Mapping early transcripts of herpes simplex virus type 1 by electron microscopy.

JOURNALOFVIROLOGY, July 1978,p. 56-73 0022-538X/78/0027-0056$02.00/0

Copyright© 1978 AmericanSocietyforMicrobiology

Vol.27,No.1

Printedin U.S.A.

Mapping Early

Transcripts

of

Herpes

Simplex

Virus

Type

1

by

Electron

Microscopy

JAMES R. STRINGER, LOUIS E. HOLLAND, ANDEDWARD K.WAGNER*

Departmentof Molecular Biology andBiochemistry, University ofCalifornia, Irvine, California92717

Receivedfor publication23September1977

RNA displacement loop patterns in intact

herpes

simplex

virus DNA and

herpessimplexvirus DNA restrictionfragmentsindicate that viral RNA

associ-ated with polyribosomes early after infection is transcribed from three major

areas of thegenome. One area ofearlytranscriptionis in the shortsegment of the

viralDNA and isroughlydelineatedby the invertedrepeat sequencesbounding

thissegment.The othertwo areasofearly mRNA

transcription

map in thelong

segment. Each ofthe three major areas ofearly mRNA

transcription

can be

further resolved into several regions offrequent loopingbordered by regionsin

which RNAdisplacement loopsare rare.Theseregionsrange in size from about 1.5kilobasestoabout9kilobases witha meansize ofabout 3.5kilobases.Although

the data do not allow precise assignmentofindividual earlygenelocations,it is

seen, even atthe lowest level ofresolution,thattheearlygenesare notcompletely

contiguous butaredistributedalong the

length

of theherpes

simplex

type1viral genome.

Theexpression ofherpessimplex virustype1

(HSV-1) during productive infection is

tempo-rallyregulated (14, 25, 26,29).Thefactors

gov-erning the control of HSV-1geneexpressionare

largely unknown.Animportant considerationin

studying such factors isa

knowledge

of the

ar-rangementofcoordinately expressed viralgenes.

PriortoviralDNAsynthesis(early),twoclasses of viralRNA,differinginabundancebyatleast 10-fold,are present onpolyribosomes. The abun-dant class of early RNA ishomologousto20 to

25% of the viral DNAsequences, and contains

two components, "a" or "immediate early"

RNA, and

",B"

or

"delayed

early" RNA. These

components are experimentally distinguishable

by the fact that a or immediate early HSV-specific RNA is found in the cytoplasmin cells treated with cycloheximide during infection, while the,B,delayed early RNA isnot (6,9, 14,

26, 29). The amount of the DNAhomologousto

a-immediate earlyHSV-specific RNAhas been

reported to be in the rangeof10 to15%, i.e., half

orlessof the totalearlyRNA (14).

Thelow-abundance class of viral RNA found incellsprior toviral DNAreplicationis

homol-ogous to a further 20 to30% of HSV DNA (14,

25, 26, 29). This class of HSV RNA is present at much higher concentrations

following

viral DNA replication and is the "y" or"late" viral RNAencodingstructuralproteins (12, 14, 25, 26, 29).

There are two fundamentally different

tech-niquesfor mapping viral transcripts onto viral

genomes. The first involves hybridizing viral

RNA to restriction endonuclease fragments

us-ing either solution hybridization or the blot

method of Southern(22). Thesecondtechnique,

termed RNA displacement loop (R-loop)

map-ping (27), utilizes the electron microscope to

directly visualize loops formed induplex DNA

through displacement of one DNA strand by

RNA moleculeshomologous to the other DNA

strand.

Bothmethods offer their ownunique

advan-tages.Hybridizationtorestrictionfragments

of-fersrelativespeed and,atleastpotentially,

quan-titation of the abundance of classes of RNA

transcriptshomologoustothefragmentin

ques-tion.R-loopingcanbeutilizedathighresolution

tovisualize individualtranscripts, thedirection

of the transcripts on the DNA, and unusual

structuressuch as"spliced" mRNAreported in

adenovirus and simianvirus 40 (2, 4, 5, 15-17).

R-looping at moderate resolution can resolve

areasoftranscriptional activity and itsabsence

withinregionsofthegenomeunresolved by

re-striction endonucleasecuts.Together,the meth-odsprovidecomplementary,aswellas

reinforc-ing, information concerningthe transcriptional activityofviral genomes. Both have been used

toresolvetranscriptional maps of the adenovirus

andsimian virus 40 genomes (1, 5, 15).

Biochemicalmapping by hybridization to

re-striction fragments has been used to locate

a-immediate early HSV transcripts. These studies showthatthe a-immediate earlygenes are not 56

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Voil. 27,1978

contiguous in the viral genome (6, 13). Since

total early HSV RNA has been shown to

hy-bridize to everyrestriction fragment examined

(6), there has been no definition in the total

earlyRNA transcriptionmap.

We have applied the R-loop mapping

tech-nique todeterminethe arrangement of the genes

codingfor totalearly HSV-1 mRNA. The data

presentedinthisreportprovidea

moderate-res-olution map of total early HSV-1 transcripts

which demonstrates areas of high, moderate,

and low or notranscriptionalactivity.

MATERIALS AND METHODS

Celisand virus. HeLa cellsweregrown in

mono-layer culture in Eagle minimal essential medium (8)

with Earle salts, 10% calf serum, and no antibiotics.

Growth conditions and assay formycoplasmic

contam-inationwere asdescribed(24, 29). TheKOS strain of

HSV-1was agift from G. CohenattheUniversity of

Pennsylvania. Virus was grown in HeLa cells at a

multiplicity of infection of0.1 PFUpercell and

rou-tinely plaque purified after four to six passages as

described (31).

RNA. Cells were infected at a multiplicity of30

PFU per cell using conditions described previously

(25, 26).Time after infection was measured after virus

absorption (0.5h). Cellswere harvestedat 3hafter

infection.

Details ofcell fractionation, polyribosome isolation,

and polyadenylated RNA purification were as

de-scribed(23). Briefly,theprocedure appliedthe

mag-nesium ionprecipitation method of Palmiter(19) to

obtainpolyribosomes. The Mg2+-precipitated

polyri-bosomepelletwassuspendedin 0.01 MNaCl-0.01 M

Tris-0.0045 M MgCl2, pH 7.4 (RSB), and briefly

di-gestedat roomtemperature with 200,gof

electropho-reticallypurified DNase(SigmaChemicals, St. Louis,

Mo.) per ml. RNAwasextracted from the

polyribo-somefractionbyproteinase K (Merck

Pharmaceuti-cals, Elmsford, N.Y.) digestion, followed by phenol

extraction as described (23). Radioactive RNA was

preparedasabove, butcellswerelabeled from2to 3

hafterinfection with15,uCiof[3H]uridineper ml(27

Ci/mmol; Schwarz Bio Research, Inc., Orangeburg,

N.J.).

