Functional limits of oriP, the Epstein-Barr virus plasmid origin of replication.

0022-538X/89/073016-10$02.00/0

Copyright© 1989, American Society for Microbiology

Functional Limits of

oriP,

the

Epstein-Barr Virus

Plasmid Origin of

Replication

THOMAS CHITTENDEN,' STEPHEN LUPTON,2 AND ARNOLD J.

LEVINE'*

Department ofBiology, Priniceton Univ,ersity, Princeton, Newv Jersey 08544-1014,1 andImmiunexCorporation,

Seattle, Washington 981012

Received 24 January 1989/Accepted 30March 1989

The Epstein-Barr virus (EBV) genome contains two cis-acting elements which are required for stable extrachromosomal plasmid maintenance in latently infected cells. The first consists of 20 30-base-pair (bp) repeats, eachof which contains a DNA-binding site for EBV nuclear antigen 1 (EBNA-1), the trans-acting

factorrequired for plasmid persistence. The second element is composed ofa65-bp dyadsymmetry,containing fourEBNA-1-binding sites. Deletionmutantswere constructed which reduce the number ofEBNA-1-binding

sites in the 30-bprepeats,alter the number of EBNA-1-binding sites in the dyad region, ortruncatethedyad element. The effect of the deletion mutations on plasmid maintenance was examined by transfecting

recombinant plasmids, containing both the mutated EBV sequences and a drug resistance marker, into D98-Raji cells. The plasmidsweretested for their abilityto generatedrug-resistant D98-Raji cell colonies and theircapacity tobe maintained in anextrachromosomal form without undergoing extensive rearrangements.

EBVplasmids with 12or 15 copies of the 30-bprepeatswerewildtypein bothassays.Plasmids with justtwo

orsixcopies of theserepeated elements failedto generate drug-resistant colonies atanormallevel, and normal

episomal plasmidswerenotdetected in theresulting colonies. Rare colonies of cells resulting fromtransfection

of these two- or six-copy mutants contained rearranged, episomal forms of the input plasmids. The

rearrangements mostoften produced head-to-tail oligomers containing a minimum of eight 30-bp repeated elements. Therearranged plasmidswere shown toberevertantforplasmid maintenance in that they yielded wild-typeor greater numbers ofdrug-resistant colonies and persisted atthe wild-typeor a greaterepisomal

copynumber. ByuseofanEBVplasmid that containedno30-bp elements,norevertantscould be isolated. One tofivecopies ofasynthetic linkercorrespondingtoaconsensus30-bp repeated element inserted intoaplasmid withno30-bp elementsnowpermitted thegenerationof oligomeric, episomal forms of themutant testplasmid. These experiments demonstratearequirement foraminimal number (sixtoeight copies) of the 30-bp repeated element. Deletions in the 65-bp dyad region had little or no effect upon the ability to generate enhanced numbers of drug-resistant D98-Raji colonies, indicating that the 30-bp repeated element is predominantly required for this phenotype. Plasmids bearing deletions in the 5' side of the dyadsequenceretainedthecapacity

toproduce stimulated numbers of drug-resistant colonies, but the extrachromosomal plasmids in these cells all contained structural rearrangements. In contrast, deletions impinging on the 3' side of the dyad element

completely abolished extrachromosomal plasmid maintenance. Simian virus 40 enhancer sequences placed adjacent (3' side) to the 65-bp dyad also abolished the ability of the EBV plasmids to be maintained in an extrachromosomal state. These studies identify the regions of the 65-bp dyad that are essential for extrachromosomal maintenance and point out a functional asymmetry which exists in the structurally

symmetrical element.

Epstein-Barr

virus(EBV)infection ofprimary B

lympho-cytes results in the "immortalization" of these cells sothat

they can replicate and grow in cell culture indefinitely (17; reviewed in reference 10). In most cases infectious virus is not produced bythesecells and only asmall fractionof the viral genome is expressed in the form ofstable cytoplasmic

EBV-encoded mRNA(7, 26, 35). The 180-kilobase (kb) viral genomepersists as acircular,extrachromosomal,multicopy

plasmid inlatentlyinfected cells (13, 24). Such autonomous

plasmids

replicate only once during the S phase of the cell

cycle

inacoordinate fashion with cellularDNAsynthesis (1,

14). Latentlyinfected cells commonly express several EBV-encodedproteinslocalizedinthe nucleus (6, 9, 16, 19-21, 23,

31, 33) or the cell membrane (18). Of these proteins, EBV nuclear antigen 1 (EBNA-1) appears to be required for

replication

of the EBV plasmid (25, 37).

Previous studies have identified a region of the EBV genome, oriP, as the putative viral replication origin

em-*Correspondingauthor.

ployed during latent infection (25, 36). Recombinant DNA plasmids containing oriP and a dominant selectable marker gene, such as G418 resistance or hygromycin resistance, wereshown to be maintained in an extrachromosomal form in cells expressing EBV proteins (25, 36, 37). Incorporation of the gene for EBNA-1 into such plasmids permitted episomal maintenance in cells not expressing other EBV-encoded proteins (25, 37), and mutations in the gene for EBNA-1 prevented extrachromosomal persistence of the plasmid (25). Deletion mapping localized two separable elements in oriP that were each required in cisfor extrachro-mosomal maintenance (25, 30). The first element consistsof 20copies of a tandemly repeated 30-base-pair (bp) sequence. Thesecond essential element, located about 1 kb away inthe viral chromosome, consists of a 65-bp sequence forming a dyad sequence symmetry. The dyad region harbors four related copies of the 30-bp repeat, with two of these copies embedded in the dyad structure itself (2). EBNA-1can bind to a12-bp palindromic core sequence which is found in each of the 30-bprepeats, as well as in the four related sequences

3016

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located in the dyad symmetry region (27), suggesting that EBNA-1 acts at these sites directly to facilitate plasmid maintenance. In some cases a segment of the EBV BamHI C fragment, including oriP, can functionally substitute forthe simian virus 40 (SV40) enhancer-promoter regulating a drug resistance gene (25), and the 30-bp repeat element can act as an EBNA-1-dependent enhancer when positioned adjacent to heterologous promoters (29). Other viruses, such as polyomavirus, have a similar organization at their origin of replication (34). A trans-acting viral protein,thelargetumor antigen, like EBNA-1, acts directly upon a replication origin

ofdyad symmetry to initiate DNA replication. Polyomavirus

replication also requires the presence of an enhancer ele-ment (in analogy to the 30-bp repeats) upstream from the dyad (8).

To study the sequence requirements within oriP, recom-binant plasmids were constructed containing deletion muta-tions in the 30-bp repeated sequences and in the 65-bp dyad

region. Between 6 and 12 of the 30-bp repeats appear to be required for extrachromosomal maintenance of these plas-mids in D98-Raji cells. Test plasplas-mids containing six or fewer

30-bprepeats weredefective for plasmid maintenance since theypersisted extrachromosomallyinrearranged form only. Isolated, rearranged plasmids were found to be revertant in thatthey had regained a higher number of 30-bp repeats and were fully wild type in maintaining the extrachromosomal copynumber. The structure of revertant plasmidsindicated

that the 30-bp repeats do not have to be organized in a tandemfashion in order to facilitate plasmid maintenance. The 65-bp dyad region alone was not sufficient forplasmid maintenance. Deletions extending into the 65-bp dyad ele-ment showed a significant asymmetry in this region.

