Herpes simplex virus thymidine kinase transcripts are absent from both nucleus and cytoplasm during infection in the presence of cycloheximide.

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0022-538X/80/1ll0361/05$02.00/0

Herpes

Simplex Virus

Thymidine

Kinase

Transcripts

Are

Absent from Both

Nucleus and Cytoplasm During Infection in

the Presence of

Cycloheximide

WAI-CHOI LEUNG,* KENNETH DIMOCK, JAMES R. SMILEY,AND SILVIABACCHETTI Department of Pathology,McMasterUniversity, Hamilton, Ontario, L8N3Z5,Canada

Nick-translated DNA from a recombinant plasmid containing the herpes

sim-plex virustype 1 thymidine kinase gene was used as a probe for the synthesis of thymidine kinase RNA. Therecombinantplasmidwas generated byinserting the 3.5-kilobase fragment derived by BamHI digestion of herpessimplex virus type 1 DNA into plaid pBR322. At 8 h after infection, cytoplasmic and nuclear RNA hybridized to 14% and 19% of the recombinant DNA probe, respectively. However, no ignificant hybridization was found with either nuclear or cytoplas-mic RNA extracted from cells infected and maintained in the presence of

cycloheximide.Thissuggeststhat nothymidinekinase-related RNA was

synthe-sized in the absence of a polypeptides, and supportsthe hypothesis that the a

polypeptides effect new thymidine kinase RNA synthesis rather than being

involved in processing or transport of thymidine kinase RNA. In cells infected andmaintained in the presence of the arginine analog canavanine, about 2 to 3% oftheplasmid DNA was found to hybridize with cytoplamic and nuclear RNA. However, when arecombinantplasmidDNAcontaining only thymidine kinase coding sequences was used, noscanthybridization was found. The inhibition ofthymidine kinase transcription by canavanine suggests that thymidinekinase

belongstothe

,B2

kineticclass.

Herpes simplex virus (HSV)-specific thymi-dinekinase (ordeoxypyrimidine kinase)belongs

tothe

f,

(orearly) kinetic group of virus-infected cell proteins since its expression requires

func-tional a (or immediate early) polypeptides (8,

15, 18, 20a).Theaction of the a polypeptides has been shown to occur at thetranscriptional level (15, 17). Inaproductive infection, both a

poly-peptidesandcellular DNA-dependent RNA

po-lymeraseIIarerequired fortranscription of the thymidine kinase gene (5, 16).

The aim of thisstudy was to determine the

mode of thymidine kinase gene transcription

effected by a polypeptides. Two different

hy-pothesesforthymidinekinase genetranscription

wereexamined. The firsthypothesispostulates

that thefunctionof apolypeptidesis toinitiate

new thymidine kinase-specific RNA synthesis.

Thishypothesisrequires that in the absence of

apolypeptides, suchaswhen cells areinfected

and maintained in the presence of cyclohexi-mide, no transcription of the thymidine kinase gene occurs. The second hypothesis assumes

thatthefunctionofapolypeptides is at the level

ofprocessing ortransport ofthymidine kinase

transcripts.Inthe absenceofapolypeptides,the

thymidine kinase RNA is transcribed but not

processedortansportedas functional mRNA.

Previous reports can be interpreted to suit

either of the above models. There is general

agreement that when cells are infected and maintained in the presence ofcycloheximide,

only the a mRNA appears in thecytoplasm (2,

3, 11, 22, 23). However, thegenetic complexity

of the nuclear RNAsynthesizedin the presence

ofcycloheximideisnotclear. Clementsetal. (3)

have utilized the Southern blot technique to

examine transcriptionof theHSV genome.

Ra-dioactive nuclear and cytoplasmic RNA

ex-tracted from infected BHK cells treated with

cycloheximide were hybridized to HSV DNA

fragmentsboundtonitrocellulosefilters.Similar

genetic complexitieswere found for both types

of RNA. These resultssuggest that in the

nu-cleus of infected cells treated with

cyclohexi-mide, RNAtranscripts other than those of the

a RNAs are absent, which indirectly supports

the "newRNAsynthesis"

hypothesis.

