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Journal of Virology, April 2005, p. 4506-4509, Vol. 79, No. 7
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.7.4506-4509.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Epstein-Barr Virus with the Latent Infection Nuclear Antigen 3B Completely Deleted Is Still Competent for B-Cell Growth Transformation In Vitro

Adrienne Chen, Matthew DiVisconte, Xiaoqun Jiang, Carol Quink, and Fred Wang*

Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts

Received 1 October 2004/ Accepted 23 November 2004


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ABSTRACT
 
The Epstein-Barr virus (EBV) nuclear antigen 3B (EBNA-3B) is considered nonessential for EBV-mediated B-cell growth transformation in vitro based on three virus isolates with EBNA-3B mutations. Two of these isolates could potentially express truncated EBNA-3B products, and, similarly, we now show that the third isolate, IB4, has a point mutation and in-frame deletion of 263 amino acids. In order to test whether a virus with EBNA-3B completely deleted can immortalize B-cell growth, we first cloned the EBV genome as a bacterial artificial chromosome (BAC) and showed that the BAC-derived virus was B-cell immortalization competent. Deletion of the entire EBNA-3B open reading frame from the EBV BAC had no adverse impact on growth of EBV-immortalized B cells, providing formal proof that EBNA-3B is not essential for EBV-mediated B-cell growth transformation in vitro.


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TEXT
 
Epstein-Barr virus (EBV) is an oncogenic herpesvirus associated with a variety of malignancies in T cells, B cells, and epithelial cells (17). Infection of primary B cells in vitro results in conversion of latently infected cells into immortalized lymphoblastoid cell lines (LCLs) and has been used as a model for how the virus might effect or contribute to malignant transformation in vivo. Following in vitro infection, a limited subset of viral gene products are expressed (six Epstein-Barr virus nuclear antigens [EBNA-1, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C, and EBNA-LP], three latent membrane proteins [LMP-1, LMP-2A, and LMP-2B], unpolyadenlyated RNAs EBER-1 and EBER-2, and BARTs [BamHI-A rightward transcripts]). However, only four of these gene products are considered essential for growth transformation (1, 8, 9, 11, 13, 15, 18, 20, 21, 23).

EBNA-3B is thought to be dispensable for EBV-mediated B-cell growth transformation in vitro. Three lines of evidence support this hypothesis. First, recombinant viruses encoding an EBNA-3B protein with a stop codon inserted after amino acid 109 were indistinguishable from wild-type recombinant viruses in their ability to infect and transform primary B cells in vitro (22). It is not known whether a truncated 109-amino-acid EBNA-3B product was expressed in those cells. Second, a naturally occurring EBV variant characterized from a patient with lymphoproliferative disease was found to have a 245-nucleotide deletion in EBNA-3B, resulting in the potential expression of the first 364 amino acids of EBNA-3B followed by 62 out-of-frame amino acids (6). Third, EBNA-3B expression was not detected in IB4, a cell line derived from B95-8 EBV-immortalized umbilical cord B cells (12), but the genetic lesion responsible for the loss of protein expression is unknown (16). We sequenced the EBNA-3B open reading frame (ORF) from multiple, overlapping DNA clones derived by PCR amplification of IB4 genomic DNA. There was an in-frame deletion of amino acids 336 to 598 and a point mutation resulting in a glutamic acid-to-lysine change at codon 330 relative to the wild-type B95-8 EBNA-3B sequence (Fig. 1B). Therefore, none of three mutations completely eliminated potential EBNA-3B expression (Fig. 1A), leaving open the possibility that truncated EBNA-3B products may be contributing to EBV-mediated B-cell immortalization in these instances.



