Acyclovir-resistant mutants of herpes simplex virus type 1 express altered DNA polymerase or reduced acyclovir phosphorylating activities.

0022-538X/81/120936-06$02.00/0

Acyclovir-Resistant

Mutants of Herpes Simplex Virus Type 1

Express

Altered DNA Polymerase or Reduced Acyclovir

Phosphorylating Activities

PHILLIP A. FURMAN,'* DONALD M. COEN,2 MARTY H. ST. CLAIR,' AND PRISCILLA A. SCHAFFER2

Wellcome Research Laboratories, ResearchTrianglePark, NorthCarolina27709,' and The Sidney Farber CancerInstitute, HarvardMedical School, Boston, Massachusetts 021152

Received 4 June1981/Accepted 8 August 1981

The biochemical properties offouracyclovir-resistant mutants are described. Twoofthese mutants, PAAr5 and

BWr,

specified nucleotidyl transferase (DNA

polymerase) activities which were less sensitive to inhibition by acyclovir

tri-phosphatethan their wild-typecounterparts. Another mutant, IUdRr, exhibited

reduced ability to phosphorylate acyclovir. The fourth mutant, ACGr4, both

induced an altered DNA polymerase and failed to phosphorylate appreciable amounts of acyclovir. BWr,a new acyclovir-resistant mutant derived from the

Patton strain of herpessimplex virus type 1, induced a DNA polymerase resistant toinhibition by acyclovirtriphosphate, but, unlike the polymerases induced by PAAr5andACGr4, stillsensitive to phosphonoacetic acid. Resistance of BWr to

acyclovirmapped close tothe PAArlocus and was separable from mutations in

the herpes simplex virus thymidine kinasegene by recombination analysis.

The nucleoside analog 9-(2-hydroxyethoxy-methyl)guanine (acyclovir, acycloguanosine) is aspecific and effective inhibitor ofherpes

sim-plex virus (HSV) replication (8,25)and

demon-strateslittlecytotoxicitytouninfected

cells

(25).

There has accumulated aconsiderable amount

ofevidence indicating that acyclovir exerts its

antiviral effect after conversiontoacyclovir

tri-phosphate (acyclo-GTP), which inhibits the

viral nucleotidyl transferase (DNA polymerase)

more efficiently than does the host cellaDNA

polymerase

(8,

12).

Biochemical evidence

indi-catesthatHSVthymidine kinase (HSV-TK) is

the enzyme

responsible

for phosphorylationof

acyclovirtoitsmonophosphate (8, 13). Host-cell

enzymes are

apparently

responsible for the

phos-phorylation

of

acyclovir

monophosphate

(acy-clo-GMP) (11, 21).

Parallel with biochemical studies are the

re-sults of genetic experiments which have impli-cated theHSV-TKand DNApolymerasegenes aslociwhich,whenmutated,canconfer resist-ance toacyclovirinthecell culture (4, 5, 7, 27). Withregardtothe TK gene, severalHSV mu-tants lackingTK

activity

exhibit resistance to acyclovir (4,5,8, 9,27),andthedegreeof resist-ance generally corresponds to the level of TK

activity(4,5).

With

regard

to the DNA polymerase gene,

severalmutantswhich areresistant to phospho-noacetic acid (PAA), a recognized marker for

the HSV

DNA

polymerase

gene

(2, 3, 16, 17,

22-24), are also resistant to

acyclovir,

yet exhibit wild-typelevels of TK activity (5, 27). Recom-bination and

complementation

analyses

ofone of thesemutants,

PAAN5,

showed that it

defmes

acodominant locus

(termed

ACGr-PAA)

distinct from the recessive

acgr-tk

locusandmuchmore

closely

linkedtothe PAA' locus than it istothe

acgr-tk

locus.

Complementation

analysis

indi-cated that another mutant,

ACGr4,

was a

pre-sumptive double mutant

containing

mutations

atboth loci that leadto a

highly

resistant

phe-notype(5).

