Simple repeat sequence in Epstein-Barr virus DNA is transcribed in latent and productive infections.

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Vol.44,No. 1 JOURNALOFVIROLOGY,Oct.1982,P.311-320

0022-538X/82/100311-10$02.00/0

Copyright C 1982, AmericanSociety for Microbiology

Simple

Repeat

Sequence in

Epstein-Barr

Virus

DNA Is

Transcribed in

Latent

and

Productive Infections

MARKHELLER, VICKY VANSANTEN, AND ELLIOTT KIEFF*

DepartmentsofMedicine, Microbiology, Immunology, andVirology, KovlerViralOncologyLaboratory, The

Universityof Chicago, Chicago, Illinois 60637

Received22April1982/Accepted10 June 1982

The BamHI K region of Epstein-Barr virus DNA is transcribed in latently infected cells from Burkitt tumors and in growth-transformed B-lymphocytes latentlyinfected withEpstein-Barrvirus. We determined the nucleotidesequence ofa1,153-basepair Hinflfragmentin BamHIfragmentKfrom the B95-8 Epstein-Barr virus isolate. The fragment contains a remarkable 708-base pair simple sequence repeatarray, designatedIR3, which iscomposedofonly three nucleo-tidetriplet elements: GGG,GCA,andGGA. Thetripletsareorganizedinto three

repeatunits: GCAGGA, GCAGGAGGA, andGGGGCAGGA. Immediately 3'of

IR3 are tandem nearly perfect direct repeats of two different 24-base pair sequences. IR3 isconservedat acolinearpositionin the DNAsof other

Epstein-Barr virus isolates, and a homologous sequence maps at the same

location in the genome ofagenetically related baboonherpesvirus, herpesvirus papio. IR3 is transcribed from lefttorightinlatentlyinfected, growth-transformed IB4 cells. Itencodes partofa2.0-kilobase exonof the 3.7-kilobase cytoplasmic polyadenylated RNA previously detected in IB4 cells (van Santen et al., Proc. Natl. Acad. Sci. U.S.A.78:1930-1934, 1981). IR3 also encodespartsof 2.4- and 1.0-kilobase RNAs inproductively infected B95-8 cells.

Epstein-Barr virus (EBV) is the usual caus-ative agentof infectious mononucleosis. Latent EBV infection stimulates the growth of B-lym-phocytes in vitro and in vivo. The virusmaybe an important etiological factor in two human cancers,Burkittlymphomaandanaplastic naso-pharyngeal carcinoma (for reviews, see refer-ence12).

EBVDNAis alinear, double-stranded mole-cule ofapproximately 175,000 base pairs (bp) (Fig. 1;forreviews, seereferences 6and 7). At both endsof the moleculearedirectrepeatsofa 0.5-kilobase (kb) sequence, TR. Direct repeats ofa3,071-bp sequence, IR1, divide thegenome intoa9,000-bpuniqueregion,

Us,

anda 150,000-bpuniqueregion, UL (4, 4a).DirectrepeatsIR2 and IR4, of123 and 103 bp, respectively, map within the left and right ends, respectively, of

UL

(T.

Dambaugh

and E.

Kieff,

J.

Virol.,

in

press).

There are three regions in EBV DNA which express cytoplasmic polyadenylated (poly(A)] RNAs in latently infected, growth-transformed cells(24, 25, 35, 49). These RNAs are encoded bythe large internal repeat(IRl) sequence and adjacentuniquesequences,by theBamHIRand K fragments at the center of UL, and by the EcoRI D het fragment at the right end of UL (Fig. 1). Atleast 50cytoplasmic poly(A) RNAs

are encoded by UL and Us in productively infected cells (22).

Under moderately stringent hybridization conditions (6xSSC[seebelow]and70°C)human and mouse cell DNAs hybridize to a 1.15-kb Hinflsubfragment ofthe BamHI Kfragmentof EBV DNA (M. Heller, A. Henderson, and E. Kieff, Proc. Natl. Acad. Sci. U.S.A., inpress). This observation, the transcription of the BamHI K fragment in latent and productive infections,and thesuggestionof rearrangements

in EBV DNA centered around the BamHI K

region (17) led us to examine this regionmore closely.Wereporthere the nucleotidesequence of the 1.15-kb Hinflfragmentfrom B95-8 EBV isolate. The fragment contains a remarkable sequence, designated IR3, which consists of interspersed direct repeatsofone6-bp andtwo 9-bp oligonucleotides. These oligonucleotides arecomposed ofonlythree nucleotide triplets.

