Isolation and localization of herpes simplex virus type 1 mRNA.

JOURNALOFVIROLOGY, June 1979,p.805-820

0022-538X/79/06-0805/16$02.00/0 Vol.30, No.

3

Isolation

and

Localization

of

Herpes Simplex

Virus

Type

1

mRNA

KEVIN P. ANDERSON, JAMES R. STRINGER,t LOUIS E. HOLLAND,AND EDWARDK. WAGNER*

Departmentof Molecular Biology and Biochemistry, UniversityofCaliforniaatIrvine, Irvine,

California

92717

Received for publication4December 1978

Herpes simplex virus (HSV) DNA boundtocellulose has been usedas areagent

to isolate viral mRNA for size analysison denaturingagarose gels. Totalviral polysomal polyadenylated RNAwasisolated from cellslate afterinfectionwhen

such RNA hassequencesencodedbyapproximately 45%of theHSV DNA.This RNA hasasizerangeof from 1.5to -8kilobases, withcertainsizes, suchas 1.7

to 1.9 kilobases, being favored. We have used the restriction endonucleases HindIIIandXbaIsinglyandtogethertogeneratevarious sizedfragmentscovering

the entire HSV-1genome.Thesefragmentshave been bound to cellulose toallow isolation of HSV-1 mRNA annealing to different regions of the viral genome.

Discrete sizes of viral mRNAareassociated with certainregionsof thegenome,

but the mRNApopulationhybridizingtoeventhesmallest restrictionfragments is complex. We used hybridization of size-fractionatedRNA toSouthernblotsof restriction fragments of HSV-1 DNAgeneratedbytheBglIIaswellasHindIII

andXbaI endonucleases to confirm the preparative hybridization data and to provide some overlap data for positioning transcripts. The data of blot and

preparative hybridization agreed very well and were combined to construct a

preliminarytranscriptionmapof HSV-1. Suchamaprevealed at least twoareas

ofthelong unique regionofthe HSV-1genomewhich annealed toalargenumber

of HSV-1 transcripts. Furthermore, discrete-sized mRNA specieslargerthan 5 kilobases inlengthwerefoundonlyin themiddle of thelong unique region.The

implications of thesedataarediscussed.

Herpessimplex virustype 1(HSV-1)isalarge DNA virus which replicates in the nucleus of infected cells. At least 48 HSV-1-specific pro-teins, atleast half of which arestructural

pro-teins, have been identified in infected cellsby using sodium dodecyl sulfate-polyacrylamide gels (11). The virus particle contains double-stranded DNA having a guanine-plus-cytosine

content of 67% (23). Themolecular weights of various strains of HSV-1 DNA have been

re-portedtobe between95x

106

and100x 106(8, 32), correspondingtoanapproximate length of 140,000 to150,000basepairs (140to 150kilobase [kb] pairs). The particular strain of HSV-1 DNA used in this study(KOS) is28.1times thelength

of the relaxed replicative form of

4X174

(34), correspondingto alengthof 150 kbpairs based on the known length of this phage DNA (3). HSV DNAisstructurallycomplex and has been shown to consist of a long region and a short region, each composed of unique sequences bounded by different inversely repeated se-quences(24). Each of thesetworegionsmay be

tPresent address: Cold Spring HarborLaboratory, Cold Spring Harbor, NY 11724.

linked to the otherby either end, resulting in fourequimolarconfigurations of the viral DNA (9, 39).

Transcription of thisgenomehasbeenstudied in our laboratory and in several others, with

general agreement on the following points. In cells infected at moderate multiplicity, HSV DNA synthesis begins about3 hpostinfection (p.i.) and reaches a maximum rate by 6 h p.i. Before theonsetofviral DNA synthesis, abun-dantRNAtranscripts from20 to25% ofthe viral genome arefound associated with polyribosomes (15, 28, 31). Both"Southern blot" hybridization and RNA displacement loop hybridization of HSV RNA have shown that these early HSV mRNAspeciesmap throughout the HSV-1 ge-nome (7, 29). The abundant, early HSV-1 mRNA species can be differentiated into two classes. One class consists of the

immediate-early or a mRNA's, which are found in high abundanceinthecytoplasmofcells treated with cycloheximide fromthe time ofinfection (15). TheamRNAspecies have been shown,by both blothybridizationandsolution hybridizationto isolated restrictionfragmentsof HSV-1DNA, to betranscribed from defined, noncontiguous

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806

gions of DNA comprising 10 to 12% of the viral genome(7, 14). The other major abundant class of mRNA species found prior to viral DNA replication is the /3 messages. These mRNA's encodeproteinswhich aresynthesized onlyafter proteinsencodedbythe a mRNAspecieshave beensynthesized (12).

Attimesafter infection when viral DNA syn-thesis is maximal (late), the majority ofearly RNAsequences continuestobesynthesized (7, 33),andtranscriptsfromanadditional 15to20% of the viralgenome are also foundon polyribo-somes in good yield. These are the y, orlate, mRNA species. At least a portion of these -y mRNA species has been detected even at the earliest timesafterinfection,butat a concentra-tion10- to 20-fold lower than thea

and,8

mRNA classes (15, 28, 30). At this time after infection (late), as much as 90% of newly synthesized polyadenylated [poly(A)] RNA on

polyribo-somesin the infected cellis viral(28). Further-more,it has beenextensivelyshown that all the sequences in viral mRNA can be isolated as poly(A) RNA (25, 28).

mRNA molecules of HSV-1 associated with the polyribosomes of infected cells resemble mRNA molecules of other DNA viruses and eucaryotic cells. HSV-1 mRNA, in addition to beingpolyadenylated,iscappedatthe 5' end(4,

20),andwehavefound itsaveragesizetobeno less than2kb(28).However,due inparttothe large size and complexity of the HSV genome, little has beenreportedconcerningthe proper-ties and origins of individual viral mRNA spe-cies.

In thisstudy, wehave usedtwoindependent

methodstodetermine the sizerangesofspecific HSV-1mRNAspeciessynthesizedlate after in-fection and tolocalize individual transcriptsto

specific regions of the viral genome. We have used restriction fragments of HSV-1 DNA boundtocelluloseto

purify

viral mRNA homol-ogous to these restrictionfragments. Size frac-tionation on denaturing agarose gels of viral mRNA obtained in thismannerrevealsa char-acteristicpattem ofspecific-sizemRNAspecies homologoustoeach restrictionfragment.In ad-ditiontosuch characteristicspecies,allregions of the viralgenomehybridizeto acomplex pat-tern of minorspecies of viralmRNA. The size distributions of viralmRNAencodedby differ-entregions of the HSV-1genomealso have been determined by first fractionating infected-cell mRNA on denaturing agarose gels and then hybridizing size fractions to Southem blots of HSV-1restrictionfragments.Themajor species and less prominent discrete species of viral mRNAseeninRNApreparatively hybridizedto

restrictionfragmentsarereadilyidentifiableby

using this procedure. The results of both types of experiments have been used to construct a preliminarytranscription map of the HSV-1 ge-nome.This mapshows theminimumnumber of discrete mRNA species consistent withour data.

MATERIALS AND METHODS Celisand virus.Monolayercultures of HeLa cells

weregrown at37°C inEagle minimal essential

me-dium containing 10% calf serum and no antibiotics.

Plaque-purifiedvirusof the KOS strain of HSV-1 was

used forall infections.

Preparationof RNA. Monolayercell cultures (2

x 107cells per T-150flask) were infected for 30min

with 10PFU of virus per cell and overlaid with

modi-fied medium 199 containingEarlesalts (Flow

Labo-ratories)supplemented with 5% fetalcalf serum. For

3H-labeledRNA, thecellswerelabeled from 5.25 to 6

h p.i. with 15 MCi of [3H]uridine (28 Ci/mmol;

Schwarz/Mann)perml inmedium 199 containing 5%

dialyzed fetal calf serum. For 3P-labeled RNA, the

cellswerelabeled from2 to 6 hp.i. with 200,uCi of

carrier-free [32P]orthophosphate (New England

Nu-clear)perml inEagle minimalessential medium

con-taining 1/10 thenormalphosphate concentration and

5%dialyzedfetal calf serum.

