Isolation and preliminary characterization of a phosphonoacetic acid-resistant and temperature-sensitive mutant of herpes simplex virus type 1.

0022-538X/82/040020-10$02.00/0

Isolation and Preliminary Characterization of

a

Phosphonoacetic Acid-Resistant

and

Temperature-Sensitive

Mutant

of

Herpes Simplex Virus Type 1

JASMINEI. DAKSIS,MARKM.PRIEMER,AND VOON-LOONG CHAN*

Department of MicrobiologyandParasitology, University of Toronto, Toronto, Ontario, Canada, MSS IAI

Received 29 July 1981/Accepted16 November1981

Agroupof 43phosphonoacetic acid (PAA)-resistant mutants of herpes simplex

virustype1wasisolated after the mutagenesis of infectedcells with nitrosoguani-dine. One of these mutants, designated

PAA1r,tsl,

wasfound tobe temperature sensitive(ts), that is, unable toreplicate at39.5°C, the nonpermissive

tempera-ture. Recombination analysis of PAAirtsl indicated that the PAA1r mutation and

the tsl mutation are loosely linked and are located on two separate genes.

PAAirtsl showeda defect in viral DNA synthesisat 39.5°C, whichpresumably

can be attributed to the production ofa PAA-resistant and thermolabile DNA

polymerase. PAAirtslwasalso defectivein the shutoff ofhost DNA synthesis at

therestrictive temperature.

The genome of herpes simplex virus type 1 (HSV-1) is composed of double-stranded DNA withamolecular weight of approximately 100 x

106 (10). This is sufficient genetic material to

code for 80to 100 proteins. One of these

pro-teins isaDNA polymerase that is essential for

thereplication of viral DNA(1).

Phosphonoacetic acid (PAA) isan

antiherpes-virus drug which acts by inhibiting viral DNA synthesis (14). It is aneffective inhibitor of the

virus-induced DNA polymerase (13) by virtue of its interaction with theenzymeattheinorganic

PPi-binding

site(12). Numerous mutantsof

her-pes simplex virus whosereplication, DNA

syn-thesis, and DNA polymerase activity are resis-tanttoPAAhavebeen isolated (2, 7-9). None of thesemutantsisolated solely for PAA resistance

(PAAD)

has been reported to be significantly

temperature sensitive (ts). However, the DNA

synthesis-deficient ts mutant of HSV-1, tsD9, which codes for the synthesis ofa DNA

poly-merase that is thermolabile in vivo (1), was

subsequently found to exhibit PAA resistance (9, 15). Revertants of this mutant that were

isolated at 39°C in the absence of PAA were

showntohavereverted bothof the phenotypes (9, 15), suggesting that the PAA-resistant and temperature-sensitive properties of tsD9can be

attributedtoasingle mutation.

Thiscommunicationreports theisolation and preliminary characterization of a

temperature-sensitive PAA-resistant HSV-1 mutant that we

designated as PAAirtsl.

PAA1rtsl,

at 39.50C, showed adefectin viral DNA synthesis and in the shutdown of cellular DNA synthesis. We also specifically examined whether the

PAA-resistant and temperature-sensitive phenotypic properties were due to a single or a double mutation.

MATERLALSANDMETHODS

Cellsand viruses. BHK-21/C13 cells wereobtained

from R. Sheinin (University of Toronto, Toronto, Ontario, Canada). CV-1 and LMTK- cells were a

generous giftfrom R. G. Hughes, Jr. (Roswell Park

Memorial Institute, Buffalo, N.Y.). Serially

propagat-ed stock cultures were routinely grown in a-MEM

tissue culture medium (19) supplemented with 10%

fetal calfserum(FCS).

HSV-1 strain KOS 1.1 was obtained from R. G.

Hughes, Jr., and was used as the wild-type virus in this

study. Thets mutants ts478and ts833werefrom the

same source.Thepermissive and nonpermissive

tem-peratureswere34WC and 39.5°C, respectively.

Howev-er,38.5°C is also nonpermissivetoall thets mutants

used in this study. Bu-El, Bu-B4, Bu-E2, and

Bu-C,

arethymidine kinase-deficient (TK-)mutantsisolated

in this laboratory from the HSV-1 KOS strain

(ob-tained from S.Bacchetti,McMasterUniversity,

Ham-ilton, Ontario, Canada) by growth in 30 1.g of

5-bromodeoxyuridine (Sigma Chemical Co., St. Louis,

Mo.) per mlaspreviously described (5). Thets mutant

tsD9was agenerousgift fromP. A. Schaffer (Sidney

FarberCancerInstitute, Boston, Mass.). Virus stocks

were grown onCV-1 cells at a multiplicity ofinfection

(MOI) of0.01PFU per cell.

