Inhibition of Herpes Simplex Virus Type 2 Replication by Thymidine

(1)

Inhibition

of

Herpes Simplex

Virus

Type

2 Replication by

Thymidine

GARY H. COHEN, MINDIE N. FACTOR, AND MANUEL PONCE DELEON

DepartmentofMicrobiology, School of DentalMedicine, Universitvof Pennsylvaniaand Center for Oral HealthResearch, Philadelphia, Pennsylvania 19174

Received for publication 13March 1974

Replication of herpes si7aplex virus type2 (HSV-2) wasimpeded inKB cells

which were blocked in their capacity to synthesize DNA by 2mMthymidine

(TdR). The degree of inhibition was dependentupon the concentration of TdR.

In marked contrast, HSV-1 is able to replicate under these conditions. The

failure of HSV-2 to replicate is probably due to the inhibition ofviral DNA

synthesis; therewas a marked reduction in therateof DNAsynthesis aswellas

the total amount of HSV-2 DNA made in the presence of 2 mM TdR. We

postulated that the effect of TdR on viral replication occurs at the level of

ribonucleotide reductase in a manner similar to KBcells. However, unlike KB

cells,an alteredribonucleotide reductaseactivity, highly resistanttothymidine

triphosphateinhibition, wasfound in extracts ofHSV-2-infected KB cells. This

activitywaspresentinHSV-2-infectedcellsincubated in thepresence orabsence

ofTdR.Ribonucleotide reductase activity inextractsofHSV-1-infected KBcells

showed asimilarresistanceto thymidine triphosphate inhibition. These results

suggest that the effect ofTdRon HSV-2 replication occurs at a stage of DNA

synthesis other than reduction of cytidine nucleotides to deoxycytidine

nucleo-tides.

We reported recently that herpes simplex virus type 1 (HSV-1) replication is unimpeded in KBcells incubated in the presence of 2 mM

thymidine (TdR)

(3). This concentration of TdRblocks host cell DNA

synthesis

and subse-quent cell division (1, 2, 3, 15, 17). These observations

suggested

that at least one aspect of DNA

replication

utilized

by

HSV differs

significantly

from that of the host cell. This difference was

explained by

the

finding

of a

"new" viral-induced ribonucleotide reductase

enzyme activity in the cell after infection and

by

the observation that thisenzymewas mark-edly resistant to inhibition

by

thymidine

tri-phosphate (TTP),anegativeeffector of

ribonu-cleotide reductase (1, 3, 9, 15). The "new"

enzymerelievedthe TdR blocktoDNA

synthe-sisby insuringa

pool

of

deoxycytidylate

precur-sors forreplication of virus.

The oral form of HSV has been shown to

differ

significantly

from the

genital

form

(HSV-2)in anumberof

biological, biochemical,

and

biophysical

characteristics

(12). Therefore,

we

investigated

the

capacity

ofHSV-2 to

repli-cateinKBcellsinthepresence ofexcessTdR.

Wepresent evidence heretoshowthatHSV-2

is unable to replicate in the presence of2 mM TdR. The failureto

replicate

is

probably

dueto

20

theinhibition ofviralDNAsynthesis. However, aribonucleotidereductase with properties

simi-lartotheenzymefound in HSV-1-infected cells

was also present in HSV-2-infected cells. This

suggeststhattheTdR block occurs at a stage of

DNA replication other than reduction of cyti-dine nucleotidesto deoxycytidine nucleotides.

MATERIALS ANDMETHODS

Cell cultures. Conditions for growth and mainte-nance of KB cells andbaby hamster kidney (BHK)

cells have beenpreviouslydescribed (3, 4).

Virus strains and titrations. Three strains of

HSV-1, HF, KOS, and CL, and three strains of

HSV-2, Savage(SAV) HSV (381) andVF, wereused. TheKOS, HSV (381), andVF strainswerekindly pro-vided by L. H. Moss III, Ohio State College of Medicine.StrainsVFand CLwererecentisolatesand were passed threetimes inBHKmonolayers priorto

use. Preparationofhigh-titeredvirusstockaswellas

assay by plaque titrations were

performed

as de-scribed previously (3).

