Effect of interferon on replication of herpes simplex virus types 1 and 2 in human macrophages.

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0022-538X/86/100037-06$02.00/0

Effect of Interferon

on

Replication of

Herpes

Simplex Virus

Types

1

and 2 in

Human

Macrophages

INGRID DOMKE-OPITZ,* PETRA STRAUB, AND HOLGER KIRCHNER

Institiute ofViruis Research, German Cancer Research Center, Heidelberg, Feder-alReputblic

of

Germany

Received 31 January 1986/Accepted 12 June 1986

Macrophages derived from human peripheral blood and cultured for 1 week were permissive for the

replicationof herpes simplex virus (HSV) types 1 and2. Low titers of interferon (IFN) were producedafter

virus infection. The yield of infectious virions was reduced by pretreatment of cells with natural and

recombinantIFN-otand naturalIFN-i.Recombinant and naturalIFN-yexhibitedverylow antiviral activity.

Treatment ofcells withIFN-ymixed with IFN-otorwith IFN-I didnotresult inasynergistic inhibition of virus

yield. We studied the synthesis of HSVtype 1- and HSVtype2-coded proteins in macrophages treated with

IFN-,. Induction of the HSV "-protein DNA polymerase was strongly inhibited in IFN-treated cells in a

dose-dependentmanner.As shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, otherI-and

-y-proteins of HSV were inhibited as well. Immunofluorescence studies revealed a strong inhibition of the

expression of immediate early a-protein ICP4. The results indicate that IFN acts early during the viral

replicationcycletoinhibitthe synthesis of HSVa-and IT-proteins.

Macrophages belong to the nonspecific defense

mecha-nisms and are known to be activated rapidly during a

primary infection with herpes simplex virus (HSV) (15). The role of mononuclear phagocytes in defense against HSV infections has been studied extensively in experimental

animals. Althoughmouse macrophages arecapable of virus

phagocytosis and of restricting virus infection by the

pro-duction of interferon (IFN) (16), they also replicate HSV

underappropriate culture conditions (4, 23).

HSV has been isolated from leukocytes of human patients

(12). In several studies,targetcells have been identified. The

virus has been shown to replicate in Epstein-Barr

virus-stimulated B cells (17) and in mitogen-stimulated T cells

expressing Ta antigen (3). HSV infection of fresh blood

monocytes leads to an abortive infection (28). However, after several days ofin vitro culture and differentiation into

macrophages, monocytes support virus replication (21). It

hasbeen shownby morphologicaland functional studies that invitro differentiation of human bloodmonocytesresembles in vivo differentiation of human peritoneal macrophages

(13). Thus,it appearsthat differentiated tissue macrophages

might replicateHSV andtherebycontributetodissemination

of the virus. Thispromptedusto testthe effect of IFNonthe

synthesis of infectious virions and viral proteins in human

macrophages.

MATERIALS AND METHODS

Cell cultures. Human peripheral blood mononuclear cells

were obtained by Ficoll-Hypaque density gradient

centrifu-gation (1) from buffycoats ofhealthy donors. Mononuclear

leukocytes were seeded at aconcentration of4 x 106/ml in

35-mmpetridishes(BectonDickinson andCo., Heidelberg,

Federal Republic of Germany) in medium consisting of RPMI 1640 (GIBCO Europe GmbH, Karlsruhe, Federal

Republic of Germany) supplemented with 10%

heat-inactivated, pooled human AB serum (Flow Laboratories,

Meckenheim, Federal Republic of Germany), minimal

es-sential medium vitamin solution (1:100), L-glutamine (2

* Correspondingauthor.

mM), sodiumpyruvate(1 mM), HEPES

(N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid) buffer (10 mM),

peni-cillin (100 IU/ml), and streptomycin (100 ,ug/ml). After incubationfor 16 hat 37°C, nonadherent cellswere washed

offby vigorouspipettingwith Hanksbalanced salt solution.

Theremainingadherentcellswerefurther cultured inRPMI

1640 medium containing 2% pooled human AB serum and thecomponents listed above. The mediumwasreplacedon alternate days. During in vitro culture, blood monocytes

were differentiated to macrophages. The cells increased in

size and assumed a spindle shape. After 6 days of culture, the numberof cellsperdishwasabout 5 x 105.The cultures

consisted of 100%macrophagesasshownby phagocytosisof latexparticles, byesterasestaining (18), and by

immunoflu-orescence assay with the monoclonal antibody OKM1

(Ortho Diagnostics, Inc., Heidelberg, Federal Republic of

Germany).

