Mutations in the major DNA-binding protein gene of herpes simplex virus type 1 result in increased levels of viral gene expression.

0022-538X/83/090478-09$02.00/0

Copyright ©1983, AmericanSocietyforMicrobiology

Mutations in the Major

DNA-Binding Protein Gene

Simplex Virus Type 1 Result in Increased Levels

Gene Expression

of

Herpes

of Viral

PAULJ.GODOWSKIANDDAVID M. KNIPE*

Department of Microbiology and MolecularGenetics,HarvardMedical School,Boston,Massachusetts02115

Received1 March1983/ Accepted 7 June 1983

We have examined the effect oftemperature-sensitive mutations in the herpes simplex virus 1 DNA-binding protein gene on viral gene expression. We have

found thatatthenonpermissive temperature, thesynthesis of certain immediate early, early, and late viral polypeptides was greater in cells infected with the temperature-sensitive mutantsthan incellsinfected with thewild-type virus. This effect was independent of the requirement for this viral protein for viral DNA

replication. The alteredrate ofsynthesis of viral proteinswas duetoa thermola-bilegeneproduct. Cells infected with thesemutants atthepermissivetemperature and then shifted tothe nonpermissive temperatureexhibited enhanced levels of viral gene expression. The addition of actinomycin D at the time of the

temperature shift prevented the alteration in viral protein synthesis. Therefore, continuing transcription is required for this change ingeneexpression. Northern blot analysis of cytoplasmic RNA showed that the steady-state level of specific viral transcripts expressed from parental virus genomes was greater in cells infected by these mutants at the nonpermissive temperature. These results indicate that the major DNA-bindingprotein of herpes simplex virustype1actsas a negative regulator of viral gene expression by affecting the abundance of

cytoplasmic viral mRNAs.

Theregulation of herpes simplex virus (HSV)

geneexpression isacomplexprocessdependent onboth cellular and viral factors.Upon infection

of the host cell, a limited portion of the viral genome is transcribed by the host cell RNA polymerase IItoyieldimmediateearlymRNAs. Thesearetranslatedtoyield the immediate early proteins. These proteins alter transcription or

processing of viral mRNA such thata newclass

of mRNAs is produced. The proteins encoded by these mRNAs, the early proteins, are

re-quired for viral DNA replication. After viral DNAreplication, the synthesis of certain viral proteins is initiated, whereas the synthesis of otherproteins is enhanced. Theseare

collective-ly termed the late viral proteins (29).

The immediateearly proteinsaresynthesized atmaximal rateswithin afew hours after

infec-tion of the host cell. Thesynthesis of the imme-diate early proteins is reduced concomitantly with theincrease in synthesis of early proteins. Theinvestigation of the kinetics and the

require-ments for synthesis of viral polypeptides led Honess and Roizman (13, 14) to propose that earlygeneproducts negatively regulate

immedi-ate early gene expression. The specific early geneproducts involved in thisprocess werenot

identified. By analysis of immediate early

tem-perature-sensitive mutants, Watson and

Clem-ents(34) and Dixon and Schaffer (9) showed that inactivation of the infected cell polypeptide 4 (ICP4)genefunction led toincreased synthesis of immediate early RNA and proteins,

respec-tively. Thus, both immediate early and early gene productsmay negatively regulate

immedi-ateearlygeneexpression.

We have been studying the maturation and function ofICP8, the major HSV DNA-binding protein. This early protein accumulates in the cell nucleus where it bindstoviral DNA(19, 20). ICP8 is an essential function for viral DNA

replication (6). ICP8 bindsmoretightlyto single-stranded DNA thantodouble-stranded DNA in vitro (18, 25).

The complementation group 1-1 is likely to

contain mutants defective in ICP8. Mutants in thisgroup havelesions that map in or near the

ICP8 coding sequences (6, 37). These mutants

encodemajor DNA-binding proteins that exhibit alteredantigenic properties (37)orDNA-binding

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HSV DNA-BINDING PROTEIN AND GENE EXPRESSION

activities at the nonpermissive temperature (20).

