Proc. Natl. Acad. Sci. USAVol. 81, pp. 1556-1560, March 1984Microbiology

Resistance of herpes simplex virus to 9-{1[2-hydroxy-1-(hydroxy-methyl)ethoxy]methyl}guanine: Physical mapping of drugsynergism within the viral DNA polymerase locus

(antiviral resistance/active center of herpes DNA polymerase)

C. S. CRUMPACKER*t, P. N. KOWALSKY*t, S. A. OLIVER*t, L. E. SCHNIPPER*t, AND A. K. FIELD§*Charles A. Dana Research Institute and the Harvard Thorndike Laboratory, Beth Israel Hospital, and the Divisions of tInfectious Disease and tOncology,Department of Medicine, Beth Israel Hospital and Harvard Medical School, Boston, MA 02215; and §Department of Virus and Cell Biology, Merck Sharp &Dohme Research Laboratories, West Point, PA 19486

Communicated by Harold S. Ginsberg, October 27, 1983

ABSTRACT A herpes simplex virus type 2 (HSV-2) mu-tant TS6 (strain HG52) induces a heat-labile viral DNA poly-merase at the nonpermissive temperature and is markedly re-sistant to 9-{[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl}-guanine [2'-nor-2'-deoxyguanosine; 2'NDG]. This antiviraldrug requires HSV thymidine kinase for phosphorylation to anactive inhibitor (2'NDG-triphosphate), and thymidine kinase-deficient mutants of HSV exhibit varying degrees of resistanceto 2'NDG, with the HSV type 1 (HSV-1) B2006 mutant (Kit)being markedly resistant. The ts6 mutation and the 2'ndg"-1mutation within the viral DNA polymerase locus have beenphysically mapped by marker rescue and generation of HSV-1/HSV-2 intertypic recombinants. The physical map limits forthe ts6 mutation and 2'ndg"-1 mutation are closely linkedwithin a 2.2-kilobase-pair region of DNA sequences and arephysically separate from the paaRl and acvR-1 mutations.Resistance to 2'NDG by HSV-2 ts6 can be overcome in thepresence of combinations of 2'NDG and phosphonoacetic acid,indicating drug synergism within the viral DNA polymeraselocus. These physical mapping studies expand the limits ofDNA sequences defining an active center in the viral polymer-ase to 3.5 kilobase pairs, indicating that regions spanning theentire polymerase polypeptide may contribute to a specializedsurface able to interact with nucleotides of different structure.

The new antiherpes nucleoside analog 9-{[2-hydroxy-1-(hy-droxymethyl)ethoxy]methyl}guanine (2'-nor-2'-deoxyguano-sine; 2'NDG) is an efficient inhibitor of herpes simplex virustypes 1 and 2 (HSV-1 and HSV-2), human cytomegalovirus,and Epstein-Barr virus replication (1-4). It is a preferentialsubstrate for the HSV-1 thymidine kinase (TK), is readilyphosphorylated to the triphosphate, and is a potent inhibitorof viral DNA polymerase (4). It inhibits herpes virus replica-tion in cell culture and has therapeutic efficacy against her-pes virus infections in mice. 2'NDG is a structural analog ofacyclovir [9-(2-hydroxyethoxymethyl)guanine; ACV].

Restriction endonuclease analysis of the genomes ofplaque-purified intertypic recombinants of HSV-1 and HSV-2, generated by marker rescue, has permitted physical map-ping of temperature-sensitive mutations and drug resistancemutations associated with HSV DNA polymerase activity.These included tsC4, tsC7, tsD9 (HSV-1, strain KOS), tsH(HSV-1, strain 17), and ts6 (HSV-2, strain HG52), which areall associated with a thermolabile DNA polymerase activity(5-9). Drug resistance mutations conferring resistance tophosphonoacetic acid (PAA), ACV, and adenine arabinonu-cleoside (araA) have also been shown to be defined by a 2.6-kilobase-pair (kbp) region of HSV-1 DNA sequences withinthe viral DNA polymerase locus (5, 10, 11). The recombi-