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  		FIG. 1. EBNA-3B mutation in IB4 and other EBNA-3B mutants. (A) Schematic diagram of known EBNA-3B mutants. These include the genetically engineered mutant reported by Tomkinson and Kieff (22), the spontaneously occurring mutant from a patient with lymphoproliferative disease reported by Gottschalk et al. (6), and the spontaneously occurring IB4 mutant from a B95-8-transformed cell line. Amino acid residue numbers relative to the wild-type B95-8 EBNA-3B sequence are shown. Residue numbering of the Gottschalk et al. mutant differs from that reported by those authors (6) and has been corrected to correspond with the predicted amino acid sequence for the cDNA. A 245-nucleotide deletion in the Gottschalk et al. mutant causes a frameshift, resulting in a unique sequence of 62 amino acids (hatched box) and a premature stop codon. (B) Predicted amino acid sequence of IB4 EBNA-3B showing a deletion from amino acids 336 to 598 and a point mutation at codon 330 (*). The amino- and carboxy-terminal coding sequences not shown in the figure are identical between IB4 and B95-8 EBNA-3B.

In order to formally demonstrate that EBNA-3B is not essential for EBV-mediated B-cell growth transformation in vitro, we set out to delete the entire EBNA-3B ORF from the virus. To facilitate the generation of recombinant viruses, the EBV genome was first cloned as a bacterial artificial chromosome (BAC). F-plasmid sequences for prokaryotic replication (19), a chloramphenicol resistance marker for prokaryotic selection, and a cytomegalovirus promoter-driven puromycin resistance cassette for eukaryotic selection were inserted into the SnaBI site of a plasmid containing EBV BamHI W DNA and then transfected into B95-8 cells. Puromycin-resistant cells were screened for homologous recombination of F-plasmid sequences into EBV episomes by Southern blotting of cell DNA separated by gel electrophoresis as described by Gardella et al. (5) and hybridization with an F-plasmid DNA probe. Hirt DNA was prepared from clones positive for F plasmid-containing episomes, transformed into Escherichia coli strain DH10B, and chloramphenicol-resistant bacterial colonies were screened. Restriction analysis of BAC DNA from clone 2-6 demonstrated the expected digestion patterns for B95-8 EBV DNA using several different enzymes, suggesting that this clone contained an intact EBV genome with an inserted F plasmid (Fig. 2).



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  		FIG. 2. Restriction digests of EBV BAC clone 2-6 and analysis by ethidium bromide staining after agarose gel electrophoresis. The predicted EBV DNA fragments from a circular genome based on the B95-8 GenBank sequence are shown in the table, and DNA fragments after gel electrophoresis are labeled to the left of each band. Fragments labeled with an asterisk represent F-plasmid vector fragments or EBV DNA fragments altered by insertion of the F-plasmid sequences. The F plasmid was targeted to the major internal repeat. Since the F plasmid contains at least one HindIII, SalI, EcoRI, and BamHI site, the EBV HindIII A, SalI A, and EcoRI A fragments spanning the major internal repeat are interrupted (A*) and additional fragments representing internal F-plasmid DNA or fusion fragments with EBV DNA are present (*). Normal BamHI W fragments are present since the F plasmid is inserted into only one of multiple copies resulting in one large fusion fragment (~8.3 kb) and one smaller fusion fragment not present on this gel.