The BamHI K

fragment

encodes a 3.7-kb cytoplasmic poly(A) RNA in latently infected, growth-transformedcells (49) and 2.2-, 1.9-, and 1.5-kb cytoplasmic poly(A) RNAs in produc-tively infected cells (22). Hybridizations with cDNA made from latently infected cell RNA have suggested that the 3.7-kb RNA is tran-scribed fromlefttorightin the genome orienta-tion indicated in Fig. 1 (49). We confirm the

311

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FIG. 1. Organizationof BamHI Kregionin EBV DNA. (A)Structure of the EBVgenome.ThelinearDNAis boundedby multipletandemdirectrepeats(TR)(0.5 kb). Tandeminternal directrepeatregions IR1(3,071bp)(4,

4a),IR2(123 bp),IR3(thispaper),and IR4(103bp)(Dambaugh andKieff,in press)separateregions of

unique-sequenceDNA(Ut toU5).EcoRIand BamHIrestrictionenzymesitesandthe locationsof DNA deletions in P3HR-1 andB95-8 virusisolatesareindicated(for reviews,seereferences 6and7).Thediscontinuityin themap coordinates in IR1 reflects variation in theaveragenumberofcopiesof the IR1 repeatamongdifferentEBV isolates.(B)RestrictionmapofthepDK2255.0-kb BamHI Krecombinant insert. A 32P-labeled terminal Hinfl-BamHI fragment (4.74 to 5.0 kb in pDK225 K) was hybridized to Southern blots of partial restriction

endonuclease digests of theBamHI Kinserttolocate the cleavage sites (44). (C)Restrictionmapof thepDK225

1.15-kbHinfl fragmentandsequencingstrategy.Restriction fragmentsweresequenced bychemicaldegradation

(28).The 5'-to-3'polarityand theextentof the sequenced strandsareindicated byarrows,and the labeled ends

areshownwithavertical mark. Partialsequenceinformation is indicated bydottedarrows.The restrictionmap

is derived from thesequence. The locations ofa776-bpHpaIIfragment anda735-bp AvaII-HpaII fragment

whichwereusedashybridizationprobesinthis studyareindicated below the scale.

312

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SIMPLE REPEAT SEQUENCE IN EBV DNA 313

directionof transcription of this RNA and find that the IR3 repeat is transcribed in latently infectedandproductively infected cells.

MATERIALSANDMETHODS

Cellsandvirus. The procedures and materials used

for cell culture and purification of EBV from the

culture fluids of producer cell lines have been

de-scribed previously (10). Intracellular viral DNAs were partially purified from sodium dodecyl sulfate-protein-ase K lysates of latently infected cell cultures by

repeated cycles of isopycnic centrifugation (17).

AG876 (34), W91, and P3HR-1 (21) are virus-produc-ing cell lines that were derived by culture of African Burkitt tumor tissue or, in the case of W91, by

infection of marmoset lymphocytes with

tumor-de-rivedvirus. Raji isanAfrican Burkitt tumor cell line

latentlyinfected with EBV (37). Cherry and Lamont

arelatentlyinfected cell lines that were established by

culture of lymphocytes of patients with infectious

mononucleosis (38; W. Henle and G. Henle, personal

communication). B95-8is a virus-producing cell line

derived by in vitro immortalization of marmoset

lym-phocytes with virus of infectiousmononucleosis origin

(31). IB4isaclonedlatently infected cell line obtained

by in vitro immortalization of human neonatal

lympho-cytes with B95-8 virus (24). Loukes is a human

B-lymphocyte cell line derived by culture of tumor tissue

from a patient with American Burkitt's lymphoma.

Loukesis EBV genome negative and antigen negative

(38, 49). Herpesvirus papio (HVPapio) was obtained

from culture fluids ofavirus-producing baboon

lym-phoid cell line, BA65, established from circulating

lymphocytesof a normal baboon, Papiocynocephalus

(14,18).

Preparation of RNA. Cytoplasmic poly(A) RNA

from B95-8orIB4cells was isolated bytwocycles of

oligodeoxythymidylate [oligo(dT)] chromatography

(22, 49). RNAs examinedby Northern blot analysis

weredenatured at60°Cin50%oformamide and 2.2 M

formaldehyde, electrophoresedin

formaldehyde-agar-osegels, and transferred to nitrocellulose filters (22,

48, 49). Nuclear RNAwas isolatedfrom IB4 cellsas

describedpreviously (2).

Recombinantplasmid. pDK225is a pBR322

recom-binantplasmidcontaining the BamHI K fragment of

the B95-8 strain of EBV (5). The Sall F fragment of

B95-8 viral DNAcontained in theplasmidpDK153 (5)

was subcloned into the plasmid pKH47 (16). The

subcloneisplasmidVRD1.

S1nucleaseanalysis ofIB4 RNA. VRD1 DNAwas

linearized withEcoRIrestriction endonuclease,

dena-tured and strand separated by chromatography on

oligodeoxyadenylate and oligo(dT) columns as

de-scribed previously (16). Unlabeled strand-separated

DNA(50to100ng)washybridized at 50°C for 3 h to 50

,ugof IB4cytoplasmic poly(A) RNA or yeast RNA in

10,ul ofasolution containing50%formamide, 0.4 M

NaCl, 40 mM PIPES

[piperazine-N,N'-bis(2-ethane-sulfonic acid)] (pH 6.4), 1 mM EDTA, and 0.1%

sodium dodecyl sulfate. The hybridization solutions

werediluted into 0.3 ml ofS1 buffer, and the

single-stranded regions of the DNA were digested at 37°C

withS1 nuclease (3). RNA was destroyed by

incuba-tion in 0.1 MNaOHat70°C. The

RNA-complemen-tary DNA waselectrophoresedonalkaline1%agarose

gels (29), transferred tonitrocellulosefilters, and

hy-bridized to nick-translated probes (40) as described

below.