nants containing the drug resistance mutations induce an al-tered viral DNA polymerase enzyme exhibiting altered en-zyme kinetics and a higher Ki in the presence of PAA, di-deoxynucleotide triphosphates, ACV-TP, ara-ATP, and (E)-5-(2-bromovinyl)-2'-deoxyuridine triphosphate (BrVdUrd-TP) (ref. 12; unpublished data). Purified viral polymerasefrom other drug-resistant mutants have also exhibited al-tered enzyme kinetics in the presence of ACV-TP (13) andara-ATP (14).The physical mapping of drug resistance mutations that

result in altered viral DNA polymerase activity in the pres-ence of these nucleotide triphosphates was used to proposean active center on the viral polymerase enzyme. This is aregion of 1.3 kbp in the DNA polymerase locus or a maximalregion of 2.6 kbp if the region of uncertainty where cross-over events occur is included (12).

In this report we describe a mutant of HSV-2 ts6 (strainHG52) that contains a mutation in the structural gene of viralDNA polymerase and is markedly resistant to 2'NDG. The2'ndgR-1 mutation has been physically mapped to a 2.2-kbpregion of HSV-2 DNA sequences within the DNA polymer-ase locus and is physically separate from the DNA se-quences containing the resistance mutations for ACV andPAA. The resistance of HSV-2 ts6 to 2'NDG can be over-come in the presence of 2'NDG and PAA, demonstratingdrug synergy within the viral DNA polymerase locus. Thesephysical mapping studies expand the limits of DNA se-quences defining the viral DNA polymerase active center to3.5 kbp. These observations suggest that through tertiaryfolding almost the entire DNA polymerase polypeptide maycontribute to a specialized surface that is able to interactwith nucleotides of different structure.

MATERIALS AND METHODSCells. A continuous line of African green monkey kidney

cells (Vero) was grown in Eagle's minimum essential medi-um supplemented with 10% calf serum. Cell monolayers (2.4x 106 cells) in 60-mm (diameter) plastic Petri dishes (FlowLaboratories) were used throughout.

Viruses. The wild-type strains of HSV-1 (KOS and 17) andHSV-2 (HG52) are PAAS and ACV'. The temperature-sensi-tive (TS) mutant of HSV-2 ts6 (strain HG52) has been de-scribed (15, 16). The HSV-1 PAA-resistant mutants of strain17 (ts+, PAAR_1) and strain KOS (ts+, PAAR-5) were ob-tained from John Subak-Sharpe and Priscilla Schaffer, re-spectively. The TK-deficient mutant (dPyK7) of HSV-1

Abbreviations: 2'NDG, 9-{[2-hydroxy-1-(hydroxymethyl)ethoxy]-methyl}guanine or 2'-nor-2'-deoxyguanosine; ACV, 9-(2-hydroxy-ethoxymethyl)guanine or acyclovir; PAA, phosphonoacetic acid;araA, adenine arabinonucleoside; BrVdUrd, (E)-5-(2-bromovinyl)-2'-deoxyuridine; kbp, kilobase pair(s); HSV-1 and HSV-2, herpessimplex virus types 1 and 2, respectively; TK, thymidine kinase.

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(strain 17) had been isolated by growth of wild-type virus inBrdUrd (5 ,ug/ml) (17) and the mutation was mapped bymarker rescue to the viral TK locus (18). The ACVR-6 mu-tant of HSV-1 was obtained by passing a clinical isolate ofHSV-1 in the presence of ACV (0.5 ,ug/ml) for six passagesto select a tk- mutant that expressed only 8% of the TKactivity of the wild-type virus (11). The markedly TK-defi-cient mutant B2006 of the Kit strain was obtained from Da-vid Knipe and its properties have been described (19).