Cellsynchronization. KB cellswere

synchronized

by the double TdR block method(3, 4).

Briefly,

KB cellsweretreated with2mM TdRfor 18to20h.The cells were washedfree ofTdRby centrifugation and

resuspended in fresh warm medium to reverse the

block, and then allowedtogrowforanadditional9to 12h. Atthistime, 2mM TdRwasaddedforanother

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INHIBITION OF HSV-2 REPLICATION

period of 18to20h.Infectionswerestarted 1 hpriorto reversal of thesecond-TdRblock.

DNA synthesis in synchronized cell cultures. Pulse-labeling experimentswerecarriedoutas

previ-ously described (3, 4) employing 0.2 ACi of

['H]hypoxanthine per ml (specific activity 5 to 10

Ci/mmol, NewEngland Nuclear Corp.).Methods for the separation of DNA and RNA as well as the

determination of radioactivity were previously de-scribed(3,4).Radioactive-labeledDNAwasobtained as previously described except that ['H ]hypoxan-thinewas employedasthe isotope (3, 4).Separation

of viral DNA fromcellular DNA was carriedout by CsCl density gradient centrifugation (3, 4).

Protein and RNA synthesis in synchronized cell cultures. Infectedoruninfected KB cells(generally2

x 10'cells/ml)wereincubated at the times indicated with0.2ACiof [3H]L-aminoacid mixtureperml(New

England Nuclear Corp.)tomeasureproteinsynthesis

or [3H]hypoxanthine (0.2 MCi/ml) to estimate RNA synthesis. Incorporation of label was stopped by

pouring the cellsontoicedphosphatebufferedsaline (PBS). The cellswerecollectedby centrifugation and

resuspended in 5 ml ofcold 5% trichloroacetic acid. Theresulting precipitatewaswashed three times with trichloroacetic acid and dissolved in 0.1 N NaOH. ThesampleswereaddedtoAquasoland the radioac-tivitywasmeasured.

Preparation ofextracts forenzyme assay. The

preparation ofcell-freeextracts has been previously reported (3).Theextractswerestoredat -100 C ina

Union Carbide liquid nitrogen refrigerator. Under thesestorageconditionsribonucleotide reductase

ac-tivity remains stableforseveralyears.

Ribonucleotide reductase assay. The conversion

ofcytidine diphosphate (CDP) to deoxycytidine di-phosphate (dCDP)wasmeasured byamodificationof

the method describedby Cohen (3) toinclude

proce-dures in the assay described by Setlow (14). The

standard reaction mixture (0.25 ml) contained: 10 mMTris, pH7.8, 2.5mMATP,1.4mMdithiothreitol (DTT); 2.8 mM MgCl,; 0.02 mM CDP, 1.5

ACi

of ['H]CDP (specific activity 13 Ci/mmol; Schwartz-Mann) and enzymeextract. Thesamples were

incu-bated at 30C for 15 min, and the reaction was

stopped by addition of 0.25 ml of 2 N HCl. The precipitated material wasseparated from the

super-natantfluid bycentrifugation. Thesupernatantfluid

was heatedat 100C for 15 min, then evaporatedto dryness in a desiccator containing solid KOH and

P20,. Sampleswereredissolvedin 50glitersofwater and a 25-gliter portionwas analyzed bypaper

chro-matography (3). The rate of formation ofdCMP in cell extracts prepared from uninfected KB cells and KB cells infected with HSV-1 or HSV-2 was linear

for at least 25 min and was proportional to extract addedinthe rangeof 0.2 to0.8 mgofprotein. Each

assay wasrun induplicate and theresults presented

are the average ofduplicate assays whichvaried by

10%. One unit ofribonucleotide reductase activity is definedasthatamountofenzymewhich catalyzes

for-mation of dCMP at a rate of1 nmol per mg per h.

