m290 clin pathol: mutations epstein-barrvirus protein-i ... · ebvlmp-1mutations in...

_3 Clin Pathol: Mol Pathol 1 996;49:M290-M297

Mutations in the Epstein-Barr virus latentmembrane protein-I (BNLF- 1) gene inspontaneous lymphoblastoid cell lines: effect on invitro transformation associated parameters andtumorigenicity in SCID and nude mice

Kristian Sandvej, Mette Munch, Stephen Hamilton-Dutoit

AbstractAims-(1) To study the frequency ofputa-tive malignancy associated point muta-tions and a 30 base pair (bp) deletion inexon 3 of the C-terminus of the Epstein-Barr virus (EBV) encoded latent mem-brane protein (LMP)-1 (BNLF-1) gene inwild type EBV strains. (2) To assess theinfluence of these mutations on the tum-origenicity of lymphoblastoid cell lines(LCL).Methods-Eight spontaneous EBV (wildtype) infected LCL were established fromseven subjects. Deletions and single basemutations in the C-terminus of theBNLF-1 gene were demonstrated usingbi-directional solid phase dideoxy se-quencing following PCR amplification ofviral DNA from the LCL. Tumorigenicityofthe LCL was assessed in SCID and nudemice. Serum dependent growth and abil-ity to form colonies in soft agarose wereassessed for representative LCL.Results-All LCL showed sequence differ-ences compared with the prototypic EBVstrain B95-8. The 30 bp deletion could bedetected in three of eight LCL and a 69 bpdeletion (including the 30 bp deletion) wasidentified in an additional LCL. A range ofsingle base mutations (including thosedescribed previously in association withEBV related neoplasias) was also seen insome of the LCL. In transformation stud-ies, the genetic variations did not seem toinfluence the in vitro behaviour of theLCL. In the tumorigenicity studies, thepresence of the 30 bp deletion had noinfluence on the behaviour of the LCLwhich were, as expected, tumorigenic inSCID mice but not in nude mice. Incontrast, the LCL carrying the 69 bp dele-tion was tumorigenic in both SCID andnude mice.Conclusions-Genetic changes describedpreviously in the C-terminus of theLMP-1 gene in various malignancy de-rived EBV strains are also presetit fre-quently in wild type viruses and do notsimply define tumour specific EBVstrains. Changes within this region may,however, still be important for the tumori-genicity of LMP-1 and thus play a role inEBV oncogenesis.(7 Clin Pathol: Mol Pathol 1996;49:M290-M297)

Keywords: Epstein-Barr virus, latent membraneprotein-1, spontaneous lymphoblastoid cell lines.

Epstein-Barr virus (EBV) is a human herpesvirus that causes infectious mononucleosis'and is associated with various malignancies,including nasopharyngeal carcinoma, Burkitt'slymphoma, Hodgkin's disease, peripheral Tcell lymphomas, and lymphoproliferative dis-orders in immunosuppressed patients.2-7

In vitro, EBV transformed lymphoblastoidcell lines (LCL) can be established by sponta-neous outgrowth of EBV infected B cells fromthe peripheral blood of seropositive subjects.8LCL consistently display a restricted pattern ofEBV gene products (recently designated la-tency type III), characterised by the expressionof EBV nuclear antigen (EBNA)-1, EBNA-2,EBNA-3a, b and c, and EBNA-LP, latentmembrane protein (LMP)-1 and LMP2a/b,and EBV small nuclear RNAs (EBERs)-1/2.9LMP-1 plays an important part in EBVinduced cell transformation. Among otherthings, it is essential for the transformation ofprimary B cells,'" it can transform rodentfibroblasts and human keratinocytes," 12 inhibithuman epithelial cell differentiation,'3 and it istoxic to cells when expressed at high concen-trations.'4 '5 In transgenic mice LMP- 1 induceshyperplastic dermatitis and abnormal keratinexpression.'6 LMP-1 induces DNA synthesis'7and upregulates expression of several lym-phocytic activation markers and adhesion mol-ecules. 8

Recently, Hu et al'8 demonstrated severalmutations in the gene, BNLF- 1, encodingLMP-1 in nude mouse propagated Chinesenasopharyngeal carcinoma (CAO) cells com-pared with the prototype EBV strain B95-8(ECACC no. 85011419). Subsequently, it hasbeen suggested that these BNLF-1 gene muta-tions characterise an EBV strain in Asia, whichis associated preferentially with nasopharyn-geal carcinoma.20 Tumorigenicity studies onSCID and nude mice indicated that the CAOLMP-1 was more tumorigenic than B95-8-LMP-1.2021