DNA.HSV DNAwaspreparedbothas areagent

tobeused in the isolation of HSV RNA andasintact

genome-sizemolecules for restriction andR-loop

anal-ysis.Partially purifiedvirionswereobtained from

in-fectedcellhomogenatesbyequilibrium centrifugation

inadiscontinuoussucrosegradientasdescribed (28,

30, 31). DNA for preparative hybridization of HSV

RNAwaspreparedfrom thepartially purifiedvirions

asdescribedandassayedforpurity byanalytical

iso-pycniccentrifugation (23, 30).

Forisolation ofintactHSV DNA molecules,

par-tially purified virions (30, 31) were obtained as

de-scribed aboveand thenfurtherpurified by

homoge-nization in RSB containing 0.5% Nonidet P-40 and

differential centrifugation. Purified virions from 4x

108to5x 108 cellsweresuspendedin 0.9 ml of 0.05 M

Tris-0.02 M EDTA(pH8.0), andthe DNAwasgently

released by the addition of0.1 ml of 10% Sarkosyl.

MAPPING EARLY HSV-1 TRANSCRIPTS 57

The DNA wasdeproteinized by digestion with 200

jig

of proteinase K per ml for 6 h at 45°C, then loaded on

anagarose A-150column (2 by 35 cm; BioRad,

Rich-mond, Calif.) equilibrated with 0.5 M NaCl-0.1 M

PIPES [piperazine-N,N'-bis(2-ethanesulfonic acid)]

-0.005 M EDTA (pH 7.8). The column eluant was

collected in 1-ml fractions, and DNA was monitored

by absorbance at 260nm. Viral DNA collected from

the agarose column was between 20 and 50 ,ug/ml and displayed a single component thermal denaturation

profile with aTm of 88°C in 0.1x SSC (SSC is 0.15 M

NaCl-0.015 M sodium citrate). This material was com-posed primarily of intact HSV DNA molecules as

determined by electron microscopy. Nocellular DNA

was present as determined by analytical isopycnic

centrifugation.

Restriction endonuclease digestion of HSV

DNA. Restriction enzymes, EcoRI, HindIII, and Xba

I, werepurchased from New England Biolabs

(Bev-erley, Mass.).Digestion conditions were: 0.10 M

Tris-hydrochloride (pH 7.5)-0.05 M NaCl-0.005 M

MgCl2-100 ,tg of bovine serum albumin per ml, for

EcoRI; 0.06 M NaCl-0.007 M MgCl2-0.007 M

Tris-hydrochloride (pH 7.4), for HindIII; and 0.15 M

NaCl-0.006 M MgCl2-0.006 M

2-mercaptoeth-anol-0.006 MTris-hydrochloride (pH7.9), for Xba I.

DNA was digested at a concentration of 25 ug/ml.

Aliquots of1to 3ugof restricted DNA were layered

onto0.7% agarose (Sigma) gels, poured in Plexiglas

tubes(0.6x 14cm).DigestedDNAwassubjected to

electrophoresis in gel buffer (0.04 M Tris-hydrochlo-ride-0.005 M sodium acetate-0.002 M EDTA, pH 7.8)

at 0.5 mA per gel for48 to 60h at 4°C. Gels were

stained with 0.5,gof ethidiumbromide per ml of gel

buffer, and bandswerevisualized by fluorescence at

360nm.

DNAwasrecovered from gels bydissolving the gel

slicecontaining the band of interest in saturated KI,

dialyzing exhaustively against 0.10 M NaCl-0.01 M

Tris-0.001 M EDTA(pH7.4), andremoving the

solid-ified agarose by centrifugation at 15,000 rpmfor 30

min.From40to50%of the DNA is released into the

supernatant by suchtreatment. The DNAwasthen

extracted with isopentyl alcoholtoremove ethidium

bromide.

TheHindIII, Xba I, and EcoRI restriction sites in

the DNA of HSV-1 (KOS) have been mapped (21).

Whendigested by either Xba I orEcoRI, ourDNA

yielded digestionproducts which,onagarosegels,were

identicaltothoseexpectedfrom thepublishedmaps.

The identity of individual Xba I digestionproducts

wasconfirmed byredigestionof each agarosegel band

with EcoRI. When digested by HindIII, our strain

produced an agarose gel band pattern that lacked

bands corresponding to fragments I and 0 of the

published map.There ispresentabandaslargeasthe

sumofI and 0 instead. SinceI and 0 are adjacent

pieces of DNA, thesimplest explanation is thatthe

cleavage site between I and 0ismissinginourstrain.

Redigestionofindividual HindIIIdigestionproducts

withEcoRI showedthis to be the case. TheHindIII,

XbaI, and EcoRI restriction sitesinthe DNA of the

virus strain usedin theseexperiments are, therefore,

the same asthose publishedfor HSV-1 (KOS) (21)

except foramissingHindIII sitebetweenfragmentsI

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58

STRINGER, HOLLAND, AND WAGNER

and0(seeFig. 5).

Preparative hybridization of HSV RNA.

Sev-eral preparations ofHSV-specific polyribosomeRNA

were prepared for R-loop hybridization as follows.

Infected-cell polyribosomalRNAlabeled with

[3H]ur-idinefrom2to 3hafter infection and isolatedatthe

end of the labeling period was added in eightfold

(wt/wt) excesstonative HSV DNA.32P-labeledHeLa

cellribosomal RNAwasaddedas aninternalmarker,

andthemixturewasethanolprecipitated. The

precip-itate was dissolved in 98% formamide and 0.1 M

NaCl-0.003 MPIPES-0.0003 M EDTA (pH 7.0) and

incubated at 72°Cfor 10minto denature the DNA.

Thesolutionwas nextadjustedto93%formamideand

0.3MNaCl-0.01MPIPES-0.001 M EDTA and

incu-batedat480Cfor24h. Under theseconditions,

rean-nealing of HSV DNA doesnotoccurand only

RNA-DNAhybridsareformed(3; L. E. Holland and E. K.

Wagner, unpublished data). At the end of the

incu-bationperiod, thehybridizationsolution wasdiluted

by the addition of2volumesof0.15Msodiumacetate

(pH 5.0) and precipitated with3volumesof ethanol.

The precipitate was suspended in 1 ml of2x SSC

containing0.020 M EDTA and treated with RNase

(10 ,ug of pancreatic + 10units ofTi per ml) for 10

minat 37°C. RNase wasremovedby digestion with

500,ugofproteinase K per ml in the presence of 0.5%

sodium dodecyl sulfateat 37°Cfor20min, followed

byphenolextraction.

Hybridswereseparated from unhybridized RNA by

gelfiltrationonSephadexG-100(Pharmacia,Uppsala,

Sweden).AG-100column (1.5by50cm),equilibrated

in0.1MNaCl-0.01 M Tris-0.001 M EDTA (pH7.4),

wasloaded with the aqueousphase fromphenol

ex-traction, and 1-ml fractions were collected. Hybrid

RNA(15% of the total 3H radioactivity) eluted in the

voidvolume well before any 32P-labeled rRNA.

