Stable transfection of Epstein-Barr virus (EBV) nuclear antigen 2 in lymphoma cells containing the EBV P3HR1 genome induces expression of B-cell activation molecules CD21 and CD23.

A set of B-cell activation molecules, including the Epstein-Barr virus (EBV) receptor CR2 (CD21) and the B-cell activation antigen CD23 (Blast2/Fc epsilon RII), is turned on by infecting EBV-negative B-lymphoma cell lines with immortalizing strains of the viruslike B95-8 (BL/B95 cells). This up regulation may represent one of the mechanisms involved in EBV-mediated B-cell immortalization. The P3HR1 nonimmortalizing strain of the virus, which is deleted for the entire Epstein-Barr nuclear antigen 2 (EBNA2) protein open reading frame, is incapable of inducing the expression of CR2 and CD23, suggesting a crucial role for EBNA2 in the activation of these molecules. In addition, lymphoma cells containing the P3HR1 genome (BL/P3HR1 cells) do not express the viral latent membrane protein (LMP), which is regularly expressed in cells infected with immortalizing viral strains. Using electroporation, we have transfected the EBNA2 gene cloned in an episomal vector into BL/P3HR1 cells and have obtained cell clones that stably express the EBNA2 protein. In these clones, EBNA2 expression was associated with an increased amount of CR2 and CD23 steady-state RNAs. Of the three species of CD23 mRNAs described, the Fc epsilon RIIa species was preferentially expressed in these EBNA2-expressing clones. An increased cell surface expression of CR2 but not of CD23 was observed, and the soluble form of CD23 molecule (SCD23) was released. We were, however, not able to detect any expression of LMP in these cell clones. These data demonstrate that EBNA2 gene is able to complement P3HR1 virus latent functions to induce the activation of CR2 and CD23 expression, and they emphasize the role of EBNA2 protein in the modulation of cellular gene implicated in B-cell proliferation and hence in EBV-mediated B-cell immortalization. Nevertheless, EBNA2 expression in BL/P3HR1 cells is not able to restore the level of CR2 and CD23 expression observed in BL/B95 cells, suggesting that other cellular or viral proteins may also have an important role in the activation of these molecules: the viral LMP seems to be a good candidate.

Geographical prevalence of two types of Epstein-Barr virus.

The Jijoye EBV strain is characterized by a substitution of 1.8 kb in the C-terminal part of the EBNA 2 gene compared to B95-8 or M-ABA virus. This made it possible to construct hybridization probes specific for M-ABA (type A) and Jijoye viruses (type B), which have been used to type the EBV genomes in 38 spontaneously established cell lines. Type A is more prevalent being found in 31 of 38 cases; type B virus was found in five cell lines (Jijoye, LY 67, QIMR-GOR, BL 16, and BL 29); and two cell lines, Daudi and EB-3, contained neither the M-ABA- nor the Jijoye-specific sequences. EBV type B appears to be less ubiquitous, since all type B isolates, including AG 876 virus, originated from Central Africa, La Réunion, and New Guinea. All the other cell lines, carrying EBV type A, were established from patients from Central Africa (4), North Africa (7), New Guinea (1), and Asia (6) and from white individuals (13). The restricted geographical localization of EBV type B in parts of the southern hemisphere and its similarity to herpesvirus papio (T. Dambaugh, K. Hennessy, L. Chamnankit, and E. Kieff (1984) Proc. Natl. Acad. Sci. USA 81, 7632-7636) could suggest that such viruses may have evolved by recombination of EBV with a related Old World monkey virus, alternatively, evolution of virus variants within the human species also being conceivable.

A complete set of overlapping cosmid clones of M-ABA virus derived from nasopharyngeal carcinoma and its similarity to other Epstein-Barr virus isolates.

DNA of the transforming, nondefective Epstein-Barr virus (EBV) strain M-ABA, which is derived from nasopharyngeal carcinoma cells, was cloned as large overlapping pieces into the cosmid pHC79 . The termini were cloned from closed circular virus DNA molecules out of M-ABA cell DNA in phage lambda L47 . The large overlapping clones were used to prepare a library of subclones with inserts of 1-15 kb. A detailed restriction enzyme map of M-ABA virus DNA reveals the close similarity to isolates from other sources. The high number of tandem repeats in EBV DNA stresses the importance of using cloning vectors that can be propagated in recA- Escherichia coli hosts.

Two deletions in the Epstein-Barr virus genome of the Burkitt lymphoma nonproducer line Raji.

The Epstein-Barr virus genome carried in the Burkitt lymphoma nonproducer cell line Raji was characterized by partial denaturation mapping and by hybridization of cloned viral fragments to filters containing separated Raji DNA fragments. Partial denaturation mapping revealed that the EBV DNA population of Raji cells is homogeneous and that two deletions are observed in distant parts of the genome compared to linear DNA isolated from virus particles of different strains. These deletions were characterized by blot analysis. One deletion of 3.15 kb lies within HindIII-E; the second is 2.4 kb and is located close to the right terminus of linear viral DNA. The two deletions were observed in several cell lines derived from the Raji line. These deletions might contribute to the inability of Raji cells to produce EBV either spontaneously or upon induction.