Analysis of the CAO BNLF-1 gene hasrevealed a 30 base pair (bp) deletion and sevensingle base mutations in the C-terminal region,between the coding triplets for amino acids322 and 366 of exon 3.19 In a recent study ofEBV positive lymphomas we found the 30 bp

Laboratory ofImmunopathology,University Institute ofPathology, AarhusKommunehospital,Aarhus, DenmarkK SandvejS Hamilton-Dutoit

Centre for MedicalMolecular Biology,Aarhus UniversityHospital and Faculty ofHealth Science, SkejbySygehus, DenmarkK Sandvej

Department ofMedical Microbiologyand Immunology,University ofAarhus,Aarhus C, DenmarkM Munch

Correspondence to:Dr K Sandvej, Laboratory ofImmunopathology,University Institute ofPathology, AarhusKommunehospital,Finsensgade 12, DK-8000Aarhus C, Denmark.

Accepted for publication9 July 1996

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EBVLMP-1 mutations in lymphoblastoid cell lines

deletion and six of the seven single base muta-tions in all peripheral T cell lymphomas fromMalaysian patients, in about 60% of peripheralT cell lymphomas from Danish patients and inabout 30% of cases of Hodgkin's disease andinfectious mononucleosis.22 The mutationswere detected in both EBV subtypes A and B,and the single base mutations and the 30 bpdeletion occurred independently in some

cases. In addition, we detected a 12 bp deletionconsisting of 12 of the nucleotides of the 30 bpdeletion in one case of Hodgkin's disease.These findings indicate that mutations in theC-terminal part of the BNLF-1 gene are notassociated specifically with either Asian naso-

pharyngeal carcinoma or EBV subtype A,although they do seem to be more common inAsia than in the West. Rather, the 30 bpdeletion and some of the sites of single basemutation may be hot spots which have mutatedindependently throughout the evolution ofEBV strains. Similar findings have beenreported independently from other laborato-

23 24

ries.

The apparent convergent evolution of EBVstrains of both subtype A and B from differentgeographical locations suggests that thesemutations impart some advantage to the virus.In vitro studies by Moorthy and Thorley-Lawson showed that the C-terminal part ofLMP-1 is essential for transformation.'5 25 Fur-thermore, their results indicated that theabsence of the amino acid sequence betweenpositions 334 and 364 generated a toxicLMP- 1 protein, suggesting that this aminoacid sequence is essential for the normal func-tion of the protein. The 30 bp deletion islocated within this sequence and may thereforeaffect the function of the LMP-1 protein. Thisis supported by recent findings from Li et al.26Their transfection study on Balb/3T3 cellsshowed that introduction of the 30 bp deletioninto the B95.8 BNLF-1 gene made these cellstumorigenic in nude mice. In the present studywe have sequenced the C-terminal part of theEBV-BNLF-1 gene in eight spontaneouslyestablished LCL and identified wild type EBVstrains that display various mutations withinthis region. The effect of these mutations on

LCL transformation related parameters invitro and LCL tumorigenicity in SCID andnude mice in vivo was investigated.

MethodsESTABLISHMENT OF LCL

As part of a separate study, LCL were

established with cells from six patients withmultiple sclerosis and from one blood donor.Two separate LCL were established (with a 42day interval) from cells from one of thepatients. In each case, 50 ml of venous bloodwas drawn into Venoject tubes. Heparinisedblood was diluted 1 in 2 in phosphate bufferedsaline (PBS)/heparin (PBS pH 7.4, 20 IU/mlheparin), before separating the mononuclearcells by Ficoll-Isopaque density gradient cen-

trifugation. The mononuclear cell layer was

isolated and washed in PBS and subsequentlyin RPMI 1640 (BioWhittaker, Walkersville,Maryland, USA). The cells were then counted

and seeded at densities between 2 x 1 06/ml and4 x 106/ml in 5 ml growth medium, which, onday 3, was adjusted to 10 ml. Growth mediumconsisted of RPMI 1640 supplemented with200 IU/ml penicillin (Leo, Ballerup, Den-mark), 0.2 mg/ml streptomycin (Sigma, StLouis, Missouri, USA), 0.29 mg/ml glutamin(Sigma), 0.O1M HEPES buffer (BioWhit-taker), and 10% heat inactivated human serumin Falcon Primaria bottles (50 ml). For eachpatient there were at least two mononuclearcell cultures, with and without 0.1 jg/mlcyclosporin A (Sandoz, Basel, Switzerland).The cyclosporin A was maintained in themedium for five weeks by regular refeeding.Half of the supernatant medium was changedthree times weekly until a LCL was establishedor all cells were dead. LCL appeared after 60 to180 days. Cultures were then split every two tothree days and reseeded at a concentration of5 x 105/ml.