Subsequent

intertypic

and

intratypic

markerrescue

experiments

with other

PAA

mu-tantshavealso

demonstrated

linkage

of

acyclo-vir resistance with the

PAAr

locus and with

temperature-sensitive

mutations within the

HSV DNApolymerasegene

(7;

D.M.Coen and P. A.

Schaffer,

unpublished

data;

D.

Knipe,

per-sonal

communication).

Toconfirmthe

implications

that theHSV-TK

andDNA

polymerase

genesareloci

which,

when

mutated,

can confer resistance to

acyclovir

in

cell culture, we examinedfour

acyclovir-resist-antmutantsderivedfrom the KOS and Patton strains of HSV type 1

(HSV-1). First,

the

sensi-tivity of thesemutants toinhibition

by

acyclovir

and PAAwas examined

(Fig.

1A and

B).

Both the PAA-resistant mutant PAAr5and the

pre-sumptive double mutant ACGr4

(5)

were less

sensitivetoinhibition

by

acyclovir

thanwasthe

936

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937

wild-type virus KOS

(Fig.

1A). Fifty percent

effective

dose

(ED50)

values for PAAr5 and

ACGr4 were 20- and 490-fold greater,

respec-tively, than that for KOS. PAAr5 and ACGr4

also showed much less susceptibility to

inhibi-tion by PAA, with ED50 values more than

10-and 5-fold greater, respectively, than that ob-tained forKOS

(Fig.

1B). Mutants of the Patton strain ofHSV-1, IUdRr (amutantcharacterized by Smithetal.[28]asbeing resistantto

acyclo-vir and iododeoxyuridine) and

BWT

were also

foundto be much less susceptibletoinhibition

byacyclovir thanwasthe wild-typevirus. The

ED50 values for IJdRr and BW'were

approxi-mately

100and 200timesgreater, respectively,

than that for their wild-typecounterparts. How-ever, sensitivity of these viruses to PAA was

considerably different from thatobservedforthe mutants derived from KOS. Both

Patton-de-rived mutants gave dose-response curves with

PAA comparable tothat.of the Patton strains. In fact, wild-type Patton consistently gave

higherED50 valueswith PAA than did the two mutants.

Cells infected with mutantviruseswere then

tested for their abilitytophosphorylate acyclo-vir.Acyclo-GTP levelsincells infectedwith the mutantsACGr4 and

IUdRF

were 0.3and4.0%of thelevels found in cells infected with their

wild-type counterparts (Table 1). The levels of

acy-A.

0

20

z 40

0

0-1: 60

z

801

0.1

a

1.0 10 100

AM

ACYCLOVIR

1000

p O

0

S%I

100 100

pM

PHOSPHONOACETIC

ACID

FIG. 1. Plaque inhibition dose-responsecurvesfor acyclovir (A) and PAA (B) in Vero cells, determined by

using wild-typeKOS(0)andPatton(0) andacyclovir-resistant PAAr5 ([1), IUdRrr(), BW' (A), and ACGr4 (A) viruses. Plaquereduction assaysto determineED50 values foracyclovir and PAA wereperformedas

describedbyCollinsand Bauer(6). Virusstockswerepreparedaspreviously described (8).

0

20

40

z

0

-z

601-80

I 0% .1

2^t%_i-1

40,

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clo-GTP in cells infected with the resistant mu-tants PAAr5 andBWrwere comparable to the leveLs in cells infected with wild-type viruses. Moreover, the total amountofphosphorylated acyclovir (all forms: mono-, di-, and triphos-phates)wasconsiderably higher in cells infected

with wild-type viruses, BWr, or PAAr5than in

cells infected with ACGr4 andIUdRr (datanot

shown). Similarly, extractsprepared from cells infected with IUdRr and ACGr4 contained much less acyclovir-phosphorylating activity and HSV-TK activity than did extracts from cells infected with wild-type virus, whereas BW0 and PAAr5 induced acyclovir-phosphorylating and HSV-TK activities comparabletothose of their wild-typecounterparts(P. Keller, personal

com-munication). Thus, the acyclovir resistance of BWr and PAAr5 cannotbeattributedtofailure of these mutants to phosphorylate acyclovir. The lack of acyclovir phosphorylation (TK expression) by IUdRr probably explains the cross-resistancetoacyclovir and IUdR observed for this virus(28).

