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

De-Simone FI, Sariyer R, Otalora YL, Yarandi S, Craigie M, Gordon J, Sariyer IK - PLoS ONE (2015)

Bottom Line: This unexpected suppression of T-antigen was mainly associated with the suppression of translational initiation.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Center for Neurovirology, Temple University School of Medicine, 3500 North Broad Street, 7th Floor, Philadelphia, PA, 19140, United States of America.

ABSTRACT

Objective: 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.

Methods and results: 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.

Conclusions: 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.

No MeSH data available.


Related in: MedlinePlus

IFN-γ suppresses JCV propagation in PHFA cells.A. PHFA cells were seeded on 2-well chamber slides and infected with JCV Mad1 at a MOI of 1. At 1, 3, and 5 dpi of post-infections, one group of the infected cells was treated with IFN-γ (100 ng/ml). Only 50% of the growth media of the cells were supplemented with fresh media during the course of each IFN-γ treatment. Cells were fixed at 8 dpi with cold acetone/methanol, and processed by immunocytochemistry for the detection of VP1 expression. B. The percentile of VP1 positive cells from panel A were calculated as fluorescence-forming-unit (FFU) and presented as bar graph. C. PHFA cells were infected with JCV Mad1 at a MOI of 1. At 1, 3, and 5 dpi of post-infections, one group of the infected cells was treated with IFN-γ (100 ng/ml). Whole cell protein lysates were prepared at 8 dpi, and analyzed by Western blotting for the detection of T-antigen and VP1. Tubulin was also probed in the same membranes as loading control. D. The growth media of the cells were also collected from the same infections presented in panel A and B, and processed by Q-PCR analysis for the detection of JCV copy numbers. Bar graph represents JCV copy numbers from three independent infection studies. The growth media of uninfected PHFA cells were also processed by Q-PCR and shown in lane 1 with no positivity for JCV. E. Effect of IFN-γ on cellular viability of PHFA cells. PHFA cells were treated with IFN-γ with the indicated dosages for three times at 0, 24 and 48hrs. MTT cell viability assay was performed at 72hrs post-treatments, and relative MTT activities were presented as bar graph.
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pone.0129694.g009: IFN-γ suppresses JCV propagation in PHFA cells.A. PHFA cells were seeded on 2-well chamber slides and infected with JCV Mad1 at a MOI of 1. At 1, 3, and 5 dpi of post-infections, one group of the infected cells was treated with IFN-γ (100 ng/ml). Only 50% of the growth media of the cells were supplemented with fresh media during the course of each IFN-γ treatment. Cells were fixed at 8 dpi with cold acetone/methanol, and processed by immunocytochemistry for the detection of VP1 expression. B. The percentile of VP1 positive cells from panel A were calculated as fluorescence-forming-unit (FFU) and presented as bar graph. C. PHFA cells were infected with JCV Mad1 at a MOI of 1. At 1, 3, and 5 dpi of post-infections, one group of the infected cells was treated with IFN-γ (100 ng/ml). Whole cell protein lysates were prepared at 8 dpi, and analyzed by Western blotting for the detection of T-antigen and VP1. Tubulin was also probed in the same membranes as loading control. D. The growth media of the cells were also collected from the same infections presented in panel A and B, and processed by Q-PCR analysis for the detection of JCV copy numbers. Bar graph represents JCV copy numbers from three independent infection studies. The growth media of uninfected PHFA cells were also processed by Q-PCR and shown in lane 1 with no positivity for JCV. E. Effect of IFN-γ on cellular viability of PHFA cells. PHFA cells were treated with IFN-γ with the indicated dosages for three times at 0, 24 and 48hrs. MTT cell viability assay was performed at 72hrs post-treatments, and relative MTT activities were presented as bar graph.

Mentions: The observed suppression of T-antigen expression by IFN-γ suggested a possible anti-JCV activity of this cytokine which may be a novel candidate to target the viral lytic infection cycle and treat PML. In order to test potential anti-JCV activity of IFN-γ, we utilized primary human fetal astrocytic (PHFA) cell cultures which are permissive for JCV replication and have been widely utilized by us and others as a cell culture model of JCV infection. PHFA cells were plated in 2-well chamber slides, infected with the Mad1 PML isolate of JCV at an MOI of 1, and treated with IFN-γ (100 ng/ml) at 1, 3, and 5 dpi. Cells were fixed with cold acetone:methanol at 8 dpi, and processed by immunostaining for detection of the viral major capsid protein, VP1. As shown in Fig 9A and 9B, IFN-γ treatment of JCV infected PHFA cells resulted in a significant reduction in the number of the cells expressing VP1. In addition to immunostaining, the impact of IFN-γ treatment on JCV gene expression was also analyzed by Western blotting. As shown in Fig 9C, IFN-γ treatment of PHFA cells infected with JCV suppressed T-antigen expression and resulted in a significant decrease in VP1 levels (Fig 9C). In parallel to VP1 immunostaining and Western blot analysis of viral gene expressions, the growth media harvested from the cells were collected and processed by Q-PCR for the detection of viral copy numbers. Consistent with the decreased levels of the viral gene expression and reduced numbers of the cells infected with JCV, IFN-γ also caused a significant reduction in viral DNA copy numbers as compared to infected controls (Fig 9D). To measure any possible toxicity of IFN-γ treatment, PHFA cells were treated with IFN-γ at different concentrations, and cytotoxicity was determined by MTT viability assay. As shown in Fig 9E, IFN-γ treatment of cells at concentrations ranging from 10 to 200 ng/ml did not have any significant effect on cellular viability. These results have suggested that IFN-γ inhibits JCV infection in glial cells with no toxicity associated, and may be a potential candidate cytokine for the treatment of PML.


