Physical mapping of drug resistance mutations defines an active center of the herpes simplex virus DNA polymerase enzyme.

0022-538X/81/090746-12$02.00/0

Physical Mapping

of

Drug

Resistance Mutations

Defines

an

Active Center of the

Herpes

Simplex

Virus DNA

Polymerase

Enzyme

KARL W.

KNOPF,"12

ELLIOT R. KAUFMAN,3AND CLYDE CRUMPACKER'4*

Medical ResearchCouncilVirology Unit,InstituteofVirology,University ofGlasgow,Glasgow,Scotland';

InstitutfurVirusforschung, DeutschesKrebsforschungzentrum,Heidelberg, FederalRepublicofGermany2 Division of HumanGenetics, Children's Hospital Medical Center, and DepartmentofMicrobiologyand MolecularGenetics,Harvard MedicalSchool, Boston,Massachusetts022153;and Divisionof Infectious

Diseases,DepartmentofMedicine,Beth IsraelHospital, and Harvard MedicalSchool,Boston, Massachusetts 022154*

Received21January 1981/Accepted20May1981

The genome structuresofherpes

simplex

virustype 1

(HSV-1)/HSV-2

inter-typic recombinants have been

previously

determined

by

restriction endonuclease

analysis, and these recombinants and theirparental strains have been employed

todemonstrate that mutations within the HSV DNA

polymerase

locus inducean

alteredHSVDNA

polymerase activity,

exhibiting

resistance tothree inhibitors

ofDNApolymerase. The viral DNA

polymerases

induced

by

tworecombinants

and theirparental strainswerepurified andshown topossess similar molecular

weights (142,000 to 144,000) and similar

sensitivity

to

compounds

which

distin-guish viral and cellular DNA

polymerases.

The HSV DNA

polymerases

induced

by the resistant recombinant and the resistant

parental

strainwereresistantto

inhibition by phosphonoacetic acid, acycloguanosinetriphosphate, and the

2',3'-dideoxynucleoside

triphosphates.

The resistant recombinant

(R6-34)

inducedas

much

acycloguanosine

triphosphate

asdid thesensitive recombinant

(R6-26),

but

viralDNAsynthesis in infected cells and the viral DNA

polymerase activity

were

notinhibited. The

2',3'-dideoxynucleoside-triphosphates

wereeffective

competi-tive inhibitors for theHSVDNApolymerase, and the

Ki

values for thefour

2',3'-dideoxynucleoside

triphosphatesweredetermined for the four viral DNA

polym-erases. The polymerases of the resistant recombinant and the resistantparent

possessedamuchhigher

Ki

for the

2',3'-dideoxynucleoside triphosphates

andfor

phosphonoacetic acidthandid the sensitivestrains.A

1.3-kilobase-pair

region of

HSV-1DNAwithintheHSVDNApolymerase locuscontainedmutations which

conferred resistance to three DNApolymerase inhibitors. This region ofDNA

sequences encoded foran amino acid sequence of42,000 molecular weight and

definedanactivecenterof theHSVDNApolymerase enzyme.

Thegrowth of herpes simplex virus (HSV) in

the presence of antiviral agents such as

phos-phonoacetic acid (PAA) or acycloguanosine

(ACG) leads to the selection ofdrug-resistant

mutants (13, 25). This resistance is genetically

stableandprovides adistinct phenotypeinHSV

designated PAArandACGr. The mutations

con-ferringresistanceprovidetwo markersforviral

gene functions and the physical locations of

thesemutations,paarand aCgr, have been

de-finedinthe herpes genome (5, 6, 8). The

selec-tion ofHSVtype 1 (HSV-1) andHSV-2 mutant

viruses which are resistant toPAA readily

oc-cursafterpassage of the virus inthepresence of thedrug, is the result of a single mutation (14),

and isdueto analteredHSV DNA polymerase

activity

(5, 13). The guanosine analog ACG

[9-(2-hydroxyethoxymethyl)guanine;

acyclovir]

re-quiresphosphorylation toexertits antiviral

ef-fect (9, 11). The virus-coded deoxypyrimidine

kinaseproduces phosphorylationtoACG

mono-phosphate, and a subsequent conversion to

di-andtriphosphatespresumably occursbycellular

pathways (11). The ACG triphosphate

(ACG-TP) is a main metabolite ofinfectedcellsand is

an effective inhibitor ofwild-type (WT) HSV

DNApolymerase(11,12, 26).GrowthofHSV in

the presence of ACG selects for resistant mu-tants; thisresistanceiscontrolled bytwo genetic loci in the HSVgenome, the viral

deoxypyrimi-746

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dinekinaseand theviralDNApolymerase loci

(7,25).

Restriction endonuclease analysis of the

ge-nomes of plaque-purified HSV-1/HSV-2

inter-typicrecombinants has permitted the physical

map limits for two distinct regions controlling

ACGtobedefinedwithin the HSVDNA

polym-eraselocus (8).One of these regions contains the

mutation for acgrclosely linkedtothe mutation

forpaar, and these mutationsarefound within

a1.3-kilobase-pair region of HSV DNA for

HSV-2and withina2.6-kilobase-pair region for

HSV-1. The other region is found within adjacent

sequences of HSV DNA and defines the

type-specific sensitivity of HSV-1 and HSV-2 ACG.

These regionsarefound inequivalent positions

within the HSV-1 and HSV-2 genome in the

areadefinedasthe HSVDNApolymerase locus.

Twoof theseintertypic recombinants contain an

HSV-1 DNA insertion in an otherwise HSV-2

genomeanddiffer significantly intheir

sensitiv-itytoACGandPAA.Theserecombinants were

derived by markerrescue of

temperature-sensi-tive (ts) mutations and resulted from a cross

between the DNA of HSV-2 ts6 (HG-52) and

HSV-1 strain 17 (ts+, paar_1) (R6 series) (5).

The R6-26 recombinant is sensitive to plaque

inhibitionbyPAA andACG,whereas the R6-34

recombinant exhibits PAA' and ACGr. In this

report the viral DNA polymerases induced by

these recombinants and their parental strains

have been partially purified and tested for

sensitivity and resistance to ACG-TP, PAA,

and the known chain-terminating compounds,

the

2',3'-dideoxynucleoside triphosphates

(didNTP's). This report correlates genome

structuresand thepresenceof thegenemarkers

for

paar

and

acgr

with the function ofpurified

HSVDNApolymerase in thepresenceofthree

compounds of dissimilar structure. A

1.3-kilo-base-pairregion of HSVDNA within the viral

DNApolymerase locuscontains a mutation or

mutationsconferring resistance to three DNA

polymerase inhibitors. The DNA sequences

within thisregion encode foranactivecenterof

theherpes

simplex

virus DNApolymerase.

(This workwaspresented inpart atthe

Inter-national ConferenceonHerpesViruses,Atlanta,

Ga.,

on 21 March 1980 and at the 5th Cold

Spring Harbor Workshop on Herpes Viruses,

Cold

Spring

Harbor, N.Y.,on31August1980.)

