Vol. 25, No. 6ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, June 1984, p. 742-7460066-4804/84/06074.-05$02.O00/0Copyright © 1984, American Societv for Microbiology

Strains of Varicella-Zoster Virus Resistant to 1-13-D-Arabinofuranosyl-E-5-(2-Bromovinyl)Uracil

TAKASHI SAKUMADepartment of Microbiology, Asahikawa Medical College, Asahikawa 078-11, Japan

Received 12 December 1983/Accepted 27 March 1984

Strains of varicella-zoster virus resistant to 1-p-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil (BV-araU)were isolated from varicella-zoster virus-infected Vero cells which were pulse-treated with 5-iododeoxyuri-dine or 5-bromodeoxyuridine or both and then treated with BV-araU. These BV-araU-resistant strains (BV-araUr) could not be isolated from varicella-zoster virus-infected cells treated with BV-araU alone and hadreduced viral thymidine kinase activity. Two offive BV-araUr strains were also resistant to 5-icdodeoxyuri-dine and 5-bromodeoxyuridine, whereas other BV-araUr strains were relatively susceptible to these drugs.All clones from the BV-araUr strain were susceptible to 1-p-D-arabinofuranosylcytosine and phosphonoace-tic acid, but 7 of 10 clones from the BV-araUr strain were resistant to 1-p-D-arabinofuranosyladenine. Thepossible mechanisms of induction of BV-araU resistance are discussed.

Molecular analysis of the viral genome and its relation toviral replication has been extensively studied in herpessimplex virus (HSV), Epstein-Barr virus, and cytomegalovi-rus of the family Herpetoviridae (8, 10, 12, 20, 22, 25, 30).Dubbs ahd Kit (9) have reported a mutant strain ofHSV thatis deficient in viral thymidine kinase-inducing activity, andrecently, many kinds of mutant strains of HSV have beenreported (4, 5, 15, 29).

Until recently, the study of the molecular properties ofvaricella-zoster virus (VZV) has been greatly hampered dueto problems in the isolation offree virions with high infectiv-ity and in the purification of viral DNA. A few results on thesusceptibility and resistance of VZV to anti-herpesvirusagents have been reported (7, 26, 32). Yokota et al. (32) haveisolated a mutant strain of VZV, which was deficient in viralthymidine kinase-inducing activity, from cells persistentlyinfected with VZV in the presence of 5-bromo-2'-deoxyuri-dine. Recently, Machida et al. (18, 19) have shown that 1-,B-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil (BV-araU) hasan extremely high antiviral index to HSV type 1 and VZV;the ratio of the 50% cytopathogenic dose (in micrograms permilliliter) for human embryonic lung (HEL)-F cells to the50% plaque reduction dose (in micrograms per milliliter) forviruses was about 106.The purpose of this work was to search for resistant

strains of VZV to BV-araU and to analyze the characteris-tics of these strains.

MATERIALS AND METHODS

Compounds. BV-araU was kindly supplied by H. Ma-chida, Research Laboratories, Yamasa Shoyu Co., Ltd.,Choshi, Japan. 5-Iododeoxyuridine (IUdR) and 5-bromo-deoxyuridine (BUdR) were purchased from Wako Chemical,Ltd., Osaka, Japan; 1-p-D-arabinofuranosylcytosine (araC)was obtained from Nippon Shinyaku Pharmaceuticals, Ltd.,Kyoto, Japan; and phosphonoacetic acid (PAA) was ob-tained from Calbiochem-Behring, San Diego, Calif. 9-13-D-Arabinofuranosyladenine (araA) was a gift from MochidaPharmaceutical Co., Ltd., Tokyo, Japan. [methyl-3H]thymi-dine (48 Ci/mmol) was purchased from The RadiochemicalCentre, Amersham, England.

Cell cultures. HEL cells were prepared by trypsinizationof HEL in our laboratory and used at passages 10 to 15.

