Structures of two spliced herpes simplex virus type 1 immediate-early mRNA's which map at the junctions of the unique and reiterated regions of the virus DNA S component.

0022-538X/81/010431-14$02.00/0

Structures

of Two Spliced

Herpes

Simplex

Virus Type 1

Immediate-Early mRNA's Which

Map at the Junctions of the

Unique and Reiterated Regions

of the Virus DNA S

Component

ROGER J. WATSON,l* MARJORIE SULLIVAN,2 ANDGEORGE F. VANDE WOUDE' Laboratoryof Molecular Virology, National CancerInstitute,'andLaboratory of Molecular Genetics, National Instituteof Child Health and HumanDevelopment,2National Institutes of Health, Bethesda,

Maryland 20205

Wehaveexaminedthe structures of two herpes simplex virus type 1

immediate-early (IE) RNAs (IE mRNA-4 andIE mRNA-5) which map at the junctions of

the unique (Us) and reiterated regions

(TRs/IRs)

of the virus DNA short

component.Hybrids between IE cytoplasmicRNA andherpes simplex virus type

1DNArestriction fragmentsweredigestedwithsingle-strand-specific nucleases

S1 and exonuclease VII, and the productswere analyzedby agarose gel

electro-phoresis. Data obtained with the nuclease digestiontechnique were confirmed by

electron microscopy of R-loopstructures formedwith polyadenylated IE RNA

and virus DNAfragments. Itwasfound thatboth IE mRNA-4 and IE mRNA-5

contained a260-base 5'-terminal cotranscript which mapped at equivalent loci

within

TRs/IRs.

These 5'-terminal sequences were shown to be spliced to

3'-terminal cotranscripts of1,450 bases (forIE mRNA-4) and 1,540 bases (for IE

mRNA-5). The 3'-terminal cotranscripts containedsequences encoded by both

TRs/IRs

andopposite ends of

Us,

indicating that the intronscontained by the IE

mRNA-4 and IE mRNA-5genes, found to be approximately 150 base pairs in

size,mapped

entirely

within the reiteratedsequences. Thedatasuggest that these

genes maycontaincommonanduniquecomponents, and theimplicationsof this

modelarediscussed.

The

transcriptional

programofherpessimplex

virus type 1

(HSV-1)

is divided into at least

three

phases,

namely,

the

immediate-early (IE),

early,

and latestages

(9,

11,15,

23).

TheIE stage

of

transcription

is

transient,

and the

accumula-tion of IE

RNAs,

whichmap withinrestricted

regions of the virus genome

(9, 14),

is

readily

apparent

only

in cellsinfected with virus in the

presence of

protein synthesis

inhibitors or in

cells infected with certain HSV-1

temperature-sensitivemutants atthe

nonpermissive

temper-ature

(26).

Synthesis

of

early

and late

RNAs,

whichmapatloci

throughout

the virusgenome

(9,15),is

continuously dependent

uponthe pres-enceofa

functional

IE

product (27).

Theswitch from

early

tolate

transcription

is

dependent

also

upon the onsetofvirus DNA

replication

(2, 9,

11, 15).

The structure of the linear double-stranded DNAgenome ofHSV-1isremarkablein that it

consists oftwo

unique

DNA

regions,

UL

(115

kilobase

pairs)

and

Us (14

kilobase

pairs),

each

flanked

by

different inverted

repeated regions,

TRL/IRL

(9

kilobase

pairs)

and

TRs/IRs

(6

kil-obase

pairs),

respectively

(20).

In

addition,

there

is ashort direct repetition (the "a" sequence)

present ateachterminus of the DNA,aswellas one or more

copies

ofthissequence atthe IRL/

IRS

joint (25). A consequence of this unusual

structure is that the unique DNA regions, UL

and

Us,

are found inverted relative to one

an-other, thus

giving

risetofourgenome

arrange-mentswhichareusuallypresentin DNA

prep-arationsin

equimolar

amounts(7, 12).Afurther

consequenceofthisunusualgenome structure is

thatgenes which map intheinverted reiterated

sequences are present in two molar amounts.

Thisisof

particular

relevancetothegenes which are

expressed

atthe IE stageofvirus

replication,

since many of these map, at least

partially,

within these reiterated

regions

(28). The

approx-imate

genome locations of the fivepredominant

IE mRNA species, designated IE

mRNA-1

through-5 (8), have been described

previously

(8, 28). IE mRNA-1 and IE mRNA-3, which have sizesof3.0kilobases

(kb)

and4.7

kb,

map

wholly

within thereiteratedsequences

TRL/IRL

and

TRs/IRs, respectively,

whereas the 2-kb

specieIE mRNA-2 maps in

UL.

Two additional

2-kb

species,

IEmRNA-4andIE

mRNA-5,

were 431

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foundtomap at,or closeto,thejunctionsofUs

andTRs/IRs. From these data,and from

anal-ysesof the orientation of these IE mRNA's (8), it was suggested that IE mRNA-4 and IE mRNA-5 containcommon 5' terminimappingin

TRA/IRs

andunique3' terminimapping at op-posite ends ofUs (28).Wereportthe results of experiments designed to fine-structure map IE mRNA-4 and IE mRNA-5, using the nuclease digestion technique of Berk and Sharp (3) and R-loop analysis (24). We show that these two

mRNA's contain a 5'-terminal sequence,

map-ping inTRs/IRs, spliced to non-colinear tran-scripts(cotranscripts)containingboth reiterated anduniquesequences.

MATERIALS AND METHODS Cells andviruses. Vero cellswerepropagated as monolayers in 150-cm2 flasks, usingRPMI 1640 me-diumsupplemented with10%calfserum.HSV-1 strain Pattonstocksweremade byinfectingVero cells at low multiplicities of infection.

