Epstein–Barr virus lacking latent membrane protein 2 immortalizes B cells with efficiency indistinguishable from that of wild-type virus

Peter Speck1, Kimberly A. Kline1, Paul Cheresh1 and Richard Longnecker1

Department of Microbiology-Immunology, Northwestern University Medical School, Room 6-231 Ward Building, 303 East Chicago Avenue, Chicago, Illinois 60611, USA1

Author for correspondence: Richard Longnecker.Fax +1 312 503 1339. e-mail r-longnecker{at}nwu.edu


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
Epstein–Barr virus (EBV) is a human herpesvirus that efficiently transforms and immortalizes human primary B lymphocytes. In this study, the role of latent membrane protein 2 (LMP2) in EBV growth transformation was investigated. LMP2 is a virally encoded membrane protein expressed in EBV-immortalized B cells previously shown to be nonessential for EBV transformation. However, a recent study reported that LMP2 may be an important determinant for efficient B cell transformation (Brielmeier et al., Journal of General Virology 77, 2807–2818, 1996). In this study a deletion mutation was introduced into the LMP2 gene using an E. coli mini-EBV construct containing sufficient EBV DNA to result in growth transformation of primary B cells. In an alternative approach, the introduction of the gene encoding the enhanced green fluorescent protein (EGFP) by homologous recombination into the LMP2 gene of EBV strain B95-8, generating the same LMP2 deletion mutation is reported. Careful quantification of B cell transformation using the EGFP+LMP2- recombinant virus determined that in liquid culture medium or in culture medium containing soft agarose there was no difference in the ability of LMP2- virus to immortalize primary human B cells when compared to that of wild-type virus.


   Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Epstein–Barr virus (EBV) is a human herpesvirus causing mononucleosis in adolescents and lymphoproliferative disease in immunocompromised humans, marmosets and SCID mice. EBV is associated with certain haematopoietic cancers, such as African Burkitt's lymphoma, Hodgkin's lymphoma and adult T cell leukaemia (Rickinson & Kieff, 1996 ). EBV is also associated with two diseases of epithelial origin, nasopharyngeal carcinoma and oral hairy leukoplakia (Rickinson & Kieff, 1996 ). EBV latently infects B cells in vitro and in vivo. In vitro latent infection is characterized by immortalization and proliferation of B cells generating lymphoblastoid cell lines (LCLs) (Kieff, 1996 ; Longnecker, 1998 ), providing an in vitro model of EBV-mediated transformation of human B cells. During EBV latency in LCLs there is expression of a subset of viral genes including EBV nuclear antigens (EBNA-1, -2, -3A, -3B, -3C and -LP) and three proteins which aggregate at the plasma membrane, designated latent membrane proteins: LMP1, and two forms of LMP2, known as LMP2A and LMP2B (Kieff, 1996 ; Longnecker, 1998 ). LMP2 mRNA is consistently detected in nasopharyngeal carcinoma biopsies and EBV malignancies (Rickinson & Kieff, 1996) ; and, along with EBNA1, is the only EBV-specific message detected in latent EBV infection (Chen et al., 1995 ; Kieff, 1996 ; Longnecker, 1998 ; Miyashita et al., 1997 ; Qu & Rowe, 1992 ; Tierney et al., 1994 ), suggesting that LMP2 plays an important role in virus replication, persistence and EBV-related disease in humans. Support for a key role for LMP2 in EBV latency is also inferred from recent studies using LMP2A transgenic mice (Caldwell et al., 1998 ). In these studies, B cells expressing LMP2A survive despite the absence of normal B cell receptor signals, indicating that LMP2A expression in latently infected human B cells may enable cellular longevity by mimicking normal B cell receptor signals.

LMP2 is transcribed by two different promoters 3 kb apart. The first exons of LMP2A and LMP2B are the only unique exons, and the remaining eight exons are common to both messages (Laux et al., 1988 , 1989 ; Sample et al., 1989 ). The LMP2A unique exon encodes a 119 amino acid cytoplasmic domain, while the first LMP2B exon is noncoding. The methionine at amino acid 120 is the first methionine in the first LMP2 common exon and is the initiation site for LMP2B. LMP2A and LMP2B share 12 hydrophobic transmembrane domains and a 27 amino acid carboxy-terminal domain (Laux et al., 1988 , 1989 ; Sample et al., 1989 ).

Analysis of LCLs infected with LMP2 mutants shows a role for LMP2A in maintaining EBV latency in EBV-infected LCLs. The 119 amino acid cytoplasmic amino-terminal domain is essential for LMP2A function. EBV+ LMP2A+ LCLs are blocked in cell surface immunoglobulin-stimulated calcium mobilization, tyrosine phosphorylation and lytic activation compared with EBV+LMP2A- LCLs (Miller et al., 1994 , 1995 ). The LMP2A cytoplasmic amino-terminal domain associates with and negatively regulates Src family protein tyrosine kinases (PTKs) and Syk PTK (Burkhardt et al., 1992 ; Fruehling et al., 1996 , 1998 ; Fruehling & Longnecker, 1997 ; Longnecker et al., 1991 ; Miller et al., 1995 ), while LMP2B may regulate LMP2A function by modulating spacing between individual LMP2A amino-terminal domains aggregated at the cell membrane (Longnecker & Miller, 1996 ). These characteristics of LMP2A indicate a potential role in blocking induction of the lytic phase of the EBV life-cycle (Longnecker & Miller, 1996 ).