Dele-tions removing the 5' side of the dyad did not abolish extrachromosomal maintenance of the plasmid DNA in

D98-Raji cells. In most cases the plasmids persisted after

undergoing structural alterations that may have compen-satedforthedeletions. In contrast,deletions truncating the 3' side of the dyad completely abolished plasmid mainte-nance, and rearranged episomal DNA was not observed.

Deletions that removed the EBNA-1 sites adjacent to the dyad structure alsodestroyedthecapacityfor

extrachromo-somal maintenance. The placement of heterologous se-quences immediately adjacent to the 3' side of the 65-bp dyad interfered with oriP function. The interference was alleviated bychangingthenatureoftheadjacentsequences.

A 7339

A:

aofm

.-9518

0 0

O-a :p

-AO

p20

EI}

p15

Ic

--pl2

-0

p6

ED

p2

[

B

-0 u

0

04

0

x

0

X

pHm pSLneo

FIG. 1. Structure ofplasmids containing deletions in the 30-bp repeatdomain.(A) Mapofthe EBVplasmid maintenance region. nt numbering is according to Baer et al. (2). Symbols:

LII,

region containingthe 20copies ofa 30-bp repeat, which is progressively truncated inthe mutants to leave15,12,6, or 2intact30-bprepeats;

0, region of 65-bp dyad symmetry;

4,

position ofan SstI linker insertion. (B) Structures ofplasmid vectors. pHm symbols: thick line, pML2sequencescontaining theColElreplication origin; ,

transposon Tn5sequencesdirecting theexpression ofthe hygromy-cin B phosphotransferasegene in E. coli;

LII,

sequencesfromthe herpes simplex virus type 1thymidine kinasegene controllingthe expression ofhygromycin resistance in mammalian cells. pSLneo symbols: thick line, pML2 sequencesencoding ,-lactamase (amp) andcontaining theColEl replication origin;

LII,

SV40 sequences controllingtheexpression oftheG418 resistancegene. Thedeletion mutations were cloned intheorientation showninto pHm(named

p20hm, pl5hm, pl2hm, p6hm, and p2hm) and pSLneo (named p20neo,pl5neo, pl2neo, p6neo,andp2neo).

MATERIALS ANDMETHODS

Cell culture.D98-Rajicellsare anadherent somatichybrid cell linecontaining multiplelatent EBVepisomes (11). Cells

were propagated in Dulbecco modified Eagle medium

con-taining 10% fetal bovine serum, 100 U ofpenicillin per ml, and100 ,ugofstreptomycinper ml.

Plasmid maintenance assay. D98-Raji cells were

trans-fected with 20 ,ug ofplasmid DNA (no carrier DNA) by a

modified calcium phosphate DNA precipitation procedure,

as described previously (25). Drug-resistant colonies arose

after 2to 3 weeks of selection in mediumcontaining either G418 (500 ,ug/ml) or hygromycin B (140 ,ug/ml). The cell culture medium was changed every 4 days. Numbers of drug-resistant colonies were estimated after staining plates with Giemsa. Colonieson remainingplates were pooled by

trypsinization and expanded in selective medium, and low-molecular-weight DNAwasprepared fromcell cultures by the method of Hirt (22). In some cases extrachromosomal plasmid DNA was recovered from Hirt preparations by

transforming competent Escherichia coli DH5

(Bethesda

ResearchLaboratories, Inc.) with thelow-molecular-weight

DNA extracted from 107 cells (15). Portions of the Hirt

extracts were digestedwith arestriction enzyme.

Digested

and undigested DNA preparations were fractionated on

0.9%

agarose gels and transferred to nitrocellulose as de-scribed previously (25, 32). Radioactive probes were

pre-paredby nick translation ofvectorplasmid DNA,

resulting

in specific activities of about 108 dpm/,ug.

Hybridization,

washing of filters,andautoradiographywere

performed

also asdetailedpreviously (25).

Plasmidconstructions. TheBamHI-CEBV DNAfrom the

SphI

siteatnucleotide(nt)7339 (nt

numbering

as inBaeret

al.

[2])

to the SstII site at nt 9518

(Fig. 1A)

contains the

cis-acting genetic elements necessary for

plasmid

mainte-nance: the 30-bp repeat domain

(LII)

and the

65-bp

dyad

region

(0).

The

SphI-to-SstII

EBVfragmentwascloned into the ClaI-to-XhoI sites of the

hygromycin

resistance-based

vectorpHm after the addition ofClaIand XhoIlinkers

(Fig.

1A). The 3.1-kb pHm plasmid containsthe gene for

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mycin resistance (12), selectable both in E. coli (TnS

pro-moter) and inmammalian cells (herpes simplex virus type 1 thymidine kinase expression signals). A single SstIl linker

wasintroduced at the SmaI site (nt8193) in the EBV DNA (Fig. 1A,

I

) to facilitate subsequent manipulations. The resulting plasmid, p20hm, represents the wild-type plasmid used in these studies containing thefullcomplement of 30-bp

repeatsandanunaltered dyadsymmetry element. Unidirec-tional deletions were introduced into the 30-bp repeat do-main by digestion with Bal 31 nuclease from the ClaI site in pHm. The fourdeletion mutations chosen for studyleave 15, 12,6, or2 intact copies of the 30-bprepeats, as determined

by gelelectrophoresis andDNAsequencing. These deletion mutations give rise to plasmids pl5hm, pl2hm, p6hm, and p2hm in the hygromycin vector. The ClaI-to XhoI region of EBV DNA from p2ohm and the deletion mutants was

subcloned into the EcoRI-to-XhoI sites of the G418 resis-tance vectorpSLneo (Fig. 1B), resulting inplasmids p20neo, pl5neo, pl2neo, p6neo, and p2neo. The construction of pSLneowasdescribed previously (25).

The pOneo vector, which lacks all 30-bp repeats, was

constructed by deletingthe entire 30-bprepeat domainfrom p20neo. The EBV DNA from theEcoRI site (nt 7339)tothe MluI (nt 8316) site was removed, and aBamHI linker was

inserted in its place. This results in aunique BamHI site700

bp upstreamfrom the dyad element. Multiple copies of the following synthetic double-stranded oligonucleotide,

corre-spondingtoa consensus 30-bp repeat, werecloned into the

BamHI site of pOneo:

GATCC AGATT AGGAT AGCAT ATGCT ACCCA GATAT A G TCTAA TCCTA TCGTA TACGA TGGGT CTATA TCTAG A head-to-tail orientation of tandem oligonucleotides was

ensured by digestion with BglII and BamHI after ligation, sinceonlyahead-to-tailarrangementofoligonucleotides will notrecreateaBamHI orBglII site. The number of inserted

oligonucleotides was determined by restriction fragment

sizingonhigh-percentage agarosegels. Plasmids withoneto

five tandem copies of the oligonucleotide were obtained

(plneothrough p5neo).

