Characterization of an aphidicolin-resistant mutant of herpes simplex virus type 2 which induces an altered viral DNA polymerase

VIROLOGY

135,87-96

(1984)

Characterization of an Aphidicolin-Resistant Mutant of Herpes Simplex Virus Type 2 Which Induces an Altered Viral DNA Polymerase YUKIHIRO

NISHIYAMA,**l SATORU SUZUKI,? MANABU YAMAUCHI,* KOICHIRO MAENO,* AND SHONEN YOSHIDAS

*Laboratory of Virology, Research Institute for Disease Mechanism and Control, Nagoya University School sf Medicine, Nagoya, TFaculty of Pharmuceu tiud Science, Hokkaa& University, Sappwro, and $Department of Bioch.emistrg, Institute for Developmental Research, Kasugai, Japan Received Novemlm 22, 1983;accepted February 17, 198.4 The replication of wild-type herpes simplex virus type 2 (HSV-2) was very sensitive to aphidicolin, a specific inhibitor of eukaryotic a-type DNA polymerases; viral DNA synthesis was strongly inhibited by 1 @g/ml of aphidicolin, but the synthesis of early viral polypeptides was not affected. Using aphidicolin as the selective agent, aphidicolinresistant (Aph’) viruses were isolated from HSV-2 strain 186. All of these plaque isolates induced altered viral DNA polymerases which were more resistant to aphidicolin than wild-type polymerase. These results clearly indicate that viral DNA polymerase is a target of aphidicolin in tivo and suggest that host cell DNA polymerase a may he not involved in the replication of HSV-2. Partially purified mutant polymerase exhibited a i’.5-fold lower apparent K,,, for dCTP and a 3-fold lower apparent Km for d!ITP than similarly purified wild-type enzyme. The apparent Ki value for aphidicolin of the mutant polymerase was 6.5-fold higher than that of the wild-type enzyme. Moreover, all Aph’ viruses isolated were also resistant to thymine-l-/3-u-arabinofuranoside (ara-T). While, they were as sensitive as wild-type virus to cytosine-1-fi-D-arabinofuranoside (ara-C), adenine-9$-Darabinofuranoside (ara-A), and acycloguanosine (acyelo-G). Interestingly these Aph’ isolates were more sensitive to phosphonoacetic acid (PAA) than the wild-type. In contrast, PAA-resistant (PAA’) viruses of HSV-2 were more sensitive to aphidicolin and were more resistant to all of four nueleoside analogs than the parental wild-type virus. These results suggest that the aphidieolin-binding site of HSV DNA polymerase may be very close to the binding sites for dCTP and dTTP and it functionally correlates with that for pyrophosphate group.

aphidicolin, a highly specific inhibitor of eukaryotic a-type DNA polymerases (IkeHerpes simplex virus (HSV) has been gami et ak, 1978; Pedrali-Noy and Spadari, shown to code its own DNA polymerase in 1979; Dicioccio et al, 1980). the genome (Hay and Subak-Sharpe, 1976; The replication of HSV is also sensitive Honess and Watson, 1977; Purifoy et a& to aphidicolin (Dicioccio et aL, 1980). Ped1977; Parris et uL, 1980; Chartrand et ah, rali-Noy and Spadari (1980) have shown 1980; Coen et aL, 1982). The viral DNA that the degree of inhibition of HSV polymerase is immunologically and bio- growth is similar to the degree of inhibition chemically distinct from cellular DNA of the isolated HSV DNA polymerase in polymerase cy,/3, and y (Weissbach et aL, vitro and suggested that the virus-coded 1973;Powell and Purifoy, 1977;Knopf, 1979; DNA polymerase may be the target for Ostrander and Cheng, 1980), but has some aphidicolin in wivo. Their studies, however, similarities to cellular DNA polymerase (Y, can not rule out a possible involvement of one of which is the high sensitivity to host cell DNA polymerase (Yor other cellular functions sensitive to aphidicolin in the replication of HSV. In order to clarify 1 To whom requests for reprints should be adthis point, we have isolated aphidicolindressed. INTRODUCTION

87

0042~6322/34$3.00 Copyright All rights

0 1984 by Academic Press, Inc. of reproduction in any form reserved.