Restrictionendonudeases,gelelectrophoresis,

radio-activelabeling of DNA,andhybridizationto

nitroceliu-lose filters. Restriction enzymes were obtained from

Bethesda ResearchLaboratoriesorNewEngland

Bio-labs. Viral DNAs or recombinant DNA fragments

werelabeled by nicktranslation with[a-32P]dCTP(500

Ci/mmol)(Amersham Corp.) forhybridizations to

ni-trocellulose filters. The specific activity of the

[I2P]-DNAs

was 1 x 108to3 x 108cpml,ugofDNA.

Forhybridization, nitrocellulosefilters were

preincu-batedfor 1 h in lOx Denhardthybridization solution

containing 0.2% polyvinylpyrrolidone, 0.2% bovine

serum albumin, and 0.2% Ficoll (9) and hybridized

with 32P-labeled probes in lx Denhardt solution as

described in the figure legends. The filters were

washed after hybridization at 65 to 70°C in 0.2%

sodium dodecyl sulfate anddecreasingconcentrations

of SSC (2, 0.5, and 0.1x SSC;1xSSCis 0.15 MNaCl

plus 0.015 M sodiumcitrate, pH 7.4) forseveral hours.

DNAfragmentswerepurifiedfor DNAsequencingby

agarose or acrylamide gel electrophoresis, 5' or 3'

labeled with 32P-nucleotide by using polynucleotide

kinase or terminal deoxynucleotidyl transferase,

re-spectively, and sequenced by chemical degradation

(28). Determination of the DNA sequence from the

middle of theregion inFig.1Crequiredtheresolution

of sequence ladders up to 475 nucleotides from a

labeled endon5%or6%polyacrylamide gels.

RESULTS

Mapping and sequenceoftheHinflfragment.

pDK225 is a pBR322-BamHI K recombinant

plasmid from a BamHI recombinant fragment libraryof EBV B95-8 (5). The size of the BamHI Kfragment in B95-8 viralDNAand in pDK225 is 5.0 kb. The positions of the Hinfl, SmaI, HindIII, and PstI sites in pDK225 were deter-mined by partial restriction endonuclease map-ping (44). Fig. 1B isamapof BamHIfragmentK in the orientation corresponding to that of the EBVgenome (Fig. 1A).

The nucleotide sequence ofa1,153-bp Hinfl subfragmentofpDK225BamHIfragmentKwas

determined by chemical degradation of end-labeled DNA strandsbyusingthe strategy indi-cated in Fig.1C(28).The sequence is presented 5' to 3' in Fig. 2, in the same orientation as in Fig. 1.Theorientationwasverifiedby correlat-ing the SmaI restriction enzyme cleavage site within theHinflsequence(Fig.2)withtheSmaI sitesmappedin BamHIfragmentK(Fig. 1).The 1,153-bp Hinfl fragment hasabasecomposition of 69.5 mol% guanosine plus cytosine. The strand shown in Fig. 2 contains 54.9% guano-sine,26.3%adenosine,14.6%cytosine,andonly 4.2% thymidine. Several kinds ofrepeated

re-gionsare present in thefragment. Most striking is a region at nucleotides 290 to 979 which consists of direct repeats of a hexanucleotide

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314 SANTEN, AND KIEFF

H1nfl

ENDI END3

AATCATGTCT GACGAGGGGCCAGGTACAGG ACCTGGAAAT GGCCTAGGAG AGAAGGGAGA CACATCTGGA CCAGAAGGCT CCGGCGGCAG TG2GACCTCAA 100

ENDIENDi

AGAAGAGGGG GTGATAACCA TGGACGAGGA CGGGGAAGAG GACGAGGACG AGGAGGCGGA AGACCAGGAG CCCCGGGCGG CTCAGGATCA GGGCCAAGAC 200 HpaII AvaIl 3 2 3 2 3 - 3 1 2 3

ENDI 3' SS

ATAGAGATGG TGTCCGGAGA CCCCAAAAAC GTCCAAGTTG CATTGGCTGC AAAGGGACCC ACGGTGGAAC AGGAGCAGGA GCAGGAGCGG GAGGGGCAGG 300 2 3 1 2 3 2 3 3 1 2 3 2 3 3 1 2 3 1 2 3 1 2 3 1 2 3 2 3 3 1 2 3 2 AGCAGGAGGG GCAGGAGCAG GAGGAGGGGC AGGAGCAGGA GGAGGGGCAG GAGGGGCAGG AGGGGCAGGA GGGGCAGGAG CAGGAGGAGG GGCAGGAGCA