Protein concentration wasdetermined by the method

of Lowry et al. (8).

RESULTS

Replication of infectiousvirus in the pres-ence and abspres-ence of2 mMthymidine.Table 1 shows a typical experiment in which the num-ber of PFUformed by three strains ofHSV-1 in theTdR-blocked KB cellswas

nearly

ashighas the number formed in the control cells (no TdR). In contrast, TdR hada

significant

inhibi-tory affect on replication of the three genital strains. Thetiters were200- to400-fold lower in KB cells containing TdR than in controls

(no

TdR) and were similar to

background

levels found in 3 h-infected cultures. Furthermore, this decrease in titer in the presence of TdRwas not due to release ofHSV-2 into the supema-tant fluid. The total amount of extracellular virus was at least one

log

lower than that of cell-associated virus. These results indicate that

HSV-2

replicationin

synchronized

KBcellswas inhibited

by

2mMTdR. In the

following

experi-ments, one strain of HSV-2

(Savage)

and one strain of

HSV-1

(HF)

was

employed.

Dose response relationship of thymidine concentration to virus synthesis. Figure 1 shows the dose response

relationship

between the amount ofHSV-2

synthesized by

KB cells and the concentration of TdR present in the medium. In the range 2

iM

to 2

mM,

every 10-fold increase in TdR concentration

produced

approximately

a one

log

decreaseinvirus titer.

Complete

inhibitionoccurredat 2mMTdR

and

no inhibition was observed at 2

jiM.

These results indicate that

HSV-2

replication

was inhibitedat aconcentration ofTdR which also inhibited cellular DNA synthesis and division in uninfected KB cells (3, 4) and that this

TABLE 1. Effect of excess thymidine on the replication of HSV types 1 and 2

PFUa HSV virus strain employed No Thymidine

thymidine (20h) (20h)

HSV-1

HF... 2.8x

107

1.6x107

KOS ... 1.6x 108 6.0x 107 CL (wild) ... 6.8 x 107 2.0 x 107 HSV-2

SAV . ... 2.6 x 108 1.0 x 104° HSV (381) ... 3.6 x 107 1.0 x 105° VF (wild) ... 4.0 x 10, 1.8 x 104°

aEach value represents the average of duplicate

samples. See legend in Fig. 1 for procedure.

°Values similar to background of PFU found at 3 h postinfection.

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106

PFU

SAMPLE

0

0~~~~~~~/

~

I}

11

0 2XIO3 2XIO-2

2XIO-l

2

THYMIDINE CONCENTRATION (mM)

FIG. 1. Dose response relationship of TdR concen-trations to virus synthesis. Cultures were svnchro-nized by the double TdR block method. The cells werewashed to remove TdR and infected at an input multiplicitv of 10PFUIcellin thepresence ofvary'ing

concentrationsof TdR as indicated in the figure. The viruswasallowedtoabsorb for1h. Unabsorbed virus waseliminated by three washes with minimal essen-tial medium containing the corresponding TdR con-centration. Twenty-four hours later the cells were harvested and washed with PBS, and cell associated virus wasdetermined. Symbols: 0, PFU per sample with varying concentrations of TdR; 0, background virus in KB cells containing TdR harvested at 3 h

postinfection.

inhibition of new virus synthesis was dose dependent.

Rate of DNA

synthesis

in HSV-2-infected and uninfected KB cellsin thepresenceand absence ofexcessTdR.

Synchronized

KBcells

were inf'ected with HSV-2 in the presence and absence of 2 mM TdR. The rate of DNA synthesis was measured by pulse

labeling

cell samples for 1 h with

[3H]hypoxanthine.

