RNA encoded by the IR1-U2 region of Epstein-Barr virus DNA in latently infected, growth-transformed cells.

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Vol.46, No.2 JOURNALOFVIROLOGY,May1983, p. 424-433

0022-538X/83/050424-10$02.00/0

Copyright01983, American SocietyforMicrobiology

RNA

Encoded

by the IR1-U2

Region of Epstein-Barr

Virus

DNA

in Latently Infected,

Growth-Transformed

Cells

VICKY VANSANTEN, ANDREW CHEUNG, MARY HUMMEL, ANDELLIOTT KIEFF* KovlerViral Oncology Laboratories, The University of Chicago, Chicago, Illinois 60637

Received 22 October1982/Accepted 28 January 1983

The IR1-U2region ofEpstein-Barr virus DNA consists of multiple copies ofa

3.1-kilobase (kb) repeatsequence,IR1, whichmaps totheleft ofa3.3-kb unique region, U2. Although hybridizations of cytoplasmic polyadenylated RNA from latently infected cells to viral DNA indicate that the IR1-U2 region encodes a

substantial fractionof the viral RNA in thesecells,onlyasinglelow-abundance 3-kb cytoplasmic polyadenylated RNA has been identified on Northern blots. Furtheranalysis of thecytoplasmicpolyadenylated RNAencoded by the IR1-U2 regionindicates that(i)the RNAis transcribed from lefttoright; (ii) thereareonly three copies ofthe 3-kb RNAper cell; and (iii) the RNA is spliced. The RNA

hybridizesto possibly contiguous 0.56- and 1.3-kb U2 domains. These domains andpartof IR1 hybridizetothe3-kbcytoplasmic RNA. DNAbetween IR1 and the 0.56-kb U2 domain does not hybridize to the 3-kb RNA. The CCAAT-34 nucleotide-TATAA sequence in IR1 may be part of the promotorfor the 3-kb cytoplasmic polyadenylated RNAsince(i)it enables left-to-right transcriptionof

IR1byaHeLacellextract, and(ii) latently infected cells contain giant

polyade-nylated nuclear RNAs which differ in size by 3 kb, as would be expected if transcription initiates in any copy of IR1 and continues through the rightward remaining copies into U2.

Infection of normal human B lymphocytes withEpstein-Barr virus (EBV) results in

persis-tentlatent infection and cellulargrowth transfor-mation (14, 36). Latentgrowth-transforming in-fection is characterized by the presence of the complete viralgenome(21, 39,50)ascovalently closedcircularepisomes(28, 34); by the

expres-sion of polyadenylated polyribosomal RNA

from the IR1-U2, IR3, and U5regions (19, 22, 24, 25, 37, 48); and by the expression ofnew

intranuclear (42) and cell membrane (45)

anti-gens. A similar stateof virus infection

character-izes latently infected B lymphocytes in normal

seropositive humans (33) and malignant African Burkitttumorcells (10).

Probes which include IR1 or U2 (BamHI fragments V, X, and H; Fig. 1) identify a

3-kilobase (kb) RNA incytoplasmicextractsofa

latently infected cell line, IB4 (24, 48). The

importance of theEBV IR1-U2regionin induc-ing growth transformation is also suggested by studies oftheP3HR-1 EBVstrain. TheP3HR-1

genome is deleted forthe entire U2-1R2 region

(7, 18, 23, 38, 40). The virus produced by

P3HR-1cellscaninfectlymphocytesbutcannotinduce growthtransformation (32, 41).

cDNA madefromthe 3' end of IB4 cell RNA hybridizes more tothe EBV DNABamHI

frag-mentH,which contains therightpartof U2 and IR2, than to BamHI fragments V or X, which contain IR1 and the IR1-U2juncture,

respec-tively, suggesting that the 3' end of the 3-kb

RNAisin U2orIR2(48). Since the cDNA also hybridizes to BamHI fragments V and X, the data are also compatible with the existence of several RNAs with different 3' ends. The

se-quence of IR1 revealsa CCAAT-34

nucleotide-TATAAA sequence and a TATA sequence

which could bepartofpromotersforrightward transcription from IR1 intoU2(8). Other

impor-tant features of IR1 include multiple potential splice donor and acceptor sites, a 1.1-kb open

reading frame, and an unusual 500-base pair

dyad symmetry which would have a profound

effect on the secondary structure ofRNA

en-codedby IR1 (8).

Theobjectiveof thisreportistodefine further (i) the sites for initiation oftranscription from

IR1 in vitro; (ii) the direction of transcription

from IR1 and U2 in latently infected

growth-transformedIB4cells; (iii) thenumber and size ofRNAs transcribed from IR1 and U2 in IB4

424

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IR1-U2-ENCODED EBV RNA IN CELLS

A.

