L. J. Marshall, L. Dunham and E. O. Major are located adjacent to both Spi-B sites on their 39 ends. Importantly, neither the Oct-6/tst-1/SCIP nor the NF-1- binding sites directly overlap the Spi-B-binding sites. Therefore, these factors would not affect Spi-B binding in the EMSA analysis presented in Fig. 3 and would not be affected by the mutations introduced in the Mad-4 L5, L4 or L5/L4 double mutants presented in Fig. 5. Pur a and YB- 1 bind opposite strands of the single-stranded JCV promoter/enhancer sequence, which overlap the L5 Spi- B-binding site, and regulate early and late viral gene expression, respectively, in cooperation with the large T antigen (Chen et al., 1995c; Chen & Khalili, 1995; Kerr et al., 1994). Nucleotide changes equivalent to the mutations in the Mad-4 L5, L4 or L5/L4 double mutants were introduced into the YB-1/pur a-binding site reported by Chang et al. (1996) and did not have any significant effect on the ability of protein from BJAB cells or PDAs to bind the authentic probe in competition experiments (data not shown). Importantly, studies on YB-1/pur a-binding sites focus on sequences present in the first tandem repeat and emphasize the importance of T-antigen binding to sites present in the origin of replication (Chen et al., 1995c; Chen & Khalili, 1995; Kerr et al., 1994). In the context of a Mad-4 JCV promoter/enhancer, the naturally occurring 19 bp deletion results in alteration of the YB-1/pur a- binding site in the second tandem repeat. In addition, YB- 1/pur a-binding site in the second tandem repeat of both Mad-1 and Mad-4 would not contain the important T- antigen binding sites in proximity because they occur directly after the first repeat. Taken together these points suggest that the YB-1/pur a-binding site would not play a role in the abrogation of early gene expression demon- strated in Fig. 5 for the L4 mutant plasmid. Spi-B is largely considered a lymphotrophic transcriptional activator (Rao et al., 1999a; Yamamoto et al., 2002) and is known to transactivate the l2–4 enhancer (Su et al., 1996), the LMP/TP2 promoter of Epstein–Barr virus (Laux et al., 1994), the SV40 enhancer (Petterson & Schaffner, 1987) and the LPV promoter (Erselius et al., 1990). JCV is capable of infecting a variety of cells of the lymphoid system. Detection of tandem repeat containing JCV DNA sequence in the absence of viral capsid protein in the bone marrow in patients prior to the development of PML suggests that these cells may act as a reservoir for latent virus (Houff et al., 1988; Tan et al., 2009). Spi-B expression in developing B cells correlates with reactivation of JCV in immune cells. Studies in mice demonstrate that Spi-B is expressed at a low level in pro-B cells, increases in pre-B cells and is highest in mature B cells (Su et al., 1996). Spi-B expression during B-cell development was confirmed in human using prototypic cell lines (Ray et al., 1992). If Spi- B is indeed a transcriptional activator of JCV gene expression, a minimal level of Spi-B expression in haematopoietic progenitors and pro-B cells may support latency of JCV, while upregulation of Spi-B expression in pre-B and mature B cells may lead to reactivation of JCV gene expression and production of viral progeny. 3048 Because the number of patients undergoing immuno- modulatory therapies that develop PML continues to rise (Major, 2009), understanding the role of Spi-B during JCV latency and reactivation is increasingly important. These therapies cause mobilization and expansion of cells that have the potential to harbour latent JCV infection. Natalizumab has also been shown to upregulate genes involved in B-cell differentiation, including Spi-B (Lindberg et al., 2008). If Spi-B is an activator of JCV gene expression it could be a contributing factor in the development of PML in patients undergoing these immuno-modulatory therapies. In addi- tion, PML-associated Mad-4 contains a duplication of the sites due to the tandem repeat nature of its promoter/ enhancer, one of which (L4) was essential for T-antigen expression demonstrated by mutational analysis in Fig. 5. Accumulation of Spi-B sequence motifs in promoters can confer tissue specificity (Gerster et al., 1987; Ondek et al., 1987; Schirm et al., 1987) as illustrated in the case of a lymphotrophic SV40 variant (Petterson & Schaffner, 1987). Accumulation of additional, or unique, Spi-B-binding sites in the promoter/enhancers of JCV RRs may contribute to higher levels of replication in lymphoid tissues that in turn lead to development of PML. Further investigation into the role of Spi-B during JCV infection and the presence of duplicated, and or unique, Spi-B-binding sites in the RR sequences derived from PML patients will offer insight into the molecular mechanism of JCV reactivation and the development of PML. METHODS Cells, plasmids and viruses. Human growth factors for cell culture were obtained from Peprotech. KG-1a cells were maintained in RPMI 1640 medium (Cellgro) supplemented with 20 % FBS (Atlanta Biologics) and 2 mM L-glutamine (Quality Biologics). BJAB and Raji cells were maintained in RPMI 1640 medium supplemented with 10 % FBS and 2 mM L-glutamine. SVG cells were maintained in minimal essential medium (MEM; Cellgro) supplemented with 10 % FBS and 2 mM L-glutamine. Human central nervous system progenitors were isolated and differentiated into astrocytes and neurons as described previously (Messam et al., 2003). A plasmid encoding the full-length human Spi-B cDNA, pDEB-Spi-B, was obtained from Francoise Moreau-Gauchelin at the Institut Curie in Paris, France (Ray et al., 1992). A plasmid encoding the full-length human NF-1X cDNA with an amino-terminal haemagglutinin (HA) tag, pCHA-NF1X, was described previously (Monaco et al., 2001). A plasmid encoding the full-length JCV Mad-4 genome, pMad4(586), was described previously (Martin et al., 1985). A plasmid encoding the full-length JCV Mad-4 genome, pM1TC, was described previously (Frisque et al., 1984). A plasmid encoding the full-length JCV archetype genome, CY, was described previously (Yogo et al., 1990). Isolation of primary lymphocytes. Mononuclear cells were isolated from the peripheral blood of normal donors provided by the NIH clinical centre blood bank by centrifugation on Ficoll-Hypaque gradients. CD34+ haematopoietic precursors, CD19+ B cell and CD3+ T-cell populations were purified using the RoboSep brand of immunomagnetic cell separation (StemCell Technologies). Antibodies. A mouse mAb for SV40 T antigen, which cross-reacts with JCV T antigen (EMD Calbiochem) was used at 5 mg ml21. The Journal of General Virology 91