DNAfragments. The HSV-1 DNA fragmentsused wereclonedincoliphageXgtWES.Band subcloned in plasmid pBR322 (10; K.Denniston-Thompson,L. W. Enquist, and G.F.VandeWoude,submitted for pub-lication). Deletions of the EcoRI Hfragment, to gen-erateNYS-8 andSRS-4,wereselectedfrom XgtWES-EcoRI-Hclones DEC24 and DEC36(10), as described byK.Umene andL.Enquist (manuscript in prepara-tion).

Restriction endonuclease digestion. The con-ditions used for cleavage of DNA with restriction endonucleases have beenreported previously (Dennis-ton-Thompsonetal., submittedfor publication).

LabelingHSV-1 DNAfragments invitro. DNA fragments were labeled at the 5' termini, using

[y-32P]ATP(-2,000 Ci/mmol)andpolynucleotide kinase

(18). BamHI DNA fragments (-1

Ag)

were labeled at the3'terminiin areactionvolumeof 100

pl

containing 60mMTris-hydrochloride (pH7.5), 6 mMMgCl2,100 yCi of[a-32P]GTP (>300 Ci/mmol), and 5 U of the Klenowfragment of Escherichia coli DNA polymer-aseI(Boehringer MannheimCorp.) by incubating for 30min at15°C. Theseprocedures enabled theSaIB5 andB4R1probes tobelabeledtospecific activities of

105to 106

cpm/,g.

32P-labelingof DNA fragments by nick translation with DNA polymerase I has been described previously (26).

Labeling HSV-1DNAfragmentsinNivo. Plas-midscontainingHSV-1 DNA inserts were32plabeled by growing5ml of E. coli cultures to an optical density at 550nmof 0.7 in a definedmediumcontaining200

,uM K2HPO4 and200,uCi ofcarrier-free 32p;per ml. Chloramphenicol wasthenadded to 100,g/ml, and thecultureswereincubatedovernightat370C. Cells

werelysed by incubationfor 5 min onice in a volume of 1.5ml containing 50 mM Tris-hydrochloride (pH 8.0),60mMEDTA,15% sucrose and 1 mg of lysozyme per ml. Anequalvolumeof10% Triton X-100-50 mM Tris-hydrochloride (pH 8.0)-60 mM EDTA was then added,and cellDNA was pelletedbycentrifugation

for 1hat30,000rpminaBeckmantype 40 rotor. The supernatantwasextracted twice withanequal volume ofphenol-chloroform (1:1) andoncewith chloroform. An equal volume of 0.3 M NaOAc was added, and nucleic acidswereprecipitated by the addition of 2.5 volumes of ethanol and storage at -20°C. Nucleic acidswererecoveredbylow-speedcentrifugation and thendigested with EcoRI. Nucleic acidswereresolved byelectrophoresison anagarosegel, and the linearized plasmid DNA bandswerevisualizedby ethidium bro-midestaining. Theappropriate DNAfragmentswere

electroeluted from the gel and thendigested with a second restrictionendonuclease, and the required frag-ments wereisolatedby gelelectrophoresisasbefore.

Virus infection and RNA extraction.Cell motio-layers, preincubated for 30 min incomplete medium containing200 jagofcycloheximideper ml, were in-fected with HSV-1atamultiplicityof infection of 25 and maintained in this medium forafurther 6 hat

37°C.CytoplasmicRNAwasthenprepared by lysisof the cells with Nonidet P-40, removal of nuclei by centrifugation, and phenol-chloroform extraction of thesupernatant fraction (28). RNAwasstored in a

small volume of distilledwaterat-70°C.Where ap-propriate, polyadenylated RNA was selectedon an

oligodeoxythymidylate-cellulose column,asdescribed previously (28).

RNA/DNA hybridization and nuclease treat-ment. To perform a typical hybridization, approxi-mately0.1,ugof theappropriateDNAfragmentwas

ethanol precipitated with 10,ug of cytoplasmic IE RNA.The nucleic acidswerepelletedinaBeckman microfuge, and the pellet was washed once with ethanol. The pelletwasdried and then dissolved in20

pl

of 90% formamide-400mMNaCl-1 mM EDTA-40 mM PIPES [piperazine-N,N'-bis(2-ethanesulfonic acid)],pH6.4.Afteroverlayingwithparaffinoil, the microfuge tubescontainingthehybridizationmixwere

incubated for 10min at 70°C. The tubeswere then transferredto a waterbath heldat56°Cand incubated overnight. Afterincubation,thesampleswerediluted with10volumes of either S1 buffer(250 mMNaCl, 1 mM ZnSO4, 5% glycerol, 30mM NaOAc, pH 4.6) or

exonuclease VII buffer(100mMNaCl,8mMEDTA,

10 mM/3-mercaptoethanol, 10 mM

Tris-hydrochlo-ride, pH7.9). S1digestionswereperformed by adding 2,000U of theenzyme(Boehringer Mannheim Corp.) andincubatingat45°Cfor30min.Exonuclease VII digestionswereperformedbyaddinganappropriate amount of the enzyme, purified from E. coli strain M0676 bythe method of Chase and Richardson (6), andincubatingat45°Cfor 1 h.Digestionswere ter-minatedbyadding 10

,tg

ofyeasttRNA and 2.5 vol-umesof coldethanol.

Gelelectrophoresis and autoradiography.The products ofS1 andexonuclease VIIdigestions were collected by centrifugationand analyzed by agarose gelelectrophoresis. Electrophoresisundernative con-ditions wasperformedon1.4% agarosegels cast in 20 mM NaOAc-2 mM EDTA-40 mM Tris-hydrochlo-ride, pH7.8.Electrophoresis underdenaturing condi-tionswasperformed on 1.4% agarose gels cast in 30 mM NaOH-2 mM EDTA. Electrophoresis buffers werethesame asthoseinwhichthegelswere cast. CertainS1-digested sampleswereanalyzedona

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dimensional gel system, as described by Kamen et al. (16).Afterelectrophoresis, gels were either dried un-dervacuum andsubjectedto autoradiography (9) or prepared for DNA transfer to nitrocellulose (22) or diazobenzyloxymethyl-cellulose paper (1) obtained fromSchleicher & Schuell Co.

