Thymidine Kinase Activity - Antimicrobial Agents and Chemotherapy

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, June 1986, 0066-4804/86/061053-06$02.00/0 Copyright © 1986, American Society for Microbiology

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Vol. 29, No. 6

Characterization of a Varicella-Zoster Virus Variant with Altered Thymidine Kinase Activity SHIRO SHIGETA,l* SYUICHI MORI,' TOMOYUKI YOKOTA,' KENJI KONNO,' AND ERIK DE CLERCQ2 Department of Bacteriology, Fukushima Medical College, Fukushima 960, Japan,' and Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Louvain, Belgium2 Received 2 December 1985/Accepted 21 March 1986

A varicella-zoster virus (VZV) strain resistant to 5-iodo-2'-deoxyuridine (IdUrd) and 5-bromro-2'deoxyuridine (BrdUrd) but sensitive to (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVdUrd) and (E)-5-(2iodovinyl)-2'-deoxyuridine (lVdUrd) was isolated. The 2'-deoxythymidine (dThd) kinase of this mutant (Ito) strain was characterized; it was much less efficient in phosphorylating dThd, 2'-deoxycytidine, and BrdUrd than were the dThd kinases from wild-type (CaQu, Kobayashi) VZV strains. The Ito dThd kinase had a markedly decreased affinity for dThd, 2'-deoxycytidine, and BrdUrd but only a slightly decreased affinity for IVdUrd than had the wild-type VZV dThd kinase. BrdUrd was incorporated to a much lesser extent in VZV (Ito strain)-infected cells than wild-type VZV-infected cells, but IVdUrd was incorporated in Ito VZV-infected cells as efficiently as in wild-type VZV-infected cells. While resistant to IdUrd and BrdUrd, the Ito strain was susceptible to inhibitors of de novo thymidylate biosynthesis such as aminopterin.

Varicella-zoster virus (VZV) is the causative agent of both chicken pox (varicella), a primary VZV infection, and shingles (zoster) upon reactivation of the latent virus. Both diseases show a relatively mild and transient course in immunocompetent individuals, but in immunocompromised patients they can be severe and occasionally fatal. Because of their immunodeficiency, these patients may not adequately respond to immunization with VZV vaccines. A live attenuated VZV vaccine was developed by Takahashi and his colleagues and repotted to be useful in the prophylaxis of chicken pox in immunocompromised patients (11, 17). However, the vaccine cannot be used therapeutically. Immunoglobulin G antibody to VZV could be considered as a means to prevent or modify the development of severe VZV infection in immunocompromised patients; however, this material is scarce and of questionable value. Recently, several antiherpes compounds have been investigated for their in vitro activity against VZV replication, and from these compounds (E)-5-2-(bromovinyl)-2'-deoxyuridine (BVdUrd) emerged as one of the most potent and selective inhibitors of VZV replication (4, 16). Its potency against VZV exceeded that of acyclovir [9-(2-hydroxyethoxymethyl)guanine] (ACV) by 2 to 3 orders of magnitude (4, 16). To be effective as inhibitors of VZV replication, BVdUrd and ACV must be phosphorylated to the monophosphate (ACV) or diphosphate (BVdUrd) by the virus-encoded 2'deoxythymidine (dThd) kinase (TK) (9, 10, 13). Upon further phosphorylation to the triphosphates by a cellular enzyme(s), BVdUrd and ACV act as inhibitors of the viral DNA polymerase and, to a lesser extent, cellular DNA polymerases. We have recently isolated a VZV strain which is deficient in TK-inducing activity and resistant to BVdUrd, ACV, and several other antiherpes compounds such as 5-iodo-2'deoxyuridine (IdUrd) and 5-bromo-2'-deoxyuridine (BrdUrd). This VZV mutant (Kanno-Kohmura) strain *