IR1OD) TR U1

,R

,IR

-ulI. U3 ,R3 K4

small segments

totalling 0.9 Kb (1.9kb) TATA" 06kb 1.3kb

IRI IRI1(D) U2

Bam V BmHI X

mature 3kb nRNA

IR2,,i U3 BamMIH

I I I

-I 0 1 2 3 4 5 6 7 8 9 i0 11

Kb

OBmHI

if P3HR- Ideletion

FIG. 1. (A) Schematic diagram of the EBV genome. The genome is divided into five regions of unique

sequencecomplexity (Ut through U5) bounded by direct tandem copies of DNAsequencesatthe termini (TR) andatinternal siteswithin the molecule (IR1through IR4). Thediscontinuity in themapbetween U4 and U5

reflects the fact that the B95-8 isolate of EBV is deleted foraregion ofthe DNA which containsafourth internal

repeat,IR4.Thedeletion hasnoknowneffectonthebiological properties of the virus. IR1(D) indicatesthefinal incompletecopyofIRL. (B) Model for the 3-kb mRNA transcribed from the EBV IR1-U2 region inIB4cells.A BamHIrestrictionenzyme mapofthe IR1, U2, and IR2regions is shown. TATAAA indicates the position of the promoter-likesequenceatnucleotide 1137. RNAs transcribed from this regionareshown above themap.Some

oftheprimary transcripts begin in copies of BamHI fragmentVtothe left of theoneshownonthemap.In this

model,splicing of long primary transcriptsgeneratesa3-kb mRNA. The 0.6- and 1.3-kb RNAsegmentscould be

acontinuous1.9-kbsegment.The uncertaincontinuityof thesesegmentsisindicatedby the dotted line in the 1.9-kbsegment.Other RNAsegmentssplicedontothe 1.9-kbsegmenthavenotbeendemonstrated andareindicated

inparentheses. Thepolyadenylic acid 3' end (not shown) probably constitutes the remaining 0.2 kb of the3-kb maturemRNA.

cells;and (iv) whether the 3-kb polyadenylated

RNA previously identified in the cytoplasm of

IB4cells is spliced.

MATERIALSANDMETHODS

Ceil culture.TheIB4cell linewasestablishedbyin

vitro transformation of human cord blood lympho-cytesbythe B95-8 isolate of EBV and isnegativefor antigens expressed earlyandlate in EBVreplication (24).Cellsweremaintainedby addinganequalvolume ofRPMI1640medium(GIBCOLaboratories) supple-mented withl1ofetalcalfserum(K.C. Biologicals)

every 3 days. RNA was prepared from cells 3 days afterfeeding.

Recombinant DNA. Recombinant EBV (B95-8) DNAswereusedforhybridizationtoRNAblots and invitrotranscription. PlasmidspDK14,pDF322, and pDK286areclones of BamHIfragmentsV,X,andH, respectively (9). ForS1 nucleaseanalysis, restriction fragments from the library were subcloned into the vector plasmid pKH47 (6). The recombinant EBV-pBR322 plasmids were cut with BamHI to free the EBV DNA. Before ligation into the BamHI site of

pKH47,either the EBV DNAwasisolatedby electro-phoresis in agarose gels or the pBR322 vector was

destroyed by cuttingwithEcoRI,anenzymethat does not cut within the EBV DNA. Ligated DNA(ligase

was obtained from Bethesda Research Laboratories)

was transfected into Escherichia coliHB-101 bythe calcium phosphate technique (15). The subclones of BamHIfragments V, X,and HwerenamedpKH47-V, pKH47-X,andpKH47-H, respectively.

PlasmidDNAwaspreparedfrom bulk cultures(12).

After ethanol precipitation, DNA to be used for S1 nuclease analysiswas suspended in 10mM Tris (pH

8.0)-i mM EDTA andwasstored at-70°C.

Restric-tionenzymes werepurchasedfromBethesdaResearch LaboratoriesorNewEngland Biolabs andwere used asrecommendedbythemanufacturers.

PreparationofRNA.Cytoplasmicandnuclear frac-tions were prepared from IB-4 cells (48). DNA was

sheared withaTekmar Tissuemizerfor the

prepara-tionofnuclear RNA(5).Vanadylribonucleoside

com-plex (10mM)wasusedas anRNaseinhibitor (4). RNA

wasstored in 20 mM Tris (pH7.4)-2mMEDTA-0.2%

sodiumdodecyl sulfateat -70°C.

Forsome experiments, nuclear RNA was further

freed of DNAbypelietingincesiumchloride.RNAin 1 ml of 10mM Tris(pH 7.4)-2 mM vanadylribonucleo-sidecomplex with CsCladdedtoarefractive indexof 1.4017 was overlaid with a CsCl solution with a

refractive index of1.4003 andwas sedimented inan

SW41 rotorat34,000rpmfor6h.The RNAwasnot

degradedduring cesium chloride centrifugation since the45S ribosomal RNAprecursor wasstillvisible in gelsofCsCl-purifiednuclear RNA.

In vitro transcription. BamHI fragment V DNA (2 Fg) was incubated at30°C for 60min with 30 ,ul of HeLacellextract(Bethesda ResearchLaboratories)in 50 p.l ofa solution consisting of 5 mM ATP, 5 mM

CTP, 5 mM GTP, 0.05 mM UTP, 10 ,uCi of

[a-32P]UTP, 10 mM creatine phosphate, and 0.14 mM EDTA (29, as specified bythe manufacturer). After completion ofthe reaction, protein was removed by

phenol andchloroform isoamyl alcohol extraction in the presence of 0.5% sodium dodecyl sulfate-7 M

urea. Nucleic acidwas precipitated, suspended, and B.

U5 TR

primary transcripts

46,

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426 VAN SANTEN ET AL.

subjected to electrophoresis in 1.4% agarose methyl mercury gels (3).

