mouse mAb GA5 for human glial fibrillary acid protein (GFAP) (Cell Signaling Technology) was used at a 1 : 300 dilution. The rabbit polyclonal antibody for GFAP (Covance) was used at a 1 : 1000 dilution. The rabbit polyclonal antibody TUJ1 for neuronal class III b-tubulin (Covance) was used at a 1 : 2000 dilution. HA-tagged human NF-1X was visualized with the rat mAb 3F10 for HA (Roche Diagnostics) at 200 ng ml21. The rabbit polyclonal antibody 331B for human nestin was used at a 1 : 200 dilution (Messam et al., 2000). The goat polyclonal antibody for human Spi-B (Santa Cruz Biotechnology Inc) was used at a 1 : 200 dilution. The mouse mAbs for b actin AC-15 and for b-tubulin TUB 2.1 (Sigma) were used at 1 : 1000 dilutions. Fluorescent-labelled antibodies qualified for multiple labelling experiments were obtained from Invitrogen and used at 1–2 mg ml21. Preparation of Spi-B-binding site mutants in a Mad-4 back- ground. Site-directed mutagenesis was performed on the pMad4(586) plasmid to alter the L5 and L4 Spi-B site cores from 59-GGAA-39 to 59- CCAA-39 using the QuikChange II XL site-directed mutagenesis kit (Stratagene) according to the manufacturer’s instructions. Sense oligonucleotides (L4: 59-GTAAACAAAGCACAAGGCCAAGGGATG- GCTGCCAGC-39; L5: 59-TCCTGTATATATAAAAAAAAGCCAAGG- GATGGCTGCCAGCCA-39) and antisense oligonucleotides (L4: 59- GCTGGCAGCCATCCCTTGGCCTTGTGCTTTGTTTAC-39; L5: 59- TGGCTGGCAGCCATCCCTTGGCTTTTTTTTATATATACAGGA-39) containing the 59-GG-39 to 59-CC-39 mutations were synthesized by Integrated DNA Technologies (IDT). To create the L5/L4 double- mutant plasmid, site-directed mutagenesis was performed on the L4 mutant plasmid using the L5 sense and antisense oligonucleotides. DNA sequencing was performed to confirm the presence of each desired mutation as well as the fidelity of Mad-4 by the NINDS DNA sequencing facility. Isolation of total RNA. Total RNA was isolated from cell lines and primary cells using the RNeasy plus mini kit (Qiagen) according to the manufacturer’s instructions. RNA was eluted from the column in nuclease-free water and quantified using a Nanodrop 8000 (Thermo Scientific). Preparation of whole-cell extracts. Whole-cell extracts were prepared by using a modification of the method of Andrews & Faller (1991) as described previously (Monaco et al., 2001). Protein blotting. Fifteen micrograms of protein was separated by electrophoresis in a 4–12 % Bis-Tris gel followed by transfer to PVDF membrane. Membranes were blocked for 1 h in 5 % non-fat dry milk in Tris-buffered saline containing 50 mg BSA ml21, 10 mg glycine ml21, 0.05 % Tween-20 (TBS-BGT). Membranes were incubated with primary antibodies diluted in TBS-BGT for 3 h. Unbound antibody was removed by washing in TBS-BGT followed by 1 h incubation in secondary fluorescent-conjugated antibodies (Invitrogen). Unbound antibody was removed by washing in TBS-BGT. The antibody– antigen complex was visualized using a FluorChem Q imager (Alpha Innotech). Immunofluorescence. Immunofluorescence was conducted on Lipofectamine 2000 transfected, nucleofected and JCV-infected cell cultures. Cells were fixed with 4 % paraformaldehyde and permea- bilized with 0.2 % Triton X-100 before indirect antibody labelling. Samples were mounted with a glycerol-based mounting medium containing the DNA dye, 49,6-diamidino-2-phenylindole (DAPI) and analysed by fluorescence microscopy using a Zeiss Axiovert 200M microscope fitted with filters appropriate for DAPI, Alexa Fluor 488 and Alexa Fluor 568 excitation. qRT-PCR. Spi-B mRNA present in cells was measured by reverse transcription followed by qRT-PCR using the TaqMan gene expression assay (Applied Biosystems) for human Spi-B (Hs00162150_m1), http://vir.sgmjournals.org Spi-B binds JCV sequence and supports viral activity human b actin (Hs99999903_m1) and human PUM1 (Hs00206469_m1). Reverse transcription (RT) of 1 mg of total RNA was performed using