Mutation Research, 267 (1992) 125-131 © 1992 Elsevier Scicnce Publishers B.V. All rights reserved 0027-5107/92/$05.00

125

MUT 05089

Analysis of toxic and mutagenic activities of antiherpesvirus nucleosides against HeLa cells and herpes simplex virus type 1 Tatsuo Suzutani a and Haruhiko Machida b a Department of Microbiology, Asahikawa Medical College, Asahikawa 078 and b Research Laboratories, Yamasa Sboyu Co., Ltd., Choshi 288 (Japan) (Received 14 May 1991) (Revision received 22 November 1991) (Accepted 26 November 1991)

Keywords: Herpes simplex virus type 1; Nucleoside; Mutagenicity

Summary The toxic and mutagenic activities of five antiherpesvirus agents to HeLa cells and herpes simplex virus type 1 (HSV-1) were investigated. 5-1odo-2'-deox3,uridine (IDU) and 9-/~-D-arabinofuranosyladenine (araA) showed very potent inhibitory effects on cell growth and the cloning efficiency of HeLa cells, whereas l-/3-D-arabinofuranosyI-E-5-(2-bromovinyl)uracil (BV-araU), E-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) arid 9-(2-hydroxTethoxTmethyl)guanine (ACV) showed less inhibitory effect. 50% inhibitory doses of BV-araU and BVDU for cell growth were 657 and 253 #g/ml, respectively. Although the growth inhibitory activity of BVDU was very weak, as above, the mutagenic activity of this drug to the cells, estimated by induction of colchicine-resistant mutants, was observed to be 4/zg/ml, which was a markedly smaller dose than the inhibitory dose for cell growth, and the highest frequency of mutation of the cells was shown at 100 ~g/ml of BVDU. This activity was more potent than that of IDU. No mutagenic activity of BV-araU, araA and ACV to cells was observed within the concentration range of 1-800/zg/ml. IDU showed high mutagenic activity to HSV-1 growing in human embryo lung fibroblasts, and IDU-resistant mutants were induced at a high frequency. BVDU also induced a small amount of BVDU-resistant mutant virus, although this drug induced many mutant cells. No mutagenic activity of BV-araU, araA and ACV to HSV-1 was observed.

1-/3-v-Arabinofuranosyl-E-S-(2-bromovinyl)uracil (BV-araU) and E-5-(2-bromovinyl)-2°deoxyuridine (BVDU) have been shown to be effective and selective antiviral drugs against herpes simplex virus type 1 (HSV-1), variceila-zoster

Correspondence: Dr. T. Suzutani, Department of Microbiology, Asahikawa Medical College, Asahikawa 078 (Japan).

virus (VZV) and Epstein-Barr virus (EBV), but have no effect against herpes simplex virus type 2 (HSV-2) and human cytomegalovirus (HCMV)in vitro (Shigeta et al., 1983; Machida, 1986; Lin and Machida, 1988; our unpublished data). Because of the insufficient antiviral activity of 9-(2-hydrox),ethoxymethyl)guanine (ACV) against VZV and EBV compared with those of BVDU and BV-araU (Machida, 1986; Lin and Machida,

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1988), it is hoped that these drugs will be clinically used against the diseases caused by VZV or EBV infection, in previous reports, differences in the mechanism of antiherpesvirus activity were shown between BV-araU and BVDU: these compounds have a similar structure, the only difference being the 2'-OH group in the arabinofuranosyl configuration of BV-araU (De Clercq et al., 1979; Machida et al., 1981). The most important difference between BV-araU and BVDU activity was that a large amount of BVDU was incorporated into viral DNA, whereas little or no BV-araU was incorporated (Allaudeen et al., 1982; Suzutani et al., 1988a). However, the biological effects that these differences have on the cell or virus are not clear. Therefore, in this study we investigated the toxic and mutagenic activities of BV-araU and BVDU to the HeLa cells and HSV-1, compared with those of 5-iodo-2'-deoxyuridine (IDU), 9-/3D-arabinofuranosyladenine (araA) and 9.(2-hydroxyethoxymethyl)guanine(ACV). Materials and methods

Compounds BVDU and BV-araU were synthesized by Yamasa Shoyu Co., Ltd., Choshi (Japan). IDU was purchased from Wake Fine Chemical Products, Ltd., Osaka (Japan). ACV was a gift from Japan Wellcome Co,, Ltd,, Osaka (Japan), and araA was kindly supplied by Mochida Pharmaceutical Co., Ltd., Osaka (Japan).