Polyribosome-associated RNA was isolated by the

magnesium precipitationmethod ofPalmiter (22) as

describedbyStringeretal. (28). After phenol

extrac-tion and ethanolprecipitation,poly(A) RNA was

pu-rified from the total polyribosome-associated RNA,

usingoligodeoxythymidylic acid-cellulose

(Collabora-tiveResearch). Poly(A) RNA was bound to the

cellu-lose in 0.5 MNaCl-0.1MTris (pH7.6)-imM EDTA,

rinsedthoroughly,and eluted in asolution containing

0.01M Tris(pH 7.6) and1mMEDTA. This procedure

results in 50% of the3Hradioactivity and 15% of the

32P radioactivity in polyribosome-associated RNA

beingrecovered aspoly(A)RNA. Asingle T-150 flask

typically yielded4 to 5ygofpoly(A) RNA with specific activities of 120,000 and 200,000 cpm/,ug, respectively,

when 3H labeled and32plabeled. Radioactivity was

measuredasa1%solution inAquasolII scintillation

fluid (NewEngland Nuclear) in a Beckman LS-230

scintillationcounter.

Thispoly(A)RNA has been shown to be intact by the following criteria. Recovery of 5' cap structure from poly(A) RNA isolated in this manner is virtually

quantitative(20),demonstratingnoappreciable

break-age between the 3'poly(A) tail and the 5' cap.

Fur-thermore, this poly(A) RNA serves as an extremely

efficient template for in vitro translation, and the

polypeptide productsmigrate in sodium dodecyl

sul-fate-polyacrylamide gelsassharp bands corresponding

toproteins synthesized in vivo, withlittleor no

back-ground of partial translation products as would be

seenwithnickedordegradedtemplate RNA

(Ander-son etal.,unpublished data).

PreparationofHSV DNA.Cellsgrown inplastic

rollerbottles (108 cellsperbottle) were infected with

10PFU of virus percelland incubated in medium 199

containing5% fetal calfserum.For32P-labeledDNA, thecellswereoverlaid at4hp.i. with phosphate-free Eagle minimal essential medium containing 5%

di-alyzedfetal calfserumand25to 50,uCi of

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HSV mRNA 807 phosphate per ml. The cells were scraped into the

mediumafter24h,collected by low-speed

centrifuga-tion (2,000xgfor 5 min), and resuspended in 0.01 M

NaCl-0.01 M Tris (pH 7.4)-3 mM MgCl2-0.5%

Noni-det P-40. Thecells were lysed by 6 to 10 strokes in a

tight-fitting Dounce homogenizer, and nuclei were

re-moved by low-speedcentrifugation. Virus waspelleted

from thecytoplasmic supernatant by centrifugation at

27,000xgfor 20 min. The viruspellet was solubilized

in 0.1 M NaCl-0.02M EDTA-0.01 MHEPES

(N-2-hydroxyethyl piperazine-N'-2-ethanesulfonic acid) (pH7.8)-1%Sarkosyl and digested with proteinase K (250,ug/ml)for3h at 55°C. The DNA was banded by

CsCl-isopycnic centrifugationaspreviouslydescribed

byWagner et al. (35), and DNA at 1.725 g/ml (>90%

of DNA ingradient)wascollected and dialyzed against

anappropriate restriction endonuclease buffer. A total

of 25 to50pgofpurified HSV DNA is recovered from

108 cells by this procedure.

Restriction endonuclease digestion of HSV-1

DNA and fractionation of resulting fragments.

Restriction endonucleases HindIII, XbaI, andBglII

werepurchased from New England Biolabs.All

diges-tionswereperformedunder conditions recommended

by thesupplier. Enzyme, at 1 U/4 pugof DNA, was

incubated with DNA for 2 h at 37°C. Double digestion

with HindIII and XbaI enzymes was performed by

digestingfirstwith HindIII and then adjusting the salt

concentration and addingB-mercaptoethanol before

digestingwithXbaIenzyme.

Restricted DNA (30 to 100pg)wasfractionated by

electrophoresisintwo 7.5-cmtracks in 0.5%agarose,

horizontal slab gels (42 by 20 by 1.2 cm) for 48hat 60

V (80 mA). Electrophoresis buffer was 0.04 M Tris

(pH7.8)-0.005 M sodium acetate-2 mM EDTA-0.5%

ethidium bromide. Bands were visualized by using

long-wave UV light.

DNAwasisolated from excised bands by dissolving

the agarose in 5 M NaClO4 at 60°C and absorbing

DNA to hydroxyapatite as described by Wilkie and

Cortini (39).After elution in 0.5 Msodium phosphate

(pH6.8), the DNAwasextracted withisopentylalcohol

toremoveanyremaining ethidium bromide, desalted on aSephadexG-50 column, and ethanol precipitated.

Binding of DNA to diazotized cellulose. Total

HSVDNA,aswell as restriction fragment DNA, was

boundtodiazotizedcelluloseby amodification of the

procedureofNoyes and Stark (21).

m-Aminobenzylox-ymethyl cellulose(Miles Laboratories) was

reprecipi-tated and diazotizedasdescribed in reference21.

How-ever,for rinses andcoupling, 10 mM potassium

phos-phate (pH 6.5) was used as a buffer instead of 0.2M

sodiumborate (pH 8.0). DNA, in 80% dimethyl

sulf-oxide and2mMpotassium phosphate at

concentra-tions of 100to200,ug/milfor total HSV DNA and 25

to 50

jg/ml

forrestriction fragment DNA, was heated

to780Cfor 5 min, cooled to 4°C, and then mixed with

thediazotizedcellulose so that thecellulose

concen-trationwas10 mg(dryweight)/ml.When lessthan 10

pgofDNA wasbeing manipulated, Escherichia coli

DNA wasadded as carrier tobring theconcentration

to 10pg.Thecellulosesuspension was mixed

contin-uouslyat40Cfor 48hand thenrinsed twice with 80%

dimethyl sulfoxide and 2 mM potassium phosphate at

50°C, four times with 0.lx SSC (0.15 M NaCl plus

0.015Msodium citrate) at250C,three times with 98%

formamide and 0.01 M HEPES (pH 8.0), and three times with hybridization buffer (80% formamide, 0.4

MNa+, 0.1 M HEPES [pH8.0],0.005MEDTA).

32P-labeled DNA was included in each reaction so that the efficiency of coupling could be monitored. A total of 35 to45%ofinput radioactivity remained bound to the

celluloseafter rinsing.DNAcellulosecontained 5 to 10

pgof DNA per mg ofcellulosefor total DNA and 1 to

3 pug/mg of cellulose for restriction fragment DNA.

DNAcoupled tocellulosewasstored inhybridization

buffer under nitrogen at40C.

Hybridization of RNA to cellulose-bound

DNA. 3H-labeled, poly(A) RNA was hybridized to

cellulose-boundDNAfor4hat57°Cinhybridization

buffer containingrecrystallized formamide. For total

HSV DNA boundtocellulose, 1to2pgof RNA (1 x

105to 2 x 105cpm)washybridizedwith5 to 10pgof

cellulose-bound DNA. For restriction fragments

boundtocellulose,10 to20pugof RNAwashybridized

to15pg-equivalentsof DNAboundtocellulose(1

pg-equivalentis theamountofrestrictionfragment

cor-respondingtodigestionof1pigof intact HSVDNA).

Thehybridizationmixturewasagitatedeveryhourto

avoid settlingof thecellulose.Afterhybridization,the

cellulosewasrinsedfour times with2xSSCat250C,

twice withhybridizationbufferatroomtemperature,

and five times withhybridizationbufferat600C.