Preparation andhybridization of RNA blots.Nuclear polyadenylated and non-polyadenylated RNAs were separated by one cycleof oligodeoxythymidylic acid [oligo(dT)]-cellulose column chromatography (2, 35). Cytoplasmic polyadenylated RNAwaspurified by one or twochromatography cycles. The yield of cytoplas-micpolyadenylated RNA was between 1 and 2 mg per 8x 109 cells.Denaturation of RNA, electrophoresis in formaldehyde-agarose gels (27), and blotting onto ni-trocellulose (46) were as previously described (48). Thesize of nuclear RNAs wasdetermined by plotting the square root of the size against the log of the electrophoretic mobility (27). Ribosomal precursor RNAsizes of 6.9 and 14 kb and ribosomal RNA sizes of5.2and 2kbwereusedas standards(49). Electro-phoresis ofcytoplasmic and nuclear RNA wasin 0.8 and 0.6% agarose gels, respectively. Specific DNA restriction fragments were recovered from agarose (HGTP, Seakem) gels by electrophoresis against a dialysis membrane. Fragmentswerethenlabeled with 32Pby nick translation (43) and hybridized to RNA blots as previously described (48) without dextran sulfate.

The number of IR1-U2 3-kb cytoplasmic polyade-nylated RNA molecules per cellwasdeterminedfrom the relative levels ofhybridization of the RNA and known amounts ofDNA to recombinant viral DNA probes and from the RNAyieldper cell.

Si nuclease analysis. S1 nuclease analysis (6) of RNAwasperformed byusing strand-separated DNA. Individual strands of EBV restriction fragments sub-cloned into the vector plasmid pKH47were purified

by binding to oligo(dT) or oligodeoxyadenylic acid

[oligo(dA)]-cellulose (16). Nonradioactive

strand-sep-arated DNA(25to100ng)washybridizedto50 ,ugof

IB-4 cytoplasmicpolyadenylatedRNAoryeast RNA

in 10 ,ulof50%'o (vol/vol)recrystallizedformamide-0.4

M NaCl-40 mM PIPES

[piperazine-N,N'-bis(2-ethanesulfonicacid),pH6.5]-1mMEDTA-0.1% sodi-um dodecyl sulfate at 50°C for 3 h. Nonhybridized DNAsequences wereremovedbydigestionin 0.3 ml of 0.28 M NaCl-30 mMNaC2H302 (pH 4.4)-4.5mM

ZnSO4-20

p.g

each of native and denatured salmon sperm DNA per ml, with S1 nuclease (Boehringer-MannheimCorp.)at1,000to2,000U/ml,for 30 minat

37°C. After ethanolprecipitation,RNAwasdestroyed by incubation in 0.1 M NaOH at 70°C. The

RNA-cDNAwaselectrophoresedon1.5% alkaline agarose

gels(31), transferredtonitrocellulose(44), and

detect-ed by hybridization to nick-translated probes made

from EBV DNA restriction fragments purified from recombinant DNAplasmids.AHinfldigestofplasmid pDK286orpKH47-HDNA wasincludedin eachgel and blot to provide size markers. In some experi-ments, the RNA-cDNA was alsoelectrophoresed in formaldehyde agarosegelsasdescribed forRNA(48). Bothtypes ofdenaturing gelsyieldedidentical results for the sizeof the protectedDNA.

RESULTS

Nuclear RNAs encoded by the IR1-U2BamHI

fragments V, H, and H. BamHI fragmentV, X,

and H probes hybridized almost exclusivelyto

the samenuclear RNAs (Fig. 2). Atraceof the

3.0-kb cytoplasmic RNA was present in the polyadenylated nuclearRNAfraction(identified

as 2.9kb in theautoradiogram shown in Fig. 2).

Two classesofRNAs larger than3 kbcouldbe distinguished. The largest nuclear RNAs were

polyadenylated and range in size from approxi-mately 22 to 14 kb in decrements of

approxi-mately 3kb.ManyRNAspecies between 5 and

13kbwerein boththepolyadenylated and

non-polyadenylated fractions. Itwas, therefore, not

possible to discern whether the stepladder of polyadenylated RNAs continues through the

rangeof 5to 13 kb.

BamHIfragment H includesnot only theright

partof U2 but also IR2and theleft2.7 kbofU3

(Fig. 1). In addition to those nuclear RNAs in

common with BamHI fragments V and X,

BamHI fragment H identifies a 2-kb nuclear polyadenylatedRNA anda moreabundant

1.1-kbnuclear non-polyadenylated RNA.Theselast RNAsare encodedby the U3 region ofBamHI fragment H, whereas the RNAs incommon with

BamHI fragments V and X donot extend into IR2 orU3. Thus, probes which include mostly the U2part ofBamHIfragmentH hybridizeto

the same RNAsasBamHI fragmentsV andX,

whereas probes from BamHI fragment H U3

hybridizetothe 1.1- and 2.0-kb RNAs butnot to

theotherRNAs (Fig. 2;other datanot shown). IR2 does not hybridize to any ofthe nuclear

RNAs of latently infected cells (11). All ofthe

nuclear RNAs were confirmed to be RNA and

not DNA by their density in neutral cesium

chloride.

Direction of transcription of the 3-kb

cytoplas-mic polyadenylated RNA. To determine the

di-rection of transcription of the cytoplasmic polyadenylated RNA, BamHIfragments V, X, and Hwere cloned intoplasmid pKH47 (16)so

that the strands could be separated. Plasmid pKH47 is a derivative of pBR322 with adenine and thymine homopolymers inserted into the opposite strands so that the strands can be separated byusingoligo(dT) and oligo(dA)

col-umns(16). To relate the separated EBV DNA

strands which were cloned into pKH47 to the

strandsofthe EBVgenome, it was necessary to

determine the relative orientation of the EBV

andpKH47 restriction endonuclease sites andto

identify which pKH47 strands contain polydeox-yadenylic acid or polydeoxythymidylic acid.

