Gene expression of herpes simplex virus. II. UV radiological analysis of viral transcription units.

0022-538X/80/06-0604/11$02.00/0

Gene

Expression

of

Herpes Simplex Virus

II.

UV

Radiological Analysis of Viral

Transcription

Units

ROBERT L.MILLETTE* ANDROSEMARIE KLAIBER

Department ofImmunologyandMicrobiology, WayneStateUniversitySchoolofMedicine, Detroit, Michigan 48201

Thetranscriptional organization of thegenomeofherpes simplexvirustype 1

was analyzedby measuringthe sensitivity of viralpolypeptidesynthesis to UV

irradiation of theinfectingvirus.Herpes simplexvirustype1wasirradiatedwith

variousdoses of UVlightandusedtoinfectxerodermapigmentosum fibroblasts. Immediate early transcription units were analyzed by having cycloheximide

presentthroughout the period ofinfection,removing the drugat8 hpostinfection,

and pulse-labeling proteins with [3S]methionine. Delayed early transcription unitswereanalyzed insimilarstudiesbyhaving9-fi-D-arabinofuranosyladenine

presentduringtheexperimenttoblockreplicationof theinputirradiatedgenome.

The viralpolypeptideswereseparatedby gel electrophoresisandquantitated by

densitometryofthegel autoradiograms. Thefollowingresultswereobtained. (i)

The UVtargetsizes for the viraltranscriptionunitsanalyzed rangedfrom 1.44 to 5.65kilobase pairs.Thisimpliesthat thecorresponding primary transcriptshave

minimummolecularweightsrangingfrom 0.46x 106to1.82x 106. (ii)Thegenes

for the four viralproteins, 165,145, 116, and 71(molecular weightx103),exhibited UV target sizes thatagreewiththeir calculatedgenesizeormeasured mRNAsize orboth and thusmustreside inpromoter-adjacentpositions. (iii) The

transcrip-tion units for theremaininggenesanalyzedshowedtarget sizesthatrangefrom 0.42to2.59kilobasepairsgreaterthan neededtoencode therespectiveproteins. Thisprobablyisareflection of their distances from promotersorthepresenceof

interveningsequencesorboth. It further suggests that thesegenesaretranscribed

asprecursorRNAmolecules thatarelargerthan their mRNA's.(iv)Theresults

indicate thatnoneofthe immediate earlygenesanalyzed canbecotranscribed,

whereassome of thedelayed early genes mightbe cotranscribed. No evidence wasfound fortheexistance oflarge, multigene transcriptionunits.

Previous studies on the characterization of

viral transcripts producedincellsinfected with

herpes simplex virus type 1 (HSV-1)

demon-strated that virus-specific transcripts found in

the nuclei ofinfected cells range from 10S to greater than 60S, whereas those found in the cytoplasm range from about 10S to 35S (31). This prompted the speculation that HSV mRNA's are synthesized as

high-molecular-weightprecursorsthatareprocessed into

lower-molecular weight cytoplasmic mRNA's (31). Subsequent sedimentation studies on HSV-1

transcripts selected by liquid hybridization

in-dicatedthatthenuclear transcriptshave onlya somewhatlargeraveragesize thanthe

cytoplas-mic transcripts (28). However,morerecent elec-trophoretic analyses of viral RNA on

methyl-mercury-agarose gels have shownthat early

nu-clearandpolyribosomalRNAshavesimilar size distributions, whereas late nuclear RNA has a considerably largeraveragesize than the

poly-somal RNA (9). Nevertheless, the true size of

the HSV-1primary transcriptsremainsin ques-tion since these studies have neither proven a precursor-product relationship between the

larger nuclear and smaller cytoplasmic HSV RNAs nor taken into account possible rapid

processing of larger nuclearprecursors.

Other recent studies have been directed

to-wardphysically mappingthe viralmRNA'sand

polypeptide coding sequences in the viral ge-nome. These haveutilizedliquid (13) and blot

hybridizationsof RNAtoHSV DNA restriction

fragments (3, 4, 9), R-loop mapping (29), and biochemicalanalysisofHSV-1xHSV-2genetic recombinants (18, 19, 21).Althoughthese stud-ies haveleadtothe mapping ofalarge number

of viralpolypeptides, RNAs,andfunctions, they

have notyetfully establishedthe number and precise location of promoters, the polarity of mosttranscription units, the existence of com-mon transcription units and intervening se-quences, orthesize andgenome mapcoordinates

of theviraltranscription units.

604

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TRANSCRIPTION

Toanswer some of thesequestionsregarding the

transcriptional

organization of the HSV-1 genome, we utilized the UVmappingtechnique developed by Sauerbier andco-workers (23,25). The principle of this method isthat the sensitiv-ityofexpression of a gene toUV irradiation isa function of the distance of that gene from its transcriptional promoter. The effect of UV ir-radiation onthe relative rate ofgene

expression

may be quantitated either directly, by

measuring

thesynthesis of specific mRNA's, or indirectly, bymeasuring the synthesis of the specific poly-peptides. From the UV sensitivity of expression of a given gene, one can calculate theUVtarget size,in base pairs, of its transcription unit. This method has been used successfully to analyze thetranscriptional organization of a variety of procaryotic, eucaryotic, and viral genomes (for review, seereference 24).