The inhibitory effect of acyclo-GTP on the

DNA polymerase of mutant and wild-type

vi-ruses was examined by using [3H]dTTP

incor-poration as a measure ofenzyme activity.

En-zyme inhibition curves (Fig. 2) demonstrated

thatDNA polymerases ofthe viruses couldbe separated intotwoclasses, asensitive class and aresistantclass,onthebasis oftheirsensitivities toinhibition byacyclo-GTP. The sensitive class, having I50 (50% inhibition) values of about0.2

,uM acyclo-GTP (Table 1), was comprised of

bothwild-type strainsand theTK-deficient mu-tant derived from Patton (IUdRr). The DNA

polymerases induced by the KOS-derived mu-tants,PAAr5andACGr4,and thePatton-derived

mutant,BWr,wereless sensitivetoinhibition by

acyclo-GTP than their respective wild types. h5o valuesfor PAAr5, ACGr4, and BWrwere

approx-imately 5-, 9-, and 25-fold higher, respectively,

thanthe

Lo

values obtained for their wild-type

counterparts (Table 1). These data confirm the

suggestionofCoenandSchaffer (5) that PAAr5 andACGr4 contain mutationsatthe DNA

po-lymerase locus conferring acyclovir resistance. The sensitivities of the DNA polymerases of PAAr5,ACGr4, andBWrtoPAA inhibitionwere

alsodetermined (Table 1). The DNA

polymer-aseofPAAr5 and ACGr4 werefoundto be

ap-8100

60

20

0.01 0.1 1.0 10

rIMACYCLO-GTP

FIG. 2. Inhibition of wild-type and mutant virus

DNA polymerases by acyclo-GTP. Virus-induced

DNApolymerasewasisolatedandidentifiedas

de-scribedpreviously (11, 29). DNA polymerase assays werecarriedoutasdescribedby Elionetal. (8) and

Furmanetal. (12). ThesubstratesdATP,dCTP, and

dTTPwerepresentataconcentrationof100pM,and

dGTPwaspresent ataconcentration of5p.M.

Sym-bolsfor polymerases: KOS (0), Patton (O), PAAr5

(0),ACGr4(A),B W (U), and IUdRr(A).

TABLE 1. Summary of the biochemical properties of acyclovir-resistant mutants and their corresponding wildtypes'

Virus Acyclo-GTP Polymerase sensitivity (range)(I50

(,4M])^

Viral

sensitivity

Vrs levels(pmol/ __

ED_____o______M

____

106 cells) Acyclo-GTP PAA Acyclovir PAA

KOS 90.7 0.23 (0.11-0.42) 0.50 (0.21-0.86) 0.7 138 PAA`5 55.4 1.17(0.82-1.62) 3.57(2.36-4.71) 14.3 >1,400d

ACG`4 0.3 2.14(1.47-3.33) 3.78(2.43-5.07) 346 >750 Patton 52.8 0.15(0.09-0.24) 1.93(0.43-4.43) 1.4 260 BWr 121.8 3.71 (3.36-4.08) 0.97(0.55-1.50) 224 195 IUdRr 2.3 0.23 (0.04-0.89) 0.50(0.01-1.93) 136 166

Experimentaldetails may be found in thelegendstoFig. 1and2.

bConcentrations of substrates for theacyclo-GTPinhibitionassayaredescribed in thelegendtoFig.2.

Ih0

values were calculatedbyusingthe Probit computer program, whichplacesmoreweightonthosepointsnear

theI50 point (10).For the PAA inhibition assay, theconcentration of all fourdeoxynucleoside triphosphateswas

100/AM.

TheED0ovaluesweredeterminedby Probitanalysis(10).

dNoinhibitionatthese concentrations.

