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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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Association of STAT2 protein with ISG promoters.ChIP analysis using a STAT2-specific antibody in bone marrow-derived macrophages isolated from WT, STAT1−/−, or STAT1−/−/2−/− mice and infected with S221 (MOI = 5) for 12 and 24 hours. Enrichment was measured by quantitative PCR, and percent of input DNA was determined by comparing cycle threshold value (Ct) obtained with immunoprecipitated DNA and Ct value obtained from input DNA. Genes analyzed were (A) Oas1a (B) Oas1b and (C) Irf7. Error bars represent the SEM, and asterisks denote statistically significant differences (*, p<0.05; ***, p<0.0005). Results are representative of two independent pull-down experiments.
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ppat-1001297-g006: Association of STAT2 protein with ISG promoters.ChIP analysis using a STAT2-specific antibody in bone marrow-derived macrophages isolated from WT, STAT1−/−, or STAT1−/−/2−/− mice and infected with S221 (MOI = 5) for 12 and 24 hours. Enrichment was measured by quantitative PCR, and percent of input DNA was determined by comparing cycle threshold value (Ct) obtained with immunoprecipitated DNA and Ct value obtained from input DNA. Genes analyzed were (A) Oas1a (B) Oas1b and (C) Irf7. Error bars represent the SEM, and asterisks denote statistically significant differences (*, p<0.05; ***, p<0.0005). Results are representative of two independent pull-down experiments.

Mentions: In response to type I IFN signaling, the STAT1:STAT2:IRF9 (ISGF3) complex translocates to the nucleus to bind ISREs found in the promoters of many ISGs. To determine whether STAT2 is directly involved in the transcriptional regulation of ISGs in the absence of STAT1, chromatin immunoprecipitation (ChIP) experiments were performed using a STAT2-specific antibody. Gene targets Oas1a, Oas1b, and IRF7 were chosen from ISGs that were expressed in wild type and STAT1−/− BMMs, but not in STAT1−/−/2−/− double deficient cells as determined by the PCR array analysis (Table S2). DNA from S221-infected wild type, STAT1−/− and STAT1−/−/2−/− BMMs was harvested at 12 and 24 hours post-infection, and quantitative PCR was performed on anti-STAT2 precipitated DNA using primer pairs specific to ISREs located in the promoters of these genes. Following DENV infection, increased binding of STAT2 to all three gene promoters was observed in wild type and STAT1−/− BMMs when compared to naïve cells (Figure 6A–C). STAT2 binding to the Oas1a promoter in STAT1−/− cells was only significantly enriched at 24 hours post-infection, while Oas1b and IRF7 had significant enrichment at both time points. In particular, the binding activity of STAT2 to the Oas1b promoter in STAT1−/− cells almost matched that of wild type cells (Figure 6B). Nearly undetectable DNA enrichment was observed for all three genes in STAT1−/−/2−/− cells, providing evidence of the antibody's specificity for STAT2. Taken together, these results demonstrate that STAT2 binds to the promoters of ISGs in the absence of STAT1, providing further evidence of a direct role for STAT2 in the regulation of antiviral responses.


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)

Association of STAT2 protein with ISG promoters.ChIP analysis using a STAT2-specific antibody in bone marrow-derived macrophages isolated from WT, STAT1−/−, or STAT1−/−/2−/− mice and infected with S221 (MOI = 5) for 12 and 24 hours. Enrichment was measured by quantitative PCR, and percent of input DNA was determined by comparing cycle threshold value (Ct) obtained with immunoprecipitated DNA and Ct value obtained from input DNA. Genes analyzed were (A) Oas1a (B) Oas1b and (C) Irf7. Error bars represent the SEM, and asterisks denote statistically significant differences (*, p<0.05; ***, p<0.0005). Results are representative of two independent pull-down experiments.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3040673&req=5

ppat-1001297-g006: Association of STAT2 protein with ISG promoters.ChIP analysis using a STAT2-specific antibody in bone marrow-derived macrophages isolated from WT, STAT1−/−, or STAT1−/−/2−/− mice and infected with S221 (MOI = 5) for 12 and 24 hours. Enrichment was measured by quantitative PCR, and percent of input DNA was determined by comparing cycle threshold value (Ct) obtained with immunoprecipitated DNA and Ct value obtained from input DNA. Genes analyzed were (A) Oas1a (B) Oas1b and (C) Irf7. Error bars represent the SEM, and asterisks denote statistically significant differences (*, p<0.05; ***, p<0.0005). Results are representative of two independent pull-down experiments.
Mentions: In response to type I IFN signaling, the STAT1:STAT2:IRF9 (ISGF3) complex translocates to the nucleus to bind ISREs found in the promoters of many ISGs. To determine whether STAT2 is directly involved in the transcriptional regulation of ISGs in the absence of STAT1, chromatin immunoprecipitation (ChIP) experiments were performed using a STAT2-specific antibody. Gene targets Oas1a, Oas1b, and IRF7 were chosen from ISGs that were expressed in wild type and STAT1−/− BMMs, but not in STAT1−/−/2−/− double deficient cells as determined by the PCR array analysis (Table S2). DNA from S221-infected wild type, STAT1−/− and STAT1−/−/2−/− BMMs was harvested at 12 and 24 hours post-infection, and quantitative PCR was performed on anti-STAT2 precipitated DNA using primer pairs specific to ISREs located in the promoters of these genes. Following DENV infection, increased binding of STAT2 to all three gene promoters was observed in wild type and STAT1−/− BMMs when compared to naïve cells (Figure 6A–C). STAT2 binding to the Oas1a promoter in STAT1−/− cells was only significantly enriched at 24 hours post-infection, while Oas1b and IRF7 had significant enrichment at both time points. In particular, the binding activity of STAT2 to the Oas1b promoter in STAT1−/− cells almost matched that of wild type cells (Figure 6B). Nearly undetectable DNA enrichment was observed for all three genes in STAT1−/−/2−/− cells, providing evidence of the antibody's specificity for STAT2. Taken together, these results demonstrate that STAT2 binds to the promoters of ISGs in the absence of STAT1, providing further evidence of a direct role for STAT2 in the regulation of antiviral responses.

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