proved completely deficient in phosphorylating BVdUrd and its congeners to their active forms (16, 19). When comparing the susceptibility of several laboratory and clinical strains of VZV to a number of antiherpes compounds, we detected an IdUrd- and BrdUrd-resistant VZV strain which was still TK positive (16). This VZV (Ito) strain was sensitive to BVdUrd, ACV, and other nucleoside analogs. The characteristics of the Ito strain and the biochemical basis for its resistance to IdUrd and BrdUrd are documented in this report. MATERIALS AND METHODS Viruses. The Kobayashi and Ito strains of VZV were isolated from patients with herpes zoster and chicken pox, respectively, and the CaQu strain was kindly provided by N. J. Schmidt, California Department of Health, Berkeley. The TK-deficient (TK-) (Kanno-Kohmura) strain of VZV was isolated as described previously (19). All VZV strains, except for the Kanno-Kohmura strain, induced TK activity in human embryo fibroblast (HEF) cultures, and the VZV was prepared as cell-free virus and stored at -80°C until used. Cell cultures. HEF cells were grown in Eagle minimum essential medium supplemented with 10% heat-inactivated newborn calf serum, 100 U of penicillin G per ml, and 100 jig of streptomycin per ml (growth medium). For the virusinfected cell cultures growth medium was replaced by minimal essential medium supplemented with 2% heat-inactivated fetal calf serum and antibiotics at the concentrations indicated above (maintenance medium). Compounds and reagents. IdUrd and BrdUrd were purchased from Sigma Chemical Co., St. Louis, Mo., and (E)-5-(2-iodovinyl)-2'-deoxyuridine (IVdUrd) was synthesized by R. Busson and H. Vanderhaeghe at the Rega Institute of the Katholieke Universiteit Leuven, Louvain, Belgium. The radiolabeled precursors 2'-deoxy-[63H]thymidine ([6-3H]dThd) (specific radioactivity, 25 Ci/ mmol), 5-bromo-2'-deoxy-[1',2'-3H]uridine ([1'-2'3H]BrdUrd) (specific radioactivity, 28 Ci/mmol), and 2'deoxy-[5-3H]cytidine ([5-3H]dCyd) (specific radioactivity, 20

Corresponding author 1053

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TABLE 1. Inhibitory effect of antiviral compounds on focus formation by different VZV strains ID50 (,uM)

Virus strain

CaQu Kobayashi Ito TK-

ID90 (,uM)

IdUrd

BrdUrd

BVdUrd

IVdUrd

IdUrd

BrdUrd

0.84 0.72 25 49

1.2 2.9 30 27

0.026 0.024 0.012 148

0.005 0.003 0.002

1.9 1.9 50 210

2.4 9.8 60

171

Ci/mmol) were from Amersham International plc, Buckinghamshile, England. ['251]IVdUrd (specific radioactivity, 32 Cilmtnol) was synthesized by C. Tulien and A. Verbruggen at the Laboratory of Nuclear Medicine and Radiopharmaty of the Katholieke Universiteit Leuven. Antiviral.activity. Inhibition of VZV focus formation was measured as described previously (16). HEF monolayers in plastic trays with 24 wells of 1.6-em diameter were infected with approximately 50 PFU of VZV. Immediately after virus inoculation, 1 ml of maintenance medium containing various concentrations of the conmpounds was added. The monolayers were incubated at 35°C for 3 days, and VZV foci were enumerated under a nmicroscope with 40-fold magnification. The concentration of compound required to inhibit the number of VZV foci by 50% was determined as the 50% inhibitory dose (ID50). Incorporation of radiolabeled nucleosides. HEF monolayers in weils of 1.6-cm diameter were infected with VZV at a multiplicity of infection of 0.16 and incubated in maintenance medium at 36°C for 24 h in a CO2 incubator. The cells were then washed with maintenance medium, and [63H]dThd, [1'-2'-3H]BrdUrd, or [1251I]IVdUrd was added at a

BVdUrd

IVdUrd

0.075 0.046 0.024

0.02 0.014 0.004 -380

.336

65

concentration of 1 ,uCi/ml in maintenance medium. The cells were further incubated at 36°C in a CO2 incubator for 0, 5,

