Bryan R. G. Williams

Activation of the interferon system by short-interfering RNAs

RNA interference (RNAi) is a powerful tool used to manipulate gene expression or determine gene function. One technique of expressing the short double-stranded (ds) RNA intermediates required for interference in mammalian systems is the introduction of short-interfering (si) RNAs. Although RNAi strategies are reliant on a high degree of specificity, little attention has been given to the potential non-specific effects that might be induced. Here, we found that transfection of siRNAs results in interferon (IFN)-mediated activation of the Jak–Stat pathway and global upregulation of IFN-stimulated genes. This effect is mediated by the dsRNA-dependent protein kinase, PKR, which is activated by 21-base-pair (bp) siRNAs and required for upregulation of IFN-β in response to siRNAs. In addition, we show by using cell lines deficient in specific components mediating IFN action that the RNAi mechanism itself is independent of the interferon system. Thus, siRNAs have broad and complicating effects beyond the selective silencing of target genes when introduced into cells. This is of critical importance, as siRNAs are currently being explored for their potential therapeutic use.

Interferon-inducible antiviral effectors

Since the discovery of interferons (IFNs), considerable progress has been made in describing the nature of the cytokines themselves, the signalling components that direct the cell response and their antiviral activities. Gene targeting studies have distinguished four main effector pathways of the IFN-mediated antiviral response: the Mx GTPase pathway, the 2′,5′-oligoadenylate-synthetase-directed ribonuclease L pathway, the protein kinase R pathway and the ISG15 ubiquitin-like pathway. As discussed in this Review, these effector pathways individually block viral transcription, degrade viral RNA, inhibit translation and modify protein function to control all steps of viral replication. Ongoing research continues to expose additional activities for these effector proteins and has revealed unanticipated functions of the antiviral response.

Molecular cloning and characterization of the human double-stranded RNA-activated protein kinase induced by interferon

The double-stranded (ds) RNA-activated protein kinase from human cells is a 68 kd protein (p68 kinase) induced by interferon. On activation by dsRNA in the presence of ATP, the kinase becomes autophosphorylated and can catalyze the phosphorylation of the alpha subunit of eIF2, which leads to an inhibition of the initiation of protein synthesis. Here we report the molecular cloning and characterization of several related cDNAs from which can be deduced the full-length p68 kinase sequence. All of the cDNAs identify a 2.5 kb RNA that is strongly induced by interferon. The deduced amino acid sequence of the p68 kinase predicts a protein of 550 amino acids containing all of the conserved domains specific for members of the protein kinase family, including the catalytic domain characteristic of serine/threonine kinases. In vitro translation of a reconstructed full-length p68 kinase cDNA yields a protein of 68 kd that binds dsRNA, is recognized by a monoclonal antibody raised against the native p68 kinase, and is autophosphorylated.

Functional classification of interferon-stimulated genes identified using microarrays

Interferons (IFNs) are a family of multifunctional cytokines thatactivate transcription of subsets of genes. The gene products inducedby IFNs are responsible for IFN antiviral, antiproliferative, andimmunomodulatory properties. To obtain a more comprehensive list and abetter understanding of the genes regulated by IFNs, we compiled datafrom many experiments, using two different microarray formats. Thecombined data sets identified >300 IFN‐stimulated genes (ISGs). Toprovide new insight into IFN‐induced cellular phenotypes, we assignedthese ISGs to functional categories. The data are accessible on the World Wide Web at http://www.lerner.ccf.org/labs/williams, including functional categories and individual genes listed in asearchable database. The entries are linked to GenBank and Unigenesequence information and other resources. The goal is to eventuallycompile a comprehensive list of all ISGs. Recognition of the functionsof the ISGs and their specific roles in the biological effects of IFNsis leading to a greater appreciation of the many facets of theseintriguing and essential cytokines. This review focuses on thefunctions of the ISGs identified by analyzing the microarray data andfocuses particularly on new insights into the protein kinaseRNA‐regulated (PRKR) protein, which have been made possible with theavailability of PRKR‐null mice.

Deficient signaling in mice devoid of double-stranded RNA-dependent protein kinase.

Double‐stranded RNA‐dependent protein kinase (PKR) has been implicated in interferon (IFN) induction, antiviral response and tumor suppression. We have generated mice devoid of functional PKR (Pkr%). Although the mice are physically normal and the induction of type I IFN genes by poly(I).poly(C) (pIC) and virus is unimpaired, the antiviral response induced by IFN‐gamma and pIC was diminished. However, in embryo fibroblasts from Pkr knockout mice, the induction of type I IFN as well as the activation of NF‐kappa B by pIC, were strongly impaired but restored by priming with IFN. Thus, PKR is not directly essential for responses to pIC, and a pIC‐responsive system independent of PKR is induced by IFN. No evidence of the tumor suppressor activity of PKR was demonstrated.

JNK2 and IKKβ Are Required for Activating the Innate Response to Viral Infection

Viral infection or double-stranded (ds) RNA induce interferons (IFN) and other cytokines. Transcription factors mediating IFN induction are known, but the signaling pathways that regulate them are less clear. We now describe two such pathways. The first pathway leading to NF-kappaB depends on the dsRNA-responsive protein kinase (PKR), which in turn activates IKB kinase (IKK) through the IKKbeta subunit. The second viral-and dsRNA-responsive pathway is PKR independent and involves Jun kinase (JNK) activation leading to stimulation of AP-1. Both IKKbeta and JNK2 are essential for efficient induction of type I IFN and other cytokines in response to viral infection or dsRNA. This study establishes a general role for these kinases in activation of innate immune responses.

