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Novel cell type-specific antiviral mechanism of interferon gamma action in macrophages.

Presti RM, Popkin DL, Connick M, Paetzold S, Virgin HW - J. Exp. Med. (2001)

Bottom Line: IFN-gamma inhibited MCMV replication in a signal transducer and activator of transcription (STAT)-1alpha-dependent manner much more effectively in BMMphi (approximately 100-fold) than MEF (5-10-fold).IFN-gamma inhibition of MCMV growth was independent of known mechanisms involving IFN-alpha/beta, tumor necrosis factor alpha, inducible nitric oxide synthase, protein kinase RNA activated (PKR), RNaseL, and Mx1, and did not involve IFN-gamma-induced soluble mediators.These studies in primary cells define a novel mechanism of IFN-gamma action restricted to Mphi, a cell type key for MCMV pathogenesis and latency.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Immunology and the Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

ABSTRACT
Interferon (IFN)-gamma and macrophages (Mphi) play key roles in acute, persistent, and latent murine cytomegalovirus (MCMV) infection. IFN-gamma mechanisms were compared in embryonic fibroblasts (MEFs) and bone marrow Mphi (BMMphi). IFN-gamma inhibited MCMV replication in a signal transducer and activator of transcription (STAT)-1alpha-dependent manner much more effectively in BMMphi (approximately 100-fold) than MEF (5-10-fold). Although initial STAT-1alpha activation by IFN-gamma was equivalent in MEF and BMMphi, microarray analysis demonstrated that IFN-gamma regulates different sets of genes in BMMphi compared with MEFs. IFN-gamma inhibition of MCMV growth was independent of known mechanisms involving IFN-alpha/beta, tumor necrosis factor alpha, inducible nitric oxide synthase, protein kinase RNA activated (PKR), RNaseL, and Mx1, and did not involve IFN-gamma-induced soluble mediators. To characterize this novel mechanism, we identified the viral targets of IFN-gamma action, which differed in MEF and BMMphi. In BMMphi, IFN-gamma reduced immediate early 1 (IE1) mRNA during the first 3 h of infection, and significantly reduced IE1 protein expression for 96 h. Effects of IFN-gamma on IE1 protein expression were independent of RNaseL and PKR. In contrast, IFN-gamma had no significant effects on IE1 protein or mRNA expression in MEFs, but did decrease late gene mRNA expression. These studies in primary cells define a novel mechanism of IFN-gamma action restricted to Mphi, a cell type key for MCMV pathogenesis and latency.

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IFN-γ inhibits MCMV replication in both BMMϕ and MEFs by decreasing viral yield per cell rather than decreasing the number of infected cells. (A) BMMϕ or MEFs were treated with or without 100 U/ml IFN-γ for 48 h, infected at an MOI of 1 for 2 h on ice, and then incubated for 2 h at 37°C to allow internalization of the virus. Cells were washed, serially diluted, and the frequency of productively infected cells determined. Data shown is the percentage of wells with cytopathic effect (24 wells/dilution/experiment) from one of two experiments with similar results. (B–D) BMMϕ were treated with medium (B) or 100 U/ml IFN-γ (C), infected at an MOI of 1 for 1 h at 4°C, or mock infected. Cells were stained with an IE1-specific mAb (B and C) or an isotype-matched control mAb (D), a FITC-conjugated secondary Ab, and counterstained with bisbenzimide. Data shown are double exposures of bisbenzimide staining (converted from the blue to red plane) and FITC staining. IE1-positive cells can be visualized as light yellow nuclei. Shown is a representative one of four experiments. For this experiment shown, two independent, randomly selected regions of each of two slides for each condition were photographed and counted. 21% (252 of 1,206 cells counted) of media-treated BMMϕ (B) expressed nuclear IE1 protein, whereas 17% (136 of 792 cells counted) of IFN-γ–treated BMMϕ (C) expressed nuclear IE1 protein. Infected cells stained with the isotype-matched mAb, HI-gamma-1-109.3 specific for DNP, were uniformly red (shown are IFN-γ–treated infected BMMϕ (D). Mock-infected cells stained with either mAb specific for IE1 or control mAB were indistinguishable from D.
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Figure 6: IFN-γ inhibits MCMV replication in both BMMϕ and MEFs by decreasing viral yield per cell rather than decreasing the number of infected cells. (A) BMMϕ or MEFs were treated with or without 100 U/ml IFN-γ for 48 h, infected at an MOI of 1 for 2 h on ice, and then incubated for 2 h at 37°C to allow internalization of the virus. Cells were washed, serially diluted, and the frequency of productively infected cells determined. Data shown is the percentage of wells with cytopathic effect (24 wells/dilution/experiment) from one of two experiments with similar results. (B–D) BMMϕ were treated with medium (B) or 100 U/ml IFN-γ (C), infected at an MOI of 1 for 1 h at 4°C, or mock infected. Cells were stained with an IE1-specific mAb (B and C) or an isotype-matched control mAb (D), a FITC-conjugated secondary Ab, and counterstained with bisbenzimide. Data shown are double exposures of bisbenzimide staining (converted from the blue to red plane) and FITC staining. IE1-positive cells can be visualized as light yellow nuclei. Shown is a representative one of four experiments. For this experiment shown, two independent, randomly selected regions of each of two slides for each condition were photographed and counted. 21% (252 of 1,206 cells counted) of media-treated BMMϕ (B) expressed nuclear IE1 protein, whereas 17% (136 of 792 cells counted) of IFN-γ–treated BMMϕ (C) expressed nuclear IE1 protein. Infected cells stained with the isotype-matched mAb, HI-gamma-1-109.3 specific for DNP, were uniformly red (shown are IFN-γ–treated infected BMMϕ (D). Mock-infected cells stained with either mAb specific for IE1 or control mAB were indistinguishable from D.