R-loop analysis. As initial steps, the EcoRI H fragment was excised from hybrid phage, and the plasmid pKL43 was linearized by digestion with re-striction endonuclease EcoRI. Approximately 0.1-pg

amountsof EcoRI-cut DNAwerelyophilized with 0.1 pg ofpolyadenylated cytoplasmic IE RNA. Nucleic acidswereredissolved in4plofR-loop hybridization buffer (75% formamide, 100 mM Tricine [Sigma]-NaOH, pH 8.0, 500 mMNaCl, 10mMEDTA) and incubatedovernightat56°C in sealed glass capillaries. Afterincubation,thesampleswerediluted and spread on ahypophase asdescribedpreviously(19). Visual-ization ofmolecules,measurementof their length, and normalization ofR-loop-containing moleculestogive fractional lengths were also performed as reported previously (19). Generally, it was found that greater than80% of the appropriate DNA fragments contained R-loops.

RESULTS

Mapping IE mRNA-5 bynuclease

diges-tion and alkaline agarose gel

electropho-resis. Theuseofsingle-strand-specificnucleases

S1 and exonuclease VII tomap the regions of

DNAprobes hybridized,under highstringency

conditions (5), to complementary RNA

se-quenceshas beendescribedby BerkandSharp

Map Units

0

0.1,,

IE mRNAs

I

It

(3, 4). The basic principle of this procedure is that nuclease Si digests all of the

single-strandedregions oftheDNAprobe unprotected

by duplex formation with RNA, whereas exo-nuclease VIIdigests single-stranded DNA only

ateither terminus ofaduplexstructure. Hence,

a single-strandedDNAloop boundedoneither

sidebyRNA/DNAduplexes,whichisformedat

the splice point of RNA, serves as a substrate

for nuclease Si but isnotdigested by exonucle-aseVII.

Comparison,

then, of the sizes of RNA/

DNAhybridsprotected againstthesenucleases

enables thepresence ofsplicedtranscripts to be

recognized.

Initially,weusedamodification oftheabove

technique toanalyze the colinearand

non-coli-nearregions ofIEmRNA-5. In thismodification,

the DNA probes whichare hybridizedto

unla-beled RNA are labeled at a unique 3' or 5'

terminus

generated

by

restriction endonuclease

digestion.Consequent requirementsof this

tech-iiique

arethat theuniquely labeledDNAstrand

is of the opposite polarity to the RNA to be

analyzed and that the labeled terminus of this

DNAprobemaps withinsequencesrepresented

by this RNA.

Previousanalyses ofIEmRNA-5 (8, 28)

indi-cated thatthe 3' terminus of this

transcript

maps

in the BamHI 6-BamHI 5 DNA

fragment

and the5'terminusmapsinBamHI4-BamHI3(Fig. 1). The probes selected foruse in this

analysis

0.8

0,9

1 3

*1

2

1.0

5

3

4

TRL

U

_L

IR,

IRS

r

I

i/

[

EctR

_Eco

_RI

FIG. 1. Approximate physicalmaplocations anddirectionsofsynthesis ofthefive predominantIEmRNA's, IE mRNA-1 through-5(8,28).Also shown arethe restriction mapsofthe Sregion ofHSV-1 DNAforthe

enzymesEcoRI and BamHI. TheBamHI sitesaredesignated BlthroughB8. ThemaplocationoftheEcoRI Hfragmentis indicated.

us

TRS

B5 4

B7E3oRB,B1

EcoRl=

I

Bi 3 as

lr,L-I

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[image:3.496.49.441.396.620.2]

were,therefore, theSall/BamHI5(SalB5)

frag-ment 3'terminally labeledatthe BamHI 5site

and the BamHI 4-EcoRI (B4R1) fragment 5'

terminally labeled atthe BamHI 4 site (Fig. 1

and 12).

Togeneratethe aboveprobes,wetook

advan-tageoftwo

plasmid

clones:

pkL63,

which carries

the permuted B1B5 fragment containing the

SaIB5sequences, and

pkL43,

which carries the

B4R1 fragment. These DNA fragments were

subcloned inpBR322 fromanHSV-1-defective

DNA EcoRI

fragment

as described previously

(Denniston-Thompsonet

al.,

submitted for

pub-lication).DNAsofpkL63 andpkL43wereeach

cleaved withBamHI and then labeled

respec-tively

at the 3' and 5' termini as described in

Materials and Methods.

Subsequent cleavage

of

pkL63 with Sal I and

pkL43

with EcoRI

yielded

the

uniquely

labeled SalB5 and B4R1

frag-ments, and these were isolated

by preparative

gelelectrophoresis. These

probes

werethen

hy-bridizedtototal

cytoplasmic

IE

RNA, using high

stringency conditions. After

hybridization,

the

sampleswerehalved; one-halfwasdigested with

nuclease S1 and the remainderwasdigested with

exonuclease VII. Theproductsofnuclease

diges-tion were

analyzed by

electrophoresis

on an

alkalineagarose

gel.

The

gel

wasthen dried and

[image:4.496.63.252.378.565.2]

subjectedto

autoradiography

(Fig. 2).

FIG. 2. CytoplasmicIE RNAwashybridizedtothe BamHI4-EcoRI(B4R1)andSalI-BamHI5(SalB5) DNAfragments 32p labeledatthe5' and3'BamHI termini, respectively. The labeled DNA sequences

protectedagainst nuclease Sl and exonuclease VII

(Exo7) digestion were analyzed by electrophoresis

through analkalineagarosegel and autoradiogra-phy. Tracks marked M contain DNA molecular-weight markers, and the sizes (in bases) of these markersaregiventotheleftandright of the figure.