Herpesviruses and virus titrations. A pool ofHSV type 1

(HSV-1) strain WAL was prepared as previously described

(33). Primary isolates of HSV-1 (numbers 214 and 337)and

HSV-2 (numbers 184 and 311) were kindly provided by K. E. Schneweis (Institut fur Medizinische Mikrobiologie

undImmunologie, Bonn, FederalRepublicofGermany)and

werepassaged onceonRITA cellsof Africangreenmonkey

kidney origin (RC-37 RITA; Italdiagnostics, Rome, Italy).

Virus titrations were carried out by a plaque assay with RITA cells. Titers in macrophages were determined after

freezingandthawing. Cellswere storedat -70°Cuntilassay

of virus yield.

IFNs and IFN assay. Recombinant

IFN-ot,

(9.2 x 106

IU/mg) was a gift of C. Weissmann to H.K. Recombinant

IFN-aX2 (1 x 107 IU/mg) was kindly given to us by S.

Hiemstra (Essex Pharma, Munich, Federal Republic of

Germany). Natural IFN-ox(2.1 x 105 IU/mg)waspurchased

from Stratech Scientific Ltd. (London, United Kingdom).

Natural IFN-13 (5 x 106 IU/mg) was supplied by Serono

GmbH (Freiburg, Federal Republic of Germany). Native

IFN-y (8.3 x 106 IU/mg) was a gift of P. von Wussow

(Medizinische Hochschule, Federal Republic ofGermany)

toI. D.-O.Recombinant IFN-y (3.3x 107IU/mg)waskindly

provided by Biogen (Geneva, Switzerland). Forall

prepara-37

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TABLE 1. Replicationof different strains of HSV in human macrophages

Virus titers Virus strain" (PFU/ml)b HSV-1 WAL... 6 x 10' HSV-1 214... 6 x 104

HSV-1 337... 7 x 105

HSV-2 184... 8 x 103

HSV-2 311... 1 x 105

aMOI, 0.002.

hCellswerefrozenat 5daysafterinfection.

tions, the titers weredeterminedin our laboratorybeforeuse

in experiments. Titers of the IFN preparations were

deter-mined against a reference standard (no. 23-902-530; National Institutes of Health, Bethesda, Md.). IFN titers were

deter-mined in a system of HEp-2 cells challenged with vesicular

stomatitis virusaspreviously described(8). Cells werefixed and stained 24 h after infection with vesicular stomatitis virus.

IFNtreatment and virus infection. Afterbeing cultured for

6days,themacrophages wereincubated with IFNfor18 h.

Before virus infection, IFN was removed by washing the

cells several times.The cellswere inoculatedwith0.2 mlof

virus for 1 h at different multiplicities of infection (MOI). After virus adsorption, the cells were washed, and 2 ml of

RPMI 1640 medium containing 20% fetal bovine serum

(GIBCO) was added. All experiments were performed in

duplicate cellcultures.

Assayof HSV DNA polymerase. Viral enzymeactivitywas

determined in postmitochondrial supernatants of cell

ex-tracts as described previously (27, 29). At indicated time

points, macrophages were washed once with Hanks

bal-anced saltsolutionand once withcold extraction buffer(10

mMTrishydrochloride [pH8.01, 2 mM2-mercaptoethanol,

20% glycerol). Cells were scraped with a rubberpoliceman

into 0.4 ml ofextraction buffer and stored at -70°C. After

thawing, cellextracts werecentrifugedat 12,000 x gfor15

min at4°C. HSV DNApolymerase activitywas assayedby

measuringtheincorporationof[3H]TTP (specific activity,50

Ci/mmol) into trichloroacetic acid-insoluble material with

activated DNA as the template primer. For suppressionof

cellular DNApolymerases, KCl(0.2 M) was included in the

assay mixtures. Residual cellular enzyme activities are

in-cluded in the data. All enzyme assays were performed in

triplicate in the linear range of enzyme activity. Protein

concentrations were determined by the Bio-Rad protein

assay(2).