MATERIALS AND METHODS Materials.Acyclovirwas agenerousgift fromJohn

Beale of Burroughs Wellcome Ltd., Beckenham, UnitedKingdom.PAA was agift from LaceyOverby of AbbottLaboratories, Inc., NorthChicago, Ill. Acti-vated salmon spermDNAprepared by mildpancreatic

DNase digestion as previously described (29) was a

kind gift of Helen Moss-Rixon, MRC Virlogy Unit, Glasgow. Deoxynucleoside triphosphates (dNTP's), didNTP's, and oligodeoxyguanidylic acid-polydeoxy-cytidylic acid [oligo(dG)1218-poly(dC)] were from P.

L. Biochemicals. Radiochemicals were from Radi-ochemicalCenter, Amersham.[3H]ACG was prepared by NewEngland Nuclear Corp.

Cells and viruses. Baby hamster kidney (BHK 21-C13) and Verocells were grown in Eagle medium supplemented with 10% tryptose broth and 10% bovine calf serum (21). Cell monolayers (107cells) in 90-mm plastic petri dishes (Falcon Plastics) were used throughout. Virusinfection was carried out with 10

PFU/cellinmedium without tryptose. All virus strains usedwerederived from stocksprepared inGlasgow by low-multiplicity passage in BHK 21-C13 cells. The temperature-sensitive mutant of HSV-2, ts6 (strain HG-52), has been previouslydescribed(27).The PAA' mutantofHSV-1 strain17 (ts+,paar_j) was isolated by passage in the presence of PAA (100mg/ml) and

wascharacterizedpreviously (13). The HSV-1/HSV-2 intertypic recombinants R6-26 and R6-34werederived by crossesbetween HSV-2 ts6 (strain HG-52) DNA and restrictionendonuclease-cleaved DNA from

HSV-1 strain 17 (ts+,paa'r1) DNA, isolated by marker rescue,andplaque purified at38.5°C (6).

Preparation of cell extracts andpurification

ofHSV DNA polymerase. All infected cells were

harvestedat 18 hpostinfectiontopreparecytoplasmic

extracts. To demonstrate that the resistant

recombi-nantR6-34 induces DNApolymerase activity which is resistanttoACG-mediated inhibition, cytoplasmic ex-tracts were prepared from confluent baby hamster kidney cells (BHK-C13) infected with WT HSV-2 (strainHG-52),R6-26,orR6-34(multiplicityof infec-tion, 10 PFU/cell), and at 7 h postinfection 20 uM ACG inwater wasaddedtothe infectedcells,orwater

alonewasaddedtocontrol dishes. The infected cells

wereincubated in the presenceorabsence of ACGat

37°C,andcultureswereharvestedat 18h postinfec-tion.Cellextracts wereprepared bylysingcells in0.25

M potassiumphosphate buffer (pH7.5) containing1

mM each ofdithiothreitol, EDTA, ethylene glycol-bis(B-aminoethyl ether)-N,N-tetraacetic acid, and 0.5% Triton X-100. After centrifugation to remove

nuclei (15,000xg for1hat4°C), aliquotsofcytosol extracts werekept in35% ethylene glycol at -20°C

until use. These extracts were employed directly to

assayHSV-induced DNApolymerase activityby

mea-suring theincorporation of[32P]dGTP into the syn-thetic DNAtemplateprimer,oligo(dG)1218.poly(dC).

Forpurification of HSV DNA polymerase, 200-1i samplesoffresh cellextracts(600 ,ugofprotein)were

layeredonlinearglycerol densitygradients (20to50% glycerol in 0.25 M potassium phosphate buffer [pH

7.5] containing 1 mM dithiothreitol and 0.1 mM

EDTA), velocitysedimentationwascarriedout ina

Sorvall TV885rotor(45,000xg for15hat2°C)with appropriate markers for molecular weight, 0.25-mi fractionswerecollected,and10,ulof eachfractionwas

assayedforDNApolymeraseactivity (17).

DNA synthesis.Infected cellsweregrown in the

absenceorpresence of5jAMACGin 10mlof medium

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748 KNOPF, KAUFMAN, AND CRUMPACKER

containing 87.5,iCi of carrier-free "Pi. Cells were harvested 18 h postinfection by scraping them into coldreticulocyte standard buffer. After being pelleted, cellswere suspended inreticulocyte standardbuffer containing 10 mMEDTA, 1% sodium dodecyl sulfate, and0.5mgofproteinase K per ml and incubatedat

500Cfor3h. Then DNAwasphenol extracted twice andprecipitated with alcoholat-20°Covernight.The washedand dried DNA pelletsweresuspended in 10 mM Tris-hydrochloride (pH 8)-10 mM EDTA con-taining 100,ugof RNaseAper mland 1,000 U ofTi RNase (Worthington Diagnostics) per ml and incu-bated for3 h at370C. The DNAwasalcohol precipi-tated, washed, and dissolved in 50 mM Tris-hydro-chloride (pH 8)-10 mM EDTA, and CsCl was added to give a final density of 1.715 g/cm3. Equilibrium density CsCl gradient centrifugation was carried out in aSorvall TV865 rotor at 43,000 rpmfor 18 h at 200C. Gradientswere dripped from the bottom, and fractionswere collected directly ontoWhatman 3M paperslips.Determinationof trichloroacetic acid-pre-cipitable radioactivitywasperformedby the Bollum technique (4).

Measurement of ACG-TP. To determine the

amountofACG-TP formed in infectedcells,[3H]ACG

(50,tCi/ml; 200,Ci/mmol; New England Nuclear

Corp.) was added at 7 h after infection, cells were incubated at370C until 18hpostinfection and then harvested, andextracts wereprepared. Medium was removed, the disheswerewashedthree times in0.5ml ofice-cold 0.85%NaCl,andsoluble nucleotideswere

extracted in 0.5 ml of 0.4 MHCl04for30min at40C. Theextracts wereneutralized with KOH and concen-tratedbylyophilization. The ACG-TP was separated

onpolyethyleneiminecellulosethin-layer chromatog-raphy sheets developed with 2.0 M LiCl-2.0 N HCOOH (1:1, vol/vol). The chromatography sheets

were fractionated, and the triphosphate was eluted from the adsorbent with1ml of0.1NHCland counted ina liquid scintillation counter. The protein in the cellular extracts was determined by the method of Lowry et al. (20), and the results are expressed as picomoles of nucleotide formed per microgram of cell protein.

The[3H]ACG wasobtained by tritiation of ACG, and then [3H]ACGwas purified by ascending chro-matography onWhatman 3M paper in

N-propanol-water(7:3).TheRf for ACG in this system is 0.53 (11); this corresponded to the onlyUV-absorbingmaterial observed on the chromatogram. The [3H]ACG was

eluted andrechromatographed before use.