400 3 3 1 2 3 1 2 3 1 32231233 23 322 1 2 3 2 3 3 1 2 3 1

GGAGGAGGGG CAGGAGGGGC AGGAGGGGCA GGAGCAGGAG GAGGGGCAGG AGCAGGAGGA GGGGCAGGAG GGGCAGGAGC AGGAGGAGGG GCAGGAGGGG

500

2 3 1 2 3 2 3 3 1 2 3 233 1 2 3 1 2 3 2 3 3 1 2 3 1 2 3 1 2 3 2 3 CAGGAGGGGC AGGAGCAGGA GGAGGGGCAG GAGCAGGAGG AGGGGCAGGA GGGGCAGGAG CAGGAGGAGG GGCAGGAGGG GCAGGAGGGGCAGGAGCAGG

600

3 1 2 32 2 3123 1 2 3 1 2 3 2 3 1 2 3 2 33 1 2 3 123 1 2 3 2 AGGAGGGGCA GGAGCAGGAG GGGCAGGAGG GGCAGGAGGG GCAGGAGCAG GAGGGGCAGG AGCAGGAGGA GGGGCAGGAG GGGCAGGAGG GGCAGGAGCA

700

3 1 2 32 3 1 2 3 2 3 1 3 22 3 1 2 3 1 2 3 2 3 1 2 3 1 2 3 2 3 1

GGAGGGGCAG GAGCAGGAGG GGCAGGAGCA GGAGGGGCAG GAGCAGGAGG GGCAGGAGGG GCAGGAGCAG GAGGGGCAGG AGGGGCAGGA GCAGGAGGGG

800

2 3 1 2 3 2 3 3 1 2 3 1 2 3 2 3 3 1 2 3 1 2 3 2 3 1 2 3 1 2 3 2 3 1 CAGGAGGGGC AGGAGCAGGA GGAGGGGCAG GAGGGGCAGG AGCAGGAGGA GGGGCAGGAG GGGCAGGAGC AGGAGGGGCA GGAGGGGCAG GAGCAGGAGG

900

2 3 1 2 3 2 3 2 3 1 2 3 2 3 3 1 2 3 2 3 1 2 3 2 3 HpaIl

GGCAGGAGGG GCAGGAGCAG GAGGGGCAGG AGGGGCAGGA GCAGGAGGAG GGGCAGGAGCAGGAGGGGCA GGAGCAGGAG GTGGAGGCCG GGGTCGAGGA

2a 2b

ENDi

2c ENDI

GGCAGTGGAG GCCGGGGTCG AGGAGGTAGT GGAGGCCGGG GTCGAGGAGG TAGTGGAGGCCGCCGGGG1

3b

GTGAAAGAGC CAGGGGGAGA GGTCGTGGAC

HInfI END3 GTGGAGAAAA GAGGCCCATG ANT

FIG. 2. Nucleotidesequenceof the1,153-bpHinflfragment.Thesequenceispresentedinthepolarity5'to3'

intheorientation of the map shown in Fig. 1. HpaIIandAvaIIrestriction enzymesitescleaved togenerate

fragments usedashybridization probesinthisstudyareindicated.Superscripts 1, 2,and 3areshownabove the

nucleotide triplets GGG, GCA,andGGA,respectively,atpositions272to979.ThisregionistheIR3repeated

sequencein EBV DNA.Repeatunits(2 3)and(233)areunderlinedinthesequenceandareinterspersedwith anotherrepeatelement, (1 2 3)n,where nvaries from 1 to4. Direct repeatregions areindicatedin boxes at

positions981to1068 and 1076to1123. Basepairmismatches in therepeatedsequencesareshownbyanasterisk

above the mismatched nucleotide (see text).Termination codonsareindicated in the first (END1)and third

(END3) translational reading frames;thesecondreadingframeiscompletelyopen.Apotential3'splicesiteisat

position 219.

andtwononanucleotidesequences.These oligo-nucleotides arecomposed of only three triplets:

GGG, GCA, and GGA. A similar region at

nucleotides 272to286 isseparated from the long tripletarrayby thetriplet GCG atpositions 287

to289. The entirerepeated regionatnucleotides 272 to 979 is designated IR3. To indicate the formatof therepeatswithinIR3, the numerical designations 1, 2, and 3 arearbitrarily assigned

to the triplets GGG, GCA, and GGA,

respec-tively, andareindicatedassuperscriptsoverthe

corresponding triplets in Fig.2(within the limits of therepeated region only). The general struc-tureof therepeatcanberepresentedas(1 2 3),

where nvariesfrom 1 to4, interspersedwith (2

3) or(23 3). The elements (2 3)and (2 3 3 )are

underscoredtoindicate thepatternof intersper-sion of theseoligonucleotidesamongthetandem

repeats of the (1 2 3) nonanucleotide. A 6-bp

sequence, 5'-GGTGGA-3', is repeatednearthe

left andrightlimitsofIR3(positions263 to268

and980to985,respectively).