10, 15, or 20 h. For each well, both culture medium and cells (3 x 105) were harvested. The samples were then treated with 0.1% trypsin and, after the addition of 1 ml of 5% trichloroacetic acid, applied onto glass fiber filters (Toyo GC-94) and washed three times with 5% TCA and twice with 95% methanol. The filters were dried and examined for radioactivity in a liquid scintillation counter. The amount of radioactivity measured at 0 h was subtracted from all subsequent radioactivity counted. TK preparations. Confluent HEF monolayers were infected with VZV at a multiplicity of infection of 0.16 and incubated at 36°C for 48 h. By then the viral cytopathogenicity had reached 90 to 100%. The cells were dispersed by glass beads and collected in cold saline, and finally suspended at 107 cells per ml in 50 mM Tris buffer (pH 7.4) contaihing 5 mM 2-mercaptoethanol, 2 mM ATP, and 2 mM MgCl2. The cells were disrupted by four freeze-thawings (-80°C and 37°C), and the cell homogenates were centrifuged at 100,000 x g for 60 min. The supernatant was collected, and the bulk of protein was precipitated with 20%

rt 10h-.'

a

Ito

* -.* CaQu

o----o T K

~0

o-o HEF

0 0

o

AI

20

40

Fraction number

FIG. 1. Polyacrylamide gel electrophoresis on dThd kinase extracts. Crude extracts from VZV (Ito, CaQu, or TK-)-infected or mock-infected HEF were applied onto the gels. After an electrophoresis of 3 h at 4°C with a constant current of 40 mA per gel, the gels were sliced into 2-mm thin sections, and TK activity was determined for each section.

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dThd

1055

lVdUrd

BrdUrd -t 0

x x 0-

CL

Kobayashi s L.

6

0

x C

0

0*

0.

L.

c

I-

Q

Qz 0 m

0 0 L

L.

u c

Hours after Start of Incorporation

FIG, 2. Incorporation of dThd, BrdUrd, or IVdUrd into VZV-infected cells. HEF monolayers infected with the CaQu, Kobayashi, Ito, TK- strain of VZV were incubated at 36°C for 24 h in the presence of [6-3H]dThd, [1'-2'-3H]BrdUrd, or ['251]IVdUrd at 1 uCi/ml. At the indicated times approximately 3 105 cells were harvested, applied onto glass fiber filters, and examined for trichloroacetic acid-precipitable radioactivity. Background radioactivity (measured at 0 h, immediately after addition of the radiolabeled precursors) was subtracted from all subsequent radioactivity counts.

or

x

ammonium sulfate. The supernatant was then again treated with 50% ammonium sulfate, and the precipitate was used as the TK material. After the addition of glycerol and bovine albumin (10%, vol/vol), the TK material was stored at -80°C until used. Affinity chromatography. VZV TK was purified by dThd affinity chromatography as described by Fyfe (9). Briefly, dThd-3'-paranitrophenylphosphate was reduced catalytically to the paraamnino compound, and dThd-3'-paraaminophenylphosphate was purified by passage through a Dowex 50 W-X8 column (H}) (Muromachi Kagaku Co., Tokyo, Japan) with H20 as the eluting solvent. The reduced compound was then linked onto activated carboxyhexylSepharose (Pharmacia, Uppsala, Sweden). The dThdagarose column was prepared in a cylinder (1 by 6 cm), and the unreacted carboxyl groups were blocked by galactosamine. Approximately 3.0 ,uM dThd was bound per ml of wet gel. After application of the TK samples, the column was washed extensively with 50 mM Tris buffer (see above) without dThd. TK was eluted from the column with a gradient of 0 to 0.6 mM dThd in 800 mM Tris buffer (pH 6.8)

containing 2 mM dithiothreitol, 10% glycerol, and 1 mM ATP. After determination of protein concentration, bovine serum albumin was added at a concentration of 0.5 mg/ml. Most of the VZV TK activity was eluted with buffer containing 0.1 mM dThd. The eluted materials were dialyzed against 50 mM Tris hydrochloride buffer (pH 7.5) containing 5 mM mercaptoethanol, 2 mM ATP, 2 mM MgCl2, and 10% glycerol. TK assay. The reaction mixture for the TK assay was essentially as described by Dobersen et al. (5), that is, 50 mM Tris hydrochloride (pH 8.0) containing 5 ,uM [63H]dThd (25 Ci/mmol), 2 mM ATP, 2 mM MgCl2, and 5 mM 2-mercaptoethanol. Enzyme extract (25 ,ul; 0.005 to 0.073 ,ug of purified enzyme protein for TK kinetic assay) was mixed with 50 ,ul of reaction buffer and incubated at 37°C for 10 min. After incubation, the reaction was stopped by adding 25 ,u1 of 50% cold trichloroacetic acid, and then 25 p.l of the mixture was applied onto Whatman DE-81 filter paper disks. The filter paper was. washed twice with a solution of 4 mM ammonium acetate containing 5 p.M dThd and then subsequently washed with 4 mM ammonium acetate, distilled water, and 95% ethanol. The radioactivity that remained on