Frac-tionscontaining hybridswerepooled and ethanol

pre-cipitated.Theprecipitatedhybridsweresuspended in

80% formamide and 0.2 M NaCl-0.02 Tris-0.001 M

EDTA(pH 7.4) and denatured by heatingat72°C for

10min. Formamide was removedby passage overa

Sephadex G-25 column equilibrated in 0.01 M

NaCl-0.01 M Tris-0.0045 MMgCl2(pH 7.4), and the

DNA wasdigestedat370C for 5 min with 100Mgof

electrophoreticallypurified DNase I per ml. The

mix-turewasthen made 0.5% sodium dodecyl sulfate 0.005

MEDTA, phenol extracted, and passed over a

Seph-adexG-100columnequilibratedin0.1MNaCl-0.01M

Tris and0.001MEDTA(pH 7.4). Excluded RNA was

collectedandethanolprecipitated. RNA obtained by

this method had aspecific radioactivity of

approxi-mately 50,000 cpm/yg and was free of 32P-labeled

rRNA. Electron microscopy showed no evidence of

DNA contamination. Rehybridization ofRNAusing

DNA excesshybridization (23) demonstratedthat it

wasatleast85%viral.

R-loop hybridization. R-loop hybridization was

performed in 75% formamide, 0.2 M NaCl-0.1 M

PIPESand 0.005 M EDTA(pH 7.8)at56°C for 20h.

Intact, native HSVDNAconcentrationswerefrom5

to10,ug/ml.Concentrationsof individualDNA

frag-ments generated by digestion withrestriction

endo-nucleaseswerenotdirectly determined, but were at

leastfivefold less than that of intact viral DNA.

Ex-,J. Viimu.

periments with total infected cellpolyribosomalRNA

wereperformedatRNA concentrations from50 to 100

times greater than the concentration of DNA.

RNA-to-DNAconcentration ratioswereapproximately2in

experiments withpurifiedHSVRNA and7in

experi-mentsusingpolyadenylatedRNA(basedon aspecific

activity of 50,000 cpm per,g ofRNA). DNAannealing

controlsweredone under thesameconditionsas

de-scribed above forR-loop experiments.Digestionswith

S1 single-strand nucleasewereperformedasdescribed

previously (19).

Electronmicroscopy. R-loop hybridization

mix-tureswerediluted 30-fold into 100

pl

of 50%

formam-ide-0.1 M Tris-0.01 M EDTA(pH 8.5) containing, per

ml,50 ugofcytochrome c and0.2Mgof the relaxed

replicative form of4X174DNA(4XRFII DNA).This

mixturewasimmediately spreadover ahypophaseof

either10%formamide plus0.01MTris-0.001 M EDTA

(pH 8.5)or20%formamideplus0.01 MTris-0.001 M

EDTA (pH 8.5). Parlodion-coated microscope grids

were touched to the cytochrome-DNA monolayer,

stained withuranyl-acetate, and rotary shadowed with

platinum-palladium. Molecules were visualized and

photographed in either the Siemens 1Aor theJeol

100C electron microscope. Intact genome-size DNA

molecules werephotographed at anominal

magnifi-cationofx2,600.DNAfragmentswerephotographed

atmagnifications ranging fromx3,000toxlO,000.

Measurement of molecules. Negatives of

R-looped HSV DNA molecules and restriction fragments

wereprojected and traced to produce

high-magnifi-cation (x20to X30)replicas of the photographic

im-ages.ThesetracingsweremeasuredonaTektronics

4956 Graphics Tablet and Tektronics 4051 computer

(Tektronics Corp., Beaverton, Ore.). All HSV DNA

wasmeasured inreferencetothe average length of at

least104XRFIIDNAmolecules present in the same

field. HSV DNA molecules measuring 28±14XRFII

DNA unitswere scoredasintactviral genomes and

normalized to a standard length (28 4XRFII DNA

units). HSV DNA restriction fragments which fell

within 5%of themeanmolecularlength for that

frag-ment wereconsideredfullsize andwerenormalized to

themeanlength. Themeanlengths of the restriction

fragments analyzed agreed quite closely with those

expected fromanalysisonagarosegels.

RESULTS

R-loop

reaction. When duplex DNA is in-cubated with homologous RNA in high

concen-trations offormamide, RNA can hybridize to its

complementarysequence in the DNA, thereby

displacing the other DNA strand and forming an R-loop. R-loops form because RNA:DNA

hybridsare morestable than DNA duplexes in

high-formamidesolvents (3, 27). The rate of the

R-loop reaction is strongly dependent on the

incubation temperature,proceedingfastest near

the melting temperature of the DNA duplex

(27). The Tmfor HSV-1 DNA under our

hybrid-ization conditions was

570C,

as deternined by

analysis with

Si

endonuclease. Our

hybridiza-tionswereperforned at

560C.

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Voi.. 27, 1978

R-loops formed in the viralDNAupon

incu-bation witheither total infectedHeLa cell

po-lyribosomal RNA, polyadenylated infected

HeLa cell polyribosomal RNA, or HSV RNA

purified by preparative hybridization. The high

concentration oftotal infected cell

polyriboso-mal RNA required to form R-loops in HSV

DNAseverely impaired thevisualization of the

DNA. Most of the data presented hereare

there-fore derived from DNA molecules hybridized

with purified HSV RNAorwith polyadenylated

RNA. R-loops on representative intact HSV

DNA molecules are shown in Fig. 1A.

Repre-sentative R-loops showing good contrast

be-tween the double-stranded DNA:RNA duplex

and the displaced single-stranded DNA are

shown in Fig. 1B.

TheR-loop reaction isabsolutely specific for

viral RNA. No loops occurred when viral DNA

was incubated alone (Fig. 1C), with

mock-in-fected HeLa cell RNA, with

ribonuclease-treated infected-cellRNA,orwith

ribonuclease-treated HSV RNA. ThatnoDNA:DNA

anneal-ing occurred under R-loopanneal-ing conditions is also

shown in Fig. 2. No DNA:DNA duplex was

fornedevenwhen the reactionwasdriventoa

DNA Cotgreaterthanfive times that attained

in R-loop experiments.

The orientationproblem. Tovisuallymap

structuressuchasR-loopsonlinear DNA

mol-ecules, the molecules must be arranged in a

standard orientation.Orienting HSV DNA

mol-eculesisaproblem exacerbated bytheintemal

rearrangements which occur in the genome

yielding four differentarrangementsof the DNA

in approximately equimolar amounts (11, 20).

This property of the HSVgenome precludeda

simple best-fit R-loop alignmentorientation

pro-cedure,and itwasthereforenecessaryto

deter-mine the R-loop patternsofdefinedregions of

the DNA to aid in orienting intact R-looped

HSV DNA.

Due to the technical difficulties of handling

molecules the size of HSVDNA,wehave found

it impracticable to work with molecules

R-loopedinalarge portion of their length.

There-fore,wehave examinedalarge numberof intact

HSV DNAmoleculesbearingalimited number

ofR-loops permolecule. Since only molecules

bearing alimited number ofR-loopsper

mole-cule couldbe obtained ingood yield,itwasbest

toemploy regionsdevoid ofR-loopsas

orienta-tion aids. Regionsof the genomewhichare

in-frequently R-looped by early polyribosomal

RNAwererevealedby (i)analysisof theR-loop

frequency distribution ofa randomly arranged

population of intact HSV DNA molecules and

(ii) theR-loop patterns onpositionally defined

restrictionfragmentsof viral DNA.