Hy-bridizedRNA waseluted in 98% formamide and 10 mMHEPES (pH8.0)at600C.Theeluantwasdiluted

to a final formamide concentration of 22% and

ad-justedto0.1 Msodium acetate, and the eluted RNA wasethanolprecipitated.

Methylmercuryagarosegels.RNAwas fraction-atedbyelectrophoresison1.4% agarosegels(15by0.6 cm)containing10mMmethylmercuryhydroxide

(Al-pha) as described by Bailey and Davidson (2). Gels

wererunfor4 to 6hat3mA/gel (2.5 V/cm). After

electrophoresis, the gels were soaked in 50 mM

,B-mercaptoethanolfor 20 min andslicedat2- or3-mm

intervals.The3H radioactivityineach slicewas meas-uredby dissolvingslices in0.3ml of7%perchloricacid

at 72°C andmixingwith4 ml ofAquasol. 32P

radio-activity of each slice was determined by Cerenkov

radiationcountingof intact slices.

Preparationandhybridizationof DNA

South-ernblots. Slabgelsof restrictionendonucleasedigests

were blotted onto nitrocellulose paper (BA 85,

Schleicher &Schuell) inlOx SSC, usingthe method

ofSouthern (27).TheDNA-containingblotswerecut into4-mm stripsfor hybridization to the RNA

con-tained inindividual slices ofmethylmercury agarose

gels. Agarosegels containing10to15

pg

of32P-labeled

poly(A)RNAweresoaked in15mM

,B-mercaptoeth-anolfor 30 min andcutinto 2-or3-mmslices. Slices

containing32P-labeledRNAweredissolvedby heating

to

720C

in asolution containing 0.4 M Na+, 0.1 M

HEPES(pH 7.8)-0.005MEDTA,and 65%formamide.

Thedissolved32P-labeled RNA thenwashybridized

for24to36 hat570Cinsealedplasticbagscontaining

individualnitrocellulose strips.Stripsthenwererinsed

forthree 2-hperiodsat

480C

inhybridizationbuffer

and 65%formamide, arranged intheir originalorder

from the blot, wrapped in Saran wrap, and pressed

next toKodakX-omat-RX-rayfilm for24to72h.

VOL. 30,1979

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RESULTS

Sizerangeof HSV-1mRNA.P poly(A) RNA, labeled in HSV-1-ir

cellswith a45-min [3H]uridine pL afterinfection, washybridized to boundtocelluloseasdescribed in I Methods. The hybridized RNA(15 input radioactivity) was eluted fr(

lose and subjectedto electrophor turing agarose gels containing m hydroxide. Such gels exhibitalinea between the log size of RNA and migrated (2). The size, in kb, ofH species was determined by compa tancemigrated by HSV-1 mRNA,,c distance migrated by 3P-labeled

species, which were added to tI

internal size markers. Figure 1 sh tinct size species of HSV-1 mRNA >8 to -1 kbarereadily distinguis isaratherlarge proportion of vira size range of 1.9 to 1.7 kb, a de mRNAthat is2.1to 2.7 kb in size significantamountofviralmRNA kb. Inalarge number ofpreparati tions, the sizerangeand discretesj

20

to 18 16 1 4

x

a 1@

.8

--6

2

12 24 16

SLICECtNE*6

FIG. 1. Size distributionofHSV-1 beledpolysomal poly(A) mRNAfrom

infection was hybridized to 15pg o)

bound to cellulose, and the HSV-sl

eluted (see text). This viralmRNA w

32P-labeled HeLa cell rRNA andfracti

trophoresis on a 0.6- by 15-cm gel

agarosecontaining10 mMmethylmer(

(2). Electrophoresiswasfor4 hat 3n

The gel was fractionated in 2-mm s

radioactivitywasdetermined. The siz

wastakentobe0.67±0.07x106daltoi

2kb)andthatof28SrRNA,1.76±0.1L

(5.2 kb) (17, 37).Sizeextrapolationswei

factthat RNAmigrationisalogarith,

RNA sizeasshown byBaileyandDa discussed in Holmetal.(10).

RNA detected were invariant, but there was

'olyribosomal

some

variability

in theamountof

radioactivity

nfected HeLa recovered in the size ranges shown. Theamount

alse

at 5.25 h of viral RNA between 10 and 2.5 kb varies HSV-1 DNA between 55 and 65% of the total viral RNA

Materials

and recovered. Such variation isnotcorrelated with

to20% of the the

specific preparation

of DNA

cellulose

used om the cellu- in a givenexperiment.

esis on dena- To establish that the viral RNA purifiedby

ethylmercury

hybridization

tocellulose-boundHSV-1 DNA is rrelationship an accurate

representation

of the actual

popu-Lthe distance lation of HSV-1 RNA labeled

during

the

pulse

[SV-1 mRNA

period,

we tested our

hybridization

procedure

iring

the dis- for

specificity

and forits

ability

to leave RNA

speciesto the

undegraded.

Two

experiments

established the HeLa rRNA

specificity

of

cellulose-bound

HSV-1 DNA for ie sample as viral sequences. When

[3H]uridine-labeled,

un-ows

that dis- infectedHeLa cell

poly(A) polyribosomal

RNA ranging from was incubated with

cellulose-bound

HSV-1

5hable. There

DNA,

only

0.05to0.10% of theradioactive HeLa 1 RNA in the RNAwasboundtothe

cellulose

afterour

stan-barth

of viral dard

rinsing procedure.

Equivalent

amounts

of , and quite a

poly(A) polyribosomal

RNA frominfectedcells larger than5 yielded from 15 to 20%

hybrids.

Analysis

of

ive

hybridiza-

nonspecifically

bound uninfected cell RNA on

peciesof viral

methylmercury

agarose gels showed no

signifi-cant peaks of radioactivity migrating in any

given sizerange.

In asecondexperimenttotestthe specificity of hybridization to cellulose-bound DNA,

poly(A)polyribosomal RNA from infected cells

.14 washybridizedtocellulose-boundE. coliDNA.

12 .Again, less than 0.1% of inputamounts of ma-terial bound to the cellulose. That which did bind showednopeaks of radioactivityon meth-ylmercury gels.

Two control experiments demonstrated that there is no appreciable degradation of HSV-1 mRNAduringthe hybridization and elution pro-cedure. First, 32P-labeled HeLa cellrRNA was

40 60 incubated under hybridization conditions and

thenrun ondenaturing gel electrophoresis.No

RA3H_

I evidence of degradation of RNA couldbe seen mRNA.

lat

by either an alteration in the ratio ofthe 28S

cells lateafter

f HSV-l DNA rRNA to 18S rRNA or the appearance of in-pecific- mRNA creased RNA

migrating heterogeneously.

Inthe

as mixed with second control experiment, 3H-labeled poly(A)

tonated by elec- polyribosomalRNAwasisolated from infected made of 1.4% cells and directly sized on a methylmercury aga-cury hydroxide rose gel. The gel wassliced,and each slice was

nA (2.5 V/cm)- assayedfor viral RNAby hybridizationto

HSV-1lices,

and

the

1DNA ina mannersimilarto that described by

es

of 000

rRNas

Weiss etal.(36).Detailed resultsof this

experi-n~s(2,0(X bases,

5x106daltons ment

will

be

presented

elsewhere

(Holland

et

re based onthe al., submitted for publication). It suffices here to micfunction of

report

that the distribution of

HSV-specific

ividson (2)and RNA detected by hybridization acrossthe gel

was essentially identical to that of HSV RNA

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HSV mRNA 809

isolated by hybridization to cellulose-bound HSV-1 DNA.

We concludefrom these controlexperiments

that preparative hybridization of viral mRNA with HSV DNA bound to cellulose under our conditions gives an accurate and reproducible

representation of thepopulationofviral mRNA species present in the isolated RNA

population.

Ashasbeen discussed inMaterials and

Methods,

our evidence that the RNA is not

degraded

during our isolation is excellent; therefore, we conclude that the DNA-cellulose

hybridization

gives us a valid representation of viral RNA

labeled during the pulse period. This is

con-firmed byourfindings(Andersonetal.,

unpub-lished data) that DNA-cellulose-hybridized

RNA isanefficienttemplatefor in vitro trans-lation, andthe proteinproductsshow no indi-cation of mRNA

degradation.