Mutagenesisand isolation of mutants.Monolayers of

BHK-21/C13 cells in60-mmdishes wereinfected with

wild-type HSV-1 strain KOS 1.1 atamultiplicity of1

PFUpercell. Aftertheadsorptionperiod, the inocula

wereaspirated andtheinfected cells werewashed with

phosphate-buffered saline (PBS).Toeach culturedish,

a-MEMcontaining

10%o

FCSwasadded for aperiod of

4h. Infected cells werethen washed with PBS and

givena1-hpulse of

N-methyl-N'-nitro-N-nitrosoguan-20

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PHOSPHONOACETIC

idine (10 rig/ml; Aldrich Chemical Co., Milwaukee,

Wis.) in a-MEMcontaining10oFCS. After the

expo-sure to nitrosoguanidine, the infected cells were

washed twice moreand fed withoa-MEM

supplement-ed with 10%oFCS forafurther incubation of 20 hat

34°C. The resultinglysateswerereplatedonBHK-21/

C13 cells in the presence of 100 ,ug of PAA(disodium

salt, from Abbott Laboratories, North Chicago, Ill.)

perml. Plaques were picked and cloned twice in the

absence of the drug. Each PAA-resistant isolate was

screenedfor temperaturesensitivity by measuring the

plaquing efficiency at 34°C and 39.5°C. Any clone

which exhibited at least a104-foldreduction in

plaqu-ing efficiency when assayed at the nonpermissive

temperature was acceptedas ats mutant.

Viral DNA synthesis. To determine whether

PAA,rtsl

was capable ofsynthesizing viral DNA at

the nonpermissivetemperature, confluent monolayers

of CV-1 cells in 60-mm disheswerewashed with PBS

and then mock infected or infected with wild-type

virus or

PAA,rtsl

at anMOI of 5. Two milliliters of

a-MEMcontaining 2% FCSwasaddedtoeachdishfor 6

h.This mediumwasthenremoved,and thecellswere

washed with PBS. Cells were refed with 5 ml of

a-MEMplus 2% FCS and 2,uCiof

[methyl-3H]thymidine

(64.0Ci/mmol, NewEnglandNuclear, Boston,Mass.)

perml.After 18h,theinfected cellswerescrapedinto

themedium andpelletedby low-speedcentrifugation.

Each batch ofcells was washedwith 10 ml of TNE

buffer (0.01 MTris-chloride,pH 7.4, 0.1 MNaCl,and

0.001 MEDTA)(16) andrepelleted. Thesupernatants

weredecanted; then thetubesweredrained and frozen

at-70°C.Afterthawing, the cellswereresuspendedin

2.0 ml of TNEbuffer and incubatedat room

tempera-ture for 10 min in the presence of 0.18 ml of 109o

Nonidet P-40 and 0.07 ml of 109o sodium dodecyl

sulfate. Self-digested nuclease-free pronase

(Calbio-chem-Behring, LaJolla, Calif.) wasadded to afinal

concentration of 50 ,ug/mlfora30-minincubationat

37°C.One milliliter of each cellextractwasaddedto4

mlof CsCl dissolved in TNEbuffer,and therefractive

indexwasadjustedto1.4001to2.Samples(5ml)were

loaded into cellulose nitrate tubesforcentrifugationat

25°C inan SW50.1 rotor at25,000rpmfor 60 h ina

Beckman L5-50centrifuge.The tubeswerepunctured

from the bottom, and8-dropfractionswerecollected.

Therefractive indices of selectedfractionswere

mea-sured with a Bausch & Lomb refractometer. The

remaining samples were precipitated with cold 5%

trichloroacetic acid and added onto Whatman GF/A

glassfiber filters. Filterswerewashed threetimes with

cold5% trichloroacetic acid andtwotimes with cold

95% ethanol beforetheywereovendried.

Acid-precip-itable radioactivity was determined by scintillation

counting.

DNApolymerase assay.Theinvitro thermolability

anddrugsensitivity of the HSV-1 DNA polymerase

weretestedbyexaminingthepropertiesofcrude

en-zymeextracts. Approximately 5 x 107 BHK-21/C13

cells grown in roller bottleswerewashed with PBS and

mockinfectedorinfected with wild-type virusorthe

putativeDNApolymerasemutantatamultiplicity of

10 PFUper cell. Infectedcellswere incubated at 34°C for9 h.Cellswerewashed twice with coldPBS; they

were then suspended in 0.75-ml volumes of 10 mM

Tris-chloride (pH 7.5)-150 mM KCl-0.5 mM

dithio-threitol andsonicated with aBransonprobeSonifier

(model S125) for 10 s, at which time cell rupture was complete. An equal volume of 3.4 M KCl, 10 mM EDTA, and 1 mg of bovine serum albumin per ml was

added to each extract, and the extracts wereincubated

at0°C for 20 min. MgCl2 was added to a final

concen-tration of 3 mM, and the preparationswereincubated

at room temperature for 30 min. The extracts were

centrifugedat30,000 x gfor 15 min in aSorvall SS/34

rotor, and the supernatants were dialyzed for 14 h

against 1 liter of 10 mM Tris-chloride, pH 7.5, and 1

mM

P-mercaptoethanol.