Deletions within the dyad symmetry region were made after first subcloning the EcoRV-to-XhoI region (for 5' deletions) or the SstI-to-HpaI region (for 3' deletions) of p20neo into Bluescript SK (Stratagene). Unidirectional de-letions from either the EcoRV or the HpaI side were

introduced using exonuclease III (Stratagene protocol) or

restrictionenzymes OxaNI, SmaI,and NdeI. Deletion

end-points were determined by the sequencing of

double-stranded plasmid DNA by a modified Sanger dideoxy

method (U.S. Biochemicals).Thedeletions chosenfor anal-ysiswere thenrecloned in p2oneo (replacingthe unaffected

dyad region of p2oneo). The resulting plasmids have struc-tures similar to that ofp20neo, except they contain either internal deletionsbetween the EcoRV site and endpointson

the 5' side of the dyad region (see Fig. 5) or deletions

extending between endpoints on the 3' side of the dyad and the XhoI site. The exonuclease-generated deletions are

namedaccordingtothe lasttwonumbers in thentposition of the deletion endpoint. For example, the 3' deletion 3.71A

removes EBV DNA between nt 9071 and the XhoI site. Similarly, the 5' deletion 5.49/removes EBV DNA between

nt9049 and the EcoRV site. Restriction enzyme-generated deletions Hpa3'A, Sma3'A, Nde3'A, andOxa5'Ahave dele-tion endpoints within the dyad region at nt positions 9136, 9108, 9062, and9008,respectively. The SstI-to-XhoI regions of 3' dyad deletion mutants were also cloned into p2Ohm

(replacing the nondeleted SstI-to-XhoI dyad region of

p20hm), forming plasmids pHpa3'Ahm, pSma3'LThm, p3.95Ahm, p3.71Ahm, and pNde3'zThm.

RESULTS

There are two EBV genetic elements, constituting oriP, that are required in cis for the extrachromosomal mainte-nanceof plasmids in latently infected cells:a30-bp sequence repeated 20 times and a65-bp sequencearranged in a dyad (25, 30). An extensive deletion analysis of these two ele-ments was undertaken in an attempt to understand the minimum sequence requirements for their function in la-tently infected cells.

Plasmid maintenance requirements for the 30-bp repeats. Therequirement for the 30-bp repeat region was first exam-ined by constructing plasmids bearing deletions which pro-gressively truncate the 30-bp repeatdomain. Bal 31 deletions were introduced into the 5' side of the repeat domain (Fig. 1),leaving 15, 12, 6, or just 2 of the original 20 repeats intact, while the dyad symmetry region was not affected in these plasmids. The deleted oriP sequences were cloned into two different vectors: pSLneo, encoding G418 resistance regu-lated by SV40 enhancer-promoter sequences, and pHm,

containingthe genefor hygromycin resistance selectable in both E. coli and animal cells. Deletion plasmids pl5hm, pl2hm, p6hm, and p2hm share the pHm vector backbone, whereas the deletion series pl5neo through p2neo contain the pSLneobackbone. Plasmids p20hm and p2Oneo contain the wild-type EBVplasmid maintenance sequences, with all 20 repeats intact, cloned into pHm and pSLneo, respec-tively. Previous studies have demonstrated that extrachro-mosomal plasmid maintenance is associated with a substan-tial (up to 200-fold) increase in the number of drug-resistant

coloniesrelative toaplasmid vector that must integrate into cellular DNA (25). Therefore, the ability of the deletion mutants topersist extrachromosomally was tested by trans-fecting the plasmids into D98-Raji cells (which supply EBNA-1 gene product in trans from the endogenous latent EBV genomes) and selecting either hygromycin- or G418-resistant colonies. Similar results were obtained with both the pHm-and thepSLneo-based test plasmids. This demon-strates that the conclusions of these experiments do not depend upon either the nature of the selectable marker employed (G418orhygromycin) or the nature of the plasmid

constructionsused.Typically, deletion mutants with 15 or 12 30-bp repeats gave rise to wild-type levels of drug-resistant colonies (Table 1). However, plasmids with just six or two copies of the 30-bp repeats were greatly impaired (10- to 40-fold) in their ability to generate drug-resistant colonies, yielding numbers comparable to a vector that must integrate into cellular DNA to persist in the cells (Table 1).

To determine whether these plasmids were maintained extrachromosomally, drug-resistant colonies were pooled and expanded in cell culture, and low-molecular-weight DNA was prepared by the method of Hirt (22). The low-molecular-weight DNA preparations were then analyzed by Southern blotting to determine whether the transfected plasmid persisted extrachromosomally in these cells. An analysis of these preparations digested withXhoI indicated that extrachromosomal DNA of the expected size was presentforp20neoand thepl5neo and pl2neo mutants (Fig. 2A, lanes a,b, and c, respectively). However, the extrachro-mosomal plasmid DNA detected in the low-molecu-lar-weight fractions of p6neo (Fig. 2A, lane d) and p2neo

transfections appeared to have undergone rearrangements,

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TABLE 1. Transfection of mutant and revertant EBV plasmids Relative no. Estimated

Plasmid ofdrug-resistant episomal

b

D98-Rajicolonies" copyno.

pSLneo 1 0

p20neo 350 1

pl5neo 330 1

pl2neo 260 1

p6neo 35 5-10'

p2neo 10 20-50C

pHm 1 0

p20hm 340 1

pl5hm 340 1

pl2hm 280 1

p6hm 30 10-20'

p2hm 10 20-50'

p2Rlhm 550 10-20

p2R2hm 220 10-20

p2R3hm 200 10-20

p2R4hm 180 10-20

p6Rlhm 650 10-20

p6R2hm 330 10-20

a Relative number of drug-resistant colonies estimated 2 weeks after transfection,ascomparedwith the numberofcolonies produced by pSLneo

orpHm(set to1).

b Approximatecopy numberdetermined fromSouthernblots, by

compar-ing the hybridization intensity with plasmid DNAstandards (as inFig.2A). ' Indicatesthepresenceof rearranged extrachromosomal plasmidDNA.

as demonstrated by additional bands of anomalous size,

particularlyin thecaseofp2neo(Fig.2A, lane e). Appropri-ately sized form I (uncut)extrachromosomal plasmidDNA

(Fig.2B,arrow) was evidentforp20neo,pl5neo,andpl2neo

(Fig.2B, lanes a,b,and c). In contrast,formIplasmidDNA

ofthe correct size was not detectable for p6neo or p2neo, and the extrachromosomal DNA present was of much greatermolecularweightthanexpected(Fig.2B, lanes d and

e).Digestion ofthe HirtextractswithXhoI,whichcuts once in the parentplasmidstructure,reducedasubstantialportion

of the

higher-molecular-weight

extrachromosomal plasmid

species, detected in the Southern blot ofundigested DNA

preparations, totheappropriate linearmonomersize(Fig. 2,

compare lane d in panel B and lane d in panel A). These results are consistent with the possibility that the high-molecular-weight plasmid species observedin these South-ernblots were

extrachromosomal,

multimeric forms ofthe

transfectedplasmid.Thereisalwaysasmall levelofsheared

high-molecular-weight chromosomal DNA present in these Hirt extracts. This raises the possibility that the plasmid species detected in Fig. 2B represent tandem, integrated copies of thetransfected plasmidDNA. This appearstobe veryunlikelyfor tworeasons. First, Hirt DNApreparations

obtained from cell lines derived by transfection with

plas-mids thatareexpected tointegrate into cellular DNAnever

produced suchprominentbandsonSouthernblots. InaHirt

extractpreparedfrompSLneo,forexample, onlyfaint bands

ofless than one copy percell were detected

(i.e.,

see

Fig.