MAPPING EARLY HSV-1 TRANSCRIPTS 59

Random-order R-loop pattern. The

fre-quencydistribution of R-loops in51intactHSV

DNAmolecules, linedupwithoutregard to ori-entation, is shown in Fig. 3. As expected, the

histogram is complex and shows symmetry

around the middleof the distribution. Because

of the internal rearrangements in HSV DNA, mostpositions in the random-order histogram

represent four different physical loci on the

DNA (see Fig. 4); therefore, the absence or

presenceofR-loopsat mostDNAloci isdifficult

toascertainfrom the random-order histogram.

The center of the histogram is an exception

because

only

two

physical

loci on the DNA contributetothis region of the histogram. Since there isalow level ofR-loops around thecenter

ofthe random-order histogram,R-loops must be

rare orabsentatthetwolociin the DNAwhich

contribute to this region of the histogram.

In-spection of Fig.4shows that thetwoDNA loci whichwill contribute to the centerof the

ran-dom-orderhistogramoccur at 30and50%ofthe

genomelength in from the long-segment end of

the DNA.

R-loops

inselected HSV DNA restriction fragments. As discussed in Materials and

Methods,

the

HinduI,

Xba

I,

andEcoRI restric-tionsites in the DNA of the HSV-1 (KOS) line used in these

experiments

arethesame asthose published for HSV-1 (KOS) by Skare and

Sum-mers (21) exceptforamissing HindIII

cleavage

sitebetween

fragments

Iand0 (Fig. 5). Three Xba Irestrictionfragments, A, F, and G, were

selected for R-loop analysis. These fragments

accountfor 58% of theDNA and can beobtained

as

single

bandsonagarose gels.As seenin Fig. 5, these

fragments

coverthe distal 8% of the

long

segmentofthe HSV-1genome

(fragment

G);

a

segment 16% of thegenome in

length

nearthe

centerof thelongsegment(fragment F); and the

terminal 36% of the HSV-1genome,

encompass-ingboth arrangements of the shortsegment of

the molecule

(fragment A). Selected fragments

bearing

R-loops

areshown in

Fig.

6.Individual molecular histograms for 25molecules ofeach of these fragments are shown in

Fig.

7A. A

composite histogram

of

R-loops

of these

frag-ments

corresponding

to their

position

on the

intactHSV-1genomeis shownin

Fig.

7B.

Inspection of selected individualAfragments (Fig. 7A) showed thatloopsmayoccur onboth

ends ofanindividualfragmentbutare rare near

the center. Since any arrangement of A

frag-ments will

produce

a

semi-symmetrical

histo-gram, the orientation problem is obviated. On

the other

hand,

since

loops

donot occur onboth

ends of every Afragment, thelack of any

align-ment criteria means the relative

peak heights

within the histogram of the A

fragments

are

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60 STRINGER, HOLLAND, AND WAGNER J. Viui-.

=~~~~

(fS;

X

-P

--{..-V~~~~~~~~~~V

FIG. 1. Electronmicrographs ofHSV-1 (KOS)DNA molecules bearing R-loops formedwith earlyRNA

Hybridizationwasfor24hat56°Cin 75%Formamideasdescribed in thetext. Thesmall circularmolecules

areOXRFIIDNA,includedas asize marker.(A)AnHSV DNA moleculeR-loopedwithpurifiedHSV RNA. The HSV DNA molecule is27.7

0XRMI

unitslong (47.1

tun).

ThebarindicatesItun. (B)R-loopsformedin HSVNAb totl ealy RA islatedfrom polyribosomes. Arrows indicate loops, Sdesignates

single-strandedDNA. The barindicates 0.1 ,um. (C)HSV DNA molecule incubatedunder conditionsidentical to

thoseforA and B but in theabsenceofRNA.Thelengthofthemolecule is 28.0

OXRFII

units(47.4 ,um).The bar indicates 1.0 ,un.

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[image:5.499.52.452.61.567.2]

MAPPING EARLY HSV-1 T'HRANSCRIIPT'S

v~~~~~

.t~~~~~~~~~~~4

r~~~c

'-"-V..,.;~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

[image:6.499.59.448.70.561.2]

~ @. ;|> w

FIG. 1B.

somewhatarbitrary.The AfragmentsinFig.7A

have been aligned to produce approximately

equivalent R-loop frequencies at both ends of

thehistogramsectioncorrespondingtofragment

A(Fig. 7B).

XbaIfragmentsFand Gboth showed

exten-siveR-loopingandcould not beunambiguously

orientedtoproduce uniquedistributions.

There-fore,inorientingR-looppatterns of intact

mol-ecules fragmentsF andGwereutilized onlyto

the extent that they established extensive

R-looping in the region of thegenomerepresented

bythesefragments.

Orientation ofR-loopedintact HSV DNA.

TheR-looppatternofXbaIfragmentA

unam-biguously established a high incidence of

R-Voi.. 27, 1978

₆₁

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62 STRINGER, HOLLAND, AND WAGNER 1Vri.

C

t

*

C

4

1'

t

FIG. 1C.

loops in DNAlocatedoneither side ofthelong segment-short segment junction and very low levels ofR-loopingin theregionof thejointitself

(Fig. 7B). Analysis of the fragment, therefore,

revealed a defined non-R-looped region in the

DNA, which could be used to orient intact

R-loopedHSV DNA.

The existence ofat least one large region in whichR-loopsarescarcein thelongsegmentof the DNA was suggested by the random-order .4

J. vilml'.

11

11 I'' .:

I

'R

I

I I A

III, * .b

I

I

I

- F

11

I i

10 .

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[image:7.499.61.450.61.588.2]

MAPIPING EARLY HSV-1 T'RANSCHIPI'TS

-1 -0.7 -6.4 -6.1 6.2 0.5 6.8 1.1 1.4 1.? 2

Log Cot

FIG. 2. Lack ofDNA renaturation under R-loop hybridization conditions. (0 ---0) 50,000 cpm of

'H-labeled HSVDNA(specificactivity, 300,000cpm/p) wasmixed with20pgofunlabeled HSVDNA which had

been shearedtoanaveragesizeof14kilobases(30). TheDNAwasprecipitated and dissolvedin 200,ulof

R-loopbuffercontaining75%formamideand0.2 MNa+.This mixturewasthenheatedto78°Cfor10minand

incubatedattheR-loopingtemperature(56°C) forvariousCot values (1Cais100pgofDNAperml incubated

for 1 h).Aliquots(10-,ul) wereremoved, diluted into 0.35

Al

ofSI digestion mix(25), and digested withSl

nuclease todetermine theamountofDNArenaturation. (X-X) Aparallel experimentwascarriedout at 30°Ctoshow thattheHSVDNAdoes,infact,renature attheproperreannealingtemperature.