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NISHIYAMA

resistant (Aph’) viruses of HSV-2 strain 186. In this study, we have characterized our Aph’ mutant extensively with respect to the viral DNA polymerase and the sensitivities to antiherpetic compounds. Furthermore, the properties of Aph’ isolates have been compared with those of PAAresistant (PAA’) isolates which have been derived from the same parental virus as Aph’ viruses. MATERIALS

AND

METHODS

Cells and viruses. Human embryonic fibroblasts (HEF) were prepared as described previously (Nishiyama et ak, 1983a), grown in Eagle’s minimal essential medium (MEM) supplemented with 10% fetal calf serum (FCS), 100 units/ml penicillin, and 100 pg/ml streptomycin, and used throughout this study. Vero cells, a stable line of African green monkey kidney cells, were used for the preparation of partially purified HSV DNA polymerase. HSV2 strain 186 was obtained from Dr. Fred Rapp, Pennsylvania State University College of Medicine, Penna. The virus stock was prepared in HEF by infecting at low multiplicities (0.01 to 0.1 PFU/cell) as described previously (Nishiyama and Rapp, 1981). Chemicals. [meth$3H]Thymidine (20 Ci/mmol), [methyZ-3H]thymidine 5’-triphosphate (89 Ci/mmol), and [5-3H]deoxycytidine 5’-triphosphate (29 Ci/ mmol) were purchased from New England Nuclear Corporation. Deoxyribonucleoside 5’-triphosphates, dATP, dTTP, dCTP, dGTP, and ribonucleoside 5’-triphosphates were obtained from Boehringer Mannheim. Adenine-9-@-D-arabinofuranoside (ara-A), cytosine-1-/3-D-arabinofuranoside (ara-C), thymine-l-/3-D-arabinofuranoside (ara-T), and phosphonoacetic acid (PAA) were purchased from Sigma Chemical Company. Aphidicolin and 9-(2-hydroxyethoxymethyl)guanine (acyclo-G) were from Wako Pure Chemicals and Wellcome Research Laboratories, respectively. Isolation of Aph’ mutants. Wild-type HSV-2 strain 186 isolated from a single plaque was mutagenized by ultraviolet (uv) irradiation (3600 ergs/mm’), and mono-

ET AL.

layers of HEF were infected with the uvirradiated viruses at a multiplicity equivalent to 0.1 PFU/cell, incubated for 48 hr, and then harvested. Infected cells were subjected to three cycles of freezing and thawing. Virus thus obtained was serially passaged at multiplicities of 0.01 to 0.1 PFU/cell in increasing concentrations of aphidicolin (0.5 to 10 pg/ml). After the last passage, viruses were plaque-purified three times in the presence of aphidicolin and then propagated in HEF as described above. Plaque inhibition tests. To determine the dose-response of antiviral activity of inhibitors, plaque inhibition tests were carried out. Confluent monolayers of HEF were infected with 150 to 200 PFU of HSV2 in 35-mm plastic dishes. After a 1 hr adsorption period at 37”, the cultures were overlaid with 2 ml of 0.5% agarose in MEM containing appropriate concentrations of inhibitors and then incubated for 1 or 2 days at 37”. The plaques were counted using a dissecting microscope at X 20 magnification after fixation with 5% formalin and stained with 0.7% crystal violet. Partial putification of HSV DNA polyme-a-seand assay of HSV DNA polymerase. Partial purification was carried out according to Knopf (1979) with some modifications. Confluent monolayers of Vero cells were infected with Aph’-1 or wildtype virus at a multiplicity of approximately 2 PFU/cell, and harvested at 12 to 15 hr postinfection. Infected cells (3.5 to 5 g) were suspended in 20 ml of extraction buffer containing 0.25 M potassium phosphate (pH 7.5), 10 mM 2-mercaptoethanol (2-ME), 1 mM ethylenediamine tetraacetate (EDTA), 0.5% Triton X-100,20% glycerol, and 0.5 mM phenylmethane sulfonyl fluoride (PMSF), and sonicated using a Branson sonifier with a microtip for 15 see, 8 times. The sonicated cell suspension was centrifuged at 12,000 rpm for 10 min and the supernatant was further centrifuged at 30,000 rpm for 2 hr, and then dialyzed against 10 mM potassium phosphate (pH 7.5) containing 10 mM 2-ME, 1 mM EDTA, and 20% glycerol. The dialysate was adsorbed onto a column (1.5 X 30 cm) of DEAE-cellulose (DE-52), previously equil-