By using host cells lacking excision repair activity, xeroderma pigmentosum (XP) fibro-blasts, toprevent the repair of the input irradi-atedviralgenome, we have been able to analyze thesensitivity of

synthesis

ofalarge number of HSV-1

polypeptides

to UV irradiation of the infecting virus. The results of these studies pro-vide values for themuiimum sizes of viral tran-scription units and their primary transcripts and the distances of viralgenes from theirpromoters and

place

limitations on which genes may be

cotranscribed.

Moreover,they show that certain viral genesarepromoter adjacent, whereas oth-ers are considerably removed from their pro-moters and therefore must be transcribed as larger precursors.

MATERIALS AND MEETHODS

Virusandcelis.HSV-1,Flstrain(7),was

propa-gatedinHEp-2 (humanepidermoid carcinoma) cells

byamodification ofpreviouslydescribed methods(26,

27).ConfluentHEp-2cellmonolayers inrollerbottles

were infected at amultiplicity of 0.02 PFU/cell in phosphate-bufferedsalinecontaining0.1%glucoseand 1%inactivated calfserum (PBS-gs).Afteradsorption at37°Cfor 2h,the mediumwasreplacedwith medium

199containing 1% inactivated calf serum, and incuba-tionwascontinuedfor2 to3daysat34°C.The cells

werescraped, centrifuged,andresuspendedinmedium

199plus1%inactivatedcalf serum,using2.5to3ml of medium per roller bottle. The cellsweredisrupted by

sonication for 2 min in an ice bath with an MSE UltrasonicPowerUnitatmaximumpower. The cell debriswasremoved by centrifugation for 30 min at

2,000xg.Viruswaspurifiedfrom thecellextractby

centrifugation through lineargradients of10to50%

(wt/vol) sucrose in VB (0.15 M NaCl-0.02 M Tris-hydrochloride[pH 7.5])for1hat25,000rpmand4°C inaSpincoSW27rotor.The virusbandwascollected,

diluted threefold in VB, and

centrifuged

for 1 h at

25,000 rpm and4°C inan SW27rotor topellet the

virus.Thevirus, dissolvedinasmall volume of

PBS-gs,had a titer of 4x109 to 6x

10i

PFU/ml. Virus was plaque assayed on Vero (African green monkey kid-ney)cells and XP cells with an overlay medium of 1%

methylcellulose inDulbecco-modified Eagle medium (DME) plus 1% inactivated calf serum. Virus and HEp-2and Vero cells were obtained from B.Roizman, University ofChicago,Chicago,Ill.

XPcells (AmericanType CultureCollection,

Rock-vile,Md.; no. CRL 1223, XP 12BE) were used for all UV mapping experiments. They were propagated in DMEplus 10% inactivated fetal bovine serum (Flow Laboratories Inc.,Rockville,Md.)andpassaged ata1:

3or 1:4dilution every 3 or4days. HEp-2 cells were

propagatedinmedium 199 plus 10% inactivated fetal bovine serum in 32-ounce (ca. 0.946-liter)bottlesor in medium 199plus5%calf serum inrollerbottles; Vero cellswerepropagated in DME plus 10% inactivated fetal bovine serum. Both kinds ofcellswerepassaged

every3 to4days at a 1:7 to 1:9 dilution.

UVirradiation of virus. Mixtures consisting of 1.8x 109to5x 109PFU of sucrosegradient-purified HSV-1 and9 x

10'0

to12 x1010PFUof bacteriophage T7(included asadose indicator) in 2.0 to 2.5 ml of PBS-gswereirradiated with constant stirring in a

12-cmwatchglassonice. A UV lamp(Gelman Instrument Co., Ann Arbor, Mich.) that provided anoutput of 0.38to0.54

J/m2

per sat apeakwavelength of254 nm

anddistance of 34 cm was used. Theviruswas irradi-ated with dosesvarying from0 to67J/m2.Samples of 20 to 50

Id

wereremoved at various times and diluted into1 to2ml ofPBS-gsonice. The UVinactivation ofphageT7wasmonitoredby removing 10-to20-ul

samplesandtitrating onEscherichia coliB., (a

re-pair-minus strain from W. Sauerbier, University of Minnesota,St. Paul, Minn.). This servedasaninternal dosestandard with 1.8 J/ m2 giving one T7 lethal hit (1).

AfterUVirradiation, alloperationsinvolving irra-diated virus and infected XP cells were performed

underyellow lightorindarkness until termination of the

experiments

toavoidphotoreactivatonof the

UV-damagedDNA.

Infecting,labeling,andharvestingcells. Mono-layersof XPcells,85to99%confluent, wereprepared

in

9.6-cm2

cluster dishes. Thecellswereinfected with unirradiatedorUV-irradiatedHSV-1at20to50PFU/

cell in0.4ml ofPBS-gs for 1 h at 37°C. Viruswas

removed, and the cells were overlaid with 2 ml of DMEplus1%inactivated calfserumcontainingeither

noinhibitors,50

Ag

ofcycloheximide (Calbiochem,La

Jolla,Calif.) perml,or100 to 150

AM

9-.8-D-arabino-furanosyladenine(ara-A)and1

jg

ofCovidarabine (2-deoxycoformycin) per ml (both were gifts from H. Machamer,Parke,Davis &Co., Detroit,Mich.).At2 to8hpostinfection (timesafteraddition of virusto

cells),thecellswererinsed three times with DMEplus 1%inactivated calfserum (when

cycloheximide

was

used) andtwotimes withMEM-1/100Met-1% DCS

(minimalessential mediumplus1%dialyzedcalfserum

andcontaining1/100theusualamountofmethionine) and then labeled with 0.4 ml of MEM-1/100 Met

containing[3S]methionine(Amersham Corp.,

Arling-tonHeights,Ill.)at20to30

1Ci/ml

for45minat

37°C.

