Infection by agnoprotein-negative mutants of polyomavirus JC and SV40 results in the release of virions that are mostly deficient in DNA content

<p>Abstract</p> <p>Background</p> <p>Human polyomavirus JC (JCV) is the etiologic agent of a brain disease, known as progressive multifocal leukoencephalopathy (PML). The JCV genome encodes a small multifunctional phospho-protein, agnoprotein, from the late coding region of the virus, whose regulatory functions in viral replication cycle remain elusive. In this work, the functional role of JCV and SV40 agnoproteins in virion release was investigated using a point mutant (Pt) of each virus, where the ATG codon of agnoprotein was mutated to abrogate its expression.</p> <p>Results</p> <p>Analysis of both viral protein expression and replication using Pt mutant of each virus revealed that both processes were substantially down-regulated in the absence of agnoprotein compared to wild-type (WT) virus. Complementation studies in cells, which are constitutively expressing JCV agnoprotein and transfected with the JCV Pt mutant genome, showed an elevation in the level of viral DNA replication near to that observed for WT. Constitutive expression of large T antigen was found to be not sufficient to compensate the loss of agnoprotein for efficient replication of neither JCV nor SV40 in vivo. Examination of the viral release process for both JCV and SV40 Pt mutants showed that viral particles are efficiently released from the infected cells in the absence of agnoprotein but were found to be mostly deficient in viral DNA content.</p> <p>Conclusions</p> <p>The results of this study provide evidence that agnoprotein plays an important role in the polyomavirus JC and SV40 life cycle. Infection by agnoprotein-negative mutants of both viruses results in the release of virions that are mostly deficient in DNA content.</p

Transcriptional regulation of human polyomavirus JC: evidence for a functional interaction between RelA (p65) and the Y-box-binding protein, YB-1.

The transcriptional control region of the human neurotropic polyomavirus JC virus contains a consensus NF-kappa B site which has been shown to enhance both basal and extracellular stimulus-induced levels of transcription of JC promoters. Here, we show that the expression of JC late promoter constructs containing the NF-kappa B site is decreased by cotransfection with the NF-kappa B/rel subunits, p50 and p52, but enhanced by the p65 subunit. However, JC promoter constructs lacking the NF-kappa B site were activated by p52 and p50 and repressed by p65. This antithetical response of the JC promoter mapped specifically to the D domain, which is a target site for the cellular transcription factor, YB-1. Band shift studies indicated that YB-1 and p65 modulate each other's binding to DNA: YB-1 augments the affinity of p65 for the NF-kappa B site, while p65 reduces the binding of YB-1 to the D domain. Results from coimmunoprecipitation followed by Western blot (immunoblot) analysis suggest an in vivo interaction between p65 and YB-1 in glial cells. Functionally, YB-1 appears to act synergistically with p65 to control transcription from the NF-kappa B site. A converse pattern is seen with the D domain, in which YB-1 acts synergistically with p50 and p52 to regulate transcription. p50 and p52 may function as transcriptional activators on the D domain by removing the repressive effect of p65 on YB-1 binding to the D domain. On the basis of these data, we propose a model in which NF-kappa B/rel subunits functionally interact with consensus NF-kappa B sites or YB-1-binding sites, with disparate effects on eukaryotic gene expression

A 23-base-pair sequence element and its role in JC virus gene transcription

JC virus (JCV) is a common human neurotropic polyomavirus and the causative agent of a fatal demyelinating disease, called Progressive Multifocal Leucoencephalopathy (PML). The virus remains latent in healthy individuals but is reactivated in those who exhibit prolonged underlying immunosupression due to immunosupressive drugs, chronic illness, cancers and AIDS. Reactivation of the virus leads to selective lytic infection of oligodentrocytes which are the myelin producing cells of central nervous system (CNS) and subsequently to PML. The regulatory region of JCV is hypervariable and several variants of which have been described. The prototype strain of JCV, Mad-1, is typically isolated from PML patients and is characterized by having two 98 bp tandem repeats within its regulatory region. The archetype strain (Cy) and some intermediate forms of JCV contain a 23 base pair sequence element (23 bpse) in their regulatory region. It is hypothesized that deletions and duplications within the regulatory region of Cy lead to a more vigorous virus capable of replicating in new cells and tissues. The role of the 23 bpse in JCV gene expression is not well understood. My thesis work centers around understanding of its role in this respect. In the following manuscripts, we provide experimental evidence that the 23 bpse play a role in JCV promoter activity. Data from our studies demonstrate that the sequences present within the 23 bpse not only serve as target binding sites for specific cellular factors and but also confer responsiveness to an extracellular stimulus such as PMA. We also investigated the effect of inducible NF-[special characters omitted]B family of transcription factors, p50 and p65, on the transcriptional activity of JCV promoters through the 23 bpse. Results from these experiments suggest that both factors indirectly involve in the transcriptional activity of JCV promoters through the 23 bpse. Previously identified cellular proteins (YB-1 and Pur[special characters omitted]) and a viral regulatory protein, JCV large T antigen (JCV T) were shown to interact with specific target sequences within the control region of JCV and modulate JCV gene transcription from the viral early and late promoters. Since the 23 bpse also confers potential binding target sequences for these proteins, we also investigated their effect on JCV gene transcription. Results from these studies demonstrate that YB-1 and Pur[special characters omitted] physically and functionally interact with each other and modulate JCV gene transcription through the 23 bpse. We also examined the physical and functional interaction between YB-1 and JCV T and its relevance to JCV gene transcription. We observed that JCV T functionally cooperate with YB-1 in transactivation of viral late genes. Taken together, these results suggest that the 23 bpse plays a role in modulation of JCV gene transcription by serving as target binding sites for specific cellular proteins

A Comprehensive Proteomics Analysis of the JC Virus (JCV) Large and Small Tumor Antigen Interacting Proteins: Large T Primarily Targets the Host Protein Complexes with V-ATPase and Ubiquitin Ligase Activities While Small t Mostly Associates with Those Having Phosphatase and Chromatin-Remodeling Functions

The oncogenic potential of both the polyomavirus large (LT-Ag) and small (Sm t-Ag) tumor antigens has been previously demonstrated in both tissue culture and animal models. Even the contribution of the MCPyV tumor antigens to the development of an aggressive human skin cancer, Merkel cell carcinoma, has been recently established. To date, the known primary targets of these tumor antigens include several tumor suppressors such as pRb, p53, and PP2A. However, a comprehensive list of the host proteins targeted by these proteins remains largely unknown. Here, we report the first interactome of JCV LT-Ag and Sm t-Ag by employing two independent &ldquo;affinity purification/mass spectroscopy&rdquo; (AP/MS) assays. The proteomics data identified novel targets for both tumor antigens while confirming some of the previously reported interactions. LT-Ag was found to primarily target the protein complexes with ATPase (v-ATPase and Smc5/6 complex), phosphatase (PP4 and PP1), and ligase (E3-ubiquitin) activities. In contrast, the major targets of Sm t-Ag were identified as Smarca1/6, AIFM1, SdhA/B, PP2A, and p53. The interactions between &ldquo;LT-Ag and SdhB&rdquo;, &ldquo;Sm t-Ag and Smarca5&rdquo;, and &ldquo;Sm t-Ag and SDH&rdquo; were further validated by biochemical assays. Interestingly, perturbations in some of the LT-Ag and Sm t-Ag targets identified in this study were previously shown to be associated with oncogenesis, suggesting new roles for both tumor antigens in novel oncogenic pathways. This comprehensive data establishes new foundations to further unravel the new roles for JCV tumor antigens in oncogenesis and the viral life cycle