26 m

0

L

E

c u L

s

L

0

0 P

E D

N

T

E R

A

L %'

18

16 14 12

18

8

6

4

2

e 16 20 36 46 56 66 76 86 90 166

[image:8.499.105.397.75.307.2]

GENOME LENGTH (M)

FIG. 3. Random-order histogram ofthe positions of R-loops formed in HSV-1 (KOS) DNA by early

polyribosomal RNA. Fifty-one R-looped, intact HSV-1 DNA molecules were aligned without regard to

orientationtoproducethishistogram(see text).Intactmoleculeswerethosemeasuring28+ I XRFIIDNA unitsinlength.

VoL. 27, 1978

188 99

63

s

I N

G L E S T

R

A H D E D D

N

A

88

78

60

58 40 30

20

16

6

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[image:8.499.106.405.394.616.2]

64 STRINGER, HOLLAND, ANI) WAGNER

5 1 5 25 35 45 50 55 65 75 85 95

95 85 75 65 55 SO 45 35 25 1 5 5

75 65 55 45 35 4o 25 1 5 5 85 95

95 85 5 15 25 4O 35 45 55 65 75

5 1 5 25 35 45 4O 55 65 75 95 85

85 95 75 65 55

JO

45 35 25 1 5 5

75 65 55 45 35 30 25 1 5 5 95 85

[image:9.499.57.450.56.296.2]

85 95 5 15 25 70 35 45 55 65 75

FIG. 4. Distributionof regions of the HSV-1genome inarandom-orderarrangement. Theeight possible

arrangementsofthe HSV-1genomeareshowndiagramatically.Numbers denote percentageofgenomelength

ofaprototypical arrangement. Cross hatches denote thelong segment-short segment junction. The dotted line

shows thetwolociofaprototypical arrangement of HSVDNA which will contributetothecenterofa

random-orderhistogram.

C;I HIIO | HJ HA HK HL HC HN HG

XG xc XF XE XA

HH HIO HJ HA dSK HL HB HN HM

-LONG SEGMENT _SHORT SEGMENT.

HD HL HK HA HJ HIO HF HN HG

XD XE XF XC XB

HlD HL HK HA HJ HIO HE HN HM

0

R D E R

1

0R D E R

FIG. 5. Sitesfor cleavage of HSV-1 (KOS) DNA by the restriction endonucleases Xba I and HindIIIas

determinedbySkare and Summers(21). Thefour arrangements oftheHSV-1genomearearrangedinto two

ordersonthe basisofthelong-segmentorientation. Thepositions of HindIIIsitesaremarkedbythe vertical

solid lines, those ofXba Ibyvertical dashed lines. The individualfragments (HA, XA, etc.) are named

followingtheterminology ofSkareand Summers (21). The sitesoftheHSV-1 (KOS) strain used hereare

identicalwith those describedbytheseauthorsexceptforthelackofaHindIIIsitebetweenfragmentsI and 0;theresultingcombinedfragmentis termed "HIO"(seethetext).

histogram.ThishistogramshowedR-loopstobe

scarce in DNA loci mapping approximately 30

and/or50% in from thelong-segmentend. Since

XbaIfragmentF could be orientedtoproduce

a non-R-looped region between 40 and 45% in

from thelong-segment end, andsince no early

polypeptides have been found to map in this

area (L. Morse, L. Pereira, B. Roizman,and P.

Schaffer, in F.Rappetal., ed.,ThirdInt.Symp.

onOncogenesisandHerpesviruses, inpress;N.

M.Wilkie,N. D.Stow,H. S.Maroden,V.Brown,

R. Cortini, M. C. Timbury, and J. H.

Subak-Sharpe, in F. Rappetal., ed., ThirdInt. Symp.

onOncogenesisandHerpesviruses,inpress),it

wasreasonabletotentativelyassumetheregion

of thegenome mappingfrom 40to 45% infrom

0 R D E R 1

0 R D E R 2

,1. viumol.

on November 10, 2019 by guest

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[image:9.499.59.448.360.506.2]

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

-S~~~~~~~~~~~~~~~~~~~~~~~~.

o.%j7

41~~~~~~~~~~~~~~~~~~~~~~~b "49

FIG. 6. HSV-1 DNA restriction fragments R-looped with earlypolyribosomal RNA. The small circular

moleculesare

4LXRFII

DNA. The barindicates0.1 Lm.(A)Xba Ifragment F; this fragment is4.5

4XRFII

units(7.8gm)inlength. (B) HindIII fragmentA; thisfragmentis7.11XRFIIunits (12.3

pmn)

in length. (C)Xba

Ifragment G; this fragment is2.4

O.XRFII

units (4.15pin)inlength. Loops are shown with arrows indicating

thesingle-strandedDNA(5).

65

on November 10, 2019 by guest

http://jvi.asm.org/

[image:10.499.45.473.49.617.2]

G F A

s * ~* ***muU 3

| |

'

|

-

:

-~~~~

*S

_________

mu*

EU

.*- I s * **

]_~~~a

_________

_*LLJLL

33,s 3: - ml

sB '

a

ls

U

* *

*

*

msa

G F

20 30 46 59 66

GENONE LENGTH (M) FIG. 7.

A

79 86 96 166

A.

1

5

16

15

26

25

B.

SS

54

46

42

36

30

24

18

12

6

6 N 0 L E C U L E S L 0 0 p E

D

I

I N T E R U

A L

X1

on November 10, 2019 by guest

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MAPPl'ING

EARLY HSV-1 T'RANSCRIPTS 67 the long-segment end of the molecule to be

largely devoid of R-loops. The validity of this

assumptionwasascertained from the results of orientationoperations based on the assumption asdiscussed further below.

If theorientationof the short segment of the

HSV-1genome is ignored,full-lengthHSV DNA

moleculescan bearranged intotwoorders

cor-responding to the two possible orientations of

the longsegment of the genome. These

proto-typicalarrangements areshowninFig.5asorder

1 and order 2. We undertook to sort out

R-looped intact HSV DNA molecules with regard

to the orientation of the long segment on the basis of (i) theR-loop distribution in the region of the DNA

represented by

Xba Ifragment A

and (ii) the strong suggestion ofa lack of R-looping in aregion nearthemiddle ofa

proto-typicalarrangementof the HSV DNA molecule.

A rarely R-looped region near the joint is

ex-pected to occurin all intact HSV DNA mole-cules,since thesequences nearthe

long

segment-shortsegment

joint

are the same

regardless

of the relative orientations of the two DNA

seg-ments. The region of low

R-looping

which

ap-peared tobe nearthemiddle ofa

prototypical

arrangementof the HSVDNAmolecule

would,

in anygiven

molecule,

occur oneither side of

the center of rotation of the

long

segment,

i.e.,

from 45 to50% for order 1molecules and from

30 to35% for order2molecules.

Moleculeswerescreened and

preliminarily

as-signedto eitherorder1 or order2onthe basis of their fitto

templates

which

designated

alack ofR-loopsinthe

regions

of theDNAdescribed above.