APHIDICOLIN-RESISTANT

ibrated with 20 mM potassium phosphate (pH 7.5) containing 10 mM 2-ME, 1 mM EDTA, and 20% glycerol (buffer A). The column was washed with buffer A and eluted with a linear gradient from 0.02 to 0.4 M potassium phosphate. HSV DNA polymerase activity was eluted as a peak at 0.23 to 0.25 Mpotassium phosphate. The peak fractions of viral DNA polymerase were dialyzed against buffer A and applied to a phosphocellulose (P-11) column (1.5 X 8 cm). Protein was eluted with a linear gradient from 0.02 to 0.5 M potassium phosphate. HSV DNA polymerase was recovered as a single peak at 0.38 to 0.40 M potassium phosphate and was completely separated from cellular DNA polymerases at this step. After phosphocellulose chromatography, the viral DNA polymerase fractions of the mutant and wild-type viruses exhibited specific activities of 3800 and 2900 units/mg protein, respectively, and were purified about loo-fold from the high-speed supernatant. One unit of activity was defined as an amount that catalyzes the incorporation of 1 nmol of 4 dNTPs into the acid-insoluble fraction in an hour. The standard reaction mixture for partial purified HSV DNA polymerase (25 ~1) contained 50 mMTris-HCl (pH 8.0), 8 mM MgClz, 0.5 mM dithiothreitol (DTT), 100 mM ammonium sulfate, 80 PM concentrations of dATP, dGTP, dCTP, and [3H]dTTP (0.5 &i/nmol), 1.25 pg activated calf thymus DNA (Yoshida et al, 1974), and enzyme. In this assay condition, the activity of DNA polymerase (Ywas inhibited by more than 95%. Incubation was carried out at 37” for 10 min, and the acidinsoluble radioactivity was measured as described previously (Yoshida et ah, 1971). DNA analysis h CsCl density gradient equilibrium centr$ugation HSV-infected HEF were labeled with 10 &i/ml of [3H]thymidine for 2 hr in the presence or absence of inhibitors, and extracted DNA was analyzed by CsCl density gradient equilibrium centrifugation as described previously (Nishiyama and Rapp, 1981). Polyacrylamide slabgel ekctrophoresis (PAGE). PAGE was carried out by the method of Laemmli (1970). Samples were dissociated in 0.0625 M Tris-HCl (pH 6.8)

MUTANT

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OF HSV-2

containing 5% SDS, 2% 2-ME, 10% glycerol, and 0.001% bromophenol blue, followed by heating at 100” for 1 min. The acrylamide concentrations were 8.5% for the separating gel and 3% for the stacking gel. RNA polymerase (165K, 155K, 39K) and bovine serum albumin (68K) were used as reference protein markers. After electrophoresis, the gels were fixed, dried and then exposed to Kodak Royal X-Omat films at -80”. RESULTS