Thelabelingmediumwasthenremoved,andthecells

were rinsed two times with ice-cold

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bufferedsaline,lysedwith 0.2or0.3 ml oflysingbuffer

(2% sodium dodecyl sulfate,0.05 MTris-hydrochloride [pH 7.0], 0.7 M 8-mercaptoethanol, 5%sucrose, and bromophenol blue),and frozen at-90°Cuntil

electro-phoresis.

Polyacrylamide gel electrophoresis. Infected celllysateswerethawedand removed from theculture dishes with the aidofascraper,sonicatedbrieflyto reduce theviscosity,and heated for 2minat100°Cto denature proteins. Polyacrylamide slab gels (140 by

160 by 1.3 mm) were prepared essentially by the method ofLaemmli (15)but with0.36%

N,N'-diallyl-tartardiamide (Eastman OrganicChemicals, Roches-ter,N.Y.) as across-linker(8).Arunning gelof8.5%

acrylamideandastackinggel of 3.5or4%acrylamide

wereused. Proteinsamplescontaining 8,000to50,000 cpmof3Sin 20to50

pl

wereappliedandsubjectedto

electrophoresis for 6.5 to 7.5h at200Vand 12to 16 mA. Gelswerestained for 4hwith0.25% Coomassie

brilliantblue (SigmaChemicalCo.,St.Louis, Mo.)in

ethanol-acetic acid-water (5:1:5), destained with wa-ter-acetic acid-isopropanol (8:1:1), dried by heating

undervacuum onWhatman3MMpaper,andexposed toKodak X-Omat RP film for 3to20daysfor

auto-radiography. Polypeptideswerequantitatedby

scan-ning filmswithaZienah soft-laserscanning densitom-eter (Biomed Instruments, Inc., Chicago, Ill.) and

measuring peak heights.Studies withlabeledprotein standardsrun ongelsshowed thatpeakheightswere

directly proportional to thequantityofproteinlabel under theconditions used.

Molecular weightsofviralpolypeptideswere deter-mined from mobilities relativeto protein standards

(Escherichiacoli RNApolymerase, bovineserum

al-bumin,ovalbumin, immunoglobulin G,E. coli DNase

I, papain, trypsin, chymotrypsin, and pepsin). Viral

polypeptideswerenumberedaccordingtotheir molec-ularweightsinthousandsandrelatedtoICP

(infected-cellpolypeptide)numbers ofHoness andRoizman(10,

11).

Calculations. The percentage of the amount of eachpolypeptide synthesized relative to the unirra-diatedcontrolsamplewascalculated andplottedon a

semilog plotasafunctionofUVdose to theinfecting

virus injoulespersquaremeter.Linesgivingthebest fittotheequationln(Rd/Ro) =-kdwerecalculated

bythe curve-fitting program on a Hewlett-Packard

(HP 97)calculator. In thisexpression, Rdistherateof synthesis of the individual polypeptideatUVdose d injoulespersquaremeter,Rois therateofsynthesis

ofthepolypeptideata zero UVdose,and k is the

first-orderrate constantorUVinactivationcrosssection in

squaremetersperjoule.Theslopesofthelines were

equal to the UV inactivation cross sections, k; the

reciprocalofkprovidedtheUVdoseyieldingonehit, or37%inactivationofpolypeptide synthesis. TheUV

target size of transcription units in base pairs was

calculatedby using the value of2.30 x 10-2 m2/Jas

theUVinactivation crosssectionfor 1,000 base pairs ofDNAunderourconditions. This valuewasobtained

fromaplot oftheUVinactivation crosssections, k,

versusthesizesofthegenesfor the individual

poly-peptidesinbasepairs (seebelow,Fig. 5). The number ofbasepairsrequiredtoencodeeachpolypeptidewas

J. VIROL. calculated from thefollowing relationship:number of basepairs=molecularweightofpolypeptidex3/115,

where115equalstheaverage molecularweight ofan

aminoacid.

RESULTS

UV radiological analysis of early viral

genes.In the first series of UVmapping

exper-iments,

weanalyzedthesensitivityof

synthesis

ofHSV-1immediate early (IE) polypeptidesto UVirradiationofthe virus. The mRNA for the

IE proteins accumulates in the cytoplasm of

infectedcellsduringacycloheximide block,and itcanbeassayedbyitstranslationinto

polypep-tidesimmediatelyafter removal of thedrug(11,

14).