Cells and titus Human embryo lung fibroblast (HEL) cells, strain HAIN-55 (Suzutani et al., 1988b), were kindly supplied by H. Okumura, National Institute of Health of Japan. HeLa cells were obtained from Flow Laboratories, Inc., McLean, VA (U.S.A.). These cells were cultured in Eagle's minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS). The VR-3 strain of HSV-1 was generously supplied by the American Type Culture Collection, Rockville, MD (U.S.A.). The methods for the preparation of the stock viruses have been described previously (Suzutani et al., 1988b).

Assayof inhibitory effect of drugs on cell growth HeLa cells were seeded in 24-well tissue cuiture plates at 2 × 104 per well. After 1 day, the cells were refed with MEM-10% FBS containing an appropriate amount of the test drug. After incubation for 2 days, the cells were harvested by trypsinization and the viable cell numbers were counted microscopically.

Assayofmutagenic activity of drugs to cells Exponentially growing HeLa cells were treated with an appropriate amount of the test drugs in MEM-10% FBS for 48 h. The cells were washed 3 times with MEM and replenished with MEM10% FBS and cultured for 5 days to allow expression of mutated genes (Gupta and Siminovitch, 1980). 1 x 106 and 1 × 10 2 treated cells were seeded in 100-ram plastic petri dishes containing 10 ml of MEM-10% FBS to evaluate the number of mutant cells and cloning efficiency, respectively. After overnight incubation, 10 ml of MEM-10% FBS containing colchicine (final concentration 20 ng/ml colchicine; colchicinemedium) was added to the dishes for counting mutant cells and the cells were incubated for 4 days. The cells were refed with 20 ml of fresh colchicine-medium, in order to maintain the concentration of colchicine, and incubated for 2 weeks. The plates, for determination of the cloning efficiency of the cells, were refed with MEM-10% FBS and incubated similarly. After fixation of cells with 10% formaldehyde neutral buffer solution, the numbers of colonies which were formed with and without colchicine selection were counted and the mutant frequency was calculated as follows: (number of mutant colenies)× (1/number of cells plated) × (l/cloning efficiency).

Assayof inhibitory and mutagenic acticity of drugs to HSV.I The inhibitory effect of each drug on the replication of HSV-I in HEL cells was evaluated by the plaque reduction assay described by Suzutani et al. (1988b). To investigate the mutagenic activity of drugs to HSV-I, the number of plaques in the overlay medium containing the test drugs was counted as follows: confluent HEL cell monolayers in a 24.

127

well plastic plate were infected with HSV-I at a multiplicity of infection of 0.05 pfu per cell. After a l-h adsorption period at 37°C, the cultures were overlaid with MEM-2% FBS containing 0.5% methylcellulose and an appropriate amount of test drug. The number of plaques, after 2 days of incubation, was counted microscopically and considered to be a spontaneously drug-resistant virus which had initially existed in the stock virus used in this experiment (referred to as spontaneous mutants). After 4 days of incubation, the number of plaques in the wells in which no plaque had been observed at 2 days of incubation were counted microscopically and considered to be the drug-resistant virus induced by treatment with the test drug (referred to as induced mutants).

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Growth inhibitory and cytotoxic effects of drugs on HeLa cells

Fig. 2. Effects of the drugs on plating efficiency of HeLa cells. BV-araU (o), B V D U (o), A C V ( • ), araA ( • ), I D U ( [] ).

The effects of the drugs on HeLa cell growth were examined. IDU and araA had potent inhibitory activities on HeLa cell growth, and 50% inhibitory doses (IDs0s) of IDU and araA were 31.6and 20.0/zg/ml, respectively(Fig. 1). BVDU and BV-araU showed low anticellular activities and ID~0s of these drugs were 253 and 6570zg/ml, respectively. In the case of ACV, weak i,hibitory effects on cell growth were observed at 40 ozg/ml, but these effects were not augmented by increasing the concentration of drug. The IDs, of ACV was over 400 ~ g / m l , which was the highest concentration in our assay system.