HSV DNA polymerase assay. When the syn-thetic template primer,

oligo(dG)12118poly(dC),

was employed, the standard reaction mixture contained (in

afinal volume of 100,l): 50 mM Tris-hydrochloride (pH 8.0), 50,ug of bovine serum albumin, 0.5 mM dithiothreitol, 7.5 mM MgCl2, 100 mM ammonium sulfate,0.01mM[32P]dGTP(1,450cpm/pmol), and 50

jig

ofoligo(dG)1218.poly(dC) per ml. With activated DNA as the template primer, thestandard reaction mixture contained (in a final volume of 100

pl):

50 mM Tris-hydrochloride (pH 8.0), 50

jig

of bovine serum albumin,0.5mMdATP, dCTP, dGTP, and[32P]dTTP (200 cpm/pmol), and 100

itg

ofactivated salmonsperm DNA. Under these conditions, all DNA polymerase

assaysexhibited linear kinetics of incorporation for 20 min.Incubationwasperformed at370C for the indi-catedtime, andsamples of the reaction mixturewere

spottedontoWhatmanGF/C glass ifiters of Whatman

no. 1filter paper,precipitated by 10% trichloroacetic acid, and treatedtodetermine acid-insoluble radioac-tivity as previously described (17, 24). One unit of HSV DNA polymerase activity is defined as the

amountof enzymecatalyzingthepolymerizationof 1 nmol ofdeoxynucleotides per h under standard assay conditions.

Alkaline DNase assay. Alkaline DNasewas

mon-itored as described by Morrison and Deir (23) by following the release of acid-soluble products from [3H]thymidine-labeled DNA. The reaction mixture contained(inafinal volume100tl):50M Tris-hydro-chloride (pH9), 5 mM MgCl2, 10 nM dithiothreitol, and 10 ,ug ofdouble-stranded, [3H]thymidine-labeled BHK21-C13 DNA (6,250cpm/,ug)and either5-, 10-,

or

20-pl

samples of cellextractin 200

A1

ofasuspension ofHyflo-SuperCel (20 mg/ml;Koch-LightLabs)in 1 Mperchloric acid. Aftermixingandcentrifugationat

10,000xgfor2.5min,150-,lI samplesof the superna-tants werecounted in10mlofAquasolcocktail. Under thegivenconditions, 1 U of alkaline DNaseactivityis definedastherelease of1 nmol ofdeoxynucleotides per hat37°C.

Thymidine kinase assay. Thymidine kinase activity was assayed by measuring the amount of [3H]thymidine phosphorylated by an extract of in-fected cellsby the method of Jamiesonetal.(15, 16). Briefly, the reaction mixture contained (in a final volume of100tl): 20mM potassium phosphate (pH 7.5), 10mM MgCl2, 10,LM [3H]thymidine (10 ,uCi/

!Lmol),

and cellextract ataconcentration of 50 to 100

,ug ofproteinperml.After incubation for 30minat

370C, radioactivity bound to Whatman DE81 filter disks wasdetermined. One unit ofthymidine kinase activity is defined as the conversion of 1 nmol of thymidine intoaphosphorylatedform within 1 hat

370Cunder the stated assayconditions. Protein

con-centrationsweredeterminedbythe methodofLowry

etal. (20).

RESULTS

Development of HSV-1/HSV-2 intertypic recombinants and determination of the

HSV DNA

polymerase

locus.

Seventy-four

HSV-1/HSV-2 intertypic recombinants have

been generated by marker rescue of

tempera-ture-sensitivemutantsexhibiting defectiveDNA

synthesisat thenonpermissive temperature (5,

6).The genome structuresoftheserecombinants

havebeen determinedby restriction

endonucle-aseanalysis ofthegenomes after plaque

purifi-cation andhavebeen employed in determining

thephysicalmap limits of five

temperature-sen-sitive mutations (ts) and mutations conferring

resistance to PAA (paar) and ACG (acgr) in

both HSV-1 and HSV-2 (5, 6, 8). The genome

structuresfor the recombinants resulting from

crossesbetween HSV-2ts6 (HG-52) and HSV-1

strain 17(ts+, paar_j) (R6series)wereanalyzed

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withsevenrestrictionendonucleases in the

re-gion from30 to 50 map units,enabling the

phys-icalmaplimitsof the HSV-2 ts6mutation and

thepaa-1 andacg-1 mutations of HSV-1 (strain

17) to be established (6, 8). Two of these R6 recombinants, R6-26 and R6-34, were selected

for further analysisto determine whether

mu-tationswithin theHSV DNA polymerase locus

which confer resistance to plaqueinhibition by

ACG and PAA induce an altered HSV DNA

polymerase activity. These two recombinants

wereselectedbecause they possesssimilarsmall

HSV-1 DNA insertions inan otherwise HSV-2

genome.ThisHSV-1DNA correctsthe ts6

mu-tationpermitting bothrecombinants toreplicate

atthenonpermissivetemperature, but these two

recombinants differ widely in their sensitivity to

PAAand ACG. The R6-34recombinant has an

HSV-1 DNA insertion extending fromthe

HSV-1 BglII i-d site to the HSV-1 EcoRI m-o site

(mapunits39.6 to 42.8, including the region of

uncertainty), is PAAr (efficiency of plaqueing,

0.6inthepresence and absence of 100lg of PAA

per ml) and markedly ACG' (50% inhibitory

dose, 17.5

,tM).

The R6-26 recombinant, on the

other hand, contains HSV-1 DNA sequences

extending fromtheHSV-1 BgllI i-d site to the

HSV-2 Bam h'-j' site (map units 39.6 to 41.0,

including theregion of uncertainty), but is still

PAA8 (efficiency of plaqueing,0.4 x 10-4in the

presenceand absence of100 ,ugofPAA per ml)

and ACG8 (50% inhibitory dose, 0.12 ,uM) (8).

The genome structures of these two recombi-nants and their restriction endonuclease sites

have been previously published (6, 8). The

re-combinants and their parental strainswere

em-ployed in all infections, and their polymerases

were

partially

purified and compared.

Effect

of

ACGonviral and cellular DNA

synthesis

ininfected cells. The recombinant

andparental strains differed markedly intheir

sensitivityto PAAandACG when analyzed in

a plaque reduction assay. This efficiency of

plaqueing in the presence of5 ,uM ACG

com-pared with that in the absence of inhibitorwas

82%forR6-34, <0.5% for

R6-26,

45% for

PAAr_1,

and 9% for HSV-2 ts6 (8). Since the effect of

PAA on DNA

synthesis

of

parental

strainsand

PAArmutants has been

previously

determined

(13),it was of interest toalsomeasurethe effects

ofACGonviral and cellular DNAsynthesisin

cells infected with the recombinants and their

parental strains. To show that the presence of

the

acgr-1

mutationaffectsthe

ability

of infected

cellstosynthesizecellular and viral DNA in the

presence of ACG, the DNA

profiles

from

in-fectedcells in the presence and absenceof ACG

were compared. Confluent cells (BHK) were

infected in the absence orpresence ofACG

(5

,uM), and theincorporation of 32p, into DNAwas

determinedat 18hpostinfection by analysis of

total cellular DNA on CsCl density gradients

(Fig. 1). In thepresenceof the drug, viralDNA

synthesisof the ts6 parent and the R6-26

recom-binant washardly detectable. Viral DNA

syn-thesis of the

PAAr_1

parent andthe R6-34

re-combinantwere resistant toACG, with20 and

23%,respectively, of viralDNAbeingpresentin

the absenceof thedrug. CellularDNAsynthesis

wasdecreased byACG in all four infections, but

40% ofcellularDNAsynthesis remainedin the

presenceof5

,uM

ACG. For both the PAAr-1 and

the R6-34 recombinant,viral DNAsynthesisis

inhibited to a greater extent by ACG (5 ,uM)

than is plaque formation,

indicating

that the

ACGr virus may be more effective in utilizing

viral DNA in the formation of infectious virus.