Guinea pig embryo cells were prepared in our laboratory andused at passages 3 to 5. Vero cells and WI-38 cells werepurchased from Flow Laboratories, Inc., Rockville, Md. Allcells were cultivated with Eagle minimum essential mediumcontaining 10% newborn calf serum, kanamycin (60 ,ug/ml),and 0.075% NaHCO3. Viral infection was carried out byusing Eagle minimal essential medium supplemented with2% newborn calf serum (maintenance medium) at 370C.

Viruses. The Oka strain ofVZV was kindly supplied by M.Takahashi, Research Institute for Microbial Diseases, OsakaUniversity, Osaka, Japan. The YS strain was isolated in ourlaboratory on HEL cells from vesicular fluid of a patientwith varicella (H. Kawai, T. Sakuma, K. Sasaki, M. Sato,H. Watanaloe, M. Honda, S. Ozaki, and M. Azuma, manu-script in preparation). BV-araU-resistant (BV-araUr) strainsYSR, 03, 07, 018, and 026 were obtained as follows. Verocell sheets (30- by 50-mm bottle) were infected with thestocked YS-infected cells (cell-to-cell infection) at a multi-plicity of 0.1. After adsorption for 1 h at room temperature,the cell sheets were refed with maintenance medium contain-ing 10 to 20 ,ug of IUdR or BUdR or both per ml andincubated for 24 h at 37°C. The cell sheets were washed withphosphate-buffered saline solution (pH 7.4), replenishedwith fresh maintenance medium, and incubated for 48 h at370C. Then, 0.1 to 1 jig of BV-araU (200 to 2,000 times theMIC for parent YS strain) per ml was added and cultivateduntil a cytopathogenic effect (CPE) was apparent. One or afew small plaque-like CPE was observed on the Vero cellsheets about 7 days after infection, and after an additional 2or 3 days, the CPE was dominant. The virus-infected cellswere harvested by trypsinization. The fresh Vero cell sheets-were inoculated with the infected cells and cultivated withBV-araU-containing maintenance medium alone until a CPEwas observed. In this step, the apparent CPE (about 20 to30%) was observed 2 or 3 days after infection. The laterpassage procedure was repeated two more times. The infect-ed cells obtained as described above were stored at -700Cwith Eagle minimal essential medium containing 10% new-born calf serum supplemented with 10% dimethyl sulfoxide.Cloning from the resistant strain was carried out by theplaquing method with free virus of VZV in a mediumcontaining 1 ,ug of BV-araU per ml.

Cell-free virus was prepared as follows. The VZV-infected

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HEL cell sheet showing about 80% CPE was scraped with arubber policeman. The cells were suspended in SPGA (0.218M sucrose, 3.8 mM KH2PO4, 7.2 mM K2HPO4, 4.9 mMsodium glutamate, and 1% bovine albumin) and sonicated ina Kubota ultrasonic cell disruptor (Kubota, Tokyo, Japan) at20 kc/s for 30 s. The suspension was centrifuged at 3,000 rpmfor 15 min, and the supernatant fluid was used as the cell-freevirus.

Assay for MIC of various test compounds. Assay forantiviral activity of compounds to VZV was carried out bythe 50% plaque reduction method as follows. Confluentmonolayers of HEL cells grown in a 24-well microplate(Corning Glass Works, Corning, N.Y.) were infected withabout 50 PFU of free virus in 0.1 ml of SPGA per well. After2 h of incubation at 37°C, SPGA was discarded, and theinfected cells were replenished with 1.0 ml of maintenancemedium containing a serially diluted test compound. Dupli-cate wells for each dilution were used. The infected cellswere incubated at 37°C for 7 days, and the number of plaquesin each well was counted with a microscope. The plaquecounts were expressed as a percentage of the numberobtained in similar control wells and were plotted against thelogarithm of the concentration of compound to give dose-response lines, from which the MIC showing 50% plaquereduction was calculated.