These crude enzyme prepara-tions were frozen and thawed once prior to use. For

enzymethermolability assays, samples of 25 ,ul were

incubated at 45°C for various periods of time and then added to reaction mixtures (total volume, 100 ,ul) for determination of the viral DNA polymerase activity. These contained 100 mM Tris-chloride (pH 8.0), 0.5

mMdithiothreitol, 100 mM (NH4)2SO4, 2mM MgCl2,

0.45mgof bovine serum albumin per ml, 10.25 ,ug of

pancreatic DNase-activated calf thymus DNA, 0.1 mMeach ofdATP, dCTP, dGTP, and TTP, and 0.5

,uCi of [methyl-3H]thymidine-5'-triphosphate (44 Ci/

mmol, Amersham Corp.). Mixtures were incubated for

30 min at 38.5°C. In the case of a drug resistance

assay,different concentrations of PAA were included

andtheincubation was at 34WC for 30 min. Samples of

50 ,ul were spotted onto Whatman 3MM filter paper

disks, washed once with cold 5% trichloroacetic acid plus 1% sodium pyrophosphate, twice with cold 5%

trichloroacetic acid, twice with cold95% ethanol, and

oncewithdiethyl ether, and then dried. Radioactivity

was measured by scintillation counting. The counts

from the mock-infected system for each assay were subtracted from those of the wild-type or the mutant virus system.

Genetic crosses and isolation of recombinants. A

number of TK-PAAS mutants were crossed with PAAlrts1. BHK-21/C13 cells were infected with 5 x

10'

PFUof each parental virus (total MOI, 10 PFU per

cell) in a-MEM with 5% FCS. Newly titrated virus

wasused for each cross to ensure an equal MOI for

each parentalstrain. After a 2-h adsorption period, the

unadsorbed virus was removed, the infected cells were

washed with PBS, and a-MEM containing 5% FCS

and a 1/5 dilution of anti-HSV serum (supplied by

FlowLaboratories,Inc.,Rockville, Md.)wasaddedto

each culture dish. After 1 h, the infected cells were

washed once more, refed with a-MEMplus5%FCS,

and allowedtoincubateat34°Cfor 20 h

postadsorp-tion. At thistime,theresulting lysateswereharvested

andreplatedonBHK-21/C13cellsunder nonselective

conditions (i.e., in the absence of PAA and

5-bromodeoxyuridine). Plaques were picked and

re-cloned twice under these nonselective conditions.

Individualrecombinantsweresubsequentlyidentified

by their ability to grow on BHK-21/C13 cells in the

presence andabsenceof 100jigof PAA per mlat34°C,

on LMTK- cells in the presence of 30 ,ug of

5-bromodeoxyuridinepermlat34°C,andonCV-1 cells at34°Cand39.5°C.

Complementationtests. Complementation tests

be-tweenpairsofts mutants wereperformed bya

modifi-cationof theprocedure of Schafferetal.(16).

Approxi-mately 2 x 105 CV-1 cells were infected at the

nonpermissive temperature with thets mutantseither

singly at anMOIof2.5PFU per cell orinpairsat a

total MOI of 5 PFU per cell. The inocula were

21

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aspirated after adsorption,and the cellswerewashed

withPBS. Each infected culturewasfed with 1.0 mlof

a-MEMplus 2%FCS, and thetestplateswere

incu-bated for 20 h at 39.5°C. Cells were collected and

subjected to three cycles of freezing and thawing.

LysateswereassayedonCV-1 cellsat34°C.

Comple-mentation indiceswerecalculatedby dividingthetiter

ofvirus obtained from the mixedinfectionbythesum

of the titers fromthesingleinfections. If this valuefor

any cross wasgreaterthanorequalto2,then

comple-mentationwasdeemedtohave occurred.

RESULTS

Isolation of mutants. The mutagenesis and selection for PAA-resistant clones resulted in the isolation of 43 PAAr mutants of HSV-1. When tested for temperature sensitivity, one

mutant, bearing the designation

PAA'rtsl,

was

foundtohaveaplaquingefficiencyof 2.9 x

10'

at 39.5°C and 3.0 x

10-5

at 38.5°C relative to 34°C in

BHK-21/C13

cells. In comparison, the

wild-type

virus had aplaquing efficiency of 2.3 x 10- at 39.5°C and 7.9 x 10-1 at 38.50C relative to 34°C. The plaquing efficiency of

PAA,rtsl

in CV-1 cells is the same as that in

BHK cells. The PAA resistance of PAAirtsl

wastestedby plaqueassayand is shown inFig. 1.

Replication efficiency of

PAA,'tsl.

The

isola--j

z

0

I~-U.

