Proc. Natl. Acad. Sci. USAVol. 86, pp. 9558-9562, December 1989Medical Sciences

Epstein-Barr virus nuclear protein 2 is a key determinant oflymphocyte transformation

(recombination/berpesvirus/latency)

JEFFREY I. COHEN, FRED WANG, JOAN MANNICK, AND ELLIOTT KIEFF*Departments of Medicine, and of Microbiology and Molecular Genetics, Harvard Medical School, 75 Francis Street, Boston, MA 02115

Communicated by Bernard N. Fields, September 12, 1989 (received for review August 30, 1989)

ABSTRACT Epstein-Barr virus (EBV) efficiently trans-forms B lymphocytes to perpetual proliferation. The EBVlaboratory strain P3HR-1 is transformation-incompetent andlacks a DNA segment that includes the EBV nuclear antigen 2(EBNA-2) gene and a portion of the EBNA leader protein(EBNA-LP) gene. These two genes are expressed in trans-formed B lymphocytes. Recombinant transformation-compe-tent EBVs were produced by transfecting P3HR-1-infected cellswith a cosmid containing the DNA deleted in P3HR-1. Deletionof 105 nucleotides from the middle of the EBNA-2 gene had nodiscernible affect on transformation. Two larger EBNA-2deletions abolished transformation but did not affect EBNA-2nuclear localization. Two naturally occurring EBV variants(EBV types 1 and 2) differ extensively in their growth-trans-formation phenotype and in their EBNA-LP, EBNA-2, andEBNA-3A, -3B, and -3C genes. Recombinant P3HR-1 carryingEBV-1 EBNA-2 has many of the EBV-1 in vitro growth-transforming effects; recombinant P3HR-1, isogenic except forEBV-2 EBNA-2, has many of the EBV-2 growth-transformingeffects including slow emergence of transformants, growth intight clumps with few surrounding viable cells, and earlysensitivity to dilution with fresh medium. Thus, EBNA-2 is anessential molecule in lymphocyte growth transformation byEBV and a major determinant of the differences betweenEBV-1 and EBV-2 in lymphocyte growth transformation.

Epstein-Barr virus (EBV) causes fatal lymphoproliferativedisease in immunodeficient patients and is a likely initiatingfactor in Burkitt lymphoma (1). EBV efficiently transformsnormal human B lymphocytes in vitro (2) and induces fatalB-lymphocyte proliferation in cottontop tamarins (3). Sixdifferent EBV-encoded nuclear proteins (EBNA-1, EBNA-2,EBNA-3A, EBNA-3B, EBNA-3C, and EBNA-LP) and alatent-infection membrane protein (LMP) are characteristi-cally expressed in growth-transformed, latently infected Blymphocytes (4-7).

Previous results indicate that EBNA-2 and LMP are im-portant for lymphocyte growth transformation. (i) LMP is adominant transforming oncogene in rodent fibroblasts (8, 9)and induces expression of activation markers, vimentin, andcell adhesion molecules in B lymphocytes (10, 11). (ii) Alaboratory-derived EBV strain, P3HR-1, which lacks a DNAsegment that includes the EBNA-2 gene and part of theEBNA-LP gene (12), will not transform lymphocytes (13)unless it has recombined with another EBV genome andreacquired the deleted DNA segment (14). (iii) EBNA-2expression in rodent cells reduces the serum requirements(15) and in EBV-negative Burkitt lymphoma cells causesgrowth in tight clumps and increases expression of B-cellactivation antigen CD23 (16); EBNA-LP has no effect inthese assays (16).