To test whether this EBV BAC clone was competent for viral replication and B-cell immortalization, the EBV BAC was replicated in 293T cells (3). In order to facilitate transfection of the large BAC DNA, we used a diaminopimelate-dependent E. coli strain (BM2710) that coexpressed the invasin gene from Yersinia pseudotuberculosis and the listeriolysin O gene from Listeria monocytogenes to allow the transfer of recombinant DNA into mammalian cells after simple coincubation (7). Since this E. coli strain was already chloramphenicol resistant, we used lambda phage Red recombinase-mediated homologous recombination to replace the chloramphenicol resistance gene in EBV BAC clone 2-6 with a kanamycin resistance gene (2) to create the EBV BAC clone MD1. The MD1 BAC was then transformed into BM2710 cells, and kanamycin-resistant bacteria were incubated with 293T cells as described elsewhere (7). Analysis of puromycin-resistant 293T cells by gel electrophoresis as described by Gardella et al. (5) and Southern blotting revealed a much higher frequency of 293T clones containing episomal BAC DNA after BM2710-mediated DNA transfer than after lipofection (data not shown). Episome-positive 293T cell clones were induced to produce virus by treatment with medium containing 20 ng of phorbol 12-myristate 13-acetate per ml and 3 mM n-butyric acid followed by infection with an rhBZLF-1-expressing recombinant adenovirus made replication incompetent by UV inactivation. Western blotting of induced cells showed that the inactivated adenovirus expressed high levels of rhBZLF-1 protein (data not shown). Monitoring of viral replication in induced 293T cells by staining for gp350 cell surface expression typically showed 5 to 40% gp350-positive cells after 4 days. Cell-free viral supernatants were used to infect human peripheral blood mononuclear cells in 96-well plates in the presence of cyclosporine. The frequency of LCL outgrowth correlated with the percent induction of gp350-positive 293T cells, and the growth rate of MD1 BAC-derived LCLs was comparable to that of LCLs immortalized with B95-8-derived virus. MD1-derived LCLs continued to grow in the presence of puromycin, and BAC DNA was recovered by transformation of Hirt DNA into E. coli (data not shown), confirming that the BAC-derived virus was able to immortalize peripheral blood B cells. MD1 BAC-derived LCLs expressed the EBV latent proteins to the same levels as wild-type-virus-infected LCLs (Fig. 3A), demonstrating that a significant amount of heterologous DNA can be inserted into the EBV major internal repeat without adversely affecting latent gene expression or B-cell growth transformation.



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  		FIG. 3. Latent infection gene expression and growth of LCLs derived from wild-type MD1 or EBNA-3B-deleted BACs. (A) Latent protein expression in wild-type MD1 BAC-derived LCLs and EBNA-3B-deleted BAC-derived LCLs. Whole-cell lysates were analyzed by Western blotting for expression of EBV latent proteins. EBV-immune human serum was used for detection of EBNA-1, -2, -3A, -3B, and -3C; EBNA-LP was detected with the JF186 monoclonal antibody (4); and LMP-1 was detected with the S12 monoclonal antibody (14). BJAB is an EBV-negative Burkitt's lymphoma line, B95-8 is an LCL infected with B95-8 virus, and MD1 is an LCL infected with the wild-type MD1 BAC-derived virus. Four independently derived EBNA-3B-deleted BAC-derived LCLs are shown. (B) Growth curves for wild-type MD1 BAC-derived LCLs and EBNA-3B-deleted BAC-derived LCLs. Wild-type MD1 ({blacksquare}) and two independent EBNA-3B-deleted BAC-derived LCLs (3B del 2A L4 [{blacktriangleup}] and 3B del 2.2F L11 [{circ}]) were seeded at an initial density of 4 x 105 cells per ml in a 24-well plate at day 0, and cell counts were taken over a period of 5 days. Data are means ± standard deviations for three replicates.

The EBNA-3B ORF was deleted from the wild-type MD1 BAC by using lambda phage Red-mediated recombination. Transient expression of lambda phage Red recombinase proteins in E. coli containing the MD1 BAC was induced from the temperature-sensitive pKD119 plasmid (2), and the EBNA-3B ORF was knocked out by homologous recombination and replacement with a chloramphenicol resistance marker. The EBNA-3B ORF was targeted by transfecting bacteria with PCR-amplified DNA containing 50 nucleotides of the EBV genome upstream of the EBNA-3B translational start site (EBV nucleotides 95,281 to 95,330), followed by a FLP recombinase target (FRT) site, the chloramphenicol resistance marker, another FRT site, and 50 nucleotides downstream of the EBNA-3B translational stop site (EBV nucleotides 98,248 to 98,297). Recombinants were screened for chloramphenicol resistance, and replacement of the EBNA-3B coding region with the chloramphenicol resistance marker and FRT sites was confirmed by restriction digestion and Southern blot analysis. The chloramphenicol resistance marker was removed after expression of the FLP recombinase encoded by the pCP20 plasmid, leaving a single 84-bp FRT scar sequence in place of EBNA-3B (2). The removal of both EBNA-3B and the chloramphenicol resistance gene was confirmed by sequencing of PCR products across the targeted recombination site with primers in EBNA-3A and EBNA-3C.

