Fig. 2. Spi-B is expressed in JCV susceptible cells. Spi-B protein was detected in JCV susceptible KG-1a, BJAB, SVG and PDA (a) and cells derived from human fetal brain progenitors, PDA and PDN (b) by protein blotting. b-Tubulin protein levels were detected to demonstrate equal loading. which indicates that protein bound to the SV40 probe in the shifted complex is Spi-B. The same EMSAs were carried out for the potential JCV Spi-B-binding sites. Probes for Spi-B sites present in archetype promoter/enhancers, L3 and L14, did not bind protein in any cell type tested (Fig. 3b). Probes for Spi-B sites present in PML-associated promoter/enhancers, L5 and L4, bound protein expressed in KG-1a, BJAB and PDAs shown in Fig. 3(c) and (d), respectively. Incubation of both the L5 and L4 probes with cellular extracts resulted in the formation of multiple protein–DNA complexes labelled shifts C1–C3 that were consistent between cell types. Formation of multiple protein–DNA complexes on Spi-B-binding sites has been described using EMSA analysis for many promoters in published reports (Dekoninck et al., 2003; Erselius et al., 1990; Garrett- Sinha et al., 2005; Laux et al., 1994; Petterson & Schaffner, 1987; Zhao & Sample, 2000). In the case of JCV L5 and L4, each complex was competed by excess of unlabelled oligonucleotide probe and alteration of the Spi-B site core to 59-CCAA-39 resulted in abrogation of formation of all complexes. Addition of Spi-B antiserum caused supershifts of the original complexes, similar to that observed for SV40. The C2 shift for the L5 site and C3 shift for the L4 site required higher excess unlabelled probe for competi- tion and were not completely supershifted upon addition of Spi-B antiserum, suggesting an abundance of these complexes over others. These results suggest that Spi-B is a component of multiple complexes at these locations on the viral promoter/enhancer. Spi-B is known to bind multiple cofactors that cooperate to affect gene expression such as cellular proteins TBP and pRB (Rao et al., 1999b), OBF-1 (Bartholdy et al., 2006) and CBP (Yamamoto et al., 2002), and the viral proteins EBNA-2 and EBNA-3C for Epstein– Barr virus (Zhao & Sample, 2000). It is possible that the multiple shifts represent separate complexes that may include cofactors necessary for activity of Spi-B. Importantly, competition with varying levels of unlabelled probe and Spi-B antiserum supershifts demonstrate that each of the complexes formed for both L5 and L4 are Spi-B specific. http://vir.sgmjournals.org Spi-B binds JCV sequence and supports viral activity Expression of exogenous Spi-B in semi- and non- permissive cells increases T-antigen expression Because Spi-B is expressed in cells that are susceptible to JCV infection, we sought to determine if Spi-B overexpression in non-permissive cells could support viral activity. Previous studies showed that exogenous expression of NF-1X in non- permissive cells by transfection prior to JCV exposure increased T-antigen expression (Messam et al., 2003). NF- 1X and Spi-B were expressed by transient transfection in semi-permissive progenitor cells and non-permissive PDNs for 24 h followed by exposure to Mad-4 JCV. Five days after JCV exposure, cells were analysed for expression of T antigen, as well as the cell-specific marker for progenitor cells (nestin) or neurons (b III tubulin). Double-positive cells for T antigen and the appropriate cell-specific marker were quantified from three separate experiments. Pre- expression of NF-1X and Spi-B resulted in a statistically significant increase in T-antigen expression in progenitors (Fig. 4a) and PDNs (Fig. 4b). NF-1X protein expression was confirmed 3 days post-transfection by immunofluorescence (progenitors: 5–10 %; PDN: 1 %). An increase in Spi-B mRNA expression was confirmed 3 days post-transfection by qRT-PCR. Spi-B gene expression was increased from 0.0005 to 1.7 in progenitor cells and from 0.05 to 1.7 in PDNs. These results demonstrate that expression of Spi-B, like NF-1X, supports early viral gene expression. Mutation of the L4 Spi-B-binding site in the Mad-4 promoter/enhancer is sufficient to abrogate JCV activity in PDAs To determine if Spi-B sites in the JCV promoter/enhancer that bind Spi-B are important for viral activity, site-directed mutagenesis was used to generate plasmids encoding mutations in the Spi-B site core of the L5 and L4 sites in Mad-4 (Table 1). Plasmids encoding archetype, Mad-1, Mad-4, L5 mutant Mad-4, L4 mutant Mad-4 or L5/L4 double-mutant Mad-4 were introduced into PDAs via nucleofection. T-antigen expression was quantified on a per cell basis by immunofluorescence, 6 days after nucleofec- tion. Fig. 5 demonstrates that T-antigen expression occurred at similar levels from the Mad-1 and Mad-4 plasmids, while expression from the archetype plasmid was rarely detected. Mutation of the L5 site resulted in a slight, but not statistically significant, increase in T-antigen expression comparable to Mad-1. Mutation of both the L5 and the L4 sites resulted in abrogation of T-antigen expression. Importantly, mutation of the L4 site alone also resulted in abrogation of T-antigen expression. These results dem- onstrate that abrogation of viral activity due to the L4 site mutation is dominant in the presence of a wild-type L5 site. DISCUSSION The promoter/enhancer of JCV is considered to be the portion of the RR that confers specific tissue tropism and supports development of PML. PML-associated variants 3045