Natural EBV isolates belong to one oftwo variants (EBV-1or EBV-2) that differ in their growth-transformation pheno-type in vitro. EBV-1-transformed cells grow more rapidly,are less concentration-dependent, and reach a higher satu-ration density when compared with EBV-2 transformants(17). EBV-1 and EBV-2 differ at their EBNA-LP, EBNA-2,and EBNA-3A, -3B, and -3C alleles (18, 19). EBV-1 (e.g.,B95-8 or W91 virus) EBNA-2 is 82-87 kDa, while EBV-2(e.g., AG876 or Jijoye virus) EBNA-2 is 75 kDa (18). EBV-1(B95-8) EBNA-2, EBNA-LP, EBNA-3A, -3B, and -3C have56%, 78%, 84%, 80%, and 72% amino acid identity with theirEBV-2 (AG876) counterparts, respectively (ref. 18; J. Sampleand E.K., unpublished data). Thus, the differences in growthtransformation between EBV-1 and EBV-2 may be due toallelic variation in EBNA-2 or other viral genes.To critically examine the role ofEBNA-2 in B-lymphocyte

transformation, we produced recombinant EBV by inducingvirus replication in P3HR-1 cells (clonal derivative of theBurkitt lymphoma line Jijoye) transfected with cosmids con-taining the DNA deleted in P3HR-1 virus (including theEBNA-2 gene and part of the EBNA-LP gene) and homol-ogous flanking sequences. By varying the EBNA-2 se-quences in the transfected DNA, we inserted EBV-1 EBNA-2, EBV-2 EBNA-2, or specific EBV-1 EBNA-2 deletions.Using this approach we have evaluated the role of EBNA-2in determining the differences in growth transformation be-tween EBV-1 and EBV-2, and we have begun to locate theEBNA-2 domains essential for lymphocyte transformation.

MATERIALS AND METHODSCell Lines, Virus, and Plasmid DNA. The P3HR-1 clone 16

cell line derived from the P3HR-1 variant of Jijoye cells (13)was kindly provided by G. Miller. B95-8 and W91 areprototype EBV-1 strains; AG876 and Jijoye are prototypeEBV-2 strains. The cosmid containing the EcoRI A fragmentfrom W91 DNA (corresponding to nucleotides 7315-69,119of B95-8; ref. 20) inserted in plasmid MUA3 (21) is hereindesignated T1EBNA-2, because it contains an EBV-1EBNA-2 gene.

Construction of Mutations. Plasmid HK-EBNA-2 was con-structed by inserting the W91 HindIII-Kpn I fragment (cor-responding to B95-8 nucleotides 48,039-52,940; ref. 20) intopBluescript KS(+) plasmid (Pharmacia). Plasmids HK-EBNA-2d200-234 and HK-EBNA-2d248-382 were obtainedby removing the internal EBNA-2 Stu I or Sph I fragmentsfrom plasmid HK-EBNA-2. (Numbers refer to amino acidsdeleted from the EBNA-2 protein based on the sequence ofB95-8 EBNA-2 in ref. 20). Plasmid HK-EBNA-2dl48-324was constructed by digestion with BstEII, synthesis of blunt

Abbreviations: EBV, Epstein-Barr virus; EBNA, EBV-encodednuclear protein (antigen); LMP, latent-infection membrane protein;ICAM-1, intercellular adhesion molecule 1; LFA-1, lymphocytefunction-associated antigen 1.*To whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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ends with DNA polymerase I (Kienow fragment), digestionwith Bal I, and ligation of the residual large fragment. Thenucleotide sequence of the BstEII-Bal I junction was deter-mined (22) to verify that the deletion preserved the openreading frame. Cosmids T1EBNA-2d200-234, T1EBNA-2d248-382, T1EBNA-2d148-324, and T2EBNA-2 were ob-tained by replacing the 4-kilobase (kb) HindIII-Rsr II frag-ment (which includes the EBNA-2 gene) from cosmidT1EBNA-2 with the corresponding HindIII-Rsr II fragmentfrom plasmid HK-EBNA-2d200-234, HK-EBNA-2d248-382, HK-EBNA-2d148-324, or pDA113 (which includes theAG876 EBV-2 EBNA-2 gene; ref. 18), respectively.

