of activity relative to the standard assay, as described under Materials and Methods. ’ Cells were harvested at 8 hr p.i. d Cells were infected with 20 PFU per cell and harvested at 8 hr p.i. e Extracts were incubated on ice for 40 min with 2 mM pyridoxal phosphate. After addition of 1.5 mg/ml NaBH, in 20 mMTris-HCI, pH 7.8, the extracts were further incubated for 1 hr and dialyzed for 2 hr against 100 mM Tris-HCI, pH 7.8.

nous infection, reached a maximum at 6 to 8 hr p.i., and then started to decline. This time course is similar to that of viral DNA replication. No differences in induction of ribonucleotide reductase activity were observed when cells were infected in the presence of cytosine arabinoside or phosphonoacetic acid, an inhibitor of ASFV DNA oolvmerase. The induction of ribonucleotide reductase activity was dependent on the multiplicity of infection (Table 1).

We could detect an induction of enzyme activity with m.o.i. as low as 1 PFU per cell. Maximum induction was obtained when the cells were infected with m.o.i. of 20 PFU per cell or higher. We routinely used throughout this work a m.o.i. of 10 to 20 PFU per cell. Effects of modifications

of the reaction

conditions

Some characteristics of the virus-induced and of the cellular enzyme were compared by testing the effect of

ASFV-INDUCED

RIBONUCLEOTIDE

77

REDUCTASE

TABLE 2 PARTIALPURIFICATIONOF ASFV-SPECIFIC AND UNINFECTEDCELL RIBONUCLEOTIDEREDUCTASES Mock

ASFV

Protein b-w) Extract Streptomycin sulfate precipitate supernatant Ammonium sulfate supernatant precipitate DEAE-cellulose

Activity (U x 103)

Specific Activity (U X 103/mg)

Protein hs)

Activity (U x 103)

Specific activity (U X 10Ymg) 195

1.2

966

805

1.2

234

0.84

422 767

913

0.78

11 172

22

0.1 1 0.0035

107 609 220

5536 62857

0.095 0.008

37 126 60

1326 7500

several modifications in the reaction mixture (Table 1). Cytoplasmic extracts were used in all the experiments. The dependence on pH was determined using 100 mM Tris-HCI buffers of different pH. Both the virus-induced and the cellular enzymes had a pH optimum of 7.8. The activity of extracts from mock-infected cells was stimulated 1.5-fold by 2.5 mM Mg2+. Higher concentrations did not result in an increase in enzymatic activity as compared to assays in the absence of the ion. In contrast, the virus-induced enzyme was inhibited by Mg2+ at all tested concentrations. Omission of NaF, which is often included in ribonucleotide reductase assays as an inhibitor of phosphatases, slightly affected both the virus-induced and the cellular enzyme. Absence of FeCI, or dithiothreitol resulted in a considerable loss of activity, which indicates that ASFV-induced ribonucleotide reductase belongs to the iron-tyrosyl class of ribonucleotide reductases like the cellular enzyme. EDTA also caused an inhibition of about 80% of both enzymatic activities, which is a further indication of the role of metal ions in the catalytic reaction. Hydroxyurea had a more marked inhibitory effect on the virus-induced enzyme than on the cellular enzyme. Both activities were inhibited by pyridoxal phosphate, an inhibitor of ribonucleotide reductase that interacts with the regulatory subunit Ml of the enzyme (Gory and Mansell, 1975). Partial purification of ribonucleotide from infected and uninfected cells

reductases

Partial purification of ribonucleotide reductases from infected and uninfected cells is summarized in Table 2. Cytoplasmic extracts from 2 X 1O7cells yielded 1.2 mg of total protein. Specific activities were 0.805 U/mg in the case of infected cells and 0.195 U/mg in the case