Drugs. 2'NDG was supplied by Richard Tolman (MerckSharp & Dohme). A stock solution of 5 mM 2'NDG was pre-pared in distilled H20 and stored at -20'C. ACV was sup-plied by Burroughs Wellcome (Research Triangle Park, NC)and a 10 mM stock solution was prepared in distilled H20and stored at -200C. PAA as the disodium salt was the gift ofAbbott.

Intertypic Marker Rescue and Analysis of Genome Struc-ture of Recombinants of HSV-1 and HSV-2. The technique ofintertypic marker rescue of ts mutations has been describedin detail (5), and the genome structures and phenotypic char-acteristics of the R6 recombinants have been published (5).In brief, dishes of baby hamster kidney cells were coinfectedat the nonpermissive temperature (38.5°C), per dish, with 0.4,g of intact DNA from the HSV-2 ts6 (HG52) mutant and 0.4pg of unseparated, restriction endonuclease-cleaved frag-ments of DNA from HSV-1 strain 17 (ts+, paar1). DNAtransfection was by use of the modified calcium phosphatetechnique (20). The ts+ virus progeny from such crosseswere plaque-purified three times at 38.5°C, and the parentalorigin of restriction endonuclease sites in the DNA was de-termined (5). Resistance to 100 pg ofPAA was present in theDNA crosses as a nonselected marker. The ts+ recombi-nants were previously tested for resistance to PAA by deter-

mining the relative efficiency of plaguing in the presence andabsence of drug and for resistance to ACV by determiningthe ID50 (,M) of drug (10).The physical maps of HSV-1 strain 17 and HSV-2 strain

HG52 for the restriction endonucleases Xpa I (1), HindIII(2), EcoRI (3), Bgl 11 (4), Hpa I (5), Kpn I (6), and BamHI (7)in the region of 30-50 map units on the HSV genome havebeen determined (5, 18). All of the restriction sites are sum-marized at the top of Fig. 1. The numbers correspond to thenumbers assigned to each restriction endonuclease, and thesites are aligned with the corresponding one for each map.The restriction sites and the corresponding fragments areshown above the line for HSV-1 and below the line for HSV-2. One map unit is equivalent to 106 daltons (1.6 kbp), andthe genome orientation is as described (18). The order andsize of the restriction endonuclease fragments for each typehave been defined (21, 22).

Inhibition of HSV Plaque Formation by 2'NDG. The antivi-ral activity of 2'NDG was assayed by a plaque-reductionmethod performed in the presence of increasing concentra-tions of drug in Vero cells. Inhibition of plaque formation by2'NDG and PAA together was determined by keeping onedrug constant (PAA) and increasing the other over a broadrange of concentrations. Both drugs were present in theoverlay medium. The ID50 (pg/mI) of each drug combinationor single drug relative to control wells without drug was cal-culated directly from the plot relating surviving plaques inthe presence of increasing concentrations of drug on semi-logarithmic paper. The relation of the percentage of survi-vors vs. the logarithm of the drug concentrations is a linearfunction in the region of 50% reduction, and the best linearplot was obtained by the method of a least squares fit (23).To determine synergism, the ID50s obtained in the presence

R6-26

R6-30

R6-34

R6-19

HSV-2ts6

30 35 40 45 50

4 5 5 4 153 3 ~~~3

6 6 6 6 6 6 6 6

71 7717 71 6X77 777

66 666 6

4 5 5 4 2 4

4

2 74 7

7 6

4 7

4 6 3

2 7

6 6

6 7

PAA -I

HSV-I (strain 17 ) WT

HSV-2 (stain HG52)WT

ID 50(Mg/m2'NDG ACV

0.05

0.08

0.07

0.10

15.50

0.10

0.34

1.60

0.03

0.05

4.00

2.30

0.32

0.92

0.13

0.27

FIG. 1. Genome structure determined by restriction endonuclease analysis (5, 10) and sensitivities to 2'NDG and ACV of R6 recombinantsand parental strains of HSV. Endonuclease cleavage sites for the enzymes given in the Materials and Methods are shown above the line forHSV-1 and below the line for HSV-2; solid regions indicate HSV-1 DNA sequences, clear regions indicate HSV-2 DNA sequences, andhatching indicates regions of uncertainty. Data on 50o inhibition with ACV are from ref. 10.