In thisreport, we examinedthe effect of these

defectsinICP8 on viralgeneexpression.

MATERIALS AND METHODS

Cells, viruses, and inhibitors. The procedures used for propagating and titrating viral stocks have been described (19). Stocks of temperature-sensitive virus-es and wild-type viruses were titrated in parallel at 33°C. The origins of HSV type 1 (HSV-1) mP and mPtsHAl have been described (6, 12). HSV-1 strains KOS1.1 andKOS1.ltsl3(reference 15; L. Holland, R. Sandri-Goldin, J. C. Glorioso, and M. Levine, manu-script in preparation) were provided by R. Sandri-Goldin and M. Levine, University of Michigan, Ann Arbor. Strains KOS and KOStsC7 (27, 28) were pro-vided by P. Schaffer, Dana-Farber Cancer Institute, Boston, Mass. Sodium phosphonoacetate (PAA) was a gift of Abbott Labs, N. Chicago,Ill.PAA was used at 400,ug/ml,aconcentration which totally inhibits viral DNA replication in Vero cells (M. Quinlan and D. Knipe,unpublisheddata). PAAwasadded at thetime of infection and maintained throughout the incubation andlabeling period. Actinomycin D was obtained from Calbiochem, La Jolla,Calif.

Labeling ofinfectedcells. Theinfection of Vero cell monolayers at amultiplicity of 20 PFU per cell and the labeling of these cultures with [35S]methionine (New England Nuclear Corp., Boston, Mass.) were per-formed in constant-emperature water baths as de-scribed previously (19). The 15-min labeling period was terminated by submerging the flasks in an ice bath. The cellswerewashed 3 times with phosphate-buffered saline at 0 to 4°C, scraped with a rubber policeman, and collected by centrifugation. The cell pelletwassuspended in electrophoresis sample buffer (19)containing 1 mM phenylmethylsulfonyl fluoride. Proteins from these sampleswereanalyzed by electro-phoresis in 9.25% diallyltartardiamide cross-linked polyacrylamide gelsasdescribedpreviously(19). Por-tions of eachsample containing equal quantities of cell lysatewereloadedontothegels. Theautoradiograms shown and those used for tracing were exposures within the linear response range for the film. The labeling of cultures for 5to30 min also showedlinear incorporation of label into viral polypeptide bands (datanotshown).Autoradiogramswerescannedwith

a Joyce-Loebl microdensitometer, and peak areas

weredetermined withaKeufel and Esser planimeter.

RNAisolationandcharacterization. Vero cell

mono-layerswereinfected with the indicated HSV-1strains andmaintained in culture mediumcontaining 400 ,ug of sodium PAA per ml. At 6 h postinfection, the cells wereharvested, and cytoplasmic RNA was isolated by

amodification of the method of Berk et al. (4). The cellswerelysedwith1%NonidetP-40(Sigma Chemi-calCo., St. Louis, Mo.) in0.15 MNaCl-0.02M Tris-hydrochloride (pH 7.9). Nuclei were separated from

thecytosol fraction bycentrifugationat2,000 xgfor

10minat4°C.Thecytosol fractionwaspoured intoan

equal volume ofasolutionof7Murea,0.35 MNaCl,

10 mMTris-hydrochloride (pH 7.4), 10 mMEDTA,

and4% SDS.This wasextracted twicewithanequal volume ofphenol (saturated with0.15 MNaCl, 10 mM Tris-hydrochloride (pH 7.6), and0.5 mMEDTA) and one-half volume ofchloroform-isoamyl alcohol (24:1).