of uninfected cells. Fractionation by precipitation with streptomycin sulfate, as used by other authors (EngStrom et al., 1979; Slabaugh and Mathews, 1986), did not result in a significant increase in specific activity. Differently, ammonium sulfate precipitates were enriched sevenfold in enzyme activity. Fractionation by ion exchange chromatography on DEAE-cellulose resulted in considerable further increases in specific activity. These were a 78-fold increase in the case of the virus-induced enzyme and 39-fold in the case of the cellular enzyme. This difference was partially due to the lower protein concentration of active fractions from infected cells, as viral and cellular enzymes were eluted at different salt concentrations. The major peak of ASFV-specific ribonucleotide reductase eluted at 430 mM NaCI, whereas the enzyme from uninfected cells and a minor peak from infected cells eluted at 340 mM NaCI. Final recoveries of virus-specific and endogenous enzymes were 22.8 and 25.6%, respectively. Kinetics

of the reaction

The effect of substrate concentration on the kinetics of CDP reduction was different for the virus-induced enzyme and for the endogenous enzyme, as shown by the Lineweaver-Burk plot depicted in Fig. 3. The K,,, determined for the purified viral enzyme was 7.5 &“. In agreement with published values (Eriksson et a/., 1979), the Kmdetermined for the purified endogenous enzyme was 30 pCLM. Effect of ribonucleotides and deoxyribonucleotides on the endogenous and induced ribonucleotide reductase activities The activity and substrate specificity of E. co/i and mammalian ribonucleotide reductases are controlled

78

CUNHA

I 01 0

0.04

0.08

0.12

0.16

0.20

l/S FIG. 3. Determination of the K,,, for CDP of partially purified ribonucleotide reductase from infected cells (solid circles) and from uninfected cells (open circles). Substrate concentrations are expressed as pM and the velocity of the reaction as nmol/mg/min.

by allosteric regulators. ATP is usually the activator of the enzyme and deoxyribonucleoside triphosphates are negative regulators (Thelander and Reichard, 1979). We studied the effects of these ,regulators on the activity of ASFV-induced and uninfected cell ribonucleotide reductase. As shown in Fig. 4A, increasing concentrations of ATP caused a marked progressive inhibition of CDP reduction by both infected and uninfected cell extracts. However, 2.5 mM ATP was required for maximum activity of the cellular enzyme when purified enzyme was used instead of crude cytoplasmic extracts. Higher concentrations of ATP, above 6 mM, caused only a slight inhibition of the purified cellular enzyme as compared to the activity in the absence of ATP. Similarly to the effect on cytoplasmic extracts from infected cells, ATP caused a decrease in the activityof partially purified viral enzyme, although this inhibition was not as marked as the inhibition of enzyme activity in cytoplasmic extracts. The noncleavable ATP analog AMP-PNP was effective as activator of the cellular ribonucleotide reductase, even when cell extracts were used. In contrast, AMP-PNP inhibited the activity of ASFV-induced ribonucleotide reductase (Table 1). The differences observed in experiments using cell extracts and partially purified enzymes could be due to side reactions that redirect the substrate into alternative pathways, namely, dephosphorylation and phosphorylation reactions. We quantitated the proportion of added CDP that migrated at different times after incubation as CMP, CDP, and CTP. For this purpose a different chromatographic procedure on polyethyleneimine-cellulose was used (Randerath and Randerath, 1967; Slabaugh and Mathews, 1984). This method separated nucleoside mono., di., a-nd triphosphates

