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, 87 (16), 9260-70

MicroRNA Profiling of Sendai Virus-Infected A549 Cells Identifies miR-203 as an Interferon-Inducible Regulator of IFIT1/ISG56

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MicroRNA Profiling of Sendai Virus-Infected A549 Cells Identifies miR-203 as an Interferon-Inducible Regulator of IFIT1/ISG56

William A Buggele et al. J Virol.

Abstract

The mammalian type I interferon (IFN) response is a primary barrier for virus infection and is essential for complete innate and adaptive immunity. Both IFN production and IFN-mediated antiviral signaling are the result of differential cellular gene expression, a process that is tightly controlled at transcriptional and translational levels. To determine the potential for microRNA (miRNA)-mediated regulation of the antiviral response, small-RNA profiling was used to analyze the miRNA content of human A549 cells at steady state and following infection with the Cantell strain of Sendai virus, a potent inducer of IFN and cellular antiviral responses. While the miRNA content of the cells was largely unaltered by infection, specific changes in miRNA abundance were identified during Sendai virus infection. One miRNA, miR-203, was found to accumulate in infected cells and in response to IFN treatment. Results indicate that miR-203 is an IFN-inducible miRNA that can negatively regulate a number of cellular mRNAs, including an IFN-stimulated gene target, IFIT1/ISG56, by destabilizing its mRNA transcript.

Figures

Fig 1
Fig 1
Small-RNA profile of uninfected and Sendai virus-infected A549 cells. A549 cells were mock infected or infected with Sendai virus (Cantell strain; 5 PFU/cell). RNA was purified 10 h later and size fractionated to yield RNA <200 nucleotides in length, and cDNA libraries were prepared for SOLiD sequencing. (A) Venn diagram illustrates the number of miRNAs identified (by greater than 100 sequence tags) that are either unique or common to mock-infected and Sendai virus-infected libraries. (B) Scatter plot indicates the abundance of each identified miRNA in the mock-infected or Sendai virus-infected library with greater than 100 sequence tags. Blue dots represent miRNAs with less than 1.5-fold change, and red dots represent miRNAs with greater than 1.5-fold change.
Fig 2
Fig 2
Validation of changes in miRNA abundance induced by Sendai virus infection. For each indicated miRNA, normalized sequence tag abundance is plotted to the left of the dashed line. Individual miRNAs were also measured in freshly prepared RNA derived from A549 cells that were either mock infected or infected with Sendai virus (5 PFU/cell; 10 h) and then subjected to specific TaqMan microRNA RT-qPCR assays; results are plotted to the right of the dashed line. Statistical significance was determined by a two-tailed t test (*, P < 0.05; **, P < 0.01).
Fig 3
Fig 3
IFN mediates regulation of miR-203. (A) A549 cells were mock infected or infected with either influenza virus A/Udorn/72 (Udorn), A/WSN/33 (WSN), or Sendai virus Cantell Strain (Sendai) (5 PFU/cell; 10 h). RNA was purified at 10 h postinfection and size fractionated to yield RNA of <200 nucleotides in length, and the abundances of miR-203, miR-449b, and miR-16 were analyzed using TaqMan miRNA RT-qPCR assays. (B) High-molecular-weight RNA was used to measure the abundance of IFN-β mRNA during Sendai virus and influenza virus infection by RT-qPCR as indicated. (C) A549 cells were left untreated (UNT) or treated with PBS or increasing concentrations of IFN-α (250 units/ml, 500 units/ml, 1,000 units/ml, or 5,000 units/ml). RNA was purified at 10 h posttreatment, and the abundance of miR-203 was analyzed by TaqMan miRNA assays. (D) A549 cells were left untreated or treated as indicated with PBS or IFN-α (1,000 units/ml) and mock infected or infected with Sendai virus (5 PFU/cell). RNA was purified at 10 h posttreatment, and the abundance of miR-203 was analyzed by TaqMan miRNA assays. (E) A549 cells were left untreated, treated with PBS or IFN-α (1,000 units/ml), or transfected with poly(I·C) (5 μg/ml). RNA was purified at 10 h posttreatment, and the abundance of miR-203 was analyzed by TaqMan miRNA assays. (F) Vero cells were left untreated or treated as indicated with PBS or IFN-α (1,000 units/ml) and then mock infected or infected with Sendai virus (5 PFU/cell). RNA was purified at 10 h posttreatment, and the abundance of miR-203 was analyzed by TaqMan miRNA assays. Statistical significance was determined by a two-tailed t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., not significant).
Fig 4
Fig 4
Activation of miR-203 by immediate and delayed signaling pathways. (A) A549 cells were infected with Sendai virus (5 PFU/cell), and the abundance of miR-203 was measured at indicated times after Sendai virus infection by TaqMan miRNA RT-qPCR. (B) A549 cells were stimulated with IFN-α (1,000 units/ml) or infected with Sendai virus (SeV) (5 PFU/cell) for 4, 10, and 24 h in the presence (+) or absence (−) of cycloheximide (CHX 100 ng/ml; 1 h pretreatment and continuous thereafter). Abundances of miR-203 and miR-449b were determined by TaqMan microRNA assays at the indicated time points. Statistical significance was determined by a two-tailed t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001. M, mock; hpt, hours posttransfection.
Fig 5
Fig 5
Identification and characterization of IFIT1/ISG56 as an miR-203 target. (A) Illustration of the 3′ UTR of IFIT1/ISG56, with boxes indicating the positions of two partially overlapping miR-203 seed matches. (B) A549 cells were transfected with 25 nM nontargeting miRNA mimic (Control) or nontargeting control miRNA inhibitor (Inhib) or miR-203-specific mimic or miR-203 inhibitor, as indicated. Cells were either untreated (−) or treated (+) with 1,000 units/ml IFN-α. RNA was purified from cells 10 h later and analyzed by RT-qPCR for the induction of IFIT1/ISG56 mRNAs. In parallel, levels of miR-203 were measured by TaqMan assay. Statistical significance was determined by a two-tailed t test (*, P < 0.05; **, P < 0.01). (C) A549 cells were transfected with miRNA mimics and inhibitors and then treated with IFN-α as described for panel B Whole-cell lysates were prepared 10 h later and analyzed by immunoblotting with antiserum specific for IFIT1/ISG56, MDA5, or GAPDH protein.
Fig 6
Fig 6
Model of the regulation of miR-203 and IFIT1/ISG56 during Sendai virus infection. Cells infected with Sendai virus generate an antiviral response by producing type I IFN, which signals through the JAK-STAT-ISGF3 pathway to induce IFN-stimulated gene products including IFIT1/ISG56, miR-203, and IFN regulatory factors (IRFs). Both IFN production and IRF induction are sensitive to treatment with CHX. miR-203 can be induced by IFN initially by preexisting proteins, but sustained miR-203 expression requires newly synthesized signaling proteins, potentially, but not limited to, IRF family members (IRF?). miR-203 accumulation functions to destabilize IFIT1/ISG56 mRNA and regulate its translation. See the text for more details of the model.

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