Another parameter that indicates the growth inhibitory and cytotoxic effects of the drugs is the cloning efficiency of the cells. The numbers of colonies formed by the cells treated with the drugs for 48 h and then incubated for 5 days without the drugs were measured (Fig. 2). All viable cells which had been treated with BVDU. BV-araU or ACV had colony-forming ability. However, the percentage of colony-forming cells among the IDU- or araA-treated cells declined in proportion to the dose of the drug.

Mutagenic acticities of the drugs on HeLa cells

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It has been reported that several kinds of nucleoside analogues have mutagenic activities on mammalian cells (Watson et al., 1987; Davidson et am., 1988). Drugs which are used for treatmeat of human diseases, especially in the case of systemic treatment, should have no mutagenic activity. Therefore, the mutagenicity of the antiherpesvirus drugs to HeLa cells was studied by estimating colchicine-resistant mutants (Lin and Machida, 1988) in the drug-treated cell population (Fig. 3). In 1.2 x 107 HeLa cells not treated

with test drugs, cells did not form colonies in the medium containing 20 ng/ml of colchicine (colchicine-medium). When treated with araA, ACV

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TABLE 1 MUTAGENIC ACTIVITY OF TEST DRUGS IN THE INDUCTION OF DRUG RESISTANCE IN HSV-1

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Mutagenic activity of test drugs to HSV-1

or BV-araU, cells did not form colonies in the colchicine-medium (Fig. 3). However, colchicine-resistant cells were induced by IDU and BVDU treatment. The peak mutation rates were observed at 40/~g/ml of IDU and 100/~g/mi of BVDU, when about 0.85 and 1.5 mutants per 106 colony.forming cells were induced, respectively, The frequency of mutation by 100 /~g/ml of BVDU was over 18 times that of the spontaneous background and about 10% of the frequency of mutation which would be induced by treatment with 150/.tg/ml of ethyl methanesulfonate (EMS). ~mc~B-~' !~~~\~U ! ~

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In the preliminary experiment for induction and selection of a drug-resistant virus, the antivi-

ral activities of the drugs were determined by the plaque reduction assay. As shown in Fig. 4, little or no HSV-1 plaque was observed in MEM containing I00/~g/ml of IDU or araA, or 1/zg/ml of ACV, BV-araU or BVDU. Following this, the experiments for induction and selection of the drug-resistant virus were performed in medium containing the above concentrations of the drugs. In the presence of these concentrations of the drugs, 0-9 plaques of drug-resistant virus, which existed in the stock virus as spontaneous mutants, were observed after 2 days' incubation (Table 1). After incubation for a further 2 days, 3.7% of the inoculum was mutated by treatment with IDU. This frequency was much higher than that in the HeLa cells. BVDU induced only 1 BVDU-resistant virus, whereas araA, ACV and BV-araU did not induce a drug-resistant virus. Discussion

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Fig. 4. AnIi-HSV-I activities of the drugs in the plaque reduction assay. BV-araU (o), BVDU (*), ACV ( , ) , a r a A ( • ) , IDU ([3).

In this study, we analyzed the toxic and mutagenie activities of five kinds of antiherpesvirus nucleoside analogs. These analyses are critical because a number of different nucleoside analogs have been reported to be mutagens (Watson et al., 1987; Davidson et al., 1988). In addition, comparative investigation of the different effects o f compounds which h a v e a similar s t r u c t u r e c a n

