Replication of equine herpesvirus type I: Resistance to hydroxyurea

VIROLDGY

67,56-67 (1975)

Replication

of Equine Herpesvirus

Type I: Resistance

to

Hydroxyurea JAMES

Department

C. COHEN,’

MICHAEL DENNIS

of Microbiology,

University

L. PERDUE,’ CHARLES J. O’CALLAGHAN of Mississippi

Accepted

April

Medical

Center,

C. RANDALL,

Jackson,

Mississippi

AND

39216

10, 1975

Hydroxyurea (HU) at concentrations that rapidly and completely inhibit mammalian cellular DNA synthesis does not prevent either equine herpesvirus type 1 (EHV-1) DNA synthesis or virus replication. Analysis by CsCl isopycnic centrifugation of DNA synthesized in HU treated, EHV-1 infected L-M cell cultures demonstrated the synthesis of only viral DNA, whereas both cellular and viral DNA were synthesized in uninhibited, infected cultures. With regard to the mechanism of HU-resistance of EHV-1 DNA synthesis, these studies showed the following: (1) Selective degradation of cellular DNA and increased nuclease activity to provide deoxyribonucleotides for viral DNA synthesis were not induced by infection and/or the inhibitor, (2) HU did not selectively inhibit cellular DNA polymerase activity as both cellular and viral DNA polymerase activities were present in HU-treated, infected cells and were not inhibited by HU added to the in vitro enzyme assay, (3) Cycloheximide inhibition of protein synthesis revealed the requirement of a protein(s) other than viral polymerase at 4 hr postinfection for viral DNA synthesis in the presence of HU. These results do not rule out the possibility that a HU-resistant, viral-induced ribonucleotide reductase activity may be responsible for the HU-resistance of this herpesvirus.

exhibit an altered ribonucleotide reductase activity. Several other modes of action, however, have been proposed to explain the inhibitory effect of HU (Yarbro, 1968; Volger et at., 1966; Rosenkranz et al., 1969). Plagemann and Erbe (1974) recently suggested that HU might deplete intracellular pools of purine deoxyribonucleotides by a mechanism other than inhibition of nucleotide reductase for they detected increased levels of pyrimidine deoxyribonucleotides (TTP and dCTP) in hydroxyurea-treated cells. Skoog and Bjursell (1974) also found increased levels of TTP in HU inhibited cells suggesting that a general depletion of intranuclear deoxyribonucleotide pools did not occur in HU inhibited cells. Hydroxyurea has been shown to inhibit completely DNA synthesis of several animal viruses including poxviruses (de Sousa, 1965; Rosenkranz, 1966), adenoviruses

INTRODUCTION

Hydroxyurea, a cancer chemotherapeutic agent which appears to be a specific inhibitor of DNA synthesis, has been widely used to synchronize cell cultures and to investigate the biochemistry of DNA-containing viruses. The mechanism(s) by which HU rapidly inhibits mammalian cellular DNA synthesis is not fully understood. Several studies have shown that HU blocks the reduction of ribonucleotides to deoxyribonucleotides and inhibits nucleotide reductase activity in in oitro assays (Young and Hodas, 1964; Adams and Lindsay, 1967; Young et al., 1967; Krakoff et al., 1968; Moore, 1969). In support of this evidence, Lewis and Wright (1974) recently reported that five mammalian cell lines resistant to hydroxyurea, ’ National Institute of Allergy and Infectious Diseases Predoctoral Trainee (AI-69). 56 Copyright@ All rights

1975 by Academic Press,Inc. of reproduction in any form reserved.

HERPESVIRUS

HU-RESISTANT

(Levy et al., 1968; Sussenbach and Van Der Vliet, 1973), and herpes simplex virus (Nii et al., 1968; Wagner et al., 1972). Epstein-Barr viral DNA synthesis, however, was reported to be resistant to hydroxyurea (Hampar et al., 1972), and this was confirmed recently by Mele et al. (1974). In this paper, we demonstrate that DNA synthesis of another herpesvirus, EHV-1, is resistant to HU at concentrations that rapidly and completely inhibit cellular DNA synthesis and that other viral-specific functions-induction of viral DNA polymerase activity and production of infectious virions-are not prevented by large concentrations of HU. In addition, we present studies which rule out the role of virus-induced and/or HU-induced degradation of cellular DNA as a rescue mechanism for resistance of EHV DNA synthesis to hydroxyurea inhibition. MATERIALS

AND

METHODS

Cell Cultures

L-M mouse fibroblast cells were cultured in suspension in YeLP medium supplemented with 3% fetal bovine serum (Microbiological Associates, Inc., Bethesda, Maryland) as previously described (O’Callaghan et al., 1968a,b). Virus infection and radiolabeling of cellular fractions with radioactive precursors were carried out in suspension culture using Eagle’s minimum essential medium (MEM; Grand Island Biol. Corp., Grand Island, New York). Virus Propagation

and Assay

Equine herpesvirus type I (EHV-l), Kentucky A strain, as adapted to L-M cells by Randall and Lawson (1962) was employed in these investigations. The conditions for virus propagation and purification were those described previously (Perdue et al., 1974, 1975). Released virus was titered by the plaque method of Perdue et al. (1974). Virus passages and cell cultures were checked routinely for Mycoplasma contamination by the culture methods described previously (Stock and Gentry, 1969). Inhibitors

Hydroxyurea

(HU,

Sigma

Chemical

57

REPLICATION

Corp., St. Louis, Missouri) was added as a concentrated solution in distilled water (dH,O) to the final concentration specified in each experiment. Cycloheximide (Sigma Chemical Company, St. Louis, Missouri) in dH,O was added to achieve a final concentration of 50 pg/ml. Radioisotopic

