5-Methoxymethyldeoxyuridine-resistant mutants of herpes simplex virus type 1

5-Methoxymethyldeoxyuridine-resistant mutants of herpes simplex virus type 1

VIROLOGY 114,576-579(1981) Wvlethoxymethyldeoxyuridine-Resistant Mutants of Herpes Simplex Virus Type 1 V. VEERISETTYANDG. Department of Microbiol...

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Mutants of Herpes Simplex Virus Type 1

V. VEERISETTYANDG. Department of Microbiology,

School of Medicine,


University of Mississippi, Jackson, Mississippi $9216

Received May 11, 1981; accepted July 17, 1981 5-Methoxymethyldeoxyuridine (MMdU)-resistant (MMdU’) mutants of herpes simplex virus type 1 (HSV-1) were isolated by a single passage of the virus in MMdU at 100 ~1 ml (3.7 X 10e4 Af). The isolates were cloned and passed in the presence of MMdU at 5 pg/ml (0.18 X lo-’ A4). All MMdU’ mutants showed considerable cross-resistance to the nucleoside analogs acycloguanosine (ACG), (E)-5-bromovinyldeoxyuridine (BVdU), and arabinosylthymine (araT), but were as sensitive as the parental strain to phosphonoacetate (PAA). One mutant, MMdU’-20, induced significant deoxythymidine kinase (dTK) activity. Because it was only 30 times more resistant to BVdU than the parental wildtype virus, while mutants MMdU’-2 and -12-3 were about 10,000 times more resistant, it was suspected that the mutation was in the dTK locus. All three mutants, however, showed a similar pattern of resistance to the other nucleoside analogs ACG, MMdU, and araT. These results suggest differences in the active sites for PAA and nucleoside analogs (with respect to the viral DNA polymerase), and also among the nucleoside analogs (with respect to the viral dTK).

SMethoxymethyldeoxyuridine (MMdU) is a nucleoside analog which inhibits herpes simplex virus replication (1). Although it is relatively nontoxic to animal cells, its value as an antiherpesviral drug has not been fully explored. During our investigation we observed virus plaques at relatively high drug concentrations and low input virus more often with MMdU than with 9-(2-hydroxyethoxymethyl)guanine (acycloguanosine, ACG), (E)-5bromovinyldeoxyuridine (BVdU), or arabinosylthymine (araT). This prompted us to investigate the possibility that MMdU played a direct role in the rapid development of drug-resistant mutants and, if so, to characterize the mutants to determine whether the mutation occurred in the viral deoxythymidine kinase (dTK) locus or in the viral DNA polymerase (Pol) locus. Because the mechanism of inhibition of viral replication is well understood in the case of araT, BVdU, and ACG [phosphorylation by viral dTK is required for antiviral activity (241 and in the case of PAA ’ To whom reprint

requests should be addressed.

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[this does not require viral dTK for antiviral activity but directly binds to the pyrophosphate binding site on DNA polymerase (S)] our studies using these drugs enabled us to infer the site of mutation in MMdU-resistant mutants. If the mutation were in the viral Pol locus, it would almost certainly be reflected in an altered binding of one or more of the various inhibitors, possibly including PAA, but not in a deletion of viral Pol. On the other hand, if the mutation were in the viral dTK locus, the mutant should in any case be sensitive to PAA, but resistant to all three of the nucleoside analogs in the case of a dTKmutant. AraT was purchased from Raylo Chemicals (Edmonton, Alta., Canada), and PAA, BVdU, and MMdU were gifts from Ronald Duff, Eric DeClercq, and Lorne Babiuk, respectively. ACG was provided by Burroughs Wellcome Company. The drugs were dissolved in distilled water, filter sterilized, and stored at -20” as stock solutions at the following concentrations: araT, 5 mg/ml; PAA, 2 mg/ml; MMdU, 17 mg/ml (50% ethanol); BVdU, 2 mg/ml;



and ACG, 2 mg/ml. Drug dilutions were made as desired in growth media (Eagle’s medium supplemented with 5% bovine calf serum) prior to use. Baby hamster kidney (BHK-21), 3T3 dTK-, and L Kit dTK- cells were cultured as previously described (5, 7). BHK cells were infected with wild-type HSV-1, strain 1’7 MP (Glasgow) at a m.o.i. of 0.002 to 0.005. Mutants of HSV-1 resistant to MMdU were isolated by infecting confluent BHK cell monolayers in 24-well cell culture trays at low multiplicities in the presence of MMdU at 100 pg/ml(3.7 X 10e4 iW) in the growth medium. After 30-40 hr each well was examined microscopically and those containing one to three plaques were marked. After a further 48 to 72 hr of incubation the contents of each marked well were harvested using a sterile rubber policeman, sonically disrupted (5), and stored at -70”. These stocks were serially diluted and used to inoculate confluent monolayers of BHK cells in wells. Those wells showing single plaques were marked, and subsequently harvested and stored as described above. All the clones were further passed until considerable viral cytopathic effect could be detected. Finally, stocks of mutants were prepared by infecting BHK cells in a 25-cm2 plastic tissue culture flask and viral titers were determined using BHK cell monolayers. In all virus passages the medium contained MMdU at 5 pg/ml(O.l8 X 10e4 ikf). Plaque reduction assays were carried out in BHK cells as described (5) with the final determination of plaque markers made at 4048 hr after infection. The percentage reduction of plaque number was plotted against log concentration of the drugs, and the concentration required for a 50% reduction (ID& was read from the graph. Altogether, 18 clones were established; however, only a few were studied in detail. The data in Table 1 indicate that the MMdU’-mutant viruses were considerably more resistant than the parent virus to MMdU, araT, ACG, and BVdU but were equally sensitive to PAA. Further, while mutant MMdU’-20 had very low resistance to BVdU compared to the other mutants, in its sensitivity to MMdU, ACG, araT, and





