ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, OCt. 1976, p. 682-686 Copyright © 1976 American Society for Microbiology

Vol. 10, No. 4 Printed in U.S.A.

Differential Effects of 5-Methylmercapto-2'-Deoxyuridine the Replication of Herpes Simplex Virus Type 1 in Two Cell Systems

on

ROBERT HARDI, R. G. HUGHES, JR.,* Y. K. HO, K. C. CHADHA, AND T. J. BARDOS

Department of Medicinal Chemistry, State University of New York at Buffalo, Buffalo, New York 14214, and Department of Medical Viral Oncology, Roswell Park Memorial Institute, Buffalo, New York 14263* Received for publication 19 April 1976

5-Methylmercapto-2'-deoxyuridine (MeMUdR), a structural analogue of thymidine (TdR), inhibits herpes simplex virus type 1 production in mouse L (Lb) cells at concentrations that are not inhibitory to vira-l growth in monkey kidney (CV-1) cells. It is moderately toxic to Lb cells but not to CV-1 cells at a concentration that causes 95% inhibition of viral replication in Lb cells. MeMUdR is incorporated into cellular and viral deoxyribonucleic acid (DNA) in both systems, but to a significantly higher level (compared with thymidine) in Lb cells. These results indicate that MeMUdR is a substrate for enzymes leading to DNA synthesis and suggest that the biological function of herpes simplex virus type 1 DNA is impaired only when the incorporation of MeMUdR into the DNA reaches a relatively high level. Nucleoside analogues represent an important family of antiviral agents (7). A member of this family, 5-methylmercapto-2'-deoxyuridine (MeMUdR) was first synthesized by Kotick et al. (3). The structure of this compound is shown in Fig. 1. It was stated by Stout and Robins (6) that MeMUdR showed significant inhibition of type 1 herpes simplex (HSV-1) in their antiviral screen, but the cell system used was not described. The present report shows that the compound has a differential effect on HSV-1 replication in two cell-virus systems, with replication in mouse L (Lb) cells being more sensitive to inhibition than replication in monkey kidney CV-1 cells. We also show that the compound is incorporated into viral and cellular deoxyribonucleic acid (DNA) in both cell-virus systems.

tific Corp.) and were infected at a multiplicity of infection of 5 plaque-forming units per cell. Virus was adsorbed for 1 h at 37°C, and the cultures were washed twice with medium and then incubated in medium containing MeMUdR. Cells were scraped into the medium 24 h postinfection, frozen, and then thawed before titrations were carried out as previously described (1). Cytotoxicity assay. Cells were seeded in 60-mm dishes at 105 cells per dish and, after attachment, were cultured in medium containing the drug. Dishes, in triplicate, were trypsinized and counted each day for 5 days. Preparation and separation of cellular and viral DNA. Cells were grown in 75-cm2 plastic tissue culture flasks (Lux Scientific Corp.) and were infected at 10 plaque-forming units per cell. Virus was adsorbed for 1 h at 37°C. Cultures were then washed twice with EM5C, and 10 ml of EM5C was replaced. Label was added 6 h postinfection {[3H]thymidine (TdR), 333 uCi/,umol, 10-5 M final concentration; MATERIALS AND METHODS [3H]MeMUdR, 100 ,uCi/,umol, 1O-5 M final concenCells and virus. The sources of CV-1 monkey kid- tration}. ney fibroblasts and Lb mouse fibroblasts have been Cells were scraped off 24 h postinfection. A 1-ml described (1, 5). These cells were maintained in a sample was frozen and later titrated to verify promodified Eagle medium (1), supplemented with non- ductive infection. Nucleic acids were extracted by a essential amino acids and 5% calf serum (EM5C). slightly modified procedure of the method of KraThe source of HSV-1, strain KOS (HSV-1, KOS 1.1), iselburd et al. (4). Cells were suspended in 0.02 M has also been described (1). tris(hydroxymethyl)aminomethane - hydrochloride MeMUdR was prepared according to Kotick et al (pH 8), containing 0.01 M ethylenediaminetetraace(3). [3H]methyl-labeled MeMUdR was synthesized tic acid, 1 mg of Pronase per ml, and 0.5% sodium via selective S-methylation of 5-mercapto-2'-deox- dodecyl sulfate, and incubated overnight at 37°C. yuridine (MUdR) with [3H]MeI (manuscript in prep- Extraction was done with phenol-chloroform (1:1), aration). and nucleic acids were precipitated with cold ethaVirus inhibition assay. Cells were grown to con- nol. The precipitate was dissolved in 0.1 x SSC (0.15 fluency in plastic tissue culture dishes (Lux Scien- M NaCl plus 0.015 sodium citrate) and dialyzed over682