AND COSTA

very distinctly, but did not allow a good resolution of ribonucleotides from deoxyribonucleotides. As shown in Fig. 5, much [3H]CDP added to cytoplasmic extracts was dephosphorylated to CMP or dCMP. The proportion of nucleoside monophosphate in the mixture containing infected cell extract (61%) was considerably higher than that in the mixture containing uninfected cell extract (34%). The proportion of labeled CMP or dCMP in the mixtures containing purified viral or cellular enzyme was lower, 31 and 220/o, respectively. Only 4 to 6% of total labeled nucleotide could be separated as CTP or dCTP. The phosphorylation reaction in the standard reaction mixtures was therefore not significant. In contrast, the addition of ATP greatly favored the phosphorylation pathway. This effect was more intense in the case of uninfected cell extracts, in wjhich 56% of the label was detected in CTP. The equivalent proportion in infected cell extracts was 42%. Phosphorylation of the substrate after the addition of ATP to reaction mixtures containing purified enzymes was less important than in the case of cytoplasmic extracts. Triphosphates represented 22 and 26% of total labeled nucleotide after incubation with purified viral and cellular enzymes, respectively. We also analyzed the effect of deoxyribonucleotides, which are negative regulators of mammalian ribonucleotide reductases. Increasing concentrations of dATP or dTTP caused a progressive inhibition of the activity of both virus-induced and cellular enzymes (Fig. 4B). The degree of inhibition of the cellular enzyme activity was almost the same for dATP and dITP but in the

E- 100 i '-

80

' z

60

$.

40

). .7 ‘i;

20

2 2

4

6

610

ATP, mM

12

3

4

5

dNTP, mM

FIG. 4. Effects of nucleotides on endogenous and virus-induced ribonucleotide reductase. Cells were infected with ASFV at 20 PFU per cell or mock-infected and extracted at 8 hr p.i. (A) Cytoplasmic extracts (open symbols) or partially purified enzyme (solid symbols) from infected (circles) or mock-infected (squares) cells were assayed for ribonucleotide reductase activity in the presence of different concentrations of ATP. (B) Effect of dATP (circles) and dTTP (squares) on ribonucleotide reductase activity of cytoplasmic extracts from infected (solid symbols) or uninfected cells (open symbols).

ASFV-INDUCED

RIBONUCLEOTIDE

% 80 60 40 20 %

80 60 40 20

no addition Eci CMP

+ ATP

+ ATP no addition

0 CDP

0 CTP

FIG. 5. Redirection of the substrate into phosphorylation or dephosphorylation pathways, Ribonucleotide reductase reaction mixtures containing cytoplasmic extracts (A, C) or partially purified enzyme (B, D) from infected (A, B) or uninfected cells (C, D) were incubated for 30 min at 37”. Aliquots of 5 ~1 were added to 10 ~1 of cold 10% trichloroacetic acid. After 15 min on ice, the precipitate was removed by centrifugation and the samples were spotted onto plastic-backed polyethyleneimine-cellulose thin-layer chromatographic plates. Chromatograms were developed for about 1 hrwith 1 M LiCI. Spots containing CMP, CDP, and CTP were located by uv illumination of markers, cut, and counted by liquid scintillation. Parallel experiments were performed with extracts to which 5 mM ATP were added. Bars represent the distribution of the radioactivity in phosphorylated cytidine nucleotides.

case of ASFV-induced ribonucleotide reductase dTTP caused a considerably more pronounced inhibition than dATP. Isolation

of hydroxyurea-resistant

79

REDUCTASE

Two plaque-purified mutants, HUR-4 and HUR-21, were selected for further characterization. In different successive passages in the presence of 5 mM hydroxyurea, the infection with these mutants resulted in yields of 3 to 5 x lo7 PFU per loo-mm dish. These yields are very similar to normal wild-type yields in the absence of the inhibitor. Titers obtained in the presence of different concentrations of hydroxyurea, up to 10 mM, were similar to those obtained in the absence of the inhibitor (Fig. 1). Plaquing efficiency was always close to 1, with minor variations from passage to passage. The resistance to hydroxyurea could be due to a mutational alteration of the enzyme rendering its active site less susceptible to inactivation by the drug, or to overproduction of ribonucleotide reductase. The assay of ribonucleotide reductase activity during the course of infection with wild-type or mutant virus showed that HUR-4 and HUR-21 induced two or three times more enzyme than wild-type virus (Fig. 6). The presence of hydroxyurea during infection did not affect the level of enzyme production or the kinetics of its synthesis. The effects of increasing concentrations of hydroxyurea in vitro on wild-type and mutant virus-induced enzymes were very similar (Fig. 7). This seems to exclude the alternative mechanism of mutational alteration of the enzyme. Mapping the gene for the small subunit specific ribonucleotide reductase