TABLE 2. Kinetic constants of purified dThd kinase from VZV (CaQu, Kobayashi, and Ito strains) with dThd, dCyd, BrudUrd, or IVdUrd as substrate Km (>LM) Vmax (nmol/min per mg)a Virus strain IVdUrd dThd BrdUrd BrdUrd IVdUrd dCyd dThd dCyd 243.0 17.24 4.17 11.62 0.45 0.23 0.09 1.03 CaQu 288.9 12.5 33.33 0.07 22.22 1.43 0.27 0.87 Kobayashi 38.0 4.2 5.6 0.23 3.3 Ito 2.22 2.67 4.00 a

Protein concentrations of each dThd kinase were as follows: CaQu and Kobayashi, 0.2

p.g/ml; Ito, 2.9 ,ug/ml.

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ANTIMICROB. AGENTS CHEMOTHER. CaQu

1001

.0

E z (n 0

501

0

4U-

u

U.

0.

0

0

10- 3

10

2

Io- 1

Concentration of Aminopterin (tpM)

FIG. 3. Effect of aminopterin on focus formation of VZV (strains CaQu, Ito, and TK-). HEF monolayers were infected.with approximately 100 focus-forming units of VZV and incubated in the presence of various concentrations of aminopterin in dThd-deprived medium. The number of VZV foci was determined 3 days after infection.

the filter was counted in a liquid scintillation spectrometer. The radioactivity obtained With [I25]IVdUrd as a substrate of TK was determined in a gamma counter. Polyacrylamide gel electrophoresis. To detect virus-specific TK activity, we fractionated the enzyme extract by 5% polyacrylamide slab gel electrophoresis following the procedure of Hackstadt and Mallavia (12). Enzyme extract (50 RIl; 50 ,ug) was applied onto the gel. Electrophoresis was carried out for approximately 3 h at 4°C at a constant current of 40 mA per slab gel (150 by 90 by 2 mm), using 2.5 M Tris buffer (pH 8.9) containing 0.192 M glycine, 1 mM MgCl2, 10 mM 2-mercaptoethanol, and 2.5 mM ATP. After electrophoresis, the gels were sliced into 2-mm sections, and the arnount of TK activity was determined for each section. TK activity was detected by incubation of each section in 125 ,1 of reaction mixture for 30 min at 37°C.

RESULTS Patient and isolation of Ito strain. Patient 1 was 13 months old when presenting with chicken pox. Two weeks before the onset of disease, her elder brother, aged 2 years 11 months, also suffered from chicken pox. One day before visiting Fukushima Medical College Hospital, patient 1 had a sudden fever (38°C) and skin eruptions including vesicles. At the time she visited the hospital, the fever had risen to 38.80C and skin eruptions were scattered over the whole body. Vesicular fluid was obtained and immediately inoculated on HEF monolayers. The fever continued for 3 days and the eruption continued for 7 days after the first visit to the hospital. By day 8 all symptoms had disappeared. No antiviral compounds were administered. The virus strain,