Transfection and Infection. Cosmids (20 ug) were precip-itated in ethanol and transfected into P3HR-1 cells by use ofan electroporator (Progenitor II; Hoefer) with a pulse of 350V, 100 AF, 1 sec. After transfection, virus replication wasinduced with phorbol 12-myristate 13-acetate (20 ng/ml;Sigma) and n-butyric acid (4 mM; Sigma). After 48 hr the cellswere lysed by three cycles offreezing and thawing and pooledwith the cell supernatant. The pooled material was filteredthrough a 0.45-,um filter and pelleted at 8800 x g for 2 hr.Each virus preparation was incubated with 107 human um-bilical cord mononuclear cells for 1 hr at 37°C and plated intomicrotiter plates at 106 cells per ml. The cells were fed eachweek with fresh RPMI 1640 medium supplemented with 1ofetal bovine serum.

Additional transfected P3HR-1 cells were analyzed forEBNA-2 by immunofluorescence microscopy using anEBNA-2-specific monoclonal antibody (23). Southern blot-ting, immunoblotting, and flow cytofluorometric analysis ofsurface expression of CD23, intercellular adhesion molecule1 (ICAM-1), and lymphocyte function-associated antigen 1(LFA-1) were performed as described (16).

RESULTSRecombinant P3HR-1 with EBV-1 EBNA-LP and EBNA-2.

Transformed cell lines were obtained when cosmid T1-EBNA-2 was transfected into P3HR-1 cells, virus replicationwas induced, and filtered cell lysates were used to infecthuman cord blood lymphocytes. Cosmid T1EBNA-2 con-tains the DNA deleted in P3HR-1 with -20 kb ofhomologousflanking DNA on either side of the deletion (Fig. 1). Lym-

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phocytes infected with cell lysates were aliquoted in micro-titer plates so that individual clones of transformed cellscould be obtained, allowing an estimate of the titer of thetransforming, putative recombinant virus. Similar numbersof clones were obtained when P3HR-1 cells were transfectedwith either covalently closed circular cosmid DNA or ex-cised, linear, EBV insert DNA (data not shown). Southernblot analysis of the EBV DNA in the lymphoblastoid cellclones indicated that all clones derived from cosmid DNAshowed evidence of homologous recombination between theP3HR-1 EBV genome and EBV cosmid DNA (examplesshown in Fig. 2).Immunoblot analysis indicated that lymphoblastoid cell

clones containing recombinant genomes expressed anEBNA-2 identical in size to EBV-1 W91 EBNA-2 and slightlysmaller than EBV-1 B95-8 EBNA-2 (Fig. 3). Clones withrecombinant genomes expressed an EBNA-1 identical in sizeto P3HR-1 EBNA-1 (Fig. 3) and slightly larger than W91EBNA-1 (data not shown). Furthermore, lymphoblastoid cellclones with recombinant genomes contained an LMP iden-tical in size to LMP from Jijoye cells (the parent of P3HR-1)and smaller than W91 LMP (Fig. 3). In contrast, P3HR-1 cells(nonrecombinant) expressed very low levels of LMP. Thesefindings confirm that the EBV genome in the lymphoblastoidcell clones is the result of homologous recombination be-tween the EBV sequences in the cosmid DNA (T1EBNA-2)and the P3HR-1 genome.

Effect of Deletions Within EBV-1 EBNA-2. Nucleotidesequence analysis of90% of the W91 EBNA-2 gene indicatedthat the predicted amino acid sequence of the EBNA-2protein in EBV-1 W91 has >96% identity with the sequenceofEBNA-2 from EBV-1 B95-8 (J.I.C. and E.K., unpublisheddata). EBV-1 W91 cosmids with one of three in-frame dele-tions in the EBNA-2 open reading frame were constructed(Fig. 1). These deletions were located in the central EBNA-2domain that is poorly conserved between EBV-1 and EBV-2,since that portion of the molecule is less likely to be essentialfor EBNA-2 function. The deletion in cosmid T1EBNA-2d200-234 removes 35 amino acids near the center of thisdomain. The deletion in cosmid T1EBNA-2d148-324 beginsbefore and ends after this domain, while the deletion incosmid TlEBNA-2d248-382 removes the carboxyl portion of