6B,lane a). Second, many

independent

multimericor

rear-rangedplasmidspecieswererescued inE. colifrom the Hirt extracts ofp6hm and p2hm transfections (see

below).

The large sizes of these rescued

plasmids

areconsistent with the

high-molecular-weight plasmid

species

apparent in the Southern blots.

One

interpretation

of these

findings

isthat, whereas 12or

more copies of the 30-bp repeat are sufficient for

plasmid

A

1 2 34 a b c d e

_^.

a

Ba

b c d e

Babo

do

__

FIG. 2. Southern blot analysis of low-molecular-weight DNA extracts prepared from pooled G418-resistant D98-Raji colonies after transfection with plasmids containing deletions in the 30-bp repeatdomain. (A)Xhol-digestedHirtextracts. Lanes 1 through 4, Markers, containing 1,000, 500, 100, or 10 pg, respectively, of XhoI-digestedp2oneoDNAmixed withlow-molecular-weightDNA preparedfromD98-Rajicells(about5 x 106cellswere used forall Hirtpreparations). Low-molecular-weightDNAwasextracted from G418-resistant cells after transfection of p20neo (lane a), pl5neo

(laneb), pl2neo(lanec), p6neo(laned), andp2neo(lane e). DNA preparations were digested withXhoI, fractionatedbyagarosegel

electrophoresis, blotted to nitrocellulose, and probed with

32p_

labeledpSLneo. (B) UndigestedHirtextracts.Lanesathroughe are

thesame asinpanelA. Thearrowindicates theexpectedpositionof form Iextrachromosomalplasmid DNA.

maintenance,

plasmids

with 6 of fewer

30-bp

repeats are

defectivefor

episomal

maintenance. Theextrachromosomal

DNA present in rare

drug-resistant

colonies

resulting

from

transfections of

plasmids

with six or fewer

30-bp

repeats

mayrepresent

structurally

rearranged

formsofthedefective

input

plasmids

that have

regained

plasmid

maintenance

function.

Rearrangements

of this type

might

have been selected for in these

experiments

because an increased

capacity

for extrachromosomal maintenance results in a

significantly

enhanced

ability

to

produce

drug-resistant

col-onies.Totestthis

possibility, rearranged

plasmids

from Hirt extracts of

p6hm

and

p2hm

transfections were isolated

by

transformation ofcompetent E. coli. Ofmore than 50

plas-mids rescued from

p6hm

and

p2hm

Hirt extracts,none were

structurally

equivalent

tothetransfectedparent

plasmids,

as

determined

by

restriction enzyme

fragment

mapping.

In

general,

the rescued

plasmids

were

extremely

large

(>20

kb). Large

plasmids

of this size transformE.

coli

with much

lower

efficiency

than do smaller-size

plasmids (15);

there-fore,

the

plasmid

rescue

findings

supporttheconclusionthat

few,

if any, of the smaller 6-kbparent

plasmids

werepresent

in the Hirt extracts.

Several

rescued,

rearranged

plasmids

were chosen for

further

analysis.

Plasmids

p2Rlhm, p2R2hm,

p2R3hm,

and

p2R4hm

were isolated from

low-molecular-weight

DNA

extracted from

hygromycin-resistant D98-Raji

cells that had been transfected with

p2hm;

p6Rlhm

and

p6R2hm

were

rescued from

p6hm

Hirt

preparations.

The

majority

of these rescued

plasmids

were

extremely

large

(>20

kb)

and their structures were not

analyzed

in detail.

However,

the

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Clal

XhoI

Clal

Clal

a

Clal

FIG. 3. Structure of plasmid p2Rlhm. p2Rlhmwasisolated from the low-molecular-weight DNA fraction of hygromycin-resistant D98-Raji cells resulting from transfection of p2hm(adeletionmutant

with just two copies of the 30-bp repeats). The structure is a

head-to-tail tetramer of the parent plasmid p2hm. Symbols: thick lines, EBV DNA; _, truncated 30-bprepeatdomains (two 30-bp

repeats); dyad symmetry regions: --* hygromycin resistance

genes. The indentation shows the position of a small deletion

(approximately 300 bp) that resultsin theloss ofasingleXl?olsite.

tureofone plasmid, p2Rlhm, wasdetermined (Fig. 3).This

20-kbplasmid, isolated fromap2hmHirtextract, consists of fourcopiesof the transfected plasmid (p2hm) linked headto

tail as a perfect tetramer (Fig. 3), with the exception of a

small deletion of approximately 300 bp. The p2Rlhm struc-ture,therefore, hasatotal of eight30-bprepeats(foursetsof twocopies).

The possibility that these rearrangements had restored plasmidmaintenancewasthen testeddirectly by transfecting

therescued plasmids back intoD98-Rajicells. It isclearthat the rearrangedplasmids havearestored capacitytoproduce hygromycin-resistant colonies, compared with the parent plasmids from which they were derived (Table 1). In some cases the rescued plasmids yielded more colonies than did

the wild-type construct with 20 repeats (Table 1, compare

p2Rlhm andp20hm). Equimolar levels of plasmidswerenot

employed in this experiment (20 jig of plasmid DNA was

transfected in all cases, regardless of the plasmid size).

Therefore, the numbers of drug-resistant colonies reported inTable 1 may, in fact, underestimate the degree of

resto-ration ofplasmid maintenance function tothe rescued plas-mids.