20 40 60 80 100

pg PAA/ml

FIG. 1. Inhibition of HSV-1 by PAA. The virus stocksweregrownat34°Cintheabsenceof PAA and

wereplaque assayed with the drug included in theagar

overlayer. The viruses werewildtype(0), PAA1rtsl

(0),and thets+ revertantof PAAlrtsl, R7-2(A).

tionof a PAA-resistant mutant that was foundto betemperaturesensitive presentedthe possibili-ty that this mutant codes for a thermolabile DNA polymerase. To begin thecharacterization of this mutant, PAAirtsl, experiments were performeddealing withtheefficiency of replica-tion ofthe mutant either in the presence of 100 ,ug of PAA per mlor at39.5°C. Theseproperties of the mutant werecompared with thoseofthe wild-type strain from which the mutant was derived.

Triplicate monolayers of 105 CV-1 cells were infected with wild-type HSV-1 KOS 1.1 or

PAA,rtsl

at a multiplicity of 1 PFU per cell. After an adsorption period of 1 h, the infected cellswere washed with PBS and incubated for 20 h at 34°C in a-MEM plus 5% FCS that contained or lacked 100 ,ug of PAA per ml. Progenyvirionswereharvested, and titers were determined on CV-1 cells by plaque assay at 34°C in the absence ofPAA. The yields from eachsetoftriplicatelysates wereaveraged. As shown in Table 1,thewild-type andthe mutant viruses yielded approximatelythe same titer of progenyin the absence ofPAA. However, in the presenceofthe drug, whereas the production of wild-type virus during growth was severely in-hibited (0.051% of the control), the titer of mutant viruswasreducedonly threefold.

A similar experimentwas performedto com-pare the growth of wild-type KOS 1.1 and

PAA,rtsl

at34°C and 39.5°C in the absence of PAA. At 34°C, both viruses had similar yields (Table 2); however,at39.5°C,the production of wild-type virus was only slightly inhibited, whereas the yield of

PAAirtsl

at 39.50C was only 0.048% of thatat340C.

Viral and celular DNA synthesis. To deter-mine whether the mutation(s) in

PAAirtsl

af-fected the ability of the virustosynthesize viral and cellular DNA in vivo at 34°C and 39.5°C, newly madeDNAin infected cells waslabeled with tritiated thymidine and analyzed by CsCl gradient equilibrium centrifugation. Extracts of mock-infected CV-1 cells (Fig. 2A and B) exhib-ited only one peak at both340Cand 39.5°C. The gradient density at thatpointwas 1.690 g/cm3, corresponding tocellularDNA. Wild-type- and

PAAlitsl-infected

cell extracts showed two peaks at both temperatures (Fig. 2C-F), the secondpeak occurringwherethe gradient densi-ty was 1.715

g/cm3,

representingviral DNA.

As shown in Fig. 2C and 2D, the amount of viralDNA synthesized at39.5°Cwas 2.1 times thatproducedat340C inthewild-typeKOS 1.1-infectedcells. The meanincreaseobtainedinsix separate experiments was 1.2. In comparison, the amount of viral DNA synthesized in the

PAA,'tsl-infected

cellsat39.5°Cwasonly5.6% ofthelevelsynthesizedat340C (Fig.2Eand 2F).

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[image:4.492.49.455.604.656.2]

PHOSPHONOACETIC ACID-RESISTANT HSV-1 23

TABLE 1. Yield ofwild-type virus and PAA' tsl after growth in the presenceorabsence of 100 ,ug ofPAA/

Mla

Viruls Virus titerb±SD(PFU/ml) +PAAtiter/

-PAA +PAA -PAA titer

Wild type 8.4 ±5.1 x 105 4.3 ±2.8 x 102 5.1 x 10-4

pAArtsl 8.7± 1.4 x 105 2.7± 0.9x 105 3.1 x 10-1

aExperiments wereperformed at 34°C.

bValues represent the averageof triplicate samples.

In three other experiments this value ranged from 0to 10%o, with a mean of4.9% (datanot

shown). This indicates that PAAirtsl is partially deficient in viral DNA synthesis atthe

nonper-missivetemperature.

Withregardtocellular DNAsynthesis,allthe viruseswere very efficient in inhibiting cellular

DNA synthesisat34°C(Fig. 2), asobservedby

other workers(1). At 39.5°C,however, the shut-down of cellular DNA synthesiswas much less pronounced in the

PAA,rtsl-infected

cells than

ithad been in these cellsat34°C. Theamountof cellular DNAproduced in the

PAA,rtsl-infected

cellsat39.5°Cwas4.5 times thatsynthesizedat 34°C, adifference significantly greater than the

1.2-fold increase observed in wild-type KOS 1.1-infected cells(Fig. 2C-F). The ratio of cellu-lar DNA synthesized at 39.5°C relative to that synthesized at 34°C in the mock-infected cells

was 1.8(Fig. 2A and 2B). Themeanscalculated from several experiments for the cellularDNA synthesized at 39.5°C relative to that synthe-sizedat 34°C in the

PAA,rtsl-,

KOS 1.1-, and mock-infected cellswere3.4(four experiments),

1.1 (six experiments), and 1.9 (four experi-ments), respectively. The difference in the meansof the cellularDNAratios between wild-type KOS 1.1 and

PAAi'tsl

was found to be significantataprobability of 0.01 (as judged bya t test). These results suggest that PAAirtsl is

temperaturesensitive for the shutdown of

cellu-lar DNAsynthesis. Therefore, PAAirtsl is de-fective in viral DNAsynthesis andinthe shutoff of host DNA synthesis at the nonpermissive temperature.

