Human JC polyomavirus (JCV) is the etiologic agent of the neurodegenerative disease progressive multifocal leukoencephalopathy. By using JCV as a model, we investigated the role of the viral early protein tumor antigen (TAg) in the binding of two cellular proteins, Pura and YB-1, to JCV regulatory sequences. Results from band-shift assays with purified YB-1, Purca, and TAg indicated that efficient binding of Pura, a strong activator ofearly gene transcription, to a single-stranded target sequence corresponding to the viral lytic control element, is diminished in the presence of the late gene activator YB-1, which recognizes the opposite strand of the Pura binding site. Of particular interest was the ability of Pura and TAg to enhance binding ofYB-1 to DNA molecules without being associated with this complex. Binding studies using a mutant peptide encompassing the N terminus ofYB-1 indicated that the C terminus ofYB-1 is important for its DNA binding activity. The ability of Purax and TAg to increase binding ofYB-1 to DNA is independent ofthe YB-1 C terminus. Similarly, results from band-shift assays using Pura variants indicated that two distinct regions of this protein contribute either to its ability to bind DNA or to its ability to enhance YB-1 DNA binding activity. Based on the interaction of Pura, YB-1, and TAg, and their binding to DNA, a model is proposed for the role of these proteins in transcription ofviral early and late genes during the lytic cycle.to cell-specific transcriptional factors, other regulatory proteins found in various cells and tissues are believed to be critical in facilitating viral gene expression during the lytic cycle (13-18). Results from this (10-12, 17, 19) and other (9,20) laboratories have indicated that the 98-bp enhancer/promoter sequence of JCV encompasses multiple cis-regulatory elements that interact specifically with nuclear proteins and modulate viral early and late gene transcription. A region designated the lytic control element (LCE) in the 98-bp repeat proximal to the origin of DNA replication contains a pentanucleotide repeat sequence, AGGGAAGGGA, juxtaposed to a poly(dT-dA) tract that displays a single-stranded configuration (21). This motif differentially affects viral gene expression by positively and negatively modulating early and late promoter transcription (17,19) and plays an important role in viral DNA replication (22,23). In this study we demonstrate that two distinct cellular proteins, Pura and YB-1, activators for the early and late gene transcription of JCV, respectively, modulate each other's binding to the LCE. Moreover, we show that the viral early protein TAg is capable of enhancing binding of YB-1 to DNA molecules. We propose a working model for the potential role of LCE-binding proteins and the viral TAg in programming the early-to-late shift of the viral gene transcription during the lytic cycle.

Initiation of polyomavirus DNA replication in eukaryotic cells requires the participation of the viral early protein T antigen, cellular replication factors, and DNA polymerases. The human polyomavirus JC virus (JCV) is the etiologic agent of the fatal demyelinating disease progressive multifocal leukoencephalopathy in immunocompromised individuals. This virus exhibits a narrow host range and a tissue specificity that restricts its replication to glial cells of the central nervous system. Restriction of viral DNA replication due to species specificity of the DNA polymerase, coupled with glial cell-specific transcription of the viral early promoter, is thought to account for the brain-specific replication of JCV. In this report we demonstrate that overexpression of Pur ␣, a protein which binds to single-stranded DNA in a sequence-specific manner, suppresses replication of JCV DNA in glial cells. Results from footprinting studies indicate that Pur ␣ and T antigen share a common binding region spanning the single-stranded ori sequence of JCV. Further, T antigen was capable of stimulating the association of Pur ␣ with the ori sequence in a band shift assay. Whereas no evidence for simultaneous binding of Pur ␣ and T antigen to single-stranded DNA has been observed, results from coimmunoprecipitation and Western blot (immunoblot) analyses of proteins derived from cells producing JCV T antigen indicate a molecular association of JCV T antigen and Pur ␣. The binding of Pur ␣ to the single-stranded ori sequence and its association with T antigen suggest that Pur ␣ interferes with the activity of T antigen and/or other regulatory proteins to exert its negative effect on JCV DNA replication. The importance of these findings in the reactivation of JCV in the latently infected individual under immunosuppressed conditions is discussed.

We have isolated a partial recombinant cDNA clone from a HeLa expression library which encodes a protein capable of binding to the central region of the human neurotropic JC virus (JCV) enhancer/promoter, termed the B region. Sequence analysis revealed a complete homology of the partial cDNA clone to the N-terminal region of a previously described DNA-binding protein, termed YB-1. Band shift analyses have indicated that the bacterially produced YB-1 interacts specifically with the double-stranded B oligonucleotide as well as the corresponding single-stranded DNA fragment representing the early promoter sequence. Further analysis indicated that the YB-1 protein binds specifically to the C/T-rich sequence of the B domain, which is located in close proximity to the TATA box within the virus enhancer/promoter. Results from cotransfection experiments demonstrated that the full-length (YB-1) but not the partial cDNA enhances expression of the JCV late (JCVL) promoter in glial cells. Cointroduction into glial cells of a recombinant expressing the YB-1 and JCVL deletion mutants indicated that removal of the C/T-rich sequence of the B domain reduces the level of activation of the virus promoter by YB-1. Further cotransfection experiments revealed that the virus transactivating protein T antigen appears to diminish the ability of YB-1 to activate JCVL gene expression. RNA studies indicated that YB-1 is expressed in several cell types and tissues. Examination of YB-1 RNA from mouse brain at various stages of development revealed high levels of YB-1 RNA at early stages of development and lower levels at all subsequent developmental stages.

The multifunctional protein of papovaviruses, Tantigen, regulates the virus lytic cycle partly by exerting transcriptional control over viral and cellular gene expression. In this study, the ability of the T-antigen of human neurotropic JC virus (JCV) to enhance expression from the virus late promoter has been further examined. By deletion analysis, a T-antigen-responsive region was mapped within the JCV 98 bp enhancer/promoter between nucleotides 139 and 168. Interestingly, T-antigen appears to mediate transactivation by increasing expression from a basal transcriptional initiation site and through a novel T-antigen-dependent initiation site (TADI). The TADI element contains a region hom-