Virus was generated from the BAC lacking the entire EBNA-3B ORF (EBNA-3B)-deleted BAC as described above. MD1 and EBNA-3B-deleted virus supernatants were obtained from BAC-containing 293T cells undergoing comparable levels of lytic replication as measured by gp350 cell surface staining and were used to infect human peripheral blood mononuclear cells in 96-well plates. A comparable number of wells containing immortalized B cells were obtained from MD1 and EBNA-3B-deleted-virus supernatants. MD1 and EBNA-3B-deleted LCLs expanded from 96-well to 24-well plates and larger flasks at similar rates, and there was no apparent difference in the growth rates of EBNA-3B-deleted versus wild-type BAC-derived LCLs (Fig. 3B). PCR amplification of cell DNA confirmed the deletion of the EBNA-3B ORF and replacement with the scar sequence in the EBNA-3B-deleted LCLs (data not shown). Immunoblotting of the EBNA-3B-deleted LCLs showed wild-type LMP1 and EBNA expression levels except for the absence of EBNA-3B (Fig. 3A). These results show that EBNA-3B is not essential for EBV-induced B-cell growth transformation in vitro. Small differences in B-cell immortalization efficiency cannot be ruled out by these semiquantitative assays.

This study shows for the first time that all of EBNA-3B can be deleted without significantly compromising EBV-induced B-cell growth in vitro. The technical approach used for previous genetic analyses could not exclude the possibility that a truncated EBNA-3B product was expressed by the recombinant viruses (22). In addition, we show that the naturally occurring EBNA-3B mutant, IB4, could potentially express a truncated EBNA-3B product. The removal of the complete EBNA-3B ORF from an EBV BAC eliminates the possibility that some portion of EBNA-3B is still required for B-cell immortalization.

In this study, we also isolated a new EBV BAC clone competent for virus replication and B-cell immortalization. Two other EBV BACs were cloned and characterized previously (3, 10). Our EBV BAC differs from these by the insertion of the F-plasmid sequences into the major internal repeat region. These experiments demonstrate that the major internal repeat can accommodate a significant amount of heterologous DNA without adverse impact on EBV latent gene expression or B-cell growth transformation. Notably, the normal expression of EBNA-LP and other EBNAs suggests that the foreign DNA sequences in the major internal repeat do not interfere with the mRNA splicing required to assemble the leader protein ORF in bicistronic transcripts with other EBNAs. The nonessential role for EBNA-3B in vitro suggests that EBNA-3B may be essential for EBV infection in vivo. Development of a similar BAC-based genetic system for the rhesus lymphocryptovirus would allow this hypothesis to be directly tested in the rhesus animal model system.


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ACKNOWLEDGMENTS
 
This work was supported by grants from the U.S. Public Health Service (CA68051 and DE14388).

We thank Greg Smith and Lynn Enquist for kindly providing assistance and the original F plasmids from which our constructs were derived, Catherine Grillot-Courvalin for kindly providing the BM2710 strain, Barry Wanner for kindly providing the lambda Red recombinase and FLP recombinase plasmids, and Eric Rubin for discussion and assistance.


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FOOTNOTES
 
* Corresponding author. Mailing address: Channing Laboratory, 181 Longwood Ave., Boston, MA 02115. Phone: (617) 525-4258. Fax: (617) 525-4257. E-mail: fwang{at}rics.bwh.harvard.edu. Back


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Journal of Virology, April 2005, p. 4506-4509, Vol. 79, No. 7
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.7.4506-4509.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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