Analyses of HSV proteins. Human macrophages cultured

on35-mmpetri dishes were infected with HSV. At 7.5 h after

infection, the medium was replaced in Eagle basal medium

TABLE 2. Production of IFN by HSV-infected humanmacrophages

IFN concn(IU/ml)

Hafterinfectiona

ExptI Expt II

4 13 21

9 16 21

12 13 35

24 13 56

48 16 21

72 <3 21

aCellswereinfectedwith HSV-1 at an MOI of 2.

without methionine, to which 40 ,Ci of [35S]methionine per

mlhad been added(Amersham Buchler, Brunswick,Federal

Republicof Germany). The cellswereharvestedafter 90 min

ofpulse-labeling. Labeled proteins were subjected to

elec-trophoresis on 8% sodium dodecyl sulfate-polyacrylamide

gels (19). For determination of molecular weights, protein

markers (Sigma Chemical Co., St. Louis, Mo.) were

in-cluded.

Immunofluorescence. For immunofluorescence studies,

macrophages grown on slides were infected with HSV-1.

Theywere fixed with methanol-acetone at -20°C 6 h after

virus infection. Cells were incubated for 30 min at room

temperature with a mixture containing phosphate-buffered

saline, 1% bovine serum albumin, and 10% fetal bovine

serum to preventunspecific binding of antibodiesto the Fc

receptorsof macrophages. Afterbeingwashed in

phosphate-buffered saline-1% bovine serum albumin, the cells were

coated for 30 minatroomtemperaturewitha1:10dilution of

amonoclonal antibody againstHSV-1 ot-protein ICP 4(30).

Afterbeing washed, slides were labeled for 30 minatroom

temperaturewith fluoresceinisothiocyanate-conjugated Fab

fragment (Dianova GmbH, Hamburg, Federal Republic of

Germany) diluted 1:25. The cellswere examinedand

photo-graphed under UV-optics with a microscope (Dialux 20;

Wild-Leitz GmbH, Wetzlar, Federal Republic of Germany).

RESULTS

Replication ofHSV andIFNproduction in human

macro-phages. Human blood monocytes cultured for 1 week

sup-portedthereplicationof HSV. As shownpreviously (20,28) susceptibilitytoHSVincreasesduringinvitrodifferentiation

ofblood monocytes to macrophages after several days of

culturing (datanotshown).ThecellswerereplicatingHSV-1

aswell as HSV-2 strains (Table 1). The yield of infectious

virusparticles after infectionat an MOIof 0.002wassimilar

with the four primary isolates tested when compared with

laboratory strain WAL.

Macrophages werecapable ofIFNproduction after infec-tion with HSV at high multiplicity. However, only small

':0

2

0~~~~

C 0~~~~~~~~

0~~~~~~~~~

0 0

2

-1 2 3 4

log10 lU/miIFN

FIG. 1. Inhibition of HSV-1 replication by different types of IFN.CellsweretreatedwithnativeIFN--y(A), recombinantIFN-y

(-), recombinant IFN-ot, (0), recombinant IFN-a2 (0), natural IFN-ox(H), or natural IFN-,B (A) for 18 h and infected with HSV-1

at an MOI of 2. Cells were stored at -70°C at 24 h after virus infection.Virus yieldin untreated cultures was 2 x 106PFU/ml.

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amounts of IFN were produced (Table 2). IFN was detected

by4haftervirus infection.The capacity for IFN production varied greatly among blood macrophages from different donors.

Effect of differentIFNs on HSV replication. Wecompared

theantiviralactivities ofdifferent types of native and recom-binant IFNs in human macrophages (Fig. 1). Inhibition of

HSV-1 replication was dose dependent with natural IFN-a and

IFN-P

as well as withrecombinant

IFN-ao

andIFN-a2. However,the natural IFNsappeared to be more active than

the recombinantIFNs. Inhibition was most pronounced with

native

IFN-P.

Pretreatment of macrophages with 10 IU of

IFN-P

per ml resulted in90% inhibition of virus growth. In

contrast,

IFN-y

exhibited very low antiviral activity in

humanmacrophages. This result was obtained with natural

IFN--y and with recombinant IFN-y. Log10reduction ofvirus

titerswas about 0.7, evenafter treatment of cells with IFN

dosesas high as

104

IU/ml.