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Table 1. Sensitivity of mutants of HSV to 2'NDG, ACV,and PAA

ID50, pg/miType 2'NDG ACV PAA

HSV-1KOS WT 0.27 0.16 12.0PAAR5 (KOS) 0.23 5.2 120.0Strain 17 WT 0.34 0.13 14.0PAAR_1 (strain 17) 0.10 0.9 150.0dPyK-7 (strain 17) 0.82 4.7 19.0ACVR-6 (clinical isolate) 11.0 14.8 4.0B2006 (Kit) 55.0 12.5 11.0

HSV-2HG52 (WT) 1.6 0.27 11.0ts6 (HG52) 15.5 0.28 12.0

The 50% inhibitory dose (ID50) is the concentration of drug thatinhibited viral plaque formation by 50% on Vero cells. Each value isthe mean of three independent assays and in each case the SD was<10% of the mean.

of various combinations of 2'NDG and PAA are plotted onan isobologram. The isobologram using ID50 concentrationof two antiviral drugs against a single virus is defined to illus-trate synergism for the combination when 50% inhibition oc-curs with only one-fourth the ID50 concentration of eachdrug alone (24).

RESULTSResistance of HSV-2 ts6 and TK- Mutants to 2'NDG. The

HSV-1 strains PAAR_1 (strain 17) and PAAR_5 (strain KOS)were derived by passage in the presence ofPAA (100 Mg/ml)and exhibit resistance to PAA, ACV, and araA (6, 10, 11,13). These two mutants are sensitive to plaque reduction by2'NDG and the ID50 is similar to that of the HSV-1 strain 17and strain KOS viruses (Table 1). The HSV-2 ts6 (strainHG52) mutant is sensitive to inhibition by PAA and ACV butit is 10-fold more resistant to 2'NDG than the parent HSV-2(HG52) (ID50 = 15.5 ,g/ml). The HSV-2 ts6 mutant inducesnormal levels of viral TK and alkaline DNase and these locican be excluded as playing a role in resistance ofHSV-2 (ts6)to 2'NDG (12, 25). These studies indicate that resistance to2'NDG is mediated by the viral DNA polymerase locus, butby a different region of DNA sequences than those contain-ing resistance mutations for paaR_1 and acvR1.

HSV-1Bgl-ll i-d

39.6

HSV-2Bgl-ll o-c

40.2

2'ndg r_,

HSV-2Bam Hi h'-j'

41.0

Resistance to 2'NDG can also be mediated by the viral TKlocus. The dPyK-7 mutant of HSV-1 (strain 17) induces onlya small amount of viral TK activity, is markedly resistant toACV, and exhibits only one-half as much sensitivity to2'NDG compared to HSV-1 strain 17 (Table 1). However,the ADVR-6 mutant, a TK- mutant derived by serial passagein ACV and inducing only 8% of the viral TK of the initialWT virus, exhibits marked resistance to both ACV and2'NDG. Mutant B2006 (Kit) expresses no TK activity, failsto induce the 43-kilodalton viral TK protein, and is the mostmarkedly resistant mutant to ACV and 2'NDG [ID50 = 55,g/ml]. Thus, resistance to 2'NDG is mediated by both theviral DNA polymerase and TK loci of HSV. A mutant ex-pressing diminished TK activity, dPyK-7 (strain 17), ismarkedly resistant to ACV but only one-half as sensitive to2'NDG, suggesting a difference in the manner in which thesetwo drugs are processed by the viral TK enzyme.