Mutational analysis of LMP2 reveals that neither LMP2A nor LMP2B are required for in vitro infection or transformation of primary B cells (Kim & Yates, 1993 ; Longnecker et al., 1992 , 1993a , b ). Studies defining the nonessential nature show that LMP2- EBV-infected LCLs appear identical to wild-type EBV LCLs in initial outgrowth, subsequent growth, sensitivity to limiting cell dilution and to low serum, and growth in soft agarose. Longnecker et al. (1992 , 1993a ) sought to demonstrate that efficiency of primary B cell transformation is quantitatively unaffected by LMP2 deletion. Their approach utilized expression of lytic antigen gp350/220 after induction of lytic replication as a crude indicator of virus production. These studies found that induction of gp350/220 occurred similarly in wild-type and LMP2-deleted LCLs, and that filtered released virus yielded similar numbers of transformed clones on infection and culture of primary B cells. Recently, however, it has been reported that LMP2 expression is important for efficient B cell immortalization (Brielmeier et al., 1996 ). The approach taken utilized mini-EBV plasmids, which are E. coli constructs containing the minimal EBV genomic sequences that when packaged into an EBV coat initiate and maintain proliferation of infected B cells (Kempkes et al., 1995a ). Compared with fully competent mini-EBV, LMP2-deleted mini-EBV plasmids were severely impaired in capacity to yield immortalized B cell clones (Brielmeier et al., 1996 ). A key characteristic of this approach is the tendency within mini-EBVs for small mutations to be introduced into sequences they encode. This is exemplified by a report that, using this approach, spontaneous deletion of a C residue in the EBNA3A open reading frame in one mini-EBV showed EBNA3A has a phenotype for initiation of B cell transformation (Kempkes et al., 1995b ). The absence of DNA sequence data of LMP2-deleted mini-EBVs or of marker rescue studies suggests that the reduction of immortalization efficiency may result from instability of DNA in mini-EBVs causing a mutation in another gene important for immortalization.

To resolve this issue we constructed a recombinant EBV, designated EBfaV-GFP, in which LMP2 was replaced with a reporter gene. We inserted the reporter gene encoding green fluorescent protein (GFP) into the LMP2 locus to enable visualization of infection. GFP was chosen because it fluoresces strongly and stably in many mammalian cells and can be monitored noninvasively in living cells (Chalfie, 1995 ; Chalfie et al., 1994 ). The enhanced form (EGFP) has modifications corresponding to human codon usage and it fluoresces 35 times more strongly than wild-type GFP (Cormack et al., 1996 ; Heim et al., 1995 ; Heim & Tsien, 1996 ). The CMV immediate early (ie) promoter was used as it is a strong constitutive promoter in a wide range of mammalian cells.

Insertion of a neomycin resistance–CMVie–EGFP cassette by homologous recombination into the LMP2 locus followed by drug selection gave rise to B95-8-derived cell lines harbouring a mixed population of recombinant (LMP2-EGFP+) and wild-type EBV. The LMP2 mutation was the same deletion mutation used by Longnecker et al. (1992 , 1993a ) and by Brielmeier et al. (1996) . These cells were stimulated with phorbol ester and butyric acid, yielding infectious EBV consisting of a mixture of wild-type virus and recombinant virus. This mixture was used to infect primary B cells at very low multiplicity of infection, yielding LCLs which were analysed for presence of recombinant (EGFP+LMP2-) or wild-type (LMP2+) virus. Separate experiments employed two differing methodologies of B cell transformation: after infection cells were maintained either in 96-well dishes with RPMI tissue culture medium or were suspended in soft agarose over a feeder layer of previously irradiated cells. The ratio of recombinant to wild-type virus in the infection mixture was determined by Southern hybridization and compared with the ratio of recombinant LCLs to wild-type LCLs resulting from infection. Existence of a phenotype for LMP2 in efficiency of B cell transformation predicts under-representation of recombinant (EGFP+LMP2-) LCLs as a proportion of all LCLs derived from this procedure. We report here that a mixture of wild-type and recombinant (EGFP+LMP2-) EBV yields wild-type EBV and EGFP+LMP2- LCLs in proportions similar to those in the infecting virus. This indicates that in the context of the complete EBV genome, as opposed to EBV mini-plasmids, efficiency of transformation of primary B cells by EBV is unaffected by deletion of LMP2.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Virus and cells.
B95-8, an EBV-infected marmoset cell line permissive for virus replication (Miller & Lipman, 1973 ; Miller et al., 1972 ) was obtained from the ATCC. BJAB (Klein et al., 1974 ) is an EBV-negative B lymphoma cell line. Daudi (Klein et al., 1976 ), an EBV-positive B lymphoma-derived line, was from the ATCC. All cells were grown in RPMI 1640 medium with 10% inactivated foetal bovine serum, penicillin (1000 U/ml) and streptomycin (1000 µg/ml; complete medium). Primary human mononuclear cells were obtained from blood samples from healthy donors and purified by centrifugation over Ficoll-Paque (Pharmacia).

