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STAT2 mediates innate immunity to Dengue virus in the absence of STAT1 via the type I interferon receptor.

Perry ST, Buck MD, Lada SM, Schindler C, Shresta S - PLoS Pathog. (2011)

Bottom Line: High viral loads correlate with disease severity, and both type I & II interferons (IFNs) are crucial for controlling viral replication.Further studies demonstrated that this STAT2-dependent STAT1-independent mechanism requires the type I IFN receptor, and contributes to the autocrine amplification of type I IFN expression.Examination of gene expression in the spleen and bone marrow-derived macrophages following DENV infection revealed STAT2-dependent pathways can induce the transcription of a subset of interferon stimulated genes even in the absence of STAT1.

View Article: PubMed Central - PubMed

Affiliation: Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, California, USA.

ABSTRACT
Dengue virus (DENV) is a mosquito-borne flavivirus, and symptoms of infection range from asymptomatic to the severe dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). High viral loads correlate with disease severity, and both type I & II interferons (IFNs) are crucial for controlling viral replication. We have previously reported that signal transducer and activator of transcription (STAT) 1-deficient mice are resistant to DENV-induced disease, but little is known about this STAT1-independent mechanism of protection. To determine the molecular basis of the STAT1-independent pathway, mice lacking STAT1, STAT2, or both STAT1 and STAT2 were infected with a virulent mouse-adapted strain of DENV2. In the first 72 hours of infection, the single-deficient mice lacking STAT1 or STAT2 possessed 50-100 fold higher levels of viral RNA than wild type mice in the serum, spleen, and other visceral tissues, but remained resistant to DENV-induced death. In contrast, the double-deficient mice exhibited the early death phenotype previously observed in type I and II IFN receptor knockout mice (AG129), indicating that STAT2 is the mediator of the STAT1-independent host defense mechanism. Further studies demonstrated that this STAT2-dependent STAT1-independent mechanism requires the type I IFN receptor, and contributes to the autocrine amplification of type I IFN expression. Examination of gene expression in the spleen and bone marrow-derived macrophages following DENV infection revealed STAT2-dependent pathways can induce the transcription of a subset of interferon stimulated genes even in the absence of STAT1. Collectively, these results help elucidate the nature of the poorly understood STAT1-independent host defense mechanism against viruses by identifying a functional type I IFN/STAT2 signaling pathway following DENV infection in vivo.

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Activation of STAT1 and STAT2 in bone marrow-derived macrophages.(A–C) Whole cell lysates were generated from bone marrow-derived macrophages that were isolated from WT, STAT1−/−, STAT1−/−/2−/−, or STAT1−/−/AR−/− mice and treated with (A) 1000U/mL recombinant murine IFN-β for 15 min, (B) serum from naïve (N) or S221-infected (I) mice for 1 hour, or (C) infected with S221 (MOI = 5) for the times indicated. Protein levels of STAT1, phosphorylated STAT1, STAT2, phosphorylated STAT2, and beta-actin were examined by Western blot. (D) Nuclear localization of STAT2 in bone marrow-derived macrophages isolated from WT and STAT1−/− mice, and treated with serum from naïve (N) or S221-infected (I) mice for 1 hour. STAT2 (red), and DAPI (blue). Original magnification, ×400. Images are representative of each strain. Data are representative of two or more individual experiments.
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ppat-1001297-g005: Activation of STAT1 and STAT2 in bone marrow-derived macrophages.(A–C) Whole cell lysates were generated from bone marrow-derived macrophages that were isolated from WT, STAT1−/−, STAT1−/−/2−/−, or STAT1−/−/AR−/− mice and treated with (A) 1000U/mL recombinant murine IFN-β for 15 min, (B) serum from naïve (N) or S221-infected (I) mice for 1 hour, or (C) infected with S221 (MOI = 5) for the times indicated. Protein levels of STAT1, phosphorylated STAT1, STAT2, phosphorylated STAT2, and beta-actin were examined by Western blot. (D) Nuclear localization of STAT2 in bone marrow-derived macrophages isolated from WT and STAT1−/− mice, and treated with serum from naïve (N) or S221-infected (I) mice for 1 hour. STAT2 (red), and DAPI (blue). Original magnification, ×400. Images are representative of each strain. Data are representative of two or more individual experiments.

Mentions: To confirm activation of STAT2 in the absence of STAT1, phosphorylation and nuclear localization of STAT2 was examined in BMMs. Type I IFN signaling activates STAT2 via phosphorylation of a tyrosine residue (Y689), which is required for its association with STAT1 and incorporation into the transcriptionally active complex ISGF3 [40]. BMMs isolated from wild type, STAT1−/−, STAT1−/−/2−/−, and STAT1−/−/AR−/− mice were stimulated with recombinant IFN-β for 15 minutes, and the phosphorylation status of STAT2 was examined via Western blot (Figure 5A). As expected, both STAT1 and STAT2 were phosphorylated in wild type cells. Phosphorylation of STAT2 was observed in STAT1−/− but not STAT1−/−/AR−/− cells, although the basal level of STAT2 was lower in both STAT1−/− and STAT1−/−/AR−/− cells than in wild type cells. These results show that STAT2 activation can occur in the absence of STAT1 following type I IFN stimulation.