Physical Map Limits of the 2'ndg"-l Mutation. To physical-ly define the map limits for the 2'ndgR-1 mutation within theviral DNA polymerase locus, HSV-1/HSV-2 intertypic re-combinants previously employed to demonstrate the closelinkage of the acvR-1, paaRJ, and araAR_1 mutations weretested for plaque inhibition in the presence of 2'NDG. Theserecombinants contain a small insertion of DNA sequencesfrom mutant PAAR_1 of HSV-1 (strain 17) in an otherwiseHSV-2 genome (ts6, strain HG52). The insertion of HSV-1DNA sequences corrects the ts6 defect, enabling the recom-binants to grow at the nonpermissive temperature of 38.5°C(5). The genome structure of these recombinants in the re-gion of 30-50 map units on the HSV genome, the restrictionendonuclease enzymes used in the analysis, and the ID50 foreach recombinant and the parental strains to 2'NDG andACV are depicted in Fig. 1.The R6-26 recombinant contains an HSV-1 DNA insertion

extending from the HSV-1 Bgl II i-d site to the HSV-2BamHI h'-j' site (maps units, 39.6-41.0, including the regionof uncertainty where crossover events occur). This is thesmallest region ofDNA sequences that all of the R6 recombi-nants have in common and this region of DNA sequencesdefines the maximal map units containing the HSV-2 ts6 mu-tation. The R6-30 recombinant contains an HSV-1 DNA in-sertion extending from the HSV-1 BamHI g-v site to theHSV-2 BamHI h'-j' site (map units, 37.8-41.0); the R6-34recombinant contains an HSV-1 DNA insertion extendingfrom the HSV-1 Bgl II i-d site to the HSV-1 EcoRI m-o site(map units, 39.6-42.8); and the R6-19 recombinant contains

HSV-1Kpn x-cI1

41.8

HSV- 1Eco RI m-n

I

42.8

ara-A r_ 1, acv r_ 1, paa r-1eVIMA/ 'S"""0X' 5

bvdu r -1

Kbp01

2.1 4.2 5.1

FIG. 2. Physical map limits of drug resistance mutations within the DNA polymerase locus of HSV. Restriction endonuclease sites andfractional location on the HSV-1 genome are shown. Hatched areas indicate regions of uncertainty where crossover events can occur. Maximallimits for the 2 ndgR-1 mutation are obtained from this study and are compared with previously derived limits for paaR-1 (5, 6), acvR-1 (10),araAR_1, and bvdu -1 (mutation for resistance to BrVdUrd) (11). Limits shown for paaR_1 , acvR-1 , and araAR_1 do not imply order but only thatresistance mutations for these three drugs are closely linked within a 2.6-kbp region of HSV DNA.

Proc. NatL Acad Sci. USA 81 (1984)

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0

z

CMj

8 12

PAA [Mug/mi]

FIG. 3. Isobologram using the 50% plaque-reduction concentra-tion (ID50) of 2'NDG and PAA (pg/ml) to illustrate the synergisticeffect of the combination of two drugs on HSV-2 ts6. The ID50 foreach combination was determined by plaque-inhibition assay inVero cells and a plot of survivors vs. the logarithm of the drug con-centration for 2'NDG with PAA held constant over several concen-trations.

an HSV-1 DNA insertion extending from the HSV-2 Kpn Ij-m site to the HSV-1 Kpn I x-c site (map units, 37.6-41.8).All of these recombinants have corrected the ts6 mutationand therefore grow at 38.50C. All are markedly sensitive to2'NDG (ID50s = 0.05-0.10 ,ug/ml) but have varying pheno-types with respect to PAA, ACV, araA, and BrVdUrd resist-ance. Recombinant R6-34 is resistant to PAA, ACV, araA,and BrVdUrd; R6-19 is resistant to PAA, ACV, and araA;and R6-26 and R6-30 are sensitive to PAA, ACV, araA, andBrVdUrd (10, 11). The physical map limits of DNA sequencecontaining the resistance mutation for 2'NDG overlap withthe DNA region containing the resistance mutations forACV, PAA, and araA (Fig. 2). However, the resistance mu-tations for these three drugs and for BrVdUrd can be trans-ferred separately from the 2'ndgR_1 mutation. This indicatesthat the resistance mutations occur in separate parts of theDNA polymerase locus, even though the mapping limits con-tain regions of overlap.Synergism of 2'NDG and PAA on HSV-2 ts6. The HSV-2