R-loop

distribution

histograms

of thetwo

populations

were

prepared

and

juxtaposed

so

that the

regions

inthe

histograms

representing

the DNA molecule

long

segments were

coli-nearly

aligned.

The success of the

preliminary

assignments

of molecules to order 1 or 2 was

determined

by

inspection

of

juxtaposed

histo-grams for

mirror-image

symmetry. The

sym-metry was strong but could be

improved

by

realigning

molecules which couldbeorientedin morethan onewayonthe basisof the

original

template trial. The final

assignment

of HSV-1 molecules into orders 1and2is shown in

Fig.

8.

Histograms of the

R-loop

frequency

in these moleculesareshown in

Fig.

9. The existence of

well-definedmirror symmetry in Fig. 9 indicates

that the template-aided orientation procedure

hassuccessfully sortedoutthe differentgenome

arrangementsand oriented thesegroups of

mol-ecules with respect to the long and short

seg-mentsof the DNA. The histograms display

max-imum symmetry when the internal end ofthe

longsegment is placed 18% in from the

short-segmentend. This locationof the jointbetween

the long and short DNA segments is in good

agreementwith direct measurements of the joint

position (11, 20).

R-loop mapof the early genes of HSV-1

(KOS) determined from restriction

frag-ments. The distributions of R-loops produced

in seven restriction fragmentswere determined

toconfirm the R-loop dataobtained with intact

molecules. Fragments were chosen to cover as

much ofthe HSV-1genome as possible. HindIII

fragmentsA,

1-0,

J, K, and L and Xba I

frag-ments A and G were used. As seen in Fig. 5,

these fragments together represent 99% of the

HSV-1genome.

All fragmentswith the exception of HindIII A

run assingle-component bandson agarosegels.

HindIIIfragmentA isamolar

fragment

which

comigratesonagarosegels withaquarter-molar

fragment, HindIII fragment B. Preparations of

HindIII fragment A are therefore 80% pure.

Analysis of30 R-looped fragments showed 25

fragments which fit a pattern consistent with

thatexpected from analysis of (i)anoverlapping restrictionfragment,Xba F, and (ii) intact HSV DNAmolecules. The five HindIIIfragments in

whichR-loops occurredinarrangements

differ-entfromthe other25 weredesignatedasHindIII fragment B andwere not included in the

com-posite histogram shown in Fig. 10. Xba I

frag-mentsAandGwerealignedastheywereinFig.

7B.TheHindIII

fragments

werealignedtobest fit the loop distribution obtained from intact DNA in order1 (Fig. 9).

Sinceonly fragments which contained R-loops

were scored when restriction fragment R-loop

patterns were determined, the relative peak

heights betweenfragmentsarenotindicative of the relative heights ofcorresponding peaks in

thehistogram derived fromintact DNA. There-fore, the composite histogram of R-loop

fre-quencyonthe HSV-1 genome shown inFig. 10

FIG. 7. (A)Graphicdisplay ofindividualXba Irestrictionfragments ofHSV-1(KOS)DNAR-loopedwith

earlypolyribosomalRNA. The restrictionfragmentsXba IA, F,and Gwererecoveredafterelectrophoretic

separationonagarosegelsandhybridizedwithpurified early HSVpolyribosomalRNA. Individual molecules

foreachfragmentwerealignedasdiscussed in thetext. The solid boxesrepresent thepositions ofR-loops.

The average lengthsforXba Ifragments A, F, and G were 9.6, 4.2, and2.4 4XRFIIunits, respectively,

corresponding to molecularweightsof33.8 x H06, 14.9x 106, and8.5 x 106(see thetext). (B) TheR-loop

frequency distributionsoftheHSVDNArestrictionfragmentsin(A).Theputative jointbetween thelongand

shortregionin XbaIfragmentAisat apositionnear orat82%genomelength. Thispartial histogramwas

usedtoalignintact HSV-1 DNA molecules andtoassignthemtoorder1ororder 2(see text).

Voi.. 27,1978

on November 10, 2019 by guest

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68 STRINGER, HOLLANI), AND WAGNER

a a

.

-'

a

a

a

I

.

-.

U

Ia

a

N

OD 0. IV

COPl( OP

,,o\j ^r, 0% 0%

OD

n

N

.

-1U

(D w -4 ob

MNomCmN ~Co-SooB I

I .

1-40

:

:

a

a

I

a

. so

a

1-I

V MN -0

4C N N aSN

a -.a-.

-40

I

a

I)

M -

I

a

a a

a

.

N N _ _N _

_J _

-x

x)

x

x

1-Is

.

.

w

w

x

.

IV

Mv

M

x

W

JZ

X

w

x

La-0

x

m

Jz

Z

J. Viliol'.

.L.

C0

.ti

CY

t4

*o

o6

00

.-

o

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*

L;qc

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0 0.

*) s

ci:to~

0. <

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+1E

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;

on November 10, 2019 by guest

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MAPP'ING EARLY HSV-1 ThANSCHIITS 69

28 ORDER 1

Ie

I

ORDER 2

is

J

FIG. 9. Frequency distributionofearlypolyribosomalR-loopsformedin intactHSV-I(KOS) DNA. The

R-loopfrequency histograms ofHSV-1moleculesoforder1 andorder 2arearrangedsothatthe long regions

arecolinear. Theputative jointbetweenthelongandtheshortregionisattheverticalaxes.

mayonlybecompared with that for full-length corresponding to the order 1 non-R-looped

re-molecules of order 1 (Fig. 9) withrespect tothe gionmappingfrom 45to50% inFig. 9.

general pattern of peaks and gaps, not with DISCUSSION

respect to relative peak heights. The general

patterns are comparable. It is most significant We have obtained histograms detailing the

that HindIlI fragment A can be oriented to frequencydistributionalong thelengthofa

pro-produceanR-loop frequencydistribution which totypicalarrangement oftheHSVgenomeof

R-shows a lack ofR-loops in the region of DNA loops formed by polyribosomal RNA isolated

0

L E C

U

L E S

L

0 0 p E

D

N

N

T

E

R U

A

L

69

Vo1.. 27,1978

on November 10, 2019 by guest

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[image:14.499.67.448.65.536.2]

70 STRINGER, HOLLAND, AND WAGNER

60

II

L 54 XGI

HIO

|

HJ|

HA

HK HL XA E

C 48 ,

U L

E 42

S

L 36

0

0

P 38

E D

I 24

N

T 1

E 1

R

U

A 6

L

,% 8

8 Is 26 38 46 56 68 78 86 98 188

[image:15.499.112.394.67.280.2]

GENOME LENGTH (IC)

FIG. 10. Composite R-loop distributioncompiled from R-loop patternsof HSV-1 DNA restriction fragments

hybridized to early polyribosomal RNA. Purified early polyribosomal HSV RNA was hybridized with

restriction endonucleasefragments,and25individualmoleculesofeachfragmentwereanalyzedasdescribed

in thelegendtoFig. 6 and in thetext.Data derivedfrom the R-loop distribution of Xba Ifragments A and G

and HindIIIfragmentsA, J,IO, K,andL,werecombinedtoformthecompositedistribution. The boundaries

ofthefragmentsareindicatedbythe solid lines. Thejoint region between the short and long segments is at

apositionnear or at82% genomelength.

early after infection

(Fig.

