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Previous studies on Epstein-Barr virus (EBV)-positive B-cell lines have identified two distinct forms of virus latency. Lymphoblastoid cell lines generated by virus-induced transformation of normal B cells in vitro, express the full spectrum of six EBNAs and three latent membrane proteins (LMP1, LMP2A, and LMP2B); furthermore, these lines often contain a small fraction of cells spontaneously entering the lytic cycle. In contrast, Burkitt's lymphoma-derived cell lines retaining the tumor biopsy cell phenotype express only one of the latent proteins, the nuclear antigen EBNA1; such cells do not enter the lytic cycle spontaneously but may be induced to do so by treatment with such agents as tetradecanoyl phorbol acetate and anti-immunoglobulin. The present study set out to determine whether activation of full virus latent-gene expression was a necessary accompaniment to induction of the lytic cycle in Burkitt's lymphoma lines. Detailed analysis of Burkitt's lymphoma lines responding to anti-immunoglobulin treatment revealed three response pathways of EBV gene activation from EBNAl-positive latency. A first, rapid response pathway involves direct entry of cells into the lytic cycle without broadening of the pattern of latent gene expression; thereafter, the three "latent" LMPs are expressed as early lytic cycle antigens. A second, delayed response pathway in another cell subpopulation involves the activation of full latent gene expression and conversion to a lymphoblastoidlike cell phenotype. A third response pathway in yet another subpopulation involves the selective activation of LMPs, with no induction of the lytic cycle and with EBNA expression still restricted to EBNA1; this type of latent infection in B lymphocytes has hitherto not been described. Interestingly, the EBNA1+ LMP+ cells displayed some but not all of the phenotypic changes normally induced by LMP1 expression in a B-cell environment. These studies highlight the existence of four different types of EBV infection in B cells, including three distinct forms of latency, which we now term latency I, latency II, and latency III.

Most Epstein-Barr virus (EBV)-positive Burkitt's lymphomas (BLs) carry a wild-type EBV genome and express EBV nuclear antigen 1 (EBNA1) selectively from the BamHI Q promoter (latency I). Recently we identified a distinct subset of BLs carrying both wild-type and EBNA2 gene-deleted (transformation-defective) viral genomes. The cells displayed an atypical "BamHI W promoter (Wp)-restricted" form of latency where Wp (rather than Qp) was active and EBNA1, -3A, -3B, -3C, and -LP were expressed in the absence of EBNA2 or latent membrane proteins 1 and 2. Here we present data strongly supporting the view that the EBNA2-deleted genome is transcriptionally active in these cells and the wild-type genome is silent. Single-cell cloning of three parental Wp-restricted BL lines generated clones carrying either both viral genomes or the EBNA2-deleted genome only, never clones with the wild-type genome only. All rescued clones displayed the Wp-restricted form of latency characteristic of the parent line and retained the original parent cell phenotype. Interestingly, Wp-restricted parent lines and derived clones were markedly more resistant to inducers of apoptosis than standard latency I BL lines. Furthermore, in vitro infection of EBV-negative BL lines with an EBNA2 gene-deleted virus generated EBV-positive converts with Wp-restricted latency and a similarly marked apoptosis resistance. We postulate that, in the subset of BLs displaying Wp-restricted latency, infection of a tumor progenitor cell with an EBNA2 gene-deleted virus has provided that cell with a survival advantage through broadening antigen expression to include the EBNA3 proteins.Epstein-Barr virus (EBV), a human B-lymphotropic herpesvirus with cell growth-transforming ability, is linked to three different B-cell malignancies, endemic Burkitt's lymphoma (BL), Hodgkin's disease, and posttransplant lymphoproliferative disease (PTLD) (36). We know relatively little about the role played by the virus in these different tumor contexts, except for those oligoclonal or polyclonal PTLD lesions which arise in the setting of profound T-cell impairment, classically in early posttransplant patients. These lesions express the same spectrum of EBV latent-cycle proteins (53) as do B cells transformed by EBV to permanent lymphoblastoid cell lines (LCLs) in vitro (19). This latency III form of infection is characterized by BamHI C promoter (Cp) and to a lesser extent BamHI W promoter (Wp) activity, leading to expression of EBV nuclear antigen EBNA1, -2, -3A, -3B, -3C, and -LP, and by activation of EBNA2-responsive promoters elsewhere in the viral genome, leading to expression of latent membrane proteins LMP1 and LMP2. The LCL-like nature of viral gene expression in PTLDs strongly suggests that EBV is the principal, perhaps the sole, driving force behind these lymphoproliferations.The situation is quite different in endemic BL, where EBV's role clearly complements that of the principal cellular genetic change, namely, activation of the c-myc oncogene by translocation to an immunoglo...

Studies of Epstein-Barr virus (EBV)-positive cell lines have identified several forms of virus latency, but the patterns of virus gene expression in EBV-positive tumour cells appear more variable. However, it is unclear to what extent these differences merely reflect the increased sensitivities of different detection methods. Here, the design and validation of novel real-time RT-PCR assays to quantify relative levels of EBV transcripts are described. When the new assays were used to screen a collection of endemic Burkitt's lymphoma tumours, abundant Qp-driven EBNA1 expression was found, whereas the other latent transcripts (with the exception of LMP2A) were either absent or detectable only at trace levels. Analysis of 12 nasopharyngeal carcinoma biopsies revealed significant levels of EBNA1 and LMP2A transcripts in almost every case but, in contrast to previous reports, LMP1 expression was undetectable. These new quantitative assays may help to provide a clearer picture of EBV gene expression in tumour material.Epstein-Barr virus, a B-lymphotropic herpesvirus with growth-transforming properties, is linked to a number of human lymphoid-and epithelial-cell malignancies, including post-transplant lymphoproliferative disease (PTLD), Hodgkin's disease (HD), endemic Burkitt's lymphoma (BL) and nasopharyngeal carcinoma (NPC) (Rickinson & Kieff, 2001). Studies of virus-positive cell lines and EBVassociated tumour biopsies have shown that EBV can adopt one of several forms of latent infection, which are distinguished by different patterns of latent antigen expression. In lymphoblastoid cell lines (LCLs), generated by the experimental infection of resting B cells in vitro, and in early-onset PTLD lesions arising in immunocompromised transplant patients (Young et al., 1989), EBV expresses the full spectrum of latent genes; these include two small nuclear RNAs (EBERs), the highly spliced BamHI A rightward transcripts (BARTs), six nuclear antigens (EBNA1, -2, -3A, -3B, -3C and -LP) and three latent membrane proteins (LMP1, -2A and -2B) (Rickinson & Kieff, 2001). This latency III form of infection is associated with the activity of two promoters, Cp and Wp, that drive the expression of all six EBNA mRNAs (Sample et al., 1986;Bodescot et al., 1987;Woisetschlaeger et al., 1989) and additional promoters in the BamHI N region that transcribe the individual LMP genes (Fennewald et al., 1984;Hudson et al., 1985;Laux et al., 1989). However, the situation is quite different in other virus-positive tumours, which, although positive for the EBER and BART mRNAs, show more restricted patterns of virus latent antigen expression. Thus, most BL tumours and derived BL cell lines that retain the original biopsy cell phenotype in vitro display a latency I form of infection characterized by expression of a single viral protein, EBNA1 (Rowe et al., 1987;Gregory et al., 1990). In this case, the EBNA1 mRNA is transcribed from a novel promoter Qp (Schaefer et al., 1995;Nonkwelo et al., 1996), whereas the Wp, Cp and LMP promoters are all silent. NPC...