of ASFV-

Highly conserved aminoacid sequences can be determined from the known sequences of the genes for

viral mutants

Hydroxyurea-resistant mutants were isolated after five passages of ASFV in the presence of 5 mM hydroxyurea. At the end of this series of blind passages, total cytopathic effect could be observed at the same time, approximately 48 hr, as in the case of cells infected in the absence of hydroxyurea. The virus titer after the last blind passage was 5 X lo5 PFU/ml, whereas the titer of wild-type virus after one passage in the presence of hydroxyurea was lower than 1O3 PFU/ ml. Plaquing efficiency is defined as the ratio between the number of plaques formed in the presence and in the absence of hydroxyurea. This ratio gives a more direct indication of the proportion of mutant virus in the total virus population. After the series of blind passages in the presence of hydroxyurea, the plaquing efficiency of the resistant virus was 0.8, whereas the plaquing efficiency of wild-type virus was lower than 0.001.

0.8

A

4B

0 0

3

6

9

12 0

3

6

9

12

hr p.i. FIG. 6. Induction of ribonucleotide reductase activity by wild-type ASFV (open circles) and by hydroxyurea-resistant mutants HUR-4 (solid circles) and HUR-21 (solid squares). Cells were infected at 10 PFU per cell in the presence (A) or absence (B) of 5 mM hydroxyurea. At different times after infection, cytoplasmic extracts were prepared and assayed for enzyme activity.

80

CUNHA

g

AND COSTA

two HindIll fragments of 3.4 and 2.6 kb (Fig. 8). Two other faint bands of 1.9 and 1.3 kb were also detected in /-/indIll digests of HUR-21 DNA. Considering the size of the probe, these results seem to indicate that the gene is at least duplicated in the mutant genome.

80

.-;

DISCUSSION

01

0

2

4

6

8

10

Hydroxyurea, mM FIG. 7. In vitro effect of hydroxyurea on ribonucleotide reductases induced by wild-type (open circles), HUR-4 mutant (solid circles), and HUR-21 mutant virus (solid squares). Cells were infected at 20 PFU per cell and extracted at 8 hr p.i. Cytoplasmic extracts were assayed in the presence of different concentrations of hydroxyurea.

both subunits of eukaryotic and viral ribonucleotide reductases (Slabaugh et a/., 1988; Tengelsen et al., 1988). These aminoacid sequences are Lys-Thr-GlyMet-Tyr-Tyr near the carboxy-terminus of the large subunit (Ml) and Thr-Asn-Phe-Phe-Glu near the carboxy-terminus of the small subunit (MZ). We used degenerate oligonucleotide probes to detect the corresponding DNA sequences in the genome of ASFV and in recombinants of ASFV DNA. No hybridization was detected with the Ml probe, even under low-stringency conditions. The M2 probe hybridized under stringent conditions (Itakura et al., 1984) to the 29.6-kb EcoRI-A fragment of ASFV DNA and to the 6.8-kb HindIll-B fragment, which is contained in the EcoRl fragment (Fig. 8). This result was confirmed by dot blot hybridization with a collection of X EMBL3 recombinants covering the viral genome (G. Ribeiro and J. V. Costa, unpublished results). Only one recombinant, XGR26, hybridized with the M2 probe (data not shown). This recombinant has a 15.9-kb insert that overlaps the right part of the EcoRI-A fragment. The recombinant plasmid pGMS, prepared by cloning the HindIll-B fragment in vector pTZ19R, was used to map the putative small subunit gene within the HindIll-B fragment (Fig. 8). Single or double digestions with Sac1 and Pstl mapped the gene to a 2.3-kb fragment in the middle of the HindIll-B fragment. HUR-21 mutant DNA cleaved with EcoRl or HindIll was also subjected to Southern blot hybridization with the probe for the small subunit gene. In both cases, the probe hybridized to more than one viral DNA fragment: four EcoRl fragments of 6.3, 2.9, 2.3, and 0.4 kb, and