Figure 2A shows therateof DNA

synthesis

in

uninfected KB cells. When KB cells were

washed free of'TdR, a wave ofDNA

synthesis

occurred. Therate ofDNA

synthesis

increased rapidly for3 to 4 h.then decreased

by

8h. The cellnumberremainedconstant for 8to10h post

reversal and then doubled. A second round of

DNAsynthesis

(corresponding

to the second S

phase) commencedat12 to 14h.When the cells

wereincubatedinthe presence of

TdR,

therate

ofhostcell DNAsynthesisremainedconstantat

less than

8%

ofthe reversed culture and there

was noincrease in cell number. Theresults are

essentially the same as previously reported (3).

The pattern of DNA synthesis in

synchro-nized cultures infected with HSV-2 was

mark-edlydifferent (Fig. 2B). In the absence of TdR,

the rateofDNA synthesis reached a

maximnum

approximately 5 h after infection and was never

greater than 30% of that found for uninfected

KB cells. DNA synthesis in the infected cells

thendeclined slowly for at least 16 h.

In contrast, the rate of DNA synthesis in TdR-blocked cells increased only slightly by 3 h after inf'ection and subsequently returned to a

lowlevel. These results show that DNA

synthe-sis in TdR-blocked infected cells occurs at a

muchreduced rate and suggests that the

inhibi-14 r A

12

10 6

5 -14

lo 4

x

UJ3 z

-2 z x 0 I = 4

3n

2

o

-B

A

/ \HSV-2 no TdR

6~~~~~

-/& oe.-HSV-2+ TdR

A A

O 2 4 6 8 10 12 14 16 18

HOURS POST REVERSAL

FIG. 2. Rate of DNA synthesis in synchronized culturesofuninfectedKBandHSV-2-infectedcells in the presence and absence of TdR. The rates were determinedby pulse labeling5.0-ml cellsamples (2x

105 cells/ml) with [3H]hypoxanthine for 1 h, as

indicated in MaterialsandMethods. Symbols: panel

A, 0,uninfected cells; 0, uninfectedin thepresence of2mM;panelB,A,infected cells, andA,infectedin thepresence of2mM TdRcells wereinfected at an input multiplicity of20PFU/cell.

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INHIBITION OF HSV-2 REPLICATION

tion of viral replication may be due to an

inhibition of viral DNA synthesis.

Before conclusions could be drawn concerning

HSV-2 productionand the level of DNA

synthe-sis, itwasnecessarytoshow thatthe radioactive

DNA made after infection was viral and to

determine how much viral DNA was made in

thepresence of TdR.

CsCl density gradient analysis of DNA

synthesizedinHSV-2-infected KB cells in the

presence or absence ofexcess TdR.

Synchro-nized KB cellsinfected with HSV-2 were

incu-bated in the presence or absence of TdR.

Tritium-labeledhypoxanthinewasaddedtothe

cells 1 h afterreversal and 9 h laterthey were

harvested. Theextracted DNAwasanalyzed by

buoyant density ultracentrifugation in CsCl.

The radioactive profiles obtained are shown in

Fig. 3. Two bands of radioactivityweredetected

in infected cells incubated in the presence or

absence ofTdR. One band (1.700 g/cm3)

corre-sponds in buoyant densityto KB DNA (3) and

thesecond (1.725g/cm3) toHSV DNA (3). The

amount ofviral DNA synthesized in the

pres-8

6

x

w

z

I

z

x 4

0~

I I

10

D-2

u

1.740 1.730

.720 , 1.710 -" 1.700 O

1.690

1.680

0 10 20 30 40 50 60 70

FRACTION NUMBER

FIG. 3. CsCl density gradient analysis of DNA synthesizedinKB cellsinthepresenceorabsenceof

excess TdR. In each case. [3H]hypoxanthine was

added at a time corresponding to I h after TdR reversal. The cells werethen incubated foran

addi-tional 9h. Symbols: 0,DNA (2.8 gg) isolated from

HSV-2-infectedKBcells whichwereincubatedinthe

absenceofexcess TdR;*,DNA (5.8 Mg)isolated from

HSV-2-infected KB cellsand incubated in the pres-enceofexcess TdR.

enceof

TdR

was

markedly reduced.