DNA polymerase assays. The finding that PAAirtsl-infected CV-1 cellswere temperature

sensitive for the synthesis of viral DNAat the nonpermissive temperature, coupled with the fact that

PAA,rtsl

is resistant to PAA, a drug

that bindstoHSV-1-induced DNApolymerase, providedastrongindication that the DNA

poly-merase was indeed affected in the mutant. To examine this possibility, crudeenzyme extracts prepared from wildtype-andPAArtsl-infected cells were heated at45°C for various times and then incubated at 38.5°C for 30 min in the

presence of the DNA polymerization reaction

mixture. The thermal inactivation of the virus-induced DNA polymerase is shown in Fig. 3. After20minat45°C,morethan 70% of the wild-type enzyme's polymerizing activity remained. On the other hand, the DNA polymerase

en-coded by

PAA,rtsl

was rapidly inactivated at

45°C. Approximately 65% of this crude

en-zyme'sactivitywasdestroyed after 4minatthe elevatedtemperature,and only 16% of the poly-merizingactivity remained after 20minat45°C, thus showing that the viral DNA polymerase activity of

PAAIrtsl-infected

cells was more

sensitivetothermalinactivation than that of the wild-type-infectedcells.

As shown in Fig. 4, similar DNApolymerase

assaysinthepresenceofPAA,at34°C, revealed

that

PAA,rtsl

codes foraDNApolymerase that

is resistant to PAA when compared with the wild-type enzymeinvitro.

*+

revertants of PAA4rtsl. The replication

efficiency, DNA synthesis, and DNA

polymer-ase experiments indicated that the mutant

PAA1rtsl

wasindeedsignificantly PAA resistant

and temperature sensitive. In apreliminary

at-tempt to determine whether the two mutant

phenotypes of PAAirtsl were due to a single

TABLE 2. Yield ofwild-type virus and PAAr tsl after growthateither 34 or38.5oCa

Virustiter't SD(PFU/ml) 38.5°Ctiter/

Virus 34°C 38.50C 340C titer

Wild type 1.4 +0.1 x 106 1.1 ± 0.1 x 106 7.9x 10-1

PAAr tsl 1.2 +0.2 x 106 5.8 t 1.2 x 102 4.8x10-4

aExperimentswereperformed in the absence of PAA.

bValues represent the averages of triplicate samples.

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mutation ortwo separate mutations, ts+

rever-tantswereisolatedby plating individual

plaque-purified lysates ofPAAirtsl at the

nonpermis-sive temperature. The two independently

derivedts+revertantsweresubsequently tested

for PAA sensitivity and plaquing efficiency at

39.5°C. Each of the revertantclones (R7-2 and Ri-i) was capable of forming plaques at the A

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FIG. 2. Separation of viral and cellular DNA synthesized at 34°C and 39.5°C by CsCl equilibrium

centrifugation.Theexperimentswere(A) mockinfected,34°C; (B)mockinfected,39.5°C; (C)wild typeinfected, 34°C;(D) wild typeinfected,39.5°C; (E) PAAlrtsl infected,34°C; (F)PAAlrtsl infected, 39.5°C.

nonpermissive temperature with an efficiency thatwas similar to that ofwild-type virus (the

plaquing

efficiencies ofwild-type KOS 1.1, R7-2,andRi-i were 2.3 x10-1, 6.4 x 10-2, and 4.6 x 10-2, respectively). However, these ts+ re-vertants werestill almost as resistant to PAA as

PAA,rtsl

(Fig. 1). These results seemedto

indi-cate that the two mutant phenotypes of PAAirtsl were caused by two separate muta-tions. However, the single-mutation model couldnotbe excludedbythese data, since true reversion mutation (return of the primary base alterationtothe base sequence ofthe wildtype) is

probably

rare

compared

with second-site re-version

(suppression)

mutations. Changes of the latter kind neednot a

priori

result inreversionof both

characteristics.

Segregation of the

PAA,

and the tsl muta-tions. The ts+ revertant studies of

PAAirtsl

suggested the presence of two separate muta-tions. To examine this question further, we crossed

PAA,rtsl

with various

PAASts+TK-strains.PAArTK-and

PAASTK+

recombinants wereidentified, and the degreeoflinkage ofthe

PAAIrtsl

putative mutations was examined by determining the frequency of

PAAirtsl+

and PAAistsl recombinants among thePAA/TK re-combinants. As shown in Table3,infour

sepa-ratecrosses,atotalof16PAA/TK recombinants were isolated, and 10 of these isolates had a crossoverbetween thePAA1r and the tsl muta-tions. Thus, the mutant

PAAirtsl

clearly con-tains twodistinct mutations.