The hygromycin-resistant D98-Raji colonies arising after transfection with the isolated, rearranged plasmids were

pooled and expanded in culture. The state of the plasmid DNA in each of these cultures was analyzed by Southern blots of low-molecular-weight DNA preparations digested with XhloI. The plasmids transfected into new cell cultures were maintained extrachromosomally (Fig. 4, lanes b through g) and at even higher copy numbers than was the

wild-type plasmid, p20hm (Fig. 4, lane a). The apparent

increase incopy number is due, in part, tothe large size of theplasmids. For example,onecopyofatetrameric plasmid

percell,whenlinearized with XlzolI, willproduce the hybrid-ization signal equivalent to four copies of a monomeric

plasmid per cell, even though they may be in equimolar

quantities. Significantly, the revertant plasmids do not

un-dergoanyfurther detectablestructural rearrangements. The bands detected on these Southern blots correspond to

re-FIG. 4. Extrachromosomal maintenance of rearranged plasmids isolated from p2hm and p6hm Hirt extractswhen transfected into D98-Raji cells. Lanem. MarkerDNA, containing 500pgofp2ohm DNA digested withX/ol and mixed with thelow-molecular-weight DNA extracted from 5x 10" D98-Rajicells(thenumberofcells uLsed for all Hirt preparaitions). Low-molecular-weight DNA was

pre-pared fromhygromycin-resistant colonies arising after transfection ofp20hm (lanea). p2Rlhm (lalneb), p2R2hm (lanec).p2R3hm(lane d). p2R4hm (lane e). p6Rlhm (lane f). and p6R2hm(lane g). Hirt

extractsweredigestedwithXl?olbefore fractionationon anagarose

gel and blotting to nitrocellulose. The filterwas probed with 32P-labeledpHm DNA.

striction fragment patterns of purified plasmid DNA visible

on ethidium bromide-stained agarose gels (not shown).

These experiments demonstrate that more than 6 (but fewerthan 12) 30-bprepeatsare required for plasmid

main-tenance inD98-Raji cells. Transfection of plasmids withsix

or fewercopies gave rise to rare drug-resistant colonies in

which rearranged formsof the transfected plasmid persisted extrachromosomally. The structural rearrangements

com-plemented orsuppressedthe plasmid maintenancedefect of the parent plasmid and restored efficientextrachromosomal

maintenance.

Insertionofthe30-bprepeatsequenceintoanEBVplasmid withonly the65-bp dyadelement.The experiments shown in Fig. 1 to4demonstrate thatprobablyat leasteight copies of

the30-bp repeat elementare requiredforboth the enhanced generation of drug-resistant colonies in culture and

extra-chromosomal maintenance. Interestingly, mutant plasmids-with as few as two such 30-bp repeated elements can

undergorearrangementsleadingtoarevertantEBV plasmid thatisasefficientasthe wildtypeinitsabilitytopersist.The questionremained whetheranEBV mutant plasmidwith no

copiesof the30-bp repeatedsequence,pOneo, could undergo

arearrangementtoproducearevertantepisome. Theentire 30-bp repeat domain was removed from p20neo (Fig. 1) to

form pOneo (the 65-bp dyad region remained intact). The

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TABLE 2. Transfection ofEBV plasmids withincreasing numbersof 30-bp repeat sequences

No.otf d-ug--esistant Estimated

Plasmid D98-Raji colonies' episomal

copy no.'

p2Oneo 1,50() 1

pSLneo 3 0

pOneo 5

plneo 25 10-20'

p2neo 17 10-20'

p3neo 10 10-20"

p4neo 14 10-20'

p5neo 9 10-20'

Number ofdrug-resistant colonies estimated2weeksaftertransfection.

Approximatecopyntumberdetermined from Southern blots.

Indicattesthe presenceof reairranged extrachromosomalpllsmid DNA.

pOneo vector failed to produce enhanced levels of G418-resistant colonies relative to pSLneo after transfection of D98-Raji cells (Table 2). Pooled colonies from this transfec-tion contained no detectable plasmid DNA in the low-molecular-weight fraction. Therefore, pOneo probably inte-grated into the host DNA and was unable to produce revertant episomal elements. One to five copies of a syn-thetic linker containing a consensus sequence of the 30-bp repeat element (25) were clonedintopOneo (plneo to p5neo). In all cases these plasmids failed to produce increasedlevels of G418-resistant colonies relative to pSLneo in D98-Raji cells(Table 2). When these colonies were expanded and the low-molecular-weight DNA fraction was analyzed, oligo-meric forms of the input plasmid DNA were found to persist extrachromosomally (Table 2). The inser-tion of as few as

7339

_.0 0 a

A: EC

-one30-bprepeat. therefore, permitted the selectionof high-molecular-weight. multimeric forms of the input plasmid and,concurrently. the selectionofthe property of extrachro-mosomal maintenance.

Plasmid maintenancerequirements within the dyad symme-try region. The results ofprevious studies (25. 30, 36) have identified a critical 140-bp region (Fig. 5, the 65-bp dyad) between the EcoRV and HpaI sites as necessary (in combi-nation with the 30-bp repeats) for EBV plasmid mainte-nance. To define the requirements within this region more

precisely, small deletions were introduced by limited diges-tion with exonuclease III or by use of restriction enzymes, resulting in the removal ofaDNA fragment (Fig. 5). The 5' (EcoRV side) deletions produced for this study share a common endpoint at the EcoRV site (upstream EBV se-quences were notaffected) and extend progressively intothe 65-bp dyad region (Fig. 5). Deletions OxaS'A, 5.25A, and 5.30A remove 0. 4.and9bp. respectively, ofthe 65-bpdyad structure, butdo not affect any of the four EBNA-1-binding sites (shaded regions of Fig. 5). The more extensive dele-tions 5.38A, 5.49A, and 5.65A remove 17, 28, and 44 bp, respectively, from the left side of the 65-bp dyad structure. Inaddition, 5.49A deletes the most 5' EBNA-1-bindingsite, and 5.65A deletes both EBNA-1 sitesembedded within the dyadstructure (Fig. 5). All 5' deletion mutants werecloned into a G418-based vector (Fig. 1), resulting in plasmid constructs identical to p2oneo, except containing the small deletions within the dyad region.

The effect of these mutations onthe ability of plasmidsto be maintained extrachromosomally was examined by trans-fectingtheplasmidsintoD98-Rajicells. All deletionmutants gave rise to high numbers ofG418-resistant colonies,

com-9518

p2Oneo

q5I

b d

t-6 5 b~65p Dyad Sy tr

9136 9518 8997

Oxa5'A 5.25A 5.30A 5.38A

5.49A

5.65A

FIG. 5. Endpoints of deletions extending into the 5' and 3' sides of the 65-bp dyadsymmetry region. (Top) Malp of the EBV plasmid

maintenance regionpresentinthewild-type (nondeleted)plasmid p2Oneo. Batsenumbering is accordingtoBaeeretal.(2). Symbols: . 20 tandem30-bprepeats: 0.dyadregion.(Bottom)Detail of theEcoRV-to-Hpaldyadregionandendpointsofdeletion mutations.Crosshaitched boxes indicate thepositionof fourEBNA-1-bindingsiteswithin thisregion. Lineswitharrowsshow theregionsof EBV DNA that havebeen

deleted in the corresponding exonuclease- or restr-iction enzyme-generated mutations. nt positions ofdeletion endpoints are detailed in

Materials and Methods.

