A 23-bp Sequence Element from Human Neurotropic JC Virus Is Responsive to NF-κB Subunits

Abstract

The regulatory region of the human neurotropic JC virus (JCV) is composed of several cis-acting motifs that confer cell type specificity to viral gene transcription and enable the viral promoters to respond to extracellular stimuli. For example, the bidirectional 98-bp tandem repeat sequences, positioned between the JCV early and late genes, were shown to be responsible for basal and activated levels of viral gene transcription in central nervous system (CNS) cells. Additionally, the NF-κB site located approximately 75 bp from the repeats on the early side of the viral genome was also found to influence both levels of viral transcription. Recently, we isolated a novel JCV variant, JCVPhila-1, from a clinical specimen that contains a 23-bp sequence element (23-bpse) within its promoter–enhancer region. Here we demonstrate that this element is responsive to an extracellular stimulatory factor, such as phorbol 12-myristate 13-acetate (PMA), and can augment the basal levels of the viral early and to a lesser degree late promoter activities in cells derived from the CNS. The 23-bpse, by associating with nuclear proteins present in uninduced cells, forms a 40-kDa DNA–protein complex. Although no direct correlation between transcriptional enhancement of the JCV promoter by PMA treatment and the level of the 40-kDa DNA–protein complex was observed, results from site-directed mutagenesis indicated that formation of this complex is critical for the transcriptional activation of the viral promoter by PMA. These observations suggested that transcriptional enhancement of the JCV promoter activity upon PMA treatment may be an indirect event and mediated by an intermediary factor(s). In this respect, we demonstrated that overexpression of the inducible NF-κB subunits, p50 and p65, enhanced transcriptional activity of the JCV promoter through the 23-bp region with no evidence for their direct association with the 23-bpse DNA. Of importance, the p50/p65-induced JCV promoter activity requires the nucleotide sequences within the 23-bpse that are critical for the assembly of the 40-kDa DNA–protein complex. Thus, it is likely that the NF-κB subunits, by recruiting the cellular factors such as those associated with the 40-kDa DNA–protein complex, influence the basal level of the viral gene transcription. The implications of these findings with respect to regulation of viral and cellular genomes by extracellular stimuli and NF-κB pathway are discussed.