Isolation of Aph’ Viruses of HSV-2 Using aphidicolin as the selective agent, we have isolated Aph’ clones from wildtype HSV-2 strain 186 as described under Materials and Methods. Figure 1 represents a dose-response experiment for the effect of aphidicolin on the plaque formation of three isolates and the wild-type. The plaque formation of wild-type virus was inhibited by 50% at 0.5 pg/ml of aphidicolin and completely suppressed at 4 pg/ ml. In contrast, the plaque formation of Aph’ viruses was entirely resistant to 1 pg/ml of aphidicolin. At 2 pg/ml of aphi-

I

0

2 1 AphidicolinI~g/ml]

0

4

FIG. 1. Effect of aphidicolin on the plating efficiency of wild-type HSV-2 strain 186 and three Aph’ isolates. The sensitivity of viruses to aphidicolin was measured by plaque reduction assays on monolayers of HEF as described under Materials and Methods, and the plaque counts were expressed as a percentage of the number obtained in control cultures. Symbols: 0, wildtype; 0, Aph’-1; n , Aph’-3; A, Aph’-6.

90

NISHIYAMA

ET AL.

dicolin, the plating efficiencies of all three plaque isolates were more than 20-fold higher than that of the wild-type. All six Aph’ isolates were also examined for temperature sensitivity and plaque size. The plating efficiency of wild-type virus was 1.03 at 38.5” relative to 34”, while those of Aph’ viruses ranged from 0.92 to 1.05. The plaque diameter of both Aph’ and wildtype viruses was 2.0 to 2.5 mm in Vero cells after 3 days at 3’7”, suggesting that the replication efficiency of Aph’ clones is not inferior to that of the wild-type. E#ect of Aphidicolin on the Synthesis of Viral DNA and Polypeptides To measure the effect of aphidicolin on DNA synthesis of wild-type and Aph’ viruses, CsCl density gradient equilibrium centrifugation was carried out. Monolayers of HEF were infected with Aph’-1 or wildtype virus at a multiplicity of approximately 5 PFU/cell, and labeled with rH]thymidine (10 &i/ml) between 3 and 5 hr postinfection in the presence or absence of 1 pg/ml of aphidicolin. Figure 2 shows CsCl density gradient profiles of labeled DNA extracted from infected cells. Viral DNA synthesis (density, 1.725g/cm3) of the wild-type was very sensitive to aphidicolin; more than 80% of viral DNA synthesis was inhibited by 1 pg/ml of aphidicolin. While the viral DNA synthesis of Aph’-1 was inhibited by only 20% at this concentration. In the absence of aphidiCohn, there was no significant difference between the wild-type and the mutant in the efficiency of viral DNA synthesis. It was also noted that cellular DNA synthesis (density, 1.695 g/cm3) of infected cells was highly resistant to aphidicolin, probably due to repair DNA synthesis induced by HSV (Nishiyama and Rapp, 1981; Nishiyama et al, 1983b). Monolayers of HEF were infected with Aph’-1 or wild-type virus at a multiplicity of approximately 20 PFU/cell in the presence or absence of 2 pg/ml of aphidicolin, and labeled with r5S]methionine (10 &i/ ml) from 2 to 6 hr postinfection in the presence or absence of the drug. The results of PAGE analysis were shown in Fig. 3.

Fraction

FIG. 2. Effect of aphidicolin on DNA synthesis of wild-type- (A) or Aph’-10 (B) infected cells. Confluent monolayers of HEF were infected with viruses (5 PFU/cell) and incubated with growth medium at 37”. Cells were then labeled with 10 rCi/ml of [3H]thymidine in the presence (0) or absence (0) of 1 pg/ml of aphidicolin between 3 and 5 hr postinfection. Extracted DNA was analyzed by CsCl density gradient equilibrium centrifugation. Fractions were collected and precipitated in 5% trichloroacetic acid, and radioactivity was determined.