Mentions: To determine why IFN-γ is more effective at controlling MCMV growth in BMMϕ than MEFs, we analyzed the effect of IFN-γ on sequential stages in the MCMV replication cycle in MEFs and BMMϕ. We first determined the frequency of cells productively infected with MCMV using limiting dilution analysis and a MEF indicator monolayer previously shown to detect 1–10 PFU of MCMV even in the presence of IFN-γ 735. IFN-γ did not alter the frequency of productively infected MEFs (Fig. 6 A). However, 100 U/ml of IFN-γ decreased the frequency of productively infected BMMϕ two- to fourfold (Fig. 6 A). In the absence of an IFN-γ–induced decrease in MCMV yield per infected cell, this difference would not explain the 100-fold decline in MCMV titer caused by the same dose of IFN-γ in BMMϕ (Fig. 1). We next quantitated IFN-γ effects on the frequency of BMMϕ expressing the IE1 protein by immunofluorescence. IFN-γ did not significantly change the frequency of BMMϕ expressing the IE1 protein (Fig. 6). This ruled out significant effects of IFN-γ on viral binding, internalization, and the frequency of cells expressing IE1 protein as an explanation for the antiviral effects of IFN-γ in BMMϕ. However, we did notice that immunofluorescent staining for IE1 was qualitatively fainter in IFN-γ–treated than control BMMϕ (see below). Results from limiting dilution analysis and staining for IE1 protein demonstrated that IFN-γ effects on MCMV titer were due to changes in the frequency of productively infected cells and viral yield per infected cell.


Novel cell type-specific antiviral mechanism of interferon gamma action in macrophages.

Presti RM, Popkin DL, Connick M, Paetzold S, Virgin HW - J. Exp. Med. (2001)