GENETIC FINGERPRINTINGTo test for cross contamination of the LCL,typing of DNA from each LCL culture wasperformed. The DNA was purified accordingto standard methods from mononuclear cellsisolated on the day ofblood sampling and fromLCL. Restriction fragment length polymor-phisms (RFLPs) of variable numbers oftandem repeat (VNTR) regions wereinvestigated.27-0 HLA-DQa typing was per-formed on PCR amplified DNA with theAmpliType kit (Roche, USA).

CYTOGENETICSApart from the following modifications, flowcytometry analysis was performed as describedpreviously.3' After washing twice in PBS, thecells were resuspended in 2 ml stainingsolution containing 10 mM Tris-HCl, 80 mMKC1, 20 mM NaCl, 3.4 mM sodium citrate,10 mM MgCl2, 0.5 mM EDTA, 0.5 mM Sper-mine, 0.5 mM Spermidine, 0.1% NonidetP40, pH 7.6, and propidiurn iodide (PI) to afinal concentration of 100 gl PI/ml. Thestaining/lysis time was 10-15 minutes. Anormal diploid reference was superimposed onthe expected normal diploid peak in the histo-gram. The DNA profile was analysed on aFACStar Plus analyser (Becton Dickinson,Burlingame, California, USA).

TUMORIGENICITYFemale SCID and nu/nu mice ofNMRI back-ground were obtained from BomholtgaardAnimal Breeding and Research Centre, Ry,Denmark, and kept under conventional condi-tions during the experiments. The mice wereseven to eight weeks old, with five to 10 mice ofeach kind being used for each cell line.Suspensions of 5.5 x 10' cells in 200 ,ul 0.9%NaCl were inoculated subcutaneously in theleft flank. The mice were inspected weekly forthe appearance and progressive growth oftumours. Tumour tissue was snap frozen andstored at -80°C. Animals without tumourswere kept for 26 weeks.

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Sandvej, Munch, Hamilton-Dutoit

IMMUNOHISTOLOGYCell pellets from cell cultures and tumourtissue from SCID and nude mice were snapfrozen. Sections were cut and stained usingAPAAP immunohistology. The LCL fromboth cultures and tumours were characterisedas B cells using monoclonal antibodies directedagainst CD19, CD20 and CD21. The sectionswere also stained for activation markersintracellular adhesion molecule- 1 (ICAM- 1)(CD54), LFA-3 (CD58) and CD23, and forbcl-2. Expression of EBV gene products wasdemonstrated using the monoclonal antibodiesPE2 (EBNA-2), CS.1-4 (LMP-1) and BZ1(BZLF-1 protein), as described previously.32

SAMPLE PREPARATION FOR PCRFive 10 ,um frozen sections were cut from themouse tumours and placed in an Eppendorftube using disposable Pasteur pipettes. Beforeand after cutting sections for PCR, histologicalcontrol sections were cut and stained with hae-matoxylin to demonstrate the presence oftumour cells. Between cases the cryostat wascleaned with 70% alcohol and for each case anew knife was used. For every tumour twotubes with sections from a pellet consistingonly of Tissue-Tek cryostat embedding me-dium (Miles, Elkhart, Indiana, USA) were cutas negative controls of the sectioning proce-dure. For PCR on cell lines, 1 o6 cells were spundown directly from cultures. Both sections andcell pellets were digested in 250 g1 proteinase K(200,g/ml) for 48 hours at 56°C. The protein-ase was inactivated by heating to 98°C for 15minutes. The remains of the tissue was spundown briefly and the supernatant was used forPCR.

PCRPCR was carried out in an automated thermalcycler (Perkin Elmer, Cetus, Norwalk, USA).All primers were synthesised by DNA Technol-ogy (Aarhus, Denmark). For EBNA-2 typing asingle step PCR procedure was used. Primersflanking a 115 bp deletion in the U2 regionencoding EBNA-2A gave rise to a 378 bpproduct from EBNA-2A EBV strains and a483 bp product from EBNA-2B strains.33 EBVcell lines B95.8 and AG876 carryingEBNA-2A and EBNA-2B, respectively, wereused as controls.34 To test for deletions in theC-terminus of the BNLF-1 gene, a PCR prod-uct was amplified from nucleotide positions168390 to 168074, according to Hudson etal.35 Hodgkin's disease cases and the cell lineAG 876 previously shown to contain theBNLF-1 30 bp deletion, and Hodgkin'sdisease cases and the cell line B95.8 shown notto contain deletions, were used as positive andnegative controls, respectively.22 PCR wascarried out in a 50 p1 reaction mixture (3.75mM MgCl2 and 5 units of AmpliTaq polymer-ase Stoffel fragment (Perkin Elmer Cetus)).Primers and PCR conditions have beendescribed in detail elsewhere.2236 The size ofthe PCR product was verified after electro-phoresis in Visigel separation matrix (Strata-gene, La Jolla, California, USA) stained withethidium bromide.