The strategy used to identify the

polydeoxya-denylic acid-containing strand of pKH47 is

shown inFig.3. Theorientation ofEBV DNA in

pKH47-V, pKH47-X, and pKH47-H was then

determined by restriction endonuclease

analy-sis.

Each of the separated strands of plasmids pKH47-V, pKH47-X, and pKH47-H was then

hybridized tocytoplasmic polyadenylatedRNA J. VIROL.

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IR1-U2-ENCODED EBV RNA IN INFECTED CELLS

PA(+)

PA(-)

V

X H

* - 22.1

69.11

- 9.

14it --

13.7

6

9

& &*

5.3tg|6

20. -2.0

H(R)

V

X H

H(L)

H(R)

-1

-13

6.9k

-6.5

5.3 g1|-4.6

-2.0

2.0.

2.... . 1

FIG. 2. Sizes ofIB4nuclear RNAs transcribedfrom EBV BamHIfragments V, X,and H.32P-labeledprobes

of BamHIfragmentsV, X,and Hwerehybridizedtoblots ofpolyadenylated[PA(+)]andnon-polyadenylated [PA(-)] nuclear RNA. The BamHIfragmentV probeisPstIfragmentb(see Fig. 5). H(L)andH(R)indicate

probesmade from the SstI-a and SstI-bfragmentsofBamHIfragmentH(see Fig. 7).Ribosomal and precursor ribosomal RNAs fromIB-4cells(A)wereusedassize standards(49). RNAs detectedby hybridizationtothe DNAfragment probes areindicatedbylines. RNAsidentified in bothpolyadenylatedandnon-polyadenylated RNAarenotindicatedonthe blotsofpolyadenylatedRNA.

and treated with S1 nuclease. RNA protected

parts of the EBV DNA R strand of all three

clones, indicating thatRNAis transcribedfrom

lefttoright from BamHI fragments V, X,and H

(see Fig.5through 7). CytoplasmicRNA did not

protectanyof the L strand from SI digestion. Invitrotranscription from IR1. The

observa-tions that cytoplasmic polyadenylated RNA is

transcribed from the R strand of BamHI

frag-mentsV, X, and H and that thesamefragments

encodelarge nuclear RNAs differing in size by

thelength of IR1 suggestthattranscription may

be initiated in any copy ofIRL. A CCAAT-34

nucleotide-TATAAA sequence similarto many

RNApolymerase II promoter sequences and a

less similar TATA sequence have been defined

in IR1 and are in the correct orientation to

promote transcription from left to right (8).

Utilization of these promoter-like sequences

wastestedby usingaHeLacellextract(29) and

BamHIfragment V DNA. BamHI fragment V is

onecompletecopyofIRL.Therelevantmapsof

BamHI fragment V are shown in Fig. 4. The

CCAAT-34 nucleotide-TATAAA sequence

would promote transcription ofa1.9-kb runoff

product, whereas the TATA sequence would

promotetranscription ofa1.4-kb RNA(ora

1.6-kb RNA if the promoterfunctions in the

oppo-sitedirection).Transcripts of approximately 1.9

kbweredetected(Fig. 4A).Thesynthesis of this

RNAwas inhibited by 0.8 ,ug ofalpha-amanitin

perml, suggesting that the 1.9-kb runoff product

is transcribed by RNA polymerase II. As

ex-H

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FIG. 3. Determination of the oligo(dA)- or

oligo(dT)-bindingstrand ofplasmid pKH47. (A) Sche-matic diagram of the pKH47-X DNA used in the experiment. The plasmid DNA was cleaved with HindIII (H), labeled with 32P at the 5' ends (-) (30),andthen cleaved with BamHI(B).The oligo(dA)-and oligo(dT)-binding sites are indicated by closed rectangles.Onlythe strand withacounterclockwise 5' to3' directioncontainsalabeled end which would be

covalently attached to oligo(dA) or oligo(dT). This plasmidDNAwasdenatured and strandseparated by

binding to oligo(dA)- and oligo(dT)-cellulose. (B) Autoradiogramofanagarosegel containingthe mate-rial recovered from oligo(dA)-cellulose (A) and oligo(dT)-cellulose (T). The data indicate that the 5' labeled end isonthepoly(A)-containing strand.

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FIG. 4. Transcriptionfrom EBVBamHI fragmentVinvitro.BamHI fragmentVDNApurified from plasmid

pDK14 (9) was transcribed bya HeLa cell extract. The labeledtranscripts were electrophoresed in methyl

mercury agarose gelsand detectedbyautoradiography. Marker human andE.coli ribosomalRNAs are shown in

lanes 1 and 2. InA, the products of transcription from BamHI fragmentVDNA are shownin lane4.Negative controls in whichnoBamHI fragmentVDNA was added(lane 3) orinwhichRNApolymeraseIIwasinhibited

by0.8 jigofalpha-amanitin perml (lane 5) are shown. Promoter-like sequences identified in the nucleotide

sequence (8)areshown in themapofBamHI fragmentV(A). The numbers indicatenucleotides. The map below

shows the expected length of runoff products if transcription begins near each promoter-like sequence.