Labeling

and Assay

The DNA of EHV-1 infected and mockinfected cultures (maintained in MEM) was labeled, utilizing [5- 3H]thymidine ( [3H]TdR; 50.8 CilmM; New England Nuclear Corp., Boston Mass.) at a concentration of 1 to 10 r.tCi/ml for varying times, depending on the experiment. Determination of incorporation of radiolabeled precursor into the acid-insoluble fraction was accomplished by trichloroacetic acid (TCA) precipitation. Briefly, aliquots of cells were collected by centrifugation and washed with cold phosphate buffered saline (0.001 M phosphate pH 7.4; 0.15 M NaCl); the cells were resuspended in cold dH,O and acid insoluble material was precipitated with cold 10% TCA. The precipitate was collected by centrifugation and washed again with 10% TCA; the supernatants containing the acid soluble fractions were pooled, while the precipitate was dissolved in 0.11 N NaOH. Aliquots of the dissolved precipitate and of the supernatants were added to Aquasol (New England Nuclear Corp., Boston, Mass.) scintillation cocktail and were assayed for radioactivity in a Beckman LS230 scintillation counter. CsCl Isopycnic

Centrifugation

EHV-1 infected or mock-infected nuclei were isolated by the method of Borun et al. (1967) using Nonidet P-40 (NP-40; Shell Oil Company, NY, NY) in isotonic buffer (0.01 M Tris, pH 7.4, 0.14 M NaCl, 0.0015 M MgCl,). Nuclei were collected by centrifugation and 1.2 x 10’ nuclei were resuspended in 7.5 ml SSC (standard saline citrate buffer; 0.15 M NaCl, 0.02 M Na citrate pH 7.4). Sarkosyl (Geigy Chemical Corp., Ardsley, NY) was added to a final concentration of l.O%, and the nuclei were lysed at room temperature for 30-60 min. Crystalline C&l (The Harshaw Chemical

58

COHEN

Company, Solon, Ohio) was added to a final density of 1.710 g/cc in 11.0 ml final volume. Separation of viral and cellular DNA was achieved by density gradient centrifugation in a SW41 rotor (Beckman Instrument Company, Palo Alto, California) at 30,000 rpm for 46 hr at 25”. Gradients punctured at the bottom were collected dropwise into 0.25 ml fractions. Samples were taken of each fraction and counted using Aquasol. ‘“C-TdR labeled Micrococcus lysodeihticus DNA (p = 1.731) was added as a density marker, and the densities of individual fractions were determined by measurement of the refractive index using an Abbe refractometer and by weighing. Enzyme Assays Cellular extracts for DNA polymerase and DNase assays were prepared from mock-infected or EHV-1 infected L-M cells by sonication (10 KC; 1 min) of 20 x lo6 cells/ml in 10 mM Tris-HCl pH 7.4 buffer containing 150 mM KC1 and 0.5 mM dithiothreitol. After sonication, the cellular debris was removed by centrifugation at 20,000 g for 30 min. The supernate from this centrifugation was collected and stored at -70” until assayed. Total protein in each extract was determined by the method of Lowry et al. (1951). Viral specific DNA polymerase activity was assayed for by the high salt method described by Weissbach et al. (1973). This method involves the addition of K,SO, (150 mM), which is required for viral polymerase activity, to the assay. The reaction mixture, total volume of 0.25 ml, consisted of 100 mM Tris-HCl, pH 8.0, 0.5 mM dithiothreitol (DTT), 2 mM MgC12, 0.45 mg bovine serum albumin (BSA; fraction V), 0.1 mM dATP, 0.1 mM dGTP, 0.1 mM dCTP (Calbiochem, La Jolla, California), 0.05 mM TTP, 150 mA4 K2S04, and 100 pg activated calf thymus DNA. Isotope ( [3H]TTP; 0.65 pM; 19.14 CilmM) was added to each reaction mixture and its incorporation into the acid insoluble fraction was determined by precipitation with 10% cold TCA. The precipitate collected on Whatman no. 3 filter discs was washed with liberal amounts of 5% TCA and was

ET AL

dried and counted by liquid scintillation. All assays were carried out at 37” for 30 min. The cellular (without K,SO,) DNA polymerase activity of infected and control cellular extracts was assayed for in a reaction mixture consisting of 50 mM Tris-HCl, pH 8.5, 0.5 mM DTT, 7.5 mM MgC12, 90 pg BSA, 0.1 mM dATP, 0.1 mM dGTP, 0.1 mM dCTP, 0.05 mM TTP, 0.65 pM [3H]TTP, and 100 pg activated calf thymus DNA. Final volume, sample size, and processing were identical to that for the viral polymerase assay. Calf thymus DNA (2.5 mg/ml; Sigma Chemical Company, St. Louis, Missouri) was activated by treatment with bovine pancreatic DNase (Sigma Chemical Company, St. Louis, Missouri) employed at a concentration of 40 ng/mg DNA; incubation was carried out for 5 min at 60” in 0.01 M Tris-HCl, pH 7.4, and 5 mM MgCl,. After incubation, the DNA was extracted with phenol and dialyzed against 10 mM Tris-HCl, pH 7.4. DNase activity was measured by a modification of the procedure of Weissbach et al. (1973). A portion of the crude cellular extract was assayed in 50 mM Tris-HCl, pH 8.5, 0.5 mM DTT, 7.5 mM MgC12, and 45 pg BSA. [3H]TdR labeled native (purified by the method of Soehner et al., 1965) or denatured (100’ H,O bath for 10 min) L-M cell DNA was added to the reaction mixture as substrate. The reaction mixture volume was 200 ~1 and an incubation time of 20 min at 37” was employed. At the end of the incubation, cold TCA was added to a final concentration of 10% to precipitate the acid insoluble material which was then removed by centrifugation at 1500 rpm for 15 min at 4’. The release of radiolabeled nucleotides into acid soluble material was used to quantitate DNase activity in each cellular extract. RESULTS

