Isabelle Marié

Differential viral induction of distinct interferon-alpha genes by positive feedback through interferon regulatory factor-7.

Interferon (IFN) genes are among the earliest transcriptional responses to virus infection of mammalian cells. Although the regulation of the IFNβ gene has been well characterized, the induction of the large family of IFNα genes has remained obscure. We report that the IFNα genes can be divided into two groups: an immediate‐early response gene (IFNα4) which is induced rapidly and without the need for ongoing protein synthesis; and a set of genes that display delayed induction, consisting of at least IFNα2, 5, 6 and 8, which are induced more slowly and require cellular protein synthesis. One protein that must be synthesized for induction of the delayed gene set is IFN itself, presumably IFNα4 or IFNβ, which stimulates the Jak–Stat pathway through the IFN receptor, resulting in activation of the transcription factor interferon‐stimulated gene factor 3 (ISGF3). Among the IFN‐stimulated genes induced through this positive feedback loop is the IFN regulatory factor (IRF) protein, IRF7. Induction of IRF7 protein in response to IFN and its subsequent activation by phosphorylation in response to virus‐specific signals, involving two C‐terminal serine residues, are required for induction of the delayed IFNα gene set.

IRF3 and IRF7 phosphorylation in virus-infected cells does not require double-stranded RNA-dependent protein kinase R or Ikappa B kinase but is blocked by Vaccinia virus E3L protein

Induction of interferon-α (IFNα) gene expression in virus-infected cells requires phosphorylation-induced activation of the transcription factors IRF3 and IRF7. However, the kinase(s) that targets these proteins has not been identified. Using a combined pharmacological and genetic approach, we found that none of the kinases tested was responsible for IRF phosphorylation in cells infected with Newcastle disease virus (NDV). Although the broad-spectrum kinase inhibitor staurosporine potently blocked IRF3 and -7 phosphorylation, inhibitors for protein kinase C, protein kinase A, MEK, SAPK, IKK, and protein kinase R (PKR) were without effect. Both IκB kinase and PKR have been implicated in IFN induction, but cells genetically deficient in IκB kinase, PKR, or thePKR-related genes PERK, IRE1, orGCN2 retained the ability to phosphorylate IRF7 and induce IFNα. Interestingly, PKR mutant cells were defective for response to double-stranded (ds) RNA but not to virus infection, suggesting that dsRNA is not the only activating viral component. Consistent with this notion, protein synthesis was required for IRF7 phosphorylation in virus-infected cells, and the kinetics of phosphorylation and viral protein production were similar. Despite evidence for a lack of involvement of dsRNA and PKR, vaccinia virus E3L protein, a dsRNA-binding protein capable of inhibiting PKR, was an effective IRF3 and -7 phosphorylation inhibitor. These results suggest that a novel cellular protein that is activated by viral products in addition to dsRNA and is sensitive to E3L inhibition is responsible for IRF activation and reveal a novel mechanism for the anti-IFN effect of E3L distinct from its inhibition of PKR.

Ringing the interferon alarm: differential regulation of gene expression at the interface between innate and adaptive immunity

Abstract The genes for type I IFNs (IFN-α and IFN-β) are rapidly induced in response to viral infection. IFN regulatory factor (IRF) proteins are key to the regulation of IFN gene expression; the early response to virus results in secretion of a subset of IFN genes through the action of IRF3 in conjunction with additional transcription factors, such as NF-κB and AP-1 (c-jun–ATF2). This early IFN acts in an autocrine manner to stimulate the production of IRF7, a transcription factor capable of activating the many additional members of the IFN-α gene family. The dependence of IRF7 on virus-induced phosphorylation for its activity ensures that IFN production is limited to virus-infected cells. Additional members of the IRF family may provide additional levels of control, in both a cell-type and virus-specific manner, particularly in dendritic cells that serve as major producers of IFN and a key interface between innate and adaptive immunity.