IFN-γ inhibits MCMV replication in both BMMϕ and MEFs by decreasing viral yield per cell rather than decreasing the number of infected cells. (A) BMMϕ or MEFs were treated with or without 100 U/ml IFN-γ for 48 h, infected at an MOI of 1 for 2 h on ice, and then incubated for 2 h at 37°C to allow internalization of the virus. Cells were washed, serially diluted, and the frequency of productively infected cells determined. Data shown is the percentage of wells with cytopathic effect (24 wells/dilution/experiment) from one of two experiments with similar results. (B–D) BMMϕ were treated with medium (B) or 100 U/ml IFN-γ (C), infected at an MOI of 1 for 1 h at 4°C, or mock infected. Cells were stained with an IE1-specific mAb (B and C) or an isotype-matched control mAb (D), a FITC-conjugated secondary Ab, and counterstained with bisbenzimide. Data shown are double exposures of bisbenzimide staining (converted from the blue to red plane) and FITC staining. IE1-positive cells can be visualized as light yellow nuclei. Shown is a representative one of four experiments. For this experiment shown, two independent, randomly selected regions of each of two slides for each condition were photographed and counted. 21% (252 of 1,206 cells counted) of media-treated BMMϕ (B) expressed nuclear IE1 protein, whereas 17% (136 of 792 cells counted) of IFN-γ–treated BMMϕ (C) expressed nuclear IE1 protein. Infected cells stained with the isotype-matched mAb, HI-gamma-1-109.3 specific for DNP, were uniformly red (shown are IFN-γ–treated infected BMMϕ (D). Mock-infected cells stained with either mAb specific for IE1 or control mAB were indistinguishable from D.
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Figure 6: IFN-γ inhibits MCMV replication in both BMMϕ and MEFs by decreasing viral yield per cell rather than decreasing the number of infected cells. (A) BMMϕ or MEFs were treated with or without 100 U/ml IFN-γ for 48 h, infected at an MOI of 1 for 2 h on ice, and then incubated for 2 h at 37°C to allow internalization of the virus. Cells were washed, serially diluted, and the frequency of productively infected cells determined. Data shown is the percentage of wells with cytopathic effect (24 wells/dilution/experiment) from one of two experiments with similar results. (B–D) BMMϕ were treated with medium (B) or 100 U/ml IFN-γ (C), infected at an MOI of 1 for 1 h at 4°C, or mock infected. Cells were stained with an IE1-specific mAb (B and C) or an isotype-matched control mAb (D), a FITC-conjugated secondary Ab, and counterstained with bisbenzimide. Data shown are double exposures of bisbenzimide staining (converted from the blue to red plane) and FITC staining. IE1-positive cells can be visualized as light yellow nuclei. Shown is a representative one of four experiments. For this experiment shown, two independent, randomly selected regions of each of two slides for each condition were photographed and counted. 21% (252 of 1,206 cells counted) of media-treated BMMϕ (B) expressed nuclear IE1 protein, whereas 17% (136 of 792 cells counted) of IFN-γ–treated BMMϕ (C) expressed nuclear IE1 protein. Infected cells stained with the isotype-matched mAb, HI-gamma-1-109.3 specific for DNP, were uniformly red (shown are IFN-γ–treated infected BMMϕ (D). Mock-infected cells stained with either mAb specific for IE1 or control mAB were indistinguishable from D.
Mentions: To determine why IFN-γ is more effective at controlling MCMV growth in BMMϕ than MEFs, we analyzed the effect of IFN-γ on sequential stages in the MCMV replication cycle in MEFs and BMMϕ. We first determined the frequency of cells productively infected with MCMV using limiting dilution analysis and a MEF indicator monolayer previously shown to detect 1–10 PFU of MCMV even in the presence of IFN-γ 735. IFN-γ did not alter the frequency of productively infected MEFs (Fig. 6 A). However, 100 U/ml of IFN-γ decreased the frequency of productively infected BMMϕ two- to fourfold (Fig. 6 A). In the absence of an IFN-γ–induced decrease in MCMV yield per infected cell, this difference would not explain the 100-fold decline in MCMV titer caused by the same dose of IFN-γ in BMMϕ (Fig. 1). We next quantitated IFN-γ effects on the frequency of BMMϕ expressing the IE1 protein by immunofluorescence. IFN-γ did not significantly change the frequency of BMMϕ expressing the IE1 protein (Fig. 6). This ruled out significant effects of IFN-γ on viral binding, internalization, and the frequency of cells expressing IE1 protein as an explanation for the antiviral effects of IFN-γ in BMMϕ. However, we did notice that immunofluorescent staining for IE1 was qualitatively fainter in IFN-γ–treated than control BMMϕ (see below). Results from limiting dilution analysis and staining for IE1 protein demonstrated that IFN-γ effects on MCMV titer were due to changes in the frequency of productively infected cells and viral yield per infected cell.

Bottom Line: IFN-gamma inhibited MCMV replication in a signal transducer and activator of transcription (STAT)-1alpha-dependent manner much more effectively in BMMphi (approximately 100-fold) than MEF (5-10-fold).IFN-gamma inhibition of MCMV growth was independent of known mechanisms involving IFN-alpha/beta, tumor necrosis factor alpha, inducible nitric oxide synthase, protein kinase RNA activated (PKR), RNaseL, and Mx1, and did not involve IFN-gamma-induced soluble mediators.These studies in primary cells define a novel mechanism of IFN-gamma action restricted to Mphi, a cell type key for MCMV pathogenesis and latency.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Immunology and the Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

ABSTRACT
Interferon (IFN)-gamma and macrophages (Mphi) play key roles in acute, persistent, and latent murine cytomegalovirus (MCMV) infection. IFN-gamma mechanisms were compared in embryonic fibroblasts (MEFs) and bone marrow Mphi (BMMphi). IFN-gamma inhibited MCMV replication in a signal transducer and activator of transcription (STAT)-1alpha-dependent manner much more effectively in BMMphi (approximately 100-fold) than MEF (5-10-fold). Although initial STAT-1alpha activation by IFN-gamma was equivalent in MEF and BMMphi, microarray analysis demonstrated that IFN-gamma regulates different sets of genes in BMMphi compared with MEFs. IFN-gamma inhibition of MCMV growth was independent of known mechanisms involving IFN-alpha/beta, tumor necrosis factor alpha, inducible nitric oxide synthase, protein kinase RNA activated (PKR), RNaseL, and Mx1, and did not involve IFN-gamma-induced soluble mediators. To characterize this novel mechanism, we identified the viral targets of IFN-gamma action, which differed in MEF and BMMphi. In BMMphi, IFN-gamma reduced immediate early 1 (IE1) mRNA during the first 3 h of infection, and significantly reduced IE1 protein expression for 96 h. Effects of IFN-gamma on IE1 protein expression were independent of RNaseL and PKR. In contrast, IFN-gamma had no significant effects on IE1 protein or mRNA expression in MEFs, but did decrease late gene mRNA expression. These studies in primary cells define a novel mechanism of IFN-gamma action restricted to Mphi, a cell type key for MCMV pathogenesis and latency.

Show MeSH
Related in: MedlinePlus