Effect of HUon DNA Synthesis in EHV-1 Infected Cells DNA synthesis in HU-treated and untreated, EHV-1 infected and mockinfected L-M cell suspension cultures was

HERPESVIRUS

HU-RESISTANT

measured by both continuous and pulse labeling with 13H]TdR. HU was added at the time of infection to a final concentration of 2.5 x 10m3 M. In continuous labeling experiments, [3H]TdR was added after virus attachment, at 2 hr postinfection. At regular intervals, aliquots of cells from each culture were collected and either pulse-labeled and processed for acid insoluble material with TCA, or in the case of continuous labeling experiments, samples were taken and processed immediately. The data in Fig. 1 illustrate the effect of HU on total DNA synthesis. HU inhibited greater than 98% of cellular DNA synthesis in uninfected cultures. In EHV-1 infected cultures, total DNA synthesis was completely inhibited through 4 hr PI; after 4 hr, a significant amount of HU-resistant DNA synthesis commenced. By 11 hr PI, total incorporation of [3H]TdR in inhibited, infected cultures was greater than seven times that of the inhibited, uninfected cultures. DNA synthesis resistant to HU was observed only in infected cultures and began at 4-5 hr PI in cultures to which HU was added either at the time of infection or prior to infection. The effect of HU on the rate of DNA synthesis was measured by pulse radiolabeling experiments (data not shown). The rate of DNA synthesis of HU treated, mock-infected cultures was reduced greater than 99% as compared to untreated mock-infected cultures. A significant increase in the rate of DNA synthesis beginning at 4 hr PI was observed in HU-treated, infected cultures. Addition of HU prior to infection had no effect on either the rate or the time of initiation of HU-resistant DNA synthesis in infected cultures. Previous work (O’Callaghan et al., 1968a, b) showed that EHV-1 DNA synthesis was initiated at approximately 4 hr PI, the time at which HU-resistant DNA synthesis commences. These results suggest that EHV-1 DNA synthesis is resistant to HU. CsCl Density

Gradient

Analysis

To confirm that HU selectively blocks the synthesis of cellular DNA in EHV-1 infected cells, the DNA synthesized in

REPLICATION

FIG. 1. Inhibition of [3H]TdR incorporation in mock-infected and EHV-1 infected L-M cells in the presence of 2.5 mM HU added at the time of infection. Replicate suspension cultures were radioactively labeled continuously from 2 hr PI with [3H]TdR (1 pCi/ml; 50.8 Ci/mM). Samples containing 15.0 x lo6 cells were collected at the specified times and were fractionated for TCA precipitable cpm as described in Materials and Methods. Duplicate aliquots of each sample were counted. Mock-infected, untreated, (A -A); Mock-infected, 2.5 x 10m3 M HU (A-A); Infected, untreated, (0-O); Infected, 2.5 x 10m3 A4 HU (04).

HU-treated, infected cultures was analyzed by density gradient centrifugation in CsCl. Untreated and HU-treated, infected cultures were pulse labeled with [3H]TdR at 7-9 hr postinfection, the time of maximal HU-resistant DNA synthesis and a time during infection when approximately

50% of the DNA being synthesized

is viral

(O’Callaghan et al., 1968b). After labeling, the cells were collected, and the viral and cellular DNA, released by lysis of isolated nuclei, were separated by isopycnic centrifugation in CsCl as described in Materials and Methods. The results of these analyses are shown in Fig. 2. In untreated, infected cultures radiolabeled DNA banded at densities of 1.716 g/cc and 1.690 g/cc, corresponding to those of EHV-1 DNA and L-M cell DNA, respectively. In HU-treated, infected cultures, however, virtually all [3H]TdR labeled DNA had a density of 1.716 g/cc, that of viral DNA. These results confirm those presented in Fig. 1 and demonstrate that equine herpes viral DNA synthesis is resistant to HU at a concentra-

60

COHEN

ET AL.

[3H ]TdR incorporation was evident prior to the initiation of viral DNA synthesis which occurs at 4-6 hr postinfection (O’Callaghan et al., 196813). At HU concentrations of 2.5 x 10e3 M or greater, DNA synthesis prior to 4-6 hr PI (cellular DNA) was negligible. At all concentrations of HU which completely inhibited cellular DNA synthesis (2.5 x 10m3 M or greater), DNA synthesis resistant to HU began at 4 hr postinfection and was maximal at 8-12 hr postinfection, the time of the greatest rate of EHV-1 DNA synthesis (O’Callaghan et al., 1968b). Results identical to these were observed when HU at these concentrations was added at times prior to infection. Thus, concentrations of HU at 2.5 x 1O-3 M or greater effectively inhibit cellular DNA synthesis but fail to inhibit the synthesis of EHV-1 DNA.

s_;:.[~~~~ 2 2

4

6

.3 IO 12 14 16 16 20 22 24 26 26 FRACTION NO.

FIG. 2. CsCl isopycnic gradient analysis of DNA synthesized in EHV-1 infected L-M cells. Cells infected at a multiplicity of 15 PFU/cell were labeled with [3H]TdR (1 rCi/ml; 50.8 Ci/mM) at 7-9 hr PI. DNA released by lysis of isolated nuclei with 1% Sarkosyl was separated into cellular and viral species by centrifugation in buffered CsCl, final average density 1.710 g/cc, for 46 hr at 30,000 rpm in an SW41 rotor, as described in Materials and Methods. [“C]TdR labeled M. ~,vsodeikticus DNA (p = 1.731 g/cc) was added as a marker, and the density of individual fractions was determined by measurement of the refractive index. (A) EHV-1 infected L-M cells, no HU (B) EHV-1 infected L-M cells treated with HU, 2.5 mM final concentration, at the time of infection.

tion (2.5 x 10e3 M) which inhibits all cellular DNA synthesis. Effect of HU Concentration Synthesis

