Epstein–Barr virus nuclear antigen-1 renders lymphocytes responsive to IL-2 but not IL-15 for survival Penelope Tsimbouri, Yazeed Al-Sheikh, Mark E. Drotar, William Cushley and Joanna B. Wilson Correspondence Joanna B. Wilson Joanna.Wilson@bio.gla.ac.uk Division of Molecular and Cellular Biology, Faculty of Biomedical and Life Sciences, University of Glasgow, Glasgow G11 6NU, UK Received 5 July 2007 Accepted 18 June 2008 Epstein–Barr virus nuclear antigen-1 (EBNA-1) is the only latent protein expressed in all virus- associated tumours. It plays a critical role in viral propagation and in the replication, episomal maintenance and partitioning of the viral genome. However, its tumorigenic potential is debated. We have previously shown that lymphocytes from a tumour-prone, EBNA-1-expressing, transgenic mouse line show increased responsiveness to interleukin-2 (IL-2). It was important to determine whether this property was unique to the transgenic line or whether it is a general consequence of EBNA-1 expression in B cells. In order to distinguish between these possibilities, explanted lymphocytes from two independent transgenic mouse lines were examined. The lymphocytes from both lines showed enhanced proliferation rates compared with controls. The transgenic lymphocytes survived for extended periods in culture, dependent on the dose of IL-2, while IL-15 (the receptor of which shares the b and c chain components of the IL-2 receptor) induced little effect. In accordance with this, transgenic B cells showed enhanced induction of expression of the IL-2 receptor a chain (CD25), which modulates affinity for the ligand. As this phenotype is evident in lymphocytes from mice of both lines, it is necessarily independent of any transgene insertion site effects and may be attributed to EBNA-1 expression. Furthermore, 10/12 tumour-bearing transgenic mice had elevated IL-2 levels in serum and 4/6 tumours were CD25 positive. IL-2 is normally produced by activated T cells in vivo; thus, chronic immune activation or modulation could elicit this unique mode of virus-infected cell survival. INTRODUCTION Epstein–Barr virus (EBV) is a human herpesvirus asso- ciated with a number of malignancies, including endemic Burkitt’s lymphoma (eBL), nasopharyngeal carcinoma (NPC), Hodgkin’s disease (HD) and lymphomas in immunosuppressed individuals (Kawa, 2000). The ability of EBV alone to induce B-cell proliferation is supported by the efficient transformation of primary B cells into permanent lymphoblastoid cell lines (LCLs) upon in vitro infection by the virus. In LCLs and tumour cells, EBV establishes a latent infection in which a subset of viral genes is expressed. Different tumour types express a different subset of these latent genes but in all cases, EBV nuclear antigen-1 (EBNA-1) is expressed; indeed, EBNA-1 is the only viral antigen consistently detected in eBL. EBNA-1 is required for stable and efficient latent EBV infection (Humme et al., 2003; Lee et al., 1999). It activates replication of the viral episomal genome and plays a critical role in viral genome segregation at cell division (Ceccarelli & Frappier, 2000; Chittenden et al., 1989; Kapoor & Frappier, 2003; Lee et al., 1999). EBNA-1 also transactivates the expression of several viral latency genes from the viral Cp promoter (Gahn & Sugden, 1995; Zetterberg et al., 2004). While EBNA-1 plays a crucial role in viral maintenance, it may also contribute to the development of malignancy. However, it is clearly not essential to the in vitro immortalizing property of the virus since a viral strain with a deletion in the EBNA-1 gene is still able to generate LCLs in culture, albeit at much-reduced efficiency (Humme et al., 2003). Nevertheless, Kennedy et al. (2003) showed that EBNA-1 contributes to the ongoing survival of EBV-positive BL cell lines, a property which was demonstrated to be independent of the viral genome maintenance function. Furthermore, EBNA-1 interacts with a ubiquitin-specific protease USP7 (Holowaty et al., 2003; Saridakis et al., 2005), which plays a role in regulating the p53 and Mdm2 pathway (Li et al., 2004). Thus, it is hypothesized that EBNA-1 could interfere with the stabilization of p53 and thereby affect cell survival and proliferation (Saridakis et al., 2005). A supplementary figure is available with the online version of this paper. Journal of General Virology (2008), 89, 2821–2832 DOI 10.1099/vir.0.83296-0 0008-3296 G 2008 SGM Printed in Great Britain 2821