The virus was irradiated with

increasing

doses of UV light and then used to infect XP cells.Cycloheximidewaspresentfrom 1 h after

additionof virus andthroughoutthe period of

infection.At7or8 hpostinfection , thedrugwas removed, the cells were pulse-labeled with

[3S]methionine, and the labeled polypeptides wereanalyzed by polyacrylamidegel

electropho-resis andautoradiography.SixIEviral

polypep-tidescanreadilybedistinguishedby the

sensi-tivityoftheirsynthesis to UV irradiation of the

infectingvirus (Fig. 1). Thesearedesignatedas

viral polypeptides 165, 145, 123, 86, 71, and 55 in termsoftheir molecular weightsx 103 as deter-mined fromgel mobilities.Accordingtothe Ho-nessandRoizmannomenclature (10, 11), these presumablycorrespond tothe a and ,B

polypep-tidesICP 4, 6, 0, 20, 22, and 27. Insome

experi-ments, a delayed early (DE) polypeptide, 116, probablythe

ft

polypeptideICP 10,wasdetected

andanalyzed. This protein usually does not ap-pearwhencycloheximide is added with orbefore

the addition of virus. However, to achieve a better host turnoff in these experiments, the drug was added at the time ofvirusremoval, 1 h postinfection(30).

When the relative amount ofeach viral pro-tein synthesized wasdeterminedby

densitome-try of theautoradiograms and its logarithmwas

plottedagainst the UV dose given to the infect-ing virus, a series offirst-orderrelationshipswas

obtained (Fig. 2). The lines represent the best-fit plots for the datumpointsasdeterminedby computer analysis. It is apparent that the UV

sensitivity of synthesis of each polypeptide is not adirect functionofitsmolecular weightas would be expected if themRNA for each protein were transcribed from a continuous promoter-adjacent sequence.

The slopes of the lines yield the first-order rate constants for UV inactivation, orthe UV inactivationcrosssections, k, which are a

meas-ureof therelativeUVsensitivityof the

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UV

Dose

(J.m-2)

U C

0

7.46

14.9

224

29.8

165

145-

123-116- ;

86 S£*

71-

Op"-i .

55

-FIG. 1. Effect of UV irradiationonHSV-1 earlygeneexpression. HSV-1wasirradiated with UV lightat the indicated doses and used to infect XP cells. Cycloheximide (50pg/ml) waspresentfrom 1 to 8 h

postinfection.Thedrugwasremoved,and the proteinswerelabeled with[3S]methioninefor45min.Proteins wereanalyzedby gel electrophoresis and autoradiography..Viralpolypeptidesarenumbered in thousands of daltons.(U) Uninfected cell proteins; (C)control, infected cellsnottreatedwithcycloheximide.

sion of eachgene. From thesevalues, using the intrinsic dose-response factor of 2.30x 10-2

M2/

J (see below, Fig. 5), we calculated the target

sizes in basepairs of the transcription units of

the early genes analyzed (Table 1). Since the

UVsensitivityassaydoes not take into account

transcription "downstream" from thegenebeing analyzed (unlessthese sequences would be

re-quiredfortheproductionof functionalmRNA), the values shown represent minimum sizes for

the early transcription units. In Table 1, the

transcriptionunittargetsizesarecomparedwith

the number ofbase pairs calculated to encode

eachpolypeptide.Valid datawerenot obtained

forpolypeptide71 since itcomigrateswithahost

protein.

From these data we conclude the following.

(i) Theminimumtargetsizes for theearlygene

transcription unitsanalyzed rangefrom 3.05 to

5.35 kilobase pairs. (ii) The genes for proteins

116 and 165 have UV target sizes that

corre-spond closelytotheirpredictedgene sizes and thusmostlikely resideinpromoter-adjacent po-sitions.(iii) Thegenesfor viralproteins 145, 123, 86, and 55 show UV target sizes thatare0.46to

2.59 kilobasepairs greaterthan needed to encode

their respective polypeptides. This could be a

reflection of their distances frompromoters or

the presence ofintervening sequences or both

and indicates that theirprimarytranscriptsare

considerably larger than the size required for

their mRNA.

UV radiological analysis of DE HSV-1

genes.Inthis series ofexperiments,weallowed

viralproteinstobesynthesized from the onset

of infection, but to prevent the production of

nonirradiated progeny DNA molecules, we

in-hibited viral DNA replication with ara-A. As

373

44.8

WA**

_4-..lg

opwo:

Q&

!.,: .:::w

4.

..p.

:1 'r

....iiiiiii6lililL 17,::.

,"Wm"

dwa

'Wr--gw

'si"

400

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[image:4.504.106.394.75.409.2]

608 MILLETTE AND KLAIBER

10

01O

XL1

231

165

3 ~~~~~~~~~~~~~~~145

10 20 30 10 20 30

UV DoW (J-m2)

FIG. 2. Relative ratesof earlyprotein synthesisas afunction ofUV dose. Relativeratesof synthesiswere

deternined bydensitometryofseveralgel autoradiograhzs, includingthat shown inFig. 1. Linesrepresent

calculated best-fitfirst-order plotsfromthe averagesofseveralexperiments. Thedifferentkindsofdatum points indicate separateexperiments.Forabettercomparison,alllineswerenormalizedso as tointersect the

ordinateat100%. Viralpolypeptidenumbersaregivenin theright-handmargins.