BI-DIRECTIONAL SOLID PHASE DIDEOXYSEQUENCINGSequence analysis was performed to demon-strate the size and the location of deletions, andto detect single base mutations, as describedrecently.22 Briefly, in all cases two parallel PCRswere performed, one for sequencing the sensestrand and one for sequencing the antisensestrand. The PCR product for sequencing thesense strand was produced with a non-biotinylated sense primer and a 5' biotinylatedantisense primer. The product for sequencingthe antisense strand was produced with a non-biotinylated antisense primer and a 5' bioti-nylated sense primer (the same primers as usedto amplify the PCR product). After PCR theamplified fragments were captured from35-50 p1 of the reaction mix (the amountneeded for sequencing was estimated byelectrophoresis), with 30 gl Streptavidin conju-gated magnetic beads (Dynabeads, Dynal,Norway), according to the manufacturer'sinstructions. After several washing steps thedouble stranded PCR product was denaturedby incubating with 20 p1 0.1 M NaOH for fourminutes at room temperature. The supernatantcontaining the PCR product generated by thenon-biotinylated primer was removed. TheDynabeads/single stranded DNA complex waswashed once in 250 mM Tris (pH 8)/0.1%Tween 20, once in TE buffer and once in dou-ble distilled water, and resuspended in 10 pldouble distilled water. The sequencing reac-tion was carried out according to the manufac-turer's instructions (PRISM Sequenase Termi-nator Single-Stranded DNA Sequencing Kit,Applied Biosystems Inc., Foster City, Califor-nia, USA), using fluorescence labelled dide-oxynucleotide terminators as described.22 Thegel was electrophoresed for 14 hours on asemiautomated ABI 373A sequencer (AppliedBiosystems), and the results were analysedusing the Seg Ed software program (AppliedBiosystems).

ANALYSIS OF SERUM DEPENDENT GROWTHRepresentative LCL with a 30 bp deletion(MS 1845), with a 69 bp deletion (MS 1859)and with no deletion (MS1851 and MS1858)in the C-terminal region of the LMP-1molecule were used for analysis of growth andsurvival at different serum concentrations.Cells were seeded in triplicate at a startingdensity of 0.4 x 106 cells per ml (day 1) in10 ml growth medium with either 0.1%, 1.0%or 10% human serum. The number of viablecells was counted daily using the trypan blueexclusion test.

COLONY FORMATION IN SOFT AGAROSESeaplaque agarose (BioWhittaker) was dis-solved in distilled water. A base layer of 0.3%agarose in 1.5 ml RPMI 1640 supplementedwith 200 IU/ml penicillin (Leo), 0.2 mg/mlstreptomycin (Sigma), 0.29 mg/ml glutamin(Sigma), 0.01 M HEPES buffer (BioWhit-taker), and 10% heat inactivated human serumwas poured into six-well multidishes (Nunc,Roskilde, Denmark).

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322+ 323 324 325 326 327 328+ 329+ 330+ 331+ 332 333 334+ 335+B95.8 CAA TTG ACG GAA GAG GTT GAA AAC AAA GGA GGT GAC CAG GGCCAO AATTG ACG GAA GAG GTT G AAC AAA GGA GGT GAC G GGC1851 CAA TTG ACG GAA GAG GTT CAA AAC AAA GGA GGT GAC CAG GGC1874 CAA TTG ACG1844 CAA TTG ACG1858 GAA TTG1847 AATTG1845 AATTG1533 AATTG

ACGACGACGACG

336CCGCCGCCG

GAA GAG GTT CAA AAC AAA GGA GGT GAC CAG GGC CCGGAA GAG GTT CAA AAC AAA GGA GGT GAC CAG GGC CCGGAA GAG GTT GAA AAC AAA GGA GGT GAC G GGC CCGGAA GAG GTT GAA AAC ACA GGA GGT GAC O GGC CCGGAA GAG GTT GAA AAC AAA GGA GGT GAC G AGC CCGGAA GAG GTT GAA AAC ACA GGA GGT GAC O GGC CCG

337 338+ 339CCT TTG ATGCCT T ATGCCT TTG ATGCCT TTG ATGCCT TTG ATGCCT T ATGCCT T ATGCCT T ATGCCT T ATG

340 341 342- 343 344ACA GAC GGA GGC GGCACA GAC G GGC GGCACA GAC GGA GGC GGCACA GAC GGA GGC GGCACA GAC GGA GGC GGCACA GAC GGGGC GGCACA GAC G GGCGGCACA GAC GGC GGCACA GAC GGC GGC