However,

late in virusinfection, symmetric

transcription

andsequestrationof viral RNAsequencesinthe

nucleus havebeendemonstrated(1,9,10, 13, 14,

20). In a more detailedstudy, Jones and

Roiz-man (12) examinedthekinetics of

hybridization

of radiolabeled viral DNA probes with excess

unlabeled RNA

by

liquid

hybridization.

They

found that viral RNA

accumulating

in the

nu-cleusandcytoplasmof

HEp-2

cells infected and maintainedinthepresence of

cycloheximide

was 361

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362

homologous to 33 and 12% of viralDNA, respec-tively. The thymidine kinase gene locus, at about 0.3physical map units, was carried by the

BglII

Mand HsuI Alfragmentsused in thestudy.Of the DNAcontained in theBglI M andHsulAI fragments, 0.5% and 1.4%, respectively, were

found to be homologous to the nuclear RNA extracted from cycloheximide-treated cells. Whetherthesetranscriptsrepresenta gene

se-quences or B and y gene sese-quences isnotclear. However, inorder fora,B genetranscripttobe expressed inthecytoplasn,newRNAsynthesis

isrequired (20). These resultscanbeinterpreted

tosuitthe"processing" hypothesis.

In thestudies citedabove,resolution of HSV transcripts detectedwasdefinedbythe

restric-tionendonuclease fragmentsusedas

hybridiza-tionprobes.Very often it isnotclear whethera

given fragment contains genes ofonly one or

more than one kinetic class. The aim of this study was to more clearly define the mode of transcriptionalcontrol,andwehaveconsidered

thymidine kinase as a representative of the 8

protein genes.Wigleretal.(24) have shown that

a3.5-kilobase BamHIfragment of HSV type 1

(HSV-1) DNA carries the viralthyniidinekinase gene. The DNAs from two recombinant

plas-mids were used as hybridization probes to

ex-amine transcription of the thymidine kinase genes.Theplasmid pTK1contained the BamHI fragment inserted into theplasmid pBR322. A

PstI fragment which included only sequences

coding for thymidine kinase was contained in the secondplasmid,pCl. Theuseof DNAsfrom these recombinant plasmids as hybridization

probes avoids possible cross-contamination which

might

have resulted fromseparationand elution of DNAfragments bygel electrophoresis.

Recently,HSV ,Bpolypeptideshavebeen

sub-classified into two groupson the basis of their

expressionin thepresencesof thearginine

ana-logcanavanine (17). The ,B1 group of

polypep-tides is expressed when cells are infected and'

maintained in medium containing canavanine; the

fP2

group ofpolypeptides is not. Attempts using pCl plasmid were also made in this study

todetermine whetherthe HSV-1 thymidine

ki-nasebelongsto the

f,l

or,82

group.

MATERIALS AND METHODS

Infection ofcells.Confluentmonolayers of human epidermoidcarcinomano. 2 (HEp-2)celLswereused.

Cellsin

150-cm2

tissuecultureflasks (Corning) were infected withHSV-1,strain KOS, at v multiplicity of infection of20PFU/cell.The virus was adsorbedfor

30min at37°C, and unadsorbed virus was decanted. Untreated cell cultures were fed with 20 ml ofEagle minimal essential medium F-17(GIBCO, Burlington,

Canada) containing 2% fetal calf serum. The canavan-ine-treated cell cultures were fed with medium supple-mented with 2.8 mM canavanine (Sigma, St. Louis, Mo.), andcycloheximide-treatedcellcultures were fed with medium supplemented with 200,tg of cyclohexi-mide(Sigma) per ml. The cells were incubated foran

additional 8 h at 37°C and then harvested.