Ribonucleotide reductase is a critical enzyme for DNA metabolism. It provides the only route for de nova synthesis of deoxyribonucleotide precursors, which is the first specific step in DNA synthesis. Ribonucleotide reductase is closely coupled to cell DNA replication and is absent or inactive in resting cells (Nordenskjold et a/., 1970). To circumvent this constraint, herpesviruses and poxviruses induce the synthesis of their own ribonucleotide reductases (Cohen, 1972; Henry et al., 1978; Lankinen et al., 1982; Slabaugh et al., 1984). The genes for herpesvirus and vaccinia virus ribonucleotide reductases have been identified and sequenced

A

ASFV -EH

HUR EH

B

FIG. 8. Mapping the gene forthe small subunit of ASFV ribonucleotide reductase using an oligonucleotide probe. (A) Southern blot hybridization of ASFV and HUR-21 DNA cleaved with EcoRl (R) or HindIll (H), and of recombinant plasmid pGMS after single or double digestion with Hindlll (H), Sacl (S), or Pstl (P). Size markers are fragments of X DNA cleaved with HindIll. (B) Diagram of the hybridization data.

ASFV-INDUCED

RIBONUCLEOTIDE

(McLauchlan and Clements, 1983; Slabaugh el a/., 1988; Tengelsen et al., 1988). In this paper we repot-t the identification of a novel ribonucleotide reductase in cells infected with ASFV. The induction of enzyme activity correlated with the multiplicity of infection and had a time course with a peak at 6-8 hr p.i. Induction occurred also in the presence of inhibitors of DNA synthesis. These results indicate that the induction of ribonucleotide reductase activity is an early viral function. Some ASFV early proteins accumulate late in infection when DNA synthesis is blocked. Other early proteins are down-regulated independently of viral DNA synthesis and are not observed late in infection of blocked cells (Esteves et a/., 1986). ASFV-induced ribonucleotide reductase belongs to the second class of early proteins, since inhibition of DNA synthesis did not cause superinduction of the enzyme activity. The enzyme is not encapsidated into the virions. This is consistent with the minimal requirement for encapsidated enzymes. The only enzymes that must be carried by the infecting virus particles are those involved in RNA synthesis and processing, which are required for early virus gene transcription. The fact that the induction of ribonucleotide reductase activity is an early viral function does not necessarily imply that the enzyme is virus-coded. ASFV has a large genome of about 180 kbp and induces the synthesis of more than 100 virus-specific proteins in infected cells (Esteves et a/., 1986). A viral gene product could be responsible for the derepression of the cell enzyme expression. However, this hypothesis is not supported by our observations. The virus-induced enzyme and the cellular enzyme respond differently to several modifications in the reaction conditions and to allosteric effecters of CDP reduction, have considerably different K,.,,for CDP reduction, and are eluted at different salt concentrations in ion exchange chromatography. The cell independence of ASFV for enzymes involved in DNA metabolism (Costa, 1990) and the similarities in biochemical strategy with poxviruses give indirect support to the hypothesis that ASFV-induced ribonucleotide reductase is indeed a direct viral gene product. This assumption is strengthened by the isolation of hydroxyurea-resistant mutants that induce the overproduction of ribonucleotide reductase. More compelling evidence for the viral origin of the induced enzyme was provided by the detection in the viral genome of sequences homologous to a conserved region of the small subunit of several eukaryotic and viral ribonucleotide reductases. The recombinant plasmid pGMS that hybridized with the M2 probe will