1859 GAA TTG ACG GAA GAG GTT GAA AGC AAA AGA GGT GA

345 346 347 348 349 350 351 352+ 353 354 355 356- 357 358 359 360 361- 362 363 364 365 366+B95.8 GGT CAT AGT CAT GAT TCC GGC CAT GGC GGC GGTCAO GGT1851 GGT CAT AGT1874 GGT CAT AGT1844 GGT CAT AGT1858 GGT CAT AGT1847 GGT1845 GGT1533 GGT1859

CAT GAT TCC GGC CAT GGC GGC GGTCAT GAT TCC GGC CAT GGC GGC GGTCAT GAT TCC GGC CAT GGC GGC GGTCAT GAT TCC GGC CGT GGC GGC GGT

GAT CCA CAC CTT CCT ACG CTG CTT TTG GGT TCTGAT CCA CAC CTT CCT ACG CTG CTT TTG GGTAGAC CCA CAC CTT CCT ACG CTG CTT TTG GGTACGAC CCA CAC CTT CCT ACG CTG CTT TTG GGT[ACGAC CCA CAC CTT CCT ACG CTG CTT TTG GGTAC

GAT CCA CAC CTT CCT ACA CTG CTT TTG GGTA

GAT CCA CAC CTT CCT ACG CTG CTT TTG GGTAC

GAT CCA CAC CTT CCT ACG CTG CTT TTG GGTACGAT CCA CAC CTT CCT ACG CTG CTT TTG GGTAC

T CCA CAC CTT CCT ACA CTG CTT TTG GGT GCT

Figure 1 Sequence analysis of the C-terminal region of the EBVBNLF-l gene which encodes LMP-l. Single base mutations identical with thosefoundin the CAO BNLF-1 sequence are underlined and in bold. Single base mutations differingfrom both the B95.8 and CAO BNLF-1 gene are underlined.Amino acid changes identical with thosefound in the CAO LMP-1 protein are indicated byframing the coding triplet. The S bp and the 11 bp repeatsequences which could be implicated in the generation of the 30 bp and 69 bp deletions, respectively, are in italics. + indicates that the single base mutationaffects the amino acid sequence; - indicates that the single base mutation does not affect the amino acid sequence.

The top layer was either 0.2%, 0.3%, 0.45%,or 0.6% agarose in 1.5 ml ofthe same medium asused for the base layer; 3000 cells were added perwell from one of the following cell lines:MS1845, MS1851, MS1858 or MS1859. Aftersetting, the agarose was covered with 1 mlmedium. All culture dishes were incubated at37°C in 5% CO2. Three independent experi-ments were carried out and each cell line wasplated in triplicate. A colony was defined as acluster consisting ofmore than 10 cells.

ResultsPCREBNA-2 type A was detected in all eight LCLand in the EBV subtype A control cell lineB95.8. EBNA-2 B was only found in the EBVsubtype B control cell line AG 876. Amplifica-tion of the C-terminal region of the BNLF-1gene indicated the presence of a deletion infour of eight LCL. In three cases (MS1845,MS 1847 and MS 1533) gel electrophoresisindicated that the deletion was approximately30 bp. In the fourth case (MS1859) it waslarger. PCR control experiments on tumoursfrom SCID and nude mice gave results identi-cal with those found in the respective inocu-lated LCL.

SEQUENCE ANALYSISThe results are summarised in fig 1. In thethree cases with electrophoretical evidence of adeletion around 30 bp, sequence analysis con-firmed the size to be 30 bp. Examination of thesequence environment surrounding the 30 bp

deletion revealed two 9 bp repeats (GGCG-GCGGT) coding for three Gly residues(amino acids 343 to 345 and amino acids 353to 355). We have proposed previously that the30 bp deletion can be generated by slippedmispairing of direct repeats.22 The sequenceanalysis suggests that this has probably beenpreceded by a single base mutation in aminoacid 342 (A to T) giving two 10 bp repeats(TGGCGGCGGT; third base of the codingtriplet ofamino acids 342 to 345 and third baseof the coding triplet of amino acids 352 to355), one of which is included in the 30 bpdeletion (fig 1). Indeed, one of the LCL with-out the deletion (MS 1858) has this mutation atamino acid 342. In addition, this cell line has amutation in the coding triplet for amino acid352 (A to G), giving two 11 bp repeats adjacentto the 30 bp deletion, a change that would fur-ther increase the likelihood of a deletion occur-ring by slipped mispairing. Thus, there iscircumstantial evidence that both of thesemutations may be present prior to the deletionof the 30 bp sequence.

In the MS 1859 LCL with electrophoreticalevidence of a deletion larger than 30 bp,sequence analysis revealed a 69 bp deletioncomprising the sequence of the 30 bp deletionand a further 39 bp. The sequence environ-ment of the 69 bp deletion contains two fivenucleotide repeats (GGTGA), one of which isincluded in the 69 bp deletion (fig 1).

