23 and 66 bp insertions (Yogo et al., 1990). Archetype is not associated with PML and is replication incompetent in tissue culture (Daniel et al., 1996). Consistent isolation of tandem repeat containing sequences in tissues obtained from PML patients strongly suggests the importance of these sequences in viral pathogenesis (Frisque et al., 1984; Jensen & Major, 1999; Martin et al., 1985; Marzocchetti et al., 2008; Vaz et al., 2000). Interestingly, JCV multiplication in susceptible cells is restricted by activation of viral gene expression by host transcription factors such as Oct-6/tst-1/SCIP (Wegner et al., 1993), pur a (Chen & Khalili, 1995), YB-1 (Chen et al., 1995c; Kerr et al., 1994) and NF-1 (Amemiya et al., 1989, 1992; Messam et al., 2003; Monaco et al., 2001; Ravichandran & Major, 2008; Shivakumar & Das, 1994; Sumner et al., 1996; Tamura et al., 1988). However, NF-1X is the only factor that has been shown to be important for JCV activity in glial (Kumar et al., 1993, 1996) and immune cells (Monaco et al., 2001). Because JCV latency is associated with cells undergoing haematopoietic development, it is probable that transcription factors previously reported as lymphoid specific regulate JCV gene expression. One such factor, Spi- B, activates gene expression from a lymphotrophic variant of SV40 (Petterson & Schaffner, 1987) and lymphotrophic papovavirus (LPV) (Erselius et al., 1990), both of which share genetic architecture and promoter regulation with JCV. In addition, Spi-B is upregulated in peripheral blood mononuclear cells in response to treatment with Natalizumab (Lindberg et al., 2008), an immuno-modula- tory therapy that has been associated with the development of PML (Major, 2010). Spi-B is an Ets transcription factor that is required for normal B-cell receptor signalling and formation of germinal centres in the spleen (Garrett-Sinha et al., 1999). Spi-B binds target sequences containing a 59- GGAA/T-39 core (Araki et al., 1988; Dorn et al., 1988; Erselius et al., 1990; Laux et al., 1994; Petterson & Schaffner, 1987; Wasylyk et al., 1993) and can cooperate with the retinoblastoma protein (pRB) to alter expression of proteins involved in B-cell maturation (Hagemeier et al., 1993; Mao et al., 1996; Rao et al., 1999b; Weintraub et al., 1995). Spi-B is involved in differentiation and maturation of B cells and is expressed at high levels in developing and mature B cells (Chen et al., 1995a, b; Ray et al., 1992; Su et al., 1996). RESULTS Spi-B-binding sites are present in JCV promoter/ enhancer sequences The Spi-B-binding site consensus sequence was identified as 59-WWWRRRGAASNDR-39 where the internal RGAA core is conserved (Laux et al., 1994). Promoter/enhancer sequences from PML-associated Mad-1 (Frisque et al., 1984), Mad-4 (Martin et al., 1985) and the non-pathogenic archetype variants (Yogo et al., 1990) were analysed for Spi-B-binding sites using the criteria of a 59-NGAA-39 core. Four potential Spi-B-binding sites (Fig. 1) were http://vir.sgmjournals.org Spi-B binds JCV sequence and supports viral activity Fig. 1. Diagram of potential Spi-B-binding sites in JCV promoter/ enhancer sequences. The non-coding viral RRs from the PML- associated Mad-1 (a), Mad-4 (b) and the non-pathogenic archetype (c) JCV variants are represented. The PML-associated variants contain 98 bp tandem repeats (light grey). A 19 bp deletion in the second repeat of Mad-4 (red) results in the loss of the second TATA box (blue). The archetype sequence contains two inserts (dark grey) in a single 98 bp unit. Sites that bind Spi-B protein in EMSA assays are shaded in yellow and sites that did not bind protein are white. identified in the JCV promoter/enhancer sequences and are listed in Table 1. Mad-1 (Fig. 1a) and Mad-4 (Fig. 1b) promoter/enhancers contain a binding site, labelled L5, adjacent to the TATA box in the first repeat. Mad-1 contains a second L5 site in the second repeat. A naturally occurring 19 bp deletion in the Mad-4 promoter/enhancer results in the loss of the second TATA box and a unique Spi-B-binding site labelled L4. The archetype promoter/ enhancer (Fig. 1c) contains 23 and 66 bp insertions within a single 98 bp unit, resulting in the loss of the L5/L4- binding sites. The 23 bp insertion results in a single base pair change, 59-AAAAGGGAAGGGA-39, at the 39 end of the L5 site to 59-AAAAGGGAAGGTA-39, labelled L3. The 66 bp insertion resulted in a unique site labelled L14. Spi-B is expressed in JCV susceptible cells JCV infects a variety of different cell types including CD34+ haematopoietic precursors, B cells, and astrocytes in culture. However, Spi-B gene expression has been described exclusively in the B cell lineage (Dahl et al., 2002; Erselius et al., 1990; Garrett-Sinha et al., 2005; Kim et al., 2003; Ray et al., 1992; Schmidlin et al., 2008; Schweitzer & DeKoter, 2004; Su et al., 1996, 1997). Northern blot analysis of Spi-B mRNA in brain tissue from mice showed no message present (Ray et al., 1992; Su et al., 1996). Using a sensitive method of quantitative real-time RT-PCR (qRT- PCR), Spi-B mRNA expression was measured in the various cell types susceptible to JCV (Table 2). Spi-B mRNA was detected above the non-template control for 3043