In cross 4

(PAAIrtsl

x

Bu-Cj),

where we obtained 11

PAA/TK

recombinants out of 44 plaques analyzed, giving a recombination fre-quency of

25%,

the PAAr/tsl and

tsl/Bu-C,

respective

recombination frequencies were 32 and 18. The PAA1r and tsl mutations were clearly loosely linked and most likely to be located intwo separate genes.Similarly, the tsl and

Bu-C,

mutations were located in two sepa-rate genes. The maximum recombination fre-quencybetweentwomutations within the HSV-1 TK gene was reported to be about 2% (18). The aboverecombination data suggesta proba-blelinkagemapofthe tsl,

PAA1r

and

Bu-C,

loci as shownin the diagrambelow.

tsl 18%

Bu-C,

25%

PAA,r

1( 32%

Complementation studies. Valuable informa-tion on the tsl mutainforma-tion present in PAAirtsl maybe obtainedfrom

complementation

studies

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>_ ~~0

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z

30-

20-10

2 4 6 10 12 14 16 13 20

HEAT INACTIVATION (min)

FIG. 3. Effect of temperature on the

HSV-1-in-duced DNA polymerase activity inextractsof infected

BHK-21/C13 cells. Extracts of cells infected with

wild-type virus (0) orPAAi'tsl (0) orofmock-infected

cellswereheatedat450Cfor the indicated times and

were assayed for viral DNA polymerase activity as

described in Materials and Methods. 3H counts

ob-tainedfrom the mock-infectedsystem(50to120 cpm)

were subtracted from those of the wild-type- and

PAAIrtsl-infectedsystems.Resultsareexpressed

rel-ativetothepolymeraseactivitiesfoundinthe

unheat-edextracts, whichgave2,500to3,200cpm.

with otherknowntsmutants. Studieshave been performed with tsD9, ts833,andts478,and with other ts mutants isolated in our laboratory. Complementationindices obtained from partof these studiesareshown in Table 4. Indexvalues listedfor eachcross weretheaveragesof dupli-catesamples.

Allof the tsmutantstested werefoundtobe complementary to one another, indicating that the mutations in each of these mutants are

located in differentgenesof the HSV-1genome.

DISCUSSION

Previous studies(1, 9, 15) reported the isola-tion of a DNA synthesis-deficient mutant of HSV-1, tsD9,whichwassubsequentlyfoundto

exhibit PAA resistance and code fora

thermola-bile DNApolymeraseinvivo.Thisstudy report-ed the isolation of a novel PAA-resistant and temperature-sensitive mutant of HSV-1 (PAAirtsl), which wasisolated asbeing PAAF.

Reversion studies and recombination analyses

of

PAA,'tsl

indicated that thePAA1F mutation

and tsl mutation were two distinct mutations located on separate genes. The tsl mutation mapped at approximately 18% recombination units to the left of the TK- mutation, Bu-C1,

whereas the PAA1r mutationmappedat approxi-mately 25% recombination units to the right of Bu-C1. The probablelocationofthe tslmutation with respect to the TK and PAA markers is tsl-TK-PAA.

ViralDNAsynthesisin the

PAAlrtsl-infected

cells was substantially reduced at 39.5°C, the nonpermissive temperature. The observation that viral DNA synthesis was nottotally blocked at39.5°Cindicates that PAAirtsl is not quite as temperature sensitive for DNA synthesis as tsD9, tsC4, and tsC7 (1). The in vivo DNA synthesisdefect and the increasedthermal sensi-tivity of the PAAirtsl-inducedDNApolymerase demonstratedin vitro are consistent witha no-tion thatthe PAA1r mutation is responsible for the production of a viral PAA-resistant DNA polymerasethat isthermolabile at39.5°C.These results support the studies withtsD9 (1, 9, 15), which showed that a PAA-resistant mutation is located inthestructural genefor the viralDNA polymerase. Thestructurallyaltered viral DNA polymerasewas stillpartly functionalthoughat 39.5°C, sinceminimal levels of viralDNA were synthesizedin

PAA,rtsl-infected

cells.

PAAirtsl was also defective in the shutoff of

110 O

100

0

:t

'S

5S.

02 13

10 20 3S sU

Jug PAA/ml

FIG. 4. Effect ofPAAon theHSV-1-inducedDNA

polymerase activity in extracts of infected BHK-21/

C13 cells. Cellswereinfected withwild-type virus(0)

orPAAirtsl (0)or weremock infected for the

prepa-ration of extracts to determine the in vitroactivityof

the viralDNApolymerase in the presence of PAA. 3H

countsobtained from the mock-infected system (50to

120cpm) weresubtractedfrom those of the

wild-type-and

PAA,rtsl-infected

systems. Results are expressed

relative to the polymerase activities found in the

absence of PAA, which gave2,500 to 3,200 cpm.

