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. 2005 Apr;79(7):4506-9.
doi: 10.1128/JVI.79.7.4506-4509.2005.

Epstein-Barr virus with the latent infection nuclear antigen 3B completely deleted is still competent for B-cell growth transformation in vitro

Adrienne Chen  1 , Matthew Divisconte, Xiaoqun Jiang, Carol Quink, Fred Wang
Affiliations

Epstein-Barr virus with the latent infection nuclear antigen 3B completely deleted is still competent for B-cell growth transformation in vitro

Adrienne Chen et al. J Virol. 2005 Apr.

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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Figures

FIG. 1.
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.
FIG. 2.
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.
FIG. 3.
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 (▪) and two independent EBNA-3B-deleted BAC-derived LCLs (3B del 2A L4 [▴] and 3B del 2.2F L11 [○]) were seeded at an initial density of 4 × 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.
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References

    1. Cohen, J. I., F. Wang, J. Mannick, and E. Kieff. 1989. Epstein-Barr virus nuclear protein 2 is a key determinant of lymphocyte transformation. Proc. Natl. Acad. Sci. USA 86:9558-9562. - PMC - PubMed
    1. Datsenko, K. A., and B. L. Wanner. 2000. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA 97:6640-6645. - PMC - PubMed
    1. Delecluse, H. J., T. Hilsendegen, D. Pich, R. Zeidler, and W. Hammerschmidt. 1998. Propagation and recovery of intact, infectious Epstein-Barr virus from prokaryotic to human cells. Proc. Natl. Acad. Sci. USA 95:8245-8250. - PMC - PubMed
    1. Finke, J., M. Rowe, B. Kallin, I. Ernberg, A. Rosen, J. Dillner, and G. Klein. 1987. Monoclonal and polyclonal antibodies against Epstein-Barr virus nuclear antigen 5 (EBNA-5) detect multiple protein species in Burkitt's lymphoma and lymphoblastoid cell lines. J. Virol. 61:3870-3878. - PMC - PubMed
    1. Gardella, T., P. Medveczky, T. Sairenji, and C. Mulder. 1984. Detection of circular and linear herpesvirus DNA molecules in mammalian cells by gel electrophoresis. J. Virol. 50:248-254. - PMC - PubMed

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