Analysis of Epstein–Barr virus latent gene expression in endemic Burkitt's lymphoma and nasopharyngeal carcinoma tumour cells by using quantitative real-time PCR assays

Abstract

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...

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