The agnoprotein of polyomavirus JC is released by infected cells: Evidence for Its cellular uptake by uninfected neighboring cells

AbstractPoliomavirus JC replicates in glial cells in the brain, and causes the fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML). PML is usually seen in patients with underlying immunocompromised conditions, notably among AIDS patients and those on chronic immunosuppressive regimens. The late leader sequence of JC virus contains an open reading frame encoding a small regulatory protein called agnoprotein. Agnoprotein contributes to progressive viral infection by playing significant roles in viral replication cycle. Here, we demonstrate that agnoprotein can be detected in cell-free fractions of glial cultures infected with JCV, transfected with expression plasmids or transduced with an adenovirus expression system. We also provide evidence that extracellular agnoprotein can be taken up by uninfected neighboring cells. These studies have revealed a novel phenomenon of agnoprotein during the viral life cycle with a potential of developing diagnostic and therapeutic interventions

IFN-Gamma Inhibits JC Virus Replication in Glial Cells by Suppressing T-Antigen Expression.

Patients undergoing immune modulatory therapies for the treatment of autoimmune diseases such as multiple sclerosis, and individuals with an impaired-immune system, most notably AIDS patients, are in the high risk group of developing progressive multifocal leukoencephalopathy (PML), an often lethal disease of the brain characterized by lytic infection of oligodendrocytes in the central nervous system (CNS) with JC virus (JCV). The immune system plays an important regulatory role in controlling JCV reactivation from latent sites by limiting viral gene expression and replication. However, little is known regarding the molecular mechanisms responsible for this regulation.Here, we investigated the impact of soluble immune mediators secreted by activated PBMCs on viral replication and gene expression by cell culture models and molecular virology techniques. Our data revealed that viral gene expression and viral replication were suppressed by soluble immune mediators. Further studies demonstrated that soluble immune mediators secreted by activated PBMCs inhibit viral replication induced by T-antigen, the major viral regulatory protein, by suppressing its expression in glial cells. This unexpected suppression of T-antigen was mainly associated with the suppression of translational initiation. Cytokine/chemokine array studies using conditioned media from activated PBMCs revealed several candidate cytokines with possible roles in this regulation. Among them, only IFN-γ showed a robust inhibition of T-antigen expression. While potential roles for IFN-β, and to a lesser extent IFN-α have been described for JCV, IFN-γ has not been previously implicated. Further analysis of IFN-γ signaling pathway revealed a novel role of Jak1 signaling in control of viral T-antigen expression. Furthermore, IFN-γ suppressed JCV replication and viral propagation in primary human fetal glial cells, and showed a strong anti-JCV activity.Our results suggest a novel role for IFN-γ in the regulation of JCV gene expression via downregulation of the major viral regulatory protein, T-antigen, and provide a new avenue of research to understand molecular mechanisms for downregulation of viral reactivation that may lead to development of novel strategies for the treatment of PML

Immune regulators secreted by activated PBMCs inhibit JCV genomic DNA replication mediated by T-antigen.

<p>A. JCV genomic DNA replication initiated by Large T-antigen is suppressed by immune mediators secreted by activated PBMCs. PHFA cells were transiently transfected with pBLCAT3-Mad1-Early reporter gene construct contains origin of viral replication and pCDNA3.1- T-antigen expression plasmids. The culture media of the cells were supplemented with conditioned medium obtained from either uninduced or induced PBMCs at a ratio of 50%. Cells were harvested at 72hrs post-transfections, and cell pellets were divided into half. One portion of the cell pellets was utilized for the purification of low molecular weight plasmid DNAs. The DNA samples were first digested with DpnI/BamHI restriction enzymes and separated on 1% agarose gel by electrophoresis. Southern blot analysis of replicated plasmid DNA was performed by DIG-High Prime DNA Labeling and Detection Starter Kit (Roche, US) as described by the Manufacturer. In lane 1, kb-ladder was loaded as molecular weight marker. In lane 2, pBLCAT3-JCV-early plasmid was first linearized with BamHI digestion and loaded as positive control (3ng). Lane 3 was just sample buffer. B. The second half portion of the cell pellets from experiments presented in panel A was used for the preparation of whole cell protein lysates, and western blot analysis were performed for the detection of T-antigen and tubulin expressions. The bar graph represents the normalized expression of T-antigen to tubulin.</p

Neither autophagy nor proteasomes are involved in neuroimmune suppression of T-antigen expression.

<p>A. T98G cells were transfected with pCDNA 3.1 plasmid encoding T-antigen. Cells were treated with conditioned media obtained from either uninduced or induced PBMCs inn the presence or absence of Bafilomycin A (1uM). Whole cell protein extracts were prepared, and processed for the detection of T-antigen, P62, and LC3 by western blotting. The same membranes were stripped off and probed for tubulin as loading control. B. T98G cells were transfected with pCDNA 3.1 encoding T-antigen. Cells were treated with either CM-induced or uninduced in the absence of DMSO or MG115 (10uM) or MG132 (10uM) as indicated. Whole cell protein lysates were prepared and expression of T-antigen was detected by western blotting. Same membranes were stripped off and probed for tubulin as loading control.</p

Soluble Immune mediators secreted by activated PBMCs limit translation initiation in glial cells.

