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IRF-3-Dependent and Augmented Target Genes During Viral Infection

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IRF-3-Dependent and Augmented Target Genes During Viral Infection Genes and Immunity (2008) 9, 168–175 & 2008 Nature Publishing Group All rights reserved 1466-4879/08 $30.00 www.nature.com/gene SHORT COMMUNICATION IRF-3-dependent and augmented target genes during viral infection J Andersen1, S VanScoy2, T-F Cheng2, D Gomez2 and NC Reich2 1Department of Biochemistry and Cell Biology, Stony Brook University, New York, NY, USA and 2Department of Molecular Genetics & Microbiology, Stony Brook University, New York, NY, USA Activation of the transcription factor interferon regulatory factor-3 (IRF-3) is an essential event in the innate immune response to viral infection. To understand the contribution of IRF-3 to host defense, we used a systems biology approach to analyze global gene expression dependent on IRF-3. Comparison of expression profiles in cells from IRF-3 knockout animals or wild-type siblings following viral infection revealed three sets of induced genes, those that are strictly dependent on IRF-3, augmented with IRF-3, or not responsive to IRF-3. Products of identified IRF-3 target genes are involved in innate or acquired immunity, or in the regulation of cell cycle, apoptosis and proliferation. These results reveal the global effects of one transcription factor in the immune response and provide information to evaluate the integrated response to viral infection. Genes and Immunity (2008) 9, 168–175; doi:10.1038/sj.gene.6364449; published online 20 December 2007 Keywords: transcription factor; host defense; innate immunity; global profiling; gene expression; signal transduction Introduction A critical cellular transcription factor that is activated both in response to viral infection and to toll-like An effective first line of defense to infection depends on receptors is the interferon regulatory factor-3 (IRF-3).1–11 the action of cells and mediators of innate immunity, IRF-3 is expressed constitutively in all cells of the body, and a specific and long-lasting defense depends on and animals with a targeted gene disruption are the collaboration of these components with cells and susceptible to viral infection.9,10 IRF-3 resides in a latent mediators of acquired immunity. Infections usually begin state primarily in the cytoplasm, but following serine in a single site or tissue, but the physiological reaction is phosphorylation by TANK-binding kinase or inhibitor of both local and disseminating. Responses include produc- NF-kB kinase-related IKKe, it accumulates in the nucleus tion of cytokines at the site that lead to recruitment of in strong association with the histone acetyl transferases phagocytes and lymphocytes, and in turn these cytokines CREB-binding protein or p300.12–14 and cellular responders disseminate to involve the entire Identification of genes targeted by IRF-3 is critical to immune system. For this reason analyzing the effects of understanding an effective defense response to viral infection on a single cell or tissue can provide knowledge infection. IRF-3 is known to regulate expression of the of a more global systemic response. type I interferon-beta gene (IFN-b), and a subset of Viral infection of cells causes the activation of a type I IFN-stimulated genes independent of the action number of transcription factors that, in turn, induce of IFN.3,11,15 IFNs are unique among the cytokines for genes whose products generate a biological response, be conferring cellular resistance to viral infections, and it survival of the infected cell or survival of the host with therefore a successful innate immune response depends the sacrifice of the infected cell. Each activated transcrip- on their production and action.16–19 IRF-3 contributes to tion factor may function distinctly to regulate expression the induction of the IFN-b gene, but it is not sufficient for of specific target genes, or they may function coopera- IFN-b gene induction and needs to cooperate in an tively to regulate expression of a common target gene. enhanceosome with NF-kB and ATF-2/c-jun transcrip- Cooperative gene induction can result from the con- tion factors.20 IFN is secreted by the infected cell and certed action of multiple transcription factors, although binds to cell surface receptors on the same cell or each transcription factor individually may not be adjacent cells stimulating a signal pathway that induces sufficient to induce the gene target. expression of IFN-stimulated genes. IFN-receptor binding activates Janus tyrosine kinases that phosphory- late the DNA-binding transcription factors, signal trans ducers and activators of transcription (STAT).16–18 Correspondence: Dr NC Reich, Department of Molecular Genetics Since IRF-3 plays a significant role in viral defense, & Microbiology, Stony Brook University, Nicolls Rd, Life Sciences efforts have been made to identify genes regulated by Bldg, Stony Brook, New York 