129

clarify the structure required for antiviral activities, non-cell toxicity and non-mutagenicity, The mechanisms of selective activity of BVaraU and BVDU against HSV-1 and VZV have been widely reported. These nucleosides are specifically phosphorylated by viral-induced thymidine kinase (TK) to monophosphate and also to diphosphate (Cheng et al., 1981; Fyfe, 1981; Chen et al., 1984; Ayisi et al., 1987; Suzutani et al., 1988a). Pho~phorylation of these nucleosides to diphosphate by viral TK is essential for antiviral activity, because these drugs have a less inhibitory effect on HSV-2 replication, and HSV-2-induced TK has low thymidylate kinase activity. Furthermore, almost all BV-araU and BVDU taken up into HSV-2-infected cells is detected in monophosphates (Cheng et al., 1981; Fyfe, 1981; Machida, 1986; Ayisi et al., 1 9 8 7 ; Suzutani et ai., 1988a). Diphosphates of BV-araU and BVDU are phosphorylated to triphosphate by nucleoside diphosphate kinase which is a cellular enzyme, and then inhibit viral DNA replication (Ruth and Cheng, 1981). it has been reported that BVDU triphosphate (BVDUTP) is incorporated into DNA by viral DNA polymerase, competing with thymidine triphosphate (TTP), whereas BV-araU triphosphate (BVaraUTP) may not be incorporated (Allaudeen et al., 1982; Suzutani et al., 1988a). The present results show that BVDU and IDU induce mutant HeLa cells, but BV-araU, araA and ACV do not. There have been many reports on the mutagenic activity and its mecisamsm ot some kinds of nucleoside analogs, especially mutation induced by S.bromodeoxyuridine (BDU) (Freese, 1959; Davidson and Kaufman, 1978; Kaufman and Davidson, 1978; Ashman et al., 1981; Watson et al., 1987; Davidson et al., 1988). The mechanisms of mutation induced by BDU are explained as follows. (i) BDU is phosphorylated to BDU triphosphate (BDUTP) and the high concentration of BDUTP inhibits the ribonucleotide reductase which catalyzes reduction of CDP to dCDP. This inhibition induces a high BDUTP/dCTP molar ratio and results in misincorporation of BDUTP opposite guanine residues in DNA, causing transitions of G . C to A. T (Davidson and Kaufman, 1978; Kaufman and Davidson, 1978; Ashman et al., 1981; Davidson et

al., 1988). (ii) BDU is incorporated into DNA as a thymidine analog and covalently pairs with adenosine. However, the base of BDU has a tendency to mispair with guanine residues during the replication of DNA, because the hydrogen atom at the N1 site of BDU shifts and bonds to the oxygen atom attached to carbon atom 6, sometimes. This mispair induces transitions of A- T to G. T (Freese, 1959; Watson et al., 1987). Davidson et al. (1988) have reported that the first mechanism is predominant in mammalian cells. Perhaps BVDU and IDU induce mutant cells in a similar way to BDU, because the structures of these nucleoside analogs are alike. However, part of the mutagenic effect of IDU against HSV-1 may be the second mechanism because viralspecific ribonucleotide reductase is induced in HSV-l-infected cells and this enzyme is not inhibited by an excess of TI'P or other deoxynucleoside triphosphates (dNTPs) (Huszar and Bacchetti, 1981; Langelier and Buttin, 1981; Makayama et al., 1982). it has been reported that, after HSV infection, the TFP pool is enlarged about 25-50 times, but such dramatic changes are not observed in other dNTP pools (Jamieson and Bjursell, 1976). It is not clear why only the TTP pool in HSV-infected cells has to be increased, and whether the frequency of error in the reading process of viral DNA polymerase is influenced by such a perturbation of dNTP pools or not. In order to be able to answer these questions, it is necessary to analyze and clarify the IDU-induced mutations. The frequencies of appearance of BVDU- and IDU-induced mutant cells were highest at 100 and 40 izg/ml, respectively, and the frequencies of mutation were decreased by excess BVDU and IDU (Fig. 3). A similar phenomenon has been observed in alkylating agent-induced mutation (Koyama et al., 1982). Therefore, it is unclear whether BVDU, BV-araU, araA and ACV have mutagenic activity against HSV-1 or not, because there is a possibility that the dose, which we used to analyze the mutagenic activity of drugs against HSV-1, was excessive for virus mutation. Though BV-araU and BVDU have a similar structure, these nucleosides have shown different effects on cell mutation. This difference might be caused by the difference in affinity to cellular