Virus strain






wt MMdU*-2 MMdU’-3 MMdU’-12-3 MMdU’-20

0.20 >lOO >lOO >lOO >lOO

0.01 0.90 0.30 0.50 1.20

lO.OO 0.50 7.50 0.03

0.05 2.50 0.50 1.50 1.00

2.0 1.5 2.0 1.5 2.75

a Sensitivities of HSV strains to various antiviral drugs were measured by plaque reduction assay in BHK cells. The number of plaques produced in the control well were arbitrarily set at 100%. The IDm dose was obtained by plotting the percentage survivors against log concentration of the drug on loglog paper and the dose required for a 50% plaque reduction was read from the graph.

PAA it was similar to the others. MMdU’2 and 12-3 differed from all other mutants in resistance to BVdU; they were about 10,000 times more resistant than the parent wild-type virus. These findings prompted us to study the induction of dTK by MMdU’ mutant viruses. BHK cells express cytosol dTK while the 3T3 and L cell lines used are dTK-negative mutants (although they express the mitochondrial dTK). This permits comparative studies of the capacity of individual MMdU’ viruses to induce dTK. For dTK assay BHK, 3T3 dTK-, and L Kit dTK- cells were grown to confluence in duplicate 5-cm plastic petri dishes and infected with mutant HSV clones at 5-10 PFU/cell. The plates were rocked slowly for 2 hr at 37”, 100% humidity, and 5% C02, and the inoculum was then replaced with 5 ml of medium. The cells were harvested at 18 hr after infection by scraping in 0.5 ml of lysis buffer (0.01 M Tris-HCl, pH 7.5, 0.01 M KCl, 0.05 mM deoxythymidine [dT], 1 mM MgC12, and 2 mM dithiothreitol [DDT]), sonically disrupted, and assayed by measuring the amount of 14Clabeled dT phosphorylated by the crude cell extracts as described (8). No MMdU’ clone except MMdU’-20 was able to induce significant amounts of dTK either in BHK






a L dTK-(KIT)


possible that the dTK induced by MMdU’20 has an altered substrate specii.city and does not phosphorylate ACG, araT, and MMdU as well as it does BVdU and dT. Recently Darby et al. (9) have isolated a dTK+ ACG-resistant mutant that could phosphorylate dT but not ACG. Since no detectable dTK could be observed in MMdU’-2 and 12-3, their increased resistance to BVdU relative to that seen with ACG and araT in MMdU’-3 could not be explained solely by the presence or absence of dTK expression, unless MMdU’3 expressed a kinase for which dT was not a detectable substrate but which would phosphorylate BVdU well enough to allow the greater sensitivity observed. It should be pointed out that the detection of phosphorylation of dT is an in vitro biochemical assay and may be less sensitive than the detection of phosphorylation of BVdU, which depends on biological activity. The other possibility is that a mutation might have occurred in the viral DNA Pol which affected the substrate specificity of the Pol; this then might confer different degrees of resistance to the triphosphates of the various nucleoside analogs. In no instance was the sensitivity to PAA affected, which indicates noninvolvement of the Pol PAA binding site in the mutations found. Our data, as well as those of Darby et al. (9), present a more complex picture for nucleoside analog-induced mutations than that which may be inferred from the mere deletion of the dTK. Because ACG, BVdU, and araT are already in human clinical trials, the information about crossresistance of mutants developed in the presence of one drug may be important, as well as the rapidity with which such mutants may arise, a phenomenon also reported by others (10-12) with ACG.

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3 4-14-~~28-19-1Y)-2M~Y)-41l-112-112~212-3 VIRUS STRAINS

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b 3T3



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nnnn nrll-lnn nil ” MI WT 1 2 3 4-14-214228-l 9-110.210dM~l1.112-112-2U-3 14 17 20 VIRUS STRAINS c

FIG. 1. Induction of deoxythymidine kinase (dTK) activity by HSV-WT and MMdU’mutants in (a) BHK cells, (b) dTK L KIT cells, and (c) dTK- 3T3 cells. Confluent cell monolayers were infected at 5-10 PFU/ cell, harvested 18 hr after infection, sonically disrupted, and assayed as described (8). Data are presented for individual mutants, parental (WT) virus, and for mock-infected (MI) cultures.

(Fig. la), dTK- 3T3 (Fig. lb), or dTK- L Kit (Fig. lc) cells. The level of dTK induction by MMdU’-20 was from 50 to ‘70% that of the parent wild-type virus in all cases. Clones 2, 12-3, and 20 maintained the same resistance pattern to araT, ACG, MMdU, and PAA, but MMdU’-20 had minimal resistance to BVdU, while 2 and 123 were highly resistant to this drug. It is

ACKNOWLEDGMENTS This work was supported by NIH Grant DE 5089 and American Cancer Society Grant CH 74. We thank J. Mize, N. Lawrence, G. Arnett, M. Meek, and M. Rehfeldt for technical assistance. REFERENCES

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