Hpa3'A

Sm&3'A

3.95 A

3.71 A

Nde3A

a --Vlrlrlv7m ---7,rV7,7,M I OA i

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TABLE 3. Deletionanalysis of EBV plasmids

Estimated

Plasmid No. of_D98-Rajidrug-resistant_colonies" episomal

b

o.

copyno.'

pSLneo 5 0

p2Oneo 1,500 1

pOxa5'Aneo 1,400 1

p5.25Aneo 1,300 1

p5.3OAneo 1,400 5-10'

p5.38tAneo 1,100 5-10'

p5.49Aneo 1,400 5-10'

p5.65Aneo 1,000 5-10'

pHpa3'Aneo 1,300 0

pSma3'Aneo 1,100 0

p3.95Aneo 1,000 0

p3.71Aneo 1,000 0

pNde3'Aneo 1,100 0

pHm 4 0

p20hm 800 1

pHpa3'Ahm 1,000 1

pSma3'Ahm 900 <0.2

p3.95Ahm 800 0

p3.7lAhm 900 0

pNde3'hrhm 800 0

"Numberofdrug-resistant colonies estimated2weeksafter transfection.

bApproximatecopynumberdetermined from Southern blots. Indicates thepresenceofrearranged extrachromosomalplasmidDNA.

parable to a wild-type plasmid with an unaffected dyad

region (p2Oneo) (Table 3). This result was expected on the basis of previous findings that plasmids lacking the entire dyad region still give rise to stimulated numbers of drug-resistant colonies, despite being incapable of extrachromo-somal plasmid maintenance (25). Stimulation of the number ofdrug-resistant coloniesinthis situationmostlikely results from the EBNA-1-dependent enhanceractivity ofthe 30-bp repeats. The G418-resistant colonies arising after transfec-tion of the 5' deletransfec-tion plasmids were pooled and expanded in cell culture. Low-molecular-weight DNAwas prepared and analyzed by Southern blotting (Fig. 6). The blot of undi-gested small-molecular-weight preparations indicated that

appropriately sized form I plasmid DNA was present for

Oxa5'Aneo (Fig. 6B, lane c), which doesnotaffect the dyad structure, and p5.25Aneo, which removes 4 bp of the dyad structure(Fig. 6B, laned). Deletion ofanadditional 5 bp of the dyad structure (p5.3OAneo) resulted in the appearanceof predominantly higher-molecular-weight episomal plasmid

species (Fig. 6B, lane e). The more extensive deletions had a similar effect (Fig. 6B, lanes f, g, and h); a substantial

portion of the extrachromosomal DNA was of altered size. Extrachromosomal plasmid DNA was detected even in the case ofp5.65Aneo (Fig. 6,lane h), which deletes more than halfof the dyad structure and the two EBNA-1 sites con-tained within it. A small fraction of the signal detected in somelanes ofFig.6 may representintegrated plasmid DNA. Thefaint signal (less than one copy per cell) detected in the pSLneo vector Hirt preparation (Fig. 6, lane a) indicated some contamination of low-molecular-weight DNAs with high-molecular-weight cellular DNA. However, the

hybrid-ization signals from the 5' deletion mutants (Fig. 6, lanes e

through h) were substantially more intense than the low-level contaminants. The detailed nature of the altered plas-mid DNA structures present in the 5' deletion mutant Hirt extracts remain to becharacterized.

Asimilaranalysis was undertaken with a series of deletion

mutations thatextendinto the 3' end(HpaI side) of the dyad

5

A

a b c

d

e

f

g

h

i

3

j

kl

I m

--

4W,.N

e

-qp

Ba

b c d e f 9

h

i j k I m

FIG. 6. Effectonplasmidmaintenance of deletionsinthe 5'and 3' sides of the dyadregionwhentested inthepSLneovector. (A) SouthernblotanalysisofXhoI-digestedlow-molecular-weightDNA preparations. Low-molecular-weight DNAwas extractedfrom 5 x

106 G418-resistant D98-Raji cells after transfection with pSLneo (lane a),p2oneo(lane b), the 5'deletionmutants(lanescthroughh: pOxa5'Aneo [c], p5.25Aneo [d], p5.3OAneo [e], p5.38Aneo [fl,

p5.49Aneo [g], and p5.65Aneo [h]), and the 3' deletion mutants

(lanesithroughm:pHpa3'/neo[i], pSma3'AneoU],p3.95Aneo [k], p3.71Aneo [1], and pNde3'Aneo [m]). XhoI-digested DNA was

electrophoresedon anagarosegel, transferredtonitrocellulose, and probed with 32P-labeled pSLneo. (B) Southern blot ofundigested low-molecular-weight DNApreparations. Lanesarethesame asin panel A; however, DNA was not digested with Xhol before frac-tionationon anagarosegel.

region.

pHpa3'zAneo

was constructedbydeletingthe400bp

of EBV DNA between the HpaI and XhoI sites of p20neo

(Fig. 5). More extensive deletions from the 3' end were introduced by use of restriction sites (pSma3'Aneo and

pNde3'Aneo) to drop a DNA fragment or were made by

exonuclease III treatment

(p3.95Aneo

and p3.7lAneo) (Fig. 5). These deletions didnotaffect the upstream 30-bprepeat domain.

The 3'-side deletions were tested in the plasmid mainte-nance assayby transfection ofD98-Raji cells. Again, all of

the deletion mutants gave rise to stimulated numbers of

OD

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G418-resistant colonies, equivalent to the wild-type plasmid p20neo (Table 3). Southern blots of low-molecular-weight DNApreparations from pooled, expanded cultures indicated that allof the 3' deletion mutants were defectiveforepisomal maintenance (Fig. 6, lanes i through m). The faint bands present in lanes corresponding to the 3' deletion mutants reflect plasmid DNA integrated into cellular DNA, since they are of extremely low intensity (less than one copyper cell) and comparable hybridization signals were obtained from the pSLneo vector alone (Fig. 6A and B;compare lanes i through m with lane a).

These results raise the possibility that DNA sequences between the HpaI and SstII sites might be involved in plasmid extrachromosomal maintenance because pHpa3'Aneo was defective for episomal maintenance in this assay (Table 3 and Fig. 6). This finding is in apparent conflict with the results of Reisman et al. (30), who showed that the HpoI-to-SstII region was dispensable for plasmid maintenance ina similar assay.

The Hpa3'A deletion, by removing the EBV DNA se-quencesbetween the HpaI and SstII sites of p20neo, brings the vector DNA sequences (SV40 sequences directing the expression of G418 resistance) immediately adjacent to the EBV dyad element. The possibility remained that the close proximity of the vector sequences, in particular the SV40 origin region, was interfering with the function ofthe EBV dyad element. To examine this possibility, the 3' deletion mutations (Fig. 5) were subcloned into a heterologous vec-tor, pHm (Fig. 1), which has a different selectable marker (hygromycin resistance) and contains no SV40 DNA se-quences. After transfection ofD98-Raji cells, hygromycin-resistant colonies were pooled and expanded in culture for

low-molecular-weight DNA preparations. Again, all plas-mids yielded high numbers of drug-resistant colonies (Table 3). Southern blots of these Hirt extracts showed that the

Hpa3'A deletion in the pHm vector background did not prevent plasmid maintenance (Fig. 7, lane b), as it did in the pSLneo background. A faint signal (less than one copy per cell) was visible for pSma3'Ahm (Fig. 7, lane c), indicating thatthis plasmid was, at best, inefficiently maintained extra-chromosomally. Deletion mutation p3.95Ahm, which re-moves two EBNA-1 sites but does not affect the dyad structure, completely abolished plasmid maintenance (Fig. 7, lane d). Similarly, the more extensive 3' deletion mutants

p3.71Ahm and pNde3'Ahm werealso incapable ofepisomal maintenance (Fig. 7, lanes e and f). These results suggest that the inability of the Hpa3'A mutation to support extra-chromosomal maintenance, when tested in the pSLneo vector (pHpa3'Aneo), was due to interference by adjacent vectorsequences, possibly the SV40enhancer-origin region. ThepHm vectorlacks theseinterfering sequences, since the

Hpa3'Adeletion facilitated extrachromosomal maintenance whentested in the pHm vector(pHpa3'Ahm, Fig. 7, lane b).