Hybridization of the 5'-labeled B4R1 DNA

probetocytoplasmic IE RNA resulted in

pro-tection ofapredominant 785-base (b) DNA

frag-mentagainstS1 digestion andtwofragmentsof

1,220and1,600bagainst exonuclease VII

diges-tion (Fig. 2). Hybridization of the 3' terminally

labeled SaIB5 probe to cytoplasmic IE RNA

resultedinprotection ofapredominant band of

570 b against S1 digestion and a predominant

band of590bagainst exonuclease VII digestion

(Fig. 2). Minor bands of approximately 900 and

1,250b werealso detectable in the SaIB5

exo-nucleaseVII-digested sample. Before

unambig-uousinterpretation of these data could be

exer-cised, further control experiments were

per-formedto ensure thatprotection of these

frag-mentsagainst nucleasedigestionwasdependent

upontheaddition of

virus-specific

RNA.

5'-labeled B4R1 and3'-labeled SalB5 probes

were

incubated,

as

before,

with either

cytoplas-mic IE RNAor anequivalentamount ofyeast

tRNA. After nuclease S1 or exonuclease VII

digestion,

the products were analyzed as

de-scribed above. The results(Fig. 3) indicated that

protection of the 785-b B4R1 and 570-b SaIB5

fragments against S1 digestionand protection of

the 1220-b B4R1 and 590-b SalB5 fragments

against exonuclease VII digestionwere

depend-ent upon the addition of virus-specific RNA.

However, protection of the 1,600-b B4R1 and

1,250-b SaIB5 fragments against exonuclease

VIIdigestionwasindependent of the addition of

virus-specific

RNA. As thesizes of these latter

fragments

were equivalent to the sizes of the

B4R1 andSaIB5 probes, failuretobe digested

by

exonuclease VIImayhave been the resultof

limited reassociation or folding ofthese DNA

fragments

atthe termini.

The protection of minorSalB5 fragments of

750and850b againstexonuclease VIIdigestion

appeared

tobedependentupon theaddition of

virus RNA (Fig. 3). If these are the result of

splicing

atthe 3' terminus ofIE mRNA-5,then

these splices must be present in a very small

proportion of transcripts.

From

datadetailed above, thefollowing

con-clusions were drawn. First, the 5' terminus of

the colinear transcript maps 785 b upstream

fromthe BamHI 4 site, and the 3' terminus of this

colinear

transcript maps 570 b downstream

from theBamHI 5site. Second, from the

differ-ence in size between the B4R1 fragment pro-tected against S1 digestion and that protected

against exonuclease VII digestion, the presence

of a non-colinear transcript

spliced

to the 5'

terminusof the majorIE

mRNA-5

sequencewas

apparent.Third, theclosecorrelation in thesizes

ofthe

SaIB5

fragmentsprotected against

S1

and

exonuclease VII digestions implied that there

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SPLICED HSV-1 mRNA's 435

A

on

a

lSw

'X

.--a

iz-- ~ ~ ~ ~ ~ ~

"'

FIG. 3. Either(A) the B4RI DNAfragment32plabeled at the BamHI 5' terminus or (B) theSalB5DNA fragment32plabeledattheBamHI3'terminuswasincubated under hybridization conditions with (+) or without(-)theadditionof cytoplasmicIE RNA. WherenocytoplasmicIE RNA waspresent, anequivalent amountofyeast tRNA wasadded. After digestion ofthese samples with nuclease Sl or exonuclease VII (Exo7), theproductswereanalyzed by electrophoresis throughanalkalineagarose gel andautoradiography. Tracks marked M containmolecular-weight markers,the sizes(inbases)of which are indicated.

was nospliceatthe 3' terminus ofIEmRNA-5: thesmalldifference in size observedwas

proba-bly

aresult of the

processive

activity of

exonu-clease VII(6),

thereby

notdigestingaway

single-stranded DNA

completely.

Fourth, assuming

that the small

(185-b)

BamHI 5-BamHI4

frag-mentsequencesarepresentin the

major

colinear

region

ofIEmRNA-5

(an

assumption

later

con-firmed), the total size of this colinear transcript

is 1,540b

(i.e.,

785+570 + 185

b).

Analyses

of

RNA/DNA hybrids.

The

ma-jor product of

Si digestion

ofaduplex formed

between a

spliced

RNA molecule and a DNA

fragment encoding that

transcript

is an RNA/

DNA

hybrid

whose size

corresponds

tothe total

length of the

spliced cotranscripts,

the RNA

strand of thehybridholding togetherthetwo or morenon-colinear exons. Inpractice,inausually

minorprotection of these RNA/DNA hybrids,

nicking of the RNAat the splice point occurs

(4). Thus,nativegel

electrophoretic

analysis

of

theSl-digested hybridsformed between the

5'-labeled B4R1 fragment and

cytoplasmic

IE

RNAshould revealtwobandsupon

autoradiog-raphy,thelargercorrespondingtothemajorIE

mRNA-5sequence

(the

3'-terminalcotranscript)

plus the 5'-terminal cotranscript and thesmaller

correspondingtothe3'-terminalcotranscript

se-quencealone. Such aresult wasobserved (Fig.

4). Native gel analysis of the S1-digested,

3'-labeled SalB5

DNA/IE

mRNA-5hybrid (Fig. 4)

revealed,

as expected, only one predominant band.

The sizes ofthese RNA/DNA

hybrid bands,

as measured with duplex DNA size

markers,

weregreater than

predicted

(Fig. 4).