Transcriptionin either direction from thepromoter-like sequence atnucleotide 1663 is shown. In B, products of

invitrotranscriptionaftercleavageof the BamHIfragmentVtemplate DNA withBglII (lane 3) orSstII(lane 5) are shown. Lane 4 shows the products oftranscriptionfrom SstIl-cleaved BamHI fragment V DNA in the

presenceof 0.8jigofalpha-amanitin perml.Adiagramoftheexpectedeffect of cleavage of the template DNA on

the size of thetranscript initiated nearthepromoter-like sequence atnucleotide 1137 is also shown (B).

pected from the restriction endonuclease sites, cleavage ofBamHIfragment V with BgIII had no effect on the length ofthe runoff product, whereascleavage withSstII shortened the

run-offproductto1 kb(Fig.4B). This indicates that

the CCAAT-34 nucleotide-TATAAA sequence

is partofafunctionalpromoter.A small amount

of1.9-kb RNAwas madewithout BamHI

frag-mentV. Thisendogenous templateactivity was

unaffected by0.8 ,ug ofalpha-amanitinperml.

Si nuclease analysis of cytoplasmic RNA

en-coded by BamHJ fragments V, X, and H. The

DNA sequencesofBamHIfragmentsV, X, and

H which encode cytoplasmic polyadenylated

RNAweremapped by Si nucleaseanalysiswith the strand-separatedDNAsofplasmids

pKH47-V, pKH47-X, and pKH47-H. The R strand of each fragment was hybridized with IB4

cyto-plasmic polyadenylated RNA, andunhybridized

DNAwasdegraded with Si nuclease. The

RNA-protectedDNAwaselectrophoresedin

denatur-ingagarosegels,blottedontonitrocellulose,and

detected by hybridization to 32P-labeled EBV

DNAfragments. The RNA protecteda 1.75-kb

segment of BamHI fragment V and the

full-length 3.1-kbBamHIfragmentV(Fig.5). Minor

protected DNAs of 1.75 to 3.1 kb were also

detected. When only DNA to the right of the

Sstl site (see map in Fig. 5) was used in the

hybridization, the full-length protected DNA

and the slightly smaller minor species were

reduced in size to 2.4 kb, and the RNA still

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429

Sstl SstI-o Y I Y I

32N #*

*< P 424

16> - 4* *4175

93-.93>

3.1 kb

1.75 kb

... ... BmHI SstI -TATAAA

'-09kb PstI-bT ifI-£- >~

r -...T T "".- s .... ,__

kbO 0.5 i.5 2_(0 2.5 3.0

Bam HTV

extends tothe rightwardBamHI site.

Further-more, the data indicate that the minor species

and thefull-length protected DNAdiffer onlyat

their 5' end, which istobe left of the SstI site.

RNAprotecteda0.56-kb segmentofBamHI fragment X and a family of DNA segments

ranginginsize from 1.1 kbtofull-lengthBamHI fragment X (Fig. 6). Cleavage ofBamHI

frag-ment X DNA with restriction endonucleases

XhoI andHpaI, which cutprogressively closer

toitsright end,progressively removed the

high-er-molecular-weight components of the family

and had no effect on the size of the 0.56-kb

protected segment. Identical results were

ob-tained by using only the parts ofBamHI

frag-mentXtothe right of the XhoI and HpaI sites.

Cleavage of pKH47 with HindlllremovesDNA

tothe left of the

HindIll

site in BamHIfragment

BonmHI X (Fig. 6) while leaving the HindIII-bfragment

...,h..._.-..;..j.-7;

1^X Y- - Y r I Y -:

FIG. 5. S1 nuclease analysis of IB-4 cytoplasmic polyadenylated RNA transcribed from the R strand of EBV BamHI fragment V. Hybridizations were done

with yeastRNA ascontrols (Y) andwith IB-4 cyto-plasmic polyadenylated RNA (I). In SstI hybridiza-tions, plasmid pKH47-Vwascleaved with SstI before

strandseparation and hybridizationtoRNA.The SstI site is shown in themapof BamHIfragmentVbelow. After SstIrestriction, EBV DNA sequences onboth

sides of the restriction site remained covalently

at-tachedtopolydeoxyadenylic acid,wererecoveredby

bindingtooligo(dT)-cellulose, andwerehybridizedto

RNA. In SstI fragment a hybridizations, the DNA was also cleaved with a restriction endonuclease

whichcleaves within thevectorsothat only the SstI

fragment a part of BamHI fragment V is recovered andhybridizedtoRNA.The blots shownweremade fromalkaline gels. The positions of marker DNAs (A) andDNAs protectedby IB-4 RNA and notbyyeast RNA (A) are shown. Sizes are indicated in kb. A

restrictionenzyme mapof BamHIfragmentV indicat-ing themap position and size of protected DNAs is shown below. TATAAAindicates the promoter-like

sequenceatposition 1137 in Fig. 3. The possiblerange

ofposition of the1.75-kb protectedDNAsegmentis indicated by parentheses. The directionof transcrip-tionofthe RNA is indicated byarrows. The 0.9-kb

PstI fragment b and Hinfl fragment a, used for

hybridization to blots of cytoplasmicRNA in Fig. 7,

arealso shown.

protected a 1.75-kb segment. Thus, the RNA

protecting the 1.75-kb segment is transcribed

entirely fromDNAtotheright of the SstI site,as

would be expected if the RNA begins nearthe

CCAAT-34 nucleotide-TATAAA sequence and

;