The synthesis of late viral polypeptides was markedly reduced in cells infected with wild-type virus by 2 pg/ml of aphidicolin, but the synthesis of early viral polypeptides was not affected. In cells infected with Aph’-1, aphidicolin suppressed slightly the synthesis of late viral polypeptides. There was no effect of aphidicolin on protein synthesis in mock-infected cells. Properties

of Viral DNA Polymmase

To determine whether Aph’ mutant-induced DNA polymerase has an altered sensitivity to aphidicolin, crude extracts of infected cells were assayed for viral DNA polymerase activity in the presence of various concentrations of aphidicolin. As shown in Fig. 4, DNA polymerases of Aph’ isolates were much more resistant to aphi-

APHIDICOLIN-RESISTANT Mock

-+

MUTANT

91

OF HSV-2

Infected

-w+

Aph’ - +

0u Aphidicolin

FIG. 3. Effect of aphidicolin on protein synthesis of mock- (a, b), wild-type(c, d) or Aph’-l- (e. f) infected cells. Confluent monolayers of HEF were infected with viruses (20 PFWcell) in the presence (b, d, f) or absence (a, c, e) of 2 wg/ml of aphidicolin and labeled with [?l]methionine (10 &i/ml) between 2 and 6 hr postinfection in methionine-free MEM. The radioactive polypeptides were analyzed by PAGE as described under Materials and Methods. Triangles (4) indicate late viral polypeptides whose production was markedly reduced.

dicolin than that of the wild-type. This result, together with those of experiments on the effects of aphidicolin on the synthesis viral DNA (Fig. 2) and polypeptides (Fig. 3), suggests that viral DNA polymerase is the only target of aphidicolin in vivo. To know the enzymological properties of viral DNA polymerase of Aph’ mutant, viral DNA polymerase was purified from infected Vero cells by DEAE-cellulose and phosphocellulose column chromatography. As shown in Figs. 5 and 6, aphidicolin inhibited viral DNA polymerase competitively with respect to both dCTP and dTTP. The apparent K, values for dCTP were ‘7 and 0.95 pikf for the wild-type and mutant polymerases, respectively. While, the apparent K,,, values for dTTP were 10 and 3.6 PM for the wild-type and mutant polymerases, respectively. The apparent Ki value (6.5 PM) for aphidicolin of the mu-

lug/ml 1

FIG. 4. Effect of aphidicolin on the activity of viral DNA polymerases of wild-type or Aph’ viruses. Confluent monolayers of HEF in 60-mm plastic dishes were infected with viruses (5 PFWcell), incubated with growth medium at 37”, and harvested 12 hr postinfection. DNA polymerase was extracted by freeze-thawing as described previously (Nishiyama et al, 1982), and aphidicolin sensitivity of viral DNA polymerases in the crude extracts was measured as described under Materials and Methods except that the concentrations of dCTP and dTTP were 10 and 50 pM concentrations of ATP, GTP, CTP, and UTP were added to prevent degradation of dNTPs (Sugino and Nakayama, 1980). The 100% values ranged from 10 to 12.5 pmol of dTMP incorporation. Symbols: 0, wild-type; 0, Aph’-1; n , Aph’-Z$ A, Aph’-6.

tant polymerase was 6.5-fold higher than that (1 PM) of the wild-type polymerase (Fig. 7). The results indicate that Aph’ mutant polymerase has an increased affin-

FIG. 5. Lineweaver-Burk plots for determination of the K,,, for dCTP with partially purified viral DNA polymerases from wild-type(A) or Aph’-l- (B) infected cells. The activity of viral DNA polymerases was assayed as described under Materials and Methods except that the concentration of rH]dCTP was varied and those of dATP, dGTP, and dTTP were constant (80 ~&f). Symbols: 0, no aphidicolin; 0, 1 pg/ml aphidicolin.

92

NISHIYAMA

A

ET AL.