RNAextraction. Cells were harvestedbyscraping themonolayers fromflasks with a rubber policeman, washed three times in ice-cold phosphate-buffered saline, and lysed in 1% Nonidet P40-10 mM Tris-hydrochloride (pH 7.4)-150 mM NaCl for 4 min at 4°C.Thecytosoland nuclearfractions were separated by centrifugation at8,000 x gfor 10 min at 4°C. The nuclearpellets were resuspended in the above buffer. To both the cytosol and nuclear fractions, proteinase K, EDTA, and sodium dodecyl sulfate were added to final concentrations of 250 Ag/ml, 5 mM, and 0.5%, respectively, and samples were incubated at 370C for 30 min. The cytosol andnuclear fractions were ex-tractedrepeatedly with equal volumes of phenol-chlo-roform(1:1) until no protein interphase was visible. Phenol was extracted from the aqueous phases with ether. Two volumes of ethanol was added, and nucleic acids were precipitatedovernightat -20°C. The pre-cipitateswerecollectedby centrifugation at 16,000 x g for 30 min at 40C and washed twice with 70% ethanol-0.1 M NaCl. Thepelletswere suspended in 3 mMMgCl2-10 mM sodium acetate (pH 5.1) and di-gestedwith 20lAgofRNase-free DNase (Boehringer-Mannheim)perml at 37°C for 30 min. The samples were deproteinized by proteinase K digestion and phenol-chloroform extraction, followed by ether ex-traction. RNA was then precipitated with ethanol overnightat-200C.The RNAprecipitates were col-lected bycentrifugation,washed, and stored at -50QC in70%ethanol until used.

Constuction of recombinant plasmids con-taining either the entire or partial thymidine kinase gene. The construction of recombinant plas-midspTK1 (7) and pXl (6) has been described. Both plasmids carry a 3.5-kilobase BamHI fragment of HSV-1 DNA, derived from strains KOS andcl.101, respectively, and contain the thymidine kinase coding sequences (Fig. 1A). They differ from one another, however, in that the viral DNA fragment is inserted withoppositeorientation into the bacterial vector.

The entire thymidine kinase gene is contained within the largestPvull subfragment of the HSV-1 BamHIfirgment (4; Fig.1A), and the thymidine ki-nase-transforming activity of HSV-1 DNA is inacti-vatedby cleavage with EcoRI (24). Thus it islikely that the PstI C fragment of pXl or pTK1 DNA consistsentirely of thymidine kinase coding sequences. Consistentwith thisinference, viable thymidine ki-nase-deficient deletion mutants ofHSV-1,lacking only thisfragment, have been obtained (20a). Accordingly, the PstI Cfragmentderived from pXl was inserted at thePstI site onpBR322 to yieldpCl (Fig.1B).This plasmld wasshown tocontainasingle insertof the PstI Cfragmentbyanalysisof the fragments produced bycleavagewithPstIandEcoRIand double digestion withPvuIIplusHindIII.

Nicktranslation of DNA. Radioactive labeling of pTK1 or pCl DNA in vitro was carried out by nick

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HSV THYMIDINE TRANSCRIPTION 363

RESULTS

The nuclear and cytoplasmic RNA harvested

at8hafterinfectionwerehybridized with

nick-translatedpTK1 plasmid DNA. As shown inFig.

2A, virus-specific RNAwaspresentin boththe

nucleus and cytoplasm of untreated infected

t cells.The cytoplasmic andnuclear RNA

hybrid-ized with 14% and 19% of the pTK1 DNA,

re-spectively. Assuming asymmetric transcription, these saturation levels suggestthat about 70% and 100%of the HSV-1 BamHI thymidine

ki-naseDNAfragmentis representedin

cytoplas-mic andnuclear RNA, respectively. No

signifi-cant amount of hybridization was found with

RNA extracted frommock-infected cells (data notshown).Similarly, in cells infected and

main-PU 11I

B) cogBomHI

RI

pCi

FIG. 1. Diagram of recombinant plasmids pXl andpCI. (A)pXl consistsofthe HSV-1 BamHI 3.4-kilobase fragment (dark portion) inserted into pBR322(white portion). The directionof transcrip-tionofthethymidinekinase(TK)gene is indicated bythearrow(22).PlasmidpTKI contains thesame BamHIfragmentbut inserted in theopposite direc-tion.(B)pCIconsistsofthe PstI Cfragment frompXl inserted intopBR322.

