The dsRNA protein kinase PKR: virus and cell control.

The IFN-induced double-stranded RNA-dependent protein kinase (PKR) is one of the four mammalian serine-threonine kinases (the three others being HRI, GCN2 and PERK) that phosphorylate the eIF2 alpha translation initiation factor, in response to stress signals, mainly as a result of viral infections. eIF2 alpha phosphorylation results in arrest of translation of both cellular and viral mRNAs, an efficient way to inhibit virus replication. The particularity of PKR is to activate by binding to dsRNA through two N terminal dsRNA binding motifs (dsRBM). PKR activation during a viral infection represents a threat for several viruses, which have therefore evolved to express PKR inhibitors, such as the Vaccinia E3L and K3L proteins. The function of PKR can also be regulated by cellular proteins, either positively (RAX/PACT; Mda7) or negatively (p58IPK, TRBP, nucleophosmin, Hsp90/70). PKR can provoke apoptosis, in part through its ability to control protein translation, but the situation appears to be more complex, as NF-kappaB, ATF-3 and p53 have also been implicated. PKR-induced apoptosis involves mainly the FADD/caspase 8 pathway, while the mitochondrial APAF/caspase 9 pathway is also engaged. As a consequence of the effects of PKR on translation, transcription and apoptosis, PKR can function to control cell growth and cell differentiation, and its activity can be controlled by the action of several oncogenes.

PKR stimulates NF-kappaB irrespective of its kinase function by interacting with the IkappaB kinase complex.

The interferon (IFN)-induced double-stranded RNA-activated protein kinase PKR mediates inhibition of protein synthesis through phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha) and is also involved in the induction of the IFN gene through the activation of the transcription factor NF-kappaB. NF-kappaB is retained in the cytoplasm through binding to its inhibitor IkappaBalpha. The critical step in NF-kappaB activation is the phosphorylation of IkappaBalpha by the IkappaB kinase (IKK) complex. This activity releases NF-kappaB from IkappaBalpha and allows its translocation to the nucleus. Here, we have studied the ability of PKR to activate NF-kappaB in a reporter assay and have shown for the first time that two catalytically inactive PKR mutants, PKR/KR296 and a deletion mutant (PKR/Del42) which lacks the potential eIF2alpha-binding domain, can also activate NF-kappaB. This result indicated that NF-kappaB activation by PKR does not require its kinase activity and that it is independent of the PKR-eIF2alpha relationship. Transfection of either wild-type PKR or catalytically inactive PKR in PKR(0/0) mouse embryo fibroblasts resulted in the activation of the IKK complex. By using a glutathione S-transferase pull-down assay, we showed that PKR interacts with the IKKbeta subunit of the IKK complex. This interaction apparently does not require the integrity of the IKK complex, as it was found to occur with extracts from cells deficient in the NF-kappaB essential modulator, one of the components of the IKK complex. Therefore, our results reveal a novel pathway by which PKR can modulate the NF-kappaB signaling pathway without using its kinase activity.

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.

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.

The role of the phosphorus BI-BII transition in protein-DNA recognition: the NF-kappaB complex.

We examined, by 1H and 31P NMR, the solution structure of a 16 bp non-palindromic DNA fragment (16M2) containing the HIV-1 NF-kappaB-binding site, in which the sequences flanking the kappaB site had been mutated. 31P NMR was particularly useful for obtaining structural information on the phosphodiester backbone conformation. Structural features were then compared with those of the two previously studied DNA fragments corresponding, respectively, to the native kappaB fragment (16N) and a fragment in which mutations have been introduced at the 5' end of the kappaB site (16M1). For the mutated 16M2 duplex, NMR data showed that the BI-BII equilibrium, previously reported for the native fragment (16N) at the kappaB flanking steps, was lost. The role of the BI-BII equilibrium in NF-kappaB recognition by DNA was then investigated by electrophoretic mobility shift assay. We found that the isolated kappaB site has the potential to bind efficiently due to the BI-BII equilibrium of the kappaB flanking sequences.

Molecular cloning of two new interferon-induced, highly related nuclear phosphoproteins.