Two other classes ofdirectrepeatsareinthe

Hinfl sequence. They are indicated as boxed

regions in Fig. 2. One class consists of three

tandemrepeats ofa24-bp sequence (sequences 2a, 2b, and 2c) at positions 994 to 1,068. The

prototypeformissequence2b. A transition from

TtoCoccursatposition 1,003incopy2a,anda

3-bp insertion, GCC, is present at positions 1,062 to 1,064 in repeat2c (indicated by

aster-isks above the mismatched nucleotides). An

*** END1i END1I

TA GAGGA

3a

kCGTGAAAGAGCCAGG GGGGGAAGTC

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[image:4.496.63.430.79.397.2]

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incompletecopyof thisrepeat sequence occurs between IR3 and 2aatpositions 981to993. The second class is composed oftwo tandem 24-bp sequences (sequences 3a and 3b) at positions 1,076 to 1,123(nonidentical basesare indicated by asterisks).

Conservation and variability of the IR3 region. Since IR3 has homology with a component of human and mouse cell DNAs (Heller et al., in press),it ispossible that IR3 has beenacquired by B95-8 virusthrough recombination with cell DNA. SomeEBVisolateswould then be expect-edtobe lackingIR3 ormight incorporateit into adifferent site. Analternative model is that IR3 isa componentofthe EBV genomefrom various sources. To determine whether the IR3 se-quence is a common feature in other EBV isolates, the 32P-labeled HpaIIfragment, which contains 57 nucleotides to the left of IR3, 708 nucleotides of IR3, and 11 nucleotides to the right ofIR3 (Fig. 1C and 2), washybridized to nitrocellulose filters containing the separated BamHI fragments ofthe W91,AG876, P3HR-1, Raji, Cherry, or Lamont isolates ofEBV (see above for adescription of the origins of these EBVstrains). The right panels of Fig. 3 show the results of this hybridization. The left panels present aportion of the sameblots whichwere rehybridized with32P-labeledEBV DNA to indi-cate all of the viral DNA fragments. The IR3 probe identifies a single fragment in all of the EBVstrains. The 32P-labeledAvaII-HpaII frag-ment(IR3plus 16 bptotheleft and11bptothe right of IR3 [Fig. 1C and 2]) and a pDK225 BamHI K probe also hybridize to the same fragments on other Southern blot strips of BamHIfragments ofeachofthe EBV DNAs (17 and data not shown). In each instance, the extentof hybridization oftheIR3-specific probe tothesingleBamHI Kfragmentwas proportion-al to the amount of viral DNA on the blot as judged from the hybridization with 32P-labeled EBVDNA.The results indicatethat much or all of the IR3 sequence is conserved within the BamHI Kfragment in each of these isolates.

The BamHI Kfragments vary insize among theEBVisolates(Fig. 3)asfollows: Cherry (5.2 kb) >AG876 (5.0 kb)> B95-8(5.0kb)> P3HR-1 (4.9kb)> W91(4.8 kb) > Lamont(4.8kb) >

Raji (4.7 kb). This variability reflects either differences among EBV isolates in the unique-sequence portions of BamHI fragment K or heterogeneity within the IR3 repeat sequence. Heterogeneity in the IR3 region might occur through intramolecular or intermolecular un-equal homologous recombination within the re-peat. Thevariability of IR3among EBV DNAs was investigated by hybridizing the 32P-labeled HpaII fragment (described above) to Southern blotsofthe separatedHpaIIrestriction enzyme

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FIG. 3. Conservationof IR3 sequences in DNAs of

different EBV strains. DNAs from virus (B95-8, W91, AG876, and P3HR-1) or from nonpermissively

infect-ed cells (Cherry [C], Lamont [LI, and Raji) were

digested with BamHI, electrophoresed on agarose

gels, andtransferred to nitrocellulose filters. The right

panels show the resultsof hybridization(in 6x SSC at

72°C) ofa32P-labeled,0.77-kbHpaII IR3 DNAprobe

to the filters. The left panels show the result of

rehybridization of the same filters to a 32P-labeled

EBV DNA probe. Only the BamHI K region of the

filters is shown.

fragments of EBV DNAs purified from virus, infected cells, or recombinant plasmids. The results established thatvariability in the size of IR3 does occur among isolates, but that IR3 is stableamongtheDNAmolecules of each isolate (Fig.4). TheIR3-HpaIIfragmentsof B95-8 viral DNA and the recombinant derivative pDK225 are0.77 kb. The fragments ofW91 viral DNA and therecombinant EcoRIBcosmid derivative are 0.68 kb, whereas the AG876 and P3HR-1 viral and the Lamont intracellular IR3-HpaII fragments are 0.80, 0.70, and 0.64 kb, respec-tively. The observed range of variation in the size of the HpaII fragment containing IR3 is about 0.16 kb (AG876 versus Lamont [Fig. 4]) and accounts for most or all ofthe variability betweenthe BamHI Kfragments ofthese DNAs (Fig. 3). Thedifference in size of theBamHI K fragmentsin theCherry and RajiDNAssuggests that more extensivevariation inthe IR3 region may occur.