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NOTES

proximatelyseventimesmoreresistantto inhi-bition by PAA than was wild-type KOS DNA

polymerase. Incontrast,BWrDNApolymerase

wasfoundtobeno moreresistanttoinhibition

by PAA than was the DNA polymerase of its

parental virus, strain Patton. The DNA

polym-erase induced by IUdR' showed notonly wild-type sensitivity to acyclo-GTP but also

wild-typesensitivitytoPAA (Table1).Theapparent

Kmvalues for thefour natural deoxynucleoside

triphosphates ranged from 1 to 4 ,uM (unpub-lished data). All viral DNA polymerase

prepa-rations exhibitedafourfold stimulation of

activ-ity in thepresenceof 50mMammonium sulfate,

whereas cellularaDNA polymerase activitywas

reduced by 50%,indicating that the polymerase preparationswere virus specific (20,29). In

ad-dition, the DNApolymerases induced by KOS, Patton, IUdRr, and BWrwere50-foldmore

sen-sitive than the a cellular DNA polymerase of

HeLa S-3 cellsto inhibition by PAA ata

con-centration of 5,uM, thus confirming their viral

origin.

Genetic experiments previously identified PAAr5as amutantwhose resistancetoacyclovir

was separable by recombination from the

acy-clovir resistance mutations in acgr-tk mutants

and closely linked tothe PAA resistance locus (5). To determine whether themutantBWr be-haved similarlyinrecombination tests,we

per-formed crosses between BW' and the acg'-tk mutant, ACGr35, which is partially acyclovir

resistant owingtoamutation whichreduces TK

activitytoabout 15% ofwild-type levels (5). The abilitytomeasurerecombination between these

twovirusesdependeduponthe factthatneither plated efficiently in400 1LMacyclovir (Table 2). However, when BW' and ACGr35werecrossed,

3.3% of the resulting progeny wereresistantto

400,uM acyclovir (Table 2). These dataimplya

recombination frequency of 6.6%, whichismuch

greaterthananyfound betweenmutantswithin thesamecomplementationgroupwhichmapin

theuniquesequencesofthe HSV-1genome(R. A. F. Dixon and P. A. Schaffer, unpublished data). Similar results (not shown)wereobtained

when BWT was crossed with the conditionally

resistant acgr_tkmutant,KG-ill,whichexhibits thermolabileTKactivity (4).

To determine whethertheacyclovirresistance

ofBWrwaslinkedtothe PAAresistance locus, we crossed BWr with PAAr5, and the progeny

were examined for their plating efficiency in

both PAA at1.4 mM and acyclovirat 100 tiM.

Each parent used in the crosses wasrelatively

resistanttooneof thesedrugsbutquitesensitive

tothe otherortothe combination of bothdrugs

at these concentrations (Table 2). A

recombi-nant of thesetwoviruses would be expectedto

plate efficiently in both drugs. However, only 0.08% of the progeny were resistant to both

drugs, implying a recombination frequency of

only 0.16% (Table 2). In contrast, in a parallel experiment, when the acgr_tk mutant, ACGr35,

TABLE 2. RecombinationofBW,ACGr35, and PAAr5a

PFU/ml EoPb

NorusdrIn

acyclovir In acyclovir RF- RF-P+

Nou

dru_g In acyclovir (100,uM)and Inacyclovir (100uM)and Ad(%) (400

ItM)

PAA (1400 (400jiM) PAA(1,400

JIM) AM)