For

exam-ple, the apparent molecular

weights

of the

B4R1/IE mRNA-5

hybrids

were

approximately

900 and 1,150

b,

whereas the smaller ofthese

twobands would be

expected

tobe785b. This

apparently

aberrant

electrophoretic

mobility

of

RNA/DNA hybridswas

consistently

noted for

duplexes of<1

kb,

although

notfor

larger

hy-brids. However, the difference in molecular

weightbetween theobserved

B4R1/IE

mRNA-5 hybrids

suggested

that the

length

of the

5'-terminal

cotranscript

was

approximately

250b.

Two-dimensional

gel

analysis

ofBamHI

4-EcoRI

DNA/IE

mRNA-5

hybrids.

To

con-firm that the two B4R1

DNA/IE

mRNA-5

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[image:5.451.81.368.61.327.2]

436 SULLIVAN, AND

-S

13S3 s

1076

9-0 so

SWr i

FIG. 4. Cytoplasmic IE RNA was hybridized to

either a 3', 32P-labeled SalB5 or a 5', 32P-labeled B4RJ HSV-1 DNAprobe, and RNA/DNA hybrids protected against nuclease Si digestion were

ana-lyzed by electrophoresis throughanativeagarosegel andautoradiography. Thepositions of duplexDNA size markersareindicatedin the tracklabeled M.

bands observed by neutralagarose gel analysis were generated by duplex formation with a

spliced RNA molecule, these RNA/DNA hy-brids were subjected to two-dimensional gel analysisasdescribedby Kamenetal. (16). The

hybridswere runfirst, under native conditions,

downone side ofasquare agarose gel. The gel wasthen soaked in alkalito destroy the RNA strands oftheduplexes and turnedthrough900, andelectrophoresiswasresumed under alkaline

conditions. Unlabeled HaeIII-digested 4X174 replicative-form DNA fragmentswere

coelectro-phoresed for size markers. After electrophoresis, the DNAfragments were transferred to nitro-cellulose. Autoradiography ofthis blot (Fig. 5) revealedtwospots.The electrophoreticmobility of thesespotsunder native and alkaline condi-tionswasdetermined withrespect tothe 4X174 DNA size markers, which were visualized by

hybridization with 32P-labeled 4X174 DNA(not

shown). Under native conditions,the mobility of thesespotscorrespondedtothe sizes of thetwo

bandsobserved withtheB4R1 probeonnative

gel analysis (Fig. 4). In the second dimension, under alkaline conditions, the electrophoretic mobility of both spotswassimilar,

correspond-ingtoasize of785b. These data indicatedthat

undernative conditions the RNAstrand,

repre-sented by the

larger

spot, held

together

non-colinearly

transcribed

DNA

fragments.

Alkali

treatment of this

RNA/DNA

hybrid

released

these DNA fragments,

only

one of which was

labeled.

Size

of

the IE mRNA-5

5'-terminal

cotran-script.

Use ofend-labeled DNA

probes

doesnot

allow direct size determination of each

non-col-inear

transcript.

Todetermine the size of the

5'-terminal

cotranscript

ofIE

mRNA-5,

aninvivo

32P-labeled

B4R1 DNA

fragment

wasusedas a

probe for the

Si analysis.

Included in this

ex-periment also were

analyses using

the in vivo

labeled

SalB5

fragment

and the

unseparated

fragments

produced by cleavage

ofB4R1 with

XhoI. Sequences

protected

against

Si

digestion

by

hybridization

to

cytoplasmic

IE RNA were

resolvedby alkalineagarosegel electrophoresis.

Autoradiography of thisgel revealed the

pres-ence of thecharacteristic 570-b SalB5 and

785-b B4R1

fragments

(Fig.

6). Protection ofaB4R1

sequence of260 b was alsoapparent, andthis thenmayrepresentthe5'-terminal

cotranscript.

Itcouldnotbeexcluded that

protection

of this

260-b fragment resulted from

hybridization

of

the B4R1

probe

to IE

mRNA-4,

which was

previously

reported to share 5'-terminal se-quenceswith IE mRNA-5

(28).

It is shown below

that IE mRNA-4 and IE mRNA-5 contain a

similar

5'-te:rminal

sequence.

Cleavage

of B4R1

withXhoI before

hybridization

resulted in

pro-tection of bands of

approximately

250and450b

(Fig. 6), as

predicted

from the restriction map

for thisenzyme

(Fig. 12).

R-loop

analysis

of IE mRNA-5 and

IE

mRNA-4.Tosubstantiate data obtained

by

the

nuclease

analyses,

the 5' terminusofIE

mRNA-5 was mapped by

using

the

R-loop technique

(24). Cytoplasmic polyadenylated IE RNA was

incubated with EcoRI-cutpkL43 (containingthe

B4R1 fragment insert) under conditions

favora-ble for

R-loop

formation. The

positions

of

R-loops in the DNA molecule were mapped by

electron microscopy: orientation of the

mole-culeswasfacilitatedby the 4.0-kbpBR322 DNA

sequence of EcoRI-cut

pkL43,

in which no

R-loopswere observed.

The two typesofR-loopstructures observed areshown inFig. 7.Some moleculescontained

asingle R-loop,witha smallspur near one end

of the loop. Other moleculescontained two

R-loops, with the

small

spur

still

present. This

indicated that the spur, which then corresponds tothe intron of the IE mRNA-5 gene, issingle

strandedand may thus form a duplexwith the

complementaryDNAsequences of the displaced

strand. Obviously, the duplex thus formed is

relativelyunstable

thernodynamically.

TheR-loops present in 19 representative

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ecules are shown schematically in Fig. 8. Also

shown is a summary ofthese data, which

indi-cates that the 5'-terminal sequence of IE

mRNA-5 isapproximately500 b from the EcoRI site ofB4R1. This summary also indicates that the sizemeasurementsofthe IE mRNA-5 com-ponents obtained by this analysis are in close agreement with those obtained by the nuclease

digestiontechniques.