0 00

-03

0

1

2 1 /dTTP,

-02 NM-'

0

02

Am-C bM]

0.4

FIG. 6. Lineweaver-Burk plots for determination of the K,,, for dTTP with partially purified viral DNA polymerases from wild-type(A) or Aph’-l- (B) infected cells. The activity of viral DNA polymerases was assayed as described under Materials and Methods except that the concentrations of [8H]dTTP were varied and those of dATP, dGTP, and dCTP were constant (80 p&f). Symbols: 0, no aphidicolin; 0, 1 rg/ml aphidicolin.

ity to dCTP and dTTP and a reduced affinity to aphidicolin compared with the wild-type polymerase. Sensitivity to Nucleoside Analogs and PAA The above results suggest that Aph’ viruses may have altered sensitivities to various antiherpetic agents of which the target is viral DNA polymerase. Therefore, we examined them for the sensitivities to four nucleoside analogs, ara-C, ara-T, araA, and acyclo-G, and to a pyrophosphate analog, PAA by plaque inhibition tests. As shown in Fig. 8, all Aph’ isolates tested were much more resistant to only ara-T

FIG. 7. Apparent Ki values for aphidicolin of partially purified viral DNA polymerases from wild-type(A) or Aph’-l- (B) infected cells. The activity of viral DNA polymerases was assayed as described under Materials and Methods except that ~HjdCI’P was used as a labeled substrate in place of [3H]d’ITP. The concentration of dCTP was 10 (0) or 66 (0) &f, Aphidicolin was added to the reaction mixture at the concentrations indicated.

0

1

2 An-A

Awl

40 bM1

cl5 1 Acyclo-G

CM]

2 [Ml

FIG. 8. Sensitivities of Aph’ and wild-type viruses to ara-C (A), ara-T (B), ara-A (C), and acyclo-G (D). For assaying drug sensitivity, plaque inhibition tests were carried out on monolayers of HEF as described under Materials and Methods. The plaque counts were expressed as a percentage of the number obtained in control cultures. Symbols: 0, wild-type; 0, Aph’-1; n , Aph’-3; A, Aph=6.

than wild-type virus. The sensitivities of the Aph’ isolates to other three nucleoside analogs were almost identical to those of the wild-type. Interestingly, all Aph’ isolates exhibited a higher sensitivity to PAA compared with the wild-type (Fig. 9). Inverse Relation between the Sensitivity to Aphidicolin and PAA In order to determine whether the sensitivity to aphidicolin correlates inversely to that to PAA, we tried to isolate PAA’ mutants from Aph’ mutant and the wildtype. Viruses were mutagenized by uv irradiation (3600 ergs/mm’), propagated in HEF, and then serially passaged in increasing concentrations of PAA. After several passages, PAA’ viruses were easily isolated from wild-type virus. These PAA’ isolates induced PAA-resistant viral DNA polymerases in infected cells (data not shown) and were more sensitive to aphidicolin than the parental wild-type (Fig.

APHIDICOLIN-RESISTANT

MUTANT

Concmtntion

0

12.5 25 PAA W/ml)

50

FIG. 9. Sensitivities of Aph’ and wild-type viruses to PAA. Plaque inhibition tests were carried out as described under Materials and Methods. The plaque counts were expressed as a percentage of the number obtained in control cultures. Symbols: 0, wild-type; 0, Aph’-1; 6 Aph=a; A, Aph=6.

10). On the other hand, we failed to obtain the Aph’ viruses which simultaneously exhibited substantial resistance to PAA; the Aph’ isolates which recovered the resistance to PAA up to the level of that of the wild-type, designated as Aph’-PAAP, appeared to lose the high resistance to aphidicolin. Since both PAA’ and Aph’ mutants were derived from the same parent clone of wildtype HSV-2, it was of interest to also examine the sensitivities of PAA’ isolates to four nucleoside analogs. As shown in Fig.

FIG. 10. Sensitivities of PAA’, Aph’-PAA*, and wildtype viruses to PAA (A) and aphidicolin (B). Aph’PAAP clones were isolated from Aph’-1 by passaging increasing concentrations of PAA after mutagenization with uv. Plaque inhibition tests were carried out as described under Materials and Methods. Symbols: 0, wild-type; A, PAX-l; v, PAA’-& n , Aph’PAAp-1; +, Aph=PAAP-2.