From these

studies

and othersweestimate that

only

10% of the HSV-2 DNA found in the controls was synthesized in the presence of TdR. These results differfromthose

previously

reported

for HSV-1 (3). In that case excess TdR had little effect on the rate or amount of viral DNA synthesis.

Viral inhibition

of

KB cell RNA

and

pro-tein synthesis. The observation thatnewviral DNA was made in

HSV-2-infected

cells in the

presence ofTdRsuggests thatsome viral

func-tions were

expressed.

Supportive

evidence was obtained

by

examining anothercharacteristic of

HSV

infection.

We asked whether HSV-2 infection of

TdR-blocked

KB cells resulted in the inhibition of RNA and proteinsynthesis. Cellular RNA and proteinsynthesis are not

markedly

inhibited in mammalian cells by excess TdR (2, 16, 17). When cellsareinfected with

HSV,

inhibition of host macromolecular synthesis is

dependent

upon production ofviral-induced protein(s) (6, 13). Therefore, inhibition of host RNA and proteinsynthesis would constitute indirect evi-dence that viral functions are

expressed.

Table 2 shows the effect of2 mM TdR on RNA and protein synthesis in uninfected and infected KB cells. When the cells were incu-batedinthe presenceof

TdR,

neither RNAnor protein synthesis were

markedly

inhibited. In

contrast, both RNA and protein synthesis in

HSV-2-infected KB cells were markedly inhib-ited, and the extent of this inhibition was enhanced

by

the presence of TdR.

Ribonucleotide

reductaseactivity in HSV-infected cells grown in the presence and absence

of

excess TdR.

HSV-1

replicates

in TdR-blocked KB cells (3). This characteristic has been ascribedtothe productionof a "new" or altered ribonucleotide reductase activity which is not subjectto allosteric regulation by TTP (3). DNA replication by this virus is not regulatedinthesame manner as host cell DNA

TABLE 2. Effect of TdRonuninfected and infected KB cell RNA andprotein synthesis

KBcell+TodrR %Inhibition

KB cell

Tdor

-___ ____

RNAa Proteina

Uninfected 0 0

Uninfected + 5 12

Infected _ 57 68

Infected + 93 88

a

[3H

lhypoxanthine

or

3H-labeled

aminoacids was addedat atimecorrespondingto1hafterTdR rever-sal. The cellswerethen incubatedforanadditional9 hinthe presenceorabsenceofTdR.

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COHEN, FACTOR, ANDPONCEDELEON

synthesis. Thus it was possible that the failure ofHSV-2 toreplicate would be reflected by the absence of a "new" ribonucleotide reductase activity similar to that found with HSV-1. Studies were undertaken to compare the ribo-nucleotide reductase activity found in unin-fected cells and in cells infected with HSV-1 and HSV-2.

Ribonucleotide reductaseactivity in synchro-nized KB cells is periodic, being highest in

extracts prepared at orjust prior to reversalof

the TdR block (3). Enzyme activity in the washedcellsremains at a maximum for 6 to 8 h post reversal, then decays rapidly during the

courseofthemitotic cycle (3). Incontrast,if the

TdR block is not reversed, the activity of the

enzyme remains essentiallyunchanged.

Synchronized KB cells were infected with HSV-1 or HSV-2 and washed free of TdR 1 h later. Cells were harvested at 3.5 and 9 h postinfection corresponding to the S and G2-M phases of the cell cycle. Figure 4A shows that reductase activity of uninfected KB cells was, as previously shown (3), highly sensitive to inhibition by TTP. Furthermore, the percent inhibition by TTP was identical for enzymes prepared at the two times in the cell cycle.