Little of the newly synthesized viral DNA

ap-pears toberequired for effective packaging into

infectious virus.Adirectcomparison of the

effi-ciency ofplaqueing and viral DNA

synthesis

in

thepresence ofACG isnot

possible

fromthese

twoexperiments because the cellswereinfected

atdifferentmultiplicities and

analyzed

at

differ-

5--S

x

E

z

05

a-C'l4

(Y)

HSV

A I

BHK

f\uj

HSV BHK

B I I

I~~~~~~~~~~~~~~~~~~~~~~~~~~~~

20 20

IC

1 5 0D I I

_v\S~~~~~~~I

10 2-0 3-0 10

Fraction number

20 30

FIG. 1. Equilibrium density gradients of DNA from infected cells in the presence and absence of

ACG. BHK cellswereinfectedwith HSV-2

(HG-52)

ts6(A), HSV-1 PAAr-l (B),R6-26(C), orR6-34

(D)

virusandgrowninthepresence(0)orabsence(0) of

5 MACG and32p;.At18hpostinfectioncellswere

harvested, and DNAwasextracted andanalyzed by

CsCIdensitygradientcentrifugation.Fractionswere

collected andprecipitatedin 5% trichloroaceticacid,

andradioactivitywasdetermined. The DNAprofiles

obtained in thepresence and absence

of

ACG are

plotted on the samefigureforcomparison. Arrows indicate thedensityofHSVDNA (1.729

g/cm3)

or

cellular DNA(1.705

gm/cm3).

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enttimes after viral infection.To radiolabelwith 32P, the cellshad to be grown in P04-free

me-dium aswell. However, the R6-34recombinant

and thePAAr-1 parentalvirusclearlyshowthe highest resistance of viral DNA synthesis and

thehighest efficiency ofplaqueing inthe

pres-enceofACG.

An altered viralDNApolymeraseconfers resistancetoACG-mediatedinhibition.The extractsfrom HSV-2 WT(HG-52)-, R6-26-,and R6-34-infected cells incubated in the presence

and absence of ACG (20

,tM)

wereprepared at

18h postinfectionwhen ACG had beenpresent (7 to 18 h). These cytoplasmic extracts were

employedas an enzyme source in the assay of

HSV DNA polymerase activity by using

oligo(dG)1218s*poly(dC)

as atemplate primerand

measuringtheincorporation of[32P]dGTPinto

acid-insoluble material after 10 min at 370C. With each of thecellular extractsobtained with-outACG treatment, thekinetics of polymeriza-tion proceeded linearly for 20 min. When

in-creasing amounts of cellular extract were

em-ployed in the DNA polymerase assay alinear

increase inincorporation of[32P]dGTPintothe synthetic template was also observed; this in-crease was almostcompletely inhibitedby

ex-tractsprepared from ACG-treatedHSV-2

WT-or R6-26-infected cells (Fig. 2A and B). The extract prepared from ACG-treated, R6-34-in-fectedcells,however,exhibitedlinearkineticsof DNApolymeraseactivitysimilartothoseofthe extract of R6-34-infected cellsprepared in the absence of ACG (Fig. 2C).

To exclude the possibility that the ACG-re-sistant DNApolymeraseactivity inducedby R6-34 was merely due to the failure of

R6-34-in-fected cells to make an active inhibitor, the

amountofACG-TP formedincellsinfected with

R6-26 and R6-34 in the presence of[3H]ACG

wasmeasureddirectlybypolyethyleneimine

cel-lulose chromatography. It has been previously shownthat theACG-TPis the activeinhibitor

of HSV DNA polymerase (11, 12, 26). The

amount ofACG-TP formedin 26- and R6-34-infected cellspermicrogram ofcellular

pro-teinwasverysimilar andwasmuchgreaterthan

thatformedinuninfected Verocells(Table1).

To confirm that the R6-34-infected cells

in-duce an active inhibitor of DNA polymerase

activity,the ability ofheat-inactivated extracts

fromACG-treated,infectedcellstoinhibit HSV

DNApolymerase wascompared. When 10,ulof

aninfected cell extractwas heatedat600C for

10min and then addedtoanactivecellextract,

little inhibition was observed (97.9% of DNA

polymeraseactivity remaining).Anextract,

pre-pared inthepresence of ACG (20,uM,7to18h

5 10 15

Cell extract(,ugprotein)

FIG. 2. HSV DNApolymeraseactivityofextracts

ofBftKcellsinfectedwithrecombinant virusesinthe

presence and absence of ACG. Cells were infected

withHSV-2(HG-52) WT(A),R6-26 (B), orR6-34(C)

in theabsence (0) orpresence(0) of ACG(20IpM), and extractsprepared at 18 hpostinfection. HSV DNApolymerase activitywasdeterminedby

measur-ing[32P]dGMPincorporatedwithanoligo(dG),1218

poly(dC) template primer in standard assayfor 10

minat370CasdescribedinTable3.Protein

concen-tration was determined by method ofLowryetal.

[image:5.499.314.408.65.280.2]

(20).

TABLE 1. FormationofACG-TP in cellsinfected

withrecombinantvirusesa

ACG-TP TotalACG-TP formed

Virus

formed(pmol)

(pmol/ug

of cellprotein)

R6-26 100.5 5.0

R6-34 81.0 4.9

Vero alone 2.0 0.14

aConfluent Vero cells in 90-mm dishes were in-fected at amultiplicity of infection 10, and at 6 h [3H]ACGwasadded(200,uCi/mmol).Cells were har-vestedat 18h andextracted withHC104, and ACG-TP was determined by polyethyleneimine cellulose chromatography. Protein in cellular extracts deter-minedbymethod ofLowryetal.(20).

postinfection) andinactivated, produced 59.9%

inhibition ofthe DNA polymerase activity

in-duced by HSV-2 WT-infected cells. This same

levelofinhibition wasobservedafter the

addi-tion ofinactivatedextractspreparedfrom

ACG-treated, R6-26-orR6-34-infectedcells (Table

2).