qScript cDNA supermix (Quanta Biosciences) according to the manufacturer’s instructions. Six 1 : 10 serial dilutions of the resultant BJAB RT reaction were prepared to generate a relative standard curve to determine Spi-B mRNA levels. Singleplex qRT PCRs were assembled using each RT reaction with 26 TaqMan universal PCR master mix and 206 TaqMan human Spi-B gene assay or 206 TaqMan human b actin or PUM1 endogenous control assay according to the manufacturer’s instructions (Applied Biosystems). Relative quantification of Spi-B gene expression was determined using the relative standard curve method described on the Applied Biosystems website (Biosystems, 2008). Spi-B mRNA levels were normalized to input template based on the endogenous control. The BJAB standard is assigned a value of one and the other cell types are reported as values relative to BJAB. EMSA. Oligonucleotides with the sequence of the SV40 Spi-B site (59-CTGAAAGAGGAACTTG-39), or the JCV Spi-B site L3 (59-AAA- AGGGAAGGTA-39), L4 (59-CAAGGGGAAGGGA-39), L5 (59-AAAG- GGAAGGGA-39) and L14 (59-TATAGTGAAACCC-39) were synthe- sized with and without 59 biotinylation by IDT. Mutated versions of the SV40 Spi-B site (59-CTGAAAGACCAACTTG-39), and JCV Spi-B L4 (59-CAAGGCCAAGGGA-39) and L5 (59-AAAGCCAAGGGA-39) were synthesized with 59 biotinylation. Oligonucleotides with sequences complementary to those listed above were also synthesized with or without 59 biotinylation as indicated above. The oligonucleotides for the authentic- or mutated-binding sites were annealed to form double- stranded probe at a concentration of 100 ng ml21. The biotin labelled probes were diluted 1 : 200 in water. Biotin labelled authentic probe or mutant probe was incubated with 5–25 mg nuclear extract from KG-1a cells, BJAB cells or PDA in the presence or absence of a 5-, 100- and 400- fold excess of unlabelled authentic probe. Supershifts were carried out by incubation of the cellular extracts with 2 ml Spi-B antiserum for 30 min on ice before the addition of probe as described previously (Laux et al., 1994). The reactions were incubated at room temperature for 20 min and electrophoresed in a 6 % polyacrylamide–TBE DNA retardation gel (Invitrogen). The complexes were transferred to a positively charged nylon membrane and detected using the LightShift chemiluminescent EMSA kit (Thermo Scientific/Pierce). Transfection and infection. Exogenous Spi-B and NF-1X were pre- expressed in human fetal brain-derived progenitor cells or PDN followed by exposure to Mad-4 JCV in a modification of the method described by Messam et al. (2003). Cells were allowed to attach to the wells of PDL-coated six-well dishes at a density of 2–46105 cells per well for 2 days. Each well of cells was transfected with 1 mg pDEB-Spi- B DNA or pCHA-NF1X using Lipofectamine 2000 according to the manufacturer’s instructions (Invitrogen). Cells were exposed to medium alone for the mock condition. Approximately 48 h after transfection each well of cells was exposed to 333 haemagglutinaton units (HAU) of Mad-4 JCV or medium alone from the mock condition. Cells were fed with half new medium every 2–3 days. Immunofluorescence was performed on days 5 and 8 post-JCV exposure and T-antigen expression was quantified versus the cell- specific marker expression of nestin for progenitors or b III tubulin for PDN. Nucleofection. Archetype, Mad-1, Mad-4, L5 mutant Mad-4, L4 mutant Mad-4 and L5/L4 double-mutant Mad-4 plasmids were introduced into PDAs by nucleofection using the Amaxa basic kit for primary neurons (Amaxa) according to the manufacturer’s instruc- tions. Briefly, 16106 cells were nucleofected with 2 mg DNA using program C13. Cells were allowed to attach for 4 h, followed by replacement of culture medium. Cells were fed with half new medium every 2–3 days. Nucleofection using the pmaxGFP (Amaxa) reporter plasmid was included to determine nucleofection success and 3049