Deletion of the nontransforming Epstein-Barr virus strain P3HR-1 causes fusion of the large internal repeat to the DSL region.

The nontransforming Epstein-Barr virus (EBV) strain P3HR-1 is known to have a deletion of sequences of the long unique region adjacent to the large internal repeats. The deleted region is believed to be required for initiation of transformation. To establish a more detailed map of the deletion in P3HR-1 virus, SalI-A of the transforming strain M-ABA and of P3HR-1 virus was cloned into the cosmid vector pHC79 and multiplied in Escherichia coli. The cleavage sites for BamHI, BglII, EcoRI, PstI, SacI, SacII, and XhoI were determined in the recombinant plasmid clones. Analysis of the boundary between large internal repeats and the long unique region showed that in M-ABA (EBV) the transition is different from that in B95-8 virus. The map established for SalI-A of P3HR-1 virus revealed that, in contrast to previous reports, the deletion has a size of 6.5 kilobase pairs. It involves the junction between large internal repeats and the long unique region and includes more than half of the rightmost large internal repeat. The site of the deletion in the long unique region is located between a SacI and a SacII site, about 200 base pairs apart from each other. The sequences neighboring the deletion in the long unique region showed homology to the nonrepeated sequences of the DS(R) (duplicated sequence, right) region. Sequences of the large internal repeat are thus fused to sequences of the DS(L) (duplicated sequence, left) region in P3HR-1 virus DNA under elimination of the DS(L) repeats. Jijoye, the parental Burkitt lymphoma cell line from which the P3HR-1 line is derived by single-cell cloning, is known to produce a transforming virus. Analysis of the Jijoye (EBV) genome with cloned M-ABA (EBV) probes specific for the sequences missing in P3HR-1 virus revealed that the sequences of M-ABA (EBV) BamHI-H2 are not represented in Jijoye (EBV). In Jijoye (EBV) the complete DS(L) region including the DS(L) repeats is, however, conserved. Further analysis of Jijoye (EBV) and of Jijoye virustransformed cell lines will be helpful to narrow down the region required for transformation.

Two distant regions of the Epstein-Barr virus genome with sequence homologies have the same orientation and involve small tandem repeats.

The two regions of the Epstein-Barr virus genome (DSL and DSR) carrying homologous sequences at distant parts of the long unique region are described. Cleavage of cloned DNA containing the DSR region with restriction endonucleases revealed a so far unrecognized small tandem repeat of approximately 120 base pairs present in approximately 20 copies. Heteroduplexes of the DNA of two clones containing DSL and DSR respectively, visualized in the electron microscope by cytochrome c spreading, revealed that the region of homology is approximately 2.5 kb long, involves small tandem repeats, and has the same orientation in the viral genome. Mica adsorption of the heteroduplex showed, that the homologous region consists of approximately 1.5 kb with only partial homology including the small internal repeats and 0.9 kb with well-matched duplexes. When DNA containing the DSL region reanneals, it can give rise to two single-stranded loops of the same size at different positions suggesting the presence of a row of tandem repeats also in this region.

Comparison of Epstein-Barr virus strains of different origin by analysis of the viral DNAs.

Epstein-Barr virus (EBV) originating from Burkitt's lymphoma (P3HR-1 and CC34-5), nasopharyngeal carcinoma (M-ABA), transfusion mononucleosis (B95-8), and a patient with acute myeloblastic leukemia (QIMR-WIL) was isolated from virus-carrying lymphoid cell lines after induction with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate. Viral DNA was analyzed by partial denaturation mapping and by use of the restriction endonucleases EcoRI, HindIII, and SalI and separation of fragments in 0.4% agarose. By using the restriction enzyme data of B95-8 (EBV) and W91 (EBV) obtained by Given and Kieff (D. Given and E. Kieff, J. Virol. 28:524-542, 1978), maps were established for the other virus strains. Comigrating fragments were assumed to be identical or closely related among the different strains. Fragments of different strains migrating differently were isolated, purified, radioactively labeled, and mapped by hybridization against blots of separated viral fragments. The results were as follows. (i) All strains studied were closely related. (ii) The number of internal repeats was variable among and within viral strains. (iii) B95-8 (EBV) was the only strain with a large deletion of about 12,000 base pairs at the right-hand side of the molecule. At the same site, small deletions of about 400 to 500 base pairs were observed in P3HR-1 (EBV) and M-ABA (EBV) DNA. (iv) P3HR-1 (EBV), the only nontransforming EBV strain, had a deletion of about 3,000 to 4,000 base pairs in the long unique region adjacent to the internal repeats carrying a HindIII site. (v) Small inserted sequences of 150 to 400 base pairs were observed in M-ABA (EBV) and B95-8 (EBV) at identical sites in the middle of the long unique region. (vi) Near this site, an insertion of about 1,000 base pairs was found in P3HR-1 (EBV) DNA. (vii) The cleavage patterns of P3HR-1 virus DNA and the results of blot hybridizations with P3HR-1 virus fragments are not conclusive and point to the possibility that in addition to the normal cleavage pattern some viral sequences may be arranged differently. Even though it is possible that small differences in the genome organization may have significant biological effects, the great similarity among different EBV strains does not favor the hypothesis that disease-specific subtypes exist.