Nucleic acids were precipitated by the addition of sodium acetate to 0.2 M and 2 volumes of ethanol followedby incubationat-20°Cfor 12 h. Samplesof each RNApreparation (10 or20Fg) were denatured andsubjectedtoelectrophoresis in 0.8% neutral

agar-ose gelscontaining 2.2 Mformaldehyde asdescribed previously (16), except thatethidium bromidewas not

included in the gel. The RNA was transferred to

nitrocellulose (Schleicher & Schuell Co., Keene, N.H.)in lOx SSC (1xSSC is 0.15 M NaClplus 15 mM sodiumcitrate [pH7.0]) (31). The filters were prein-cubated in 50% formamide (Fluka, Hauppauge, N.Y.)-5 x SSC-0.02% Denhardt solution (8)-S50 mM potassium phosphate buffer (pH 6.5)-250 ,ugof yeast RNA perml-0.25% sodiumdodecylsulfate for 17 hat 42°C. Hybridizationswereperformedfor 48 h with 32p-labeled DNAprobes (5 x 106 cpm pernitrocellulose strip) under these same conditions. The filters were

washedtwice in 2xSSC-0.1% sodium dodecyl sulfate

at roomtemperaturefor 30 min andoncein 0.1x SSC-0.1% sodium dodecyl sulfate at 50°C for 1 h in a circulating water bath. Autoradiography was per-formed withKodak XRP-5 filmat room temperature

or at -70°C with intensifying screens. Plasmid pBamHI-A' (7)was agiftof R.Costa and E. Wagner, UniversityofCalifornia,Irvine. Thisplasmidcontains the HSV-1 BamHI A' DNA fragment (map

coordi-nates 0.255 to 0.266) inserted in the BamHI site of pBR322. PlasmidpSG18SalA(20)containsanHSV-1 Sall DNAfragment(map coordinates 0.385to0.419, equivalenttotheSail 0fragmentof HSV-1 strain F DNA[21])inserted in the Sall site ofpBR325. Plasmid pSG-1 (10) was generously provided by M. Levine, University of Michigan, Ann Arbor. This plasmid contains the HSV-1 EcoRI JKDNAfragmentinserted in theEcoRI site of pBR325. The BamHI SPfragment is contained within the EcoRI JKfragmentandarises from thejunctionof the L and S components ofviral DNA(21).

Plasmid DNAs were isolated by CsCl equilibrium density gradient centrifugation oflysates from plas-mid-containing cells (5). The HSV-1 DNA inserts in theseplasmidswerereleased from thevectorDNAby digestion with the appropriate restriction enzyme. Restriction endonuclease digestions were performed under conditions specifiedby the supplier (New En-glandBiolabs, Beverly, Mass.). The DNAfragments containingtheHSV-1-specific sequenceswere isolat-ed by electroelution from neutral agarose gels (23). These were labeled by nick translation (26) with [32P]dCTP(800Ci/mmol;AmershamCorp., Arlington Heights,Ill.)to aspecific activityof 5 x

107

to7 x

107

cpm/,lg.

RESULTS

Synthesisofviralproteinsincellsinfected with aDNA-binding proteinmutant.Themajor

DNA-binding protein, ICP8,encoded by the group 1-1 mutant tsl3 is thermolabilefor binding toviral DNA (20). Others have shown that group 1-1 mutants aredefective forviral DNAreplication (6,37;L. Hollandetal.,manuscriptin

prepara-tion). Viral DNAreplication enhances late gene

expression, and late gene products inhibit early

VOL.47,1983 479

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480 GODOWSKI AND KNIPE

B

39.50C

M ts ts

*A:i4

330C

M

t s

ts-

--.e.-,w

et'-r.

,:,... 1

-49Pk,, ~~~~I.I -!- .::4

..

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..:

FIG. 1. Comparison of theratesofsynthesis of viral proteinsincellsinfected with HSV-1 tsl3orts+virus in

the presence ofPAA. Cells were infected with tsl3 (ts) or KOS1.1 (ts+)virus ormock-infected (M)in the presenceof PAA. At the times indicated, the cellswerelabeledfor 15minwith 6,uCiof[35S]methionineperml.