Thus, infectionwith theWT HSV-2orthetwo

recombinants containing a single HSV-1 DNA

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[image:6.499.52.243.99.377.2]

TABLE 2. Effect of ACG on HSV DNA polymerase activityofextractsofBHK cellsinfectedwithHG52,

R6-26, and R6-34virus

HSV DNA

Cell _Additionsb polym-_{erase inhibition}Relative

extracta activity (%)C

(% of control)

HG52 100.0

HG-52 97.9 59.9

HG-52 plusACG 38.0

R6-26 91.3

R6-26plus ACG 37.5 53.8

R6-34 99.4

R6-34plus ACG 35.3 64.1

R6-26 100.0

HG-52 115.3 84.7

HG-52plusACG 30.6

R6-26 138.3 98.3

R6-26plus ACG 40.0

R6-34 119.6

R6-34plus ACG 30.2 89.4

R6-34 100.0

HG-52 105.0 16.6

HG-52plus ACG 88.4

R6-26 95.0 9.9

R6-26plus ACG 85.1

R6-34 66.1

R6-34plus ACG 793 -132

aBHK

cells

wereinfected with the indicated virus and grown in the absenceorpresence ofACG(20,M) from 7 hpostinfection, andcellextract wasprepared

at 18 h postinfection. Heat-inactivated extract was

prepared by heatingat

600C

for30mintoinactivate DNApolymerase.Samples(10

pd)

ofextractaloneor

with10

pd

of inactivatedextract wereincubated for 10 minat

370C

under HSV DNApolymeraseassay

con-ditionsusing50

Mg

ofoligo(dG)12-18-poly(dC) as

tem-plateand0.01mM

['P]dGTP

(1,450cpm/pmol).The values for 100% represent theincorporation of71.4, 23.5, and12.1pmolof dGMP per,g ofcellproteinfor HG52;R6-26; and R6-34-infectedcells, respectively.

b InactivatedcellextractandACG-treated cell ex-tract wereaddedasindicated.

eRelative inhibitionbyACG-treated inactivated ex-tractsisthedifference obtained inpolymeraseactivity ofagivenextractassayedwithmock-treatedor ACG-treated inactivatedcellextracts.

insertion in an otherwise HSV-2 genome induced

similar amounts ofan active inhibitor of HSV

DNA polymerase

activity.

The DNA

polymer-aseactivity induced

by

the R6-26recombinant

was extremely sensitive to inhibition

by

the

heat-activated extracts,

revealing

an 84to 98%

decrease in DNA

polymerase

activity.

The DNA

polymerase

activity

of the HSV-2 ts6 mutant

wasinhibitedby58.9%

by

inactivated extracts,

afigure verysimilarto the inhibitionobserved

for HSV-2 WT DNA

polymerase.

Extractsfrom

infectedcells alone

produced

noinhibition.

The DNA

polymerase

activity

induced

by

the

ACG-resistant recombinant R6-34or the PAAr_

1HSV-1parentviruswas

markedly

resistantto

inhibition byinactivatedextracts fromcells

in-fectedinthepresence of ACG. In the absence of

anyinhibitor, 12.1pmolof[32P]dGTPper

lag

of

proteinwas incorporated intoDNA

by

the

ex-tract from R6-34 infected cells; this was de-creased by only 16.6 to 9.9% after the addition of extractsfrom ACG-treated HSV-2 WT-, or R6-26-infected cells (Table 2). In the presence of an

inactivatedextractfrom ACG-treated,

R6-34-in-fected cells,the R6-34-induced DNApolymerase

was actually stimulated by 13%. The PAA-r

HSV-1 parent DNA polymerase wasinhibited

by only 11.7%.Thus, the R6-34 recombinant and

its

PAAr_1

parent induced HSV DNA

polymer-ase activity which was resistant to the

ACG-induced inhibitorpresentin infected cell

cyto-plasma.This cytoplasmic inhibitor is ACG-TP,

and the resistance of the R6-34-induced DNA

polymerasetoACG-TPis due to an altered viral

DNA polymerase.

The

altered

viralDNApolymeraseis

re-sistanttoinhibition by didNTP's.The HSV

DNA polymerasewas purified from BHK

cells

infected withthe recombinants and their

paren-tal strainsby

sedimentation

in glyceroldensity

gradients (Fig.3).Allfoursedimentation

exper-iments revealed a major peak of DNA

polym-erase activity sedimenting only slightly slower

than the

sedimentation

standard,

glyceralde-hyde-3-phosphate dehydrogenase (molecular

weight, 144,000). The peak has a molecular

weightofapproximately 142,000 to 144,000, the

estimated molecular weight of the HSV DNA

polymerase (17, 24). The major alkalineDNase

activity and the dNTP'swereclearly separated.

Thepeak fractions possessingthe DNA

polym-erase activity were further examined for their properties in high salt and with inhibitors of

DNA polymerase activity, N-ethylmaleimide

and PAA, two inhibitors previously shown to

provide sensitive criteria for discriminating HSV

DNA

polymerases

fonn cellulara and ,B DNA

polymerases (28). All four of theDNA

polym-eraseswere

clearly

stimulatedin the presenceof

100

MuM

ammoniumsulfateby5- to10-fold when

activatedsalmonsperm DNAwas

employed

as

a

template

(Table

3).Aconcentration of2.5

,uM

N-ethylmaleimide

inhibited the observed DNA

polymeraseactivityobtained fromallfour

infec-tions by greater than 95%

(Table 3). By

the criteriaof molecular

weight,

stimulation

by

high

salt,and inhibition

by

N-ethylmaleimide,

there-fore,theobservedDNA

polymerase activity

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752 KNOPF, KAUFMAN, AND CRUMPACKER

1 2 3 4

5A

I D

[image:7.499.62.252.62.252.2]

10 20 30 10 20 30 Fraction number

FIG. 3. Purification ofHSVDNApolymerasefrom parental and recombinant virus-infected cells.

Ex-tractsfrom cellsinfectedwith HSV-2(HG52)ts6(A), HSV-1 PAA'-I (B), R6-26(C), and R6-34 (D) were

layeredonlinearglycerol gradients(20to509%)and centrifuged (Sorvall TV885rotor, 45,000 rpm,2°C,

15h). Fractionswerecollected,and 10-,ilsamplesof eachfractionwereassayedforHSVDNApolymerase with activated salmon sperm DNA as a template (-).Alkaline DNaseactivity(0)wasalso determined under standard incubation conditions in10-Ml

sam-ples ofeach fraction. Sedimentation markers were

included in eachgradientandareindicatedas fol-lows: 1,catalase; 2,glyceraldehyde-3-phosphate de-hydrogenase; 3, bovineserumalbumin; 4, cytochrome c;and 5,amixtureof[32P]dATPand[32P]dGTP.

sessed the properties of the HSV DNA

polym-erase enzyme,andthe DNApolymerase

activity

obtained after all four infections

appeared

to

have asimilar molecularweight.

The structureofACG-TP is similartothat of

the didNTP's in that these nucleoside

analogs

lacka3'-OHgroup,preventingchainelongation

(2). The didNTP's are well-established

inhibi-tors ofDNA

synthesis

which become

incorpo-rated intotheprimer andproduce chain

termi-nation. The four didNTP's were compared for

theabilitytoinhibit the viralDNA

polymerase

from the two recombinants and their

parental

strains. Initial experiments

employing

oligo-(dG)12

18-poly(dC)

as a

template primer

to

mea-sure the

incorporation

of

[32P]dGTP

indicated

that theR6-34-inducedDNA

polymerase

in

cy-tosol extracts was resistant to inhibition by

didGTP. ViralDNApolymerase activity in

ex-tracts of HSV-2 WT (strain

HG-52)-,

R6-26-,

andR6-34-infected cellswascompared, and the

concentration of didGTPrequiredtoinhibit 50%

of theDNApolymerase activity (50% inhibitory

dose) wasdetermined.The 50%inhibitory dose

values forthe

viral

DNApolymerase induced by

HSV-2, R6-26, andR6-34 were5, 1, and 74,uM

ofdidGTP,

respectively (data

not

shown).