Identification of HSV-1 mRNA

species

encoded by different

regions

of the viral genome. The

HindIII, XbaI,

BglIl,

and

HindIII/XbaIdouble-digestrestriction endonu-clease sites in the

prototypical

arrangement of the DNA of our strain

(KOS)

of HSV-1 are shown inFig.2.Theseenzymes cleave the viral DNA molecule into a number of

fragments

LR Lu

Bgl J K IOIPINIMI I

which migrate as individual bands on agarose

gels. Gel bands were shown to contain

single

DNAfragments by blotting thegelsonto

nitro-cellulose (27) and hybridizing with 32P-labeled overlapping fragments from different restriction

endonuclease digests. Sites generated by the otherthree arrangements of the viralDNA are described in thelegendtoFig.2.

The restriction fragments of HSV-1 DNA, shown in Table 1, were isolated from agarose

gelsand boundtocellulose(Materialsand Meth-ods). Individual cellulose-bound HSV-1 DNA restrictionfragments then were used toisolate viral RNA molecules containing sequences ho-mologous to that DNAfragment. We have used

15

,ug-equivalents

of restrictionfragmentbound

tocelluloseas astandardamountof DNA inour

hybridizations. We define the amount of any givenrestriction fragment derived from1,ug of total HSV-1 DNA as 1 ,ug-equivalent of that fragment.Inthismanner, the concentrations of DNA sequences in the cellulose-bound restric-tionfragmentswerekeptinthesameproportion

aswouldoccurinahybridization using15,ug of totalHSV-1 DNA boundtocellulose.

Three controls accompanied each

hybridiza-tiontocellulose-bound HSV-1 DNA restriction

LR SR SU SR

D | G | F H IL

Hind H 10 J A I K L I D

Xba G I C F E

H/X XG II 10 J 1.I XF AE I K I L 11

AC

D (SHORT) D 'M

INI

G

A

A

A

80 90 100

GENOME LENGTH (%)

FIG. 2. Map of restriction endonucleasefragments of the KOSstrain of HSV-1. Thesemapsarebasedon

publishedmapsforthese sites(18,26,38). We have confirmedthemforourstrain (KOS) ofHSV-1asdescribed

previously (29). The fragmentsgenerated bycleavage by the restriction endonucleasesBglII,HindIII,XbaI,

andHindIII/XbaI doubledigestsareshown fortheprototypicalarrangement(P) ofourHSV-1 strain. The

solidboxesatthe endsof the longsegment representthe longrepeat(Lit)sequences;theopen boxesonthe

short segment represent the shortrepeat(Sit)sequences.Three otherarrangementsoccur(9,39). TheseareII

where thelongsegmentisinvertedrelativetothe short; Is,where the shortsegmentisinvertedrelativetothe

long; and ILs, where both the long and short segments are inverted relative to the prototype. These

arrangementshavenoeffectonthe yield offragmentswhicharetotally contained by cleavagesites in either the longorshortsegment, but do leadtosubmolar bands for fragments coming fromthe ends of molecules and thosebridgingthejunction between the long and shortsegments.Submolarfragments for the

endonucle-asesshown are asfollows.Half-molar fragments arise from ends of molecules: BglIIL andHindIIIGare

from P and IL. BglIIJ,HindIIIH, XbaI A (XbaI D+short region),andXbaI Garefrom P and Is. BglII F,

HindIII D,XbaI B(XbaI G+short region), and XbaI Darefrom IL and Its. BglII HandHindIIIMarefrom

Is andILs. Quarter-molarfragments arise asthe sumof the half-molar fragmentsfrom either sideof the

junction foreacharrangement:BglII A is F+H,andHindIIIC is D+M. These regionsarecontiguous in

P. BglIIB is J+H, andHindIII FisH+M. These regionsarecontiguous in IL. BglII C isF+ L, and

HindIII B is D+G. These regions arecontiguousin Is. BglII E is J+ L, andHindIIIE is H+ G. These regions arecontiguous in I-s. XbaIhas no cleavagesites in the short segment; therefore, the half-molar

fragmentsXbaI Aand XbaI B have botharrangementsof the shortregion.

MINI G

VOL. 30,1979

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A

A

A

A

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810 ANDERSON ET AL.

TABLE 1. HSV-1 endonucleasefragments isolated

for DNA-cellulosebinding

Fragmenta Mol wt

Size'

Molar- Region of ge-(source) (x1lo6) _pairs)(kb ity nome_sented'

repre-(X)G 6.9 10.5 0.5e Longrepeat

(H)IO 9.5 14.4 1

(H)J 7.5 11.4 1

(H/X)AC 3 4.5 1

(X)F 15.5 23.5 1 Long unique

(H/X)AE 7.3 11.1 1

(H)K 6 9.1 1

(H)L 5.4 8.2 1

Long unique (H)C 22.2 33.6 0.25 jLongrepeat

Shortrepeat

(H)M 4.7 7.1 0.5e Shortrepeat

(H)N 3.1 4.7 1 Shortunique

(H)G} 8.9 13.5 0.5e _ShortShortunique_repeat

H(F) } 12.8 19.4 0.25e Long repeat_Shortrepeat

a (X) isderived froman

XbaI

digest; (H) is froman

HindIII digest; (H/X) is fromaHindIII/XbaIdouble

digest.

bBasedon1 kbpair havingamolecularweight of 6.6x 105.

' BasedonthefourfoldarrangementoftheHSV-1

genomeasdescribedinthelegendtoFig.2.

dSee

Fig.2.

'Submolar fragments are derived from the four

,ossiblearrangementsof theHSV- 1genomedescribed

inthelegendto Fig.2.Thus, X(G) is fromPandIs,

H(C) is from P,H(M)isfromIs andus,H(G) isfrom

PandIL, and H(F)isfromIL.

fragments. (i) Asmallamount of the RNA

sam-pleto behybridizedwasfractionatedinparallel with the preparatively hybridized RNA on de-naturingagarosegels; (ii) asample of the RNA

to be hybridized to cellulose-bound restriction fragment DNA was hybridized to total HSV-1 DNA bound to cellulose and the hybrids were fractionated in parallel with the hybridstothe restriction fragment; (iii) 32P-labeled HeLa rRNA wasaddedto the hybridization mixture, and radioactivity which did not bind to the cellulose also was fractionated in parallel with thehybridized RNA at the end of the hybridi-zation period. The quality of each sample of RNA obtained by each preparative hybridiza-tion was evaluated on the basis of the control

experiments. The RNA wasdeemed intact and representativeifnodegradationof the unhybrid-ized RNA and 32P-labeled rRNA was apparent and ifthe RNApurifiedbyhybridizationtototal cellulose-boundHSV-1 DNA lookedtypical(see Fig. 1). All DNA fragmentsshow hybridization efficienciesroughlycomparabletofragmentsize (Table2).

The size distributions of RNA purified by

hybridizationtodifferentHSV-1 restriction frag-ments are shown in Fig. 3. The panels are ar-ranged to represent regions of the HSV-1 ge-nomestarting withthe leftend of the L region through the S region of the P arrangement. Fromthisfigure, it is evident that certain viral mRNA species are associated with certain re-striction fragments. However, in all cases, the mRNA population homologous to a given re-striction fragment is complex. Some RNA spe-cies migrate as major sharp bands, suggesting very defined sizes for these mRNA species, which are synthesized inhigh, or at least mod-erate, abundance. RNA migrating sharply at a size of 6 kb hybridizes to HindIII fragment J and HindIII/XbaI double-digest fragment AC (Fig. 3C and D). A discrete 9.6-kb species is foundinRNAhybridizedtoHindIII/XbaI dou-ble-digest fragmentAE (Fig. 3F). HindIII frag-mentKhybridizesefficientlywith threediscrete RNA species, 7.2, 5.2, and 3.8 kb in size (Fig. 3G). Sharply migrating RNA species 3 and 1.9 kb insizehybridize toHindIIIfragmentL (Fig. 3H). Other RNA bands migrate broadly, sug-gesting a heterogeneity of sizes. A relatively large amount of such material is seen in RNA that hybridizes to XbaI fragment F (Fig. 3E). The RNA species that hybridizes to HindIII fragmentG (Fig. 3K) also isillustrative of such sizeheterogeneity.Note inparticularthe

broad-nessof thepeakcenteredat 1.9kb,which covers

arangeofsizesfrom 1.6to2.2kb.Otherdiscrete andheterogeneoussizeclassesof RNA homolo-gous to HSV-1 restriction fragments are indi-catedinTable 2.