93

--i14

-~. 6 -56 .5

0.56kb -1.9 kb11het

...__.__... ... .. ~~~~~~~~~~~~~~~~o

Bam HI XhoT HpaI Hindm

,I I~~~~~~~~~~~~~~~~

Barn HIT

H,r I-b---h - Hird

kb 05 0 .5 2 0

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BormHI X

FIG. 6. S1 nuclease analysis of IB-4 cytoplasmic polyadenylatedRNAtranscribed from the R strand of EBVBamHI fragment X. In the lanes labeled XhoI andHpaI, DNAonbothsides of the restriction sites

was used in the hybridization to RNA. In the lanes labeledXhoIL andXhoIR, only DNA to the left or

right, respectively, was used. Since HindlIl also

cleaves thevector,only HindlIl fragmentbwasused

in the lanes labeled HindlIl. The brackets indicatea

family ofprotectedDNAsegmentsof different sizes. Theblots shownweremadefromformaldehyde

agar-osegels.BamHIfragmentXcontainsapartialcopyof IR1, shownasIR1(D),andpartofU2.ThepartofIR1 PstIfragmentbwhich is within BamHIfragmentXis indicated below the BamHI fragment X restriction endonuclease map. Thetransition from IR1 to U2is 123basepairstotheright of the XhoIsite shown. The HindIIIfragmentsaand bofBamHIfragmentXused for hybridization to blots ofcytoplasmic RNA (see Fig.7)areshown. For furtherexplanation,seelegend

toFig.4and5. uncut

Y I

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Y I

41 N

3 2

>1-6.> E

1

413

93-< ., 4

.93>z,Io

13 kb BamHI

-...

S...

sta Hinf I c dgf

kbO 2

Bamr U2

:2

FIG. 7. SI nuclease a] ized to the R strand of Hybridizations with IB4 RNA (N) are included. 1 from alkaline gels. TheSs

Hinfl fragments c ofBc

hybridizationtoblotsofc

are shown. As indicated below the restriction mat

tains partof U2, all of IR abbreviations andsymbol

jst :;: Sst-.- Ss6.-- of BamHI fragment H. Since the

BamHI-H-y Sst-I-b fragment doesnothybridizeto the 3-kb

cytoplasmic polyadenylated RNA (see below) but does identify an abundant 1.1-kb nuclear

33 non-polyadenylated RNA, Si nucleaseanalysis

was performed with nuclear non-polyadenylated

4i

3 A g RNAtoseewhether the 1.1-kb

protected

DNA

* 41 l w l

sobserved

in Fig.7couldresultfrom

contamina-tion of

cytoplasmic

RNA with nuclear

non-polyadenylated RNA. A 1.1-kb protected DNA

resulted (Fig. 7, lane N). S1 nuclease analysis with cytoplasmic polyadenylated RNA which was less contaminated with nuclear RNA

re-Akb vealed only the 1.3-kb protected segment (data

ajT HJ notshown). Protectionof theRstrandof BamHI

Sst b- fragment H by the nuclear non-polyadenylated RNAindicates that this RNA is transcribed from the same strand as the cytoplasmic

polyade-4. 5 nylated RNA.

tHi.

Identification of DNA segments which encode

U ~ the 3-kb RNA. To determine which DNA

seg-ments identified in S1 nuclease analysis encode nalysis of IB4 RNA hybrid- the 3-kb or other possible cytoplasmic RNAs

EBV BamHI fragment H. and which are likely to have been identified non-polyadenylated nuclear because of contamination of the cytoplasmic

rhe blots shown were made RNA with nuclear RNA, small fragments of ;tlfragments a and b and the BamHI fragments V, X, and H were hybridized

7mHI fragment H used for to Northern blots of cytoplasmicpolyadenylated ,ytoplasmic RNA (see Fig. 8)

by the schematic diagram ornuclear RNA (see maps of BamHIfragments

ps,

BamHIfragment H con- V, X, and H in Fig. 5 through 7). DNA from the

2, and part of U3. For other Hinflfragment aof BamHIfragmentV

hybrid-Is,seelegendtoFig.4and 5. ized to the 3-kb RNA (Fig. 8); DNA from the

attached to pKH47 homopolymer. RNA-pro-tected DNA segments of 0.56 and 0.9 kb were

identified usingthe Rstrandof HindIII-b. These data confirm that the 0.56-kb protected DNA segmentisentirelytotheright of the HindlIl site within the BamHI-X-HindIII-b fragment and the family of larger protected DNA segments hasacommonright end at or near the right end of BamHI fragment X. The data also indicate that the family of protected DNA segments arisesbecause ofheterogeneity beginning1.1kb from the right end of BamHI fragment X and extending to the left end of BamHI fragment X (Fig. 6).

Cytoplasmic polyadenylated RNA protected 1.3- and 1.1-kb segments of BamHI fragmentH DNA(Fig. 7). Use of the SstI fragments a and b