130 enzymes and the m o d e o f inhibitory effects on D N A replication. In a previous report, it was shown that B V D U had a higher binding affinity to mitochondrial TK, but not to cytosolic TK, and that BV-araU had lower binding affinity to both mitochondrial and cytosolic T K ( C h e n g et al., 1981). F u r t h e r m o r e , the amounts taken u p into the D N A differed markedly between B V D U and BV-araU, as described above ( A l l a u d e e n et al., 1982; Suzutani et al., 1988a). Larsson et al. (1983) described that a nucleoside analog such as an antiherpes drug was ideal to contain a normal base and act as a chain terminator, if incorporated into the D N A , in o r d e r to decrease the risk o f mutagenic effects. By replacing the deoxyribose in B V D U with arabinose, producing BVaraU, selective antiherpesvirus activity was increased and mutagenic activity to cells was decreased. We hypothesize that these desirable reSUitS may be caused by BV-araU functioning as a chain terminator or an inhibitor of D N A polymerase, Acknowledgements

We gratefully thank Professor M. Kuwano for helpful discussion and C. Hatanaka for the excel-

lent assistance in this study, References Allaudeen, H.S., M.S. Chen, J.J. Lee, E. De Clercq and W.tl. Prusoff (1()82) Incorporation of ~.5-(2-halogenovinyl).2', deo~uridines into deoxyribonucleic acids of herpes s i m pies virus type I-infected cells. J Biol. Chem, 257, 603-606. Ashman, C.R., G.P.V. Reddy and R,L. Davidson (It)81) Bromodeoryuridine mutagenesis, ribonucleofide reductase activity, and deoryribonucleofide pools in hydroxyurea-resistant mutants, Somat. Cell Genet., 7. 751-768. Ayisi. N,K.. R.A. Wall, R.J. Wanklin, H. Machida, E. De Clercq and S.L. Sacks (1987) Comparative metabolism of ~-5-(2-bromovinyl)-2'.deoxyuridine and I-p-warabinofuranosyI-u-5-(2-bromovinyl)uracil in herpes simplex virus-infected cells, Mol. Pharmacol,, 31,422-429. Chen. M.S., L.A. Amico and D.J. Speelman (1984) Kinetics of the interaction of monophosphates of the antiviral nucleosides 2'-fluoro-l-#-D-arabinofuranosylpyrimidine and (~,)5-(2-bromovinyl)-2'-dcoxyuridinewith thymidylate kinases from veto cells and herpes simplex virus types I and 2, AntimicrobialAgentsChemother.,26, 778-780. Cheng, Y..C., G. Dutschman, J.J. Fox, K,A. Watanabe and H, Machida (1981) Differential activity of potential antiviral nucleoside analogs on herpes simplex virus-induced and