{blacksquare} Plasmids.
Plasmid PS196, to target the CMV promoter–EGFP/SV40 promoter–neomycin resistance cassette into the viral genome, is based on pRL60 (Longnecker et al., 1993a ), which contains a SacI (B95-8 bp 163473)-to-KpnI (B95-8 bp 3502) fragment from EBV B95-8 DNA. The 3848 AseI–EcoO109I fragment from pEGFP.N1 (Clontech Laboratories) was end-filled using DNA polymerase Klenow fragment and ligated into end-filled EcoRI/SalI-digested and phosphatase-treated pRL60. Restriction enzyme analysis confirmed the rightward direction of the EGFP–neomycin resistance cassette in the plasmid, the same direction as LMP2. Plasmid pSVNaeZ, to induce lytic replication, has been described (Marchini et al., 1991 ; Swaminathan et al., 1991 ).

{blacksquare} Transfection and drug selection of B95-8 cells.
DNA for transfection was banded twice on CsCl density gradients. Recombinant cell lines were generated by transfecting 107 B95-8 cells per 0·4 ml complete medium with 15 µg pPS196 and 5 µg pSVNaeZ. Cells in a 4 mm cuvette were pulsed at 230 V, 960 µF with a Gene Pulser (Bio-Rad), diluted in 5 ml complete medium, cultured for 48 h and transferred to 96-well plates at 50000 cells per well in complete medium plus G418 (Gibco-BRL) at 700 µg/ml. Half the medium was replaced every week until colonies emerged (3–4 weeks).

{blacksquare} Southern blot hybridization.
Two DNA probes were used in separate Southern hybridizations. As a probe for EGFP a 791 bp BglII–NotI fragment of pEFGP.NI was gel-purified. As a probe for LMP2 a 2862 bp SalI–KpnI fragment (cutting at B95-8 sequence positions 644 and 3506, respectively) of pRL60 was used, which hybridizes to EBV DNA adjacent to the sequences deleted in EBfaV-GFP. 32P-radiolabelling used Ready-To-Go DNA labelling beads (Pharmacia). Genomic DNA was prepared as described (Wang et al., 1991 ). DNA from wild-type or recombinant EBV cell lines was BamHI-digested, electrophoresed, transferred to nylon (GeneScreen Plus, NEN Life Sciences) and probed as described (Wang et al., 1991 ). DNA from cell lines rather than virus stocks was analysed as there is no evidence that LMP2 has a virus packaging phenotype. Intensity of probe hybridization to bands was quantified by an Alpha Image 2000 image analysis system (Alpha Innotech) and confirmed using a Storm Phosphoimager (Molecular Dynamics).

{blacksquare} Production of virus stock.
Cells harbouring EBfaV-GFP were stimulated to release virus by 4 days culture in complete medium plus phorbol ester TPA (12-O-tetradecanoyl phorbol-13-acetate, 20 ng/ml) and butyric acid (3 mM; Sigma). Cells were centrifuged (5 min/1500 r.p.m.) and supernatant filtered through a 0·45 µm cellulose–acetate filter and stored at -140 °C.

{blacksquare} Infection of cells and production of LCLs.
Cells were infected by incubating virus with cells (105 cells for titration of virus stocks; 107 cells per 96-well plate for LCL production) in 1 ml medium for 1 h at 37 °C with agitation. Cells were centrifuged (5 min/1500 r.p.m.), supernatant discarded and cells resuspended in complete medium. Virus stocks were assayed for infectious `green' units on Daudi cells, which are readily infectable with EBV. Stocks of EBfaV-GFP typically contained sufficient virus for 1 µl to yield 250–500 EGFP-expressing cells. Infection of human cells was in cyclosporin A (1 µg/ml) to suppress donor EBV-directed T cells (Chang & Lung, 1994 ; Okano et al., 1990 ). Half the medium was replaced every week until colonies emerged (3–4 weeks).

{blacksquare} Growth of infected cells in soft agarose.
Immortalization of primary B cells yielding either wild-type or recombinant LMP2-infected LCLs was by plating in soft agarose over an irradiated (4000 Rads) fibroblast feeder layer as described (Sugden & Mark, 1977 ). Primary human B cells were infected with EBfaV-GFP at very low multiplicity, yielding 3–10 LCLs per 96-well plate. Cells were plated in 0·3% low melting point agarose (Fisher Biotech) in 96-well plates at approximately 3x105 cells/ml. After 5–7 weeks, the wells were scored for macroscopic cell growth and EGFP fluorescence.

{blacksquare} Visualization of GFP.
Fluorescence in infected cells was measured 24–36 h post-infection using a Zeiss Axioskop microscope or by flow cytometry. For photomicroscopy, unfixed infected cells were placed on a slide and overlaid with a coverslip.

{blacksquare} Flow cytometry.
Flow cytometry used a FacsCalibur (Becton Dickinson) with CellQuest software. The instrument was adjusted so that fluorescence of uninfected cells fell within the first decade of the logarithmic scale on which the emission is displayed. The mean fluorescent intensity of negative controls ranged from 2·5 to 3·5. Plots show at least 10000 events.

