L. J. Marshall, L. Dunham and E. O. Major Fig. 3. Spi-B binds unique sites present on JCV promoter/enhancers. An EMSA was performed using BJAB cell extract and biotin-labelled oligonucleotides for the SV40 Spi-B site as a positive control for Spi-B-DNA complex formation (a). Authentic- binding site probe was incubated with cell extract alone or in combination with 5-, 100- or 400-fold excess unlabelled oligonucleotide competitor, or Spi-B antiserum. Mutant probe was incubated with cell extract to demonstrate specificity for the Spi-B-binding site core. Identical EMSAs were performed using BJAB, KG-1a, and/or PDA cell extract and biotin-labelled oligonucleotides for the archetype JCV L3 and L14 (b) and JCV L5 (c) and L4 (d) Spi-B sites as probes. Mad-1 and Mad-4 contain promoter/enhancers that exist as tandem repeats, which contain TATA boxes and duplications of essential transcription factor-binding sites including Spi-B. In this study, Spi-B-binding sites that actively bound Spi-B protein expressed in JCV susceptible cell types are present in promoter/enhancer sequences from Mad-1 and Mad-4, but not the non-pathogenic archetype (Table 2, Figs 2 and 3). Mad-1 contains two identical Spi- B-binding sites (L5) within each of the 98 bp repeats, while Mad-4 contains the same site as Mad-1 (L5) in the first repeat followed by a unique site (L4) in the second repeat that results from a naturally occurring 19 bp deletion. 3046 Journal of General Virology 91