Extractswereprepared, and the viral proteinswereanalyzed by gel electrophoresisasdescribed in thetext.An

autoradiogram of the gel is shown. Someviralpolypeptides (ICP)areindicated. Thetwoforms ofICP8, differing

intheirdegree of intramolecular disulfide bonding (17),arealso indicated.

gene expression (29). Thepurposeof this study

was to determine whether ICP8 affects viral

gene expression independently of the require-mentof thisprotein for viral DNAreplication.

We compared the amounts of viral proteins synthesized during pulse-labeling periods in cells infected with tsl3 or its wild-type parent,

strain KOS1.1. To control for any effect that DNA replication might have on viral gene

expression, the infected cultureswereincubated

withPAA, an inhibitor of the viral DNA poly-merase. Replicate cultures were infected with

KOS1.1 ts+ or tsl3 viruses and maintained in

mediumcontaining PAAat330 or39.5°C. At 1-h

intervals between 3to8 hpostinfection, the cells

werepulse labeled with [35S]methionine, and the

labeled viral polypeptides were analyzed by

electrophoresis and autoradiography. At 39.5°C, cells infected with tsl3 synthesized increased

amountsof certain viralproteins relative tothe cells infected with thets+ virus (Fig. 1A). These included the early ICPs 6, 8, and 36 and the late ICPs 5, 11, 15, and 25. In addition, the

immedi-ate early ICP4 was synthesized in greater

amountsandatlater timesin cellsinfectedwith tsl3. At the permissivetemperature (33°C), the

amounts of viral proteins synthesized in cells infected with tsl3 and thets+ virusweresimilar (Fig. 1B). Thus, under conditions in whichviral DNA replication was uniformly inhibited in all cultures, the effect of thetemperature-sensitive mutationwastoincrease the level ofexpression of several viralgenes.

From this experiment, we quantitated the

magnitude of the difference in thepeakratesof synthesis of viral polypeptidesinthemutantand wild-type infected cells. By microdensitometry and planimetry of the autoradiograms (Fig. 1),

we determined theamount of labeled ICP8. At 39.5°C,theamountofsynthesisof ICP8 in cells infected with ts13 wasgreater at all times than that observed in cells infected with ts+ virus. Thepeak rate ofsynthesisof ICP8 (5 h postin-fection) was approximately threefold greater in

the mutant-infectedcells(Fig. 2A).At33°C,the

amount of ICP8 synthesized in cells infected with themutant and ts+ viruswas similar (Fig.

2B).

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HSV DNA-BINDING PROTEIN AND GENE EXPRESSION 481

A B

200

160

120

80

;40 =

0

IL_ ,,

0 c D

c

120-40

CL ~~~~~~a.a A C a a

HoursPostInfection

FIG. 2. Ratesofsynthesisof ICP8 in cells infected withDNA-negative mutants orts+ viruses. Infected cultureswerelabeledfor 15 minatthe timesindicated, and viral proteins were analyzed as in Fig. 1. The intensityof the ICP8 bandswasdeterminedby micro-densitometry andplanimetry. (AandB)Symbols: 0,

cells infected with tsl3; *, cells infected with ts+ virus. (C and D)Symbols:0,cells infected withtsC7; *, cellsinfected with KOS ts+. Cellswereincubated

at39.50C (A andC)or33°C (BandD).

Comparison of late gene

expression

by ICP8

mutants and replicating wild-type virus. The

expression

of HSV lategenes is- inhibited when

viral DNA replication is blocked (29). The

re-sultspresented abovedemonstratethatthe

inhi-bition of late gene

expression

from

parental

DNAmoleculeswasatleast

partially

overcome

by mutations in the

major

DNA-binding

protein.

It wasof interest tocomparetheamountof late

gene

expression by

ICP8mutantswiththatofa

replicating wild-type virus. Replicate cultures were infected at

39.5°C

with the ICP8 mutant

tsHAl (6,

20)

or its parent strain mP in the presence orabsence ofPAA. At 6 h

postinfec-tion, viralpolypeptide synthesiswas

analyzed.