STAT2 mediates innate immunity to Dengue virus in the absence of STAT1 via the type I interferon receptor.

Perry ST, Buck MD, Lada SM, Schindler C, Shresta S - PLoS Pathog. (2011)

Activation of STAT1 and STAT2 in bone marrow-derived macrophages.(A–C) Whole cell lysates were generated from bone marrow-derived macrophages that were isolated from WT, STAT1−/−, STAT1−/−/2−/−, or STAT1−/−/AR−/− mice and treated with (A) 1000U/mL recombinant murine IFN-β for 15 min, (B) serum from naïve (N) or S221-infected (I) mice for 1 hour, or (C) infected with S221 (MOI = 5) for the times indicated. Protein levels of STAT1, phosphorylated STAT1, STAT2, phosphorylated STAT2, and beta-actin were examined by Western blot. (D) Nuclear localization of STAT2 in bone marrow-derived macrophages isolated from WT and STAT1−/− mice, and treated with serum from naïve (N) or S221-infected (I) mice for 1 hour. STAT2 (red), and DAPI (blue). Original magnification, ×400. Images are representative of each strain. Data are representative of two or more individual experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3040673&req=5

ppat-1001297-g005: Activation of STAT1 and STAT2 in bone marrow-derived macrophages.(A–C) Whole cell lysates were generated from bone marrow-derived macrophages that were isolated from WT, STAT1−/−, STAT1−/−/2−/−, or STAT1−/−/AR−/− mice and treated with (A) 1000U/mL recombinant murine IFN-β for 15 min, (B) serum from naïve (N) or S221-infected (I) mice for 1 hour, or (C) infected with S221 (MOI = 5) for the times indicated. Protein levels of STAT1, phosphorylated STAT1, STAT2, phosphorylated STAT2, and beta-actin were examined by Western blot. (D) Nuclear localization of STAT2 in bone marrow-derived macrophages isolated from WT and STAT1−/− mice, and treated with serum from naïve (N) or S221-infected (I) mice for 1 hour. STAT2 (red), and DAPI (blue). Original magnification, ×400. Images are representative of each strain. Data are representative of two or more individual experiments.
Mentions: To confirm activation of STAT2 in the absence of STAT1, phosphorylation and nuclear localization of STAT2 was examined in BMMs. Type I IFN signaling activates STAT2 via phosphorylation of a tyrosine residue (Y689), which is required for its association with STAT1 and incorporation into the transcriptionally active complex ISGF3 [40]. BMMs isolated from wild type, STAT1−/−, STAT1−/−/2−/−, and STAT1−/−/AR−/− mice were stimulated with recombinant IFN-β for 15 minutes, and the phosphorylation status of STAT2 was examined via Western blot (Figure 5A). As expected, both STAT1 and STAT2 were phosphorylated in wild type cells. Phosphorylation of STAT2 was observed in STAT1−/− but not STAT1−/−/AR−/− cells, although the basal level of STAT2 was lower in both STAT1−/− and STAT1−/−/AR−/− cells than in wild type cells. These results show that STAT2 activation can occur in the absence of STAT1 following type I IFN stimulation.

Bottom Line: High viral loads correlate with disease severity, and both type I & II interferons (IFNs) are crucial for controlling viral replication.Further studies demonstrated that this STAT2-dependent STAT1-independent mechanism requires the type I IFN receptor, and contributes to the autocrine amplification of type I IFN expression.Examination of gene expression in the spleen and bone marrow-derived macrophages following DENV infection revealed STAT2-dependent pathways can induce the transcription of a subset of interferon stimulated genes even in the absence of STAT1.

View Article: PubMed Central - PubMed

Affiliation: Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, California, USA.

ABSTRACT
Dengue virus (DENV) is a mosquito-borne flavivirus, and symptoms of infection range from asymptomatic to the severe dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). High viral loads correlate with disease severity, and both type I & II interferons (IFNs) are crucial for controlling viral replication. We have previously reported that signal transducer and activator of transcription (STAT) 1-deficient mice are resistant to DENV-induced disease, but little is known about this STAT1-independent mechanism of protection. To determine the molecular basis of the STAT1-independent pathway, mice lacking STAT1, STAT2, or both STAT1 and STAT2 were infected with a virulent mouse-adapted strain of DENV2. In the first 72 hours of infection, the single-deficient mice lacking STAT1 or STAT2 possessed 50-100 fold higher levels of viral RNA than wild type mice in the serum, spleen, and other visceral tissues, but remained resistant to DENV-induced death. In contrast, the double-deficient mice exhibited the early death phenotype previously observed in type I and II IFN receptor knockout mice (AG129), indicating that STAT2 is the mediator of the STAT1-independent host defense mechanism. Further studies demonstrated that this STAT2-dependent STAT1-independent mechanism requires the type I IFN receptor, and contributes to the autocrine amplification of type I IFN expression. Examination of gene expression in the spleen and bone marrow-derived macrophages following DENV infection revealed STAT2-dependent pathways can induce the transcription of a subset of interferon stimulated genes even in the absence of STAT1. Collectively, these results help elucidate the nature of the poorly understood STAT1-independent host defense mechanism against viruses by identifying a functional type I IFN/STAT2 signaling pathway following DENV infection in vivo.

Show MeSH
Related in: MedlinePlus