A KS B HSV-I C HSV-2

80 5X

<40.r E o TT o

ac y 9in,ea- of u e n i

20 Aur

~~~~L

of5 cs snh 35the V10-410 3 10-2 10-1 1010-4 10-3 12 tol 1010-4 103 IU2 1ol

CON\CENTRATION\OF TTP(mM)

FIG. 4. Effect ofTTPonribonucleotide reductase

activity in extracts of uninfected and infected KB

cells. A cultureofKB cellswass,vnchrovnizedbythe double TdR blockmethod. One hourpriortoreversal,

onethirdofthe culturewasinfectedwithHSV-1,one third with HSV-2, and the other third was

mock-infected. Cellswere infectedat aninputmultiplicity

of25PFUIcell, for both viruses.After 1hof incuba-tion the cultures were washed by centrifugation,

re-suspended in fresh medium, and allowed to grow.

Samplesweretakenat3.5and9hafterTdR reversal. Theactivitiesofeachpreparationweredetermined in the presence ofvarying concentrations ofTTP. The standard assay conditionswere used as described in Materials andMethods, except that TTPwasadded

to samples as indicated in the figure. One hundred

percent activity was determined by performing the assay in theabsence ofTTP. Symbols: 0, 3.5h;*, 9 h. PanelA, ribonucleotide reductaseactivity in ex-tracts prepared from uninfected cells (3.5 h = 1.94 U,9h = 1.84U); panel B, enzymeactivityinextracts

prepared fromHSV-1-infected cells (3.5 h = 1.42 U,

9h = 0.76 U);panel C, enzymeactivity inextracts

prepared fromHSV-2-infectedcells(3.5h = 1.52U,9

h = 0.91 U).

Figure 4B and C show that enzyme activity in

extracts prepared at 3.5 h postinfection either

with HSV-1 or HSV-2 had the samesensitivity

to TTP as did the enzyme prepared from KB

cells. However, by 9 h postinfection (Fig. 4B and C) ribonucleotide reductaseactivity in both HSV-1 and HSV-2 infected cell extracts was markedly resistanttoinhibition.More than 70%

ofthe maximum activity was present even in

the presence of 4.0 mM TTP. These findings indicate that a "new" reductaseactivity is also present in extracts obtained from the HSV-2-infected cells. Moreover, the extent of TTP inhibition was the same for both viral-induced enzymes.

Even though HSV-2 induceda"new" ribonu-cleotide reductase, itwas possible that the virus

was unable to induce the enzyme in the

pres-enceofTdR. Therefore, KB cells were infected

with HSV-2 in the presence of TdR. We ob-served that the presence of TdR during infec-tionhadnoeffect onthe specific activity of the

enzyme and no effect on the sensitivity ofthe

enzymetoTTP.Moreover, the observation that dialysis had no effect on thereductase activity

suggests that the insensitivity of the isolated

HSV-2 enzyme to exogenous TTP was not due to an alteration in nucleotide pool size. No synergistic effect on ribonucleotide reductase

activity wasdetectablewheninfected and

unin-fected extracts were mixed. The activity of a mixture of equal amounts of the two extracts wasequivalenttothesum of the activity of each

extract measured separately. From these

stud-ies, we conclude that the failure of HSV-2 to replicate in the presenceofexcess TdR cannot be explained by the inability of this virus to inducea "new" TTP-insensitive ribonucleotide reductaseactivity.

DISCUSSION

The present

study

compared

the pattern of

replication of HSV-1 and HSV-2 in KB cells growing in the presence or absence of excess

TdR. Ourfindingssuggestthat TdRhas

differ-ent effects on the

replication

of HSV-1 and

HSV-2. This observation adds an additional

biochemical

criterion which

distinguishes

HSV-1 from HSV-2

(12).

Evidence has been

presented

that this difference is not due to a

failure of HSV-2 to infect TdR-blocked KB cells. This evidence includes detection of new

viral DNA albeit in low

quantity,

inhibition of hostcell RNA and

protein

synthesis,

and

induc-tion ofan altered ribonucleotide reductase