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FIG. 1. Prototype EBV genome, P3HR-1 genome, and cosmids used for transfections. The EBV genome (top line) consists of unique (U),internal repeat (IR), terminal repeat (TR), oriLyt (origin of lytic replication), and oriP (origin of plasmid replication) DNA domains. The P3HR-1virus is deleted (solid bar) for DNA corresponding to bases 45, 644-52, 450 in the EBV B95-8 genome (20). T1EBNA-2, the EBV-1 W91 EcoRlA-fragment cosmid used in transfections, contains EBV DNA corresponding to bases 7315-69,119 of the EBV B95-8 genome (20).TlEBNA-2d200-234, TlEBNA-2d248-382, and TlEBNA-2d148-324 are in-frame deletion mutants of cosmid T1EBNA-2 made by using StuI, Sph I, or BstEII/Bal I, respectively. Cosmid T2EBNA-2 is isogenic with T1EBNA-2 except that it has an EBNA-2 gene derived from EBV-2(AG876). The notations above T1EBNA-2 indicate the polyproline region (PP), the area of highest divergence between EBV-1 and EBV-2 aminoacid sequences, the arginine-glycine motif (RG), the negatively charged region (NEG), and the putative nuclear localization signal (N) inEBNA-2.

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FIG. 2. Southern blot of DNAs from P3HR-1, B95-8, and W91Burkitt lymphoma cells and from lymphoblastoid cells containingrecombinant genomes derived from cosmids. Total cell DNA wasdigested with BamHI and probed with a radiolabeled EBV BamHI Hfragment to demonstrate that the recombinants derived from thecosmids have aBamHI H fragment similar in size to that ofW91 (6.9kb), while B95-8 and P3HR-1 have smaller fragments (6.0 and 4.1 kb,respectively). Lymphoblastoid cells derived from cosmid T1EBNA-2d200-234 contain a 6.8-kb fragment due to the 105-base-pair dele-tion. A faint band at 9.7 kb is seen in some ofthe lanes correspondingto the BamHI B1 fragment of P3HR-1 [which contains the oriLytsequence also present in BamHI H (ref. 24 and Fig. 1)]. Numbersrefer to size of DNAs in kilobases; TlEBNA-2d refers to T1EBNA-2d200-234.

this domain and extends past the arginine-glycine repeatmotif (Fig. 1). Two independent constructs of each cosmidwere prepared to eliminate the possibility of additional mu-tations occurring during cloning.

Transfection ofP3HR-1 cells with each of the four cosmids(including the three cosmids with the deletions) inducedEBNA-2 antigen in similar numbers of cells (about 0.1%) 48hr after electroporation. Cells transfected with cosmidT1EBNA-2 or TlEBNA-2d200-234 and then stained withmonoclonal antibody to EBNA-2 (23) showed fine nucleargranules indistinguishable from the pattern seen in B95-8

cells. In contrast, staining of cells transfected with cosmidscontaining more extensive deletions (T1EBNA-2d148-324 orTlEBNA-2d248-382) showed coarser nuclear granules.Cosmid T1EBNA-2 yielded an average of 9 transformed cellclones (range 1-30, n = 9), while cosmid T1EBNA-2d200-234 yielded an average of 6 clones (range 5-18, n = 6)per experiment. Cosmids containing more extensive dele-tions (EBNA-2d248-382 or EBNA-2d148-324) failed to yieldany transformants in six separate experiments despite main-tenance of the infected cord cells for 10 weeks (see below).The difference in the number of transformants betweencosmid T1EBNA-2 and either of the two more extensiveEBNA-2 deletion cosmids was highly statistically significant(P < 0.01, Wilcoxon sum rank test). Clones derived fromcosmid TlEBNA-2d200-234 could be expanded as rapidly asclones derived from cosmid T1EBNA-2 or from B95-8 virus.Thus, EBNA-2 is essential for initiation of lymphocytegrowth transformation. Furthermore, within EBNA-2 a cen-tral divergent domain is dispensable, while an adjacent do-main(s) is indispensable for transformation.Many Type-Specific Transforming Differences Are Attrib-