{blacksquare} PCR.
A standard protocol was employed (Vahey et al., 1995 ). Primers detecting LMP2 sequences were PS003, CTTCTTGCCCGTTCTCTTTCTTAG and PS004, CTTCTGTACGCTAGTATCAGGAGC. These primers amplify a 546 bp fragment.

Primers for EBV gene BHRF1 were BHFR1-C, GTGCATGGAAATGGTA and BHRF1-D, AAGGCTTGGGTCTCC. These primers yield a product of 239 bp.

{blacksquare} Electron microscopy.
EBfaV-GFP virus, diluted 1:25 in water, was pelleted onto Formvar-coated grids (Ted Pella) by ultracentrifugation in a Beckman Airfuge as described (Herold et al., 1991 ). Grids, fixed in 1% glutaraldehyde and stained with 1% phosphotungstic acid, were examined in a JEOL 1220 electron microscope. Counts were the sum of at least six grids, examined on two separate occasions. The number of nucleocapsids per virion was scored only when clearly discernible.


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
Construction and characterization of EBfaV-GFP
The CMV promoter-driven EGFP gene and the SV40 promoter–neomycin-resistance gene cassette was inserted into the EcoRI–SalI region of plasmid RL60. To allow a direct comparison of results of this study with earlier studies, the same LMP2 null deletion mutation was used as in previous studies by Longnecker et al. (1993b ) and Brielmeier et al. (1996) . This plasmid contains the EBV KpnI–SacI region spanning the viral terminal repeats (Fig. 1a, line 1). The resulting clone was transfected into B95-8 cells which were cultured in 96-well plates in G418 at 700 µg/ml. From a total of 6720 wells seeded, approximately 650 G418-resistant clones developed, of which 402 contained green fluorescing cells. As these clones grew they were progressively transferred to 48-, 24- and 6-well plates, and then into 25 cm2 flasks, with continued G418 selection. Approximately 1 month after picking from 96-well plates, DNA was prepared from EGFP-positive clones. Southern hybridization on BamHI-digested DNA from these 402 clones revealed that six contained the EGFP gene targeted by homologous recombination into the viral genome (data not shown). At this time approximately 30–50% of cells in these six clones expressed EGFP by fluorescence microscopy. After culture in G418-containing medium for another 2 months this percentage increased to virtually 100% by fluorescence microscopy (Fig. 2b, panel 2) or by flow cytometry (Fig. 2b, panel 3). In parental B95-8 cells there was no discernible background signal by fluorescence microscopy (Fig. 2a, panel 2). By flow cytometry the average signal in cells harbouring EBfaV-GFP (Fig. 2b, panel 3) was approximately three orders of magnitude brighter than in parental B95-8 cells (Fig. 2a, panel 3). In initial experiments the presence of EGFP-expressing EBV was shown by infecting B cell clones (BJAB or Daudi, data not shown) with filter-sterilized stocks of virus and monitoring for fluorescent green cells, first evident about 12–15 h after infection. Fluorescent cells were enumerated by flow cytometry 24 h after infection, with the clone yielding the highest number of green cells designated EBfaV-GFP and used in all subsequent experiments. Characterization of this clone will be further discussed below.



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Fig. 1. (a) Schematic representation of plasmid used to insert enhanced green fluorescent protein (EGFP) and the neomycin-resistance gene (neor) into EBV. Line 1 is a map of part of the EBV genome including the terminal repeat portion (shaded box), showing EcoRI and SalI restriction enzyme sites which define the region into which the EGFP–neor was inserted. Plasmid PS196 (line 2) includes sequences encoding EGFP–neor flanked by the EBV KpnI–SacI genomic fragment spanning the viral terminal repeats, targeting the EGFP–neor cassette into the viral genome (line 3), positioned to direct transcription rightwards, i.e. in the same orientation as LMP2. In recombinant EBfaV-GFP, digestion with BamHI yields a 6·5 kbp fragment encompassing EGFP (line 4). Line 5 shows the BglII–NotI fragment of EGFP used as a probe for EGFP. Line 6 shows the SalI–KpnI fragment of pRL60 used as a probe for EBV. (b) Southern blot analysis of EBfaV-GFP using a probe for EGFP. Five µg of cellular DNA was BamHI-digested, separated by gel electrophoresis, blotted onto nylon membrane and hybridized with a 32P-labelled EGFP-specific probe. Lanes: 1, recombinant DNA; 2, DNA from cells bearing the parental wild-type B95-8 DNA; 3, 100 pg BamHI-digested PS196. Since the SacI–KpnI fragment used in the targeting vector does not extend beyond the right-hand BamHI site, BamHI digestion of PS196 does not result in the same fragments as present in the recombinant EBV. (c) Southern blot analysis of EBfaV-GFP using a probe for EBV sequences. Five µg of cellular DNA was digested with BamHI, separated by gel electrophoresis, blotted onto nylon membrane and hybridized with a 32P-labelled probe consisting of the 2862 bp fragment from SalI to KpnI as shown in (a). Lane 4 contains BamHI-digested DNA from cells harbouring a mixture of wild-type EBV and EBfaV-GFP. The probe hybridizes to a 9·6 kb fragment from wild-type EBV DNA and a 6·5 kb fragment from EBfaV-GFP DNA. Densitometric analysis of these bands, with the combined hybridization in both bands at 100%, showed the upper band contained 82·4% of total signal and the lower band contained 17·6%, which was confirmed by phosphoimage analysis of these bands. Lane 5 contains DNA from the EBV-negative B lymphoma cell line BJAB, and lane 6 contains DNA from wild-type B95-8-infected cells.