Resistance

of EHV-1

Replication

to HU

The results shown in Fig. 3 demonstrate that EHV-1 DNA synthesis is resistant to

virtually

on Viral DNA

To determine the effect of various concentrations of HU on viral DNA synthesis, individual infected cultures were treated at the time of infection with HU at final concentrations of lo-” M, 5 x 10e3 M, 2.5 x 10M3 M, and 2.5 x 10m4 M. At various times after infection, samples were pulselabeled with [3H]TdR and processed as previously described for acid precipitable radioactivity. The results are shown in Fig. 3. As may be seen, all concentrations of HU reduced significantly the rate of total DNA synthesis in infected cultures as compared to that of untreated, infected cultures. It appeared that some host DNA synthesis occurred in cultures treated with the lowest concentration of HU, 2.5 x lo-’ M, for a significant, although reduced, level of

OJ

;

I

I

I

2

4

6

6

HOURS

1

IO

I

I

12 14

I

16

I

I

I

16 20 22

POST-INFECTION

FIG. 3. Effect of HU at various concentrations on DNA synthesis in EHV-1 infected L-M cells. The inhibitor was added at the time of infection. Infected cells were labeled for 30 min with [3H]TdR (1 &i/ml; 50.8 Ci/mM) at various times PI and processed for acid insoluble cpm as described in Materials and Methods. Untreated (O---O), 2.5 x lo-’ M HU (x-x), 2.5 x 1O-3 it4 HU (-1, 5 x 10m3 M HU (m---m), lo-* M HU (A-A).

HERPESVIRUS

HU-RESISTANT

HU at concentrations as high as lo-* M. It was therefore of interest to ascertain the effect of HU on the production of EHV-1 infectious virus. HU at various concentrations was added to L-M cell cultures and the cells were then infected at a multiplicity of 15 PFU/ml; after the virus attachment period of 1.5 hr, the cells were collected by centrifugation and washed twice with medium containing the appropriate concentration of HU. The cells, resuspended at a concentration of 2 x 106/ml in medium supplemented with HU, were incubated at 37” and at various times, samples for plaque titrations were collected in triplicate and stored frozen at -70” until assayed. As may be seen in Fig. 4, concentrations of HU of 2.5 x 10m3 A4 or greater resulted in a delay of 2 hr in the replicative growth cycle and a reduction in total virus production of less than one log at 20 hr PI. These concentrations of HU, however, failed to prevent virus productions and relatively large quantities of virus were produced in these inhibited cells. In cultures treated with 2.5 x 10m4 M HU, total virus production was similar to that of untreated cultures and maximal virus titers were obtained at 12 hr as was the case in untreated cultures. Experiments carried out at a lower multiplicity of infection (3 PFU/cell) gave identical results. Thus production of infectious virus as well as viral DNA synthesis, occur at concentrations of HU which completely inhibit cellular DNA synthesis. Effect of HU on the Stability DNA

of Cellular

Replication of the viral genome in the presence of HU may be explained by the presence of large pools of deoxyribonucleotides produced by degradation of cellular DNA during infection. Recent studies in our laboratory (Perdue et al., 1975) suggested that during EHV-1 infection, cellular DNA is not degraded and utilized in the synthesis of viral DNA. It is possible, however, that nuclease activity is stimulated or induced by HU to provide large pools of deoxyribonucleotides for viral DNA synthesis. To examine whether this is

61

REPLICATION

f\. 2

.\ ‘.

‘. 8 12 HOURS POST-INFECTION

20

FIG. 4. Effect of various concentrations of HU added at the time of infection on production of infectious EHV-1. L-M cell suspension cultures were infected with EHV-1 at a multiplicity of 15 PFU/cell. Triplicate samples were collected at the indicated times PI, and released virus was titered as described in Materials and Methods. Untreated (O--O), 2.5 x 10-‘MHU (x-x). 2.5 x 10m3MHU (O-TO), 5 x 1O-3 M HU (M-a), 10.’ M HU (A-A).

a possible mechanism of resistance of viral DNA synthesis to HU, two series of experiments were carried out. First, extracts of untreated and HU-treated, infected cells were assayed in uitro for DNase activities using both purified native and denatured radiolabeled L-M cell DNAs as substrates. Secondly, the lability of radiolabeled cellular DNA was monitored in untreated and HU-treated, control and infected cultures during infection. The results of the first series of experiments are summarized in Fig. 5. Low levels of nuclease activities were detected in mock-infected cultures, whereas both the untreated and HU-treated, infected cultures exhibited significant levels of DNase activity after 2 hr PI. The levels of nuclease activity specific for both native and denatured DNA in HU-treated, infected cells were not significantly elevated above those of the untreated, infected cultures. In fact, nuclease activity specific for native host DNA appeared to be significantly lower in the HU-treated cultures after 4 hr, the period during which viral DNA synthesis resistant to HU commences (Fig. 1).

62

COHEN

ET AL.

thesis, studies examining the effect of cycloheximide, a potent inhibitor of protein synthesis, on the rate of [3H]TdR incorporation in HU-treated, infected cultures were carried out. Cultures infected with EHV-1 in the presence or absence of 2.5 x 10e3 M HU were treated with cycloheximide (50 &ml final concn) at various times, and the rates of DNA synthesis during infection were determined. The results are summarized in Fig. 6. In cycloheximide inhibited cultures not treated with HU (open circles), the rate of DNA synthesis declined rapidly throughout the experiment; this DNA being synthesized in the presence of cycloheximide is cellular because (1) its synthesis occurs before the initiation of viral DNA synthesis and (2) it is not made in the presence of HU (closed triangles) which selectively inhibits cellular DNA synthesis. In HU-treated, infected cultures, addition of cycloheximide at 2 hr FIG. 5. DNase activities in untreated and HUtreated, EHV-1 infected L-M cells. Cellular extracts were prepared at various times after infection and were assayed for nuclease activities specific for native or denatured DNA, as described in Materials and Methods. (A) Double-stranded F3H]TdR labeled L-M cell DNA as substrate, (B) Single-stranded [3H]TdR labeled L-M cell DNA as substrate. Mock infected (O-O); infected, untreated (04); infected in the presence of 2.5 mM HU (x-x).