utable to EBNA-2. To evaluate the contribution ofEBNA-2 tothe differences in growth transformation between EBV-1 andEBV-2 transformants, the EBV-1 EBNA-2 gene in cosmidT1EBNA-2 was replaced by the EBV-2 EBNA-2 gene fromAG876 DNA (18). The resulting hybrid cosmid, T2EBNA-2,was transfected into P3HR-1 cells to generate recombinantvirus; recombinants derived from parental cosmid T1EBNA-2 were generated in parallel. EBV-2 (Jijoye) was one control,since P3HR-1 is a laboratory mutant ofthe Jijoye cell line (13)and the Jijoye EBNA-2 amino acid sequence is identical withthat of AG876 (reviewed in ref. 26). EBV-1 (B95-8) wasanother control. Cosmid T2EBNA-2 yielded an average of 1transformed cell clone (range 0-3, n = 6) in each experiment,markedly lower than the number obtained with cosmidT1EBNA-2 (P < 0.01, Wilcoxon sum rank test). Lympho-blastoid cell clones derived from cosmid T2EBNA-2 werevisible -macroscopically only after 6-10 weeks in culture,whereas clones derived from cosmid T1EBNA-2 were visibleafter only 3-4 weeks. Cell clones derived from T2EBNA-2grew in tighter clumps, with poorer cell viability outside ofthe clumps as compared with clones derived from T1EBNA-2(Fig. 4). Moreover, in contrast to clones derived fromT1EBNA-2, clones derived from T2EBNA-2 could not besubcultured or expanded beyond the initial microwell stage

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FIG. 3. Immunoblots of proteins from EBV-positive (P3HR-1) or EBV-negative (BJAB) Burkitt lymphoma cells and lymphoblastoid cell linescontaining EBV (Jijoye, B95-8, IB4, W91) or recombinant EBV derived from cosmids. (A) Antiserum from a donor infected with a type 1 EBVstrain shows an 87-kDa EBV-1 EBNA-2 in B95-8 or IB4 cells, an 84-kDa EBNA-2 in lymphoblastoid cells derived from cosmid T1EBNA-2,and a 79-kDa EBNA-2 in cells derived from cosmid TlEBNA-2d200-234. The open arrow indicates EBNA-1. (B) Antiserum from a donorinfected with an EBV-2 strain shows a 74-kDa EBV-2 EBNA-2 in Jijoye cells and in lymphoblastoid cells derived from cosmid T2EBNA-2, butlittle or no detectable EBNA-2 in cells derived from cosmids T1EBNA-2 or TlEBNA-2d200-234. (C) Monoclonal antibody to LMP (25) showssimilar levels of LMP in cells containing EBV-1, EBV-2, or recombinant EBV derived from cosmids. P3HR-1 cells express very low levels ofLMP, while W91 cells express an LMP that is slightly larger than LMP from P3HR-1 recombinants or Jijoye cells. Numbers refer to size ofproteins in kilodaltons; T1EBNA-2d refers to TlEBNA-2d200-234.

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FIG. 4. Phase-contrast photomicrographs of lymphoblastoid cells with recombinant P3HR-1 genomes. Cells derived from cosmid T1EBNA-2(Left) grow in loose clumps with a carpet of viable cells between the clumps, whereas cells derived from T2EBNA-2 (Right) grow in tight clumpswith few viable cells between clumps. (x65.)

for several weeks after the appearance of clumps. Earlierattempts to expand the cultures resulted in uniform celldeath. The growth of T2EBNA-2 P3HR-1 recombinant-infected clones was similar to that of EBV-2 (Jijoye)-infectedclones derived in parallel. Immunoblots confirmed that thelymphoblastoid cells derived from cosmid T2EBNA-2 ex-pressed the EBV-2 EBNA-2 gene (Fig. 3).Lymphoblastoid cell lines obtained from EBV-1 (B95-8 or