 


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Fig. 2. EGFP fluorescence viewed by microscopy and flow cytometry. (a) B95-8 cells, shown by phase-contrast illumination (1), the same field with fluorescence illumination, showing lack of background signal (2) and a dot plot of EGFP fluorescence measured by flow cytometry (3). The instrument was adjusted to place the mean fluorescence signal in the middle of the first decade of the logarithmic EGFP scale. (b) EBfaV-GFP cells by phase contrast (1), the same field by fluorescence illumination (2) and a dot plot of flow cytometry showing an average of ca. three orders of magnitude increase in brightness (3). (c) Daudi cells infected with EBfaV-GFP, shown by phase contrast (1), the same field by fluorescence illumination, showing ca. 20% of cells infected (2) (two EGFP-expressing cells on the right-hand side of the field are identified by arrows in panel 1) and flow cytometry of Daudi cells showing the maximal infection of these cells seen in EBfaV-GFP infection, with approximately 30% of cells expressing EGFP (3). FSC, Forward scatter.

 
Presence of the neomycin-resistance–EGFP cassette in place of the EcoRI–SalI region of LMP2 was confirmed by PCR on DNA from cells of EBfaV-GFP. Primers were designed to detect sequences unique to EBfaV-GFP, using a primer specific for neomycin resistance and another for an EBV genomic sequence adjacent to the region deleted. PCR yielded a band of the anticipated size in EBfaV-GFP cells with no amplification product produced in reactions on control cell lines (data not shown). Control PCR reactions using primers specific for the BHRF1 gene gave bands of the expected size, 239 bp, as seen in Fig. 3(b), as a positive control for efficacy of the PCR reactions. PCR with LMP2 primers on DNA from cells with wild-type EBV also gave a band of the expected size, 546 bp, as seen in Fig. 3(a). PCR using LMP2 primers on EBfaV-GFP DNA also amplified a 546 bp band, showing that cells of clone EBfaV-GFP contained recombinant (LMP2-EGFP+) and wild-type EBV genomes.



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Fig. 3. PCR analysis of LCLs derived using recombinant virus EBfaV-GFP and wild-type EBV. (a) Ethidium bromide-stained PCR products from amplification of LCL genomic DNA using primers PS003 and PS004, specific for LMP2 sequences in the EcoRI–SalI region which is deleted from recombinant virus EBfaV-GFP. PCR product from PS003 and PS004 is 546 bp in size. Shown are representative LCLs transformed either by recombinant EBfaV-GFP virus or by wild-type (B95-8) EBV present in the mixture of virus used to infect primary B cells. LCLs derived from recombinant virus are BG10-F10, SF10-D3, LL2-B5, LL2-G10 and LL2-G3, all of which express EGFP and by PCR are here shown to lack LMP2. LCLs derived from wild-type EBV are BG10-D5, RS2-D4, PS2-A8, LL2-C2 and LL2-C9, none of which express EGFP. Genomic DNA from EBfaV-GFP cells contains DNA from wild-type and recombinant viruses. BJAB is a negative control containing DNA from the EBV-negative BJAB cells. Positive control for PCR is DNA from B95-8 cells. PO indicates control with primers only and no cellular DNA. (b) Efficacy of PCR reactions shown in (a) above is demonstrated by inclusion in the same reaction tube of control primers BHRF1-C and BHRF1-D which amplify a 239 bp DNA product from the BHRF1 region of EBV. The presence of comparable amounts of the control PCR product confirms the efficacy of the polymerase reaction in each of the amplifications shown in (a) above. (c) To determine sensitivity of detection of wild-type EBV DNA using LMP2 primers PS003 and PS004, dilutions of genomic DNA from wild-type-infected LCLs (diluted with DNA from BJAB cells) were added to a constant amount of genomic DNA from LMP2- LCLs and amplified as above using LMP2 primers. Results indicate that a single wild-type EBV genome is readily detected in a background of 10–100 mutant EBV genomes.

 
Southern hybridization confirmed that neomycin-resistance and EGFP genes were inserted into the viral genome by homologous recombination. DNA from cells bearing wild-type EBV and from cells of clone EBfaV-GFP was BamHI-digested, blotted and hybridized to a radiolabelled probe for EGFP (Fig. 1a, line 5). Fig. 1 shows the diagnostic 6·5 kbp band (Fig. 1a, line 4 and b, lane 1) resulting from fusion of EGFP and neomycin-resistance genes with the EBV genome in EBfaV-GFP cells. Southern hybridization also utilized a 2862 bp probe for the EBV genome (Fig. 1a, line 6) which hybridizes to BamHI fragments of predicted size 9622 bp (Fig. 1c, lane 6) in wild-type EBV and 6506 bp (Fig. 1c, lane 4) in EBfaV-GFP. Bands of both these sizes were present in DNA from cells of clone EBfaV-GFP (Fig. 1c, lane 4), confirming the presence of a mixture of wild-type and recombinant virus. Densitometric analysis of these bands, in duplicate on separate blots, showed 82·4% of EBV genomes present in EBfaV-GFP stocks to be wild-type and 17·6% recombinant, and phosphoimage scanning confirmed these proportions (data not shown).