Certain trends can be discerned from exami-nation of Fig. 3and Table2.Forexample,RNA ranging in size from 1.6 to 2 kb is a major component of RNA hybridizing to total HSV DNA and hybridizes to all of the restriction fragments shown. Such RNA is a major

com-ponent ofthe RNAhybridizing toall but three of these restriction fragments (HindIII

frag-mentsJ and K and HindIII/XbaI double-digest

fragment AE). Conversely, only one fragment (HindIII fragment IO) has sequences homolo-gous to a well-resolved RNA species (2.6 kb) between2.1 and2.7kb. Discrete peaks of RNA

>5.5 kb are found to hybridize to only four

fragments (HindIII fragments J and K and

HindIII/XbaI double-digest fragments AE and AC). It is clear, however, that heterodisperse RNA, upto about8kb in size, can be detected in RNAhybridizing to most fragments.

Some of the restriction fragments examined have commonDNAsequences. This is a conse-quence of the rearrangement of the long and shortsegmentsgenerating the four isomeric con-figurations of the genome and the presence of

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TABLE 2. HSV mRNA species hybridizing to restriction fragment DNA-cellulose

Size(kb)of' Proportionof

radio-Restrictionfragment" ficiency (%)b Prominent mRNA Resolvable mRNA activitymigrating

species species >5kb

(X)Gd

(H)IO

(H)J

(H/X)AC

(X)F

(H/X)AE

(H)K

(H)L

(H)Cd

(H)N

(H)Gd

(H)Md

(H)Fd

1.3

1.4

1.6

0.6

3.9

1.2

1.1

0.83

1.35

0.8

1.2

0.36

1.1

2.8 1.9

4.4±0.4

2.6 1.9 6

4.8±0.3

4.2±0.2

6 2.9

1.9±0.15

4.2±0.2

3.3±0.2 1.9 1.3 4.1

2.8±0.3

1.6±0.25 7.2 5.2 3.8 2.8 1.9

1.9

1.9 1.5

3.1±0.2

1.9±0.3

1.9

2.0

4.3 1.4

0.16(B)e

0.15

3.0±0.2 2 1.8 1.5 5.2 4.5 3.9 8.4f

9.6 6.5 5.2 2.9 1.9

5.2 4.4 1.6 4.8 4.1 2.8

4.5±0.4

2.7±0.2 1.0 4.2±0.2

4.4

2.8±0.2

4.3 2.8 1.6

0.24

0.24

0.32(B)

0.19

0.32

0.12

0.14

0.2(B)

0.13(B)

0.10

0.09

aSeeFig.2.

bIn

all

cases, 1 x 106 to 2 x

106

cpm of

'H-labeled

polysomal poly(A) RNA was used. Hybridization was

carriedoutwith15,ig-equivalentsof the fragment DNA bound tocelluloseand RNA fractionated as described

inFig.3.

'Size of RNAspecies is basedonthe positionof 28S and 18S rRNA markers (see Fig. 1). When a region of

migratingRNA wasbroad enough to indicate size heterogeneity, the range of size values is indicated.

Fragments (X)G, (H)C, (H)G, (H)M, and (H)F are generated by only certain arrangements of the HSV-1

genomeasdescribed inthelegendtoFig.2and inTable1.

'(B)indicates that the RNA species>5kb in size migrate as a polydisperse range.

fThis band isonly partially resolved.

the invertedrepeat sequences. Forexample,the entiresequence of HindIIIfragmentM is con-tained withinHindIII fragments C and F, and the short repeat sequence whichcomprises bet-ter than 90% of HindlIl fragment M also is contained in HindIlI fragment G. All three of thesefragmentsexhibithomologousRNA of the samesizes found forHindIIIfragmentM. The sizes of RNA hybridizing toXbaI fragment G

and HindIlI fragments C and F are similarly

consistent with the sequences shared by these fragments.

Localization ofspecific viral transcripts on the HSV-1 genome. As an independent

check for the DNA-cellulose fractionation of HSVmRNA,aswellas toprovidesomelimited overlapdata for mappingHSV mRNAspecies, wecarriedout anumber of DNAhybridizations

VOL. 30,1979 HSV mRNA 811

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812 ANDERSON ET AL.

456 400 350 308

251

200 1Se 16i 50 0

K U K

362

C.

Hiind

III (J)

280

246, 206, 166, 120

46

6

w---4501 4660 351 361 250 206 150

50 0

980 E. Xba I (F) 5F. HX (AE)

206

70e, ~~~~~~~175

69e, ~~~~~~~158,

509, ~~~~~~~125,

l O 20 30 40 f O 75 t 0 28 30 XO

lee ~~~~~~~25

315

G.Hin~d

III (K) 315 H.

Hivnd

III (L)

2818 C.288

245 245

216, 216,

1-5, 175,

146, 146,

185, 185,

78 78

35 35

16 28 36 46 56 10 26 38 48 58

SLICE NUMBER

FIG. 3. Size distributionofHSV-1 mRNAhybridizedtorestrictionfragmentsof viral DNA. (A) through

(M) are denaturing agarose gels ofviral RNA hybridized to HSV-1 restriction fragments fromdifferent

regionsof the genome (see Table 1). Thefragmentsarearrangedso as torepresentregions beginning from the

left ofthe Lregion throughthe shortregion ofthe P arrangement. In eachpanel, polysomal poly(A) RNA

from 4 x 107 to8 x 107 cells washybridized to 15pg-equivalents of restriction fragment DNA cellulose.

SpecificamountsofDNAcelluloseused andhybridizationefficienciesareshown in Table2.Detailsof RNA

labeling and gelfractionationare as described in thelegendtoFig. 1 and in the text. The arrows on each

panel show thepositionof the32P-labeledHeLa rRNA includedas aninternal-size marker.

of size-fractionated RNA to Southern blots of restricted DNA.Polysomalpoly(A) mRNA was labeled for 4 h with 32P, isolated from infected

cells, and fractionated on methylmercury aga-rosegels.Thelongerlabelingtimewas necessary toget RNAofsufficientspecific activityforblot hybridization. Gelswere sliced into 2- or 3-mm

slices, and Cerenkov radiationwasdetermined. Radioactivityprofiles obtainedwerequite repro-ducible, and a typical one is shown in Fig. 4.

Parallel gels with a small amount of the 32P-labeled RNA and 3H-labeled HeLa cell rRNA showed that the two peaks of indicated radio-activity migrated with sizes of 6.0 and 1.9 kb. These two peaks were used as internal size markersin all blotting experiments. Theslices weredissolvedinhybridizationbuffercontaining 65% formamide (see Materials and Methods), andRNA from individualgel sliceswas hybrid-ized tonitrocellulose stripscontainingrestriction

A. Xb@ I (C)

_ ._ . _..

9. Hind III (10)

.A i.