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

De-Simone FI, Sariyer R, Otalora YL, Yarandi S, Craigie M, Gordon J, Sariyer IK - PLoS ONE (2015)

IFN-γ suppresses JCV propagation in PHFA cells.A. PHFA cells were seeded on 2-well chamber slides and infected with JCV Mad1 at a MOI of 1. At 1, 3, and 5 dpi of post-infections, one group of the infected cells was treated with IFN-γ (100 ng/ml). Only 50% of the growth media of the cells were supplemented with fresh media during the course of each IFN-γ treatment. Cells were fixed at 8 dpi with cold acetone/methanol, and processed by immunocytochemistry for the detection of VP1 expression. B. The percentile of VP1 positive cells from panel A were calculated as fluorescence-forming-unit (FFU) and presented as bar graph. C. PHFA cells were infected with JCV Mad1 at a MOI of 1. At 1, 3, and 5 dpi of post-infections, one group of the infected cells was treated with IFN-γ (100 ng/ml). Whole cell protein lysates were prepared at 8 dpi, and analyzed by Western blotting for the detection of T-antigen and VP1. Tubulin was also probed in the same membranes as loading control. D. The growth media of the cells were also collected from the same infections presented in panel A and B, and processed by Q-PCR analysis for the detection of JCV copy numbers. Bar graph represents JCV copy numbers from three independent infection studies. The growth media of uninfected PHFA cells were also processed by Q-PCR and shown in lane 1 with no positivity for JCV. E. Effect of IFN-γ on cellular viability of PHFA cells. PHFA cells were treated with IFN-γ with the indicated dosages for three times at 0, 24 and 48hrs. MTT cell viability assay was performed at 72hrs post-treatments, and relative MTT activities were presented as bar graph.
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Related In: Results  -  Collection

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pone.0129694.g009: IFN-γ suppresses JCV propagation in PHFA cells.A. PHFA cells were seeded on 2-well chamber slides and infected with JCV Mad1 at a MOI of 1. At 1, 3, and 5 dpi of post-infections, one group of the infected cells was treated with IFN-γ (100 ng/ml). Only 50% of the growth media of the cells were supplemented with fresh media during the course of each IFN-γ treatment. Cells were fixed at 8 dpi with cold acetone/methanol, and processed by immunocytochemistry for the detection of VP1 expression. B. The percentile of VP1 positive cells from panel A were calculated as fluorescence-forming-unit (FFU) and presented as bar graph. C. PHFA cells were infected with JCV Mad1 at a MOI of 1. At 1, 3, and 5 dpi of post-infections, one group of the infected cells was treated with IFN-γ (100 ng/ml). Whole cell protein lysates were prepared at 8 dpi, and analyzed by Western blotting for the detection of T-antigen and VP1. Tubulin was also probed in the same membranes as loading control. D. The growth media of the cells were also collected from the same infections presented in panel A and B, and processed by Q-PCR analysis for the detection of JCV copy numbers. Bar graph represents JCV copy numbers from three independent infection studies. The growth media of uninfected PHFA cells were also processed by Q-PCR and shown in lane 1 with no positivity for JCV. E. Effect of IFN-γ on cellular viability of PHFA cells. PHFA cells were treated with IFN-γ with the indicated dosages for three times at 0, 24 and 48hrs. MTT cell viability assay was performed at 72hrs post-treatments, and relative MTT activities were presented as bar graph.
Mentions: The observed suppression of T-antigen expression by IFN-γ suggested a possible anti-JCV activity of this cytokine which may be a novel candidate to target the viral lytic infection cycle and treat PML. In order to test potential anti-JCV activity of IFN-γ, we utilized primary human fetal astrocytic (PHFA) cell cultures which are permissive for JCV replication and have been widely utilized by us and others as a cell culture model of JCV infection. PHFA cells were plated in 2-well chamber slides, infected with the Mad1 PML isolate of JCV at an MOI of 1, and treated with IFN-γ (100 ng/ml) at 1, 3, and 5 dpi. Cells were fixed with cold acetone:methanol at 8 dpi, and processed by immunostaining for detection of the viral major capsid protein, VP1. As shown in Fig 9A and 9B, IFN-γ treatment of JCV infected PHFA cells resulted in a significant reduction in the number of the cells expressing VP1. In addition to immunostaining, the impact of IFN-γ treatment on JCV gene expression was also analyzed by Western blotting. As shown in Fig 9C, IFN-γ treatment of PHFA cells infected with JCV suppressed T-antigen expression and resulted in a significant decrease in VP1 levels (Fig 9C). In parallel to VP1 immunostaining and Western blot analysis of viral gene expressions, the growth media harvested from the cells were collected and processed by Q-PCR for the detection of viral copy numbers. Consistent with the decreased levels of the viral gene expression and reduced numbers of the cells infected with JCV, IFN-γ also caused a significant reduction in viral DNA copy numbers as compared to infected controls (Fig 9D). To measure any possible toxicity of IFN-γ treatment, PHFA cells were treated with IFN-γ at different concentrations, and cytotoxicity was determined by MTT viability assay. As shown in Fig 9E, IFN-γ treatment of cells at concentrations ranging from 10 to 200 ng/ml did not have any significant effect on cellular viability. These results have suggested that IFN-γ inhibits JCV infection in glial cells with no toxicity associated, and may be a potential candidate cytokine for the treatment of PML.

Bottom Line: This unexpected suppression of T-antigen was mainly associated with the suppression of translational initiation.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Center for Neurovirology, Temple University School of Medicine, 3500 North Broad Street, 7th Floor, Philadelphia, PA, 19140, United States of America.

ABSTRACT

Objective: 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.

Methods and results: 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.

Conclusions: 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.

No MeSH data available.


Related in: MedlinePlus