Six of the seven single base mutationsdetected in the CAO BNLF-1 gene betweenamino acids 322 and 388 were found in all

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0.4

0.3

0.2

0.1% Human serum

0.1

0

0 1 2 3 4 5 6 7 8 9 10

Figure 2 The ability of the cell lines MS1845, MS1851, MS1858, and MS1859 to

survive in low serum concentrations was analysed by daily counts of viable cells. The serum

concentrations (0.1 %, 1.0% and 10% human serum, respectively) are indicated on thegraphs. The y-axis indicates the number of live cellslml divided by 106 and the x-axis thenumber of days in culture. Each LCL was cultured in triplicate. The curves represent themean (SD). The labelling of the curves are as follows: *, MS1845; *, MS1851; *,

MS1858; and A, MS1859. The horizontal line at 0. 4 x 1O live cellslml indicates the cellconcentration at day 1.

three cases containing the 30 bp deletion, themutation affecting amino acid 328 being theone absent in all cases (fig 1). Two of theseLCL (MS1847 and MS1533) had an addi-tional mutation affecting amino acid 330 (A toC: Lys to Thr) and the third (MS1845), a

mutation affecting amino acid 335 (G to A:Gly to Ser) (fig 1). The case with the 69 bpdeletion (MS 1859) contained none of the sin-gle base mutations found in this region of theCAO BNLF-1 gene. However, single basemutations were demonstrated in the coding

triplets of amino acids 322 (C to G: Gln toGlu), 329 (A to G: Asn to Ser), 331 (G to A:Gly to Arg), 356 (T to C: no amino acidchange), 361 (G to A: no amino acid change),and 366 (T to G: Ser to Ala) (fig 1).Three of the LCL without deletions

(MS1851, MS1874 and MS1844) showed anucleotide sequence identical with B95.8except for amino acid 328 (G to C: Glu to Gln)and amino acid 366 (T to A: Ser to Thr). Thefourth cell line (MS 1858) contained mutationsat amino acids 322 (C to G: Gln to Glu) and352 (A to G: His to Arg) (fig 1), together withthe single base mutations affecting amino acids334, 338, 342, and 366 detected in the CAOBNLF- 1 gene and in the three LCL with the30 bp deletion. In the sequence from aminoacids 366 to 388 no single base mutations weredetected in any of the LCL (data not shown).

GENETIC FINGERPRINTINGIn all eight LCL individual genetic fingerprint-ing patterns were demonstrated excludingcross contamination with cells from flask toflask. Six of the eight virus isolates containedindividual BNLF-1 sequences (see earlier)essentially ruling out virus cross contamina-tion. The LCL MS1844 and MS1851 wereidentical in the sequenced part of the BNLF-1gene, raising the possibility of identical EBVstrains. These LCL were established independ-ently and cultured at separate locations,strongly arguing against cross contamination.

TIME REQUIRED FOR LCL ESTABLISHMENTTransformation of B lymphocytes leads tophenotypic changes and immortalisation. De-velopment of LCL was characterised by freefloating spherical or oval clusters of more orless tightly packed cells increasing in numberover time. The time interval from seeding themononuclear cell cultures to the appearence ofLCL varied between 60 and 180 days. Therewas no correlation between the time taken toestablish a LCL and the presence of the 30 bpor the 69 bp deletion in the BNLF-1 gene.

SERUM DEPENDENT GROWTHThe number of live cells as a function of days inculture is shown in fig 2. There was no signifi-cant difference between the serum dependentgrowth of the cell lines. However, there was atendency for a more serum dependent growthfor the cell line MS 1859 (containing the 69 bpdeletion), most clearly seen in the experimentwith 1% human serum in the culture medium.The number of live cells in the MS 1859culture decreased after day 0 compared withthe other cell lines which proliferated untilabout day 7. The same tendency was seen forMS1859 with 0.1% human serum where thecells died very quickly and with 10% humanserum where the cell line proliferated moreslowly and reached the maximum number oflive cells later than for the other LCL.

CLONING IN SOFT AGAROSEThe ability to form colonies in soft agarosewith low agarose concentrations (0.2% and0.3%) was not associated with the presence of

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Table 1 Incidence of tumourformation by inoculation oflymphoblastoid cell lines subcutaneously in SCID and nudemice

Mouse type Cell line Tumour incidence

SCID mice MS1845 3 of 7MS1851 4 of 5MS1858 4 of 5MS1859 6 of 10

Nude mice MS1845 0 of 5MS1851 0 of 5MS1858 0 of 5MS1859 3 of 5

mutations. Only the number of live and deadcells in the culture flasks counted before seed-ing in agarose influenced colony formation. Arelatively high number of live cells (about1 x 106 cells/ml) and a fraction of dead cellsunder 0.1 increased colony formation.