During the molecular cloning of the human dsRNA activated-p68 kinase (PKR), polyclonal antibodies against PKR selected, in addition to cDNAs corresponding to PKR, another cDNA presenting only slight homology with PKR cDNA. This cDNA recognized an mRNA species of 2 kilobases induced by both alpha- and gamma-interferons. Its transcription did not require protein synthesis. On further library screening, it selected two highly related cDNAs, referred to as 75 and 41, displaying perfect homology over 612 base pairs and divergent at both ends. In addition, cDNA 75 presents an insertion of 150 base pairs highly homologous to a region common to both sequences. The 75 and 41 peptidic sequences are very hydrophilic, rich in basic amino acid residues, and contain several potential phosphorylation sites for different serine/threonine kinases. Furthermore, they present two protamine- and histone-like nuclear targeting sequences as well as some homology with helix-loop-helix motifs of some DNA-binding proteins. The 75-encoded product, which resolved as a 52-kDa protein after in vitro expression in rabbit reticulocyte lysates, was found to migrate as a 65-67-kDa protein after in vivo expression in insect cells. In accord with sequence data, this 65-67-kDa protein was found to be phosphorylated in vivo in the insect cells and was recovered from the membrane/nuclear pellet. In contrast, the 41-encoded product (30-kDa protein in reticulocyte lysates) could not be expressed in vivo, as it provoked a rapid and severe shut-off of protein synthesis in insect cells. The function of the 75 and 41 proteins and their relation to PKR remains to be determined. However, the presence of nuclear targeting sequences, phosphorylation sites, and helix-loop-helix motif is consistent with a role of these proteins in the mechanism of transduction of the interferon action.

Tumor suppressor function of the interferon-induced double-stranded RNA-activated protein kinase.

RNA-dependent protein kinase is a M(r) 68,000 protein in human cells (p68 kinase) or a M(r) 65,000 protein in murine cells (p65 kinase). p65/p68 is a serine/threonine kinase induced by interferon treatment and generally activated by double-stranded RNAs. Once activated, the known function of this kinase is inhibition of protein synthesis through phosphorylation of the eukaryotic initiation factor 2. Here we have investigated the potential for tumorigenicity in mice of murine NIH 3T3 clones expressing human p68 kinase, either the wild-type or a mutant inactive kinase with a single amino acid substitution in the invariant lysine-296 in the catalytic domain II. Expression of the mutant p68 kinase was correlated with a malignant transformation phenotype, giving rise to the production of large tumors of at least 1 cm in diameter within 7-12 days in all inoculated mice. In contrast, no tumor growth was observed for several weeks in mice inoculated with NIH 3T3 cell clones expressing either the wild-type recombinant p68 kinase or only the endogenous p65 kinase, the murine analogue of the p68 kinase. These results suggest that functional p65/p68 kinase (recently called PKR), by a still undefined mechanism, may also act as a tumor suppressor. Consequently, one of the pathways by which interferon inhibits tumor growth might be through its capacity to induce the enhanced expression of this kinase.

Characterization of an interferon-induced 48-kD protein immunologically related to the double-stranded RNA-activated protein kinase PKR.

Polyclonal antibodies raised against purified and urea-denatured double-stranded protein kinase (PKR) from human origin cross-reacted by immunoblotting with a 48-kD protein (p48) induced by the three types of interferon (IFN), alpha, beta, and gamma. The induction of p48 is IFN dose dependent and its accumulation occurs a few hours after the addition of IFN. The induction of p48 is blocked by actinomycin D. Analysis by two-dimensional gel isoelectric-focusing, revealed p48 as a single spot with an isoelectric point (pI) of 6.8. In the same experiment the PKR was revealed as several subspecies with pI values in the pH range of 7.4-8.0. Cell fractionation experiments indicated that PKR and p48 have different subcellular localizations: PKR was found to be associated with the microsomal pellet as shown previously whereas p48 was recovered in the microsomal supernatant fraction. In addition to these differences, PKR and p48 were found to be differentially expressed in some human cells treated with the three types of IFN. For example, in HeLa cells, IFN-alpha or IFN-beta induced similarly both PKR and p48 whereas IFN-gamma induced mainly p48. In U937 cells in which PKR was not expressed with or without IFN treatment, p48 was strongly induced by all three types of IFN. These results suggest different mechanisms for the induction of PKR and p48. In view of its presence in different types of human cells and its induction by different types of IFN, it is possible to suggest that p48 might play an important role in mediating some of the action of IFN.

Double-stranded RNA-dependent protein kinase mediates c-Myc suppression induced by type I interferons.