human cellular thymidine kinases, Antimicrobial Agents Chemother.. 20, 420-423. Davidson,R.L., and E.L. Kaufman (1978) Bromodeoxyuridine mutagenesis in mammalian cells is stimulated by thymidine and suppressed by deoxycytidine, Nature (London), 276. 722-723. Davidson,R.L., P. Broeker and C.R. Ashman (1988) DNA base sequence changes and sequence specificity of bromodeoxyuridine-induced mutations in mammalian cells, Proc. Natl. Acad. Sci. (U.S.A.), 85, 4406-4410. De Clercq, E., J. Descamps, P. De Somer, P.J. Barr, A.S. Jones and R.T. Walker (1979) (E)-5-(2-Bromovinyl)-2'-deoxyuridine: a potent and selective anti-herpes agent, Proc. Natl. Acad. Sci. (U.S.A.), 76, 2947-2951. Freese, E. (1959) The specific mutagenic effect of base analogues on phage T4, J. Mol. Biol., I, 87-105. Fyfe, J.A. (1981) Differential phosphorylation of (E)-5-(2bromovinyi)-2'-deoryuridine monophosphate by thymidylate kinases from herpes simplex viruses types 1 and 2 and varicella zoster virus, Mol. Pharmacol., 21,432-437. Gupta, R.S., and L. Siminovitch (1980) Genetic markers for quantitative mutagenesis studies in Chinese hamster ovaq., cells. Characteristics of some recently developed selective systems, Mutation Res., 69, 113-126. Huszar, D., and S. Bacchetti (1981) Partial purification and characterization of the ribonucleotide reductase induced by herpes simplex virus infection of mammalian cells, J. Virol., 37, 580-588. Jamieson, A.T., and G. Bjursell (1976) Deoxyribonucleoside triphosphate pools in herpes simplex type i infected cells, J. Gen. Virol., 31, 101-113. Kaufman,E.R., and R.L. Davidson (1978) Bromodeoxyuridine mutagenesis in mammalian cells: mulagenesis is independent of the amount of bromourucil in DNA. Proc. Natl, Acad. Sci. (U.S.A.), 75, 4982-4986. Koyama, H., I). Ayusawu, M. Okuwa. A. Takatsuki and G. Tamura (1982) Tunicamycin-resistant mutations in mouse FM3A cells, Mutation Res., 42. 243-258. Langelier, Y., and G. Buttin (1981) Characterization of ribonucleotide reductase induction in BHK-21/CI3 Syrian hamster cell line upon infection by herpes simplex virus (HSV), J. Gen. Virol., 57, 21-31. Larsson,A., B. Oberg, S. Alenius, C.-E. Hagberg, N.-G. Johans~n, B. Lindborg and G. Stening (1983) 9-(3,4.Dihydro~butyl)guanine, a new inhibitor of herpesvirus multiplication, Antimicrobial Agents Chemother., 23, 664-670. Lin, J.-C., and H. Machida (1988) Comparison of two bmmovinyl nucleoside analogs, 1o/3-D-arabinofuranosyl-~-5(2-bromovinyl)uracil and E-5-(2-bromovinyl)-2'-deoxyuridine, with acylovir in inhibition of Epstein-Barr virus replication, Antimicrobial Agents Chemother,, 32, 1068-1072. Ling, V., and L,H. Thompson 0974) Reduced permeability in CHO cells us a mechanism of resistance to colchicine, J. Cell Physiol,, 83, 103-116, Machida, H, (1986) Comparison of susceptibilities of varicella-zoster virus and herpes simplex viruses to nucleoside analogs, Antimicrobial Agents Chemother,, 29, 524526.

131 Machida, H., S. Sakata, A. Kuninaka and H. Yoshino (1981) Antiherpesviral and anticellular effects of l-/3-D-arabinofuranosyI-E-5-(2-halogenovinyl)uracils, Antimicrobial Agents Chemother., 20, 47-52. Nakayama, K., J,L. Ruth and Y.-C. Cheng (1982) Differential effect of nucleoside analog triphosphates on ribonucleotide reductases from uninfected and herpes simplex virus-infected HeLa cells, J. Virol., 43, 325-327. Ruth, J.L., and Y.-C. Cheng (1981) Nucleoside analogues with clinical potential in antivirus chemotherapy. The effect of several thymidine and 2'-deoxycytidine analogue 5'-triphosphates on purified human (a, [J) and herpes simplex virus (type 1, 2) DNA polymerase, Mol. Pharmacol., 20, 415-422. Shigeta, S., T. Yokota, T. lwabachi, M. Baba, K. Konno, M. Ogata and E. De Clercq (1983) Comparative efficacy of

antiherpes drugs against various strains of variceila-zoster virus, J. Infect. Dis., 147, 576-584. Suzutani, T., H. Machida, T. Sakuma and M. Azuma (1988a) Effects of various nucleosides on antiviral activity and metabolism of l-~-D-arabinofuranosyI-E-5-(2-bromovinyl) uracil against herpes simplex virus types 1 and 2, Antimicrobial Agents Chemother., 32, 1547-1551. Suzutani, T., H. Machida and T. Sakuma (1988b) Efficacies of antiherpesvirus nucleosides against two strains of herpes simplex virus type 1 in vero and human embryo lung fibroblast cells, Antimicrobial Agents Chemother., 32, 1046-1052. Watson, J.D., N.H. Hopkins, J.W. Roberts, J.A. Steitz and A.M. Weiner (Eds.) (1987) Molecular Biology of the Gene, Benjamin/Cummings, Menlo Park, CA, pp. 343-345.

Analysis of toxic and mutagenic activities of antiherpesvirus nucleosides against HeLa cells and herpes simplex virus type 1.

The toxic and mutagenic activities of five antiherpesvirus agents to HeLa cells and herpes simplex virus type 1 (HSV-1) were investigated. 5-Iodo-2'-d...
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