11794, USA. E-mail: [email protected] IRF-3. One approach evaluated the effects of a consti- 21 Received 7 September 2007; revised 8 November 2007; accepted 13 tutively active IRF-3 mutant. Serine amino acids November 2007; published online 20 December 2007 were replaced with aspartic acid to mimic serine IRF-3 target genes J Andersen et al 169 phosphorylation and the active IRF-3 conformation. Only a handful of genes were found to be induced by overexpression of the constitutive IRF-3 mutant and this group did not include IFN. The low number of induced genes may be due to the fact that IRF-3 often functions cooperatively with other activated transcription factors, or that the constitutive mutant does not fully simulate the active form of IRF-3. Another approach evaluated the response to viral infection of mutated cell lines that were selected to either overexpress IRF-3, or express IRF-3 below normal levels.22 Cells with reduced IRF-3 levels underexpressed some known IFN-stimulated genes, but there was no effect on expression of IFN genes. Interpretation of these results is limited since low levels of IRF-3 are still expressed and activation of other transcription factors may be sufficient to induce ISG expression independent of IRF-3. To provide knowledge of the global contribution of IRF-3 to host defense in viral infection, it is essential to evaluate the profile of genes regulated in the presence or absence of IRF-3. Genes induced after viral infection can thereby be compared using controlled parameters to Figure 1 Gene expression in virally infected cells in the presence or absence of IRF-3. (a) Northern blot analysis. MEF cultures were identify those genes that are independently or coordina- generated from the IRF-3 knockout (KO) and wild-type (WT) sibling tely regulated by IRF-3. In this study, we used a systems mice and used between passages 20–35.9 Cells were mock infected biology approach to identify the global set of cellular (À) or infected with 100 hemagglutination units (HAU) mlÀ1 genes influenced by IRF-3 during the course of viral Newcastle Disease Virus (NJ-LaSota-1946) ( þ ) for 6 or 12 h.23 infection by employing microarray technology and Neutralizing antibody to type I IFN (IFN Ab) (313 units mlÀ1; virally-infected murine embryo fibroblasts (MEFs) Biosource International Inc.) was added one hour prior to infection þ derived from IRF-3 knockout (irf-3À/À) or wild-type of half the 6 h samples as indicated ( ). RNA was isolated with þ / þ 9 RNeasy reagents (Qiagen, Valencia, CA, USA). DNA probes were (WT) (irf-3 ) sibling mice. We used Newcastle labeled with a-32P-dCTP using PrimeIT (Stratagene, Cedar Creek, Disease Virus as our model infectious agent, because TX, USA) with human b-actin 700 bp NotI/BamHI cDNA fragment, this negative strand paramyxovirus leads to efficient murine GAPDH cDNA (gift of Dr Jizu Zhi) and murine ISG54 activation of latent IRF-3, and viral replication is limited cDNA generated by RT-PCR with primers 50-AAACACCAGTGGG 0 to one round of infection in murine cells. GATGAAG and 5 -CGTCTCATACTGGGCCCACTT. The reverse image of ethidium bromide-stained 18S rRNA on the membrane is shown. (b) Western blot analysis. Cells were infected for 0, 6, or 12 h with Newcastle Disease Virus similarly to (a) and were lysed with Results and discussion 0.5% NP40 buffer.14 Molecular mass marker (m) or 70 mg protein from each sample were separated by SDS-PAGE. Proteins were Cells and RNAs used for microarray analysis transferred to membrane and reacted with anti-ISG54 antibody Cultures of IRF-3 knockout (KO) (irf-3À/À/MEF6) and WT (peptide 26–50: Genemed Synthesis Inc., South San Francisco, CA, þ / þ USA) and Alexa Fluor 680 goat anti-rabbit secondary antibody sibling (irf-3 /MEF7) MEFs used in this study were (Molecular Probes, Eugene, OR, USA) before visualization with an evaluated for mRNA expression prior to and following Odyssey infrared imaging system (Li-COR Biosciences, Lincoln, 6 or 12 h infection with Newcastle Disease Virus.9 NE, USA). Northern blot hybridization was used to ensure RNA integrity of samples used in the microarray assays (Figure 1a). Following infection there was a clear increase system, we evaluated the expression of the ISG54 protein in a known IRF-3 target gene, IFN-stimulated gene 54 in WT and IRF-3 KO cells following viral infection (ISG54), in the WT cells but not the IRF-3 KO cells. The (Figure 1b). Western blot analysis demonstrated that addition of exogenous neutralizing antibody to type I expression of the ISG54 protein accurately reflected the IFN in the culture resulted in approximately a twofold induction of ISG54 mRNA in WT cells and not in decrease in the ISG54 levels in the infected WT (lane 8). KO cells. This is not unexpected since autocrine IFN produced during the course of infection can induce ISG54 via the Experimental design for microarray analysis Janus tyrosine kinases/STAT pathway and thereby A comparison of the genes expressed in infected WT increase the levels.
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