Fig. 4. Expression of exogenous Spi-B in semi- and non- permissive cells increases T-antigen expression. The fraction of T antigen and nestin double-positive progenitor cells (a) or b III tubulin double-positive PDN (b) is represented along with the SD. An asterisk denotes the following statistically significant change in value. Progenitors: NF-1X P50.0006, Spi-B P50.0063; PDN: NF-1X P50.0004, Spi-B P50.0012. Importantly, mutation of the unique Mad-4 L4 Spi-B site and not L5 resulted in abrogation of T-antigen expression in PDAs. The locations of the L5- and L4-binding sites are of particular interest in relation to JCV activity. Abrogation of T-antigen gene expression in response to mutation of the L4 site within the viral promoter/enhancer suggests that an Spi-B site in the second tandem repeat is important for early viral gene expression (Fig. 5). Strikingly, the presence of a functional Spi-B site in the first repeat (L5) is incapable of compensating for the loss of the Spi-B site in the second repeat (L4). Mutation of the L5 site alone did not significantly alter the ability of the virus Fig. 5. Mutation of the L4 Spi-B-binding site in the Mad-4 promoter/enhancer is sufficient to abrogate JCV activity in PDA. The fraction of T antigen-positive cells as a fold change over the wild-type Mad-4 plasmid for archetype, Mad-1, Mad-4, L5 mutant Mad-4, L4 mutant Mad-4 or L5/L4 mutant Mad-4 plasmids is represented along with SD. An asterisk denotes the following statistically significant change in value. Archetype, P50.039 ; L5/ L4 mutant, P50.039 ; L4 mutant, P50.039. http://vir.sgmjournals.org Spi-B binds JCV sequence and supports viral activity to express T antigen; however, this could be due to compensation by the L4 site. The L4 site may be dominant over an initial L5 site in the context of the tandem repeat. Due to its deletion in a variety of RR sequences from PML patients including Mad-4, Her-1, Mad-7, Mad-8, Mad-9 and Mad-11 (Major et al., 1987; Martin et al., 1985; Matsuda et al., 1987) the second TATA box was not thought to be necessary for virus multiplication (Lynch & Frisque, 1990; Martin et al., 1985). In fact, molecular studies on the function of the Mad-1 RR often utilize JCV sequences that only contain a single 98 bp unit and, therefore, a single TATA box and Spi-B site (Kerr et al., 1994; Sunden et al., 2007). However, the maintenance of tandem repeat nucleotide sequences throughout PML- associated variants illustrates their importance in the pathogenesis of JCV (Gosert et al., 2010). Utilizing RRs with a single 98 bp unit may miss the importance of measuring the relationship between tandem repeats. Future studies should include full-length RR to include the effects of a functional second repeat in the context of JCV multiplication and pathogenesis. Spi-B-binding sites within the first repeat of the promoter/ enhancers of PML-associated JCV variants are situated directly downstream of the viral origin and TATA box elements, which are essential for T-antigen binding and activation of viral gene expression (Daniel & Frisque, 1993; Kenney et al., 1986a, b; Khalili et al., 1986; Krebs et al., 1995; Vacante et al., 1989). Activation of gene expression from the basal promoter is an essential event for JCV multiplication and reactivation from latency. SV40 T antigen interacts with proteins that recruit the basal transcriptional apparatus (TFIID) to the viral promoter/ enhancer including TATA-binding protein (TBP) and transcription-enhancing factor 1 (Damania & Alwine, 1996; Gruda et al., 1993; Zhai et al., 1997) and some evidence suggests similar interactions for JCV T antigen (Rekvig, 1997). Importantly, initial events of JCV infection occur in the absence of T-antigen protein, suggesting that other factors regulate this process. The amino-terminal portion of Spi-B binds TBP and is capable of recruiting the TFIID complex to promoters (Rao et al., 1999b). Therefore, a potential mechanism for activation of JCV early gene expression in the absence of T protein could involve Spi-B binding to TBP on the JCV promoter/ enhancer and recruitment of the TFIID complex. It has been demonstrated that TBP containing transcription factor complexes form on TATA-less promoters and are required for transcription (Pugh & Tjian, 1991). Therefore, Spi-B bound to the L4 site in the TATA-less second repeat of Mad-4 could be capable of recruiting TBP and TFIID to initiate transcriptional activation in the absence of a TATA box. In this case the L4 site is positioned to compensate for the lack of the TATA box in the Mad-4 variant. In addition to TATA boxes, these areas are bound heavily by other host factors that regulate viral activity. Oct-6/tst- 1/SCIP-binding sites are located adjacent to both the L5 and L4 Spi-B sites on their 59 ends and NF-1-binding sites 3047