of BamHI fragment H in hybridizations with

RNA showed that the 1.3-kb protected DNA

maps to theleftoftheSstI site, and the 1.1-kb

protected DNA maps to the right (see map of

BamHIfragmentHin Fig. 7).3 P-labeledHinfl

fragment c (the leftmost Hinfl fragment of

BamHI fragment H) hybridized to the 1.3-kb

protected DNA (data not shown), confirming

thatthis protectedDNA mapsnear theleftend

; Hinf1-G HlndIFo --Vd:d'7,Rt-n,, I- S -_, H1lff-c

; | 0 ¢ 33

* .E

~~~~~~

FIG. 8. Hybridizationoffragmentsof BamHI frag-mentsV, X,and Htothe 3-kb RNAon blots of IB-4

cytoplasmic polyadenylated RNA. The DNA frag-mentsindicated above the laneswerelabeled with32P

and were hybridized to RNA blots. The fragments usedare indicated onthe maps in Fig. 4through 6. Ribosomal RNAmarkers(A)andhybridizationof the DNAprobestothe3-kb RNA(A)areshown. Sizesare

indicated in kb. The bracket indicateshybridizationof the PstI fragment b probe to nuclear RNAs which contaminated thecytoplasmicRNApreparation.

52:>,

i 43 1

2 ) I(

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PstI-b fragmentof BamHIfragment V did not,

although ithybridizedwelltonuclearRNA(see Fig. 2) andidentifies asmallamount ofnuclear polyadenylated RNA in the cytoplasmic poly-adenylated RNA preparations. Since the PstI

fragment b sequences are notrepresented in the 3-kb cytoplasmic RNA but are represented in nuclear RNA, thefull-lengthand 1.75-to3.1-kb minor BamHI-Vfragments thatwere protected in theSinuclease analyses of DNA (seeFig.5)

cannotbeexonsof the 3-kb RNA andarelikely

tobepartofcontaminating nuclear RNAs. Simi-larly, the HindIIIfragment bofBamHIfragment

Xhybridizestothe 3 kb RNA,but theHindlIl fragmentadoesnot(Fig. 8). This indicates that the 0.56-kb segment of RNA transcribed from theHindIII fragment b (see Fig. 6) is probably

part of the 3-kb RNA. The longer protected DNAs of BamHI fragment X, in addition to

being heterogeneous, contain the HindIII

frag-ment aand PstI fragment bsequencesof BamHI

fragmentX.Therefore, theycannotbeexonsof the 3-kb RNA since the HindIII-a and PstI-b portionsofBamHIfragmentXdonothybridize

to the 3-kb RNA. Also, as would be expected from theS1 experiments, theleftward SstI

frag-ment aandHinflfragmentcof BamHIfragment

Hand thepartof theHinflfragmenta totheleft of IR2hybridize tothe 3-kb RNA, whereas the rightward SstI fragment b and IR2 donot(Fig.7 and data notshown). EcoRI fragmentJ, which

maps totheleft of IR1 in Ul, doesnothybridize

to any of the IR1-U2 RNAs.

Reconstruction experiments inwhich the hy-bridization of BamHI-X-HindIII fragment b to

the 3-kb RNA wascompared withhybridization

to known amounts of BamHI-X under similar conditions indicate that there is approximately 0.0038ngof the BamHI-Xpartof the3-kbRNA

per 5 ,ug of IB4 cytoplasmic polyadenylated

RNA. This estimate and the polyadenylated

RNA yield per cell indicate that there are

ap-proximately three copies of the 3-kb RNA per

cell.

DISCUSSION

The simplest model (Fig. 1) which would

account for the accumulated data describing

RNA from IR1 and U2 is that the CCAAT-34

nucleotide-TATAA sequence in IR1 is part of

thefunctional promoterfortranscription ofthe

IR1-U2 R strand in latently infected cells. The

smallesttranscript would startin the last

com-pletecopyofIR1, extendthrough0.9kbofIR1,

continue through the BamHI site between

BamHIfragmentsVand Xintothe 0.6 kb of IR1

and 1.4kb ofU2inBamHI fragmentX, and then

continue through at least 1.3 kb of BamHI

fragment H U2. Afterpolyadenylation at a site

about 1.3 kb intoBamHI fragment H U2, this

transcript would be approximately 5.3 kb. Other polyadenylated RNAs would begin inmore left-ward copies of IR1 and would vary in size

upwards from5kb in3-kb increments. The

1.1-kb U3 non-polyadenylated RNA could be a 3'

cleavage product of the putative primary

tran-script since it is also transcribed from the R strand of EBV DNA. However, no RNA

en-coded by IR2 has been detected. The 3-kb cytoplasmic polyadenylated RNA would be spliced from the large IR1-U2 polyadenylated RNAs andconsist (3' to 5')ofa polyadenylate tail (probably less than 0.2 kb), segmentsof1.3

and 0.56 kb from BamHI-H and -X U2 which

may be continuous, and small exons as yet

undetected from the left end of U2 and IR1,

totaling0.9kb (Fig. 1).Alternativelythe1.75-kb RNA-protectedIR1 DNA segmentcouldencode anexon of the 3-kbRNA.

Data in support of this model include the

following. (i) There are large nuclear RNAs which differ in length by approximately 3 kb (Fig. 2). IR1 isa3-kb tandem repeat. S1

nucle-ase analysis shows that some RNA molecules includeatleastonefull-lengthcopyof IR1 (Fig. 5). (ii) RNA hybridizes onlytothe R strand of

EBVIR1orU2DNA,indicating thatmost orall of the RNA is transcribed from leftto right as

shown in Fig. 1. (iii) There is a CCAAT-34 nucleotide-TATAAAsequence which ispart of

a promoter for in vitro transcription of the R

strand of IR1 (8; Fig. 4). In latently infected cells, an RNA segment was detected which is complementarytoandnearly equalinlengthto

the partof the IR1Rstrand 3'totheCCAAT-34 nucleotide-TATAAA sequence (Fig. 5). This 1.75-kb segment could be the 5' end of the

precursorRNA. ThecytoplasmicRNA