93

OF HSV-2

WI

FIG. 11. Sensitivities of PAA’ isolates to ara-C (A), ara-T (B), ara-A (C), and acyclo-G (D). Plaque inhibition tests were carried out as described under Materials and Methods. Symbols: 0, wild-type; A, PAA=l; 7, PAA’-2.

11, PAA’ isolates, unlike Aph’ isolates, exhibited higher resistances to all four nucleoside analogs than the wild-type. These results suggest that the aphidicolin-binding site of HSV DNA polymerase functionally correlates with the pyrophosphate exchange-release site. DISCUSSION

Aphidicolin, a tetracyclic diterpenoid, effectively inhibited the replication of wildtype HSV-2 strain 186. The synthesis of early viral polypeptides was not affected by aphidicolin but the viral DNA synthesis was inhibited. Aph’ clones isolated from wild-type virus induced altered viral DNA polymerases which were more resistant to aphidicolin than wild-type polymerase, and the sensitivities to aphidicolin of the mutant and wild-type polymerases in vitro were similar to those of the mutant and wild-type viruses in viva. These results strongly suggest that viral DNA polymerase is the only site of action of aphidicolin in v&o. However, mapping studies on Aph’ phenotype of the mutant will be necessary to confirm it. The isolation of Aph’ viruses also suggest that host cell DNA polymerase (Yis not required for the normal life cycle of HSV, although a-polymerase may be involved in host cell reactivation of uv-irradiated HSV (Nishiyama et aL, 1984). To compare the properties of mutant polymerase with those of wild-type polymerase, both viral polymerases were sep-

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NISHIYAMA

arated from host cell DNA polymerases by DEAE-cellulose and phosphocellulose column chromatography. The inhibition of viral DNA polymerase by aphidicolin was apparently competitive to both dCTP and dTTP, and the apparent J&, values for dCTP and dTTP of mutant polymerase were at least several-fold lower than those of wild-type polymerase. The apparent Ki value for aphidicolin was 6.5-fold higher than that of wild-type enzyme. The results indicate that mutant polymerase has an increase affinity to dCTP and dTTP and has a reduced affinity to aphidicolin compared with wild-type enzyme. Sugino and Nakayama (1980) have reported an aphidicolin-resistant DNA polymerase (Y of Lkxophda rndmmgmtw of which apparent Ki for aphidicolin is lo-fold higher than that of wild-type polymerase but the apparent K, for dCTP is the same as that of the wild-type enzyme. More recently, Liu et al. (1983) have described an aphidicolinresistant Chinese hamster cell mutant which is characterized by slow growth and hypersensitivity to uv-induced mutation. Their mutant cells have DNA polymerase (Ywhich shows a higher affinity to dCTP than wild-type enzyme. The viral DNA polymerase of an Aph’ mutant was unique with respect to having the alterations in both Ki for aphidicolin and the K,,, for dCTP and dTTP. Moreover, it was shown that there were no significant differences between mutant and wild-type viruses in the efficiency of either viral growth or DNA synthesis although Aph’ isolates induced altered viral DNA polymerases. It appears that HSV DNA polymerase can tolerate alterations in the aphidicolin-binding site without obvious consequences for the normal activities of the enzyme, as suggested by Honess (O’Hare and Honess, 1983). Aph’ isolates also exhibited altered sensitivities to other inhibitors of which the target has been known to be HSV-induced DNA polymerase. As summarized in Table 1, Aph’ isolates were more resistant to araT and more sensitive to PAA than wildtype virus, but their sensitivities to araC, ara-A and acyclo-G were almost identical to those of the wild-type. Because Aph’ isolates were as sensitive to acyclo-

ET AL. TABLE

1

SUMMARY OF SENSITIVITIES OF APH’ AND PAA’ MUTANTS TO VARIOUS INHIBITORS~ Inhibitors

Virus

Aphidicolin

PAA

Ara-C

Ara-T

Ara-A

Acyclo-G

Aphr PAA’

1 f

f 1

+ 1

I 1

1

1

a The sensitivities of mutants to various inhibitors were compared with those of the parental wild-type virus. Arrows 0, 1, -) indicate the increase, decrease, and no change in the sensitivity, respectively.