To investigate the relationship between the

structureofIE5 and that of IE

mRNA-4, afurther R-loopanalysis wasperformed,this

time using the HSV-1EcoRI Hfragment cloned

inXgt.WES.B. TheEcoRI H fragment contains

thewholeof the Usregion flanked on either side

by

TRs/IRs

sequences (Fig. 12), and thus the

completetemplates for both IE mRNA-5 andIE

mRNA-4 are presentin this DNA. Asummary

of dataobtained in this experiment is shown in

Fig.9. Themajority of unnicked EcoRIH

frag-ments contained R-loops arranged

symmetri-cally at either end of the molecule. In many

instances,introns were noted atboth ends of the

fragments, andthe positions of these were the

same relativetoeach end. This madeit

impos-sible to orient the DNA molecules, indicating

that the structures of IE mRNA-5 and IE mRNA-4 were very similar. A number of

ran-domly occurring and ranran-domly sized R-loops

were noted in some molecules in addition to

those R-loops notedatthe ends.In no instance

weretheserandom R-loops observedtocontain

splice points.

Sequences of IE 4 and IE

mRNA-1353

w-078

-872 '

0

603

'W

310 >

~~~~~~

4~~~~~~~~~~~~~~~A~~~ ~~~ ~~~ ~~~ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

FIG. 5. Nuclease SI-resistant RNA/DNA hybrids formed between cytoplasmic IE RNA and a 5',

32P-labeled B4RI HSV-1DNAfragmentwereanalyzedbyelectrophoresisintwodimensionsthroughanagarose

gel,followed by autoradiography. Thefirstdimensionofelectrophoresiswasunder nativeconditions. Thegel

wasthen soaked inalkali,turnedthrough90',andrununder alkaline conditions. Thepositions ofunlabeled DNAfragmentsize markersareindicated.

37,1981

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438 WATSON, SULLIVAN, AND VANDE WOUDE

FIG. 6. CytoplasmicIE RNAwashybridizedtothe in vivo 32P-labeled HSV-1 DNA fragments SalB5, B4R1, and B4RIfurthercleavedwith XhoI.Hybrids

so formed were treated with nuclease Sl, and the productswereanalyzedby alkaline agarose gel elec-trophoresis andautoradiography. Thepositions and sizes(inbases)ofmolecular-weight markersare in-dicated in the track labeled M.

5.Tofurther

investigate

thestructural

related-ness of IE mRNA-4 and IE

mRNA-5,

it was necessary to determine which sequences these

transcripts share in common. The strategy

adopted was togenerate unlabeledSl-digested

RNA/DNA hybrids between cytoplasmic IE

RNAand DNAfragments comprisingopposite ends of the EcoRI Hfragment. These subfrag-ments weregenerated by selectingsemispecific

deletions of the EcoRI H

fragment

cloned in

Xgt.WES.B,

asdescribed

by

Umene and

Enquist

(inpreparation), andwere

given

the

identifica-tion codes SRS-4 and NYS-8.

SRS-4, then,

con-tained the entire geneof IE

mRNA-5,

whereas

NYS-8 contained that of IE mRNA-4

(see

Fig.

12). The

Sl-digested RNA/DNA

hybrids

so

formed were separated on native or

alkaline

agarose

gels,

and DNAs were transferred by

blotting

to eithernitrocellulose or

diazobenzyl-oxymethyl-cellulose

paper. To these blots

spe-cific nick-translatedDNAfragment probeswere

thenhybridized.

Autoradiographs obtained

by hybridization

of

B4R1 andEcoRI-Hprobestoprotected NYS-8

and SRS-4 DNAsequences,resolvedonanative

agarose gel, are shown in

Fig.

10. As

expected,

B4R1 DNA

hybridized

strongly

to the

RNA-protected sequences of

SRS-4;

the two bands

apparent

correspond

to the

spliced

5'- and

3'-terminal cotranscript sequences (upper band)

and thesequencesof the3'-terminalcotranscript

alone (lower band). This DNA fragmentprobe

hybridized also to the equivalent IE mRNA-4

sequences generated with NYS-8 DNA. This

indicated thatnotonly does the 5'-terminal

co-transcriptsequenceofIEmRNA-4map in

TRs/

IRs,

but so also does part of the 3'-terminal

cotranscript (corresponding

tothe lower of the

two

bands). By

extrapolation,

the intron of the

IE mRNA-4gene, and thus of the IE mRNA-5

gene, maps

wholly

within TRs/IRs. From the

low autoradiographic intensity of the lower

NYS-8 band obtained upon

hybridization

with

the B4R1

probe,

we inferthat only limited

se-quences of the IE mRNA-4 3'-terminal

cotran-script map in reiterated DNA: the control

ex-periment,

hybridization

to this blot of

nick-translated EcoRI-H DNA (Fig. 10), indicated

that the DNA sequence mass in each ofthese

hybridswas

approximately

equivalent, thus

val-idating this inference.It wasalsonoted that the

3'-terminal cotranscript of IE mRNA-4 is

smaller than that ofIEmRNA-5.

Theresults of hybridizationof nick-translated

B4R1 DNA tothe diazobenzyloxymethyl-cellu-lose blot of these RNA/DNA hybrid species, separated on an

alkaline

agarose gel,

confirmed

some of the above observations (Fig. 11). In

addition, the probe hybridized notonly to the

3'-terminal cotranscript sequences of both IE

mRNA-4 and IE mRNA-5, but also to a 260-b

fragmentobtained with both NYS-8 and SRS-4

FIG. 7. Polyadenylated cytoplasmic IE RNAwashybridized to EcoRI-cut plasmid pkL43 (containing a B4R1 HSV-1 DNAfragmentinsert),andR-loopssoformedwereanalyzed by electron microscopy. Represented abovearethetwotypesof R-loopstructures seen.Spurs, corresponding to the IE mRNA-5 gene intron, are indicatedbyarrows.Double-stranded(ds)regions of the hybridsarerepresented by thethickerline,andthe displaced single-stranded(ss)DNA strands arerepresented by the thinnerline.