translation essentially as described by Rigby et al. (19), except that final concentrations of 25 mM potas-siumphosphate, 10 ,uM dT'T, 10uMdATP,and 20

AM

dGTP were used. The specific activity of the

[aeP]dCTP-labeled

DNA was about 2 x108 cpm/pg. liquidhybridization.The DNA-RNA hybridiza-tion wasperformedaccording to Jones and Roizman (12)with slight modifications. The reaction mixture contained5mgofnuclear or cytoplasmic RNA per ml,

0.5ngof heat-denatured [3P]DNA probe per ml, 0.04 Msodium phosphate (pH 6.8), and 0.23 M NaCl in a finalvolume of 20p1.The reaction mixture was incu-bated at 75°C for varying lengths of time. A 15-pl

sample of the reaction mixture was then withdrawn and diluted to 200

i1

in25 mM sodium acetate (pH 4.8),0.15MNaCl,1 mMZnSO4,and 50pgof single-strandedOX174DNAper ml. One half of thereaction mixture was digested withSinuclease for 1 h at 37°C. Thetrichloroacetic acid-precipitable counts per min-uteof theSInuclease-digested and undigested reac-tion mixtures were then measured. The results of hybridization were expressed as percent of DNA in hybrid and have been corrected for the self-association of the DNA probe, which was about 2 to 3% of the input counts per minute.

a

CL

m

z

< 20

z

a IL 10

0

I.-z

CL 20

10

400 800

1200

[image:3.499.47.241.59.350.2]

Rot

FIG. 2. Hybridization of nick-translated 32P-la-beledpTKI DNA tocytoplasmicand nuclear RNA

from virus-infected ceUs. Thefiguresshow the

frac-tion of32P-labekd pTKI DNA probe driven into

RNA-DNAhybrids by nuckarRNA (0) andby

cy-toplasmicRNA(0) as afwnction ofRNA concentra-tionandtime(Rot- molesofnuclebtidesxsecond

perliter). (A) Untreatedinfected cells; (B)

cyclohexi-mide-treated infected cells; (C) canavanine-treated

infected ceUs.

-B)

C)

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[image:3.499.274.424.256.571.2]

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364

tained in thepresenceofcycloheximide, no de-tectablevirus-specific RNAwasfound in either the nuclear or cytoplasmic fraction (Fig. 2B), suggesting thatunprocessed nuclear RNA

tran-scripts donotaccumulate in the absence of viral protein synthesis.

When the virus infection was initiated and maintained in thepresenceofcanavanine, both thenuclear andcytoplasmicRNAhybridizedto 2 to 3% of the pTKl plasmid DNA (Fig. 2C). The BamHIfragment istwo tothree times the size of the DNAsequencescoding for thymidine kinaseand is expectedto contain atleast

por-tions ofother viralgenes. Whetherornot this small amount of hybridization to RNA from canavanine-treated cellswasduetothepresensce

ofnon-thymidine kinase sequencesinthe

tran-scripts wasascertainedbyconstructingaDNA

probe which contained only thymidine kinase

sequences.ThePstICfragment (0.8kilobase)of pXlis located within thethymidinekinasegene

and probably contains most of the thymidine

kinase codingsequence (Fig. 1). Thisfragment

wasexcised from the pXlplasmid and recloned into pBR322. The DNA from theresulting plas-mid, pCl, was then used as a hybridization

probe.

Asshown in Fig.3C,nosignificant

hybridiza-tion was found when using the pCl DNA to

probe the nuclear and cytoplasmic RNA from cells infected and maintained in thepresenceof canavanine. The lack of hybridization to the thymidine kinase codingsequences suggeststhat thymidine kinase transcription did not occur.