Dilution of the EBfaV-GFP virus stock and measurement of the number of EGFP-positive cells after infection yielded a linear relationship, with a maximum of 30–45% of cells being positive. Fig. 2(c) shows infected Daudi cells viewed by phase-contrast illumination (panel 1), the same field by fluorescence (panel 2) and panel 3 shows approximately 30% of Daudi cells expressing EGFP, as measured by flow cytometry.

Characterization of LCLs derived from infection with mixed EBfaV-GFP/wild-type EBV
To quantify a potential phenotype of LMP2 in efficiency of immortalization of B cells, virus stock EBfaV-GFP, containing wild-type EBV and EGFP+LMP2- recombinant virus in proportions 82·4:17·6, was used to infect primary B cells from healthy donors. The rationale of this experiment was that if LMP2 has no effect on efficiency of immortalization of B cells, the ratio of wild-type to recombinant genotypes of resulting LCLs should resemble the ratio in the infecting virus. Infected cells were placed in 96-well plates at 105 cells per well and incubated at 37 °C, 5% CO2 until clones emerged, (4–5 weeks) then transferred to 24-well plates. Fewer than 10 clones grew on each 96-well plate. After further growth, cells were harvested and subjected to PCR analysis of the EBV genomes present. LMP2 primers PS003 and PS004 amplify a 546 bp fragment. As a control for efficacy of PCR, primers detecting EBV gene BHRF1 yielding a 239 bp band were included in each reaction. A total of 94 LCLs were evaluated for EGFP expression and genome structure validated by PCR, with Fig. 3(a) showing representative PCR results. All PCR reactions were duplicated on different days. Fig. 3(b), showing EBV BHRF1 sequences in representative samples, confirms efficacy of PCR. To assess sensitivity of detection of wild-type EBV DNA with primers PS003 and PS004, dilutions of DNA from 105 wild-type-infected LCLs in DNA from EBV-negative BJAB cells were added to DNA from 105 LMP2- LCLs (Fig. 3c). Primers PS003 and PS004 yielded a detectable amplification product with DNA diluted to 1:100 (Fig. 3c). These results demonstrate that a single wild-type EBV genome is readily detected in a background of 10–100 mutant genomes.

Of 94 LCLs established by infection with EBfaV-GFP, PCR using LMP2 primers showed that wild-type EBV sequences were present in 83 (88·3%), and that in 11 LCLs (11·7%) LMP2 was deleted (Table 1), each of which expressed EGFP. Southern hybridization was performed to verify the LMP2 mutation and the absence of wild-type LMP2 in the pure recombinant infected cells. Using representative mutant- and wild-type-infected LCLs, with probe specific for LMP2, only the 6·5 kbp fragment was detected, characteristic of insertion of the EGFP–neomycin resistance cassette and deletion of LMP2 sequences (data not shown). Even on long exposure, no 9·6 kbp fragment characteristic of wild-type EBV was detected. Absence of LMP2 protein expression or any truncation product was confirmed by immunofluorescence. As expected LMP2A expression was not detected in newly derived LMP2-deleted LCLs, or the previously derived LMP2 deletion mutant LCLs (Longnecker et al., 1993b ), whereas it was readily detected in wild-type-infected control LCLs (data not shown). Taken together with the results from PCR showing that a single copy of wild-type EBV is detectable in a background of 100 mutant genomes, these data confirm the absence of wild-type EBV genomes in LMP2- mutant LCLs. LMP2-deleted LCLs not expressing EGFP did not arise. Interestingly, 25 of 94 LCLs (26·6%) showed a mixed infection (Table 1) with wild-type EBV infection and EGFP expression in the same cell line. The low multiplicity at which primary B cells were infected (<=1 transforming unit of virus/106 B cells) and the high rate at which LCLs were co-infected prompted us to examine EBfaV-GFP virus stocks by electron microscopy to determine whether appreciable numbers of virions contained multiple nucleocapsids, as it has been reported that EBV virion envelopes may contain multiple capsids (Hummeler et al., 1966 ; Toplin & Schidlovsky, 1966 ).


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Table 1. Genotypes of LCLs resulting from infection of primary B cells with virus EBfaV-GFP and growth in 96-well plates

 
Stocks of virus EBfaV-GFP were on two separate occasions centrifuged onto electron microscope grids and examined using a JEOL 1220 electron microscope. The number of nucleocapsids present within a virion was scored only when the virion envelope and its contents were clearly discernible. Empty particles were ignored. A total of 245 virions were scored for the presence of multiple nucleocapsids (data not shown). Similar results were obtained from both experiments. The presence of multiple nucleocapsids in virions was frequently observed, consistent with the high frequency of apparently co-infected LCLs resulting from infection with EBfaV-GFP. The frequency of single nucleocapsids was 116/245 (47·3%), while two nucleocapsids per envelope were observed in 100/245 cases (40·8%). Three nucleocapsids per envelope were observed in 29/245 cases (11·8%).