In cultures incubated with PAA, the amount

of

synthesis

ofearly

polypeptides

(e.g., ICPs

6,

8, and 36), late

polypeptides

(e.g., ICPs

5,

11, 15, and 25) and the immediate

early

ICP4 was greater in cellsinfectedwith tsHAl

(Fig.

3,lanes 2and4). Thus, this ICP8 mutant also overpro-duced viral

polypeptides

at 39.50C. In cultures

incubatedwithout PAA, synthesis of early pro-teins and of the immediate early ICP4 was greater in cells infectedwith tsHAl than in cells

infected with the ts+ virus. However, synthesis of the late ICPs 5, 15, and 25 was greatest in cells

infected with the ts+ virus (Fig. 3). Thus, al-though the leveloflate gene expression by ICP8 mutants was greaterthan that observed with a nonreplicating wild-type virus, it was not as great as that observed in cells in which viral DNA replication was occuring. These results extendearlier studies by Conley et al. (6)

show-ingthat ICP8 mutants are defective in the

accu-mulation oflategene products.

Characterization of other mutantsdefective for DNA replication. To determine whether an in-creased level of viral protein synthesis was a general characteristic of all mutants defective

forDNA replication, we examined the kinetics

.sL

+5

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FIG. 3. Synthesisof viralproteinsincellsinfected with HSV-1 tsHAl or is+ virus in the presence or absence of PAA. At 6 h postinfection at39.5'C, the cellswerelabeled for 15 min with

(35S]methionine,

and viral

proteins

wereanalyzedasin

Fig.

1.Thesamples

shownwerefrom infection with tsHAl (ts)ormP(ts') with(+)orwithout(-)PAA.

VOL.47, 1983

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of protein synthesis in cells infected with the

HSV-1 mutantKOStsC7. Thismutanthas been shownto encode athermolabileDNA

polymer-ase, and it fails to replicate its DNA at the

nonpermissivetemperature(2). Asamemberof complementationgroup1-3,itcomplements the

ICP8 mutant tsHAl (36). At 39.5 or 33°C, the amount of synthesis of ICP8 directed by the mutant orthe wild-type virus was similar(Fig. 2CandD). The ratesofsynthesis of otherviral

proteins in cells infected with tsC7 andts+virus

werealsosimilar(datanotshown). Theseresults

indicatethat amutational block in DNA

replica-tion alone is not sufficient to account for the increased rates of viral protein synthesis de-scribed above.

Changes inviralprotein synthesisafter a tem-perature shift. The major DNA-binding protein

encoded by tsl3 and tsHAl is thermolabile for

binding

toviral DNAin vivo. TheICP8 encoded by these mutants is released from viral DNA

within 30minaftera shifttothenonpermissive

temperature (20). We determined whether the

genefunction involved inregulating the

expres-sion of viral genes could be inactivated by shifting cellstothe

nonpermissive

temperature.

CellswereinfectedwithtsHAl orts+ virus and

incubatedat33°C.At 4h

postinfection,

after the initiation of early viral

protein synthesis,

the cellswere shiftedto39.5°C. Infected cells were

labeledatvarious times after the shift. Immedi-ately after the shift to

39.5°C,

viral protein synthesis was similar in cells infected with tsHAlorts+ virus

(Fig.

4). At1hafter the shift

to 39.5°C, the rates of viral

protein

synthesis

were increased in cultures infected with either

virus(Fig.4).This increasewasalso observed in cultures left at 33°C (not

shown),

and thus this change wasnotrelatedtothetemperature shift.

The levels of viral

protein synthesis

did not

increase further in cultures infected with ts+

virus (Fig. 4). However, the synthesis of viral proteins didincrease further in cultures infected with tsHAl. By 3 h afterthe temperature shift, thesynthesis of

certain

viral

proteins

(e.g., ICPs

4, 5, 6, 8, and 11)was enhanced in the

mutant-infected cells. The synthesis of ICP8 was

ap-proximately threefold greater than in cells in-fected with ts+ virus. Synthesis of ICP4 was detected only in cultures infected with tsHAl

and only after3 to 5 h at 39.5°C. These results indicate that tsHAl encodes a gene product

which either is thermolabile or has a very short half-life. Thedeficiencyof thisproductresults in

increased ratesof viral protein synthesis. Simi-lar results were obtained in temperature shift experiments involvingtsl3 (data notshown).

