Journal of General Virology (2010), 91, 3042–3052 DOI 10.1099/vir.0.023184-0 Correspondence Eugene O. Major majorg@ninds.nih.gov Received 30 April 2010 Accepted 2 September 2010 Transcription factor Spi-B binds unique sequences present in the tandem repeat promoter/enhancer of JC virus and supports viral activity Leslie J. Marshall, Lisa Dunham and Eugene O. Major Laboratory of Molecular Medicine and Neuroscience, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892-1296, USA Progressive multifocal leukoencephalopathy (PML) is an often fatal demyelinating disease caused by lytic infection of oligodendrocytes with JC virus (JCV). The development of PML in non- immunosuppressed individuals is a growing concern with reports of mortality in patients treated with mAb therapies. JCV can persist in the kidneys, lymphoid tissue and bone marrow. JCV gene expression is restricted by non-coding viral regulatory region sequence variation and cellular transcription factors. Because JCV latency has been associated with cells undergoing haematopoietic development, transcription factors previously reported as lymphoid specific may regulate JCV gene expression. This study demonstrates that one such transcription factor, Spi-B, binds to sequences present in the JCV promoter/enhancer and may affect early virus gene expression in cells obtained from human brain tissue. We identified four potential Spi-B-binding sites present in the promoter/enhancer elements of JCV sequences from PML variants and the non-pathogenic archetype. Spi-B sites present in the promoter/enhancers of PML variants alone bound protein expressed in JCV susceptible brain and lymphoid-derived cell lines by electromobility shift assays. Expression of exogenous Spi-B in semi- and non-permissive cells increased early viral gene expression. Strikingly, mutation of the Spi-B core in a binding site unique to the Mad-4 variant was sufficient to abrogate viral activity in progenitor-derived astrocytes. These results suggest that Spi-B could regulate JCV gene expression in susceptible cells, and may play an important role in JCV activity in the immune and nervous systems. INTRODUCTION The human polyomavirus JC is the aetiological agent of the fatal demyelinating disease, progressive multifocal leukoen- cephalopathy (PML), that occurs in patients in states of immune suppression or modulation. PML is an AIDS- defining illness that occurs in approximately 3 % of HIV- infected patients (Major, 2010). Recently, PML has been reported in patients undergoing immuno-modulatory therapies (Major, 2010; Marshall & Major, 2010). PML is caused by lytic multiplication of the JC virus (JCV) in oligodendrocytes, which results in the loss of myelination of neurons and loss of neuronal function. Despite its lytic capability, JCV can persist in a variety of cell types including CD34+ haematopoietic precursors and B cells present in bone marrow (Houff et al., 1988; Marzocchetti et al., 2008; Tan et al., 2009), brain, tonsil and in circulation (Sabath & Major, 2002). Detection of JCV DNA in the absence of viral capsid protein in these cell types from patients prior to the development of PML suggests that such cells may act as a reservoir for latent virus (Houff et al., 1988; Tan et al., 2009). Trafficking of JCV-infected B cells, or haematopoietic precursors, 3042 between the bone marrow and brain is a possible method of viral dissemination. The JCV genome is a closed, circular, supercoiled DNA chromosome that permits temporal expression of early and late genes that are physically separated by the non-coding viral regulatory region (RR) (Frisque et al., 1984). RR is the most highly variable sequence among JCV isolates but always contains the origin of replication, one or more TATA boxes and a variety of enhancer elements (Ault & Stoner, 1993; Frisque, 1983; Frisque et al., 1984; Iida et al., 1993). The RR from the original isolate of JCV, Mad-1, contains a promoter/enhancer that exists as 98 bp tandem repeats containing two TATA boxes and multiple cellular transcription factor-binding sites (Frisque, 1983). TATA boxes in the tandem repeat sequence are essential for transcription of early and late viral genes (Daniel & Frisque, 1993; Kenney et al., 1986a, b; Khalili et al., 1986; Krebs et al., 1995; Vacante et al., 1989). RR variants containing tandem repeats have been isolated from tissues of patients with PML (Martin et al., 1985). The RR of a naturally occurring variant of JCV shed in urine, referred to as ‘archetype’, contains a single 98 bp unit with internal 023184 Printed in Great Britain