9and

10).

The

align-mentofintact

R-looped

HSVDNAwas

accom-plished by an iterative, subjective

procedure

aidedby

templates

which

designated regions

of the DNAin which

R-loops

are rare.These

tem-plates

wereconstructedonthe basis of

(i)

infor-mation

unambiguously

derived from

analysis

of restriction

fragments

and

(ii)

the inference from therandom-order

R-loop

distribution thata

sec-ond

non-R-looped region

existednearthemiddle of the

prototypical

arrangement of the HSV

DNAmolecule. Thesuccessof this

approach

to

categorizing

and

aligning

thedifferent

arrange-ments of

R-looped

HSV DNA molecules was

established

by

three criteria:

(i)

the

R-loop

his-tograms of the two orders of HSV-1 molecule

show

mirror-image

symmetry when the

histo-grams arealignedsothatregions

corresponding

to the DNA long segments of each order are

colinear (Fig. 9); (ii) the junction between the

longand short DNA segments can beaccurately

located by analysis ofthe symmetry; and (iii)

the R-loop distribution of a prototypical

ar-rangement ofHSVDNAcanbe reproduced by

analysisofR-loops generatedonrestriction

frag-ments which unambiguously represent defined

portionsof the DNA(Fig. 10).

Ourspecificaim inthisstudyistodetermine

the arrangement ofearly viral genes from the

position ofR-loops. In a qualitative sense, the

positions ofearly genes are delineated by the general pattern of peaks in the R-loop

histo-gram.However,there are several factorswhich

complicate the interpretation of R-loop

histo-grams. First, relative peak heights inanR-loop

histogramare afunctionofmore than the

rela-tive concentrations ofdifferent RNAspeciesin

the reactionmixture. TherateofR-loop

forma-tionat any one sitedepends solelyon the

con-centration ofcomplementaryRNA (27); butthe

relative rates ofR-loop formation at different

sites are dependent not only on the relative

concentrations of thetwocomplementaryRNA

species,but also on theirbase composition.The rateofR-loop formation declinesrapidly asthe

reaction temperature deviates from the

Tm

of

the sequence. Sites which differ by 6% inguanine

plus cytosine (G+C) content will exhibit a two-folddifferenceinR-looplevelseven thoughthe

concentrations of their complementary RNAs

are the same (27). Thus, the relative peak

heightsinFig. 9 cannotbe interpreted as

com-pletely indicative of the relative abundance of

different viral RNAspecies. Around 90% of early

viral RNA isolated frompolyribosomes

hybrid-izes to viral DNA withsingle-component

kinet-ics (23). Since RNA classesthat differ two- to

threefold in abundance would have exhibited

readily detectable multicomponent

hybridiza-tion kinetics (25), the two- to threefold

differ-,J. Viliol'.

on November 10, 2019 by guest

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MAPPING EARLY HSV-1 TRANSCHIPT'S 71

encesin themajor R-loop peak heightsseen in

Fig.9 areprobably primarilydue todifferences

in base composition and not to differences in RNAspecies abundance.

While base composition can definitely influ-enceR-loopfrequencies, its effect is insufficient toexplain all thegapsandareas of rareR-loops

which appear in the R-loop histogram. This

conclusion is basedonthefollowing. (i) Notall regions of the HSV genome with higher than

average G+C content exhibit low R-loop

fre-quencies. Comparison of Fig. 9with partial de-naturation mapsof HSV DNA (7, 10) reveals that whilesomehigh-G+C regions exhibitalow frequency ofR-looping, R-loops occur athigh frequency in thehigh-melting regions at 5 and 67% of thegenomelength. (ii) The region

with-outR-loops which extends from43 to50% of the genomelength in Fig.9includes low-meltingas

wellashigh-meltingsequences (7). (iii) Russell and Wilkie (personal communication) have

re-cently directly determined the G+Ccontent of

many HSV-1 DNA restriction fragments by nearest-neighbor

analysis.

The DNA fragment correspondingtotheregion running from46 to

50% of thegenomelength lacks R-loops (Fig.9),

but exceeds theaverageG+Ccontentof HSV-1 DNA by only 2%. According to the data of Thomas et al. (27), if significant amounts of RNA

complementary

tothisregion of the

HSV-1 genome were present onpolyribosomes early

after infection, we would expect to see 70% as many

R-loops

in this region as occur in

se-quencescontaining theaverage amountofG+C

forHSV-1 DNA.

Thereis, however,oneregion of HSV-1DNA

lacking

R-loops

in whichG+C contentmay be a

significant

factor.

Nearest-neighbor analysis

(Russell

and

Wilkie, personal

communication) has shownthe

repeated

sequenceswhichbound the short

unique

segmenttocontain the

highest

amount ofG+C in the HSV-1 genome, 72.8%.

The dataof Thomas et al. (27)

predict

that, if

complementary

RNA concentrations were

equal, R-loops

inthe

repeated

sequencesofthe short segment would be 30% as

high

as the frequency of

R-loops

formed in sequences

con-taining the averageamount ofG+C for HSV-1

DNA. Therefore,

R-loops

in theshort-segment

repeated

sequences may not

accurately

reflect

the amount of early mRNA transcribed from

this part of the viralgenome.

Just as R-loop histogrampeak heights must

be

interpreted

as afunctionof

multiple

factors, R-loop histogram peak widths must be

inter-preted in the

light

of the

following

considera-tions. The major component of early viral

mRNA is

homologous

to20 to 25%of the viral

DNA,with the best value on the orderof 22 to

23% (23, 25). Since the length ofduplex DNA

involved in R-loops shouldbe twice the amount

ofviral DNA driven into hybridbyearlyRNA,

thisRNA should produce a histogram with

ma-jor R-loop peaks covering40 to50% of itslength.

However, measurement inaccuracies tend to

broadenR-loop peaksand increase the portion

of the histogram covered by R-loops. The

pri-mary source of measurement inaccuracies in

these

experiments

is thelength uncertainty

in-herentinworking with large linearDNA

mole-cules.Apopulation of linear DNA molecules

will

exhibit a rangeof lengths, evenin referenceto

internal

length standards. Sincethere is no

ab-solute length criterion, molecules falling within

a narrow range of lengths are accepted a

full

size. HSV-1

(KOS)

DNA hasameanmolecular length of28.1±0.7

4XRFII

DNAunits (30). In

this study,wehave acceptedasintactany mol-ecule measuring between 27 and 29

4XRFII

DNA units. Mostof these moleculeswereintact

HSVgenomes, andanyvariation inlengthwas

corrected by

normalization

to a standard size. However,someof themoleculesacceptedasfull size may in fact contain less than a full HSV genome. The

R-loops

inthesemolecules willbe

laterally

displaced from their true positions, thereby tendingtobroaden thepeaksin the R-loop histogram.