The results of experiments which monitored the effect of HU on the in uiuo stability of cellular DNA in control and infected cultures are shown in Table 1. It is evident that addition of HU to control and infected cultures did not result in marked release of radioactivity from prelabeled cellular DNA. At 4 hr PI, the time at which viral DNA synthesis resistant to HU commences, the change in the radiolabeled acid-soluble pool of HU-treated, infected cultures as compared to untreated, infected cultures was less than 0.10%. Thus, it seems unlikely that HU-induced degradation of cellular DNA provides a sufficient deoxynucleotide pool to allow viral DNA synthesis in the presence of this inhibitor. Requirement of Protein Synthesis for HUResistant Viral DNA Synthesis To evaluate the requirements of protein synthesis for HU-resistant viral DNA syn-

TABLE STABILITY

Treatment

Control

o/o acid soluble

465 370

166,545

0.28

228,886

0.16

383

218,810

0.17

209,641

0.19

224,467

0.22

4

399 493 416

228,973

0.18

6

543

213,314

0.25

8 2

814 368

207,597

0.39

214,661

0.17

4 6

425

0.19

621

229,439 229,459

8 2

729

227,504

456

4

611 948

222,439 230,933

0.32 0.20 0.26

239,160 242,574

0.48

2 6 8

Infected

Infected hydroxyureab

INFECTION:

wm acidinsoluble

Hour postinfec-

4

Control hydroxyureab

1

OF CELLULAR DNA DURING EFFECT OF HYDROXYUREA”

2

6 8

cpm acidsoluble

1160

0.27

0.39

n Cellular DNA was radioactively labeled by incubating L-M cell suspension cultures with [3H]TdR (3 /rCi/ml) for 12 hr prior to infection. At 2 hr before infectioti, cells were washed extensively with unlabeled medium and were incubatqd at 37” in unlabeled medium until the time of infection. Acid-soluble and acid-insoluble cpm were determined by TCA fractionation of samples taken at the indicated times. b HU at a final concentration of 2.5 x 10m3 M was added at the time of infection.

HERPESVIRUS

HU-RESISTANT

postinfection or earlier resulted in complete inhibition of viral DNA synthesis. If, however, protein synthesis was inhibited at 4 hr PI, viral DNA synthesis resistant to HU was not inhibited and continued at a rate twofold greater than that of cultures inhibited with cycloheximide at an earlier time PI. Reversal at 4 hr PI of the cycloheximide inhibition of protein synthesis in HU-treated cultures (open squares), failed to allow significant viral DNA synthesis to occur. This suggests that the transcripts coding for proteins necessary for viral DNA (HU resistant) synthesis are not produced or are not expressed in the presence of cycloheximide. These findings are in agreement with those of Ben-Porat et al. (1974), who showed that early functions involved in synthesis of pseudorabies virus DNA are not expressed after the removal of cycloheximide added early in infection. The results of these experiments are consistent with the concept that a viruscoded or virus-induced protein synthesized early during infection is required for viral DNA synthesis in the presence of HU. However, these data could reflect the requirement for the production of a protein which is necessary for viral DNA synthesis but which is not directly responsible for the HU resistance of EHV-1 DNA synthesis.

DNA Polymerase Activities in HU-Treated Cultures Weissbach et al. (1973) described a “high salt” (150 mM K,SO,) dependent viral specific DNA polymerase activity in herpes simplex virus infected cells. Recently, we have observed a similar polymerase activity in isolated nuclei from hamster liver of animals infected with the in uiuo strain of EHV-1 (Kemp, Cohen, O’Callaghan, and Randall, unpublished). A possible mechanism that would explain viral replication in the presence of HU inhibition of cellular DNA synthesis would be the selective inhibition of cellular DNA polymerase activity. Therefore, we investigated the DNA polymerase activities of control and infected L-M cells and the effect of HU on these activities. In the first series of experiments, we measured “low salt” and “high salt” DNA

63

REPLICATION

0’ 2

7 “0”~s

I 6

PO&INF:CT106N

FIG. 6. Inhibition of DNA synthesis in 2.5 mM HU treated, EHV-1 infected cultures by cycloheximide. 12 x 10’ L-M cells were radioactively labeled by pulsing with [3H]TdR (1 &i/ml, 50.8 CilmM) for 30 minutes and the cpm incorporated into acid insoluble material was measured as described in Materials and Methods. Cycloheximide, at a final concentration of 50 pglml, was added to specific cultures at the times indicated. No HU, cycloheximide at 0 hr (O-O); HU at 0 hr, no cycloheximide (.A); HU and cycloheximide at 0 hr (A-A); HU at 0 hr, cycloheximide at 2 hr (A-A); HU at 0 hr, cycloheximide at 4 hr (m-m); HU at 0 hr, cycloheximide at 0 hr and removed by washing with medium containing 2.5 mM HU at 4 hr (O-0).

polymerases in untreated and HU treated, control and infected cells by the methods of Weissbach et al. (1973) as described in Materials and Methods. As shown by the data presented in Fig. 7, a “high salt” DNA polymerase activity is present in EHV-1 infected L-M cells; this activity was not present in uninfected cells. The “high salt” DNA polymerase activity was first detected at 4 hr PI, the time of initiation of viral DNA synthesis, and this activity increased as the infection progressed. HU added at the time of infection did not affect the production of this enzyme; in fact, higher levels of activity were observed in the treated cultures. The “low salt” activities of the untreated and HU treated, control and infected cultures did not differ.