prepara-tions were contaminated with nuclear RNAs. The discrepancy in size between the 1.9-kb runoff product of in vitro transcription and the 1.75-kb RNA segment detected byS1 nuclease analysis with RNA from latently infected cells could be due to the different types of gels (methylmercury versus formaldehyde) and dif-ferent markers (RNA versus DNA size mark-ers). Alternatively, the 1.75-kb RNA segment

could bepartofapartiallyorcompletelyspliced

RNA. The1.75-kb segmentisunlikely tobean exon of the 3-kb RNA since there are two other

RNA-protected DNA segments totaling 1.9 kb

(seebelow), and althoughtheBamHI-V-HinfI-a

partofthe1.75-kbDNA segmenthybridized to the 3-kb RNA, Hinfl fragmente, therightmost

part of the 1.75-kb segment, did nothybridizeto

the 3-kb RNA (data not shown). This suggests

that not allof the IR1 sequence totheright ofthe

promoter becomes part of the 3-kb RNA. (iv)

The 3-kbcytoplasmic polyadenylated RNAis a

spliced RNA since it is encoded partly by

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BamHI-V-Hinfl-a and by BamHI-H-Hinfl-a, whichare 3.5kbapart inthe EBV genome (Fig. 1, 5, and 7), and the intervening BamHI-X-HindIII-a fragment doesnothybridize tothe 3-kb RNA(Fig.1and7 anddatapresented above). The IR1 sequence (8) reveals many potential splicedonorandacceptorsites in thepartof IR1

to theright oftheputativepromoter. (v) AllIR1

and U2 DNA fragments tested, including the BamHI-V-PstI-b fragment (much of which is included in the BamHI-X-HindIII-a fragment), hybridize tothe large nuclearRNAs, aswould be expected if these RNAs are unspliced or

partially splicedprecursors tothe3-kb cytoplas-mic RNA(Fig. 1). (vi)Cytoplasmic polyadeny-latedRNAis complementarytopossibly

contin-uous 0.56- and 1.3-kb segmentsof theR strand of BamHIfragmentXandHU2 DNA.

Further-more, these DNA segments hybridize to the

3-kb cytoplasmic polyadenylated RNA. (vii) The large nuclear RNAs and the 3-kb cytoplasmic RNAdonot extend intoIR2 or U3; thus, they have similar 3' ends.

RNAs having common 3' and variable 5' domainswere detectedfrom IR1 and U2(Fig.5 and 6). Since the cytoplasmic preparations in

which these RNAsweredetectedwere

contam-inated with nuclear RNA, these RNAs could be partially spliced precursors to the 3-kb RNA. Alternatively, they could be mature, less-abun-dant RNAs which are spliced differently from the 3-kb RNA. Partiallyoranomolously spliced globin precursor RNAs have been detected in thenuclei of Friend erythroleukemia cells (13). Thevariablyspliced EBV IR1-U2 polyadenylat-ed RNAs are, in aggregate, as abundant asthe putative fully spliced RNAs. These variably splicedmolecules could inaggregate accountfor the seemingly discrepant finding that there are

fewercopiesof the 3-kb RNAperlatently infect-ed transformed cell than of the RNA specified by the EBV U5 region (van Santen et al., manuscript in preparation), whereas cytoplas-micpolyadenylated RNAhybridizesmore tothe IR1-U2region thanto the U5region.

The modelproposed for generation of the 3-kb

EBV RNA is similar to that proposed for the

formation of polyoma viruslatemessenger RNA

(26, 47). The 5,560-base pair circular polyoma virusDNAencodes giant RNA molecules which are tandemly repeated whole genomic

tran-scripts (1, 26, 47). Multiple copies of a

57-nucleotidesequence which is presentonly once

inpolyomavirusDNAare present at the 5' end

ofpolyomalatemessenger RNAs (26,47). Each copy of the 57-nucleotiderepeat is spliced from

acomplete genometranscript(47).

Sincethe P3HR-1 virusis deleted forU2 and IR2and is unable toinitiategrowth transforma-tion (7, 17, 18, 24, 38, 40), the IR1-U2 RNA is

believed to be important in initiating or

main-taining latent infection or growth transforma-tion. The IR1-U2 RNA would, therefore, be

expected to be a constant feature of latent

growth-transforming infection. However, the U2regionwhich encodes 1.9 kb of the 3' end of

the IR1-U2 RNA varies extensively among

transformation-competent EBV isolates (11, 23).

Alow-abundance 2.0-kb nuclear butnot

cyto-plasmicpolyadenylatedRNA wasalso identified

in this studyto be encoded by the right part of

BamHI fragment H. A more abundant 1.9-kb

polyadenylated early RNA encoded by BamHI fragment H has been previouslydetectedin the

cytoplasm ofproductively infected B95-8 cells

(20). A small amount of this RNA may be

transcribed and polyadenylated but not

trans-ported in latent infection.

ACKNOWLEDGMENTS

Wearegrateful for the gift ofrecombinant EBV DNAs from TimothyDambaugh and Victor Razkowski and for the assist-anceof JuliaDowling in tissue culture. Initial in vitro tran-scription experiments with IR1 DNAweredone byTimothy Dambaugh and Roberto Weinman.

This research was supported by Public Health Service grantsCA 19264 andCA17281 from theNational Institutes of Health and by grant MV 32G from the American Cancer Society. E.K. isafacultyresearchawardee of theAmerican Cancer Society. V.v.S. was supported by aPublic Health Service National Research ServiceAward CA09267from the NationalInstitutes of Health. M.H. is supported by Public HealthService grant AI 07182 from the National Institutes of Health.

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