ac-tivity. Taken

together,

these results

suggest

that the failureof HSV-2 to

replicate

in

TdR-blocked KB cells occurred at the level ofviral DNA

replication

or later. Evidence that the

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INHIBITION OF HSV-2REPLICATION

failure of HSV-2 to replicate in TdR-blocked

cells is at the level of DNA replication was

provided by the finding that both the rate of

DNA synthesis as well as the total amount of

viral DNAmadeweredrastically reducedinthe

presence ofexcessTdR.

A problem raised by this study concerns the

mechanism by which TdR inhibits HSV-2

repli-cation. It would be reasonable thatTdRaffects

viral replication at the level ofribonucleotide

reductase as it does in the host cell (1, 2, 15).

However, this explanation is not borne out by

ourresults. Unlike the uninfected hostcell, and

similarlytoHSV-1-infected cells,a"new"

ribo-nucleotide reductase activityhigflyresistantto

TTP inhibition is induced in HSV-2-infected

cells regardless of the presence or absence of

TdR.

Assuming that the ribonucleotide reductase is

functional in HSV-2-infected cells in the

pres-enceofTdR, what function(s) is inhibitedsoas

toprevent viralreplication?

TheTdR block may occurata stageof DNA

replication other than reduction of cytidine

nucleotide todeoxycytidine nucleotide. Recent

evidenceindicates that theproperties ofsomeof

theviral-inducedenzymesfound inHSV-1- and

HSV-2-infected cell extracts differina number

ofcharacteristics including inhibition by T1P.

Cooper (5) showed that TTP (0.1 mM)

inhib-ited the phosphorylation of deoxycytidine by

85% in extracts ofHSV-2-infected cells.

How-ever, TTP concentrations of 0.2 mM did not

significantly inhibit deoxycytidine

phosphoryl-ation in extracts of uninfected or

HSV-1-infected cells. Ogino etal. (10) showeda

differ-enceintheeffect of TdRnucleotideson

HSV-1-and HSV-2-induced TdR kinase. HSV-2 TdR

kinasewasmuchmoresensitivetoinhibition by

TdR nucleotides thanHSV-1 kinase.

Another possibility is that replication of

HSV-2 may be dependent upon somephase of

the host cell cycle. The replication of some

viruses including equine abortion virus (7) and

SV40 (11) is dependent upon some function(s)

(reviewed in ref. 7 and 11) present only during

the early S phase of the cell cycle. However,

HSV-1replication isindependent of the KB cell

cycle (3).

Experiments are now in progress to

investi-gate the mode of action of TdR on HSV-2

replication.

ACKNOWLEDGMENTS

Thisinvestigationwassupported by PublicHealthService grant DE-02623 from the National Institute of Dental Re-search.G.H.Cohenwassupported byaPublic Health Service

Research Career Development award A1-23801 from the National Institute of Allergy and Infectious Diseases.

WethankRoselyn J. Eisenberg, Lewis Pizer, and Weslev Wilcox for their generous help in the preparation of'this manuscript. Weacknowledge the excellent technical

assist-anceofMadeline Cohen.

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3. Cohen, G. H.1972. Ribonucleotidereductaseactivityof synchronized KB cells infected with herpes simplex virus.J. Virol. 9:408-418.

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Virol.7:783-791.

5. Cooper, G. M. 1973. Phosphorvlation of 5-bromodeox-ycytidine incells infected withherpes simplex virus.

Proc.Nat.Acad. Sci. U.S.A. 70:3788-3792.

6. Kaplan,A.S. 1973.Abriefreviewofthe biochemistry of herpes virus host-cell interaction. Cancer Res. 33:1393-1398.

7. Lawrence, W. C. 1971. Evidence for a relationship

be-tweenequine abortion(herpes)virusdeoxyribonucleic acid synthesis and the Sphaseofthe KB cellmitotic

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