TABLE 1. UVsensitivityandtargetsizesof early

HSV-Ipolypeptides

UVinacti- Base

Viral vationa Target pairs'

poly- crs e-

SZb

required Difference

peptide tion,k (base to en- (base

(mol t tion2k bas code pairs)

x10-3)

(xl0-2)

pairs)

pe°yd

(x102)

~~peptide

165 9.66 4,200 4,300 -100

145 12.3 5,350 3,780 1,570

123 8.44 3,670 3,210 460

116 7.02 3,050 3,030 20

86 11.8 4,830 2,240 2,590

55 7.57 3,290 1,430 1,860

ak

values

weredeterminedbycomputerfrom the

slopesof the UV inactivation plots. The values

repre-sentaveragesof threeorfour separateexperiments.

bTarget sizeswerecalculatedbydividingthe UV inactivation crosssection values, k, bythe intrinsic calibrationfactor,2.3 x10-2

m2/J

per1,000basepairs.

'Number of basepairs=molecularweightof

poly-peptidex(3/115).

previously shown by Drach and Shipman (6), this drug, in conjunction with the adenosine deaminase inhibitorconvidarabine,

will

prefer-entially

andeffectively block HSV-1 DNA

syn-thesis. Futhermore,wehave found that at

con-centrations of these drugs that quantitatively

block HSV-1 DNA synthesis,

all

but the late classes of viral proteins are synthesized in

in-fectedXP

cells

(Pedersen etal., submitted for

publication).

Monolayers of XP

cells

were infected with

virus that had been

irradiated

withvarious doses of UV

light

and then further incubated with medium

containing

ara-A and

covidarabine.

The infected cellswere

pulse-labeled

for45minwith

[3S]methionine

at 2 to 5 h

postinfection,

and the viral

polypeptides

were

analyzed

by

polyac-rylamide

gel

electrophoresis

and

autoradiogra-phy.

Alarge number of viral

polypeptides

canbe

identified

by

correlating

their mobilities with

previously

published gel

data

(10,

11) and

from the

sensitivity

oftheir

synthesis

toUV irradia-tion of the

infecting

virus

(Fig. 3).

From the kineticsof

synthesis

of these

polypeptides

(Ped-ersen et

al.,

submitted for

publication),

most

appear to

belong

to the

DE,

or

fl,

polypeptide

class (11). Since ara-A also causes a

delayed

turnoff

ofmanyof the

early

proteins

(Fig.

3and Pedersenet

al.,

submitted for

publication),

sev-eralIEpolypeptidesalso

appeared

inthese

anal-yses. In

addition,

a late

polypeptide,

154, was

consistently

observed.

The "survival"curves for the UV

sensitivity

of

synthesis

ofthesepolypeptidesare

plotted

in Fig.4. As inthepreceding IEgenestudies,one canagain observethat nodirect correlation

be-tweenUVsensitivity and polypeptide size exists.

From the kinetic data, we calculated the UV inactivationcrosssections andtranscription unit

targetsizes for the viralgenesexpressed in the

presence of ara-A(Table 2). The resultsmaybe

summarized

as

follows.

(i) All transcription units analyzed in the ara-A experiments have

mini-mum target sizes that range from 1.44 to 5.65

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609

UV

Dose

(J.m-2)

U

0

9.5

19

2&5 38

475

57

66.5

U

.~~~~~~

w

1

e"

_t

_S.

Ins. ~ ~ ~ f

-~ ~

-1P#.usar -S

n - -.

--32

34-FIG. 3. Effect of UVirradiationonHSV-1earlyandlatepolypeptides synthesizedinthepresenceof ara-A. HSV-1 wasirradiatedwith LWlightatthedoses indicated and usedtoinfect XP cells. Ara-A (150 gA)

andcovidarabine(1 pg/ml)werepresentfromIto5.75hpostinfection. Cellswerelabeledwith[35rJmethionine