64

COHEN

60

A

:o 60 ?.i zw 40 I

ET AL.

tion of cycloheximide. This seems unlikely for cycloheximide at this concentration (50 pg/ml) causes an immediate inhibition of protein synthesis in EHV-1 infected L-M cells (O’Callaghan et al., 1968a). Measurement of the “high salt” activity before and after the addition of cycloheximide at 4 hr PI (Table 3), confirmed that cycloheximide prevented any further increase in the level of viral DNA polymerase. In fact viral TABLE DNA

5

POLYMERAS HYDROX~UREA

2

ACTIVITIES: EFFECT ON In Vitro ASSAI.~

OF

60

s 40

Enzyme preparation

2.5 mM hydroxyurea

Incorpt!zed/mg protein x 10e3 “Low salt” (no KSO,)

“High salt” (150 KS:,,

13.5 24.5 23.5 22.8 20.1 23.3

Mock-infected 7. Viral specific DNA polymerase activity in 2.5 mM HU treated, EHV-1 infected L-M cell cultures. Cellular extracts were prepared and assayed for DNA polymerase activity in the presence (viral) or absence (cellular) of 150 mM K,SO, as described in Materials and Methods. (A) “Low salt” DNA polymerase activities, (B) “High salt” (150 mM K&SO, dependent) DNA polymerase activities. Mock infected, untreated (O---O); Infected, untreated (04); Infected in the presence of 2.5 mM HU added at the time of infection (x-x 1. FIG.

Thus, HU had no effect on the level (production) of cellular polymerase. To confirm that HU did not selectively block cellular DNA polymerase activity, the effect of HU added to the reaction mixtures for the assay of “low salt” and “high salt” polymerase activities was determined. The data summarized in Table 2 show that HU does not inhibit either of these activities assayed in vitro. The data shown in Fig. 6 demonstrated that inhibition of protein synthesis at 4 hr PI in HU-treated, infected cultures did not cause the inhibition of viral DNA synthesis (HU-resistant DNA synthesis); viral DNA continued to be synthesized for several hours at a rate greater than that observed prior to inhibition of protein synthesis. This observation could be due to increased levels of viral DNA polymerase after addi-

+ Infected + -

Infected in presence of 2.5 mM HUh

+

9.0 6.8 47.6 43.0 35.0 28.1

0 Cellular extracts were isolated at 8 hr PI from each culture and were assayed for “low salt” and “high salt” DNA polymerase activities in the presence or absence of 2.5 x 10m3 M HU, as described in Materials and Methods. h HU was added at the time of infection. TABLE VIRAL

DNA

POLVMERASE

CYCLOHEXIMIDE

Enzyme

ADDITION

preparation

3 ACTIVITY:

EFFECT

AT 4 HOURS

Enzyme

OF

PI” activity

[3H]-TTP incorporated/ mg protein

No cycloheximide 2.5 mM HU; 0 hr 50 pg/ml Cycloheximide, 4 hr PI; 2.5 mM HU, 0 hr

4hrPI

6hrPI

57,660

71,882

65,035

54,974

a Cellular extracts were prepared from HU-treated, EHV-1 infected cultures; half of the cultures were treated with cycloheximide, 50 pg/ml final concentration, at 4 hr PI. Extracts were assayed for “high salt” DNA polymerase activity as described in Materials and Methods.

HERPESVIRUS

HU-RESISTANT

DNA polymerase activity decreased by 15% by 2 hr after the addition of cycloheximide, whereas a 25% increase in the level of this enzyme was observed in untreated cultures. Therefore, the increased rate of HU resistant viral DNA synthesis in the presence of cycloheximide is not due to increased levels of viral DNA polymerase, but the enzymatic reaction may increase in rate as a result of elevated pools of deoxyribonucleotides at this time of infection or because of increased availability of primer and/or template, as progeny DNA becomes parental. DISCUSSION

The results presented in this paper describe for the first time the resistance of a lytic herpesvirus infection to HU, a widely employed inhibitor of DNA synthesis. Equine herpes virus type 1 can replicate with only minor alterations in the growth cycle in the presence of HU at concentrations that completely inhibit host DNA synthesis. Recently Hampar et al. (1972) and Mele et al. (1974) demonstrated a similar phenomenon in that Epstein-Barr viral DNA synthesis could be induced in the presence of HU by pyrimidine analogues that activate the EBV genome present in Burkitt lymphoblastoid cells. In contrast to these findings, Nii et al. (1968) and Wagner et al. (1972) reported that herpes simplex viral DNA synthesis was inhibited by HU. The resistance of EHV-1 DNA synthesis to HU is supported by several observations. Measurement of [3H]TdR incorporation in infected cells treated with HU revealed that DNA synthesis resistant to HU began at 4 hr PI and reached a maximal rate at 8-12 hr PI. Previous findings by O’Callaghan et al. (1968a,b) had demonstrated similar kinetics for viral DNA synthesis in EHV-1 infected L-M cells. In addition, CsCl isopycnic analysis of DNA synthesized in untreated and in 2.5 mM HU treated, infected cells revealed that only DNA with a density equivalent to that of viral DNA, 1.716 g/cc, was synthesized in the presence of HU, whereas both cellular and viral DNA were synthesized in untreated, infected cultures. Further, HU at a

