L. J. Marshall, L. Dunham and E. O. Major Table 1. Potential Spi-B-binding site sequences present in the promoter/enhancers of JC virus variants Spi-B-binding site SV40 SV40 mutant L4 L4 mutant L5 L5 mutant L3 L14 Viral variant SV40 – Mad-4 – Mad-1/Mad-4 – Archetype Archetype Sequence (5§–3§) CTGAAAGAGGAACTTG CTGAAAGACCAACTTG CAAGGGGAAGGGA CAAGGCCAAGGGA AAAAGGGAAGGGA AAAAGCCAAGGGA AAAAGGGAAGGTA TATAGTGAAACCC background detection in all cell types tested. Spi-B mRNA expression was measured at low levels in two cell types, HeLa cells and primary CD3+ T lymphocytes, in which Spi-B protein has not been detected. Immortalized B cells (Raji and BJAB) and primary CD19+ B cells expressed 100-fold and greater levels of Spi-B mRNA in comparison. A CD34+ cell line (KG-1a) and primary CD34+ haematopoietic precursors expressed less Spi-B mRNA than B cells, which is consistent with published reports (Su et al., 1996). The human fetal brain-derived SVG cell line, which supports robust JCV multiplication, expressed Spi-B mRNA comparable to CD34+ haematopoietic precursors. Human fetal brain-derived cells (progenitors, astrocytes and neurons) expressed Spi-B mRNA at levels similar to HeLa and primary T lymphocytes. Spi-B protein expres- sion was measured by protein blotting using an antibody that detects a single 43 kDa band for Spi-B (Arguello et al., 2003). Spi-B protein was detected in BJAB cells and slightly lower in KG-1a cells (Fig. 2a) consistent with mRNA levels. Spi-B protein was detected in SVG cells and in progenitor- derived astrocytes (PDA), but not progenitor cells or progenitor-derived neurons (PDN) (Fig. 2b). Spi-B protein expression in the absence of high levels of mRNA in PDAs may be due to instability and/or turnover of the mRNA. Stability of the highly related Spi-1 mRNA has been demonstrated to be extensively regulated at the post- transcriptional level (Hensold et al., 1996), which may also be the case for Spi-B. Spi-B binds unique sites on JCV enhancers Because potential Spi-B-binding sites are present in JCV promoter/enhancer sequences and cells susceptible to JCV infection express Spi-B, the association of Spi-B with potential binding sites was measured by electromobility shift assay (EMSA) using whole-cell extracts from KG-1a cells, BJAB cells and PDAs (Fig. 3). Cell extracts were incubated with double-stranded oligonucleotide probes encoding the Spi-B-binding sites described in Table 1, or the SV40 Spi-B site described previously (Petterson & Schaffner, 1987) as a positive control. Fig. 3(a) illustrates that protein present in BJAB cell extract binds the SV40 probe and causes a shift that was competed by a 5-, 100- and 400-fold excess of unlabelled probe. Alteration of the Spi-B site core from 59-GGAA-39 to 59-CCAA-39 abrogated protein binding to the mutant probe, indicating that complex formation was specific for the Spi-B core. Addition of Spi-B antiserum, suitable for distinguishing Spi-B from other related proteins in an EMSA assay (Laux et al., 1994), caused a supershift in the original complex, Table 2. Spi-B mRNA expression in immune- and brain-derived cells types Cell type Spi-B mRNA, relative level Standard deviation Category Control B cells Haematopoietic precursor Brain-derived cells HeLa CD3+ lymphocytes Raji BJAB CD19+ lymphocytes KG-1a CD34+ lymphocytes SVG Human fetal brain progenitor cells PDA PDN 0.01 0.01* 2.17 1.66 1.16* 0.31 0.75* 0.30 0.02 0.01 0.10 *Spi-B mRNA value is a mean of cell purified from multiple blood donors. 3044 0.0002 0.0061 0.1262 0.0014 0.1616 0.0082 0.3661 0.0273 0.0001 0.0013 0.0036 Journal of General Virology 91