The antiviral enzymes PKR and RNase L suppress gene expression from viral and non-viral based vectors.
(25/1374)
Expression of transfected genes is shown to be suppressed by two intracellular enzymes, RNase L and protein kinase PKR, which function in interferon-treated cells to restrict viral replication. RNase L(-/-) or PKR(-/-) murine embryonic fibroblasts produced enhanced levels of protein from transfected genes compared with wild-type cells. Increased expression of exogenous genes in RNase L(-/-) cells correlated with elevated levels of mRNA and thus appeared to be due to enhanced mRNA stability. Plasmid encoding adenovirus VA RNAs was able to further enhance accumulation of the exogenous gene transcript and protein, even in cells lacking PKR. In contrast to the increased expression of transfected genes in cells lacking RNase L or PKR, expression of endogenous host genes was unaffected by the absence of these enzymes. In addition, a dominant-negative PKR mutant improved expression from a conventional plasmid vector and from a Semliki Forest virus derived, self-replicating vector. These results indicate that viral infections and transfections produce similar stress responses in mammalian cells and suggest strategies for selectively increasing expression of exogenous genes. (+info)
Translational control by the La antigen. Structure requirements for rescue of the double-stranded RNA-mediated inhibition of protein synthesis.
(26/1374)
The La antigen is a protein which can bind both single-stranded and double-stranded forms of RNA and has regulatory effects on gene expression at the levels of transcription and translation. It was previously shown to inhibit the activation of the dsRNA-dependent protein kinase PKR by sequestering and/or unwinding double-stranded RNA. Here, we demonstrate that, as predicted by these properties, the La antigen can rescue protein synthesis in the reticulocyte lysate system from inhibition by low concentrations of dsRNA. This effect is reversed by higher concentrations of dsRNA. Using a series of deletion mutants we have investigated the structural features of the La antigen that are required for these effects. The ability to bind dsRNA is influenced by regions within both the previously characterized N-terminal RNP motif and the C-terminal half of the protein. La mutants with either N-terminal or C-terminal deletions retain the ability to inhibit the protein kinase activity of PKR and to rescue protein synthesis from inhibition by dsRNA. It is notable that sequences in the C-terminal half of the La antigen, including a phosphorylation site at Ser366, which are needed for other regulatory effects of the protein on gene expression are dispensable for the effects of La on PKR. We suggest that La regulates PKR activity solely as a result of its ability to act as an RNA-binding protein that can compete with PKR for limiting amounts of dsRNA. (+info)
The interferon-induced double-stranded RNA-activated protein kinase PKR will phosphorylate serine, threonine, or tyrosine at residue 51 in eukaryotic initiation factor 2alpha.
(27/1374)
The family of eukaryotic initiation factor 2alpha (eIF2alpha) protein kinases plays an important role in regulating cellular protein synthesis under stress conditions. The mammalian kinases PKR and HRI and the yeast kinase GCN2 specifically phosphorylate Ser-51 on the alpha subunit of the translation initiation factor eIF2. By using an in vivo assay in yeast, the substrate specificity of these three eIF2alpha kinases was examined by substituting Ser-51 in eIF2alpha with Thr or Tyr. In yeast, phosphorylation of eIF2 inhibits general translation but derepresses translation of the GCN4 mRNA. All three kinases phosphorylated Thr in place of Ser-51 and were able to regulate general and GCN4-specific translation. In addition, both PKR and HRI were found to phosphorylate eIF2alpha-S51Y and stimulate GCN4 expression. Isoelectric focusing analysis of eIF2alpha followed by detection using anti-eIF2alpha and anti-phosphotyrosine-specific antibodies demonstrated that PKR and HRI phosphorylated eIF2alpha-S51Y on Tyr in vivo. These results provide new insights into the substrate recognition properties of the eIF2alpha kinases, and they are intriguing considering the potential for alternate substrates for PKR in cellular signaling and growth control pathways. (+info)
Alternative splice variants of the human PKR protein kinase possessing different 5'-untranslated regions: expression in untreated and interferon-treated cells and translational activity.
(28/1374)
The double-stranded RNA-dependent protein kinase PKR is an interferon-inducible enzyme that possesses antiviral and antiproliferative activities. We examined expression of PKR transcripts in human placenta tissue and cultured human amnion U cells. Alternative exon 2 structures were identified and characterized that possess different functional activities. Cloning and sequence analyses of 5'-RACE cDNAs from human placenta established a linkage between exon 1 and three alternative exon 2 structures that constitute, together with part of exon 3, the 5'-untranslated region of the PKR mRNA. The alternative splice variants of exon 2 were designated Ex2alpha (83 nucleotides), Ex2beta (167 nucleotides), and Ex2gamma (401 nucleotides). All three exon 2 variants were present in placenta tissue. However, only the Ex2alpha and Ex2beta forms were detectable in the amnion U cell line. Nuclease protection analysis revealed that the Ex2beta form was slightly more abundant than the Ex2alpha form, in both placenta tissue and U cells. Interferon treatment of U cells increased the level of both Ex2alpha and Ex2beta RNA by approximately 5-fold. The translational activities, measured in a luciferase reporter assay, of RNA transcripts possessing the Ex2alpha and Ex2beta forms of the PKR 5'-UTR were comparable to each other and more efficient than those with the Ex2gamma form. (+info)
PKR; a sentinel kinase for cellular stress.