To furtherexamine the mechanismby which

ICP8 affects viral gene expression, we deter-mined whether the alteration in viral protein

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hrc) ovlowino4 shift 5 FIG. 4. Synthesis of viral proteins in infected cells after a temperature shift. Cells were infected with tsHAl(ts) ormP (ts+) at 33°C in the presence of PAA. At 4 h postinfection, the cultures were shifted to 39.50C.Atthetimes indicated after the shift, the cells were labeled with [35S]methionine, and the labeled proteins wereanalyzed as in Fig. 1.

synthesis after a temperature shift could be blocked by inhibiting transcription. In this

ex-periment, cells were infected with tsl3 or ts+

virusandincubatedat33°C for5h.Actinomycin

D was added to one halfof the cultures. All cultures were thenshiftedto39.50C. Theratesof

synthesisofICP8 were determined atthis time orafter a 4-hincubation periodatthis tempera-ture. Cells infected with ts+ virus synthesized

similar amountsof ICP8

immediately

after and 4 hafter the shift to39.5°C(Table 1). Cells

infect-ed with tsl3 showed athreefoldincrease in the rate ofsynthesis of ICP8 after a4-h incubation

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HSV DNA-BINDING PROTEIN AND GENE EXPRESSION 483

TABLE 1. Effect of actinomycin D on the overproduction of ICP8 after a temperature shifta

Virus Actinomycin Amt of

ICP8b

D Oh 4h

tsl3 - 80 263

ts+ - 99 119

tsl3 + 76 53

ts+ + 64 41

aCellswere infectedat 33°C for 5 h. At this time,

actinomycin D was added to one half of the cultures to aconcentration of 10 ,ug/ml. All cultures were then shifted to39°C for either 0 or 4 h and labeled for 15 min with[35S]methionine at 39°C.

b Theamountof ICP8 in arbitrary units determined by planimetry. These values represent the average of triplicate cultures.

period at39.5°C. However,theaddition of acti-nomycin D tocultures infected withts+ ortsl3 virus caused the synthesis of ICP8tobereduced to 64 and 70%o, respectively, of the initial rate by 4hafterthe temperatureshift.Thus,continuing transcription is needed fortheenhancedratesof ICP8 synthesis in cells infected with tsl3 at

39.5°C.These resultsalsoindicate that the

func-tional half-life of the ICP8 mRNA is similarin cellsinfected with tsl3 and ts+ viruses.

Accumulation of viral mRNA. Theobservation thattheinhibitionof transcription prevented the increased rates of protein synthesis in

mutant-infected cells suggested that the ICP8 function

may affect the level of viral mRNA accumula-tion. Weassayed the level of

viral-specific

cyto-plasmic RNA from cells infected with the mu-tant orts+ virusby Northern blot

hybridization.

Ashybridization probes,weused cloned HSV-1

DNA fragments derived from regions of the

genomethatencode ICPs

4,

5,

or8. The BamHI SPDNAfragment

hybridizes

witha4.7-kilobase (kb)

transcript

that encodes ICP4

(1, 22,

35).This probe alsocontainssequences

homolo-gous tothe3.0-kbtranscript encodingthe imme-diate

early

ICPO (1, 22). The BamHI A' DNA

fragment hybridizes

to a major transcript that encodes ICP5 and to several minortranscripts

(reference

7; E. Wagner,

personal

communica-tion).

ThepSG18SalA DNA fragment hybridizes

to a

major transcript

of4.5kb. Invitro

transla-tion of mRNA that hybridizes to the pSG18SalA DNAfragment yieldsa polypeptidewith a

mo-lecular weight of approximately 125,000. This

polypeptide is immunoprecipitatedwith a

mono-colonal antibody directed against ICP8 (L.