REPLICATION

65

concentration of four times that required to completely inhibit cellular DNA synthesis had no effect on viral DNA synthesis. Lastly, the resistance of EHV-1 DNA synthesis to HU was confirmed by the demonstration that HU did not inhibit the production and release of infectious virus, although a delay in the time course of virus production was observed. Several mechanisms could explain the inhibition of mammalian DNA synthesis by hydroxyurea. Selective inhibition of the host DNA polymerase could occur; however, no previous report has suggested this mode of inhibition, and in these studies HU did not inhibit cellular DNA polymerase activity (Table 2). The most widely accepted (and studied) effect of HU has been the inhibition of the activity of the enzyme ribonucleotide reductase, which converts ribonucleotides to deoxyribonucleotides required for DNA synthesis (Young and Hodas, 1964; Adams and Lindsay, 1967; Young et al., 1967; Krakoff et al., 1966; Moore, 1969). If this were the mechanism of action of HU, rescue from HU inhibition would require that EHV-1 either induce or code for the production of some product that will replenish the deoxynucleotide pools of the infected cell. A possible mechanism that would provide deoxynucleotides for viral DNA synthesis in the presence of HU is the enzymatic degradation of the host DNA by a viral induced DNase. Weissbach et al. (1973) have reported the existence of a viral induced DNase activity in herpes simplex virus infected cells. Perdue et al. (1975), however, showed that during EHV-1 infection of L-M cells, host DNA remained stable throughout the infection and did not contribute deoxynucleotides for the synthesis of viral DNA. It is possible, however, that the presence of HU during infection results in increased DNase activity and/or decreased stability of the host DNA. In this paper, it was shown that increased nuclease activity does not occur in HU-treated, infected cells, as determined by in vitro enzyme assay. Furthermore, significant increases in the deoxynucleotide pool during infection due to degradation of preradiolabeled host DNA were not detected in the HU

66

COHEN

treated cultures as compared to untreated cultures. In light of these results it seems unlikely that a viral and/or HU-induced degradation of cellular DNA is responsible for HU resistance of EHV-1 DNA synthesis. Consideration of the possibility of a viral specified or induced ribonucleotide reductase resistant to HU inhibition as an explanation for HU resistance is warranted in view of the recent work of Lewis and Wright (1974). They demonstrated the induction of a HU-resistant ribonucleotide reductase in HU-resistant mammalian cell cultures. Evidence suggesting the presence of a HU resistant ribonucleotide reductase in EHV-1 infection was obtained by studying the role of protein synthesis in the HU treated infection. One would expect that inhibition of protein synthesis prior to 4 hr PI would prevent viral DNA synthesis since the virus induced DNA polymerase is synthesized at this time (Weissbach et al., 1973; Ben-Porat et al., 1974; Fig. 7). As expected, viral DNA synthesis was completely inhibited by addition of cycloheximide prior to 4 hr postinfection (Fig. 61. When protein synthesis was inhibited at 4 hr PI, however, HU-resistant, EHV-1 DNA synthesis continued for several hours (Fig. 6). This synthesis of viral DNA was not due to an increase in the level of viral DNA polymerase as shown by measurement of high salt polymerase activity in HU-treated, infected cultures assayed before and after addition of cycloheximide (Table 3). Therefore, the observed increase in the rate of viral DNA synthesis in cultures after treatment with cycloheximide at 4 hr PI, may reflect increased levels of deoxynucleotides for viral DNA synthesis. These findings are consistent with the possibility that HU resistant ribonucleotide reductase activity occurs during EHV-1 infection. Studies are now in progress to evaluate the role of ribonucleotide reductase in EHV infection. Another mechanism which may explain HU resistance is the alteration of iron metabolism in the infected cell. As discussed by Moore (1967), iron at low levels serves to stimulate the activity of ribonucleotide reductase in an in vitro assay. Further-

ET AL.

more, Moore (1969) demonstrated that increased iron would partially rescue this enzyme from inhibition by HU. A recent report by Lambert et al. (1973) showed in HSV infection of HEp-2 cells that a 12-fold increase in iron uptake occurred by 20 hr postinfection with initial increases observed at 4 hr postinfection. Thus, it is possible that by increasing the iron concentration within the infected cell, partial restoration of the reductase activity would replenish the deoxynucleotide pools for viral DNA synthesis. The role of iron metabolism in EHV-1 infection and its effect on HU-resistance is currently being investigated. The question whether HU-resistance of viral DNA synthesis is due to virus altered and/or iron-rescued ribonucleotide reductase activity, remains unanswered. It should be noted that neither of these mechanisms would seem to explain the observation that only viral DNA is synthesized in HU-treated cultures. However, study of the HU resistant EHV-1 infection offers a unique situation; one has the opportunity in this infection to observe and evaluate virus-cell interaction in the absence of a major host biosynthetic process, that of genetic replication, with only slight alteration in the viral replicative cycle. ACKNOWLEDGMENTS This investigation was supported by Public Health Service research Grants AI 02032 and AI 08421 and by Training Grant AI-69 from the National Institute of Allergy and Infectious Diseases. Support was also obtained from a Brown-Hazen Research Grant awarded to one of us (DO’C) from the Research Corporation. We thank Drs. L. G. Gafford, G. A. Gentry, D. S. Lyles, and C. L. Woodley for many helpful comments in the preparation of this manuscript. REFERENCES ADAMS, R. L. P. and LINDSAY, J. G. (1967). Hydroxyurea reversal of inhibition and use as a synchronizing agent. J. Bio/. Chem. 242, 1314-1317. BEN-P• RAT, T., JEAN, J. H., and KAPLAN, A. S. (1974). Early functions of the genome of herpesvirus. IV. Rate and translation of immediate early viral RNA. Virology 59, 524-531. BORUN, T. W., SCHARFF, M. D., and ROBBINS, E. (1967). Preparation of mammalian polyribosomes