ts6 mutant exhibits marked resistance to 2'NDG (ID50 =

15.5 ,ug/ml) but this resistance can be overcome in the pres-ence of small amounts of PAA. The isobologram of the ID50sfor HSV-2 ts6 in the presence of both drugs indicates that thedecrease in amount of 2'NDG required to inhibit plaque for-mation by 50% in the presence ofPAA is greater than wouldbe expected for an additive relationship (Fig. 3). In the pres-ence of 1 Ag of PAA per ml, only 4.4 pAg of 2'NDG per ml isrequired to inhibit plaque formation by 50% compared to18.0 ug/ml with 2' NDG alone. For the purposes of thisstudy, synergism is defined to occur when the concentrationof drug that produces 50% inhibition is reduced to one-fourthof the drug concentration required when acting alone (24).By this definition 2'NDG and PAA are able to act synergisti-cally to overcome the resistance to 2'NDG exhibited byHSV-2 ts6. The resistance mutations to these two drugs oc-

cur in physically distinct regions of the viral DNA polymer-ase locus. This indicates that these two drugs can act syner-gistically to inhibit viral DNA polymerase, probably by theirinteraction with distinct regions of the viral polymerase en-zyme.

DISCUSSIONThe HSV-2 ts6 mutation had been physically mapped to theviral DNA polymerase locus and this mutation has been as-

sociated with a thermolabile HSV DNA polymerase activity(5, 7). In this report we establish that the physical location ofthe ts6 mutation is either identical or closely linked to the

resistance mutation for 2'NDG. The HSV-2 ts6 mutant ismarkedly resistant to 2'NDG, and correction of the ts6 muta-tion by insertion of a 2.2-kbp region of DNA sequences ex-tending from the HSV-1 Bgi II i-d site to the HSV-2 BamHIh'-j' site (map units, 39.6-41.0) also results in recombinantsthat are sensitive to 2'NDG. The mapping limits for the2'ndgR_1 mutation are identical to those obtained for the ts6mutation. An alternative explanation that a "second site" re-arrangement of sequences results in intergenic or intragenicsuppression of the mutant phenotype is unlikely because ofthe wide range in the length of the rescuing DNA sequence.This argues against crossovers that result in intragenic sup-pression.The assertion that the HSV-2 ts6 mutation and the 2'ndgR_1

mutation are identical or very closely linked within the DNApolymerase locus is analogous to the assertion for the HSV-1tsD9 (strain KOS) and the paaR mutation (26, 27). The HSV-1 tsD9 mutant has a mutation in the structural gene for DNApolymerase, is unable to replicate viral DNA at 38.50C, andis resistant to PAA (8, 26, 27). A revertant that is able togrow at the nonpermissive temperature also becomes sensi-tive to PAA (26, 27). Physical mapping of the tsD9 mutationby marker rescue in 14 HSV-1/HSV-2 intertypic recombi-nants indicates that the physical map limits for the tsD9 andthe paaR_1 mutations are contained in a 2.6-kbp region ofDNA sequences extending from the HSV-2 Bgl II o-c site tothe HSV-1 Kpn I x-c site (map units, 40.2-41.8) (6). Thisregion of DNA sequences also contains acvR_1 and araAR_1mutations (10, 11) and overlaps with the region of DNA de-fining the 2'ndgR-1 mutation, but the phenotype for resist-ance to 2'NDG can be physically separated from resistanceto the other three drugs. All of these drug resistance muta-tions taken together are defined by a 3.5-kbp region ofDNAwithin the DNA polymerase locus.Synergy between 2'NDG (referred to as BIOLF-62) and