Signal Integration via PKR

The vital role of interferons (IFNs) as mediators of innate immunity is well established. It has recently become apparent that one of the pivotal proteins in mediating the antiviral activity of IFNs, the double-stranded RNA (dsRNA)-activated protein kinase (PKR), also functions as a signal transducer in the proinflammatory response to different agents. PKR is a member of a small family of kinases that are activated by extracellular stresses and that phosphorylate the α subunit of protein synthesis initiation factor eIF-2, thereby inhibiting protein synthesis. The activation of PKR during infection by viral dsRNA intermediates results in the inhibition of viral replication. PKR also mediates the activation of signal transduction pathways by proinflammatory stimuli, including bacterial lipopolysaccharide (LPS), tumor necrosis factor α (TNF-α), and interleukin 1 (IL-1). PKR is a component of the inhibitor of κB (IκB) kinase complex and plays either a catalytic or structural role in the activation of IκB kinase, depending on the stimulus. The activities of the stress-activated protein kinases p38 and c-Jun NH2-terminal kinase (JNK) are also regulated by PKR in a pathway that leads to the production of proinflammatory cytokines. This review will focus on the role of PKR in nuclear factor κ B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways, because these have been the subjects of a series of publications over the past year that have reported conflicting findings. Although the conflicts may not be resolved in this review, suggestions are made for experiments that could lead to a clearer understanding of the mechanisms involved. Interferons (IFNs) are secreted proteins that signal the presence of a virus infection to surrounding cells. Cells respond to IFNs by establishing an antiviral state that prohibits virus replication, thereby limiting infection. One of the mediators of this antiviral state is an enzyme, double-stranded RNA-activated protein kinase (PKR). Recently, in addition to its role as an antiviral protein, PKR has been shown to be essential in the development of normal cellular inflammatory responses to the products of both bacterial and viral infections. In this role, PKR functions as a transducer of signals from the extracellular milieu to the nucleus, a process which enables activation of expression of genes encoding molecules important for inflammation and immune responses. PKR performs this role by directly interacting with other intracellular components of signaling pathways and regulating their activity. When the activity of PKR is disrupted, cells become defective in their programmed death responses to different signals.

A structural basis for discriminating between self and nonself double-stranded RNAs in mammalian cells

Nonspecific effects triggered by small interfering RNAs (siRNAs) complicate the use of RNA interference (RNAi) to specifically downregulate gene expression. To uncover the basis of these nonspecific activities, we analyzed the effect of chemically synthesized siRNAs on mammalian double-stranded RNA (dsRNA)-activated signaling pathways. siRNAs ranging from 21 to 27 nucleotides (nt) in length activated the interferon system when they lacked 2-nt 3′ overhangs, a characteristic of Dicer products. We show that the recognition of siRNAs is mediated by the RNA helicase RIG-I and that the presence of 3′ overhangs impairs its ability to unwind the dsRNA substrate and activate downstream signaling to the transcription factor IRF-3. These results suggest a structural basis for discrimination between microRNAs that are endogenous Dicer products, and nonself dsRNAs such as by-products of viral replication. These findings will enable the rational design of siRNAs that avoid nonspecific effects or, alternatively, that induce bystander effects to potentially increase the efficacy of siRNA-based treatments of viral infections or cancer.

Deficient cytokine signaling in mouse embryo fibroblasts with a targeted deletion in the PKR gene: role of IRF‐1 and NF‐κB

The interferon (IFN)‐induced double‐stranded RNA (dsRNA)‐activated Ser/Thr protein kinase (PKR) plays a role in the antiviral and antiproliferative effects of IFN. PKR phosphorylates initiation factor eIF2α, thereby inhibiting protein synthesis, and also activates the transcription factor, nuclear factor‐κB (NF‐κB), by phosphorylating the inhibitor of NF‐κB, IκB. Mice devoid of functional PKR (Pkr°/°) derived by targeted gene disruption exhibit a diminished response to IFN‐γ and poly(rI:rC) (pIC). In embryo fibroblasts derived from Pkr°/° mice, interferon regulatory factor 1 (IRF‐1) or guanylate binding protein (Gbp) promoter–reporter constructs were unresponsive to IFN‐γ or pIC but response could be restored by co‐transfection with PKR. The lack of responsiveness could be attributed to a diminished activation of IRF‐1 and/or NF‐κB in response to IFN‐γ or pIC. Thus, PKR acts as a signal transducer for IFN‐stimulated genes dependent on the transcription factors IRF‐1 and NF‐κB.

p38 MAP kinase is required for STAT1 serine phosphorylation and transcriptional activation induced by interferons.

Activation of cytosolic phospholipase A2 (cPLA2) is a prerequisite for the formation of the transcription factor complex interferon‐stimulated gene factor 3 (ISGF3) in response to interferon‐α (IFN‐α). Here we show that p38 mitogen‐activated protein kinase (MAPK), an activator of cPLA2, is essential for both IFN‐α and IFN‐γ signalling. SB203580, a specific inhibitor of p38, was found to inhibit ISGF3 formation but had no apparent effects on signal transducer and activator of transcription (STAT)1 homodimer formation. Regardless of this, the antiviral activities of both IFN‐α and IFN‐γ were attenuated by SB203580. Treatment with either IFN led to rapid and transient activation of p38. Both IFNs induced STAT1 Ser727 phosphorylation, which was inhibited by SB203580 but not by an extracellular signal related kinase (ERK)1/2 inhibitor (PD98059). In an inducible 3T3‐L1 clone, expression of dominant‐negative p38 led to defective STAT1 serine phosphorylation and diminished IFN‐γ‐mediated protection against viral killing. Reporter activity mediated by ISGF3 or STAT1 homodimer was diminished by SB203580 and enhanced by a constitutively active mutant of MKK6, the upstream activator of p38. Therefore, p38 plays a key role in the serine phosphorylation of STAT1 and transcriptional changes induced by both IFNs.