from 5to 5.75hpostinfection. Proteinswere analzed by gelelectrophoresis andautoradiography. Viral peptidesaredesignated by molecular weightx10-.(U) Uninfectedcellcontrols.

~~~~~~~~~~~~~~~~~~A

ae~~~~~~~~~~

10 20 30 10 20 30 10 20 0

[image:6.504.108.396.73.341.2]

VDor (j m2)

FIG. 4. Relativeratesofsynthesisofviralpolypeptides,synthesizedin thepresenceofara-A,as afunction ofUV dose.Linesplottedrepresentcomputer-determined,best-fitfirst-order plots ofdatafromtwoorthree

experimentssuch astheoneshown in Fig.3. Thedifferent symbols representdifferent experiments. Viral

polypeptidenumbersareindicatedintheright margins.

kilobase pairs. (ii) The UV targetsizes for the

expressionof mostgenesanalyzedare

consider-ably larger than the number of base pairs

cal-culated to encode theirrespectivepolypeptides. Thedata indicate that thesegenesareremoved

from their transcriptional promoters by about 145

154-126

130:

116 119=

86

92-

71-

61-

55-_

go

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610 MILLETTE AND KLAIBER

TABLE 2. UVsensitivity and target sizesofearly andlateHSV-1 genesexpressed in the presenceof

ara-Aa UV

in-Viral activa- Tat Basepairm

poly- tion

sairzge

required Difference peptide cross (b toencode (base (mol wt section, ase poly- pairs) x10-3) k(m2/J)

pairs)

peptide

(x10-2)

165 7.97 3,465 4,300 -840

154 13.0 5,650 4,020 1,630

145 10.5 4,570 3,780 790

130 10.2 4,430 3,390 1,040

126 10.4 4,520 3,290 1,230

119 9.61 4,180 3,100 1,080

116 7.07 3,070 3,030 40

92 6.48 2,820 2,400 420

86 6.94 3,020 2,240 780

71 4.24 1,850 1,830 20

61 7.21 3,130 1,590 1,540

55 3.31 1,440 1,430 10

34 7.63 3,320 890 2,430

32 7.74 3,370 840 2,530

aForcalculations,seeTable1andtext.The values

shown represent averages of four or five separate

experiments.

0.42 to 2.53 kilobase pairs and are, therefore,

mostlikely transcribed as larger precursor RNA

molecules.

(iii)

Thegenesfor viral

proteins

116, 71,and55,

however,

showaUV

sensitivity

com-mensuratewiththeir predictedgenesize in base pairs. Thus these genes most likely reside in promoter-adjacent positions.

Itshould be notedthat the UV target sizes for

several IE genes were about 450 to 1,800 base

pairs smaller in these studies thaninthe

preced-ing

cycloheximide

experiments. From kinetic

analyses of the polypeptides synthesizedin the presenceandabsenceofara-A (Pedersenetal.,

submitted for publication) and from studies on

thepartial proteolytic peptidesofseveralofthe

IE polypeptides (S. Talley-Brown, unpublished

data),

itseemsthatwewereobservingthe same IEpolypeptidesinboth experiments.There are two

likely

explanations for this apparentshift in theUVsensitivityofthese genes: (i) expression

ofthe DEgenesmight allowtranscriptionofthe IE genes from a more proximal promoter; and (ii) since DE geneexpressionisrequiredfor the turnoff ofthe IEgenes (11, 12), UVinactivation

ofthe DEgenes might cause adecreasedrate of turnoff and a lower apparent UVsensitivity of the IEgenes. With thepresentdata, we cannot

distinguish between these possibilities.

How-ever, it is clear from these considerations and from thehigherlevel ofIEgeneexpressionafter

cycloheximidereversal that the more valid data on the IE genes come from the cycloheximide experiments.

J. VIROL.

ConversionofUV inactivationcross sec-tions intotranscriptional distances.To con-vert UV inactivation cross sections into

tran-scriptional distances, ideally,oneshould haveas

a standard an internal transcription unit of known size that is expressed under the same

experimentalconditions.Lackingawell-defined

viraltranscriptionunit in thissystem,we estab-lished anintrinsiccalibrationby usingseveral of the genes analyzed. This is based on the

as-sumption that geneshaving minimum ratiosof UV crosssection togenesizereside in promoter-adjacent positions. Thus,in aplotof UV inacti-vation cross section versus RNA size in base

pairsormolecularweight,the genesadjacentto promoterswill establish aline of intrinsic cali-bration for the UV dose response under these

experimentalconditions (foradiscussion of

cal-ibration methods, see reference 24). In such a

plot with the present data, the genes for four

viralproteins, 55, 71, 116, and 165, establishan intrinsic calibration line(Fig. 5). Further support

for this calibration is obtained if we use the

actual measured sizes of several IE mRNA's

instead of the gene sizes calculated from poly-peptide molecularweights. Byelectrophoresisin

denaturing gels, wefound that the mRNA'sfor

viralproteins 165and145havesizes of3.8and

5.2kilobases, respectively (S. Talley-Brownand

R.

Millette,

submitted for

publication).

Using these values in the calibrationplot (open

sym-bols, Fig. 5),wefound that the UVinactivation

crosssectionsforthesegenesfallvery near the

intrinsic calibration line. This line shows that

1,000 base pairs of HSV-1 DNA have a UV

inactivation cross section of 2.30 x 10-2 m2/J under theseconditions. In other

words,

it takes

43.5 J/m2 to produce one

transcription-termi-nating lesion per 1,000 base pairs of HSV-1

DNA. We used this standard cross section to

calculatethetranscriptional distances.

DISCUSSION

In these studies we have demonstrated that XPcells that lackdetectable DNA excision re-pairactivitycan be usedeffectively toperform UVmapping experimentsonHSVgene expres-sion. These cells offer several advantages for such experiments. (i) They allow UV mapping

studies to be carried out at the translational level, providingthatviral DNAreplicationand

possible virus-induced DNA repair can be

blocked.Toobtainvalidresultsbythismethod, however, therate ofpolypeptide synthesismust be proportional to the amount of mRNA syn-thesized. This seemed to be the case in the presentexperiments since the UV inactivation