DISCUSSION

Todefinethe core orminimal EBVreplication originused inlatent infection,asetofdeletion mutants was constructed to map at higher resolution the requirements for EBV

plasmid maintenance withinthe two essential oriP regions:

the 30-bp repeat domain and the region of 65-bp dyad

symmetry. The genetic analysis ofthe repeat domain pre-sentedheredemonstrates that between6and12copiesofthe 30-bp repeat are required for plasmid maintenance. These

findings are consistent with theprevious results ofReisman et al., whofound 10 to 12 30-bprepeats to be sufficient for

2

3

a

b

c

d

e

f

FIG. 7. Plasmid maintenance capacity of deletion mutations in the3'endof thedyadregionwhen testedin thepHmvector. Latnes 1to3, MarkerDNA, containing 500, 50,and 5 pg,respectively, of

X/iol-digested p20hm, mixed with the low-molecular-weight DNA extractedfrom 5 x 10' D98-Raji cells (all extracts were prepared from this number ofcells). Low-molecular-weight DNA was pre-pared from hygromycin-resistant D98-Raji cells that arose after transfection with the 3' dyad deletion mutants(lanes a through f: p20hm

[a].

pHpa3'Ahm Ib], pSma3'Ahm [c], p3.95Ahm Id],

p3.71Ahm le], and pNde3'Ahm

Ifl).

DNA preparations were di-gested with XlioI, fractionated on an agarosegel, blotted to

nitro-cellulose, andprobed with 32 P-labeledpHm DNA.

plasmid maintenance (30). Deletions that left six or fewer

30-bprepeatsin an EBV plasmid orplasmids reconstructed

with uptofive copiesofa30-bp repeat oligonucleotidewere defective forplasmid maintenance in that they gave rise to

greatlyreduced numbersofdrug-resistant D98-Rajicolonies and persisted only in rearranged form. Southern blot

analy-sis and plasmid rescueexperiments indicatedthat a

substan-tial

proportion

of the extrachromosomal, rearranged DNA

was in the form ofhigh-molecular-weight, multimeric

plas-mids derived from the transfected DNA. Several rescued, rearranged plasmids were subsequently shown to have re-storedplasmidmaintenance capacity to awild-type level,as determined by their ability to produce

greatly

stimulated numbers of

drug-resistant

colonies and to be

efficiently

maintained extrachromosomally without further rearrange-ments. These

findings

indicate that, at a low frequency, plasmid rearrangements arise the restore plasmid mainte-nance capacity to transfected

episomes

originally defective forextrachromosomal maintenance.

Atpresent it is not known whenor howthese

rearranged

plasmid structures are

generated during

the

plasmid

mainte-nance assay. It is possible that they are

generated

predom-inantlyby recombination

during

transfection of the

plasmid

DNAintoD98-Rajicells. There is precedent for recombina-tion and rearrangement of transfected DNA

during

the calcium

phosphate

transfection

procedure

(4, 28).

Presum-ably, similarrearrangementsoccur

during

thetransfection of

all

plasmids,

including

the

wild-type

plasmid,

but these

events are not detected because the parent

plasmid

is

capableofefficient

plasmid

maintenance inan

unrearranged

form.

Rearranged

extrachromosomal DNAwas notdetected in a Hirt extract derived from cells transfected with a

plasmid containing

a

dyad

element but

lacking

all of the

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30-bp repeats (Table 2). Thus, the presence ofatleastoneor two 30-bp repeats appears to be required in order for rearrangementsof this type to restore plasmid maintenance (Table 2). Alternatively, the presence ofat least one or two repeatsis required tofacilitatethe rearrangement or recom-bination of the transfected plasmidDNA.

It is not clear what structural features of the

rearranged

plasmids are responsible for the restoration of plasmid maintenance. The structure of one rescued

plasmid

with restoredepisomal maintenance,p2Rlhm(Fig. 3),wasfound to be a tetramer of the transfected plasmid p2hm. The restoration ofplasmid maintenance in p2Rlhm may bedue to the regeneration of a critical number of 30-bp repeats linked in cis on the plasmid (four sets oftwo repeats,fora total ofeightrepeats). The30-bprepeats may,

therefore,

not need to be contiguous in the plasmid tofunction, sincethey

are widely spaced in the p2Rlhm structure. p2Rlhm also

contains four dyad symmetry

regions,

and it remains

possi-ble that the core EBNA-1-binding sites within the dyad

elements act to partially substitutefora

deficiency

of

30-bp

repeats. Inany casethe EBVplasmidmaintenance elements can function with considerable versatility with respect to their relative organization. Reisman et al. showed that the

spacing and orientation of the 30-bp repeats relative tothe dyad element were not critical for their function (30). The revertantplasmids isolated in this studyattest tothe remark-ableflexibilitywithregardtothe typesofstructuresthatcan

constitute afunctional

plasmid

maintenance system.

Deletion mutationswithin the

dyad

symmetry

region

were not associated with substantial reductions in

drug-resistant

colony number, even in the absence of

episomal

mainte-nance. The sequences between the EcoRV site and the left side of the65-bp dyad structure are

dispensable

for

plasmid

maintenance inD98-Rajicells. A mutation thatremoves4bp

of the 5' end ofthe65-bp dyad structurealso

permits

normal

plasmidmaintenance. Deletion ofanadditional 5

bp (total,

9

bp) of the left side of the dyad structure, or even more extensive 5' deletions, resulted in the appearance of

episo-mal DNA of altered size relative to the transfected test

plasmid. The rearranged DNA detected in transfections of the 5' dyad deletions differs from the rearranged DNA presentin the 30-bprepeat deletion mutants in at least two respects. First,asubstantial fraction ofthe

episomal

5'dyad deletion mutant plasmid DNA is not of extremely high

molecularweight (Fig. 6). Second, the appearance of rear-ranged 5' dyad deletion plasmid species is not associated with a large drop in the numberof drug-resistant D98-Raji

colonies.Itisdifficult, therefore,tointerpretthe presenceof rearranged DNA in the 5' dyad deletion plasmid Hirt ex-tracts as an infrequent, spontaneous rearrangement that restores plasmid maintenance capacity (as appears to hap-penin thecaseof extensive deletions within the30-bprepeat domain). A possible explanation for these results could be that the 5' dyad deletions do, in fact, destroy plasmid maintenance and give rise to integrated forms of DNA. Episomal, oligomeric plasmidDNA species could be gener-ated by recombination between multiple, tandemly inte-grated plasmids. Alternatively, small-molecular-weight het-erogeneousDNAfragments could result from replication out of the chromosome in an onionskin fashion (3). Another

explanation forthese results may be that the moreextensive 5'dyaddeletionsdestroy plasmidmaintenance,yetgiverise to numeroustransientlyresistantD98-Raji colonies. Indeed, Reisman et al. (30) have shown that some plasmids with altered numbers of the 30-bp repeats, introduced into

D98-Raji cells, yield abortively resistant colonies that score

positive

in a2-week

drug-resistant

assay, but not a4-week

assay (see Tables 1 to

3,

which were scored at 2

weeks).

