Ribosomal protein L22 inhibits regulation of cellular activities by the Epstein-Barr virus small RNA EBER-1.

Epstein-Barr virus (EBV) is a potent mitogenic and antiapoptotic agent for B lymphocytes and is associated with several different types of human tumour. The abundantly expressed small viral RNA, EBER-1, binds to the growth inhibitory and pro-apoptotic protein kinase R (PKR) and blocks activation of the latter by double-stranded RNA. Recent evidence has suggested that expression of EBER-1 alone in EBV-negative B cells promotes a tumorigenic phenotype and that this may be related to inhibition of the pro-apoptotic effects of PKR. The ribosomal protein L22 binds to EBER-1 in virus-infected cells, but the significance of this has not previously been established. We report here that L22 and PKR compete for a common binding site on EBER-1. As a result of this competition, L22 interferes with the ability of the small RNA to inhibit the activation of PKR by dsRNA. Transient expression of EBER-1 in murine embryonic fibroblasts stimulates reporter gene expression and partially reverses the inhibitory effect of PKR. However, EBER-1 is also stimulatory when transfected into PKR knockout cells, suggesting an additional, PKR-independent, mode of action of the small RNA. Expression of L22 prevents both the PKR-dependent and -independent effects of EBER-1 in vivo. These results suggest that the association of L22 with EBER-1 in EBV-infected cells can attenuate the biological effects of the viral RNA. Such effects include both the inhibition of PKR and additional mechanism(s) by which EBER-1 stimulates gene expression.     

Interactions between double-stranded RNA regulators and the protein kinase DAI.

The interferon-induced protein kinase DAI, the double-stranded RNA (dsRNA)-activated inhibitor of translation, plays a key role in regulating protein synthesis in higher cells. Once activated, in a process that involves autophosphorylation, it phosphorylates the initiation factor eIF-2, leading to inhibition of polypeptide chain initiation. The activity of DAI is controlled by RNA regulators, including dsRNA activators and highly structured single-stranded RNAs which block activation by dsRNA. To elucidate the mechanism of activation, we studied the interaction of DAI with RNA duplexes of discrete sizes. Molecules shorter than 30 bp fail to bind stably and do not activate the enzyme, but at high concentrations they prevent activation by long dsRNA. Molecules longer than 30 bp bind and activate the enzyme, with an efficiency that increases with increasing chain length, reaching a maximum at about 85 bp. These dsRNAs fail to activate at high concentrations and also prevent activation by long dsRNA. Analysis of complexes between dsRNA and DAI suggests that at maximal packing the enzyme interacts with as little as a single helical turn of dsRNA (11 bp) but under conditions that allow activation the binding site protects about 80 bp of duplex. When the RNA-binding site is fully occupied with an RNA activator, the complex appears to undergo a conformational change.     

Comparative analysis of the regulation of the interferon-inducible protein kinase PKR by Epstein-Barr virus RNAs EBER-1 and EBER-2 and adenovirus VAI RNA.

The interferon-inducible protein kinase PKR interacts with a number of small viral RNA species, including adenovirus VAI RNA and the Epstein-Barr virus-encoded RNA EBER-1. These RNAs bind to PKR and protect protein synthesis from inhibition by double-stranded RNA in the reticulocyte lysate system. Using a peptide phosphorylation assay we show here that EBER-1, like VAI, directly inhibits the activation of purified PKR. A second Epstein-Barr virus RNA, EBER-2, also regulates PKR. EBER-1, EBER-2 and VAI RNA exhibit mutually competitive binding to the native or recombinant enzyme, as assessed by U.V. crosslinking experiments and filter binding assays. The affinities of all three RNAs for PKR in vitro are similar (Kd = ca. 0.3 nM). Since this protein kinase has been proposed to exert a tumour suppressor function in vivo, the ability of EBER-1 to inhibit its activation suggests a role for this small RNA in cell transformation by Epstein-Barr virus.     

Possible involvement of double-stranded RNA-activated protein kinase in cell death by influenza virus infection.

We reported previously that influenza virus infection induces the apoptotic death of HeLa cells associated with activation of the Fas gene. In this report, we show that transfection with a PKR having a point mutation in the catalytic domain of K at 296 to R suppressed both the augmented expression of Fas and cell death by influenza virus infection. These results suggested the involvement of PKR in influenza virus-induced cell death.     