G as wild-type virus, it is unlikely that the resistance to ara-T is due to a secondary mutation in viral thymidine kinase. At present, it is not known why Aph’ isolates showed normal sensitivity to other arabinose analogs, e.g., ara-C, while Aph’ mutant polymerase showed reduced K,,, for dCTP. A more comprehensive kinetics study is in progress. In contrast, PAA’ isolates whose parental virus was the same as that of Aph’ isolates were more resistant to all four nucleoside analogs and more sensitive to aphidicolin than the parent. Bastow et al (1983) have reported that phosphonoformic acid-resistant variants of HSV-1 were more sensitive than the parent strain (KOS) and cross-resistant to ara-T. More recently, Coen et al. (1933) have also shown that 10 of the 11 mutants which have been shown or likely to contain mutations in HSV DNA polymerase gene were resistant to PAA and hypersensitive to aphidicolin, but one mutant (ts C4) were hypersensitive to both PAA and aphidicolin. Our observations confirmed their results and indicated that the sensitivities to aphidicolin and PAA of HSV DNA polymerase tended to correlate inversely. These results suggest that the aphidicolin-binding site is very close to, or overlapping with, not only the binding sites for dCTP and dTTP but also the pyrophosphate exchange-release (PAA-binding) site. In this study we also tried to isolate Aph’-PAA’ mutants of HSV-2, but we failed to obtain the Aph’ viruses which simultaneously exhibited high resistance to PAA. The Aph’-

APHIDICOLIN-RESISTANT

PAAP viruses, which were isolated by passaging a mutagenized Aph’ clone in the presence of PAA, recovered the resistance to PAA up to the level of that of the wildtype but lost high resistance to aphidicolin. It appears that HSV DNA polymerase permits amino acid alterations which give some degree of resistance to aphidicolin without affecting its sensitivity to PAA. However, the possibility that the remaining resistance to aphidicolin of Aph’-PAAP isolates is due to a mutation lying outside the polymerase gene cannot be ruled out. HSV DNA polymerase has been shown to be associated with a virus-induced polypeptide of molecular weight of 140,000150,000 (Powell and Purifoy, 1977; Knopf, 1979) and mutations responsible for the PAA’ phenotype have been mapped to an 0.8-1.3 kilobase-pair region in the HSV DNA polymerase locus (Knopf et aL, 1981; Coen et uL, 1983). The determination of DNA sequences coding for HSV DNA polymerase will clarify the structure of the active center of polymerase which will be identified as mutated region. The precise knowledge about interaction sites of inhibitors as well as about affinity and kinetics of interaction of drugs may also provide the basis for choosing the best drug and devising combination of drugs for treating herpes infection. ACKNOWLEDGMENTS We would like to thank E. Iwata and T. Tsuruguchi for their technical assistance. This work was supported in part by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science and Culture, Japan. REFERENCES BASTOW, K. F., DERSE, D. D., and CHENG, Y.-C. (1983). Susceptibility of phosphonoformic acid-resistant herpes simplex virus variants to arabinosylnucleosides and aphidicolin. Antimicrob. Agents Chemu ther. 23, 914-91’7. CHARTRAND, P., CRUMPACKER, C. S., SCHAFFER, P., and WILKIE, N. M. (1980). Physical and genetic analysis of the herpes simplex virus DNA polymerase locus. Virw 103,311-326. COEN, D. M., FURMAN, P. A., GELEP, P. T., and SCHAFFER, P. A. (1982). Mutations in the herpes simplex virus DNA polymerase gene can confer resistance

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