Thus,

theR6-34DNApolymerase activity requirad74

timesmoredidGTP for 50% inhibition

tha_lid

theDNA

polymerase

inducedby R6-26.

The inhibition constants

(Ki)

for all four

didNTP's with activatedDNA weredetermined

with the

purified

viral DNA polymerase from

TABLE 3. Template characteristics and apparent kineticconstantsofthepartiallypurifiedHSV DNA polymeraseenzymesofparental and recombinantviruses

HSV DNApolymerase activity (pmol

in-corporated) under thefollowingcondi- Km(AM) Ki(ILM)b

tionsa:

Virus SS

SSDNA DNA Oligo(d

SS DNA plusAS plusAS G)1218. dNTP's dGTP didNTP's PAA

plus poly(dC)

NEM

ts6 11.2 57.1 1.4 27.5 10.8 22.3+5% 22.6 8.2

PAA'-_1 13.5 128.2 6.1 57.0 11.4 22.3±5% 122.5 37.5

R6-26 9.1 85.2 1.0 25.8 11.9 22.3+5% 14.7 4.5

R6-34 14.6 142.9 2.4 69.9 10.9 22.3+5% 135.1 39.0

a HSVDNApolymeraseactivity was determined under standard incubation conditions (20 min at

370C)

with either 100,ugofactivatedsalmonsperm(SS)DNApermlor 25

jg

ofoligo(dG)*poly(dC) per ml with or without 100mMammoniumsulfate (AS) and 2.5 mM N-ethylmalemide (NEM). The source of viral DNA polymerase wasthepeak fraction ofenzyme activity from the glycerol gradient shown in Fig. 3.

bTheKmdata fordGTPanddNTP's and theKidata fordidNTP's were derived from Fig. 4 and 5. The amountsof protein usedwere6.2, 6.4, 5.9, and 6.9 gforts6,PAAr_1,R6-26, and R6-34,respectively. For theKm

andKidetermination,one-halftheamount of enzyme was used. TheKifor PAA wascalculated fromgraphsby

plottingPAAconcentrationversus

Vm.,/v

on a Dixon plot. TheKmfor dGTP wasderived from Fig. 5 by using 25

jug

ofoligo(dG)1218 poly(dC)permlas a template.

4 0

x3

02

0

.1

.84

OL 3

I

2

(L

cn 1

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[image:7.499.65.455.445.562.2]

HSV

therecombinants and theparentalstr

4 and Table 3). The units of initial

velocity (v) were expressed as pico

dTMP incorporated per microgram c

duringa20-min assay; enzyme

activit3

ear over 20min. Inthe Lineweaver-Bi

ble-reciprocal plot of1/v versus

1/(dN

competitive inhibition-type kinetics

exy

a chain-terminating

compound

were

(Fig. 4). All four of the didNTP's had

Ki

for activated DNA. The

Ki

for ea

didNTP's with viral DNA

polymerase

estfor R6-26

(Ki,

14.5,iM) followed b

ts6

(Ki,

22.6,M) andwas

highest

with

fied DNApolymerases from HSV-1, Pi

122.5

,M)

and R6-34

(Ki,

135.1

,uM).

0.051

-01

0.1

A

a

I

VI

B

0.05-0.1 0.2 -0.1

C

lip-1

V

ains (Fig. The peak fractions ofpurified DNA

polym-I enzyme erase werealso employedto testforthe

sensitiv-imoles of ityof theenzyme activity to inhibition by PAA.

)fprotein Activatedsalmxon spermDNA was employed as y was lin- a template; the reactions were linear over 20

arke dou- min,and the

Ki

for PAA was determined from

iTP), the the Dixon plot of

V./v

versus PAA. The

Ki

for

pected

for PAA was again lowest for R6-26

(Ki,

4.5

,iM)

observed followed by HSV-2 ts6

(Ki,

8.2

AM)

and was

thesame highest with the purified polymerases from

ch of the HSV-1

PAAr_1

(Ki,

37.5,M) and R6-34

(Ki,

39.0

!was low- tiM) (Table 3). The comparative

Ki

values for

yHSV-2 PAA and for the four didNTP's with the

paren-Lthepuri- tal and recombinant DNA polymerases corre-A,A 1

(Ki,

lated extremelywellwith the resistance of these

enzymes to ACG-TP, to the resistance of viral

DNA synthesis in thepresence of ACG, and to

theresistance of HSV plaqueformationto ACG

(8).

To exclude the possibility that resistance of

viral DNA polymerase to ACG-TP, PAA, and

* didNTP's is due to an altered

affinity

of the

resistantpolymerasefor dNTP's, theKmvalues

* for each of the dNTP's were determined by

using

activated salmon

sperm

DNA or

oligo-0.1

0'2

(dG)1218

poly(dC)asatemplate. TheKmvalues

were

essentially

identical forall four dNTP's or

for dGTP with the synthetic template for all

four viralpolymerases (Table3and Fig. 5). The

altered viralDNApolymerase activity was not

dueto analtered Km.

0.051

0.1 0.2 -01

I

dNTP

]-1

,

(

pM)-1

D

1

V

1

0.1 02 FIG. 4. Determination ofKm fordNTP's andKi

for didNTP's of parental and recombinant HSV DNApolymerases. HSV DNA polymerase activity

wasassayedonthepurified peak fractions from Fig.

3 under standard incubation conditions (20min at

37°C) with 100pgofsalmonspermDNApermlby usingallfourdNTP'satequivalentconcentrations. Therateof incorporation of[32PJdTTPin percent in the absence(0) orpresence(0) of50 mMofeachof

thefourdidNTP'swasusedasthe initialvelocity (v)

in thedouble-reciprocalLineweaver-Burkeplot.The

enzymeactivitywaslinearover20min.In the absence

of inhibitor, 100% represents specific activity of 9.2, 18.4, 15.4, and 22.8pmol ofdTMPperpgof protein for ts6 (A), PAArX1 (B), R6-26 (C), and R6-34 (D), respectively.

0.01

I I

-0.04 0.01 0.02 0.03 0.04

(dGTPJ1.

(UM)1

FIG. 5. DeterminationofKmfordGTP. Thepeak fractions from Fig. 3wereemployedtomeasurethe HSV DNApolymerase activityin theparentaland recombinantviruses. HSV DNApolymerasewas

as-sayed understandard conditions (10min at 37°C)

witholigo(dG)1218.poly(dC) (25 pg/ml)asatemplate,

and therateof incorporation of [32P]dGTPin per-centageof complete incorporation wasused to cal-culateinitial velocities inthedouble-reciprocol plot.

The specific activities for the DNApolymerase of

HSV-2ts6(0),HSV-PAAr_l (0),R6-26(A),and R6-34 (J) were 0.23, 0.78, 0.33, and 0.19 pmol/,ug of proteinpermin, respectively.

0 05.