Theoretical outcomes of infection in terms of wild-type or recombinant genomes delivered by a single virion from virus EBfaV-GFP, based on proportions of wild-type and recombinant EBV genomes and numbers of nucleocapsids per envelope, are calculated in Table 2. In summary the data shows that 73·3% of virions contain pure wild-type EBV DNA, 9·6% of virions contain pure recombinant LMP2-EGFP+ EBV DNA, and 16·9% of virions contain a mixture of wild-type and recombinant viral DNA. These numbers agree well with the actual results of 61·7%, 11·7% and 26·6%, respectively (Table 1).


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Table 2. Theoretical virion compositions

 
Growth in soft agarose of LCLs derived from infection with mixed EBfaV-GFP/wild-type EBV
To address the possibility that growth of B cells immortalized by LMP2-deficient virus is different in soft agarose from that of B cells in tissue culture medium, we repeated, using soft agarose suspension, the experiments in which infection of primary B cells with virus EBfaV-GFP generated LCLs. Primary B cells from healthy donors were infected with EBfaV-GFP, mixed with tissue culture medium and soft agarose, and plated over previously irradiated feeder layers of adherent cells. Multiplicity of infection was such that 3–10 clones developed per 107 infected cells or 96-well plate. Approximately 6 weeks after infection, the agarose contents of wells with clones was removed and by vigorous pipetting dispersed into a larger volume of medium in a well of a 24-well plate, after which cells were grown for another 5–7 days before isolation of DNA for PCR. Table 3 shows percentages of clones that expressed EGFP and which were deleted for LMP2. Of the 49 clones that developed, 11 (22·4%) expressed EGFP and of these 4 (8·2% of total clones) were deleted for LMP2 as shown by PCR (Fig. 4). Thus, 77·6% were infected with wild-type EBV, 8·1% were infected with LMP2 mutant virus only and 14·3% were co-infected with wild-type and LMP2 mutant virus (Table 3). Again this agrees well with the predicted values of 73·3%, 9·6% and 16·9%, respectively.


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Table 3. Genotypes of LCLs resulting from infection of primary B cells with virus EBfaV-GFP and growth in soft agarose in 96-well plates

 


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Fig. 4. PCR analysis of LCLs derived in soft agarose by immortalization of primary B cells using recombinant virus EBfaV-GFP and wild-type EBV. (a) Ethidium bromide-stained PCR products resulting from amplification of LCL genomic DNA using primers PS003 and PS004, specific for LMP2 sequences in the EcoRI–SalI region which is deleted from recombinant virus EBfaV-GFP. PCR product from PS003 and PS004 is 546 bp in size. Shown are representative LCLs transformed either by recombinant EBfaV-GFP virus or by wild-type (B95-8) EBV. LCLs derived from recombinant virus are 1BQ, 1AL, 2C CC and 11.1, all of which express EGFP and by PCR are here shown to be deleted for LMP2. Examples of LCLs resulting from mixed infection with wild-type EBV and EBfaV-GFP are 1AG and 2CY, both of which express EGFP and contain LMP2 sequences. Positive control for PCR is genomic DNA from B95-8 cells. DNA from LCL BG10-F10, infected with EBfaV-GFP alone, is a control for the presence of BHRF1 sequences and the absence of LMP2. PO indicates primers only and no cellular DNA. (b) Efficacy of PCR reactions shown in (a) above is demonstrated by inclusion in the same reaction tube of control primers BHRF1-C and BHRF1-D, which amplify a 239 bp DNA product from the BHRF1 region of EBV. The presence of comparable amounts of the control PCR product confirm efficacy of the reaction in each amplification shown in (a) above.

 
Growth of LMP2-deleted LCLs and wild-type-infected LCLs following initial outgrowth
As the readout for LCL development is the appearance of a visible clone, we examined post-immortalization growth of LMP2-deleted LCLs and wild-type EBV LCLs, as the LCLs expanded to larger cultures, to ensure there was no difference in growth rates. Newly transformed mutant and wild-type control LCLs, and established wild-type and LMP2-deleted LCLs, were plated at cell concentrations of 105, 2·5x104 or 5x103 cells per well and in medium supplemented with 10%, 5%, 2%, 1%, 0·1% or 0% foetal calf serum. Growth was assayed at days 5 and 10 (data not shown) by microscopic and macroscopic examination and by pH of the medium. There was no difference in growth of mutant- and wild-type-infected LCLs when plated at low cell density or in medium supplemented with reduced amounts of serum.


   Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
This study shows that EBV immortalization of primary B cells occurs efficiently without LMP2. It is established that LMP2 is not absolutely required for this process (Kim & Yates, 1993 ; Longnecker et al., 1992 , 1993a , b ); however, whether LMP2 influences efficiency of transformation has remained unresolved. While previous studies (Longnecker et al., 1992 , 1993a ) implied that LMP2-deleted EBV transformed B cells with efficiency similar to wild-type EBV, these studies used indirect evidence. In studies by Longnecker and colleagues, wild-type and LMP2- LCLs were induced to lytic replication and similar expression of lytic antigen gp350/220 indicated release of similar amounts of virus, which was used to infect and immortalize primary B cells. LMP2-deleted mutant and wild-type EBV were similar in number of wells positive for LCL outgrowth, in time to first macroscopic outgrowth, and in growth characteristics of the LCLs as they expanded to larger cultures. Similar studies with the same general conclusions were performed by Kim & Yates (1993) .

