U2 region of Epstein-Barr virus DNA may encode Epstein-Barr nuclear antigen 2.

Abstract

(5) or integrated DNA (6). Expression of the viral genome is limited (7-10). However, at least three sites on the viral genome are characteristically transcribed. Three transcripts-from IR1 into U2, from U3 through IR3 into U4, and from U5-are each spliced into a cytoplasmic polyadenylylated RNA that is presumed to encode protein (11)(12)(13)(14)(15). The U3-IR3-U4 RNA is known to encode an Epstein-Barr nuclear antigen (EBNA) (EBNA1), and the U5 RNA encodes a membrane protein (12,14,(16)(17)(18)47).No protein has been assigned to the IR1-U2 RNA. This RNA is initiated by a promoter in any of the copies of IR1, transcribed through subsequent copies of IR1, terminated in U2, polyadenylylated, and spliced into a 3-kb cytoplasmic RNA (11,13). Hybridizations with the separated DNA strands of IR1 and U2 indicate that 5' exons of the RNA must be encoded by IR1 (13). These putative 5' exons have not been identified, however. Furthermore, the principal open reading frame in IR1, identified by DNA sequence analysis (19), does not hybridize to the 3-kb cytoplasmic RNA, suggesting a complex splicing program for the 5' exons. The principal 3' exons have been mapped to contiguous DNA fragments near the center of the U2 domain (13). The importance of this gene in growth transformation is suggested by the finding that a nontransforming EBV isolate, P3HR-1 (20), has undergone a deletion of the U2 region (21-24). Other reportedly transformation-competent isolates are, however, known to vary in their central U2 sequence (22).An EBNA-positive human serum that reacts poorly with the EBNA1 protein detected a second nuclear protein, designated EBNA2, in nuclei of each of five latently infected cell lines that were tested (16). EBNA2 was absent from noninfected continuous B-cell lines (16). The EBNA2 protein is 85 kDa, while EBNA1 varies in size from 68 to 85 kDa, depending on the length of the EBV IR3 repeat in each EBV isolate (14, 16). Antisera raised in rabbits against EBNA1-lacZ or EBV membrane-lacZ fusion proteins react with either EBNA1 or the membrane protein (16, 47) and not with EBNA2, indicating that EBNA2 is not encoded by the U3-IR3-U4 or U5 RNA.We now report the sequence of the U2 domain of the B95-8 and AG876 isolates of EBV. The B95-8 U2 DNA sequence reveals a 1545-base-pair (bp) open reading frame. The U2 domains of some isolates such as W91, Raji, Lamont, and Cherry are quite closely related to the B95-8 U2, while others such as AG876 and Jijoye are divergent from B95-8 U2 (22). These nucleotide sequence differences among EBV isolates correlate with differences in their EBNA2 proteins, indicating that the U2 open reading frame encodes EBNA2. MATERIALS AND METHODSCell Cultures and Viral DNAs. Viral DNA was purified from the supernatant virions of induced AG876 (26), or HVPapio (27) cultures or from cells of Jijoye (20) or HVPan (28) cultures by isopycnic centrifugation (21).Electrophoretic Blotting. Nuclei were prepared by resuspending washed cells in 2% sucrose/10 mM Tris-HCI, pH 7.5/10 mM NaCI/3 mM Mg(OA...

Immortalization of Epstein--Barr virus-infected CD23-negative B lymphocytes by the addition of B cell growth factor

“…These cells may be at a point on the B cell differentiation pathway different to those directly susceptible to EB virusinduced proliferation and require the addition of BCGF to induce immortalization. Although EBNA 2 is necessary for immortalization (Dambaugh et al, 1984;Hammerschmidt & Sugden, 1989) these results clearly show it is not alone sufficient to cause immortalization of all B cells.…”

“…The functions of these proteins are for the most part unknown, but EBNA 1 is essential for maintenance of the viral episome (Yates et al, 1985). EBNA 2 and LMP are suggested to have a role in the immortalization process since EBNA 2 deletion mutants of EB virus are non-immortalizing (Dambaugh et al, 1984) and LMP expression induces a tumorigenic phenotype in Rat-1 cells (Wang et al, 1985). Immortalized B ceils also express cellular activation antigens consistent with their lymphoblastoid stage of differentiation.…”

Appearance of a different clone of Epstein–Barr virus genome in recurrent tumor of pyothorax-associated lymphoma (PAL) and a mini-review of PAL

“…22,23 Primer sequences were 5′-CCACCAGCAGCACCAGCACA-3′ and 5′-GGTGGCCACCATGGTGGCCC-3′. 23 PCR was performed, and amplified products were electrophoresed in 2% agarose gels, and Southern blotted as previously described.…”

Identification of type A and B isolates of Epstein-Barr virus by polymerase chain reaction

“…Specific primers were synthesized based on the published DNA sequences of Dambaugh et al (1984) (for EBNA 2A and 2B genes), and of Baer et al (1984) (for the BamZ and BamR regions).…”