Conservation of IR3 in another herpesvirus. EBV and HVPapio are B-lymphotrophic pri-mate herpesviruses whichare endemic in their respective host species, i.e., humans and ba-boons(13-15).Fortypercentof the sequencesof

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Restriction enzyme maps of the BamHI K

re-gion of EBV DNA and the colinear rere-gion of HVPapio DNAareshown inFig. 5. The hybrid-ization results established that HVPapio DNA containsa sequencerelatedtoIR3 whichmaps

in EcoRI fragment G and XbaIfragment H and is colinear with EBV BamHI fragment K (Fig. 5).

m

.VPaPIO

-~

a Y i:. C1S fl N

a a

I

D- Ua

x,-S3 _a

H- 0

S

a

FIG. 4. Variability in the size of IR3 among EBV isolates. DNAs from (left to right) recombinant

plas-mid pDK225 containing the B95-8BamHIK fragment,

B95-8 virus, a recombinant cosmid containing W91

EcoRI fragment B, W91 virus, P3HR-1 virus, AG876 virus, AG876 transformant (a cell line of neonatal lymphocytes transformed in vitro by AG876 virus),

and the nonpermissively infected cell line Lamont

were digested with HpaII restriction enzyme. The

resultof hybridization of a32P-labeled,0.77-kbHpaII

probe in 6x SSC at 72°C to a Southern blot of the

separated HpaII fragments is shown. The

hybridiza-tion to higher-molecular-weight fragments in the

AG876 transformant and Lamont cell DNAs is due to partial resistance of these methylated DNAs to cleav-agebyHpaII.The ethidium bromide-stained gel insert

showsadenovirus type 2 EcoRI and pBR322

BamHI-Hinflfragment size markers.

EBV and HVPapio DNAs are well conservec

between the viruses (13, 15, 19). The

homolo-goussequencesarecolinear among the genomes and aredispersed alongalmost the fulllengthof the DNAs(20, 26). Thecolinear relatedness and common DNA organization indicate that these primateviruses evolvedfroma common

ances-tor. An IR3 AvaII-HpaIIprobewas hybridized to Southern blots of the separated restriction

enzyme fragments ofHVPapio DNA to deter-mine whetherahomologoussequence is present at a colinear point in this primate genome.

0

N'- a

B.

-LvPcpo

G

H

A M LE EcoRI

D XboI

B

U/__>._1 /-;t-"B- -nBomHl

FIG. 5. IR3-related sequences in another

herpesvi-rus. (A) Identical blot strips of viral DNA from the

relatedprimateherpesvirus HVPapiohybridized in 6x

SSC at 720C to 32P-labeled viral DNA, pDK225 BamHI fragment K, and AvaII-HpaII IR3 probes.

Viralfragments which hybridized to labeled pDK225

or IR3 are indicated. Designations of the fragment

namesandtheir map locations inHVPapioDNAhave

beendescribedpreviously(18, 20,26). (B) EBV DNA

BamHl K region and the colinear region from

HVPapioDNA.Thearrowsbelow the EBVDNAmap

indicate the location of the Sall Ffragment in viral

DNAandplasmid VRD1.

.1-"... .,.r .-.

rn) Ct:

a

Ak

", f..

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Transcription of ER3. Since a 3.7-kb poly(A) cytoplasmic RNA is transcribed from the BamHI Kfragment in latently infected, growth-transformed IB4 cells (24, 49), we investigated whether IR3 encodes part of this RNA. 32P_ labeled IR3 DNA hybridized to the 3.7-kb IB4 RNA on Northernblotsand also identified 5.8-and 4.5-kboligo(dT)-selected IB4 nuclearRNAs (Fig. 6A). The direction of transcription ofthe 3.7-kb IB4 RNA and thepotential slicing ofthis transcript in the IR3 regionwereexamined by Si nuclease analysis (3) of strand-separated DNA hybridized to IB4 RNA. The 7.9-kb Sall F fragment of EBV DNA (62 to67 megadaltons) which contains IR3was subclonedinto plasmid pKH47, and the separated DNA strands were isolated (16). Eachseparated strandwas hybrid-ized to IB4 cytoplasmic poly(A) RNA. After digestion with Si nuclease, the RNA-comple-mentary DNA waselectrophoresed on denatur-ingagarosegels and transferredtonitrocellulose filters. IR3 and SalI F probes hybridize to a

2.0-kb RNA-complementary fragmentfrom the SalI F strand opposite in polarity to the

se-quence shown in Fig. 2. Neither the SalI F nor

the IR3 probe hybridized to blotsfrom experi-ments done with the SalI F strand identical in polarity tothe sequence shown in Fig. 2 (data notshown). Thus, the 3.7-kbRNA is transcribed from lefttoright inEBVDNAand contains the IR3sequence(Fig. 2). Since Northern blot anal-ysis showed that thelatently infected cell RNA is 3.7 kb whereas the RNA-protected DNA segmentisonly 2.0kb, the datasuggest that the cytoplasmic poly(A) RNA encoded by IR3 is spliced. Nootherfragmentsweredetectedwith the SalI F probe. Therefore, either multiple splicing events join small exons from Sall F DNA which total 1.7 kb in size, orpart ofthe 3.7-kb RNA is transcribed from DNA which maps outside of SalIfragmentF.