Ra-field and D. Knipe, manuscript inpreparation). With the ICP8 mRNA probe, weobserved hy-bridizationto amajor transcript of4.5 kb as well aswithseveralminor transcripts.The amountof all these transcriptswasgreaterincellsinfected

with

tsl3

at39.50C than in

cells

infected with the

ts+ virus. At 33°C, the amount of these tran-scripts was greaterin cells infected with the

ts'

virus (Fig. 5). Similarly, theamountsof the 6.6-kb transcript detected with the ICP5 mRNA probeandthe4.7-kbtranscript detected with the ICP4mRNAprobeweregreaterin cells infected

with tsl3 at 39.50C (Fig. 5). With the ICP4 mRNA probe, we detected very low quantities ofthe3.0-kb mRNA that encodes ICPO in RNA isolated from cells infected with tsl3 or

ts+

virus.Therefore,wecouldnotdetermine

wheth-erthis mRNAwasalsooverproduced. Whenwe

compared the levels of

cytoplasmic

polyadeny-lated transcripts isolated from these cultures with these same probes, similar results were

obtained (P. J. Godowski and D.

Knipe,

data

notshown).Thus,mutations in the

major

DNA-binding protein result in an increased

steady

statelevel of the mRNA for ICPs

4,

5,

and8.

DISCUSSION

Wehaveshown that cells infected with certain

HSV-1 group 1-1 mutantsexhibit increased lev-els of viral geneexpression.The members of this groupcontain mutations thatmapinor near the

coding sequences of the major DNA-binding protein. These mutations lead to alterations in

thecellular localization,DNA binding activities, orantigenic properties ofthe major

DNA-bind-ingprotein(20, 36). It therefore seems likely that

the alterations in viral gene expression are a

resultof the defect intheDNA-binding protein. As reported previously, ICP8exerts apositive

effect

on late gene expression, probably by promoting viral DNA replication (6). However,

when viralDNAreplication was inhibited in all

infected cultures by incubation with PAA, the

DNA-bindingprotein inhibited the expression of

representative immediate early (ICP4), early

(ICPs6,8, and 36), and late (ICPs 5, 11, and 15)

proteins. Thus, under these conditions ICP8

acted as a negative regulator of viral gene

expression.

Wemeasured the rates of protein synthesis in

infected cells by determining the amount of incorporation of

[35S]methionine

into viral

poly-peptide bands

during

a short pulse

period.

The

altered levelsof viralgeneexpressionobserved

in cells infected with ICP8 mutants probably

reflect true differences in the amount of synthe-sis ofthese proteins because (i) viral proteins

accumulated to higher levels in these cells as

determined by Coomassie blue staining ofthe

gels (data not shown), and (ii) the level of cytoplasmicviral mRNA was increased in these cells. Thus, the differences in labeling of

poly-peptides are probably not explained by trivial

differences such as the uptake oflabelfromthe extracellular medium.

Role ofICP8 in the cascade regulation of HSV VOL.47,1983

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!CPS mr-4A

3

333°

39.5)

ICP4 nRINA

33'

'.39..

.5.

2.0-*S . ..' '

FIG. 5. Northern blot analysis of specifictranscripts from cells infected withwild-type or mutant virus. Samples of total cytoplasmic RNA from cells infected with tsl3 (ts) or KOS1.1 (ts+) at 33 or 39.5°C were

subjectedtoelectrophoresis informaldehyde-agarosegels and transferredtonitrocelluloseasdescribed in the

text.Todetect ICP8 mRNA, 20-,ugsamples ofRNA onthe blotwereprobed with32P-labeledpSG18SalADNA.