While previous studies indicate that LMP2 does not affect efficiency of B cell transformation, they do not constitute formal analysis of this efficiency. The recent report by Brielmeier et al. (1996) suggests that LMP2 greatly influences the efficiency of this process. In their study with EBV mini-plasmids to immortalize primary B cells, the LMP2-deleted mini-plasmid yielded 52 clones, of which 50 showed the presence of co-infecting helper virus. The remaining two carried a wild-type LMP2 and mutant allele in the EBV mini-plasmid, which was packaged as a dimer, indicating recombination of the mini-EBV genome with a wild-type genome restoring the LMP2 mutation and which could have restored additional second-site mutations. Thus Brielmeier and colleagues could not demonstrate B cell immortalization by EBV mini-plasmids bearing pure LMP2-deleted genomes. In contrast, wild-type LMP2 mini-plasmids readily yielded LCLs. This study showed that loss of LMP2 causes an extreme difference in immortalization efficiency, with complete inability of the LMP2-deleted mini-plasmid to immortalize B cells, contrasting with earlier findings that LMP2 is not absolutely required for B cell immortalization. Here we report an alternative methodology in which a mixture of wild-type EBV and LMP2-deleted EBV was used to immortalize primary B cells. Measurement of the ratio of wild-type to LMP2-deleted virus in the inoculum and comparison with that ratio in resulting LCLs permits careful determination of any phenotype for LMP2 in B cell immortalization efficiency. If LMP2 does not affect immortalization efficiency, the ratio of wild-type to recombinant genomes in the input virus together with counts of viral envelopes with one, two or three nucleocapsids, predicts that pure wild-type EBV LCLs, pure recombinant LCLs and LCLs with mixed infection will make up 73·3%, 9·6% and 16·9%, respectively, of the products of immortalization.

In this study infection of primary B cells with this mixture of wild-type and LMP2-deleted virus and subsequent growth of these cells in liquid tissue culture medium yielded pure wild-type EBV LCLs, pure recombinant LCLs and LCLs with mixed infection in the proportions 61·7%, 11·7% and 26·6%, respectively (Table 1), which are indistinguishable from the predicted result.

To address the possibility that the anchorage-independent culture of primary B cells in soft agarose might reveal a phenotype for LMP2 that is too subtle to be observed in liquid tissue culture conditions, and to duplicate conditions used by Brielmeier and colleagues, immortalization of primary B cells was repeated using soft agarose. Pure wild-type EBV LCLs, pure recombinant LCLs and LCLs with mixed infection arose in proportions 77·6%, 8·2% and 22·4%, respectively, again resembling the predicted result. The similar rates of post-immortalization outgrowth of newly transformed wild-type and LMP2-deleted LCLs confirm previous results (Longnecker et al., 1993a , b ) and show that macroscopic detection of LCLs is unaffected by the absence of LMP2.

Several explanations may account for differences between our findings and those of Brielmeier and colleagues. First, genetic manipulations involved in production of the EBV mini-plasmid used in their studies may have introduced mutations into other EBV genes important for immortalization. A simple marker rescue of the LMP2 mutation should exclude this possibility. Second, orientation of the selectable marker in the system of Brielmeier and colleagues is the opposite to that used here, suggesting a downstream transcriptional effect on other genes, such as LMP1, required for B cell immortalization (Kaye et al., 1993 ). A third possibility which logically cannot be excluded is that redundancy exists between LMP2 and an unknown EBV gene product not encoded in the mini-plasmid, such that immortalization efficiency is unaffected by deletion of either of these components but diminished in the absence of both.

Of the three explanations for the discrepant results, the first arguing for incorporation of another mutation into the LMP2-deleted mini-EBV seems the most plausible. In fact, in a previous publication from the Hammerschmidt laboratory (Kempkes et al., 1995b ), the appearance of an unintended mutation occurred in the EBNA3A gene which adversely affected immortalization of primary B cells, making the addition of a second-site mutation a real possibility.

The careful analysis of LMP2 function in B cell immortalization conducted here shows that LMP2 has no role in B cell immortalization by EBV using standard cell culture techniques. Interestingly, a recent report by Caldwell et al. (1998) , shows in some situations LMP2 expression affects cell survival of B cells in LMP2A transgenic mice. In that system, LMP2A was the only viral gene expressed in the murine B cells. In tissue culture, there are nine additional proteins expressed in EBV-infected lymphocytes which may be dominant over potential LMP2A functions. Cell culture systems able to separate LMP2A function from other viral genes may allow identification of specific LMP2 functions, but in the context of primary B cell immortalization by the entire EBV genome, it is clear that LMP2 has no role.


   Acknowledgments
 
R.L. is supported by Public Health Service grants CA62234 and CA73507 from the National Cancer Institute. R.L. is a Scholar of the Leukaemia Society of America.


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Received 6 January 1999; accepted 24 March 1999.