The degreeto whichmeasuring inaccuracies broaden R-loop peak widths can be estimated from

R-loop

studieson adenovirus hexon

mes-senger RNA. The size of

R-loops produced

by

purified hexonmessageis known (2). Individual R-loop sizes correlate well with the size of the

message, butthepeak inthe R-loop histogram

corresponding to the hexon gene is two times widerthan the hexonmessagewhen the

peak

is measuredattheabscissa. The hexonpeak width isequaltothat

expected

from the hexon

R-loop

size when the

peak

ismeasuredatone-third its height (17). It is therefore reasonableto

regard

the

peak

widths atone-third their

heights

as a

more accurate measure of the amount of the

genome

represented

as

R-loops

than the

peak

widthsatthehistogram abscissa.

Whenthepeak widths of the order1 ororder

2molecules of HSV-1 in

Fig.

9aremeasuredat

one-third

peak height,

the totalamount ofthe

genome

represented

as

R-loops

is about 50%of

thelength of the HSV-1genome.Thismaybe a

high

estimate

of theactual

length

of thegenome

abletoform R-loopswith the

major

component

ofearly

polyribosomal

RNA, since the width of theR-looppeakin theshort segment is inflated

bythe short segment

being

represented

inboth

itspossible orientations.With this

qualification,

the extent of the HSV-1 genome

forming

R-loopswith the

high-concentration

classof

early

Vo.. 27,1978

on November 10, 2019 by guest

http://jvi.asm.org/

72 ST'RINGER, HOLLAND, AND WAGNER

polyribosomal HSV RNA is ingood agreement

with valuespredicted by DNA-RNA

hybridiza-tionexperiments.

Apart from a loss ofresolution, the factors

that complicate the interpretation of R-loop

datain quantitative termsdo notinfluence the

qualitativesignificance of the data. The resolu-tion reported here issufficient to establish the

distribution ofearly viral genesand tosuggest

theirspecific locations. The patterns generated

by R-looping intact HSV DNA with

polyribo-some-associated viral RNA present early after

infection indicate that this class of viral mRNA is transcribed from genes distributed through

the genome.

Inthe short segment of theR-loophistogram,

there appears a single peak which is roughly

symmetrical about the centerof the short

seg-mentand extendsslightly into the inverted

re-peat sequences which bound this part of the

DNA. This symmetry isexpected,sincewehave

notdiscriminated betweendifferentorientations

of the short segment. It is apparent that the

unique sequences in the short segment are

ex-tensively transcribed. Sincewe are not dealing

with asingle orientation of the short segment,

theR-loop histogramdoesnotreveal thedetails of the distribution of the early genes in this

segment.However, wecaninfer fromanalysisof

the individual molecules shown inFig.8thatthe

earlygenesintheuniquesequencesof the short

segmentarenotcompletely confinedtooneend

of the segment, but are distributed across its

length.

The distribution ofR-loop peaks in the long

segment of the DNA is complex. Atthe lowest

level ofresolution, the segment is divisible into

twolargeareasoftranscriptionalactivity. These

regions span the length of the long segment,

indicating that the early genes which occur in

this segmentaredistributedalongitslength and

are notcompletely contiguous. The resolution of

thelong-segment R-loop pattern is increased if

the pattern is examinedatone-third the

individ-ualpeakheightsorifloopsoccurring inonly one

molecule (4%) are considered a "background"

,1. Vilmlwo.

level. Severallarge R-loop peaks arediscernible,

two ofwhich fall in the long-segment repeated

sequences while the restoccurin theunique part

of thelong segment. Some of these peaks

prob-ably represent more than oneearly gene, since

they are much larger than the average viral

message size, which is 20S, corresponding to

2,000 nucleotides in length (18, 23). Although

theearly genesare clearlynot completely con-tiguous, they may be clustered in groups. Broad

R-loop peaks may indicate where tightly

clus-teredearly genesoccur.

The R-loop data ofFig. 9 are interpreted in

Fig. 11 as an early transcription map of the

prototypical order 1 of HSV DNA. Fourteen

regions of the DNA are R-looped at relatively

high frequencies. Theseregionsrepresent35% of

the total DNAlength; R-loopsoccurwith

mod-erate frequency in regions representing an

ad-ditional 15% of the HSV-1 DNAlength. Of the

50% of the DNA length in which R-loops are

infrequent, 35% show some R-looping and 15%

show no R-loopsat all. Some of the

contribu-tions tothe regions with low numbers ofloops

areduetopositional uncertainties,while others may well be a result of the low-concentration speciesof viral RNAfoundearlywhich

hybrid-izes to more viral DNA than does the major

componentofearly RNA.

While the transcription map in Fig. 11 does

not resolve the locations of individual early

genes, it definitely differentiates regions of the

DNA that are notrepresented as major

compo-nentsofearlymRNAfrom regions of DNA that

are represented as early mRNA in high

abun-dance. As a moderate-resolution transcription

map,then, R-looping definitely establishes that

theearlygenesarescattered about the genome. This conclusion agrees with results obtained by

liquid hybridizationofasubset of the totalearly

viral transcripts (a HSV RNA) to different

re-strictionfragments(13).Furthermore, Clements

and co-workers(6),using the blot hybridization

technqiues ofSouthern (22), have shown that

early HSV transcripts hybridize to regions

throughout the HSV-1 genome. Our resultsare

IuOOuO

oD O

mm

mm

no

xc xc

1 XF I XE I

Oe

6 is 26 36 40 56 60 76 86 96 168

GENOKE LENGTH (Ic)

FIG. 11. Schematic summary ofthe early polyribosomal RNAR-loopdistribution on the order 1prototypical

arrangement ofHSV DNA. The positions of Xba I endonuclease cleavage sites are indicated. The arrow marks theposition ofthelongsegment-short segmentjunction. Closed and open boxes delineate regions of

highandmoderateR-loopingasdetermined fromanalysis of the orderIhistogram displayed in Fig. 9. (-)

Regionsin which greater than 8% of themolecules bearR-loops; (O) regions inwhich4 to8% of the molecules

bearR-loops.

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[image:17.499.112.395.544.602.2]

MAPPING EARLY HSV-1 TRANSCRIPTS

also consistent with the results of polypeptide

mapping studies which have shown the early

genes to be distributed throughout the HSV

genome (Morseetal., inpress; Wilkieet al., in

press). When the histogram is interpreted as a

higher-resolutiontranscription map,significant

regions of the DNA are revealed as not being

represented as large amounts of mRNA early

after infection.

ACKNOWLEDGMENTS

This research was supported by Public Health Service

grantCA-11861fromtheNational CancerInstitute andan

institutionalgrantfromtheAmericanCancerSocietytoJ.R.S. J.R.S.wassupported bypredoctoral traininggrant CA-09054 from theNational CancerInstitute.

Wethank S. Malan forprovidingcomputerexpertise,M. Rice for excellent technical assistance, and N. Wilkie for communicatingnot-yet-publisheddata. Thehelpof L. Howell is alsogratefully acknowledged.

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