W91) infection or from cosmid T1EBNA-2 or T1EBNA-2d200-234 P3HR-1 recombinant infections were cryopre-served soon after outgrowth so that they could be comparedwith EBV-2 (Jijoye) or cosmid T2EBNA-2 P3HR-1 recom-binant-infected clones in parallel, when the latter clonescould be expanded. After 4 months, when lymphoblastoidcell clones derived from cosmid T2EBNA-2 or from EBV-2(Jijoye) could be expanded sufficiently for analysis, the levelsof expression of LMP, B-cell activation antigen CD23, andcell adhesion molecules were compared to those in cell linesderived from cosmid T1EBNA-2 or T1EBNA-2d200-234 orfrom EBV-1 (B95-8). Levels of LMP, ICAM-1, or LFA-1were similar in all lymphoblastoid cell clones tested (Fig. 3and data not shown). Cell clones derived from cosmidT1EBNA-2 or T1EBNA-2d200-234 or from EBV-1 (B95-8)showed similar, high-level expression of CD23; clones de-rived from cosmid T2EBNA-2 or EBV-2 (Jijoye) showedmore variable expression of CD23, with some cells express-ing high levels of CD23 (similar to clones derived fromEBV-1) and other cells expressing low levels of CD23.

DISCUSSIONThe ability to construct recombinant EBV P3HR-1 genomesby using cosmid clones ofEBV DNA that differ only in theirEBNA-2 genes significantly extends the range of EBV ge-netic analysis. The P3HR-1 deletion was known to include

the EBNA-2 gene and part of the EBNA-LP gene. Priorexperiments showed that when Burkitt lymphoma cells con-taining EBV-1 genomes were infected with P3HR-1 virus,P3HR-1 viruses were released that had undergone homolo-gous recombination with the endogenous EBV genomes,restoring the EBNA-LP and EBNA-2 DNA segment and thetransforming phenotype (14). The use of EBV cosmid DNAenables us to formally exclude the possibility that the trans-forming phenotype in these earlier experiments may havebeen due to additional recombination events elsewhere in theP3HR-1 genome.

Further, these experiments with EBNA-2 deletion mutantcosmids more precisely identify EBNA-2 to be essential forEBV initiation of lymphocyte growth transformation. Thefailure of two cosmids (TlEBNA-2d248-382 and T1EBNA-2dl48-324) to yield transformed cell lines while two othercosmids (T1EBNA-2 and TlEBNA-2d200-234) producedtransformants provides strong evidence that EBNA-2 isessential. In addition, the inability of the former two cosmidsto yield transformants indicates that the middle third of theEBNA-2 gene includes an essential domain for growth trans-formation. The expression of appropriate quantities of im-munoreactive EBNA-2 in the nuclei of cells transfected withthese former two cosmids indicates that their deletions do notmarkedly interfere with EBNA-2 processing or stability. Thepresence of coarse granules in cells transfected with thesetwo cosmids, in contrast to the fine granules in cells trans-fected with T1EBNA-2 or TlEBNA-2d200-234, suggeststhat the deletions in the former two cosmids destroy trans-forming ability by altering the macromolecular interactions ofEBNA-2. Since the EBNA-2 protein expressed with cosmidTlEBNA-2d248-382 translocates to the nucleus even thoughit lacks the arginine- and glycine-rich domain that is the onlypotential nuclear translocation signal other than that near the

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carboxyl terminus (Fig. 1), the latter is almost certainlyresponsible for EBNA-2 nuclear translocation. The observa-tion that a 35-amino acid deletion within this most divergentpart of EBNA-2 does not affect transformation is consistentwith our working hypothesis that sequences essential fortransformation are likely to be conserved between EBV-1and EBV-2. These findings are similar to those for other viraltransforming proteins [e.g., adenovirus ElA (27, 28) orpapillomavirus E7 (29)] in which domains required for trans-formation are conserved among different strains of the samevirus.These studies also indicate that EBNA-2 is responsible for