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HSV mRNA 813

360 K. Hind III (G)

315

278,

225,

135

96

45

l-360 320 280 240 200 160 126

88

40 0

369 326 28 240 286 166 126

86

48 0

135 129 105 90 75

60 45 38 15

IM. Hind III (F)

I

16 20 30 48 56

SLICE NUMBER

FIG. 3-continued

FIG. 4.SzedsriuinpbAKBfplyo

9S

66 56

40

3. 26

le

0

is Is 26 25 36 33 46

SL ICEMUSSEl

FIG. 4. Size distribution of polysomal poly(A)

RNAfromHSV-1-infectedHeLa cells.Inthe

experi-ment shown, [32PJRNA from 8 x 107 cells labeled

from2to6h p.i. with[32P]orthophosphatewas

frac-tionatedon adenaturingagarosegelasdescribed in

the legendtoFig. 1. Thegelwasdivided into 2-mm

slices,andradioactivitywasdeterminedby counting

Cerenkov radiation. Thesizerangeofthe RNA was

determinedasdescribedin Fig. 1,using 6- and

1.9-kb peaks (arrows) described in the text as size

markers. Only the top two-thirds oftheprofile is shown.

1 20 30 45 56

SLICE NUMBER

fragments of HSV-1 DNA. After hybridization, the strips were rinsed, assembled in order of

RNA size, and autoradiographed. Representa-tive blothybridizationstoHindIII/XbaI double-digest fragments and BglII fragmentsareshown

in Fig. 5. Hybridizations to blots of HindIII fragments alsowereperfonned (not shown).

Ta-ble 3 summarizes theresults ofourblot

hybrid-ization data. We have shown the size and loca-tion of the RNA species which hybridized to

defined fragments of the HSV-1 genome. The qualitative correlation between preparatively hybridized RNA and blot-hybridized RNA is excellent(cf. Tables 2 and 3). For example, RNA migratingat6kb,whichhybridizedto

cellulose-bound HindIII fragments J and HindIII/XbaI fragment AC (Fig. 3C and D), appearsashighly

radioactive spots in blots of those same

frag-ments (Fig. 5A) and in the overlapping BglII

fragments N and P (Fig. 5B). The discretely migrating RNA species isolated by hybridization

toHindIIIfragment K (Fig. 3G) alsoappearas

discrete spotsonblotsof thisfragment (Fig. 5A).

Therare 2.6-kb size mRNA species inHindIII fragmentIOis found inblotstothisfragmentas

I J. Hind III (N)

0L

6-;~

L. Hind III (N)

I

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A 10 6 5 1.9 1.6 I RNASize (KB)

-~-- D

- E X)F

-F

-_10

-G

-J

_- (H/X)AE

_(X)G

-K

-N

- -(H/X)AC

78_r 6.0 4.1 2.8 1.9 RNA Size

(KB)

-A

-B)c

-D

-E -F -G,H

-I

=J

-K

-L

M

--N

l

0

I-p

B

3.5

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HSV mRNA 815

wellas toBglII fragment 0, which is contained in it. The congruence betweenthe DNA-cellu-lose data and the blot hybridization data also holds forthebroadly migrating viral RNA spe-cies. Examination of the RNA homologous to HindIIIfragment G (Fig.3K) shows a large and broad peakof radioactivity that is2.2 to 1.6kb insize. In the blots,there is extensive

hybridi-zation of RNA in this size range to HindlIl fragment G and BglII fragment L. Inaddition to the 6-kb species, the RNA hybridizing to HindIllfragmentJ boundtocellulosemigrates

as acomplex mixture ofpartiallyresolved sizes thatrangefrom5 to 3and2 to 1.5kb (Fig.3C).

RNA hybridizing to blots of this fragment, as wellas tothecomponentBglIIfragments P and N, show a similar range of sizes. A significant portion of the RNA hybridizing toXbaI

frag-ment FDNA-cellulose migratesbroadlyin the size range of 5 to 3 kb (Fig. 3E); and RNA

hybridizingtoblots ofBglIIfragments I andD,

which overlap XbaI fragment F, also migrates broadlyin this sizerange.

Quantitative comparisonbetweentheamount of RNA ofaparticularsizehybridizingtoagiven

blot ofarestriction fragmentand that isolated from the same fragment bound to cellulose is difficult. This is because the RNA for blot

hy-bridization waslabeled for 4 hcomparedto45 min for the preparative hybridization, and be-causeexactquantitation of theamountof radio-activityhybridizing to ablot is difficult due to differential efficiency of transfer of large and small DNAfragments. However,itis clearthat,

at afirstlevel ofapproximation,the two meth-odsgivesimilar results.

There is, however, one notable quantitative

differencebetween the methods. Inexperiments

withblotted DNA,verylittle RNAhybridizesto the long repeated sequences present in XbaI

fragment G and

BglII fragment

J. In contrast, RNApulsedfrom5.25 to 6hp.i.hybridizedwell tocellulose-bound XbaIfragment G.In

experi-mentsusing labeling times of0to 2 hp.i.,

de-scribed elsewhere (Holland et al., submitted),

several discrete sizes of RNA hybridized effi-cientlytoblots ofXbaIfragmentG. The result is consistentwith there being reduced transcrip-tion from theseregionslate, comparedtoearly, after infection.Asecondpossibility is that RNA

transcribed fromthis region from 2 to 6h p.i. hasarapid turnover rate, resulting in alower steady-state concentration of this RNA in the infected-cell compared to other RNA species. This RNA would be represented to a greater extent in short pulse-labeled RNA (used for preparativehybridization) than in long-term la-beled RNA (used in blot hybridization).

Clem-ents etal. (7) also describedareduced efficiency ofhybridizationtoblots of the longrepeatregion with RNAlabeled from0to 7hp.i.

DISCUSSION

Complexity of HSV-1 mRNA. We have measured the size of HSV-1 RNA species syn-thesized during a time late in infection when viral DNA synthesis is proceedingat arapid rate and when80 to90% of the single-strand equiv-alent of the viralgenomeis expressedasmRNA inatleast moderate abundance. There are few

significantlylabeledspecies smaller than 1.5kb and little viral mRNA which migrates as a dis-crete sizespecieslargerthan 8 to 9kb (Fig. 1). Within thisrange,therearecertain favoredsizes, suchas6, 5.4,3,andespecially1.8 to 2.2and1.5 to 1.6kb.Also,onesizeclass, that between2and 3 kb, is rare for HSV-1 mRNA. The lack of HSV-1 mRNA 2to 3 kb in size appears to be

peculiar to viral RNA since uninfected HeLa cells donotlack mRNA in this sizerange (data notshown). HSV proteins have been reportedto range in size from 15,000 to at least 250,000 daltons (11). The largestsize of HSV-1 mRNA isolated in reasonable yield (8 to 9 kb) could

readily encode thelargestpolypeptides. On the other end of thespectrum, it isinterestingthat the smallest HSV mRNA species seen in any

quantityare nosmaller than 1.2kb,a sizewith

FIG. 5. Hybridization of size-fractionatedHSV-1 mRNA toDNA restriction fragment blots. Total

32P-labeledpolysomalpoly(A) RNA from infected cells -was fractionated as described in the legend to Fig. 4.

Individual slices were dissolved in 0.4 MNa',0.1M HEPES (pH7.8),5mMEDTA, and 65%formamide(see

text) andhybridizedtostripsof Southern blots ofHSV-1 DNA, either double-digested with HindIII and XbaI

endonucleases (A) or digested withBglII endonuclease (B). The locations of the cleavage sites for these

enzymesonHSV-1 DNA are shown in Fig. 2. After hybridization and rinsing (see text), strips were assembled

inorderof size of RNA used and radioautographed with Kodak X-omat-R film. Both radioautographs were

exposedfor24hat-70°C withaKodak intensifying screen, and contact prints of the X-ray film are shown.

SizeofRNAinthe slices wasdeterminedfrom the migration of the 6- and1.9-kb RNA species as described

in thelegendtoFig.4.Positions shown for restriction fragments in the blot weredeterminedbyvisualization

of the fractionated fragments of DNA before blotting. Resolution after blotting was checked by incubating a

strip with total32P-labeledRNA (not shown). In the case of thedouble-digest blot, the bands are as described

inthefootnotes to Table1;allbands areHindIII fragments except when indicated as an Xba fragment (X)

or adouble-digest fragment(HIX).