CpG-methylation silences the activity of the RNA polymerase III transcribed EBER-1 promoter of Epstein-Barr virus

CpG-methylation blocks the activity of RNA polymerase II transcribed promoters in most cases. In contrast, the role of DNA methylation in the regulation of RNA polymerase III transcribed promoters is less clarified. There are two untranslated viral RNAs (EBER-1 and EBER-2) in most malignant cells carrying latent Epstein-Barr virus (EBV) genomes. We found that in vitro methylation blocked binding of the cellular proteins c-Myc and ATF to the 5'-region of the EBER-1 gene, and silenced the expression of the EBER-1 and EBER-2 genes, transcribed by RNA polymerase III, in transfected cells.     

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.     

Semliki forest virus capsid protein inhibits the initiation of translation by upregulating the double-stranded RNA-activated protein kinase (PKR)

We investigated the possible translational role which elevated concentrations of highly purified Semliki Forest virus (SFV) capsid (C)-protein molecules may play in a cell-free translation system. Here we decomonstrate that in the absence of double-stranded RNA high concentrations of C protein triggered the phosphorylation of the interferon-induced, double-stranded RNA-activated protein kinase, PKR. Activated PKR in turn phosphorylated its natural substrate, the subunit of eukaryotic initiation factor 2 (eIF-2), thereby inhibiting initiation of host cell translation. These findings were further strengthened by experiments showing that during natural infection with SFV the maximum phosphorylation of PKR coincided with the maximum synthesis of C protein 4–9 hours post infection. Thus, our results demonstrate that high concentrations of C-protein molecules may act in a hitherto novel mechanism on PKR to inhibit host cell protein synthesis during viral infection.     

Expression of unphosphorylated form of human double-stranded RNA-activated protein kinase in Escherichia coli

Interferon (IFN)-inducible, double-stranded (dsRNA)-activated protein kinase (PKR) is a key mediator of the antiviral and antiproliferative effects of IFN. PKR is present within cells in a latent state. In response to binding dsRNA, the enzyme becomes activated, causing autophosphorylation and an increase in specific kinase activity. In order to study PKR and its inhibitors, a large amount of the enzyme in its latent, unphosphorylated state is required. When PKR is fused to glutathione S-transferase (GST-PKR) and the fusion protein is expressed in Escherichia coli, the PKR obtained is fully activated by autophosphorylation. Therefore, we have developed an expression plasmid in which both GST-PKR and bacteriophage lambda protein phosphatase (lambda-PPase) genes were placed downstream of a T7 promoter. After induction of expression, unphosphorylated GST-PKR was obtained in good yield, and purified to near homogeneity. The purified enzyme has dsRNA-dependent activation and phosphorylates the translation initiation factor eIF2 alpha. Using the recombinant protein, we analyzed the inhibition mechanisms of two viral inhibitors, vaccinia virus K3L protein and adenovirus virus-associated RNA I (VAI RNA). K3L inhibited both autophosphorylation of PKR and phosphorylation of eIF2 alpha, whereas VAI RNA inhibited only autophosphorylation. The separation of autophosphorylation and catalytic activity shows that the recombinant PKR is useful in analyzing the functions of PKR, its inhibitors, and its regulatory molecules. The coexpression system of protein kinase with lambda-PPase described here will be applicable to obtaining unphosphorylated and unactivated forms of other protein kinases.     

Central role of double-stranded RNA-activated protein kinase in microbial induction of nitric oxide synthase

NO synthase 2 (NOS2) is induced in airway epithelium by influenza virus infection. NOS2 induction late in the course of viral infection may occur in response to IFN-gamma, but early in infection gene expression may be induced by the viral replicative intermediate dsRNA through the dsRNA-activated protein kinase (PKR). Since PKR activates signaling pathways important in NOS2 gene induction, we determined whether PKR is a component in the signal transduction pathway leading to NOS2 gene expression after viral infection of airway epithelium. We show that NOS2 gene expression in human airway epithelial cells occurs in response to influenza A virus or synthetic dsRNA. Furthermore, dsRNA leads to rapid activation of PKR, followed by activation of signaling components including NF-kappaB and IFN regulatory factor 1. NOS2 expression is markedly diminished and IFN regulatory factor 1 and NF-kappaB activation are substantially impaired in PKR null cells. Strikingly, NOS2 induction in response to LPS is abolished in PKR null cells, confirming a central role for PKR in the general signaling pathway to NOS2.     

Nuclear factor 90 is a substrate and regulator of the eukaryotic initiation factor 2 kinase double-stranded RNA-activated protein kinase.

