Identification of Epstein-Barr virus sequences that encode a nuclear antigen expressed in latently infected lymphocytes.

Abstract

The Epstein-Barr virus (EBV) BamHI restriction endonuclease fragment K (B95-8 strain) was introduced into a polyoma virus expression vector and used to transfect murine NIH 3T3 cells. An EBV-associated nuclear antigen was detected in these cells in an indirect immunofluorescence test using anti-EBV nuclear antigen-positive human sera. These sera recognized a Mr 88,000 polypeptide in 3T3 cells transfected with the BamHI fragment K-containing polyoma virus plasmid by radioimmunoelectrophoresis. A Mr 88,000 polypeptide also was detected in a B-cell line latently infected with the B95-8 strain of EBV. Plasmids containing insertion and deletion mutations in BamHI fragment K directed the synthesis of truncated forms of the Mr 88,000 polypeptide in 3T3 cells. These data directly demonstrate that the polypeptide identified in EBV-infected lymphocyte lines by anti-EBV nuclear antigen-positive human sera is encoded by the viral genome.Infection of human B lymphocytes with Epstein-Barr virus (EBV) results in the establishment of permanent cell lines (1). These immortalized cells contain most, if not all, of this herpesvirus genome as a multicopy episome (2). B-cell lines that are EBV genome-positive may also be established from the blood of individuals who have had a prior EBV infection (infectious mononucleosis or inapparent infection; ref.3) and from tumor tissue in 90% of the cases of Burkitt's lymphoma in Africa (4). EBV remains in a latent state in most of these lymphoblastoid cell lines with 10% or less of the viral DNA expressed as cytoplasmic polyadenylylated RNA (5); only rare cell lines express late viral functions and produce infectious virus. Very little is known about the proteins encoded by the EBV genome in latently infected lymphocytes, but potential functions include immortalization of B lymphocytes, control of EBV gene expression, and maintenance of the viral genome as an episome.All EBV-infected cells contain a nuclear antigen (EBNA) that may be detected in an anti-complement immunofluorescence (ACIF) test using serum from individuals who have had a prior EBV infection (6). This antigen is not detected in EBV-negative lymphoblastoid cell lines using the same sera (6). DNA transfer experiments have demonstrated that the BamHI restriction endonuclease fragment K of EBV DNA is able to induce the expression of a new nuclear antigen in mouse fibroblasts that may be detected with EBNA antibody-positive but not EBNA antibody-negative human sera (7). In addition, anti-EBNA sera identified polypeptides varying between Mr 65,000 and Mr 73,000 in EBV-positive lymphoblastoid cell lines by radioimmunoelectrophoresis (RIE); these species were not detected in EBV-negative lines (8). It has been suggested that the BamHI fragment K contains at least part of the coding sequences for the antigen detected by RIE and that variation in the size of this protein between different EBV-infected cell lines is due to variation in the size of a repeated DNA sequence (internal repeat 3, IR3) contained within that fragment (9)...

Mapping genetic elements of Epstein-Barr virus that facilitate extrachromosomal persistence of Epstein-Barr virus-derived plasmids in human cells.

“…2) (42), contains the information encoding EBNA-1 (7,12,15,16,29,37). A series of insertion and deletion mutations has been constructed in the EBV BamHI-K fragment, resulting in the production of altered EBNA-1-related proteins (12). The nature of three of these mutations is reviewed in Fig.…”

“…A linker insertion mutation, S2, and two deletion mutations, S1 and S3-1 (plasmids carrying these mutations were obtained from J. Hearing) (12), which disrupt the sequences within the BamHI-K fragment encoding EBNA-1 were built into p410+, generating pCK(S2), pCK(S1), and pCK(S3-1) (Fig. 1B).…”

“…The EBV BamHI-K fragment (nucleotides 107565 to 112625 of the B95-8 strain), which has been shown to be important in the extrachromosomal maintenance of EBV-derived plasmids in HeLa (EBV negative) cells (Table 1, Fig. 2) (42), contains the information encoding EBNA-1 (7,12,15,16,29,37). A series of insertion and deletion mutations has been constructed in the EBV BamHI-K fragment, resulting in the production of altered EBNA-1-related proteins (12).…”

“…EBNA-1 is encoded within the EBV BamHI-K fragment (7,12,15,16,29,37) EBNA-1) had little or no effect on the ability of an EBVderived plasmid to persist extrachromosomally in EBVnegative cells (42). In contrast, two different mutations in EBNA-1, Si and S2, in p410+ gave rise to plasmids, pCK(S1) and pCK(S2), respectively, which failed to be efficiently maintained in an extrachromosomal form in HeLa (EBV negative) cells.…”

“…Hearing and A. J. Levine, Virology, in press). Gene transfer experiments have determined that this protein is encoded by the BamHI-K fragment of the EBV genome (7,12,15,16,29,37).…”