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PHOSPHONOACETIC ACID-RESISTANT HSV-1 MUTANT

TABLE 3. Three-factor crosses between PAAr tsl and TK- PAAS mutants

Cross No. of Genotype' %Recombination'

progeny Progeny

No. Parent1 Parent 2 any PAA ts TK PAA/tsc PAA/TK ts/rKc

1 PAArtsl Bu-E1 16 16 1 1 2

2 PAArtsl Bu-B4 58 44 1 1 2

4 1 2 2

41 1 2 2

3 PAAr tsl Bu-E2 44 6 1 2 2

4 PAAr tsl

Bu-C,

44 1 1 1 2 32 25 18

6 1 1 2

3 1 2 2

4 1 2 2

20 1 2 2

22 1 2 2

2 2 2 1

33 2 2 1

8 2 1 1

14 2 1 1

18 2 1 1

a Thenumbers 1 and 2 denote the genotypes of parents 1 and 2,respectively.

bRecombinationfrequencieswere calculated from the data obtained from cross 4only.

cOnly one-halfof the recombinants between the

PAAT/tsl

and

tsl/TK

loci were detected in ouranalysis. Therefore,inthe calculations of the recombination frequencies between these loci, the numbers of recombinants

detectedweremultipliedby afactor of 2.

cellular DNA synthesis at the restrictive

tem-perature, as evidenced by the4.5-fold increase

in cellular DNA synthesized in the

PAA,rtsl-infectedcellsat39.5°C compared with that

syn-thesized at34°C. Wild-type-infectedand mock-infected cells exhibited onlya1.2-fold anda 1.8-foldincrease, respectively. The contributions of

the

PAA,r

mutation and the tsl mutation in

affecting the levels of viral and cellular DNA synthesizedat34°C and 39.5°C have been

exam-ined with the various

PAA,Stsl

and PAAirtsl+ recombinants (see Table 3) and

PAA,rtsl+

re-vertants.

PAA,rtsl+recombinants

are not significantly

defective in viral DNAsynthesis and growthat

39.50C. ThePAArtsl+ revertants(R7-2 and

Rl-1) exhibit intermediate to wild-type levels of viral DNA production at 39.50C. PAAistsl

re-combinants also exhibit wild-type viral DNA synthesisat39.50C.However, they haveavery

lowplaquingefficiency at39.5°C, ranging from

3.0 x 10-6 to4.1 x 10-6(J. I.Daksis and V.L.

Chan, unpublished data). The observation that the

PAA,rtsl

mutant is defective in viral DNA synthesisat39.5°C, whereas the PAAirtsl+ and

PAA,stsl

recombinants arenotsignificantly

de-fective, suggests the possibility thatthePAAr and tsl gene productsmay be components ofa

DNA polymerase enzyme. Alternatively, they

maybeseparateenzymesexistingas acomplex

thatis involved in DNAreplication. The altered

PAA1r and tsl polypeptides perhaps form a

thermolabile complex.However,when the

com-plex consists of either the PAA1r and tsl+orthe

PAA'S

andtslgeneproducts, it isnotdefective in viral DNAsynthesis at 39.5°C. Presumably, the defective interaction of the altered viral PAA1r DNA polymerase with the altered tsl

geneproduct was responsible for the observed thermolability of the PAAirtsl-induced DNA polymerase (see Fig. 3). Determination of the thermolability of the DNA polymerases ofthe

PAA,stsl

andPAArts1 + recombinants, aswell

as the

PAA,rtsl+

revertants, should provide valuableinformation.At themoment,wedonot

have an explanation for the defect in plaquing

TABLE 4. Complementation betweentsmutants

Complementation index' frommixed tsmutant infection with tsmutant

PAAirtsl tsl-8 tsD9 ts199 ts833

PAA,rts1

1.2 13.3 3.9 24.1

tsl-8 4.6 26.2 27.5

tsD9 4.5 4.0

tsl99 8.1

ts833

aComplementation index = (Ax

B)39.5C/A39.5-c

+

B39.s-C.

assayed at34°C.Values greater than 2.0 were

considered to bepositive.

VOL.42, 1982 27

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efficiency atthe nonpermissivetemperature for the PAA1'tsl recombinants, which are not de-fective in viral DNA synthesis, butaredefective in the shutdown of host DNA synthesis at 39.50C.