Nuclear factor 90 (NF90) is a member of an expanding family of double-stranded (ds) RNA-binding proteins thought to be involved in gene expression. Originally identified in complex with nuclear factor 45 (NF45) as a sequence-specific DNA-binding protein, NF90 contains two double stranded RNA-binding motifs (dsRBMs) and interacts with highly structured RNAs as well as the dsRNA-activated protein kinase, PKR. In this report, we characterize the biochemical interactions between these two dsRBM containing proteins. NF90 binds to PKR through two independent mechanisms: an RNA-independent interaction occurs between the N terminus of NF90 and the C-terminal region of PKR, and an RNA-dependent interaction is mediated by the dsRBMs of the two proteins. Co-immunoprecipitation analysis demonstrates that NF90, NF45, and PKR form a complex in both nuclear and cytosolic extracts, and both proteins serve as substrates for PKR in vitro. NF90 is phosphorylated by PKR in its RNA-binding domain, and this reaction is partially blocked by the NF90 N-terminal region. The C-terminal region also inhibits PKR function, probably through competitive binding to dsRNA. A model for NF90-PKR interactions is proposed.     

Double-stranded RNA-activated protein kinase PKR of fishes and amphibians: Varying the number of double-stranded RNA binding domains and lineage-specific duplications

Background  

Double-stranded (ds) RNA, generated during viral infection, binds and activates the mammalian anti-viral protein kinase PKR, which phosphorylates the translation initiation factor eIF2α leading to the general inhibition of protein synthesis. Although PKR-like activity has been described in fish cells, the responsible enzymes eluded molecular characterization until the recent discovery of goldfish and zebrafish PKZ, which contain Z-DNA-binding domains instead of dsRNA-binding domains (dsRBDs). Fish and amphibian PKR genes have not been described so far.     

Purification and activation of the double-stranded RNA-dependent eIF-2 kinase DAI.

The double-stranded RNA (dsRNA)-dependent protein kinase DAI (also termed dsI and P1) possesses two kinase activities; one is an autophosphorylation activity, and the other phosphorylates initiation factor eIF-2. We purified the enzyme, in a latent form, to near homogeneity from interferon-treated human 293 cells. The purified enzyme consisted of a single polypeptide subunit of approximately 70,000 daltons, retained its dependence on dsRNA for activation, and was sensitive to inhibition by adenovirus VA RNAI. Autophosphorylation required a suitable concentration of dsRNA and was second order with respect to DAI concentration, which suggests an intermolecular mechanism in which one DAI molecule phosphorylates a neighboring molecule. Once autophosphorylated, the enzyme could phosphorylate eIF-2 but seemed unable to phosphorylate other DAI molecules, which implies a change in substrate specificity upon activation. VA RNAI blocked autophosphorylation and activation but permitted the activated enzyme to phosphorylate eIF-2. VA RNAI also blocked the binding of dsRNA to the enzyme. The data are consistent with a model in which activation requires the interaction of two molecules of DAI with dsRNA, followed by intermolecular autophosphorylation of the latent enzyme. VA RNAI would block activation by preventing the interaction between DAI and dsRNA.     

Activation of double-stranded RNA-activated protein kinase by mild impairment of oxidative metabolism in neurons

Thiamine (vitamin B1) deficiency (TD) causes mild and chronic impairment of oxidative metabolism and induces neuronal death in specific brain regions. The mechanisms underlying TD-induced cell death, however, remain unclear. The double-stranded RNA-activated protein kinase (PKR), has been well known for its anti-viral function. Upon activation by viral infection or double-stranded RNA, PKR phosphorylates its substrate, the α-subunit of eukaryotic initiation factor-2 (eIF2α), leading to inhibition of translation. In response to various cellular stresses, PKR can also be stimulated by its protein activators, or its mouse homologue, PKR activator (RAX). We demonstrated that TD in mice induced phosphorylation of PKR at Thr446 and Thr451 and phosphorylation of eIF2α at Ser51 in the cerebellum and the thalamus. TD caused phosphorylation of PKR and eIF2α, as well as nuclear translocation of PKR in primary cultures of cerebellar granule neurons. PKR phosphorylation is necessary for its nuclear translocation because TD failed to induce nuclear translocation of a T446A/T451A PKR mutant. Both PKR inhibitor and dominant-negative PKR mutant protected cerebellar granule neurons against TD-induced cell death. TD promoted the association between RAX and PKR. Antioxidant vitamin E dramatically decreased the RAX/PKR association and ameliorated TD-induced cell death. Our results indicate that TD-induced neuronal death is at least partially mediated by the activation of PKR.