VOL. 30,1979

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TABLE 3. LocalizationofHSV-1 mRNAbyblot

hybridization

Size of RNA

hybridiz-ingb(kb)

2.8-3(-)

1.9(-)

3.2-4(-)

1.9

4-4.6 2.5(+)

1.9

6

4.1

1.5 6.7

6(+) 2.8-5.2

1.9(-)

4.6(-)

2.8-3.2

1.9

1.5

9.8(-)

3.2-4.6(+)

1.9

1.2

9.8(-)

7.8

5.2(-)

4.1

2.8-3.6

1.6-1.9

HindIII/XbaI

double-di-gestrestrictionfragmente

X(G)

H(IO)

H(J)

H/X(AC)

H/X(AE)

H(K)

H(L)C

4(-) 2.8-3.2 1.9(+) 1.5

1.9(+)

1.5(+)

H(D)

H(M)c

{ L H(N)

H(G)

Size of RNAhybridizing'

(kb) 2.8(-) 1.9(-)

3-4.6 2.6 2.0(+)

1.6-1.9

6.5 6(+) 5.2 4(+) 3.5(-) 1.9-2.1 1.5-1.7(+)

6(+) 4.3 3.2 1.9-2.1

8.6 3.8 2.8(-) 1.7

7.1(+) 5.2-5.6(+) 3.5-3.8

1.7-1.,q(-)

2.8-3.1(+) 1.9(+) 1.5-1.7

4-4.8(+) 3-3.5(+) 2.5(-) 1.5-2.1(+)

2.8(-) 1.9-2.1(+)

1.7

3 1.9-2.1(+) 1.6(+)

4(-) 3-3.2 1.5-2.3(+)

aFragments arearrangedin relativeorderontheprototype arrangement(seeFig.2).Data areshownonly

for thosefragmentswhichareclearlyresolved.

bBasedonthepositionof 6-and 1.9-kbRNA ingels(seetext). Slicesyieldasize±7%ofthe statedvalues.

Veryintensespotsareindicatedwitha(+)and faintones are indicatedwitha(-).

'BasedonblotsofHindIIIdigestswhich resolvefragmentsL andM.

816 BglIIrestriction

fragmente J

K

0

p

N

M

I

D

A

L

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VOL. 30, 1979

atleast twice the coding capacitynecessaryfor translation ofa 15,000-dalton protein. Whereas about half of the identified viral polypeptides are smaller than 60,000 daltons, most mRNA species are 1.8 to 1.9 kb and greater in size,

providing more coding capacity than required. Since littleposttranslational processinghas been observed (11), this suggests that manyHSV-1 molecules can contain significant amounts of

untranslatedsequences.

Theviral mRNA whichhybridizestoeventhe smallest restriction fragments of HSV DNA isa complex mixture of sizes which, in some cases, exceeds the asymmetric coding capacityofthe fragment evenwhen only the mostprominent speciesareconsidered. HindIll fragment K(9.1 kb pairs in length) hybridizes efficiently with threehighlylabeled viral mRNAspecieswhose aggregatesize isover16kb.HindIII/XbaI

frag-ment AC is only 4.5 kb pairs in length, yet

hybridizeswith mRNAspecies totalingwell over 10 kb. Also, there are at least four species of viral RNA >3.5 kb in size hybridizing to the regionencompassed by BglII fragments Pand N, whosetotallengthisapproximately9kb.One explanationforcomplexpatternsof RNAspecies

bindingtoagiven region is thatsomespecies of mRNA hybridizingtospecific DNArestriction

fragments are encoded by a DNA sequence which bridges a restriction endonuclease site. Thisappears tobe thecasefor the 6-kb mRNA which hybridizes to both HindIII fragment J and HindIII/XbaI fragment AC (see below).

However, most cases cannot be explained this

simply. Forexample,withHindIIIfragment K,

we know from the hybridization patterns of neighboring DNA fragments thatatleasttwoof the large RNA molecules which hybridize to HindIIIfragmentKdonotsignificantlyextend into the sequencesadjacent tothissmallpiece

of DNA (Table 3). The aggregate size of these two species is 12 kb, which is 3 kb more than could be asymmetrically transcribed from HindIII fragmentK. Again, the localization of the RNAspecies>3.5kbhybridizingtoHindIII fragment J andBglII fragments P andN cannot be explained by RNA overlapping into

neigh-boring fragments. Suchanapparent "transcrip-tional overload" in a given region can be ex-plained by a combination of the following: (i)

symmetric transcription, (ii) asymmetric tran-scriptionandprocessing ofRNA-producing spe-cies which have overlapping nucleotide se-quences, (iii) RNA splicing eventsresulting in themRNAmoleculesbeinghomologousto more thanonelocaleonthegenome.Symmetric tran-scription probably doesnotsignificantly contrib-utesince <2% of1-hpulse-labeled poly(A) pol-yribosomalRNA frominfected cellsis

self-com-HSV mRNA 817

plementary (13; Stringer and Wagner, unpub-lished data).Since both RNA species containing shared sequences and spliced transcripts have been observed in papova- and adenoviruses (1,

5,6),it is reasonabletoexpectherpesvirusesto exhibit similar complexity in transcription and processing of viral RNA. Overlapping mRNA sequenceswould be indicatedexperimentally if 3'ends ofseveral specific mRNA sizes could be localized intoasmallregion of thegenome con-tiguouswith the restrictionfragment containing the bulk of the sequences homologous to the RNA.Preliminary results of such studiessuggest

overlappingsequences of HSV-1 mRNA do, in fact,occur(Hollandetal., submitted).

In addition to the large number of specific-sized mRNA speciesfound in localized regions of theHSV-1genome,therearealsoappreciable amounts of heterogeneously migrating RNA found hybridizing to all restriction fragments. This RNA isviral since it isnotfound in control

experiments using uninfected cells or E. coli DNAboundtocellulose. Thereare anumber of

possiblesourcesof this heterogeneously migrat-ing viral RNA. Certainly, some could arise by

degradationof RNA during workupor hybridi-zation. We have already shown that such deg-radation isquite limited, however. Furthermore, inseveralcases,similarpatternsof such

heter-ogeneously migrating RNA are seenno matter whether the RNA is size fractionated beforeor afterhybridization. Therefore,we feel this pre-cludesdegradationbeing the onlysourceofsuch

heterogeneously migrating RNA. Some RNA species may well be present in small amounts andwouldnotresolvewell frommoreabundant

species. Smallamountsofspecific-sized mRNA species hybridizing to a given restriction frag-mentcould also arise from low-efficiency hybrid-ization of RNA whosesequencesbarelyoverlap

into the fragment. Another possible source of small amounts of RNA of heterogeneous size would be thepresenceinthepolysomalpoly(A)

RNA ofpartially processed viral RNA species derived from a larger precursor. Such species have beenfound for both adenovirus and simian virus40 (5, 16).Afifthsourceofsmallamounts ofspecificsizes of RNA couldresult from

tran-scription of all four arrangements of the HSV genome (see Fig. 2). It has been reported that onlyone or twoof the arrangements of HSV-1 DNA areabletoyield viableprogenyvirus(18); however, other arrangements could be tran-scribed to produce viral RNA species whose processing could be abortive. Therelative con-tribution ofanyof thesepossiblesourcesofthe viral RNA speciespresent in lowabundancewill

beresolvedbycontinuedanalysisof HSV-1

tran-scription.

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818 ANDERSON ET AL.

Preliminarytranscriptionmap of HSV-1. Thecombined DNA-cellulose and blot

hybridi-zation data allow ustolocalizeanumber of viral mRNA sizespecies in restrictionfragments.We have constructed a preliminary transcription

mapbasedon ourdataasshown inFig.6.This map is asimplification of the data summarized inTables2 and3 in that it showsonly readily

identifiable mRNA species. We have included those mRNA's of discrete sizes whichwere ob-served from both DNA-cellulose hybridization

and blot hybridization data. Because of the greaterresolution of RNAprepared by

hybridi-zationtoDNA-cellulose,wehave used thesizes ofTable2for individual RNA

species.