-0.1

0

I

I I I I

VOL. 39,1981

0.02r

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[image:8.499.49.240.249.502.2] [image:8.499.255.447.396.527.2]

754 KNOPF, KAUFMAN, AND CRUMPACKER

To exclude thepossibility that the recombi-nantsdiffered in the expression of viral deoxy-pyrimidine kinase, theenzyme required for the

phosphorylation of ACG (11, 25), this enzyme

activity was measured directly in the extracts whichwere employed in the DNA polymerase assay. Viral deoxypyrimidine kinase activity (units per microgram of protein) was 4.4, 4.0,

and 3.2 for the HSV-2WT-, R6-26-,and R6-34-infected cell extracts, respectively, in the

ab-senceofACG. In thepresenceofACG(20 ,uM),

7.2, 6.8, and 8.0 U/,ug of protein were detected

forthe HSV-2 WT-,R6-26-, and R6-34-infected

cellextracts,respectively. TheACG-treated

cel-lular extracts exhibited a twofold increase in

thymidine kinase activity in allcases. Since

al-kaline DNase activity could interfere with the template in the DNA polymerase assay and

perhapsaccountforapparentdifferences in viral DNA polymerase activity induced by different viruses, the alkaline DNase activity induced by therecombinant viruseswasalsodetermined. It

had previously beenshown, however, that under the assay conditions employed here an

oligo-(dG)12-81

poly(dC) templatewasnothydrolyzed

by alkaline DNase (16). Alkaline DNase activity

wassimilar in allextracts,and theextracts

pre-pared with R6-26 and R6-34-infectedcells inthe

presenceand absence of ACG exhibited identical

specific activities. The activities of these two viralspecific enzymes appearedtobe identical

incellsinfected with theserecombinant viruses,

and it isunlikely thatthey playanysignificant

role in the differencesobserved in the viral DNA polymerase activities induced by these

recom-binants.

DISCUSSION

Inthisstudywehaveemployed

HSV-1/HSV-2 intertypic recombinants with well-character-izedgenomestructures todemonstrate thatthe mutations which confer resistance of HSV to ACG or PAA produce an altered HSV DNA

polymerase activity which exhibits resistanceto three inhibitors of DNApolymerase.

Two recombinants, R6-34 and R6-26, which containHSV-1 DNA insertions withinthe DNA polymerase locus ofanotherwise HSV-2genome

were selected forfurther analysis and

purifica-tion ofthe viral DNApolymerase. R6-34hasan

HSV-1 DNAinsertion extending frommapunits

39.6to42.8 including the region ofuncertainty and isACGr and PAAr. The R6-26recombinant containsanHSV-1 DNAinsertion startingfrom

the same HSV-1 BglII i-d site on the left

ex-tending from map units39.6 to 41.0, including the region of uncertainty, and is PAAS, and ACGS. The mutations in HSV-1 DNA which

confer resistancetoPAAandACGinthe R6-34

recombinant must be contained in the DNA

sequencesdefinedbymapunits41.0 to 42.8.By

analysis oftwoother

recombinants,

R6-19 and

R6-30,these limitscanbefurther narrowed to

the DNA sequences defined

by

the HSV-2

BamHIh'-j' sitetotheHSV-1Kpnx-csite

(map

units41.0 to41.8) or 1.3kilobase

pairs (5,

6,

8).

Thisissufficientgenetic informationtoencode

for an amino acid sequence of 42,000 daltons

either as a separate

polypeptide

or part of a

largerprotein. This

region

of DNAsequencesis

within the

mapping

limits of the HSV-1

(Kos)

tsD9 mutation

defining

the structural genefor

HSV DNA polymerase (5) and is

likely

to

in-clude the DNA sequence

defining

the active

centerof the HSVDNA

polymerase.

These two recombinants induce identical

amounts of

thymidine

kinase and alkaline

DNase activityin thepresence and absence of

ACG (8), and the differences in

sensitivity

to

ACG andPAA cannotbe

explained

bya

differ-ence in the activities of these two enzymes.

These two recombinants induce identical

amountsof

ACG-TP,

theactive

cytoplasmic

in-hibitor ofviralDNApolymerase,inthepresence

ofACG. TheR6-34recombinantinducesaviral

polymerase which is resistant to

ACG-TP,

whereastheR6-26recombinant and theHSV-2

WT induce DNA

polymerase

activities which

aresensitivetoACG-TP.

The

purified

DNA

polymerase

induced

by

thesetworecombinants and the

parental strains,

HSV-2 ts6 (strain

HG52)

and HSV-1 strain 17

(ts+,paar_j),possessasimilar molecularweight

of 142,000-144,000onsedimentationin

glycerol

density gradients. The

polymerase

activities

were stimulated by high salt, inhibited by

N-ethylmaleimide,andpossessed the properties of

HSV DNA polymerase. The peak fractions of

the

purified

viral DNA polymerase were

em-ployed to testfor the sensitivity of HSV DNA

polymerasetoPAA, and theviral DNA

polym-erasesinducedbyR6-34 andthePAAr_1parent

wereresistanttoinhibition by PAA,possessing

ahighKi.

Partially

purifiedviralpolymeraseswerealso

tested for sensitivity to inhibition by the four didNTP's. Thesedideoxy analogshave been in-structiveregardingthe mechanism of polymeri-zationbytheEscherichiacoli DNApolymerase, Pol I (2). The didNTP serves as polymerase substrate toelongateaDNAchainby one

resi-due. Achain terminated with such a

dideoxy-nucleoside is inert to further elongation and

relatively inert to exonuclease action at the

primer terminus or to attack by PPi. The

didNTP,

lacking

a 3'-OH group vital for chain

growth, is still boundtothe dNTP bindingsite

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on the enzyme with the same

affinity

as the

natural triphosphate

and formsa

polymerization

complex

protecting

the

primer

terminus from

nucleolytic attack. With theHSVDNA

polym-erases induced by the R6-26 recombinant and

theHSV-2ts6 parent,the didNTP'sact as

effec-tive

competitive inhibitors,

revealing

low

Ki

val-ues(Table 3).Itisof interest that theapparent

Ki

of the human leucocyte polymerase for

didTTPis 9.6,uM (1), avalue ingeneral

agree-ment for the

Ki

values of the sensitive HSV

DNA polymerases for didNTP's (14.7 to 22.6

,M).