Low-abundance cytoplasmic poly(A) 2.2-, 1.9- and 1.5-kb RNAs are expressed from the BamHIKregioninproductivelyinfected B95-8 cells (22). The IR3 probe did not hybridize to these RNAsonNorthernblots, but dididentify previously undetected 2.4- and 1.0-kb RNAs (Fig. 6C).

The RNAs detected in B95-8 and IB4 cells with the IR3 probe represent viral transcripts and are not cellular RNAs which might be identified due to homology between the IR3 sequence and cellular DNA. The pDK225 BamHI K probe did not identify any RNA on Northern blots of poly(A)RNAfrom Loukes,an EBV genome-negative B-lymphocyte cell line (49). Also, labeled cDNA made from Loukes cytoplasmicRNAdidnothybridizetoSouthern blots of viral or recombinant DNA fragments containingthe IR3region (datanot shown).

DISCUSSION

IR3 and a closely related sequence are con-servedat acolinear position in the genomes of HVPapioand all EBVisolates examined. At its simplest level of organization, IR3 is a tandem repeat array composed of only three nucleotide triplet elements: GGG, GCA, and GGA. The triplets are further organized as a hexanucleo-tidesequence, GCAGGA (H), and two

nonanu-A. IB4 POLY B. 52P-IR3 2P-SaII F C 895

aRNA i. A RN

CYTO NUT CYTr

_ CY.To_

>-t a.

-745

-

U.6

~-i.45

-4.0

-- .92

-POLY

A

O

-.51

-FIG. 6. Transcription of IR3 sequences in B95-8

and IB4 cells. (A) Northern blots(48) of cytoplasmic

and nuclear poly(A) RNA from nonpermissively

in-fected IB4 cells hybridized to a 32P-labeled

AvaIl-HpaIIIR3probe.Hybridizations were done at 52°C in

5x SSC and50%oformamide. The positions of 5.2-kb

(28S) and2.0-kb (18S)rRNAsize markersare shown

(>). (Theband which isslightly smaller than the 4.5-kb

nuclear RNA is an artifact anddid not hybridize to the

IR3 probe in several other hybridizations.) (B) S1

nucleaseanalysis of RNA from IB4 cells. Unlabeled

separatedDNA strands of a Sall F-pKH47

recombi-nant plasmid, VRD1 (62 to 67 megadaltons in EBV

DNA [Fig. 1]), were prepared by chromatography of

denatured linearized plasmid DNA on

oligodeoxya-denylate and oligo(dT) columns. A 50-ng amount of

the strand complementary to the sequence shown in

Fig. 2 was hybridized to 50 ,ug of IB4 cytoplasmic

poly(A) RNAoryeast RNA, digested withSi

nucle-ase,incubated in NaOH, electrophoresed on

denatur-ing agarose gels, and transferred to nitrocellulose.

Identical blots of theRNA-complementary DNA were

hybridized to32P-labeledIR3 andSall Fprobesin 6x

SSC at 72°C. The background hybridization near the

topof the blots probably represents reassociatedSall

F DNA formed due to slight contamination of the

RNA-coding strand with the complementary strand.

Size markersaresingle-strandedHinflfragments from

the pDK286 EBV recombinantplasmid (5). (C)

Auto-radiogram of a Northern blot of oligo(dT)-selected

cytoplasmic RNA from permissively infected B95-8

cells which was hybridized to an IR3 probe as

de-scribedfor (A).

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cleotide sequences, GCAGGAGGA (Ni) and

GGGGCAGGA (N2). Atahigher level of

organi-zation, H or Ni sequences are interspersed

between N2 sequences ortandemcopies of N2.

There is no apparenthigher order ofsymmetry

orrepeat unit in IR3. IR3probably has evolved into an extended repeated region as a conse-quenceof unequalcrossoverwhichexpandedan

ancestralsetofsequencesrelatedtoH, Ni, and N2 (43).The polymorphism in size of IR3among

EBV isolates probably has been generated by this samemechanism. Thepreservation of

sym-metrythrough therepeatarraycould reflect the tendency of unequal crossoverto maintain the

fundamental repeat pattern.

Repeat sequences, such as IR3 and the two

tandem24-bprepeats, could be recognition sites for a protein. The origin and function of IR3

might be predicted bysequencerelatedness with

a known genetic element. We compared the

nucleotidesequence ofIR3 toeucaryotic cellu-lar and viral nucleotide sequences recorded at

the Nucleic Acid Sequence Database (8). Ran-dom perfect 9 to 12-bp matches were found in many sequences, including coding and

noncod-ing regions of mRNA and interspersed and mid-dlerepetitive (Alu family) elements (23).

Homol-ogyof theseother DNAstoIR3 does notextend beyond 12 bp since therelatedsequences arenot

tandemly repeated.