Pulse-labeling experiment and glycerol gradient analysis ofviral DNA. HEL cell monolayers grown in petri dishes(diameter, 52 mm) were infected with VZV at a multiplicityof infection of 0.16 per cell. After 18 h at 37°C, the mediumwas replaced with a maintenance medium containing[3H]thymidine (4 pCi/ml). The radioactive medium was

removed 2 h later, and the cells were washed twice andincubated with fresh medium for 6 h. The VZV DNA was

obtained by the Hirt extraction method as described previ-ously (16, 24). Briefly, the cells were washed twice withphosphate-buffered saline and lysed by adding 1 ml ofsolvent (0.6% sodium dodecyl sulfate, 0.01 M Tris [pH 7.5],and 0.01 M EDTA) to each culture. After 15 min at room

temperature, the lysate was poured into a centrifuge tube,and 0.25 ml of 5 M NaCl was added, gently mixed within thetube, and maintained at 4°C for over 8 h. The supernatantfluid was separated by centrifugation at 10,000 x g for 30min at 4°C and centrifuged on a 10 to 30% neutral glycerolgradient (1.0 M NaCl, 0.02 M Tris [pH 7.5], and 0.001 MEDTA) at 105,000 x g for 5 h at 20°C in a Hitachi preparativeultracentrifuge (65P-7) equipped with a RPS-27-2 rotor.Forty-drop fractions were collected, and 180 of eachfraction was added to 20 of cold 50% trichloroacetic acid.The precipitates were collected and washed with 5% tri-chloroacetic acid on glass microfiber filter papers; the tri-chloroacetic acid-insoluble radioactivity was measured in a

Packard 2650 liquid scintillation counter.Thymidine kinase assay. Preparation of enzyme extracts

and thymidine kinase assay were performed as describedpreviously (14, 17, 21, 28). VZV-infected cells and mock-infected cells were harvested by trypsinization and pouredinto a tube. The cells (107) were washed three times withphosphate-buffered saline and once with TMT buffer (50 mMTris [pH 7.5], 5 mM mercaptoethanol, and 5 p.M thymidine)by centrifugation at 1,500 rpm for 5 min at 4°C. The cellswere suspended in 1 ml of TMT buffer and disrupted byultrasonic vibration for 30 s in a Kontes sonicator at a powerof 8 and a tune of 2.5. The sonicated cells were centrifuged at100,000 x g for 30 min at 4°C. The supernatant fluid wasused as the crude enzyme material.The reaction mixture contained 13 mM ATP, 13 mM

MgCl2, 0.26 M Tris-hydrochloride (pH 8.0), and 1 jiCi of[3H]thymidine (0.2 p.M) plus 25 ,ul of enzyme extract in atotal volume of 75 ,ul. After 15 min at 37°C, the reaction wasterminated by the addition of 20 ,u1 of cold 50% trichloroace-tic acid. Reaction products were separated by spotting 20 ,u1of the terminated reaction mixture onto Whatman DE-81 25-mm paper disks. These disks were then washed by suctionfiltration twice with 4 ml of 4 mM ammonium acetatecontaining 5 ,uM thymidine, once with 4 ml of 4 mMammonium acetate, once with 5 ml of distilled water, andonce with 4 ml of 95% ethanol. The radioactivity retained onthe disks was measured with a liquid scintillation counter.

RESULTSSusceptibility of parent VZV strains and BV-araUr strains

to DNA synthesis inhibitors. Susceptibility of parent strainsof VZV, Oka and YS, which were passaged in different cellcultures to various drugs is shown in Table 1. No change insusceptibility to drugs was observed in these parent strains.The YS strain was passaged three times in Vero cells in

the presence of IUdR or BUdR or both for a short period oftime, and in the presence of BV-araU as described above.Five strains obtained from 10 experiments, YSR, 03, 07, 018,and 026, had a high resistance to BV-araU (hereafter re-ferred to as the BV-araUr strain) (Table 2). MICs of BV-araU to these strains were 4 to 10 ,ugIml. No BV-araUr strainwas isolated from the infected cells treated with BV-araUalone (data not shown).