The viral DNA polymerase of the R6-34

re-combinant orthe HSV-1

PAAr_i

parent,

how-ever, wasmarkedly resistanttoinhibition by all

four didNTP's. Thissuggeststhat themutation

which confersresistance of these viral

polymer-ases to ACG-TP may confer resistance to all

four didNTP's as well. The data presented in

Fig. 2 indicating the effect of ACG-TP in the

cellularextract oninhibiting dGTP

polymeriza-tionby the viral polymerases of26 and

R6-34with the oligo(dG)12-18-poly(dC) template is

instructive concerning the role of ACG-TP in

binding to the viral polymerase. ACG-TP has

previously been shownto bind to the HSV DNA

polymerase and act as a competitive inhibitor

(11, 26). The R6-34 DNA polymerase is not

affected by the presence of ACG-TP and is

probably not bound by the inhibitor, whereas

the DNA polymerase ofR6-26 ismarkedly

in-hibitedby thepresenceofACG-TP, presumably

bybindingtothe viralpolymerase (Fig.2B and

C). Since the didNTP bindstothetriphosphate

binding siteontheE.coli PolI enzyme,and the

HSV DNA polymerase presumably also

pos-sessessuchasite,amutationintheDNA

polym-eraselocusonHSV which resultsinpreventing

anyanalog lacking a3'-OH groupfrom binding

with the same affinity as the natural

triphos-phatetothetriphosphate binding sitemayallow

polymerizationtoproceed. This mutation could

result in a more stringent

recognition

by the

triphosphate

binding

site of the enzyme and

could result in a failure to

incorporate

com-pounds

lacking

a 3'-OH group. Since the

pro-posed site of action ofPAA onthe HSV DNA

polymerase is at the pyrophosphate exchange

site,amutation whichconfersresistance to PAA

and ACG-TP and didNTPsuggests that the

PPi

exchange site is very

closely

adjacent to the

triphosphate bindingsite in the enzyme. These

results provide additional supportfor the

pro-posalthat HSV DNA polymerase conforms to

the model of DNA polymerase advanced by

Kornberg,

i.e.,that DNApolymerase possesses

anactive centercontainingmultiple sites, each

with adifferent function (18). Amutation

pro-ducinganalteration inone function of the active

center mayaffectthe others aswell.It has been

shownthat thesefunctionsare closelylinkedin

theE. coli PolI enzyme, and aconsequence of

chain terminationby didNTP's is the inhibition

of

PPi

exchangeand exonuclease action (2). In

HSV DNA polymerase, the 3'--5' exonuclease

and

PPi

exchangefunctions may beclosely

as-sociated because PAA, aninhibitorof

pyrophos-phateexchange,inhibits

3'-.5'

exonuclease

func-tion to a similar degree (17). Unlike didNTP's

orACG-TP,however, PAA does not get

incor-porated into DNA, and it does not serve as a

substrate forHSVDNApolymerase (3, 22).

An alternative explanation for the observed

resistance of the R6-34 and PAAr_1 DNA polym-erases to inhibition by didNTP is that these

altered polymerasespossess anenhanced ability

toexcisethese analogsby the

3'-.5'

exonuclease,

or"editing,"function of HSVDNA polymerase.

After incorporation of an altered analog, the

3'- 5' exonuclease function catalyzes the

re-moval of3'-terminalnucleosides from the primer

templateas

well

asthe template-dependent

con-version of dNTP's to monophosphates (17). A

DNApolymerase doesnot require such an

exo-nucleasefunction to be resistanttothe

inhibi-toryeffects of didNTP'sasbotheucaryotic

po-lymerases of CV-1 cells(10)and leukocytesfrom

patients with human myelogenous leukemia (1)

areresistanttoinhibition by theanalogdidTTP.

The cellular a polymerase from myelogenous

leukemia cells is sensitive to inhibition by

didTTP, however (1). Since neither the

eucar-yotica nor

,8

polymerasepossessesexonuclease

function,

and sincethey differ in their sensitivity

todidTTP, the absenceof the exonuclease

func-tion doesnotexplain resistance. The mechanism

of resistancetodidTTP exhibited bya

polym-erase most

likely

is duetoits

ability

to

discrim-inate between didTTP and dTTP,

preventing

incorporation

of the didTTP.

A

potential

objection

to the use of

hybrid

genomesfor the purpose of

genetic analysis

of

herpes viral functions is that the recombinant

genomewill

produce

newrecombinant

polypep-tideswhichare soaltered from

parental proteins

that conclusions about

parental

functions are

notvalid. This appearstobean

unlikely

possi-bilityinthepresent case,

however,

becausethe

altered resistanceof the DNA

polymerase

of the

R6-34recombinant isobserved in the

parental

HSV-1 PAA-r virus as well. The

apparent

Ki

values for thedidNTP'sareverysimilar for the

DNA

polymerase

of the R6-34recombinant and

the HSV-1 PAAr_1 parent

(Ki,

135.1 and 122.5

1tM,

respectively).

In

addition,

the

Ki

values for

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756 KNOPF, KAUFMAN, AND CRUMPACKER

the DNA polymerase of the R6-26 recombinant and the HSV-2 ts6parentarealsoverysimilar,

andthesetwostrainsare sensitivetoPAA and ACG. The molecular weights of all four viral DNApolymerases are identical in the glycerol

gradient analysisandarethesameasthe molec-ular weight of the HSV-1DNApolymerase (17, 24).

The HSV-1 sequences which definethe

mu-tations for acgr and paar map in equivalent positions in both HSV-1 and HSV-2 (5, 6, 8). The type-specific sensitivity toACG in HSV-1 and HSV-2 canbe transferred separatelyfrom

theacgr mutation (8).Theseargumentssuggest that the viral functions in the parentalstrains

are represented faithfully in the recombinants

and that the recombinantgenomesprovide

use-fulprobes for theseparationandanalysisof viral

genefunctions.

Thisreportillustrates theutilityofcombining finestructuremappingofthe HSV DNA

polym-eraselocus withtheuseofspecificinhibitorsof

DNApolymerase functionstoseparatethe func-tions of the HSV DNA polymerase. This is a

goal which has previously been realized only withprocaryotic polymerases.Indefininga

1.3-kilobase-pair region of DNAsufficient to code for an amino acid sequence of42,000 daltons, which contains mutationstothree DNA

polym-eraseinhibitors ofdissimilarstructure,wehave

defined theregion coding for the active center of theHSV DNApolymerase molecule. PAA,a

PPi

analoginhibitingPPiexchange(19); didNTP analogs lacking a 3'-OH group and shown to bindtothetriphosphate binding siteonE. coli

Pol I (2); and ACG-TP, a compound which serves as asubstrate for the HSV DNA

polym-erase,isacompetitive inhibitor ofdGTP (11, 12,

26), and hasmanyfeatures incommonwith the

didNTP's,actonseparatefunctions inthe HSV

DNApolymerase; all inhibit the polymerization reaction. Whetheronemutationorthree

sepa-rateonesaccountforthe HSV DNA polymerase

resistancetothesecompounds, thefact that the resistancemutationsmapin suchasmall region

ofthe DNA provides supportthat this region definesanactive centerfor the HSV DNA

po-lymerase. Kornberg has describedanactive

cen-ter as a specially adapted polypeptide surface

whichrecognizes and accommodates several

nu-cleosidestructures(18), and thesestudies define such a region on the HSV DNA polymerase

polypeptide.IftheHSV DNA polymerasehasa

molecularweight of140,000to150,000,the

larg-estlimits of this active centerwould be molec-ularweight 42,000, aboutathird of theenzyme

molecule. Further definition and subdivisionof this region will require similar studies on

mu-tants and recombinants which separate resist-ancemutations to inhibitors of HSV DNA po-lymerase.

ACKNOWLEDGMENTS

Wegratefullyacknowledge the excellent technical assist-anceofMaryMurphy inpreparingthe virus stocks.

C.S.C. is therecipient of Public Health Service Research CareerDevelopment award 5-K04-CA--139 from the National CancerInstitute. K.W.K. is therecipient of aEuropean Sci-enceExchangefellowship from the Royal Academy of Science, UnitedKingdom,sponsoredbytheDFG,FederalRupublicof Germany. E.R.K. wassupported by Public Health Service grants CA16751 and CA26195 from the National Cancer In-stitute.

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