Figure 1.
Survival of mice following infection with DENV.
Survival was monitored for at least 30 days in mice infected i.v. with DENV2 strain S221. (A) Mice were infected with 1010 GE (WT n = 8; STAT1−/− n = 12; STAT2−/− n = 17; STAT1−/−/2−/− n = 14; AG129 n = 13). Differences in survival were statistically different (**, p<0.005) between STAT1−/−/2−/− and AG129 mice. (B) Mice were infected with 1011 GE (n = 9) or 1012 GE of DENV (n = 5). (C) Mice were infected with 1010 GE (STAT1−/−/AR−/− n = 12; STAT1−/−/GR−/− n = 8).
Figure 2.
Viral RNA levels in DENV-infected mice.
Mice were infected i.v. with 1010 GE of S221. (A–C) Quantification of DENV RNA in the (A) serum and (B) spleen at 6, 12, 18, 24 and 72 hours post-infection, and (C) liver, kidney, and small intestine at 72 hours post-infection as determined by quantitative RT-PCR (6h n = 4; 12h n = 8; 18h n = 8; 24h n = 12; 72h n = 4). Data are shown as GE per mL of serum or GE per copy of 18S RNA for tissues. The dotted line represents the limit of detection of the assay for each tissue. (D) Levels of TNF in the serum of DENV-infected mice at 72 hours post-infection (n = 7 per group). Error bars represent the SEM and asterisks denote statistically significant differences (*, p<0.05; **, p<0.005; ***, p<0.0005; ns = not significant). Results are representative of two independent experiments.
Figure 3.
Levels of type I IFN in the serum of DENV-infected mice.
Mice were infected i.v. with 1010 GE of S221, and serum collected at 6, 12, 18, or 24 hours post-infection. Levels of (A) IFN-α and (B) IFN-β in the serum were determined by ELISA (n = 4–6 mice per group). Error bars represent the SEM. Results are representative of two independent experiments.
Figure 4.
Comparison of ISG Induction in DENV-infected mice.
Gene induction evaluated using the RT2 Profiler PCR Array (Mouse IFN-α/β Response Array; SABioscience). (A,B) Annotated gene expression of transcripts with a difference in expression of 3-fold or more over naïve (upregulation-black; downregulation-white) at 12 and 24 hours post-infection of (A) spleens isolated from wild type, STAT1−/−, STAT2−/−, and STAT1−/−/2−/− mice infected with S221, and (B) BMMs isolated from wild type, STAT1−/−, and STAT1−/−/2−/− mice infected with S221 (MOI = 5). (C, D) Gene induction in the spleen expressed as fold-increase over naïve for each strain at (C) 12 hours and (D) 24 hours post-infection. Data represent mean fold-induction of three animals per strain at each timepoint.
Figure 5.
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.
Figure 6.
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.