Other simple repeated sequences have been

describedpreviously. Examples includetandem

5-bp sequences in the recombinational class

switch region of immunoglobulin heavy-chain

genes (33); tandem repeats in the terminal a

sequence ofherpes simplex virus type 1 DNA

which mediates site-specific inversion events

(32); repeated dinucleotide arrays inintron 2 of

human gamma globin genes (42), in the

inter-genic spacer of sea urchin histone DNA (47),

and itn an "evolutionarily conserved" repeated

sequence family (30); an irregular repeat se-quence(CnT)matthetermini of linear extrachro-mosomal rDNA in Dictyostelium discoideum (11); and polymorphic tandemarrays of related 14to 15-bp sequencesin the5'-flanking region

of the human insulin gene(1). Unlike IR3,none

of these simple repeats encodes cytoplasmic RNA.

Northern blothybridizations showed thatthe

IR3 region encodes different cytoplasmic poly(A) RNAs in permissively infected B95-8

cells and in latently infected IB4 cells. In IB4 cells, IR3 is part ofa 2.0-kb exon, transcribed

left to right, which encodes part ofthe 3.7-kb

RNA identified on Northern blots (49; Fig. 6). IB4cell 5.8- and 4.5-kb nuclearpoly(A) RNAs were identified by an IR3 probe (Fig. 6). The

larger nuclear RNAs may be precursors ofthe

3.7-kb cytoplasmic poly(A)RNA. Sincewe

ex-amined onlysteady-state RNAin IB4 cells, our analysis cannot establish processing relation-shipsamongtheseRNAs. The DNAsequence in Fig. 2, correspondingto the RNA strand of the 2.0-kb exon, has a

Wor

potential splice accept-orsite, 5'-TGTCCGGAG-3', whichhas one mis-matched nucleotide (indicatedby the asterisk), compared with theconsensus sequence 5'-PyN-PyPyPyNPyAG-3' (41). Sites corresponding to theconsensus sequencesfortranscription initia-tion (M. Goldberg, Ph.D. thesis, Stanford Uni-versity, Stanford,Calif. 1979) and polyadenyla-tion(36) are not present. Itisnot knownwhether thesplice site is utilized in transcriptional proc-essing of viral RNA in IB4 cells. Termination codons occur in two of the three translational reading frames in the putative intron preceding the splice site (Fig. 2). The remaining reading frame has no stop codons through the entire Hinfl sequence.

Itis likely that the 3.7-kb cytoplasmicpoly(A) RNAis translated intoaprotein.Polyribosomal poly(A)RNA(presumably mRNA) is expressed from the sameregion ofEBV DNA in IB4 cells and in latently infected cell lines established from Burkitt tumors (24, 25, 35). If the IR3 sequence in the 3.7-kb RNA is translated into protein, the nucleotide sequence (Fig. 2)in the three possible reading frames predicts a simple coding domain of more than 200 amino acid residues composed ofonly (i) serine, arginine, and glycine; (ii) glycine and alanine; or (iii) glutamine, glutamate, and glycine.Thevariation in length of IR3 among EBV isolates predicts that the putative translation products of IR3 mRNAs would correspondingly differin size.

Growth-transformed cells latently infected with EBV contain a nuclear antigen, EBNA (39). EBNA varies in sizeamongdifferent latent-ly infected cell lines, and the relative sizes of EBNA in Namalwa, B95-8, P3HR-1, and Raji cells(46) correlate with the relative sizesof IR3 in each of these viralDNAs.Therefore,IR3 may encodepart ofEBNA. It should be noted that variation intheIR3tripletarraywhichpreserves the repeat pattern (expected from an unequal crossover mechanism) will maintain a unique translationalreading frame for codons3' toIR3. It is of interest in this regard that the single divergent region in IR3 at positions 287 to 289 (Fig.2)preservestripletsymmetryandtherefore the translational reading frame. Genetic and protein polymorphisms occurwithin the repeti-tivesequencesoftheglycine-alanine-serine sim-pleamino acid-coding domain of silk fibroin(27, 45). Alleles of silk fibroin differ in length as a consequence of unequal recombination ex-changes withinthe repetitive coding domain of the gene. These alleles encode fibroins which differcorrespondingly in size.

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SIMPLE REPEAT SEQUENCE IN EBV DNA 319 ACKNOWLEDGMENTS

We thank Victor Raczkowski for subcloning the Sall F

fragment from pDK153 into pKH47. We also thank Mary Hummel forthegenerousgift of nitrocellulose filters contain-ing B95-8 cytoplasmic poly(A) RNA. Chris Beiseland Susan Fennewaldwerehelpful in thecomputeranalysisof the DNA

sequence.

Thiswork wassupported by Public Health Servicegrants

CA17281 and CA 19264from the National Institutes ofHealth andbygrantMV 32 G from the American Cancer Society. V.V.S. isapredoctoraltrainee(CA 09268).E.K. isafaculty research awardee of the AmericanCancer Society.

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