HERPESVIRUS

HU-RESISTANT

with the detergent Nonidet P-40. Biochim. Biophys. Acta 149, 302-304. DESONOA, C. P. (1965). Inhibition of shope fibroma virus with N-hydroxyurethane and related compounds. Nature (London) 206,688-689. HAMPAR, B., DERGE, J. G., MARTOS, L. M., TAGAMETS, M. A., and BURROUGHS, M. A. (1972). Sequence of spontaneous Epstein-Barr virus activation and selective DNA synthesis in activated cells in the presence of hydroxyurea. Proc. Nat. Acad. Sci. USA 69, 2589-2593. KRAKOFF, I. H., BROWN, N. C., and REICHARD, P. (1968). Inhibition of ribonucleotide diphosphate reductase by hydroxyurea. Cancer Res. 28, 1559-1565. LAMBEHT, D. M. and HUSAIN, S. S. (1973). A study of the response of herpesvirus-infected HEP-2 cells to iron uptake and ferritin biosynthesis. Lab. Znoest. 29, 737-742. LEVY, J. A., HUEBNER, R. J., KERN, J., and GILDEN, R. V. (1968). High titer T antigen in adenovirus infected cells treated with hydroxyurea. Nature (London) 217, 744-745. LEWIS, Q. H., and WRIGHT, J. A. (1974). Altered ribonucleotide reductase activity in mammalian tissue culture cells resistant to hydroxyurea. Biothem. Biophys. Res. Commun. 60, 926-933. LOWRY, 0. H., ROSEBROUGH, N. H., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with Folin phenol reagent. J. Biol. Chem. 193, 265-275. MELE, J., GLASER, R., NONOYAMA, M., ZIMMERMAN, J., and RAPP, F. (1974). Observations on the resistance of Epstein-Barr virus DNA synthesis to hydroxyurea. Virology 62, 102-111. MOORE, E. C. (1967). Mammalian ribonucleoside diphosphate reductase. In “Methods in Enzymology,” (L. Grossman and K. Moldave, eds.), Vol. 12, pp. 155-164. Academic Press, New York. MOORE, E. C. (1969). The effects of ferrous ion and dithioerythritol on inhibition by hydroxyurea of ribonucleotide reductase. Cancer Res. 29, 291-295. NII, S., ROSENKRAN~, S., MORGAN, C., and ROSE, H. M. (1968). Electron microscopy of herpes simplex virus. III. Effect of hydroxyurea. J. Viral. 2, 116331171. O’CALLAGHAN, D. J., CHEEVERS, W. P., GENTKY, G. A.. and RANDALL, C. C. (1968b3. Kinetics of cellular and viral DNA synthesis in equine abortion (herpes) virus infection of L-M cells. Virology 36, 104-114. O’CALLAGHAN, D. J., HYDE, J. A., GENTRY, G. A., and RANDALL, C. C. (1968a). Kinetics of viral deoxyribonucleic acid, protein, and infectious particle production and alterations in host macromolecular syntheses in equine abortion (herpes) virus infected cells. J. Viral. 2,793-804. PERDUE, M. L., COHEN, J. C., KEMP, M. C., RANDALL, C. C., and O’CALLAGHAN, D. J. (1973). Characteriza-

REPLICATION

67

tion of three species of nucleocapsid of equine herpesvirus type-l (EHV-11. Virology 64, 187-204 PERDUE, M. L., KEMP, M. C.. RANDALL, C. C.. and O’~ALLAC.HAN, D. J. (19741. Studies of the molecular anatomy of the L-M cell strain of equine herpesvirus type 1: Proteins of the nucleocapsid and intact virion. Virology 54, 201-216. PLAGEMAN, P. G. W., and ERBE, J. (1974). intracellular conversions of deoxyrihonucleoside by Novikolf’ rat hepatoma cells, and effects of hydroxyurea. J. Cell. Physiol. 83, 321-826. RANDAI.I., C. C., and LA\\SON, L. A. (1962). Adaptation of equine abortion virus to Earle’s L-cells in serum free medium with plaque formation. Proc. Sot. Ewp. Biol. Med. 110, 487-489. ROSENKRANZ, H. S.. POLLAK, R. D.. and SCHMII~I.. R. M. (1969). Biologic effects of isohydroxyurea. Cancer Res. 29, 209-218. ROSENKKANZ, H. S., ROSE, H. M.. MOK(:AX, C., and HSA, K. C. (1966). The effect of hydroxyurea on virus development. II. Vaccinia virus. Virology 28, 510-519. SKOOC, L.. and BJURSEI.L, G. (1974). Nuclear and cytoplasmic pools of deoxyribonucleotide triphosphates in Chinese hamster ovary cells. J. Biol. Chem. 249, 6434-6438. SOEHNEK, R. L., GENTR\, G. A., and RAND.~LL, C. C. (19651. Some physiochemical characteristics of equine abortion virus nucleic acid. Virology, 29, 394-405. STOCK, D. A., and GENTRY, G. A. (1969). Mycoplasmal DNase activity in virus infected L-cell culture. J. Viral. 3, 313-317. SUSSENBACH, J. S., and VAN DEH VLIET, P. C. (1973). Studies on the mechanism of replication of adenovirus DNA. I. The effect of hydroxyurea. Virolog>, 54, 299-303. VOGLEH, W. P., BAIN, J. A., and HLXXLE~, C. M., JR. (1966). In uiuo effect of hydroxyurea on erotic acid synthesis. Cancer Res. 26, 1827-1831. WAGNEH, E.. SWANSTROM, R., and STAFFONI), M. (1972). Transcription of the herpes simplex virus genome in human cell. J. Viral. 10, 675-682. WEISSHACH, A., HONC, S. L., AUCKER, J., and MUI.I.EN, A. (1973). Characterization of herpes simplex virusinduced deoxyribonucleic acid polymerase. J. Rio/. Chem. 248, 6270-6277. YARBKO, J. W. (1968). Further studies on the mechanism of action of hvdroxyurea. Cancer Res. 28, 1082-1087. YOVNC, C. W., and HODAS, S. (1964). Hydroxyurea: Inhibitory effect on DNA metabolism. Science 146, 1172-1174. YOUNG, C. E., SCHOCHETMAN, G., and KARNOFSKY, D. A. (1967). Hydroxyurea-induced inhibition of deoxynucleotide synthesis: studies in intact cells. Cancer Res. 27,526-534.