VOL. 10, 1976

INHIBITION OF HSV-1 BY MeMUdR

is incorporated into DNA. Therefore, it was of interest to see if TdR could reverse the inhibi-

0

HN HOCH2

0

-SCH3 N

HO FIG. 1. The structure of 5-methylmercapto-2'-

deoxyuridine. night against the same buffer. Two optical density units of nucleic acid in 0.7 ml of solution were layered on top of 10 ml of CsCl. Samples were centrifuged for 72 h at 50,000 rpm at 250C in a Beckman 65 rotor. Fractions were collected and measured for absorbance at 260 nm and for radioactivity. Identification of MeMUdR in DNA. The enzymes used for the digestion of DNA were deoxyribonuclease from beef pancreas (3,000 units/mg), venom phosphodiesterase (20 units/mg), and alkaline phosphatase from Escherichia coli (34 units/mg). Enzymes were purchased from Worthington Biochemical Corp., Freehold, N.J. A solution in a final volume of 2 ml containing 70 Ag of MeMUdR-labeled DNA isolated from Lb cells infected with HSV-1 (3

x

683

103 cpm/ g), 40 ;Lg of

deoxyribonuclease, 30 mM MgCl2, 250 mM acetate buffer, pH 6.6, and two drops of CHCl3 was incubated at 370C for 48 h. Subsequently, 1 ml of ammonium acetate buffer (200 mM, pH 9.2) was added followed by 0.1 ml of venom phosphodiesterase (1 mg/ml). The reaction mixture was incubated at 37°C for 6 h after which 0.1 ml of alkaline phosphatase (0.22 mg/ml) was introduced, and incubation was continued for another 20 h. The digested mixture was purified on a Sephadex G-10 column and analyzed by thin-layer chromatography. Separation was carried out on Eastman sheet, 13254 cellulose, with two solvent systems: A, n-butanol-ammonium hydroxide-water (86:5:14); and B, ammonium hydroxide (1.5 M)-2-ethoxyethanol-n-butanol (3:2:1).

RESULTS

Inhibition of viral replication. The results in Table 1 show that in the presence of 10-5 M MeMUdR, HSV-1 production in Lb cells was inhibited by about 95%, whereas in CV-1 cells at this drug concentration little, if any, inhibition was observed. Thus, at the given concentration, MeMUdR can almost completely block virus replication in Lb cells and, since the same effect is not observed in CV-1 cells even though all viral-specific processes are presumably the same in permissive cells, this effect must be exerted through some cellular process. MeMUdR bears a structural resemblance to TdR and, as data in the next section will show,

tory effect of MeMUdR. TdR at 10-4 M inhibited virus growth by 38% relative to virus growth in its absence. However, virus growth was inhibited by only 16% in a mixture of 10-' M TdR and 10-- M MeMUdR relative to virus growth in 10-4 M TdR alone (Table 1). These data indicate that MeMUdR acts as an antimetabolite of TdR and that it effectively competes with the latter at less than a 1:1 ratio. No studies were carried out on the effect of MeMUdR on ribo-

nucleic acid metabolism. The cytotoxicity of MeMUdR, although lower, paralleled its antiviral activity. As Fig. 2 shows, 10-5 M MeMUdR inhibited the growth of Lb cells by about 60%, but had no effect on the rate of growth of CV-1 cells. These results suggest that the cellular process that results in MeMUdR being actively antiviral in Lb cells also results in the compound being cytotoxic for the same cells. Incorporation of MeMUdR into viral and cellular DNA. Figures 3 and 4 show the results of CsCl equilibrium density gradient centrifugation of DNA extracted from HSV-1-infected CV-1 and Lb cells cultured in [3H]MeMUdR or [3H]TdR. These data show that [3H]MeMUdR was incorporated both into HSV-1 DNA (denTABLE 1. Inhibition by MeMUdR of the replication of HSV-1 and its reversal by TdRa Concn (M) of compound:

HSV-1

titer (PFU/ml

x

10-7) in:

MeMUdR

TdR

Lb cells

0 10-6 10-5 10-s 10-5 10-5

0 0 0 10-6 10-5 10-4 10-4

7.3 4.4 0.36 1.8 2.4 3.8 4.5

CV-1 cells

42.0 58.0 36.0 42.0 32.0 28.0 22.0 a Lb or CV-1 cells were infected with HSV-1 at 5 plaque-forming units (PFU)/cell. After 1 h at 370C, cell layers were washed, medium containing the indicated concentrations of compounds was added, and the cultures were reincubated at 37°C. Compounds were added simultaneously where mixtures are indicated. Some cultures were harvested at 6 h postinfection as eclipse samples. The virus titers of these samples are near the minimum of the HSV-1 growth cycle. Values in excess of the eclipse values at later times in the growth cycle indicate the production of new infectious virus by the infected cells. Eclipse samples were taken in the presence of 10-5 M MeMUdR; Lb cells, 5 x 105 PFU/ml; CV-1 cells, 1.3 x 106 PFU/ml. The samples whose titers are given in the table were harvested 24 h postinfection.

684

ANTIMICROB. AGENTS CHEMOTHER.

HARDI ET AL.

sity, approximately 1.71 g/cm3) and cellular DNA in infected Lb and CV-1 cells. Since TdR 0 nucleotide pool sizes were not known in the 0 infected cells, the amounts of incorporation reflect the numbers of nucleoside residues incorporated from the TdR or MeMUdR in the medium and may represent less than the true values of TdR or MeMUdR incorporation due to by the endogenous TdR nucleotide dilution O D._ pools. However, if the TdR nucleotide pools in a given infected cell type do not differ in cells we .a 0 -a exposed to MeMUdR from those exposed to i: TdR, the relative numbers of TdR or MeMUdR residues incorporated per unit of viral DNA can be compared. Although there was no difference in the incorporation of [3H]MeMUdR and [3H]TdR in infected CV-1 cells (Fig. 4), in Lb cells [3H]MeMUdR was incorporated to a greater extent than [3H]TdR (Fig. 3). These %O 1 O 1 2 3 4 5 2 3 4 data indicate that the number of MeMUdR resiDAYS dues per viral DNA molecule, i.e., the extent of FIG. 2. Cytotoxicity ofMeMUdR. (A) Lb cells; (B) modification of the viral DNA, is higher in the CV-1 cells. Symbols: *, control; 0, 10-6 M Me- Lb cells than in CV-1 cells. Y

B

A

// /1 j~~~~~

i/ /

MUdR; *, 10-5 M MeMUdR.

w

a e a

0 w

m1

30

z

0

I 0

a

ia

FRACTION NUMBER FIG. 3. Equilibrium density gradient separation of cellular and viral DNA from HSV-1-infected Lb cells. (A) cells treated with [3H]MeMUdR; (B) cells treated with [3H]TdR. Symbols: absorbancy at 260 nm; *, picomoles of 3H-labeled nucleoside residues in DNA. 0,

VOL. 10, 1976

INHIBITION OF HSV-1 BY MeMUdR

685

w

a

0 w

U0

Imi

I3

0 S

0

U

I

0.

FRACTION NUMBER FIG. 4. Equilibrium density gradient separation of cellular and viral DNA from HSV-1-infected CV-1 cells. (A) Cells treated with [3H]MeMUdR; (B) cells treated with [3H]TdR. Symbols: O, absorbancy at 260 nm; *, picomoles of 3H-labeled nucleoside residues in DNA.