Incorporation of [3H]thymidine into VZV-infected cells andVZV DNA. Earlier reports (1, 9, 11) have indicated thatseveral halogenated nucleoside-resistant strains of herpessimplex virus were defective in viral-specific thymidinekinase. To determine whether the BV-araUr strain of VZVwas defective in VZV-specific thymidine kinase, incorpo-ration of [3H]thymidine into infected cells and into viralDNA was investigated. Incorporation of thymidine wasseven times greater in strain YS-infected cells than in mock-infected cells, indicating an induction of viral thymidinekinase (Table 3). However, thymidine was incorporated intocells infected with strain YSR, which is BV-araUr, at thesame level as mock-infected cells. Thymidine incorporationinto strain YS DNA was significant, but not into YSR strainDNA (Fig. 1).Thymidine kinase activity in VZV-infected cells. The results

presented above suggest that strain YSR was defective inviral thymidine kinase. Therefore, the thymidine kinaseactivity in infected cells was measured. In the wild-type YSstrain- and Oka strain-infected cells, viral thymidine kinaseactivity increased markedly, but reduced viral thymidinekinase activity was observed in cells infected with BV-araUrstrains (Table 4).

TABLE 1. Susceptibility of Oka and diverse passage lines ofVZV to various test compounds

MIC (pLg/ml) for 50% PFU reduction for:Straina

BV-araU araA araC IUdR BUdR PAA

Oka-H12 0.0006 1.2 0.13 0.4 0.5 7.4YS-W1 H13 0.0005 1.0 0.13 0.3 0.5 7.0YS-V1O H12 0.0005 0.9 0.20 0.3 0.4 7.1YS-W3 G19 H3 0.0006 0.8 0.15 0.4 0.5 3.7

a Abbreviations: H12, passage 12 in HEL cells; Wl, passage 1 in WI-38cells; H13, passage 13 in HEL cells; V10, passage 10 in Vero cells; W3,passage 3 in WI-38 cells; G19, passage 19 in guinea pig embryo fibroblast cells;H3, passage 3 in HEL cells.

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TABLE 2. Establishment of BV-araUr strains of VZV

Treatment witha: MIC (pg/ml) ofStrain BV-araU for

IUdR BUdR BV-araU 50%t PFU reduction

03 20 0.lb 5.207 20 0.1 4.7

018 20b 0.1 7.4026 10 10 0.1 10.0YSR 10 10 1.0 5.8YS 0.0005

a Establishment procedures are as described in the text.b Drug concentration (in micrograms per milliliter) in maintenance medium.

The susceptibility of these strains to BV-araU, IUdR, andBUdR is also shown in Table 4. Strains YSR and 018 of BV-araUr strains showed high resistance to IUdR and BUdR andthe same thymidine kinase activity to that of mock-infectedcells. Other BV-araUr strains had low resistance to IUdRand BUdR and had two to three times the activity ofthymidine kinase than that of mock-infected cells.

Cloning of BV-araUr strains and thymidine kinase activityin cells infected with clones. Cloning of BV-araUr strains 03,07, 018, and 026 was carried out, and all 10 clones obtainedwere resistant to more than 0.6 ,ug of BV-araU per ml,suggesting that the characteristics of BV-araUr are a stablemutation. Thymidine kinase activity in cells infected withthese clones was assayed. Again, all clones obtained hadreduced viral thymidine kinase activity (Table 5).

Susceptibility of clones of BV-araUr strains to various DNAsynthesis inhibitors. To obtain some information on thecharacteristics of BV-araUr strains, their susceptibility toother DNA synthesis inhibitors was next examined. Allclones of BV-araUr strains were sensitive to araC and PAA;however, MICs of araA for 7 of 10 clones were more thanthree times that for strain YS, and these araAr clones wereobtained randomly from the parent BV-araUr strains (Table6).