During

the

subsequent

process of

pooling

the colonies and

expanding

theculture forHirt

preparations,

the

transiently

resistantcolonies

might

then die andrare

colonies,

contain-ing

rearranged plasmid

DNA with restored

plasmid

mainte-nance

activity,

would dominate the culture. In eithercase it

is

particularly

surprising

thatextrachromosomal

DNA,

even

if

rearranged,

is present for

pS.65Aneo,

since this

plasmid

has lost more than halfof the

dyad

structure and the two EBNA-1 siteswithin it.

Previous deletion

mapping

localizedacritical

requirement

for

plasmid

maintenance within the

dyad region,

but it remained

possible

that the

episomal

maintenance

require-mentsin this

region

weredue

solely

to thecontributionsof the four

EBNA-1-binding

sites within it. Deletion mutant

5.30A

provides

evidence that the

dyad

structureitself

plays

a role in

plasmid

maintenance. The 5.30A

mutation,

which removes 9

bp

ofthe 5' endofthe

65-bp

dyad

symmetrybut

does not

directly

affect any of the four

EBNA-1-binding

sites,

results in the

persistence

ofextrachromosomal DNA

predominantly

of altered size.

The initial attemptstodefine the 3' limitforEBV

plasmid

maintenance were

complicated by negative

interference

from the

pSLneo

vector sequences. There are several rea-sonstosuspect that theinterference ofEBV

origin

function

maybe duetoSV40

enhancer-origin

sequences.Interference

by

SV40enhancer sequences onDNA

replication

has been

reported

previously

(5).

Deletion of the 400

bp

ofEBVDNA

between the

HpaI

and SstIl sites

destroys

plasmid

mainte-nance when tested in the

pSLneo

vector, but not in the

hygromycin

vector

pHm.

This deletion

places

the

SV40

origin region

immediately adjacent

to the

65-bp

dyad

in the

SLneo vector.

Furthermore,

a

negative

effect of the

SV40

origin

upon EBV-derived

plasmid

copy numberwas

demon-strated in a

previous study

(25).

A

plasmid

containing

the BamHI-C elements in the

pSLneo

backbone

p404

was main-tainedat 1to 10

copies

percell. A

plasmid

identicalto

p404,

except

harboring

a deletion of the SV40

origin region

(p404A),

was maintained at

reproducibly higher

copy

num-bers,

20 to 40

copies

percell

(25).

These

findings

mayhave

relevancefor

previous

studies that have

attempted

to

func-tionally

substitute the

plasmid

maintenance

requirement

for

the

30-bp

repeat domain with the SV40

enhancer-origin

region (29).

From the

analysis

of the

hygromycin-based

3' deletion mutants, the 3' end of the

dyad

region

has been showntobe

quite

sensitive to mutation. Deletions

extending

past the

SmaIsite

completely

eliminated

episomal

maintenance

and,

in contrast to the 5'

deletions,

did not result in the

persis-tenceof extrachromosomal DNAofaltered size. Theresults

reported

here confirm that EBV sequences 3' to the

HpaI

site are

dispensable

for

plasmid

maintenance in

D98-Raji

cells

(30).

Deletion to the SmaI site resulted in a loss of

plasmid

maintenance or, at

best,

very inefficient

plasmid

maintenance

(less

than one copy per

cell).

The failure of

p3.lAhm

to be maintained

extrachromosomally

demon-strates that the

65-bp dyad

structure alone is not sufficient

for

plasmid

maintenance

and,

furthermore,

suggests a

criti-cal role for the

EBNA-1-binding

site sequences adjacent to

the

dyad

structure. This is reminiscent of the SV40

origin,

where sequences

adjacent

to, butnot

within,

theSV40

dyad

structureare an

important

partof thecore

replication

origin

(34).

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2. Baer, R., A. T. Bankier, M. D. Biggin, P. L. Deininger, P.J. Farrell,T.J. Gibson,G.Hatful,G. S. Hudson,S. D.Satchwell, C. Seguin, P. S. Tuffnell, and B. G. Barrell. 1984. DNA se-quence andexpression ofthe B95-8Epstein-Barrvirus genome. Nature(London)310:207-211.

3. Botchan, M., W. C. Topp, andJ. Sambrook. 1979. Studieson

SV40 excision fromcellularchromosomes. ColdSpringHarbor Symp. Quant. Biol. 43:709-719.

4. Calos, M. P., J. S. Lebkowski,and M. R. Botchan. 1983. High mutation frequencyin DNA transfected into mammalian cells. Proc. Natl. Acad.Sci. USA 80:3015-3019.

5. Campbell,B.C.,and L.P.Villareal. 1986. Lymphoidandother tissue-specific phenotypes ofpolyomavirus enhancer

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6. Dambaugh,T.,K.Hennessy,L.Chamnankit,and E. Kieff.1984. The U2regionofEBV DNA may encode EBNA2. Proc. Natl. Acad. Sci. USA81:7632-7636.

7. Dambaugh, T.,K. Hennessy,S. Fennewald,and E. Kieff. 1986. The genomeand itsexpressionin latentinfection,p. 105-140.In M. Epsteinand B.Achong(ed.),TheEpstein-Barrvirus:recent advances. Heineman, London.

8. de Villiers, J., W. Schaffner, C. Tyndall, S. Lupton, and R. Kamen. 1984. Polyoma virus DNA replication requires an enhancer. Nature (London)312:242-246.

9. Dillner, J.,B.Kallin,H.Alexander,I.Ernberg,M.Uno,Y.Ono, G. Klein, and R. Lerner. 1986. An Epstein-Barr virus deter-minednuclearantigen(EBNA5) partlyencodedbythe transfor-mation associatedBam W-Y-H regionofEBV DNA: preferen-tial expression inlymphoblastoid cell lines. Proc. Natl. Acad. Sci. USA83:6641-6645.

10. Epstein,M.A.,and B.G.Achong (ed.). 1979.TheEpstein-Barr virus. Springer-Verlag, Berlin.

11. Glaser, R., and M. Nonoyama. 1974. Host cell regulation of inductionofEpstein-Barrvirus.J. Virol. 14:174-176.

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