designated as Ito, was isolated 5 days after inoculation of the HEF cells by the vesicular fluid specimen. Susceptibility of Ito VZV strain toward nucleoside analogs. The CaQu, Kobayashi, Ito, and TK- (Kanno-Kohmura) strains of VZV were compared for their susceptibility to IdUrd, BrdUrd, BVdUrd, and IVdUrd (Table 1). Based on the ID50 of the compounds for the VZV strains, the Ito and TK- strains were, respectively, 32 and 68 times less sensitive to IdUrd and 15 and 13 times less sensitive to BrdUrd than were the CaQu and Kobayashi strains. On the other hand, the Ito strain was as sensitive as the CaQu and Kobayashi strains to BVdUrd and IVdUrd, while the TKstrain was virtually resistant to BVdUrd and IVdUrd (Table 1). TK activity of Ito VZV strain. In preliminary experiments, a relatively high TK activity was detected in extracts of VZV (Ito strain)-infected HEF cells. When the TK activity of Ito, CaQu, and TK- VZV (Kanno-Kohmura)-infected and uninfected cell extracts was analyzed by polyacrylamide gel electrophoresis, three peaks of TK activity were noted (Fig. 1). The slow- and fast-moving peaks (relative mobility, 0.1 and 0.6, respectively) were identified as cellular TK, and the middle peak (relative mobility, 0.45) was identified as viral TK, since extracts from uninfected HEF cells did not show the latter peak. The electrophoretic mobility of the viral TK was similar to that reported by Hackstadt and Mallavia (12) using the same polyacrylamide gel electrophoresis procedure. Extracts from TK- VZV-infected HEF cells did not express any viral TK activity (19). Marked viral TK activity was found in extracts from HEF cells infected with either the CaQu or Ito strain of VZV. Incorporation of dTha, BrdUrd, and IVdUrd in VZVinfected cells. HEF cells infected with either the CaQu VZV or Kobayashi VZV strain incorporated [6-3H]dThd to a similar extent, whereas the cells infected with the Ito strain incorporated dThd to only 30% of the amount incorporated by the CaQu and Kobayashi VZV-infected cells. On the other hand, the cells infected with the TK- (KannoKohmura) strain did not incorporate dThd or incorporated very little (Fig. 2, left panel). [1'-2'-3H]BrdUrd was incorporated efficiently in both CaQu and Kobayashi VZV-infected cells, whereas the cells infected with the Ito or TK- strain scarcely incorporated BrdUrd. (Fig. 2, center panel). In these experiments only 0.04 ,uM each radiolabeled dThd and BrdUrd was used, and at this concentration neither dThd nor BrdUrd had an inhibitory effect on the replicative cycle of VZV. In fact, during the course of the assay, cytopathogenic effect developed progressively in the CaQu, Kobayashi, Ito, and TK- VZV-infected cell cultures to reach almost 100% after 20 h of incubation in the presence of the radiolabeled nucleosides. [125I]IVdUrd at a concentration of 0.03 ,uM was inhibitory for the further development of cytopathogenic effect in CaQu, Kobayashi, and Ito VZV-infected cell cultures but was not inihibitory in TK- VZV-infected cell culture. However, the incorporation of [1251]IVdUrd was contrary to the development of cytopathogenic effect, that is, CaQu, Kobayashi, and Ito VZV-infected cells incorporated ['251]IVdUrd efficiently, but TK- VZV-infected cells did not incorporate it at all (Fig. 2, right panel). Kinetics of phosphorylation of dThd, BrdUrd, dCyd, and IVdUrd. The kinetics of the phosphorylating activity of the dThd kinases isolated from Ito, CaQu, and Kobayashi VZVinfected cells was monitored with [6-3H]dThd, [1'-2'3H]BrdUrd, [5-3H]dCyd, and [1251]IVdUrd as substrates. From Lineweaver-Burk plots (not shown) it was clear that the phosphorylating activity of the Ito VZV dThd kinase was