Autoradiographywasperformedat roomtemperature.TodetectICP5mRNA orICP4 mRNA, 10-,ug samples of RNA onblots were probed with labeledBamHIA' DNA orBamHlSP DNA,respectively. The size of themajor RNA species detected with eachprobe is indicated in kilobases ontherightof eachpanel. These sizeswere

determined relativetomigration of28S(5.2 kb)and18S (2.0kb)rRNAmarkersindicatedontheleft of each panel.

geneexpression. Intheir model forcascade

regu-lation of HSV gene expression, Honess and Roizman (13, 14) proposed that early

proteins

act as

negative

regulators of immediate

early

gene expression. We report evidence here that the ICP8 gene function affects the level of expression ofICP4, animmediateearlyprotein. Thus, this isthe firstearly genefunction tobe

identified that regulates the immediate early to

earlygenetransition.

ICP8is neededfor viral DNAreplicationand late gene expression. However, ICP8 inhibits

late gene expression from parental viral ge-nomes when viral DNA replication cannot oc-cur. Thelate genes encode the

majority

ofthe

virion structural polypeptides (29). Thus, this

functionprevents the synthesis ofvirion struc-turalproteins untilprogeny DNAmolecules are

available forencapsidation.

Mechanism of action of ICP8 on viral gene

expression. We have shown that theamount of

mRNA encoding ICPs 4, 5, and 8 is higherin cellsinfectedwithICP8mutants.Thus,the ICP8

functionregulates thecytoplasmicaccumulation

ofviral mRNAs. We did not detectadifference inthefunctionalstability of viralmRNAsin cells

infected with these mutants in the presence of

actinomycinD.These data suggest that the ICP8

function regulates the transcription or

metabo-lism ofnewly synthesizedmRNA. Alternative-ly,this result couldreflectaneedfor continuous synthesis ofa product that acts together with ICP8 to control viral geneexpression.

There are two general models to explainthe

effect ofthe ICP8functiononviral gene

expres-sion.Inthefirst, ICP8 inhibits theexpression of

the immediate earlygenes. Lower levelsofthe

immediate early proteins wouldthen cause

low-17 ,PR %.^-,p

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[image:7.492.114.401.78.387.2]

HSV DNA-BINDING PROTEIN AND GENE EXPRESSION

erlevels of expressionofallthe laterviral gene products. In this model, ICP8 would affect its

own expression only indirectly. In the second model, ICP8inhibitstheexpression of the

differ-ent viralgenes by a common mechanism. This

could be mediated by the bindingof ICP8 to viral

DNA or to mRNA transcripts, for example. In thismodel, ICP8affects its own expression more directly. This second model is supported by the observation that after a temperature shift, the expressionof several viral genes, including that encoding protein 4, increased coordinately. Ei-therofthe two models proposed could

incorpo-ratetheobservationthatafter a shift to39.5°C of cells infected with tsHAl or tsl3, ICP8 is re-leased from viral DNA (20).

Similar alterations in the level of viral gene expressionareobserved instrains of adenovirus that encode a defective 72K DNA-binding pro-tein. This protein, like the HSV-1 ICP8, is essential forviralDNAreplication (33) and has a high affinity for single-stranded DNA (30). The 72Kprotein plays amultifunctional role in

con-trolling adenovirusgeneexpression. Itrepresses the transcription ofearly region 4 (24) and has been implicated in controlling the stability of

early viral mRNAs (3). Mutations that alleviate

the block ofadenovirus lategene expression in monkey kidney cells map in the viral gene for

the 72K protein (17). The simian virus 40 T antigen, an essential protein for simian virus 40 DNA replication, alsonegatively regulatesearly

viral transcription. Both oftheseevents involve the binding of T antigen to specific sequences

near the origin ofreplication (11, 32). Further

studies are in progressto determine the precise

mechanism by which ICP8 affects HSV gene

expression.

ACKNOWLEDGMENTS

We thank LoriRafield for technical advice concerning the

Northernblothybridizations. We also thank Anne Spang and Margaret Levin for their assistance and Rosemary Bacco for thepreparationof the manuscript.

This work was supported by Public Health Service grant CA26345 from the National Cancer Institute. P.J.G. was a

predoctoral trainee supported by the National Institutes of Health training grant 5T32GM071%. D.M.K. is a Cancer Research Scholarof the Massachusetts Division of the Ameri-canCancer Society.

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