TUMORIGENICITYThe incidence of tumour formation in SCIDand nude mice is shown in table 1. Tumourswere generated in SCID mice by all four celllines tested (MS 1845, MS 1859, MS 1851, andMS 1858). Invasive growth into the skin result-ing in necrotic ulcers was evident in two of fourmice with tumour inoculated with the LCLMS 1851 and MS 1858, two of three mice withtumour inoculated with MS 1845 and in five ofsix mice inoculated with MS1859. Tumourswere generated in three of five nude miceinoculated with the LCL MS 1859 (69 bp dele-tion) and grew non-invasively up to a size of1 cm' over the course of three to four weeks.Thereafter, the tumours regressed leaving atumour of only 0.5 cm' in two of the mice after26 weeks, at which time the mice weresacrificed. In the third mouse no residualtumour was found. None of the nude miceinoculated with the other cell lines showed evi-dence of tumour formation over the 26 weeksof follow up.

CYTOGENETICSThe four cell lines used for tumorigenicitystudies (MS1845, MS1859, MS1851, andMS 1858) were all shown to be normal diploidcell lines by flow cytometry.

IMMUNOHISTOLOGYAll cell lines displayed characteristic LCLimmunophenotype. No differences in theexpression of the adhesion molecules ICAM-1and LFA-3 or the activation marker CD23could be demonstrated between cell lines withand without mutation of the BNLF-1C-terminus. Comparable concentrations ofEBV gene products EBNA-2, LMP-1 andBZLF- 1 were detected in all LCL. The LMP-1staining pattern indicated cytoplasmic andmembrane staining with focal positivity, sug-gesting patching in the membrane. No differ-ences in staining patterns between cell lineswith and without mutations of the BNLF-1C-terminus could be detected. Bcl-2 wasexpressed by all cell lines at comparable levels.SCID mice tumours displayed a stainingpattern similar to LCL, except for CD21 andCD23 which were slightly down regulated inall tumours from SCID mice. In two tumours

from nude mice EBNA-2 expression was dem-onstrated in the nuclei of tumour cells,confirming the presence of EBV infected lym-phocytes rather than a non-specific reaction.

DiscussionRecent studies have shown that single basemutations and the 30 bp deletion detected inthe C-terminus of the CAO BNLF-1 gene canbe detected frequently in EBV associatedmalignancy in vivo. 23 36 37 The CAO BNLF-1isolate shows increased tumorigenicity inSCID and nude mice compared with proto-typic EBV strains, and it has been suggestedthat mutant LMP-1 is preferentially associatedwith aggressive forms of EBV malignancy.'6The present study shows that similar geneticchanges occur frequently in wild type EBVstrains present in spontaneously arising LCLand do not by themselves characterise atumour specific genotype. Clearly, care mustbe taken in interpreting the results of thisstudy. Only viruses carrying LMP- 1 capable ofsupporting transformation will be present inLCL. This selection bias means that thevariants we have studied may not be com-pletely representative of wild type viruses as awhole.Recombinant EBV molecular genetic ex-

periments with specifically mutated LMP-1genes have demonstrated that LMP-1 is essen-tial for LCL outgrowth.'0 Our results indicatethat neither the 30 bp nor the 69 bp deletionsare essential for LMP-1 mediated primary Bcell transformation. Furthermore, comparisonof transformation related parameters indicatedthat the deletions did not affect the in vitrobehaviour of the LCL significantly. However,there was a tendency for the LCL with theEBV strain containing the 69 bp deletion to bemore serum dependent than the other LCL.

It is tempting to ascribe the increasedtumorigenicity of MS 1859 to the 69 bpLMP-1 deletion. Clearly, however, this may bebecause of changes elsewhere in the viralgenome not identified by our analysis, and for-mal proof will require further transfectionstudies.

In vitro evidence that the 30 bp deletion maystill play a role in tumorigenesis has come froma recent study by Li et al.26 They found differ-ences in the behaviour of Balb/3T3 cells whentransfected with B95.8 BNLF-1 gene in whichthe 30 bp sequence had been deleted, com-pared with the prototypic B95.8 BNLF-1gene. The transfected cells became tumori-genic in nude mice, the transforming capacityof the virus increased, and the gene was toxic tothe cells at lower expression levels comparedwith the wild type B95.8 BNLF-1 gene. Theapparent discrepancies between this study andour findings may be partly due to differences inthe behaviour of the BNLF- 1 gene in rodentcompared with human cell lines, as has beendescribed in other circumstances.38

Alternatively, failure to detect an effect of the30 bp deletion may be because of a lack of sen-sitivity in our assay. In any event, the results ofour study and those of Li et al'6 suggest that the

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sequences of the 30 bp and 69 bp deletionsmay have an important role in LMP-1 proteinfunction.