<p>A. PHFA cells were transfected with an expression plasmid encoding T-antigen and treated with conditioned media from either uninduced or induced PBMCs. Western bot analysis of protein extracts were performed to assess phosphorylated and total protein levels of p70S6K, 4EBP1, AKT, p44/42 MAPK and mTOR. Tubulin was probed as loading control in the same membranes. B. Normalized expressions of phosphor-p70S6K, phosphor-4E-BP1, and phosphor-AKT are shown as bar graph. The phosphorylated protein signals were normalized to total protein levels of each protein from the experiments presented in panel A.</p

Cytokine array with conditioned media from uninduced and induced PBMCs.

<p><b>A</b>. Cytokine profiles of conditioned media from either uninduced (CM-Uninduced) or induced (CM-Induced) PBMCs were compared by cytokine arrays as described by the Manufacturer (RayBiotech, Human Cytokine Array C1, CODE: AAH-CYT-1) Red boxes denote the cytokines with increased levels in CM-induced compared to CM-uninduced. Green boxes denote the cytokines with decreased levels in CM-induced compared to CM-uninduced. B. Quantification of relative expression levels of selected cytokines from the cytokine array studies are represented as bar graph with relative expression.</p

Immune mediated suppression of JC virus propagation in glial cells.

<p>A. PHFA cells were transfected/infected with the Mad1 strain of JC virus as described in Materials and Methods. At 48h post-infection, culture media of the infected cells were changed with fresh media consist of 1/1 ratio of PHFA media and conditioned media obtained from either uninduced (CM-unind.) or induced (CM-Ind.) PBMCs. The treatment was repeated at 4, 8, and 12 day post-infections. Whole cell protein lysates were prepared at 8 and 15dpi, and analyzed by Western blotting for the detection of T-antigen and VP1. B. Bar graph representation of relative T-antigen and VP1 expression normalized to tubulin from panel A. C. Q-PCR analysis of viral DNA copies in growth media of PHFA cells infected with JCV. Growth media was collected in parallel to whole cell protein extracts from the infections presented in panel B. JCV DNA copies in culture media was analyzed by Q-PCR as described in Materials and Methods.</p

Neuroimmune suppression of T-antigen by IFN-γ is mediated by Jak signaling.

<p><b>A</b>. PHFA cells were transfected with pCDNA 3.1 plasmid encoding T-antigen. Cells were treated with either CM-induced or uninduced, and also incubated with a Jak inhibitor (EMD Millipore, #420099, 500 nM). Whole cell protein lysates were processed by Western blot for the detection of T-antigen, phosphorylated-Stat1, and total-Stat1 expressions. B. Normalized T-antigen expression signal from the experiments shown in panel A were represented as bar graph. C. PHFA cells were transfected with pCDNA 3.1 plasmid encoding T-antigen. Cells were treated with IFN-γ and also incubated with the Jak inhibitor. Whole cell protein lysates were processed by Western blot for the detection of T-antigen, Jak1, Jak2, phospho-Stat1, and total-Stat1 expressions. D. Normalized T-antigen expression signal from the experiments shown in panel C were represented as bar graph.</p

Soluble immune mediators suppress T-antigen protein levels in glial cells.

<p>A. T98G cells were transfected with an expression plasmid encoding T-antigen, and directly treated with conditioned media (50% volume) from either uninduced or induced PBMCs obtained from three different donors. Whole cell protein lysates were analyzed by Western blot for T-antigen and tubulin expression. B. Bar graph representation of T-antigen expression normalized to tubulin from panel A. C. Northern blot analysis of JCV-early transcript. T98G cells were transfected with pCDNA3.1- T-antigen expression plasmid which encodes all the possible early genes expressed by alternative splicing including T-antigen and sm t-antigen antigens. The culture media of the cells were supplemented with conditioned media obtained from either uninduced or induced PBMCs at a ratio of 50%. Cells were harvested and for the extraction of total RNA analyzed by Northern blot for the detection of JCV-early mRNA by using DIG Northern Starter Kit as described by the Manufacturer (Roche, US). D. Soluble immune mediators secreted by induced PBMCs do not effect expression of JCV early mRNAs. Total RNA samples from the same experiments presented in Panel C were used for cDNA synthesis by reverse transcriptase reaction. JCV-early region gene products (pre-mRNA, sm t-antigen, and T-antigen) were amplified and separated on a 3% agarose gel and stained with ethidium bromide. Lane 2 was pcDNA3.1-T-antigen plasmid DNA amplified as positive control of unspliced pre-mRA. Lane 3 was untransfected PHFA cell extracts used as a negative control. Schematic structure of JCV early region unspliced and spliced RNAs and, the size of the expected amplification products with a primer set (PF and PR), used for the amplification of JCV gene products are schematized at the top panel. Band intensities of pre-mRNA and mRNA for T-antigen in lanes 3–5 were quantified and presented as bar graph.</p