To obtain direct evidence for the incorpora- MUdR and its intermediate metabolites, retion of MeMUdR into the DNA of cells infected spectively, must be substrates in all of the enzywith HSV-1, radioactive DNA was isolated matic steps of the pathway leading from TdR to from infected Lb cells which had been cultured DNA. The higher level of incorporation into in the presence of MeMUdR. This DNA was viral DNA in infected Lb cells suggests that one digested enzymatically, and the nucleosides ob- of these cellular enzymes in Lb cells recognizes tained were separated by thin-layer chromatog- MeMUdR (or its appropriate metabolic derivaraphy. Only one radioactive peak was observed tives) as a better substrate than TdR (or the in each case with an Rf value identical to that of corresponding metabolic derivative of TdR). an authentic sample of MeMUdR. The Rf of This enzyme is probably not the cellular TdR MeMUdR in solvent system A was 0.33, and in kinase, since MeMUdR was as effective in insolvent system B the Rf was 0.85. These data hibiting virus replication in Ltk- cells, a line of indicate that MeMUdR was incorporated intact L cells lacking TdR kinase (2), as it was in Lb into the DNA. cells (data not shown). As an alternative explanation, one should consider the possibility that DISCUSSION the pool size of one of the intermediate metaboMeMUdR inhibits HSV-1 production in Lb lites of TdR might be altered by MeMUdR to a cells, but at the same concentration has no different degree in Lb cells than it is in CV-1 inhibitory effect on the replication of the virus cells. If such were the case, this too could acin CV-1 cells. As expected on the basis of its count for both the differential incorporation structure and as shown by the data in Table 1, and differential inhibitory effects of MeMUdR MeMUdR acts as a TdR analogue. Because it in the two cell systems. appears in both viral and cellular DNA, MeThe difference in susceptibility to the inhibi-

686

HARDI ET AL.

ANTIMICROB. AGENTS CHEMOTHER.

tory effect of MeMUdR on HSV-1 replication in CV-1 and Lb cells is not absolute. In the presence of 10-4 M MeMUdR, HSV-1 replication in CV-1 cells was inhibited by 97% (data not

shown).

In summary, the data indicate that MeMUdR is an inhibitor of HSV-1 replication and that it behaves as an analogue of TdR. The compound is incorporated into viral and cellular DNA both in infected CV-1 and Lb cells. Lb cells seem to incorporate the compound into viral DNA to a greater extent than do CV-1 cells, and it may be the excessive replacement of thymidine by MeMUdR residues in the viral DNA that results in the loss of its biological function in producing infectious progeny virus. ACKNOWLEDGMENTS This work was supported by Public Health Service grants CA-13114, CA-14801, and CA-06695 from the National Cancer Institute and by grant CH-20C from the American Cancer Society. Y.K.H. was a Research Fellow of the National Cancer Institute (IF22 CA02065).

LITERATURE CITED 1. Hughes, R. G., Jr., and W. H. Munyon. 1975. Temperature-sensitive mutants of herpes simplex virus type 1 defective in lysis but not in transformation. J. Virol. 16:275-283. 2. Kit, S., D. Dubbs, L. Piekarski, and T. Hsu. 1963. Deletion of thymidine kinase activity from L cells resistant to bromodeoxyuridine. Exp. Cell Res. 31:297-312. 3. Kotick, M. P., C. Szantay, and T. J. Bardos. 1969. Synthesis of 5-S-substituted 2'-deoxyuridines. Study of the factors influencing the stereoselectivity of the silyl modification of the Helbert-Johnson reaction. J.

Org. Chem. 34:3806-3813. 4. Kraiselburd, E., L. P. Gage, and A. Weissbach. 1975. Presence of a herpes simplex virus DNA fragment in

L cell clone obtained after infection with irradiated herpes simplex virus 1. J. Mol. Biol. 97:533-542. 5. Sanford, K. K., and W. R. Earle. 1948. The growth in vitro of single isolated tissue cells. J. Natl. Cancer Inst. 9:229-246. 6. Stout, M. G., and R. K. Robins. 1972. The synthesis of some 5-methoxypyrimidine nucleosides. J. Heterocycl. Chem. 913:545-550. 7. Tilles, J. G. 1974. Antiviral agents. Annu. Rev. Pharmacol. 14:469-489. an