Patterns of dose response of clone 07-1 and 018-1 tovarious inhibitors are shown in Fig. 2. Clones 07-1 and 018-1are typical strains resistant to BV-araU and araA. Clone 018-1 is also resistant to IUdR and BUdR, although clone 07-1 isless resistant to these drugs, indicating characteristics ofparent strains 07 and 018 as shown in Table 4. These resultsraise the question as to whether there is some relationshipbetween the mechanism of resistance to BV-araU and araA.The thymidine kinase activities of cells infected with clones07-1 and 018-1 in the presence of BV-araU or araA wereexamined. No competitive inhibition of thymidine kinaseactivity with araA was observed either in the cells infectedwith strain araAr or in the cells infected with strain YS (data

TABLE 3. Incorporation of [3H]thymidine into VZV-infectedcellsa

Incorporation of [3H]thymidine into VZV-infected cells at theCells following temp:

infected 33°C 37°C 39°Cwith

cpm Ratiob cpm Ratio cpm Ratio

Mock 4,692 1.00 10,222 1.00 17,563 1.00YS 25,046 5.34 76,901 7.52 55,921 3.18YSR 5,846 1.25 10,147 0.99 17,672 1.01

a Infected cells were incubated at 33, 37, and 39°C for 18 h and pulse-labeledat the same temperature for 4 h and then acid-insoluble radioactivity wasmeasured.

b Ratio of [3Hlthymidine incorporation in VZV-infected cells to that inmock-infected cells.

301

201[CV)

lo0

II

I

Os

III

I

IQKI

lIIIIsII

30 ci0

101Qz-

la C2

10 20Fraction no.

FIG. 1. Incorporation of [3H]thymidine into VZV DNA. HELcells were infected with the YS and YSR strains at a multiplicity of0.1. After 18 h of incubation 4 ,Ci of [3H]thymidine per ml wasadded to the cultures, incubated for 2 h, and then washed withphosphate-buffered saline. The cells were harvested, and the viralDNA fraction was extracted by the Hirt method as described in thetext. The viral DNA fraction was laid on 10 to 30%6 (vol/vol) neutralglycerol gradient and centrifuged at 105,000 x g for 5 h at 20°C in aHitachi preparative ultracentrifuge (65P-7) equipped with a RPS-27-2 rotor. Symbols: 0, YS DNA; *, YSR DNA.

not shown), indicating that the mechanisms of resistance ofBV-araUr and araAr are different.

DISCUSSION

The results reported here show that the BV-araUr strain ofVZV was not obtained from the wild-type strain-infectedcells in the presence of BV-araU alone, whereas BV-araUrmutants appeared easily when the infected cells were treatedwith IUdR or BUdR or both for a short term beforetreatment with BV-araU. BV-araU may be an effectiveantiviral drug for HSV and VZV infection if used alone. Themechanism of the action of IUdR and BUdR is through itscompetitive inhibition of the enzymes involved in the syn-thesis of DNA (thymidine kinase, thymidylate kinase, andDNA polymerase [27]), and the most significant mechanismof action of these drugs is thought to be their incorporationinto DNA (23). The results of this study show that all of theBV-araUr strains were reduced in viral thymidine kinaseactivity. BV-araU had a high antiviral index (18, 19), sug-

TABLE 4. Thymidine kinase activity in cells infected with VZVstrains and their susceptibilities to various drugs

Cells Relative thymidine MIC (pg/ml) for 50% PFUinfected kinase activity in reduction for:with infected cells BV-araU IUdR BUdR

Mock 1.0Oka 67.2 0.0006 0.4 0.5YS 64.6 0.0005 0.3 0.5YSR 1.2 5.8 1.9 8.003 2.2 5.2 0.78 1.507 3.0 4.7 0.39 3.0018 1.0 7.4 1.6 7.6026 2.7 10.0 0.78 3.0

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TABLE 5. Thymidine kinase activity in cells infected with clonesderived from BV-araUr strains

MIC (ug/ml) of Relative thymidineClone BV-araU for kinase activity in

50% PFU reduction infected cells

Mock 1.0YS 0.0005 64.603-1 3.6 1.503-2 8.0 2.003-3 1.0 3.203-4 10.0 1.803-5 0.9 3.003-6 0.6 1.807-1 4.7 3.0018-1 7.0 1.2018-2 10.0 0.7026-1 10.0 2.7