Wehave

indicatedsinglespeciesof RNA whichwere re-solved after DNA-cellulosehybridizationevenif such couldnotbe resolvedbyusing blot

hybrid-ization. When RNAmigratedas arange of sizes inbothDNA-celluloseand blot

experiments,

we have shownonespecies,with bracketsindicating

the extremes invalues.

Inmostcases, viraltranscriptsshown inFig.

6 are aligned in the middle of the restriction fragment containing homologoussequences. In

some cases, however, enough information is available to localize mRNA species more pre-cisely. The rationale forlocalization of the indi-vidual RNAspecies shown isasfollows. (i) The low-efficiency hybridization of both XbaI frag-mentG andBgIII fragmentJ madeconfirmation of the results of DNA-cellulose hybridization difficult. We have indicatedonlythetwospecies detectedbyblothybridizationonthemap. It is clear from both theDNA-cellulose hybridization and RNA displacement loop hybridization of early RNA (29) that there is considerable RNA hybridizing to this region in addition to the two species shown. We have rather arbitrarily lo-cated these twospecies tothelongrepeat, be-causetheunusually lowefficiencyof RNAblot hybridization suggests an unusual feature of the hybridization. (ii) RNA hybridizing toHindIII

fragment 10cellulose hybridizes mainly toBglII fragment0,which maps tothe right of most of this region. (iii) There are a large number of transcripts hybridizing to the region 20 to 30% of thetotalgenomelength in from the left end of the prototype arrangement. The size distri-butions of RNAhybridizing to BglII fragments

(M/X)

XG HIO H) AC _LL I I I

I0 9

71

6

5 a] 4 '14

LO 3

z

E2 I

(H/X)

XF AE HN NL HD (4M)KN HG

IJ

IEJ-I IEJ-I _ }J

BJ OK BOBPBN BM SI BD 8L

t II~~~~~

(25%) (50%) (75%)

FIG. 6. Preliminary minimaltranscriptionmap ofHSV-1 RNA. The data of Tables 2 and 3 and Fig. 3 and

5have been reduced to a minimal transcription map as described in the text. Only prominentlylabeledRNA

species areincluded. The location of the XbaI, BglII, andHindIIIfragmentsscored are shown for theP

arrangementof HSV-I DNA. The percentlength fromtheleftend of the genome is shown with arrows. When

RNAof thesamesize was found in neighboring restrictionfragments,it is shown as asingle speciesbridging

therestriction cleavage site, provided the radioactivity in bothfragmentsisequivalent,such as for the6-kb

RNAspecies in HindIII fragment J andHindIII/XbaIdouble-digestfragment AC. The length of the linesis

proportional to the relative length of the RNA in question. The RNA species with bracketed sizes indicate

RNA which migrates broadly in the range shown. The RNA species shown under the region spanned by

HindIIIfragmentD, which arisesfromthe IL andILsconformationsof the viral DNA, are based on blotdata

andareshown in the unique region due to the low hybridization of the long repeat region found in XbaI

fragmentG andBglIIfragmentJ.

I i

6 - - Ir 4

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[image:14.505.120.406.345.558.2]

HSV mRNA 819

0, P, andN,which subdivide thisregion, indi-catethatmostofthespecies of RNAhybridize to the region on either side of the right-hand

HindIlI site generating fragment J. (iv) The RNA species of size around 8 to 9 kb found hybridizingtoXbaIfragment F cellulose corre-sponds to RNA of this size found in blots of BglII fragments I and D and HindIII/XbaI

dou-ble-digest fragment AE. For clarity, we have shown thisas a single species, since one RNA molecule of this size islarge enoughtobridge all the restriction sites generating the fragments.

However,preliminary experimentssuggestthat twodiscrete 3'ends for RNA of this size occur inthisregion(datanotshown). We have shown RNA species on either side of thejunction be-tweenXbaIfragmentF andHindIII/XbaI dou-ble-digest fragment AE, dependingon whether RNA of this size alsohybridizestoblots ofBglII

fragment I. (v) Three RNA species have been shownmapping in HindIIIfragmentK because of the resolution of these after preparative hy-bridization (Fig. 3G). However, the midsized RNA hybridizing toblots of HindIII fragment

Kconsistentlyshowsabroader size distribution than the7.4-and 3.8-kbspecies, suggestingthat more than one RNA species may befound in this size range. (vi) The RNA hybridizing

effi-cientlytoblots ofHindIlI fragmentDhave been localized totheunique regiontothe left of the long repeat sequences since RNA hybridizes verypoorlytoboth XbaI fragment G andBglII fragment J, which also contain the longrepeat sequence. Finally, (vii) the 4-kb species,aswell as arangeofsmaller species of viral RNA species foundhybridizingtoHindIIIfragmentG cellu-lose and blots, are shown in the short unique region of thisfragmentsincethey donot

hybrid-ize to blots of HindIII fragment M or BglII

fragment L, which, like HindIII fragment G,

contains theshortrepeatregion.

Thetranscriptionmapshown inFig.6 under-estimates the number of significantly labeled HSV-1 mRNA species. This is especially true forsmaller RNA species found in large restric-tionfragmentssuchasXbaIfragmentF. Butin

spite of this and otherlimitations, certain pat-terns in the location of HSV-1 mRNA on the viral genome can be seen. The region of the genome from 10to 15% in from the left end of theprototypearrangement (BglIIfragment K) hybridizes mainly to RNA of size 1.9 to 2 kb. RNA displacement loop hybridization of early HSV RNAalsoshowslowhybridizationinthis area (29). At least two regions ofthe genome, theregion20 to30%infrom the left end(HindIII fragment J) and the region 50 to 60% in from that end(HindIII fragmentsKandL),hybridize to numerous RNA species. Higher-resolution

mapping ofRNAspeciesin theregion covered byXbaIfragment F mayreveal other such re-gions. Whereas RNA of 1.6 to 2 kb in size is

found throughout the genome, a significant

amountof RNA smaller than 1.5kb (1.2 kb) is found only in BglII fragment I. Well-resolved RNA larger than 5 kb is found in significant

amounts only toward the center of the long uniqueregionof thegenome(25to60% from the left end); furthermore, little viral mRNA>4 kb in size isfound in the shortregionand in either thelongorshortrepeatsequencesof the HSV-1genome.Specific features of HSV-1 RNA

me-tabolism which result in thesepatternsare un-known.

Correlation of the transcription map with the HSV-1 genetic map. Of the nearly 50proteins whichhave beenidentifiedasviral, about half have been localized on the HSV-1 genome by using intertypic recombinants be-tween HSV-1 and HSV-2 (19). Itis interesting to note that structural (y) polypeptides map throughout the long unique region,with a large number mapping between 20 and 30% and an-otherlargenumber between50and 60% in from the left end of the genome in the prototype arrangement. The very large structural poly-peptide, ICP-2, which would require a large mRNA speciestoencode it, has beenlocalized

inthis latter region.These areas of the genome correspondtoregions contained by HindIII frag-ment J and HindIII fragments K and L; both regions encode large mRNA species. Itshould be noted that thesetworegionsaresymmetrical around the rotation axis ofthe longsequenceof theHSV-1genome(see reference 29)sothat the question of whether the prototype arrangement or onewiththe long region invertedis

biologi-callyactive doesnotinterfere with the correla-tion.Further correlation of the HSV-1 transcrip-tion map with polypeptide maps must await studies on the biological activity of the RNA

specieswehaveidentified in thisreport.

ACKNOWLEDGMENTS

Wethank B. Gaylord and L. Tribble for excellent technical help.We also thank J. Holland for the gift of HeLa cells.

This workwassupportedby Public Health Service grant CA 11861 from the National Cancer Institute. K.P.A. was supported byPublic Health Servicepredoctoraltraining grant GM07311 from the National Institutes of Health.

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