Additionally, we tested the effect of combinations of

different IFNs on the replication of HSV-1 (Table 3).

Syn-ergistic activitiesbymixturesof IFN--y and other IFNtypes

havebeendescribedforinhibition ofHSV in mouse

macro-phages (9) or cell growth inhibition (7, 10). However, we

could notfind apotentiation of antiviral activities ofIFN-a or IFN-1 by

IFN-y.

Effect of IFN on induction of HSV DNA polymerase. To

determine the stage of virus replication that is blocked by

IFN, we tested the effect ofIFN on the induction ofHSV

DNA polymerase. The virus-coded enzyme belongs to the

class of ,B-proteins and is expressed early during the viral

replication cycle. We tested the induction of the enzyme aftertreatmentofhumanmacrophages with different doses

ofnative

IFN-3

(Fig.2).HSVDNApolymeraseactivitywas

strongly reduced with IFN doses as low as 5 IU/ml. The

induction of HSV-2DNApolymerasewas moresensitiveto

IFN than was thatofHSV-1 DNA polymerase. After

treat-mentof cells withhighIFNdoses,theHSV-2-codedenzyme wasreduced nearlyto thecellular background level.

As described above, macrophages can be induced to

produce IFN. To analyze theeffect ofendogenous IFN on

theinduction of HSVDNApolymerase, wetreatedthecells

with

poly(I):poly(C).

Only moderate IFN titers were

mea-sured in the supernatants of poly(I):poly(C)-treated cells

(Table 4). Subsequent infection with HSV-1 or HSV-2

re-sultedinamarked inhibition of HSVDNApolymerase. The

degree of inhibition by endogenous IFN wascomparable to

that observed with exogenous IFN. Again, inhibition of

HSV-2 DNApolymerase was morepronounced.

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The effect of IFN on the kinetics of induction ofHSV DNA polymerase was analyzed in human macrophages