Phosphorylation-Induced Dimerization of Interferon Regulatory Factor 7 Unmasks DNA Binding and a Bipartite Transactivation Domain

ABSTRACT Interferon regulatory factor 7 (IRF7) is an interferon (IFN)-inducible transcription factor required for activation of a subset of IFN-α genes that are expressed with delayed kinetics following viral infection. IRF7 is synthesized as a latent protein and is posttranslationally modified by protein phosphorylation in infected cells. Phosphorylation required a carboxyl-terminal regulatory domain that controlled the retention of the active protein exclusively in the nucleus, as well as its binding to specific DNA target sequences, multimerization, and ability to induce target gene expression. Transcriptional activation by IRF7 mapped to two distinct regions, both of which were required for full activity, while all functions were masked in latent IRF7 by an autoinhibitory domain mapping to an internal region. A conditionally active form of IRF7 was constructed by fusing IRF7 with the ligand-binding and dimerization domain of estrogen receptor (ER). Hormone-dependent dimerization of chimeric IRF7-ER stimulated DNA binding and transcriptional transactivation of endogenous target genes. These studies demonstrate the regulation of IRF7 activity by phosphorylation-dependent allosteric changes that result in dimerization and that facilitate nuclear retention, derepress transactivation, and allow specific DNA binding.

Acetylation of Interferon Regulatory Factor-7 by p300/CREB-binding Protein (CBP)-associated Factor (PCAF) Impairs its DNA Binding

Interferon regulatory factor 7 (IRF7) is an interferon-inducible transcription factor required for induction of delayed early interferon α genes and the onset of a potent antiviral state. After induction of IRF7 by autocrine interferon, latent IRF7 is activated by virus-induced phosphorylation on serine residues within the C-terminal regulatory domain. Although it is likely that IRF7 is subjected to a cascade of events responsible for regulating its biological activity, to date no mechanism other than phosphorylation has been reported to modulate IRF7 activity. Here, we report that IRF7 is acetylated in vivo by the histone acetyltransferases p300/CBP-associated factor (PCAF) and GCN5. The single lysine residue target for acetylation, lysine 92, is located in the DNA-binding domain and is conserved throughout the entire IRF family. Mutation of lysine 92 resulted in complete abolition of DNA binding ability. However, a mutant that cannot be acetylated by PCAF due to a change in the surrounding amino acid context of lysine 92 showed increased DNA binding and activity compared with wild type IRF7. Conversely, we showed that acetylated IRF7 displayed impaired DNA binding capability and that over-expression of PCAF led to decreased IRF7 activity. Together, our results strongly suggest that acetylation of lysine 92 negatively modulates IRF7 DNA binding.

The 100-kDa 2′,5′-Oligoadenylate Synthetase Catalyzing Preferentially the Synthesis of Dimeric pppA2′p5′A Molecules Is Composed of Three Homologous Domains

The 2–5A synthetases represent a family of proteins implicated in the mechanism of the antiviral action of interferon. When activated by double-stranded RNA, these proteins polymerize ATP into 2′-5′-linked oligomers with the general formula pppA(2′p5′A) n , n ≥ 1. Three forms of human 2–5A synthetases have been described corresponding to proteins of 40/46 (p40/p46), 69/71 (p69/p71), and 100 kDa (p100). Here we describe the molecular cloning and characterization of p100. By screening a cDNA expression library with a specific p100 polyclonal antibody, we first isolated a 590-nucleotide cDNA fragment which was subsequently used to isolate the full-length 6365-nucleotide cDNA. This cDNA recognizes a distinct interferon-induced messenger RNA of 7 kilobases. It has an open reading frame encoding a protein of 1087 amino acids including the sequence of seven peptides obtained by microsequencing of the natural p100 protein, which was purified from interferon-treated human cells. p100 is composed of three adjacent domains, each homologous to the previously defined catalytic unit of 350 amino acids, which is present as one unit in p40/p46 and as two units in p69/p71. The recombinant p100 synthesized preferentially dimeric 2′,5′-oligoadenylate molecules and displayed parameters for maximum enzyme activity similar to the natural p100. These results confirm that the enzymatic activity of p100 is distinct compared with that of p40/p46 and p69/p71.

How Viruses Elicit Interferon Production

Studies of IFN gene regulation in virus-infected cells have contributed significant information to our overall understanding of transcriptional control mechanisms in vertebrate cells. Enhanceosome theory was largely established by the analysis of IFNβ gene expression, and similarly important concepts for differential regulation of closely related genes will likely emerge from future studies of the IFNα locus. This system has also contributed to the understanding of mammalian signaling systems, with the identification of a variety of pathways that impact on gene induction through novel mechanisms. Signal transduction dependent on the action of RNA helicases and on ubiquitin-directed transcription factor phosphorylation are just some of the novel concepts emerging from the study of IFN gene induction.