gesting that this drug may have a higher affinity to viralthymidine kinase than to cellular thymidine kinase. Theseresults suggest that IUdR and BUdR were incorporated intoDNA as a thymidine analog which may cause misreading inits replication process. If so, when BV-araU is used ascombination therapy with IUdR and BUdR, BV-araU mightselect the negative mutants of viral thymidine kinase fromseveral kinds of mutants. When BV-araU is used alone, itmay incorporate more effectively into viral DNA. It thenacts as a chain terminator like other arabinosyl nucleosides(13) and causes a lethal mutation, making it difficult for theBV-araUr strain to appear. BV-araUr strains may containseveral mutants in viral DNA synthesis enzymes, such asdeletion of thymidine kinase, altered substrate affinity ofthymidine kinase, and others. Veerisetty and Gentry (29)have shown that the resistant mutant of HSV type 1 to 5-methoxymethyldeoxyuridine had an altered substrate affini-ty of thymidine kinase. Darby et al. (6) and Weinmaster et al.(31) have reported similar phenomena about the affinity ofthymidine kinase induced by herpesvirus. It may be assumedthat in strain YSR and clone 018, coding for thymidinekinase is deleted because of high resistance to BV-araU,IUdR, and BUdR and the same thymidine kinase activitylevel as that in mock-infected cells (Table 4 and Fig. 2).Meanwhile, clones 03, 07, and 026 may be mutations in theiraltered substrate affinity for thymidine kinase, because theseclones show high resistance to BV-araU but less resistanceto IUdR and BUdR, and they have a slightly higher thymi-dine kinase activity than that of mock-infected cells (Table 4and Fig. 2).

TABLE 6. Susceptibility of BV-araUr clones to various DNAsynthesis inhibitorsMIC (,ug/ml) for 50% PFU reduction for:

CloneBV-araU araA araC PAA

YS 0.0005 1.0 0.13 7.0YSR 5.8 1.2 0.15 4.703-1 3.6 0.7 0.20 7.403-2 8.0 3.2 0.28 5.403-3 1.0 3.2 0.19 4.503-4 10.0 0.8 0.20 3.903-5 0.9 3.1 0.21 7.403-6 0.6 3.2 0.16 4.407-1 4.7 3.2 0.18 3.7018-1 7.0 5.1 0.08 3.9018-2 10.0 1.1 0.15 4.1026-1 10.0 3.4 0.12 8.0

Several virus-specific enzyme activities have been identi-fied in cells infected with the herpesviruses (2). The enzymesare biochemically and kinetically distinct from their host cellenzymes and become potential targets for selective antiviralcompounds. Therefore, some mutation of these enzymegenes may cause an appearance of virus resistant to thecompound. Many resistant strains of herpes simplex virushave been reported, and the mechanisms of their formationhave been discussed (3-6, 15, 29). However, in only a fewreports has a drug-resistant mutation ofVZV been discussed(7, 26, 32). The data presented here demonstrate differentkinds of VZV mutant strains. All of these mutants signifi-cantly reduced thymidine kinase activity, but 7 of these 10clones were resistant to araA. This high ratio of cross-resistance between BV-araU and araA suggests that the viralthymidine kinase-coded gene may be closely linked to araAresistance.

Further biochemical and molecular studies of the viralDNA synthesis enzymes of these BV-araUr strains are inprogress.

ACKNOWLEDGMENTSI would like to thank M. Azuma, Department of Microbiology,

Asahikawa Medical College, for instructive discussions and advice;D. C. Burke, Allelix Inc., Ontario, Canada, for critical reading of the

11

0° 101c

-ol

co

'r 10(co

-3 -2 -1 0 1 2 -2 -1 0Log10 ,uyg/mI of drug

1 2

FIG. 2. Patterns of dose response of the wild-type YS strain andBV-araUr strains (clone 07-1 and 018-1) to various drugs. The cellswere inoculated with 50 PFU of VZV. PFU in the presence of BV-araU, IUdR, BUdR, araC, araA, and PAA were compared withPFU in control culture. Symbols: 0, YS strain; 0, 07-1 clone; A,018-1 clone.

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manuscript and advice; and H. Machida, Research Laboratories,Yamasa Shoyu Co. Ltd., Choshi, Japan, for generously supplyingBV-araU. The technical assistance of C. Hatanaka and T. Aoyanagiis most appreciated.

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