TABLE 3. Inhibition of HSVreplication by combinations of differenttypesofIFN

a

~~~~~~~~~~~Logl0

reduction of

IFNtype" IFN dose(IU/ml) virusyieldb

a 300 1.2

,B 300 2.1

y 300 0.3

+ ,8 150 + 150 2.0

a+y 150 + 150 1.3

+ -Y 150+ 150 2.1

aCells were pretreated with natural IFN-a, natural

IFN-P,

and natural

IFN-,yasindicated.

bCellswereinfected with HSV-1at anMOIof2 andstoredat-70°Cat24 hafter infection. Virusyieldin untreated cultureswas9 x105PFU/ml.

0

D"

_5.0

15

E

z

5 0

1 2 3

10910

lU/miIFN FIG. 2. Dose response of IFN for inhibition of HSV DNA polymerase. Aftertreatment withIFN-P, cells were infected with HSV-1 (O) or HSV-2 (0) at an MOI of 6. Macrophages were harvestedforassayofHSVDNApolymeraseactivityat9hafter infection. Residual cellularenzyme activity inuninfected cultures was1.834cpm/,Lgof protein.

infected with HSV-1 or -2 (Fig. 3). Kinetics of enzyme

inductionweresimilar with bothvirustypes. Enzyme

induc-tion wasdelayedin IFN-treated macrophages. Inuntreated

cultures, the first increase of viral enzyme activity was

observedat6 hafterinfection. Atthis time,DNA

polymer-ase activity in macrophages pretreated with IFN was

com-parable tocellularbackgroundlevels.

Synthesisof HSVproteinsinIFN-treatedmacrophages. As

shown by the results of the induction ofHSV DNA

poly-merase, IFN acted on

p-protein

synthesis. Sodiumdodecyl

sulfate-polyacrylamide gelelectrophoresiswas

performed

to

analyzethepatternof

synthesis

ofadditionalHSV

proteins.

Macrophageswere

pulse-labeled

with

[35S]methionine

at7.5 hafter infection. Proteinswereidentified

by

comparing

their

molecular weights with

published

data (24). The results

presented in Fig. 4 show a strong inhibition of HSV

P-proteins, namely ICP6 and ICP8. The synthesis of several

-y-proteins (ICP5, ICP11, and ICP25) were also inhibited in

IFN-treated cells. The resultsweresimilar for HSV-1 and -2.

We tried to analyze the effect of IFN on HSV

a-protein

synthesis by overinduction with

cycloheximide

and

actinomycin D. However, it was not

possible

to detect

a-proteins

on

gels.

In

addition,

detection of ICP4 was

complicated bya

comigrating

cellular

protein.

Therefore,

we

studieda-protein

synthesis by

immunofluorescence

by

using

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TABLE 4. Inhibition of HSV DNA polymerase by endogenous andexogenousIFN

DNApolymerase

IFNconcn activity(cpm/,ug

Treatment"l (IU/ml)b ofprotein)"

HSV-1 HSV-2

<3 15,140 16,067 Poly (I-C) (100 jig/ml) 60 11,432 6,039

Poly (I-C)(200 jig/ml) 100 6,958 3,298

IFN-P(100 lU/mI) 7,630 5,193

aPoly(I):poly(C)wasaddedfor 1.5 h, and the cells werewashedand further

culturedfor 16.5 h.

bSupernatantswereharvested beforeHSVinfection.

'Cellswereinfectedat anMOI of6and harvestedat8 hafterinfection.

Enzyme activity in uninfectedcultures was 1.235cpm/,lgof protein.

amonoclonal antibody against HSV-1 ICP4. About 40% of

thecells werepositive fortheexpression of ICP4at6hafter

infection(Fig. 5A). In contrast, after IFN treatmentonly4%

of the cells showed a fluorescent signal. All the results

_25

.',I

CL

0

o20

SO

E 9-26 15 1

15

z

10

10~~~~~~~~~~~~~~

U-.-- ---o

3 6 9 12 15 18

Hoursafter infection

FIG. 3. Kineticsof induction of HSV-1- and HSV-2-coded DNA polymerase inIFN-treated macrophages. Untreated cells(0,*) or cellstreatedwith103IUof IFN-,B per ml(0,O) were infected with HSV-1(0, 0)orHSV-2 (-, O) at an MOI of 6. Cells were harvested

at the indicated time points for assay of HSV DNA polymerase activity. Residual cellular enzyme activity in uninfected cultures was204

cpm/Vjg

ofprotein.

indicate that IFN inhibits HSVat anearly stage of the viral

replication cycle.

DISCUSSION

The present studywasperformed toanalyze the effect of

IFN onHSVreplication in human primary cells that might

play akeyrole in thecourseofanHSV infection. Linnavuori

and Hovi (21) have shownarestriction ofHSVreplicationin

undifferentiated blood monocytes by high titers ofIFN that

wereinducedafter virus infection. However, only low titers

ofIFN wereproducedafter HSV infection of differentiated

macrophages, and the cellswere permissivefor virus

repli-cation. Thus, the role of endogenous IFN induced by the

virus itself seems to be negligible in differentiated

macro-phages. However, if IFN was induced in macrophages

before HSV infection, it was veryeffective in

inhibiting

the

virus.

Humanmacrophages weresensitivetothe antiviral

activ-ity ofexogenous IFN-o and

IFN-P,,

whereas

IFN--y

exhib-itedonlyverylittleantiviral activity.Theeffectwas

macro-phage specific, because inhibition of HSV replication on

HEp-2 cellsbyIFN--ywascomparabletovirus inhibitionby

other IFN types(datanotshown). Inaddition, experiments

with human cell lines have revealed that similar

concentra-tions of

IFN-y

or IFN-ot or IFN-, were necessary for