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439

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5 .0 E z z

0

.3

U

.5

2a

10

15

h.I

Fractional Length

0-5 1

P-5OOb iSOb

1-~~~~~~~~~~~~~~~

±54 ±56 238b 793b *60 *127

FIG. 8. Diagrammaticrepresentation ofa number ofpKL43moleculescontaining R-loops producedas described in thelegend toFig. 7. The measured length of pkL43DNA has been standardizedfor each molecule. The relativepositions of R-loopsare shown above the continuous linerepresenting pkL43DNA. The summary indicates the mean average size (in bases) ofthe two R-loops observed, made with the assumption that the length of pkL43DNAis 5.7kb. Also indicated are the distancefrom the EcoRI-cut terminusof pkL43DNA to thestartofthefirstR-loopandthe sizeofthe intron. Standarddeviationsare

given foreachmeansize estimate in bases.

.0

a

E

z

U

0 2

5 10

[image:10.496.103.395.75.359.2]

Fractional Length 0-5

FIG. 9. Diagrammatic representation of a number of R-loop structures formed between the EcoRI H fragmentandpolyadenylated cytoplasmic IE RNA. The measured lengths of the EcoRI H fragment, which is 15.4kb in size, werestandardized. The relative positions of R-loops are shown above the continuous line representing the DNA fragment. No attempt was made to orient these molecules.

DNA. This sequence, then, corresponds to the 5'-terminal cotranscript. Hybridization to this sequence was not observed with the blot of the nativegel,since DNA fragments of this size did

nottransfertonitrocellulose.Thesizeestimates

of the 3'-terminal cotranscripts obtained with

the denaturing gelwere 1,450 b for IE mRNA

and 1,540 bfor IE mRNA-5. The sizes and map

locationof these mRNA species are summarized

inFig. 12.

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[image:10.496.73.433.441.571.2]

-~~~~~~~.~~

,qt

a"so d

FIG. 10. Cytoplasmic IE RNA washybridized to DNAs of coliphage A (isolates NYS-8 and SRS-4) containingdeletions ofthe HSV-1 EcoRI H fragment. TheHSV-1DNAsequencescarriedin these deleted

phageDNAsareshown inFig. 12.After nuclease S1 digestion, theprotected RNA/DNA duplexes were

coelectrophoresed with

0X174

and pBR322 DNA fragment markers (tracks marked M) on a native

agarose gel. The gelwas then treated with alkali,

andDNAfragmentsweretransferredbyblottingto

anitrocellulosemembrane.Amixtureof32P-labeled nick-translatedpkL43 DNA (containing the B4R1 insert)and

OX174

DNA washybridizedtothe blot, andhybridizationwas visualizedby

autoradiogra-phy. Subsequently, nick-translated EcoRI (Ri H) DNAwashybridizedtotheblot.The sizes(in bases) of DNA marker bandsareindicated.

DISCUSSION

Wehavedescribedthe structures of two HSV-1 IE mRNA's and have provided the first evi-dence that these virus transcripts are spliced.

These datawereobtained with three

independ-ently cloned HSV-1 DNA fragmentsas probes

inthe hybridization reactions, thus miIniizing thepossibilitythat theputativeintrons observed reflectedspecific additionsorrearrangementsof the virus DNA fragments on cloning. Specific

deletions ofthe clonedDNAfragmentsarenot consistent with the experimental data, for

ex-ample, the observation of introns by electron microscopy of R-loopstructures.

The datawe obtained indicate that both IE

mRNA-4 and IE mRNA-5 contain a 260-b

5'-terminal cotranscript. Thesecotranscripts map

atequivalent locationswithin

TRs/IRs,

suggest-ing that the 5'-terminal sequencesof IE

mRNA-4 and IE mRNA-5 are

similar.

More sequence data are required to establish whether these

cotranscripts are identical or, indeed, whether

microheterogeneityexists within the5'-terminal

sequence of each IE mRNA. The 260-b se-quences werefoundtobesplicedto

3'-terminal

cotranscripts of 1,450 b (for IE mRNA-4) and

1,540 b (for IE mRNA-5). These 3'-terminal

cotranscripts share some, butlimited, sequence

homology, indicating that they are encoded in

partby

TRs/IRs

and in partbyopposite endsof

Us. It

follows

that the introns of the IE

mRNA-4andIEmRNA-5geneslieentirely within

TRs/

IRs.

The size of theseintrons wasdeterminedby

R-loopanalysis to be 150 ± 56 b. Thenuclease

analysesindicated that the intron size was 175

b, that is, the size of the B4R1 fragment

pro-tected againstexonuclease VII digestion (1,220

-0* __724

_* _ -

~~4~s

ts ~ ~ l' *m

FIG. 11. Cytoplasmic IE RNA was hybridized to

the HSV-1 DNAEcoRI Hfragmentortothe DNAs

of coliphageA containingdeletions oftheEcoRI H

fragment (isolatesNYS-8andSRS-4). Afternuclease Si digestion,theprotectedDNA sequenceswere

coe-lectrophoresed withpBR322 and

OX1

74DNA

frag-mentsizemarkers(tracksmarkedM)on analkaline agarosegel.DNAfragmentswerethentransferred by blotting to diazobenzyloxymethyl-cellulosepaper. A mixture of 32P-labeled nick-translatedpkL43 DNA

(carryingtheB4RJDNAinsert)and

OX1

74DNAwas

hybridized to the blot, and hybridization was visu-alized by autoradiography. The sizes (in bases) of DNAfragmentsizemarkersareindicated.

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Kilobases

0 5

SRS(4) NYS(a)

B

Sol S B4R I

RI Hir Hind Sol XhoXho RI

I