ACGr35 1.1X107 <5.0X 102 <5.0X 102 <4.6X 10-5 <4.6X 10-5

BWr

2.6X

107

1.6x

105

1.0X

104

6.2x

10-3

3.8x

10-4

PAAr5 2.4X107 1.0X103 5.0x 102 4.0X 10-5 2.0x 10-5

BWTxACGr35 3.3X 107 1.1x 106 3.3x 10-2 6.6

PAAr5x

BWT

3.2x107 2.5X 104 8.0x 10-4 0.16

PAAr5xACGr35 7.2x106 7.5x 104 1.0x 10-2 2.0

aRecombinationanalysiswasperformed

essentially

asdescribedby Schafferetal.(26),exceptthat Vero

cells

wereused instead ofHELcells, and recombinationwasperformedat37°C. Duplicatetubecultures of Vero cells containingapproximately2x105cellsperculturewereinfected either withpairsof mutants, eachat acalculated

multiplicityof2.5plaque-formingunits(PFU)per cell inatotal volume of0.2ml,orwithsingle parentalvirus controls at a multiplicity of5 PFU per cell in 0.2 ml. Simultaneous assays of inoculum suspensions were

performedtoconfirmcalculatedinput multiplicities; if the actualmultiplicity variedmorethantwofold from the calculatedmultiplicity,results oftestswiththesemutantswereexcluded.

bEOP, Efficiency ofplating.EOP= (PFUpermilliliter in presence ofdrug)/(PFUpermilliliter in absence ofdrug).

'RF - A, Recombination frequency. RF- A = [(PFU per milliliter in presence ofacyclovir)/(PFU per

milliliterin absenceofacyclovir)]x 2x 100%.

dRF - P + A, Recombination frequency. RF -P + A = [(PFU per milliliter in presence of PAA and

acyclovir)/(PFUpermilliliter in absence of PAA andacyclovir)] x2 x100%c.

VOL. 40,1981

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andPAA'5were crossed andthe progenywere analyzed under identicalconditions,the

recom-binationfrequencywasmorethan 12-foldhigher

(Table 2).

Thus,bothgenetic and biochemicalevidence supportthenotion thatPAAF5, ACGr4,andBWT contained mutations in their DNApolymerase genes which conferred resistance to acyclovir. The resultsofthisstudyindicate thatmutations

can occurintheDNApolymerasegenethatwill confer resistance to bothacyclovirand PAA or

to acyclovir but not PAA. The latter result would beexpected ifthe HSV DNApolymerase conforms to the model proposed by Kornberg

(18) for other DNApolymerases;i.e.,DNA po-lymerase hasanactive center that iscomposed

ofmultiple sites, eachwithadifferentfunction.

Therefore, amutation which affects theprotein

atasiteother than thepyrophosphateexchange

site (the presumptive site of PAA inhibition

[19]) will not necessarily affect the pyrophos-phate exchange site (resistance to PAA). Nev-ertheless, thesimplest explanationforthedata

obtained for the mutantsPAAr5 andACGT4is

thatasinglemutationcanaffectmorethanone site.

Thenewmutantdescribedhere, BWr,which was acyclovir resistant but PAA sensitive,

de-fines yet another phenotype within the DNA

polymerase locus and separatesthe domain of

the DNA polymerasemolecule whichspecifies acyclovir sensitivity from the domain which

specifies PAA sensitivity. Thus, mutants asso-ciatedwith the HSV DNApolymeraselocuscan be temperatureresistant, drugresistant,orboth

(1, 14, 15, 17, 23), the degree of resistance to

both PAA and acyclovir varying. A detailed understanding of the molecular basis for the

widerangeofphenotypeswithin the HSVDNA

polymerase locus awaits further fine-structure

mapping and additionalbiochemical studies of

itsgeneproduct(s).

(This work waspresented in partat the 5th

Cold Spring Harbor Workshop on Herpes

Vi-ruses, ColdSpring Harbor, N.Y., on 31 August

1980.)

Wethank C.Lubbers,P. A.Temple,L. B.Sandner,and P. T. Gelep for excellent technical assistance, J. A. Fyfe for valuable discussion, G.B. Elionfor criticalreading of the manuscript and for continuous support andinterestduring thiswork,andK.0.Smithforsograciously providinguswith hismutants.

ThisstudywassupportedinpartbyPublic Health Service researchgrantCA20260 andprogramprojectgrantCA21082 fromtheNationalCancer Institute. D.M.C.wastherecipient ofpostdoctoralfellowship AI05817from the National

Insti-tutesof Health.

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