VARICELLA-ZOSTER VIRUS VARIANT

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markedly reduced as compared with that of the CaQu and Kobayashi VZV dThd kinases. The Km and Vmax values of the CaQu, Kobayashi, and Ito VZV dThd kinases were calculated with dThd, dCyd, BrdUrd, or IVdUrd as the substrate (Table 2). The Km of the Ito VZV dThd kinase was 15 to 17 times higher than the Km of the CaQu and Kobayashi VZV dThd kinases with BrdUrd as the substrate. With dThd and dCyd as the substrates, the Km of Ito VZV dThd kinase was 5 to 6.5 times higher than the Km of the CaQu VZV dThd kinase. When IVdUrd was used as the substrate, the Km of the Ito VZV dThd kinase was only three times higher than those of the CaQu and Kobayashi dThd kinases. Also, the Vmax of the Ito TK was about 1/10th to 1/70th of the Vmax of the two other dThd kinases. Thus, the affinity of the Ito TK for dThd, dCyd, and BrdUrd appears to be significantly lower than those of CaQu and Kobayashi. For IVdUrd the affinity of the Ito TK is also reduced by to a lesser extent than for dThd, dCyd, and BrdUrd. Effect of aminopterin and fluorodeoxyuridine on focus formation of VZV. Aminopterin and fluorodeoxyuridine are well-known 2'-deoxythymidylate synthase inhibitors. In the presence of 0.001, 0.01, and 0.1 1xM aminopterin in dThddeprived medium, focus formation by the TK- (KannoKohmura) strain of VZV was suppressed to about 10, 5, and 3% of the control value, respectively, whereas under the same conditions, focus formation by the CaQu strain was not markedly affected. At 0.01 jxM, aminopterin inhibited focus formation by the Ito strain to about 50% of the control value (Fig. 3). With 0.1 ,uM fluorodeoxyuridine a similar result was obtained (data not shown). The inhibitory effects of 2'deoxythymidylate synthase inhibitors on the replication of the VZV Ito strain suggest that the Ito strain, like the TKKano-Kohmura strain, depends at least in part on the de novo biosynthesis of 2'-deoxythymidylate. DISCUSSION

For herpes simplex virus (HSV), three classes of drugresistant mutants have been described. The first is TK deficient (TK-), as originally reported by Dubbs and Kit (6). The second class of mutants has TK-inducing activity, albeit with altered substrate specificity; these mutants may be resistant to some nucleoside analogs (i.e., ACV) but not to others (3, 7, 14). ACV-resistant mutants belonging to this class have particularly low phosphorylating activity for ACV (3, 14). The third class of drug-resistant HSV mutants is based on an altered DNA polymerase activity. These mutants may be resistant to ACV, adenine arabinoside, and antiviral drugs that interact directly with DNA polymerase such as phosphonoformic acid and phosphonoacetic acid (1, 2, 15). In this report, we describe a VZV mutant strain which is TK positive but resistant to IdUrd and BrdUrd. This mutant (Ito strain) has an altered TK with low affinity for dThd, dCyd, and, in particular, BrdUrd but relatively high affinity for IVdUrd. The low affinity of the Ito VZV dThd kinase for BrdUrd apparently accounts for the lack of incorporation of BrdUrd into Ito VZV-infected cells. The Ito strain is the first documented example of a VZV variant possessing a TK with altered substrate specificity. All other examples of mutants with altered TK substrate specificity that have been described in the literature concern HSV mutants. The clinical importance of drug-resistant HSV and VZV mutants is the subject of further study. A comparable virulence of TK- HSV-1 and wild-type TK+ HSV-1 for newborn mice was reported by Tenser (18); however, TK-