~~~III I I

88

26 1450

_ - IE RNAs

IEmRNA-4

FIG. 12. Summary ofthemaplocationsofIEmRNA-4 and IE mRNA-5. Apartialrestrictionmapofthe EcoRI Hfragmentisshown, indicatingthecleavagesitesofBamHI(B4-B8), HindIII (Hind),andrelevant SalI(Sal)and XhoI(Xho)sites. Also indicatedarethemaplociof fragmentsused inanalyses reported here, SalI-BamHI 5(SalB5)and BamHI 4-EcoRI(B4R1),and the HSV-1 DNAsequencescontained in deletedA

isolatesNYS-8 and SRS-4. The sizes(in bases) ofthecotranscripts ofIEmRNA-4and IEmRNA-5aregiven,

and their directionsof synthesisareindicatedbyarrows.

b)minus the total of the two B4R1cotranscripts protected against Si digestion (785+260b). As theR-loop analysis indicatedthat the 5' termini of bothIE mRNA-4 andIE mRNA-5 were lo-cated 500 bfromthe EcoRI site,thereiterated DNA sequences of the S region must extend further than 910 b from that restriction site.

Nuclease analyses indicate that the sizes of the transcribed regions ofIE mRNA-4 and IE mRNA-5 are 1,710 and 1,800 b, respectively.

Whenconsiderationistaken ofaddition ofa

3'-terminalpolyadenylate tail of 150to200b(21), it is apparent that these size estimates are in

close agreement tothesize estimates of 2.0 kb for each ofthesemRNA'sdetermined by methyl

mercury agarosegelelectrophoresis (28).

No evidence was found for the presence of

unspliced transcripts partially colinear with IE mRNA-4 and IE mRNA-5, either in polyaden-ylatedorintotalcytoplasmic IE RNA samples.

Presumably,then, the splicingeventswhich

gen-erate these mRNA'stake place in the cell

nu-cleus, and unspliced transcripts are not

trans-portedto,orare veryrapidlyturnedoverin,the

cytoplasm. Furthermore, alternative splicing patterns which generate anumber of mRNA's

from asingle transcriptional unit, as has been

observedwith theearly transcripts of adenovirus 2(4) and simian virus40 (3),werenotobserved

with these HSV-1 IE mRNA's. Consistent with these observations, use of the Northem blot

technique (1) to analyze wild-type HSV-1 IE transcripts hasnotrevealedthepresenceof ad-ditional transcripts partially colinear with IE

mRNA-4and IEmRNA-5 (R. J. Watsonetal., manuscript in preparation). Thepossibility that these additionalcytoplasmic IE transcripts are

presentinveryminoramounts cannot,however, beentirelydisregarded.

From thesequencescontainedbyIE

mRNA-4 and IE mRNA-5, it is implicit that both S

reiterated regions are transcribed, at least in

part. As has beensuggested previously (8, 28), thesemRNA'smaybe transcribedbyusing lo-cationally distinct promoters of identical se-quencemappinginTRsandIRs.DNAsequence

datato identify possible promoter regions and analyses of possible nuclear mRNA precursor

moleculesarerequiredtosubstantiate this

pro-posal. The observation, on R-loop analysis, of

randomtranscripts mappinginUs (Fig. 9) may

indicatethat theIE mRNA'sdescribed indetail abovearegenerated fromlargeprecursors

con-tainingtheseadditional RNAsequences.

Alter-natively, these random RNA speciesmayhave

been transcribed byusingpromoters normally recognizedatearlyorlatetimespostinfection. It

mustbeemphasized that these additional

tran-10 15

I1

87 6 B584II

1540 260

IE mRNA-5

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scriptswererandomas tosize andlocation,were

found in minoramounts, andwerepresentina

cytoplasmic fraction selected by binding to an

oligodeoxythymidylate-cellulose column.

IE mRNA-4 and IE mRNA-5 specify virus

polypeptides of apparent molecular weights

68,000and 12,000 (28), and these proteins have

been designated Vmw 68 and Vmw 12,

respec-tively. In vitro translation of IE mRNA-4,

se-lected by hybridization toHindIII-n, indicated

that this mRNA

specified

Vmw 68 (2). The

transcribed region of IE mRNA-4 was shown

heretobe 1,710b, sufficient tocode fora

poly-peptide ofa little more than60,000 molecular

weight. Although phosphorylation of Vmw 68

(17) mayhave ledto an overestimation of the

molecular weight,theclosecorrelation between

thecoding capacity ofIEmRNA-4 and the size

of itsputativeprotein productsuggeststhat the

transcript doesnotcontainextensive noncoding

regions. It is possible, then, that the 260-b

5'-terminal cotranscript contains codingsequences

and isnotmerelyaleader RNA. If this

cotran-script

is

translated,

then itmaybe anticipated thatthe

polypeptides specified

by IE mRNA-4

and IE mRNA-5, Vmw 68 and Vmw 12, share

commonN-terminalpeptides and that the

ter-minationsignals for those proteinsare encoded

by-'opposite

ends of

Us.

Whereas further data

arerequiredtoprovethishypothesis, it remains

aninterestingwayin whichtogenerate

polypep-tidescontaining bothcommonandunique

pep-tides.

ACKNOWLEDGMENTS

We thankKatherine Denniston-Thompson and Lynn W. Enquist forcritical evaluation of this manuscript and for helpful suggestionsduringthecourseof this work.Also,we thank Kenichi Umeneforprovidinguswith SRS-4 and NYS-8DNA.

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