Animportantnewobservationhasbeen made inthe presentstudies.Itis thatthe

PAAirtsl

and PAAistsl mutants werealldefective in the shut-off of cellularDNAsynthesisat39.5°C(only the results of

PAAirtsl

are presented here). These observations imply the involvement of the tsl geneproduct intheshutoff of hostDNA synthe-sis. The intriguing observation that

PAA,rtsl+

recombinants were also defective in the shut-down of cellular DNA synthesis implies the involvement of the PAA1r gene product in the shutdown of host DNA synthesis. We propose that the shutoff of cellular DNA synthesis by HSV-1requiresaninteraction between the viral DNA polymerase and the tsl+ polypeptide. Modification of the structure of either gene productmaygiverisetoimproper interaction in the complex,leadingtoadefect in theshutoff of cellular DNA synthesis. Augmentation of this effectmay occurwhenboth

proteins

are mutat-ed. One of the

PAAirtsl

+ revertants(R7-2)was normal in the shutoff of host DNA synthesis. Since all the

PAAirtsl+

recombinants were de-fective in the shutoff of host DNAsynthesis, the above resultimplies that thetsl+revertantgene product could interact efficiently with thePAA1r geneproducttoallow normalshutoff of

cellular

DNA synthesis.

ts+

revertantsof the PAA1'tsl recombinants will beisolated and tested for their

ability

toshutoff cellular DNAsynthesis. Itwill also be interesting to determine whether other

PAAF

HSV-1 mutants aredefective in the shut-down of host DNA synthesis at an elevated temperature.

No tsHSV-1 mutantsdefective in the shutoff of cellular DNA

synthesis

have

previously

been reported. Two ts viralDNAsynthesis-deficient mutantsof HSV-2 (ts9 and tsll) intwodifferent

complementation

groups have been

reported

to be defective in the shutdown of host DNA synthesisatthe restrictivetemperature (6, 20).It was not clear whether the viral DNAsynthesis defect of thesetwoHSV-2mutantswas aresult ofthedefective shutoff of cellularDNA synthe-sisatthenonpermissive temperature. Our find-ingthat the

PAA15tsl

and

PAArtsl

+ recombi-nant HSV-1 mutants synthesizednormal levels of viralDNA at

39.50C,

while stillbeing defec-tive in shutdown ofhost DNA synthesis, indi-catesthat theshutoffof cellularDNAsynthesis isnotessentialfor viralDNAreplication.

Clear-ly,

PAA,rtsl

is invaluable in elucidating the

mechanismof inhibitionof

cellular

DNA synthe-sisby HSV-1.

Previous fine

mapping

studies of two PAAr

mutantsof HSV-1 indicated that they may pos-sessmutations resident inseparate genes(4,11). More recently, however,

Chartrand

et al.

(3)

presented evidence for the existenceof onlyone PAArgenein HSV-1 and HSV-2.Itwill, there-fore, be of interest to

physically

mapboth the PAA1r and the tsl mutations of PAAirtsl, by markerrescue

experiments.

As shownin Table 4,

PAA,'tsl

(or thePAA1'tsl recombinant, tsl-8) wasfound to complement the ts DNA poly-merase mutants,tsD9and ts833(which is in the same

complementation

group as

tsC4

[17]),

indi-cating'that

thetsmutations in thesemutants are locatedin differentgenesof the HSV-1genome. A more extensive

complementation

study with otherts mutantsisolated from other laboratories would benecessary todetermine whether thetsl mutation of PAA1rtsl represents anovel com-plementation group.

ACKNOWLEDGMENTS

We thank RoseSheininfor the BHK-21/C13 cells and for valuable criticism during the course of this work, L. Simino-vitch for interest andvaluable suggestions on thepreparation of thismanuscript,R.G.Hughes, Jr.,P. A.Schaffer,andS. Bacchettifor theirgiftsof cellsandvirus, ChristinaA.van't Hof fortheisolationof PAA-resistantHSV-1mutants,andS. Guttman for expert technicalassistance.

Thisworkwassupported bythe NationalCancerInstitute and theMedical Research Council of Canada. V.L.C. is a Research ScholaroftheNational Cancer Institute ofCanada.

LITERATURE CITED

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PHOSPHONOACETIC ACID-RESISTANT HSV-1 MUTANT

10. Kieff, E. F., S. L. Bachenhelmer, and B. Roizman. 1971. Size, composition andstructureof thedeoxyribonucleic acid of herpes simplex virus subtypes 1 and 2. J. Virol. 8:125-132.

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14. Overby, L. R., E. E. Robahaw, J. B. Schlekcher, A. Rueter,N. L.Shipkowitz,andJ. C.-H.Mao.1974. Inhibi-tion of herpes simplex virus replicaInhibi-tion by phosphonoace-tic acid. Antimicrob. Agents Chemother. 6:360-365. 15. Purlfoy,D. J.M.,andK.L.Powell.1977.Herpes simplex

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16. Schaffer,P. A., G. M. Aron, N.Biswal,andM. Benyesh-Melnlck. 1973.Temperature-sensitive mutantsof herpes simplex virus type 1: isolation, complementation and partial characterization. Virology 52:57-71.

17. Schaffer,P. A., V. C. Carter, and M. C.Timbury. 1978. Collaborativecomplementation study of temperature-sen-sitivemutants of herpes simplex virustypes1 and 2. J. Virol. 27:490-504.

18. Smnley,J.R.,M.J. Wagner,W.P.Summers,andW. C.

Summers. 1980. Genetic and physical evidence for the polarity of transcription of the thymidine kinasegeneof herpes simplex virus. Virology 102:83-93.

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