(29/1374)
The double stranded RNA (dsRNA)-activated protein kinase PKR is a ubiquitously expressed serine/threonine protein kinase that is induced by interferon and activated by dsRNA, cytokine, growth factor and stress signals. It is essential for cells to respond adequately to different stresses including growth factor deprivation, products of the inflammatory response (TNF) and bacterial (lipopolysaccharide) and viral (dsRNA) products. As a vital component of the cellular antiviral response pathway, PKR is autophosphorylated and activated on binding to dsRNA. This results in inhibition of protein synthesis via the phosphorylation of eIF2alpha and also induces transcription of inflammatory genes by PKR-dependent signaling of the activation of different transcription factors. Along with RNaseL, PKR constitutes the antiviral arm of a group of mammalian stress response proteins that have counterparts in yeast. What began as adaptation to amino acid deprivation and sensing unfolded proteins in the endoplasmic reticulum has evolved into a family of sophisticated mammalian stress response proteins able to mediate cellular responses to both physical and biological stress. (+info)
Activation of p38 mitogen-activated protein kinase and c-Jun NH(2)-terminal kinase by double-stranded RNA and encephalomyocarditis virus: involvement of RNase L, protein kinase R, and alternative pathways.
(30/1374)
Double-stranded RNA (dsRNA) accumulates in virus-infected mammalian cells and signals the activation of host defense pathways of the interferon system. We describe here a novel form of dsRNA-triggered signaling that leads to the stimulation of the p38 mitogen-activated protein kinase (p38 MAPK) and the c-Jun NH(2)-terminal kinase (JNK) and of their respective activators MKK3/6 and SEK1/MKK4. The dsRNA-dependent signaling to p38 MAPK was largely intact in cells lacking both RNase L and the dsRNA-activated protein kinase (PKR), i. e., the two best-characterized mediators of dsRNA-triggered antiviral responses. In contrast, activation of both MKK4 and JNK by dsRNA was greatly reduced in cells lacking RNase L (or lacking both RNase L and PKR) but was restored in these cells when introduction of dsRNA was followed by inhibition of ongoing protein synthesis or transcription. These results are consistent with the notion that the role of RNase L and PKR in the activation of MKK4 and JNK is the elimination, via inhibition of protein synthesis, of a labile negative regulator(s) of the signaling to JNK acting upstream of SEK1/MKK4. In the course of these studies, we identified a long-sought site of RNase L-mediated cleavage in the 28S rRNA, which could cause inhibition of translation, thus allowing the activation of JNK by dsRNA. We propose that p38 MAPK is a general participant in dsRNA-triggered cellular responses, whereas the activation of JNK might be restricted to cells with reduced rates of protein synthesis. Our studies demonstrate the existence of alternative (RNase L- and PKR-independent) dsRNA-triggered signaling pathways that lead to the stimulation of stress-activated MAPKs. Activation of p38 MAPK (but not of JNK) was demonstrated in mouse fibroblasts in response to infection with encephalomyocarditis virus (ECMV), a picornavirus that replicates through a dsRNA intermediate. Fibroblasts infected with EMCV (or treated with dsRNA) produced interleukin-6, an inflammatory and pyrogenic cytokine, in a p38 MAPK-dependent fashion. These findings suggest that stress-activated MAPKs participate in mediating inflammatory and febrile responses to viral infections. (+info)
A cis-acting element in the 3'-untranslated region of human TNF-alpha mRNA renders splicing dependent on the activation of protein kinase PKR.
(31/1374)
We report a role for the 3'-untranslated region in control of mRNA splicing and show that human TNF-alpha 3' UTR harbors a cis-acting element that renders splicing of precursor transcripts dependent on activation of PKR, the RNA-activated protein kinase that phosphorylates eukaryotic initiation factor 2 (eIF2). When this element, designated 2-APRE, is present, splicing becomes sensitive to inhibition by the PKR inhibitor, 2-aminopurine, or by coexpression of transdominant-negative mutant PKR. Our results reveal that activation of PKR is required for splicing of mRNA when precursor transcripts contain the 2-APRE and that increased expression of wild-type PKR enhances their splicing efficiency. Thus, PKR responds as trans-acting factor to the 2-APRE. 2-APRE RNA forms a stable, 17-bp stem-loop structure and strongly activates PKR in vitro, inducing eIF2alpha phosphorylation. Despite its ability to activate PKR during splicing, the 2-APRE within the 3' UTR does not affect translation efficiency of the resulting TNF-alpha mRNA in transfected cells. PKR and the 3' UTR thus interact during mRNA splicing to confer a novel type of regulation on expression of the TNF-alpha gene. (+info)
JNK2 and IKKbeta are required for activating the innate response to viral infection.
(32/1374)
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. (+info)