curves showed first-order inactivation kinetics toa 15% survivallevel or less inmost cases. (ii)

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[image:7.504.62.255.102.299.2]

-1

II

'I

UV 1noc*tvtionCrossSection, kx102 (m2/J)

FIG. 5. Plot ofgene sizeversus UV inactivation

crosssection. The datumpointsaretakenfrom

Ta-bles1and2. Thelinerepresentsanintrinsic calibra-tionforrelating UVdoseto

transcription-terminat-inglesionsproduced byUV irradiation in the viral

DNA,where k=2.3x10-2m2/Jper1,000basepairs

ofDNA.Symbols:0,datumpoints fromthe

cyclohex-imideexperiments; A,datumpointsfrom the ara-A

experiments; 0andA,datumpointsfor thegenesof proteins145 and 165replotted byusing the measured

sizesoftheirmRNA's.

In contrast to UV mapping studies in which infected cell complexes are irradiated, studies

with XP cellscan beperformed by irradiating

onlythe free virus. Thisassuresthatthe

meas-uredUV effectsare adirect result of UV lesions

in the viral genomeand allows a moreprecise

measurementof theactual UV dose delivered to

the DNA. (iii) XP cells permit UV mapping studiestobeperformedunderessentiallyDNA

repair-free conditions.With other celllines,this

condition can be approximated only by

pulse-labeling the infectedcellcomplexes immediately

after irradiation. However, this again involves dosemeasurementproblemsandprecludesUV mappingstudies at the translational level.

Themainconclusionsderived fromthese

ex-periments arethefollowing. (i) The UV target sizes for the transcription of all HSV-1 genes

analyzedrangefrom 1.43to5.65 kilobase pairs. This implies that the corresponding primary

transcriptshaveminimumsizes thatrange from

about 0.46 x

106

to 1.82 x

106

daltons of RNA. (ii) Several of the genes studied, those for

pro-teins 165, 146, 116, and 71, exhibit UV target

sizes that coincide with their calculated gene sizes or measured mRNA sizes or both. This suggeststhatthesegenes are promoter adjacent

and do not have extensive intervening

se-quences. (iii) The UV target sizes for the

tran-scription ofmost ofthe genes studied are larger

than calculatedtocodefor their respective

poly-peptides. Assuming thatthe transcription of

se-quencesdownstream froma gene is notrequired for the production of its mRNA,theexcess base

pairs probably reflect the distances from pro-moters orthepresenceof interveningsequences orboth. (iv)Acomparison ofthemeasured UV targetsizes with the number of basepairs needed tocode for the corresponding polypeptides

(Ta-bles1and2)reveals that few,ifany, of the HSV-1 genesanalyzed could reside incommon

tran-scription units. However, overlapping transcrip-tionunits, suchasthose observed foradenovirus

and simian virus40, cannot be ruledout.

Absence of HSV DNA repair and

repli-cation.

To

successfully

apply

the UVmapping technique to the analysis of viral gene

expres-sion, it is essential that the irradiated viral ge-nomebe neitherrepaired norreplicatedduring

the experiment. The following considerations show that these requirements have been satis-fied.

(i) Hostcell DNA repair is negligible in this system. The XP cell line used in the studies

(CRL

1223,XP12BE) belongstotheA comple-mentation group, themost UV sensitive of the

XP cell lines. Studies showing that these cells

have less than 2% of the rate of DNA repair found in normal human fibroblasts (22) have been verified by S.Talley-Brownin our

labora-tory with repair assays in

cell

lysates by the

method ofCiarrochi and Linn (2) (unpublished data).

Furthermore,

studies on the

prolonged

survival of XP cells after UV irradiation by Maheretal. (17) and on therepair of UV-irra-diated adenovirus

by Day (5)

have shown that there is

virtually

noDNA

repair

activity

in these cells.

(ii) Viral DNA

repair

was not detectable in

the presence of

cycloheximide.

In the

experi-ments on IE viralgeneexpression,thepresence of cycloheximide

prevented

synthesis

of the HSV-1 DNA

polymerase

and

potential

virus-specified repair enzymes. After removal of the

drug, thebrief 45-min

pulse-labeling

period

al-lowedmainlythe IE

proteins

tobe

synthesized.

In

experiments

not

shown,

the UV

sensitivity

of IE polypeptide

synthesis

wasdetermined after the irradiated viral genomes had been in the

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[image:8.504.53.248.53.354.2]

MILLETTE

cells for4,8, or 12h inthepresenceof

cyclohex-imide. For any given IE gene, no significant changeoccurred inthe

slope

of the UV inacti-vationcurvewithincreasing times postinfection (datanotshown). Thus,therewas nodetectable

repair or

replication

of viral DNA under these conditions.

(iii) Viral DNA repair and replication were not

detectable

inthepresenceof ara-A. To

study

theUV inactivation of DE viralgenes, weused ara-A to block viral DNA

replication.

Under theseconditions, viralDNA

synthesis

was

neg-ligible (less than 0.15% of the control levels [Pedersen et

al.,

submitted for

publication]).

In additional UV

experiments,

viral polypeptides

were

pulse-labeled

for45minafter2to 12hof infection in the presence ofara-A.

During

the first5hafter

infection,

theUV inactivationcross sections for each of the DE genes remained

constant (data not

shown).

This indicates that there was no detectable loss of

transcription-terminating

lesions from the viral DNA

during

thisperiod.

Implications in the

mapping

of HSV-1

genes. The measured UV target sizes

place

a number of limitationsonthe

transcriptional

or-ganization of theHSV-1genome. A

comparison

ofthe UVtargetsizes of the IE geneswith the number of nucleotides needed tocode for their polypeptides

(Table

1) indicates that it is not possible foranyof theIE genesthatwe

analyzed

tobe contranscribed. This conclusion would be validevenifweoverestimated themagnitude of thedose-response calibration factor.