many of the differences in growth phenotype between EBV-1- and EBV-2-transformed lymphocytes. Thus, P3HR-1 re-combinants that differ only in having an EBV-1 or EBV-2EBNA-2 gene differ dramatically in growth transformation.EBV-2 EBNA-2 P3HR-1 recombinants mimic EBV-2 (Jijoye)in their slow emergence as transformed clones, their earlysensitivity to dilution with fresh medium, and their growth inlarge tight clumps. In contrast, EBV-1 EBNA-2 P3HR-1recombinants, which are otherwise isogenic with EBV-2EBNA-2 P3HR-1 recombinants, emerge rapidly and grow ina similar fashion to EBV-1 (B95-8) transformants. Thus,while the sequences of other genes (EBNA-LP, EBNA-3A,EBNA-3B, EBNA-3C) are known to differ in EBV-1 andEBV-2, the EBNA-2 allele is responsible for many of thedifferences in the growth-transformation phenotype.The finding that EBNA-2 is essential to lymphocyte growth

transformation by EBV is not unexpected in view of previousevidence that EBNA-2 induces clumping of B-lymphomacells and expression of CD23 (16). However, EBNA-2 mostlikely does not act alone in lymphocyte transformation andprobably requires other EBV genes, especially that encodingLMP. The LMP gene is an oncogene in rodent fibroblasts andinduces expression of activation and adhesion molecules in Blymphocytes (8-10). The studies reported here make it pos-sible to examine the interaction of EBNA-2 and LMP inB-lymphocyte transformation within the context of otherEBV genes. Interestingly, while LMP, ICAM-1, and LFA-1expression was similar in the EBV-1 and EBV-2 recombinanttransformed clones, CD23 differed slightly. These data sug-gest a possible role for CD23 in mediating the type-specificdifferences in transformation.The ability to produce isogenic EBVs differing only in their

EBNA-2 type will aid ongoing studies on the role ofEBNA-2in trans-induction of EBV LMP or lymphocyte CD23 as wellas the role of EBNA-2 type-speciflic differences in T-cellkilling of EBV-transformed lymphocytes (30).The ability to produce recombinant EBV genomes by ho-

mologous recombination of transfected cosmid EBV DNAwith endogenously replicating P3HR-1 EBV DNA provides anopportunity for a detailed genetic analysis of EBV transfor-mation. These experiments relied on the transforming capa-bility of recombinant virus to positively select recombinantfroni nonrecombinant virus-infected cells. This approachcould be extended to construct additional mutations in thetransfected DNA either within the segment deleted fromP3HR-1 (e.g., EBNA-LP) or in the adjacent homologousflanking regions. Additional genetic material could be linked tothe sequences necessary for restoring transformation, and theeffects of these new sequences could be used to study viralfunctions or to introduce new genes into B lymphocytes.Alternatively, other positive selection markers could be in-serted into transfected EBV DNA, which could then be usedto select for recombination in other areas of the EBV genome.This would permit extension of these experiments to otherEBV genes in other EBV genomic backgrounds.

Note. After this work was completed, Hammerschmidt and Sugden(31) reported that recombinant transforming virus could be obtainedby transfection of P3HR-1 cells with a plasmid containing thesequences deleted from P3HR-1 along with flanking sequences,origins of plasmid and lytic replication (oriP and oriLyt), and EBVterminal repeats. Insertion of a stop codon into EBNA-2 abolishedtransformation, while deletion of the carboxyl portion of EBNA-LPdid not abrogate transformation.

Caroline Alfieri, David Liebowitz, Andy Marchini, and AlanRickinson provided helpful advice or reagents. The nurses andphysicians ofthe Obstetrical Unit of Brigham and Women's Hospitalassisted in obtaining human umbilical cord blood. This research wassupported by Grant CA47006 from the National Cancer Institute ofthe U.S. Public Health Service. J.I.C., F.W., and J.M. have Fel-lowships CA01417, CA01395, and A107061 from the United StatesPublic Health Service, respectively. E.K. is partially supported bygrants from the Sandoz Corporation and the Baxter Foundation.

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