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HSV mutants have been shown to be less pathogenic for adult mice (8). In contrast, TK+ HSV mutants retain their pathogenicity for adult mice (3). The Ito strain was isolated from a patient with the clinical symptoms of chicken pox, which implies that the virus had retained its pathogenicity. The Ito strain was resistant to IdUrd and BrdUrd, yet sensitive to several other drugs such as ACV, IVdUrd, and BVdUrd (16), which is encouraging since it suggests that, should one or another antiviral drug fail, alternative forms of therapy may be successful. However, as a consequence of the increased use of antiviral drugs in the treatment of HSV and VZV infections, the virus may progressively acquire multiple resistance to antiherpes compounds. We therefore recommend that clinical isolates of HSV and VZV be routinely monitored for their in vitro sensitivity to antiviral drugs. Such drug sensitivity monitoring would seem particularly useful when prolonged or repeated courses of treatment are required, and in this case as wide a variety of antiherpes drugs as possible should be evaluated for their in vitro activity against the isolated HSV or VZV strains. ACKNOWLEDGMENTS This work was supported by grants from the Belgian F.G.W.O. (Fonds voor Geneeskundig Wetenschappelyk Onderzock, project no. 3.0040.83) and the Belgian G.O.A. (Geconcerteerde Onderzoeksacties, project no. 85/90-79). LITERATURE CITED 1. Coen, D. M., P. A. Furman, P. T. Gelep, and P. A. Schaffer. 1982. Mutation in the herpes simplex virus DNA polymerase gene can confer resistance to 9-p-D-arabinofuranosyladenine. J. Virol. 41:909-918. 2. Coen, D. M., and P. A. Schaffer. 1980. Two distinct loci confer resistance to acycloguanosine in herpes simplex virus type 1. Proc. Natl. Acad. Sci. USA 77:2265-2269. 3. Darby, G., H. J. Field, and S. A. Salisbury. 1981. Altered substrate specificity of herpes simplex virus thymidine kinase confers acyclovir-resistance. Nature (London) 289:81-83. 4. De Clercq, E., J. Descamps, M. Ogata, and S. Shigeta. 1982. In vitro susceptibility of varicella-zoster virus to E-5-(2-bromovinyl)-2'-deoxyuridine and related compounds. Antimicrob. Agents Chemother. 21:33-38. 5. Dobersen, M. J., M. Jerkofsky, and S. Greer. 1976. Enzymatic basis for the selective inhibition of varicella-zoster virus by 5-halogenated analogues of deoxycytidine. J. Virol. 20:478-486. 6. Dubbs, D. R., and S. Kit. 1964. Mutant strains of herpes simplex deficient in thymidine kinase-inducing activity. Virology 22:493-502. 7. Field, H., A. McMillan, and G. Darby. 1981. The sensitivity of acyclovir-resistant mutants of herpes simplex virus to other antiviral drugs. J. Infect. Dis. 143:281-285. 8. Field, H. J., and P. Wildy. 1978. The pathogenicity of thymidine kinase-deficient mutants of herpes simplex virus in mice. J. Hyg. 81:267-277. 9. Fyfe, J. A. 1982. Differential phosphorylation of (E)-5-(2bromovinyl)-2'-deoxyuridine monophosphate by thymidylate kinases from herpes simplex viruses types 1 and 2 and varicella zoster virus. Mol. Pharmacol. 21:432-437. 10. Fyfe, J., and K. Biron. 1980. Phosphorylation of acyclovir by a thymidine kinase induced by varicella-zoster virus, p. 1378-1379. In J. D. Nelson and C. Grassi (ed.), Current chemotherapy and infectious disease, vol. 2. American Society for Microbiology, Washington, D.C. 11. Gershon, A. A., S. P. Steinberg, L. Gelb, G. Galasso, W. Borkowsky, P. LaRussa, and A. Ferrara. 1984. Live attenuated varicella vaccine. J. Am. Med. Assoc. 252:355-362. 12. Hackstadt, T., and L. P. Mallavia. 1978. Deoxypyrimidine nucleoside metabolism in varicella-zoster virus-infected cells. J. Virol. 25:510-517. 13. Kallander, C. F. R., J. S. Gronowitz, and E. G. Torfason. 1982.

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Human serum antibodies to varicella-zoster virus thymidine kinase. Infect. Immun. 36:30-37. 14. Larder, B. A., and G. Darby. 1982. Properties of a novel thymidine kinase induced by an acyclovir resistant herpes simplex virus type 1 mutant. J. Virol. 42:649-658. 15. Schnipper, L. E., and C. S. Crumpacker. 1980. Resistance of herpes simplex virus to acycloguanosine. Role of viral thymidine kinase and DNA polymerase loci. Proc. Natl. Acad. Sci. USA 77:2270-2273. 16. Shigeta, S., T. Yokota, T. Iwabuchi, M. Baba, K. Konno, M. Ogata, and E. De Clercq. 1983. Comparative efficacy of

ANTIMICROB. AGENTS CHEMOTHER.

antiherpes drugs against various strains of varicella-zoster virus. J. Infect. Dis. 147:576-584. 17. Takahashi, M., T. Otsuka, Y. Okuno, Y. Asano, T. Yazaki, and S. Isomura. 1974. Live vaccine used to prevent the spread of varicella in children in hospital. Lancet ii:1288-1290. 18. Tenser, R. B. 1983. Intracerebral inoculation of newborn and adult mice with thymidine kinase-deficient mutants of herpes simplex virus type 1. J. Infect. Dis. 147:956-960. 19. Yokota, T., M. Ogata, and S. Shigeta. 1983. A mutant strain of varicella-zoster virus deficient in thymidine kinase inducing activity. Fukushima J. Med. Sci. 29:101-111.