Essential role of IPS-1 in innate immune responses against RNA viruses

Abstract

IFN-beta promoter stimulator (IPS)-1 was recently identified as an adapter for retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (Mda5), which recognize distinct RNA viruses. Here we show the critical role of IPS-1 in antiviral responses in vivo. IPS-1-deficient mice showed severe defects in both RIG-I- and Mda5-mediated induction of type I interferon and inflammatory cytokines and were susceptible to RNA virus infection. RNA virus-induced interferon regulatory factor-3 and nuclear factor kappaB activation was also impaired in IPS-1-deficient cells. IPS-1, however, was not essential for the responses to either DNA virus or double-stranded B-DNA. Thus, IPS-1 is the sole adapter in both RIG-I and Mda5 signaling that mediates effective responses against a variety of RNA viruses.

Figure 1.

Generation of IPS-1–deficient mice. (A) The genomic structure of murine IPS-1 gene, the targeting vector, and the predicted mutated allele are shown. Black and white boxes denote the coding and noncoding exons, respectively. E, EcoRI. (B) Southern blot analysis of offsprings from the heterozygote intercrosses. Genomic DNA was extracted, digested with EcoRI, electrophoresed, and hybridized with a probe indicated in A. The blotting gave a single 11.3-kb band for wild-type (+/+), a 7.0-kb band for homozygous mutants (−/−), and both bands for heterozygous mice (+/−). (C) Northern blot analysis for the expression of IPS-1 gene. Total RNA (30 μg) was extracted from MEFs prepared from wild-type (+/+) and homozygous mutant (−/−) mice, electrophoresed, and hybridized with an IPS-1 (Ips-1) or β-actin (Actb) probe. (D) Immunoblot analysis for the expression of IPS-1 protein. Four independent MEFs were lysed and immunoprecipitated with anti–IPS-1 antibody (IP) and then blotted with anti–IPS-1 antibody (IB). The whole cell lysates (WCL) were simultaneously blotted with anti–extracellular signal-regulated kinase 1/2 antibody.

Figure 2.

Impaired responses of IPS-1–deficient cells to various RNA viruses. (A and B) MEFs from wild type (+/+) and IPS-1–deficient mice (−/−) were infected with NDV (multiplicity of infection [moi] = 1.0), VSV NCP mutant (VSV; moi =0.1); or SeV Cm mutant (SeV; moi = 0.1). After 24 h, the concentration of the cytokines in the culture supernatant was measured by ELISA (A). After incubation for the indicated period, total RNAs was extracted and subjected to Northern blot analysis to determine the expression pattern of the respective genes (B). Asterisks indicate not detected. Indicated values are means ± SD of triplicates. (C) B220⁻ CD11c⁺ FL-DCs were infected with VSV NCP (moi = 1.0) or stimulated with 3 μM D35. After 24 h, cells were fixed and stained with anti-CD86 antibody. (D) MEFs from wild-type (+/+) and IPS-1–deficient mice (−/−) were stimulated with the indicated length of dsRNA. After 24 h, the concentration of IFN-β in the culture supernatant was measured by ELISA. Asterisks indicate not detected. Indicated values are means ± SD of triplicates. (E) Splenocytes were stimulated with D35. After 24 h, the concentration of the cytokines in the culture supernatant was measured by ELISA. Asterisks indicate not detected. Indicated values are means ± SD of triplicates. (F) MEFs were stimulated with 10 μg/ml LPS. After 2, 4, and 6 h, the expression of Cxc110 and IL-6 was determined by Northern blot analysis.

Figure 3.

Impaired responses of IPS-1–deficient cells to EMCV and poly (I:C). (A and B) PECs isolated from wild-type and IPS-1–deficient mice were infected with EMCV (moi = 0.1). After 24 h, cytokine production in the culture supernatant was measured by ELISA (A). After 6 and 9 h, total RNA was extracted and subjected to RT-PCR analysis for the expression of respective genes (B). Asterisks indicate not detected. Indicated values are means ± SD of triplicates. (C) MEFs were transfected with the indicated concentrations of poly I:C (pIC). After 24 h, cytokine production in the culture supernatant was measured by ELISA. Asterisks indicate not detected. Indicated values are means ±SD of triplicates. (D) MEFs were transfected with 10 μg/ml poly I:C and incubated for the indicated periods. Total RNA was subjected to Northern blot analysis for the expression of relevant genes. (E) IPS-1^+/− and IPS-1^−/− mice were intravenously challenged with poly I:C (200 μg/body). Sera were prepared before injection (0 h) or after 2, 4, and 8 h, and the concentration of the cytokines was measured by ELISA. Similar results were obtained from three independent experiments.

Figure 4.

Impaired NF-κB and IRF-3 activation in response to NDV in IPS-1–deficient cells. (A–E) Wild-type and IPS-1–deficient MEFs were incubated for the indicated periods after NDV infection (moi = 0.1) or LPS (10 μg/ml) stimulation. Nuclear extracts were subjected to electrophoretic mobility shift assay using an NF-κB–specific probe (A) and immunoblot analysis using anti-RelA antibody (B). Whole cell lysates were subjected to native page analysis using anti–IRF-3 antibody (C) and immunoblot analysis using anti-phospho STAT1 (p-STAT1) and anti-STAT1 antibodies (D). Total RNA was subjected to RT-PCR analysis for the expression of Irf7 and Actb (E).

Figure 5.

Role of IPS-1 in in vivo antiviral responses. (A) Wild-type and IPS-1–deficient mice (n = 2) were intravenously injected with EMCV (10⁷ PFU). Sera were collected 4 h after injection, and the cytokine level was measured by ELISA. Data are presented from one representative experiment of two independent experiments. (B) Wild-type and IPS-1–deficient mice (n = 7) were intraperitoneally infected with EMCV (10² PFU). The survival of these mice was monitored for up to 4 d. (C) IPS-1^+/− and IPS-1^−/− (n = 3) were intraperitoneally infected with EMCV (10² PFU). After 48 h of infection, virus yield in the heart was quantified by plaque assay. (D) Wild-type and IPS-1–deficient mice (n = 6) were intranasally infected with VSV (4 × 10⁶ PFU). The survival of these mice was monitored for up to 9 d. (E) Wild-type and IPS-1–deficient mice (n = 2) were intranasally infected with VSV (4 × 10⁶ PFU). After 72 h of infection, virus yield in the liver and brain was quantified by plaque assay. Asterisks indicate not detected.

Figure 6.

Role of IPS-1 in B-DNA–mediated responses. (A and B) Wild-type and IPS-1–deficient MEFs were transfected with 10 μg/ml B-DNA. After 24 h, the concentration of IFN-β, IP-10, and IL-6 was measured by ELISA (A). After 2, 4, and 6 h, total RNA was extracted and subjected to Northern blot analysis to determine the expression pattern of the respective genes (B). Asterisks indicate not detected. Indicated values are means ± SD of triplicates. (C and D) MEFs were infected with 10⁷ PFU MVA ΔE3L. After 24 and 48 h incubation, the concentration of IFN-β and IL-6 was measured by ELISA (C). Whole cell lysates were subjected to immunoblot analysis using anti-MVA antibody (D).