PAA or phosphonoformate has been demonstrated for HSV-1 and HSV-2 (28). A combination of 2'NDG and ACV didnot exhibit synergy in this system. In this report, we haveshown that 2'NDG and PAA can interact in a synergisticmanner to overcome the resistance of HSV-2 ts6 to inhibi-tion by 2'NDG alone. Synergistic antiviral activity has beenobserved for araA and PAA acting in combination to inhibitherpes virus replication (29). In addition to the physical map-ping studies indicating that the paaR_1 and 2'ndgR_1 muta-tions are defined by different regions of the viral DNA poly-merase locus, the demonstration of synergism between PAAand 2'NDG strongly suggests that these drugs interact withdifferent parts of the viral DNA polymerase. An alternativeexplanation could be that PAA stimulates error-prone viralDNA synthesis, which is more susceptible to internucleotideincorporation or chain termination by 2'NDG-TP.These studies extend the region of DNA sequences in

HSV-1 that contain mutations conferring resistance to nucle-oside analogs (Fig. 2). The previously described 2.9-kbp re-gion containing the bvduR-1 mutation (HSV-2 BamHI h'-j'site to HSV-1 EcoRI m-o site; map units, 41.0-42.8) and the2.6-kbp region containing the paaR-1, acvR_1, and araAR_1mutations (HSV-2 BgI II o-c site to HSV-1 Kpn I x-c site;map units, 40.2-41.8) are now joined by a 2.2-kbp regioncontaining the 2'ndgR_1 mutation (HSV-1 Bgl II i-d site toHSV-2 BamHI h'-j' site; map units, 39.6-41.0). This ex-tends the region ofDNA sequence containing mutations thatconfer resistance to nucleoside analogs leftward to the HSV-1 Bgl II i-d site (map units, 39.6) and rightward to the EcoRIm-o site (map units, 42.8). The right-hand limit can be re-duced further to the HSV-1 Kpn I x-c site (map units, 41.8)by considering the limits previously used to define an activecenter on the viral DNA polymerase and where the limits forthe paaR_1, acvR_1, araAR_1, and bvduR-1 mutations overlap(map units, 41.0-41.8) (11, 12).

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The region of DNA extending from the HSV-1 Bgl II i-dsite to the HSV-1 Kpn x-c site (map units, 39.6-41.8) com-prises a 3.5-kbp region of DNA containing all of the drugresistance mutations mapping within the DNA polymeraselocus and is in close agreement with the region of DNA se-quences determining the potential mRNA for the viral DNApolymerase. This is a 4.2- to 4.3-kbp transcript, whose 5' endbegins near the HSV-1 BamHI v-r site, spans the HSV-1 BglII i-d site, and extends to a 3' end between the HSV-1BamHI k'-w site and the HSV-1 Kpn I x-c site (L. Hollandand M. Levine, personal communication).The fact that resistance mutations can be shown to physi-

cally map along the entire length of DNA transcribing thispotential mRNA suggests that an active center or several ac-tive centers of polymerase function are determined by tertia-ry folding of the entire DNA polymerase polypeptide. A de-tailed picture of this tertiary folding must await the aminoacid sequence analysis of the viral DNA polymerase enzymeor be deduced from the nucleotide base sequence analysis ofthe viral DNA polymerase gene.We thank Drs. John Subak-Sharpe, Priscilla Schaffer, David

Knipe, and Ian Hay for kindly providing stocks of mutant virusesand also thank Drs. Lou Holland and Myron Levine for permissionto cite their work prior to publication. We gratefully acknowledgethe assistance of Ms. Eileen Poe in the preparation of this manu-script. This work was supported by Contract AI-52530 from theAntiviral Substances Program of the National Institute of Allergyand Infectious Disease and National Dental Institute, Grant NIA-1-P01-AG0059 from National Institute of Aging, and a grant fromMerck Sharpe & Dohme Research Laboratories.

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