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be sequenced to characterize the gene for the small subunit of ASFV ribonucleotide reductase. We were unable to detect the gene for the large subunit using an oligonucleotide probe for a conserved sequence. It is possible that the virus codes only for the small subunit, which would then form a functional enzyme by complexing with the cellular large subunit. If this is true, one would not expect modifications in allosteric regulation, because allosteric sites are located in the large subunit (Thelander and Reichard, 1979). The differences in response to the allosteric effector ATP between the virus-induced and the endogenous enzymes seem to indicate that ASFV also specifies a large subunit. The lack of detection of its gene by the M 1 probe suggests that the ASFV gene for the large subunit differs from other viral enzymes in the conserved sequence. The large EcoRI-A fragment of ASFV DNA was not observed in DNA digests from one of the hydroxyurearesistant mutants. The fragment was split into smaller fragments and each of these fragments hybridized with the oligonucleotide probe for the small subunit gene. So, the overproduction of ribonucleotide reductase by the mutants is probably due to gene amplification, as described for hydroxyurea-resistant vaccinia virus mutants (Slabaugh and Mathews, 1986). We are further analyzing the mutant DNA to characterize the structure of the mutant gene. E. co/i and eukaryotic cell ribonucleotide reductases are tightly regulated by allosteric effecters. The purified enzyme is strictly dependent on ATP for activation (Larsson and Reichart, 1966; Eriksson el a/., 1979). As previously described (Kucera and Paulus, 1982), we observed that ATP was inhibitory for the endogenous enzyme when crude cell extracts were used instead of purified enzyme. This is probably due to the utilization of the substrate by a strong phosphorylating activity of the cell extracts in the presence of ATP. Slabaugh and Mathews (1984) could eliminate the effect of the side reaction by using as an activator a noncleavable ATP analog, adenylylimido-diphosphate (AMP-PNP). These authors also observed that AMP-PNP stimulates the vaccinia virus-specific ribonucleotide reductase. We confirmed the stimulation of the cellular enzyme activity by AMP-PNP and used this activator routinely in all assays for the cellular enzyme. In contrast, and differently from vaccinia virus, AMP-PNP inhibited ASFV-induced ribonucleotide reductase activity. ASFV-induced ribonucleotide reductase activity was not dependent on activation by ATP and was even depressed by ATP. This effect was also observed with partially purified enzyme preparations, under conditions that enabled the detection of activation of the cellular enzyme by ATP. In this sense, ASFV-induced

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ribonucleotide reductase looks more similar to the herpesvirus and bacteriophage T4 enzymes, which do not require ATP as activator (Berglund, 1972; Averett et al,, 1983). Herpesvirus and bacteriophage T4 ribonucleotide reductases differ also from their cellular counterparts in that they are not negatively regulated (Berglund, 1972; Langelier et al., 1978), whereas the cellular enzymes are inhibited by deoxyribonucleoside triphosphates (Thelander and Reichart, 1979). The reduction of CDP by vaccinia virus-induced ribonucleotide reductase is also inhibited by dATP and by dlTP (Slabaugh and Mathews, 1984). This was also observed in the case of ASFV-induced ribonucleotide reductase. Deoxyguanosine triphosphate also inhibited the reduction of CDP (data not shown). The reduction of each of the four ribonucleoside diphosphates by cellular ribonucleotide reductases is differently regulated by deoxyribonucleoside triphosphates (Eriksson et al., 1979). In this work we studied only the reduction of CDP by ASFV-induced ribonucleotide reductase. A complete analysis for all four ribonucleoside diphosphates by ASFV is intended. ACKNOWLEDGMENTS We are grateful to M. Adelaide Figueira for her advice on the chromatographic techniques and to Zilda Carvalho for reviewing the manuscript. Plasmid pGMS was prepared by Margarida Meireles.

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