Mapping data from several laboratories have shown that thegenefor IEprotein 165(ICP 4)

maps in the terminal and internal repeat se-quences

(TRs

and

IRs) bracketing

the small

unique sequence (Us) and that the gene for

protein 123 (ICP 0) maps in the repeats (TRL andIRL) adjoining the large unique (UL) DNA sequence (3, 18-20). The data presented here eliminate the possibility of contranscription of

the genes acrossthe IRL-IRs junction and also preclude transcription of the IE genes from a

singlepromoterregionon acirculartemplate, as wasproposed recently by Clements et al. (3).

Acomparison of the UV target sizes with the number of base pairs needed to code for theDE

polypeptides (Table 2) reveals that there are

onlyafewpossibilities for contranscription. Al-though the data are compatible with the gene for protein 71 being promoter

proximal

tothat of32 or 34 and the gene for protein 55 lying promoter proximalto either 154, 61, 34, or 32, additional physical and UV mapping data will be needed to prove any cotranscriptions.

Fur-thermore,the measured UV target sizesprovide

noindication for the existence oflarge multigene

transcription units suchas that found for ade-novirus latetranscription.

UVinactivation of

IE

genesdoesnot af-fect the UV senstivity data of the DEgenes.

Thereisnowconsiderable evidence thatatleast

oneof the IEproteins is required for the

expres-sionof the DE and late viral

polypeptides (11,

12, 16,20). Itis

conceivable, therefore,

that the UV inactivation curves observed for the DE

genes representasummation oftwoeffects: the inactivation ofone or moreof the IEgenesand the inactivation of the individual DE genes. If this were the case, one can envision several possible modes of action of the IE gene prod-uct(s) onDEtranscription.

(i) An IE gene product is required in very

smallorcatalyticamountsfor theturn onof DE

genes.Letus assumefor thisdiscussion that this isanIEgenehavinganaverageUVtargetsize,

gene 165.Sinceweinfected thecells at a multi-plicity of30andinactivated thegene for poly-peptide 165 to about 10% of the controlatthe maximumUVdose, there remainedatthislevel ofinactivationapproximately three good copies ofgene 165productpercell.Accordingto

Pois-son distribution, the fraction of cells receiving

nointactcopies in thiscasewould be

e-&,

or0.05.

Thiswouldcause, at most,

only

aslight down-wardcurvature totheobserved UV inactivation

curves atthehigher doses. The valuesat adose givinga90% reduction in

polypeptide

synthesis (28

J/m2)

would be lowered by

only

about 5%. If the

expression

of three different IEgenes were

required, assuming forsimplicity that each has the UV sensitivity of gene 165, the DE UV inactivationcurves at 28J/m2would be lowered by only 15%. This doesnotappear tobe thecase since nosignificant downward curvatures were

observed in theDEUVinactivationcurves.

(ii) A certain threshold level ofthe IE gene

product is required for maximum DE gene

expression; below that level theamount ofDE

expression isproportional tothe amount of IE geneexpression. In this case ourobserved DE geneinactivationcurveswould bebiphasic, first

showingaslope

intrinsic

totheUV inactivation

of theDEgene and then exhibiting a slope that is the sum of that of the DE gene and that of the IE gene.Such a mechanism is very unlikely sincebiphasic curves for the UV inactivation of DE gene expression were not observed, and the target sizes for the inactivation of most DE genes are not large enough to include additional IE gene target sizes.

(iii) The amount of DE gene expression is

directly proportional to the amount of the IE gene expression. In this most extreme case we

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wouldexpect to see first-order UVinactivation

curvesfor theDEgenes, but their slopes would

be equalto thesum of the k values for the IE

plus the DE gene. This would yield apparent target sizes for the DE genes that would be at least 4,200 base pairs (the average target size for an IE gene) in excess oftheir coding require-ments. Inthe case in which more than one IE geneproduct wouldberequired for DE

expres-sion, the apparent DE gene target sizeswould beevenlarger. Theresults giveninTable 2 for

the DE genesshow that thiswas not thecase. From these considerations, we must conclude

that the UV inactivation parameters that we

observed for theDE and lateHSV-1 genes are notsignificantly alteredby the UVsensitivityof

the IEgenes.

In conclusion, we have shown that by using

exogenously irradiated virus withXPcells,one cananalyze the transcriptional organization of the HSV-1 genome by UVmapping. The data generated by this method have provided values for the minimum sizes of viraltranscription units

anddistancesof genesfrom theirpromoters and haveplaced restrictionsonwhichgenes maybe cotranscribed. This method should be

equally

applicableforstudying the transcription ofother

animal viruses, aslongasviral gene expression

canberestrictedtotheinput,unrepaired

paren-tal genome. The transcriptional distances

ob-tainedbythismethod shouldcomplement those obtained by

physical

and genetic mapping in that they should include all nontranslated se-quences required for gene expression.

Futher-more, they can provide direct evidence for

co-transcription,

polarity

oftranscription, and pro-motershifts.

ACKNOWLEDGMENTS

WearegratefultoSueTalley-BrownandMargaret

Ped-ersenforenlightening discussions,invaluable assistanceinthis

research, and many hours devotedtoreading manuscripts and drawing figures. Special thanks are due Walter Sauerbier, University of Minnesota, forhishelpfulcriticisms and

discus-sionsof this work.

Thisinvestigationwassupportedby Public Health Service grantCA21065awardedbytheNational Cancer Institute.

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