induction of the antiviral state measured via induction of

(2'-5') oligoadenylate synthetase (32). A recent study

de-scribes differences in biological activitiesofIFN--yonhuman

monocytes and WISH cells; a comparison of the IFN-y

receptor on both cell typesrevealed different binding

char-acteristics (26). Thus, the lack of antiviral activity of IFN-Y

againstHSV on macrophages couldbe related to a different

receptor onthese cells.This is inagreementwith thefinding

that IFN-y is ineffective in potentiating the antiviral

activi-m HSV-1 HSV-2

ICP

Mol.wt. 205.

I 5

_ *6

,s

_s

*6

116 _ _ *11

97*

t *f-l..:

66-

_ _

mr.Tt>

._ mora

4

*25

FIG. 4. Analysis of HSV proteins in IFN-treated macrophages by sodium dodecyl sulfate-polyacrylamidegel electrophoresis. Un-treated cells (-) or cells Un-treated with1,000 IU of

IFN-P

per ml (+) wereinfectedwith HSV-1 or HSV-2 at an MOI of 6. m, The protein pattern ofmock-infected cultures. Cells were pulse-labeled for 90 min with[35S]methionine, and each lane received an equal amount ofcell protein extracts. Molecular-weight markers and ICPs are marked.C, Amajorcellularprotein.

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FIG. 5. Effect of IFN on the expression of HSV-1 a-protein ICP4. Untreated cells (A) or cells pretreated with 1,000 IU ofIFN-Pper ml (B)wereinfected with HSV-1 at an MOI of 6. Cells were fixed at 6 h after infection and subjected to immunofluorescence with a monoclonal antibody against HSV-1 a-protein ICP4.

ties ofIFN-a or

IFN-P.

Weplan to analyze the induction of

enzymes supposed to be related to the antiviral state by

differenttypesofIFN inhuman macrophages.

Itwastheaim ofthis study to characterize the stage of the

viral replicationcycle that is mainly blocked by IFN. In our

experimental system, the synthesis of

P-

and -y-proteins

codedforby HSV-1or-2 wasclearlyinhibited. Inductionof

HSV DNA polymerase was delayed in IFN-treated cells.

This suggests that IFNmight effect HSV a-protein synthesis

becauseexpressionof,-proteins dependsonthe presenceof sufficient amounts of

at-proteins

(14). We have shown a

reduction in the a-protein level in IFN-treated cells by immunofluorescence with a monoclonal antibody against

HSV-1 a-protein ICP4. Similar findings were reached by

Gloger and Panet (11) withHeLa cells. Lipp and Brandner

(22) describe inhibition of HSVprotein synthesis inprimary

African green monkey kidney cells treated with poly(I): poly(C). Recent studies in ourlaboratory revealeda strong

inhibition of HSV-1

at-

and ,-protein

synthesis

in

IFN-treated splenic mousemacrophages (9, 31). Compared with

humanmacrophages, thesecells weremore sensitivetothe

antiviral activity of IFNagainst HSV. One reason mightbe

that the mouse macrophages werecultured in the presence

of colony-stimulating factor, which causes activation and

proliferation ofthecells.Nosimilargrowthfactor was used

in ourstudies of human macrophages.

Other studies have reached contradictory findings

con-cerningthe stageofHSVreplicationthatisblockedby IFN.

Munoz and Carrasco (25) as well as Chatterjeeet al. (5, 6)

claim that IFN inhibits alate stage duringthe viral replica-tion cycle.Whereas theformerauthors suggest that IFN acts

via the production of defective virions,the latterclaimthat

IFN inhibits the release of virus particles. Chatterjee etal.

(6) find noinhibition of HSVDNAsynthesisin IFN-treated

fibroblasts. Thesynthesis of normal levels of viral DNA in

IFN-treated cells should not be possible, given the strong

inhibition of HSVDNApolymerasedescribed in the present

studyand shown

previously

(9, 11). Comparingthe studies, it is

unlikely

thatdifferentIFNdosesor

preparations

arethe

reasonfor thecontradictory findings.Moreover,Glogerand

Panet(11)aswellasMunozand Carrasco(25)used thesame

cell line. Nonetheless, it cannot be denied that HSV

repli-cationis inhibitedbyIFN in different cell types

by

different

mechanisms.

Furtherstudies remaintobe doneto

analyze

theantiviral

activity ofIFN

against

HSV. We willcontinue this work

by

comparingthe

synthesis

of viral

transcripts

as well asviral DNA

synthesis

in IFN-treated

macrophages

and fibroblasts.

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The results of the prescnt study indicate that IFN blocks a

stepbefore HSV DNA synthesis. The mainblock during the

viral replication cycle occurs either during or before the

synthesis of immediate early(x-proteins.

ACKNOWLEDGMENTS

We thank M. Zweig for providing the antibody against HSV a-protein ICP4and H. Jacobsenfor helpful discussions.The excel-lent technical assistance of P. Hagendorn and the outstanding editorial work ofM. Kasamaschareacknowledged.

This research was supported by grant DO 301/1-1 from the DeutscheForschungsgemeinschaft.

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