Likewise,nosignificant hybridizationwasfound

withRNA from cycloheximide-treatedcells (Fig. 3B). As acontrol, in untreatedcells8h postin-fection, the cytoplasmic and nuclear RNAswere

foundtohybridize,respectively, with 7% and 8% ofthepCl DNA (Fig. 3A).

DISCUSSION

This study concerns the molecular controls that HSV a proteins might have onthe

tran-scription of the thymidine kinasegene, a repre-sentative of the

f

protein genes. The a proteins canpossibly act on the inhibition of new RNA

synthesis or on RNA processing or transport.

The latter hypothesissuggests that transcription ofRNA occurs in the absence of functional a

proteins,whereas in the new-RNA-synthesis

hy-pothesis, there is no RNA transcription. When weused eitherpTKlorpCl DNA as probes in

liquid hybridization, no RNA sequences were

detectablein either the nucleus or cytoplasm of

cells infectedand maintained in the presence of

cycloheximide. This result is best

accommo-dated by the hypothesis that the a proteins

effect newRNAtranscription from the

thymi-10o

5F

a

m

z

10 z

a 5

0

z

XU

5

-B)

C)

I l59=00-Ina Of0 Yp1e

400 800

'1200

Rot

FIG. 3. Hybridization of nick-translated 32P-la-beledpCI DNA to cytoplasmic and nuclear RNA

from virus-infected cells. The figures show the frac-tionof 32P-labeled pCI DNA probe driven into RNA-DNA hybrids by nuclear RNA (0) and by cytoplasmic RNA(0)as afunction of Rot. (A) Untreatedinfected

cells; (B) cycloheximide-treated infected cells; (C) canavanine-treatedinfected cells.

dinekinasegene.A second possibility is that the

thymidine kinase-specific RNA synthesized in the presence of cycloheximide is quickly

de-graded, andapolypeptides functiontostabilize

thymidine kinase RNA. On the basis of the presentdata,we areunabletoexclude this

pos-sibility. However, even if degraded thymidine

kinase RNA is not able to direct synthesis of thymidine kinase protein,aconsiderable degree

of hybrid formation with the plasmid DNA probe would be expectedtooccur.The

molecu-lar probes used in these experiments are

esti-matedtobeabletodetectatleastone copyof

thymidine kinase RNA per infected cell. We

believethat the lack of significant hybridization between RNAfromcycloheximide-treatedcells

andthe DNAprobes is best explained byablock

atthe level of transcription.

A)

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[image:4.499.284.441.57.400.2]

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The geneshavebeen subdivided into,1and ,B2groupsaccordingtotheir sensitivityto inhi-bitionby canavanine (17). The fl1groupof

poly-peptides, butnot,82,is expressed in thepresence

ofcanavanine. WithpTK1 DNA usedas aprobe

in liquid hybridization, a small but consistent

amountofhybridizationwasfound. The pTK1

DNAcontains the total thymidine kinase gene

sequence aswellasothernon-thymidine kinase codingsequences.Whenthe pClplasmid, which contains only thymidine kinase coding

se-quences,wasused,nosignificantamountof

hy-bridizationwasfound.This result suggests that

the transcription of the thymidine kinase gene

wasinhibitedby canavanine, andthymidine ki-nase cantherefore beclassifiedasamember of

the,B2 group.Thesedataareinagreementwith

the previous observation that expression of the thymidine kinase polypeptide is inhibited by canavanineinthecistron-specific enzyme-form-ingassay(15).

ACKNOWLEDGMENTS

This studywas supported by grantsfrom the National CancerInstitute of Canada.W.-C.L. isaMedical Research

CouncilScholar,K.D.is aMedical ResearchCouncil Postdoc-toralFellow,andJ.R.S. and S.B.areNational CancerInstitute ofCanadaScholars.

We are indebtedto Maria F. K. L.Leung forexcellent technical assistance and to William E. Rawls forcritically reviewingthemanuscript.

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