Recent studies by Moorthy and Thorley-Lawson may offer an explanation for theapparent increased tumorigenicity of EBVvariants with the 30 and 69 bp deletions inBalb/3T3 cells and in our LCL, respectively.They showed that deleting 23 (A 364-386) or

81 amino acids (A 306-386) from theC-terminus resulted in a LMP-1 protein thatwas non-transforming, but turned over nor-

mally in Rat-1 fibroblasts. In contrast, deleting64 (A 323-386) or 53 (A 334-386) amino acidsfrom the C-terminus generated toxic LMP-1mutants, suggesting that the toxicity was

caused by the absence of the amino acidsequence 334-364.1' The 69 bp deletiondescribed by us constitutes about two thirds ofthis amino acid sequence. Thus, the toxicity inRat-1 cells and the increased tumorigenicity ofLCL may be caused by similar changes infunction of the LMP-1 protein. The 30 bpdeletion variant has lost about one third of the334-364 amino acid sequence, and this may

account for its increased toxicity in Balb/3T3cells26 when driven by a weaker promoter com-

pared with the B95.8 BNLF-1 gene. Trivediet aP9 have shown that the LMP-1 proteinencoded by the CAO BNLF-1 gene (whichcontains the 30 bp deletion) is less immuno-genic than B95.8 LMP-1. This offers a

possible explanation for the increased tumori-genicity of variants lacking the 30 bp and 69 bpsequences, although it cannot account for theincreased transforming capacity of the 30 bpdeletion variant found by Li et al comparedwith B95.8.26 LMP-1 has been shown toinduce the expression of bcl-2 in B cells andthis response was found to be delayed relativeto NF-KB activation, suggesting that NF-KBmight mediate this response.40 The 30 bp andthe 69 bp deletions are located close to or pos-

sibly overlap the NF-KB activation region ofLMP-1, suggesting that the bcl-2 response of

LMP-1 expression could be affected by thedeletions. However, we found no immunohis-tological evidence for this.The 69 bp deletion is rare in vivo, having

been demonstrated previously in only one

AIDS related lymphoma, in one case ofchronic lymphoproliferative disease in a child,and in a single case of Hodgkin's disease in a

Liberian patient but in none of some 100European Hodgkin's disease cases investi-gated.22 36 37 We have previously proposed thatthe 30 bp deletion can be generated by slippedmispairing of direct repeats.22 If a similarmechanism is responsible for the 69 bpdeletion, then the relative rarity of thisvariation compared with the 30 bp deletionmay simply be explained by the size of thedeletion and the direct repeats. The directrepeats present in the sequence environment ofthe 30 bp and the 69 bp deletions are of 11 bpand 5 bp, respectively. The frequency ofslipped mispairing has been shown to beproportional both to the length of the directrepeat motif and to the extent of homologybetween the direct repeats, but inversely

proportional to the distance between them.4'As a consequence of this the 69 bp deletionshould be generated much less frequently thanthe 30 bp deletion.

In conclusion, these results and our recentlypublished data on Hodgkin's disease, periph-eral T cell lymphoma and infectious mononu-cleosis show that mutations in the C-terminalregion of the BNLF-1 gene occur frequently inboth malignant and benign EBV infected cellsand are more widespread among wild typeEBV strains than originally thought.22 Whilethese mutations clearly do not simply definetumour specific EBV strains, our study sug-gests that the LMP-1 amino acid sequence334-356 (or part of this sequence) is ofimportance for the tumorigenic effects ofLMP-1, and that changes in the function ofLMP1 may result in altered tumorigenicity ofEBV infected LCL. Studies on larger numbersofLCL from normal EBV seropositive subjectsare needed to investigate which mutationsoccur regularly in the BNLF-1 gene. Furthermutational studies in vitro are needed to definethe effect of the 69 bp deletion described inthis study.

This work was supported by the Danish Cancer Society grant95 120-01 and the Danish Multiple Sclerosis Society.

We wish to thank Niels Gregersen and Brage Andresen from theCentre for Medical Molecular Biology, University Hospital ofAarhus, for assistance with sequence analysis, Professor LarsBolund, Institute of Medical Genetics, University of Aarhus, forassistance with the flow cytometry analyses, and ProfessorS0ren Mogensen, Department of Medical Microbiology andImmunology, University of Aarhus, for critical review of themanuscript. We also wish to thank Tom Nordfelt, MarianneRasmussen, Margit Schjerven, Inga Knudsen, and MargretheKjeldsen for excellent technical assistance.

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