The antiproliferative functions of interferons result from specific effects that these cytokines exert on several cell cycle-controlling genes. The possible coupling between the interferon-responsive genes that are directly transactivated by the interferon signaling and the genes that constitute the basic machinery of the cell cycle is not clear yet. We report in this work that interferon-induced double-stranded RNA-activated kinase (PKR) is one of the specific mediators of the antiproliferative effects of the cytokine. Transfections of M1 myeloid leukemia cells with two catalytically inactive mutant forms of PKR abrogated the ability of interferon to suppress c-Myc without interfering with the pRB/cyclin D responses. As a consequence, these genetically manipulated cells displayed a small but significant reduction in their growth sensitivity to interferons, a phenotype that characterizes a single pathway disruption. Transfection of the parental M1 cells with the functional wild-type human PKR restricted their proliferation in the absence of interferons. This PKR-mediated growth inhibition could be efficiently rescued by the ectopic expression of deregulated c-myc. Taken together these results prove the existence of direct or indirect links between PKR and c-Myc suppression, thereby placing this gene along one of the complementary growth suppressive pathways that are triggered by interferons.

Human PKR transfected into murine cells stimulates expression of genes under control of the HIV1 or HTLV-I LTR.

We have analyzed the effect of transfection into murine NIH/3T3 cells of the human dsRNA-activated kinase PKR on the expression of the beta-galactosidase reporter gene, placed under control of the HIV1 or the HTLV-I LTR. beta-Galactosidase expression is stimulated when the reporter plasmids are cotransfected with wild-type PKR but inhibited when cotransfected with a catalytically inactive mutant PKR. In the case of HIV1, beta-galactosidase expression was not stimulated when cotransfection was carried out with PKR harboring mutations in the dsRNA binding domains, indicating that stimulation depends on the classical mode of PKR activation through dsRNA binding. In contrast, the dsRNA binding mutants of PKR could still partially stimulate beta-galactosidase expression from the HTLV-I LTR, suggesting that PKR activation in this case may involve different/additional mechanisms. These results show that, in addition to the known down-regulation of protein synthesis through elF2 phosphorylation, PKR can also positively stimulate gene expression in vivo, most probably through phosphorylation of a substrate distinct from elF2.

Molecular mechanisms responsible for malignant transformation by regulatory and catalytic domain variants of the interferon-induced enzyme RNA-dependent protein kinase.

Double-stranded RNA-dependent protein kinase (PKR) is suggested to play an important role in both the antiviral and antiproliferative arms of the interferon response. To gain insights into the molecular mechanisms underlying PKR's growth regulatory properties, we examined the biological and biochemical properties of PKR variants containing either a mutation in catalytic domain II (PKR-M1) or a deletion of RNA binding domain I (PKR-M7) in both reticulocyte translation extracts and in vitro kinase assays with purified reagents and compared these results with those using the same mutants stably expressed in vivo. While wild-type PKR (PKR-WT) efficiently inhibited mRNA translation in a reticulocyte extract, the inactive PKR-M1 had no effect. The PKR-M7 mutant was modestly inhibitory in this assay. The PKR-M1 variant was able to reverse the translational inhibitory effects and increased eukaryotic initiation factor (eIF)-2 alpha phosphorylation levels caused by addition of double-stranded RNA to reticulocyte extract, whereas PKR-M7 could not. Both PKR-M1 and PKR-M7 functioned as transdominant inhibitors of PKR-WT in our in vitro kinase assays. While the inhibition by PKR-M1 required a vast excess of mutant to shut down PKR function, PKR-M7 inhibited PKR-WT at approximately stoichiometric levels. To complement these experiments, we compared growth rates and alpha phosphorylation levels in transformed cell lines overexpressing either PKR-M1 or PKR-M7. Levels of endogenous eIF-2 alpha phosphorylation were significantly more diminished in PKR-M7 overexpressing cells compared with PKR-M1. These paradoxical data will be discussed in terms of the potential molecular mechanisms underlying malignant transformation caused by the PKR variants.

Colocalization within the nucleolus of two highly related IFN-induced human nuclear phosphoproteins with nucleolin.

We have previously reported the identification of two interferon (IFN)-induced cDNAs which code for two proteins, named 41 and 75, which have homology to a number of proteins involved in regulating gene expression. Here we establish that these cDNAs correspond to in vivo synthesized mRNAs. Expression of the 41 and 75 cDNAs, both in vitro and in vivo, generated proteins of 30 and 68 kDa, respectively. In a variety of mammalian cells, 41 and 75 were found to be located in the nucleus, with 41 being localized to the nucleolus, whereas 75, although it is mainly concentrated at the periphery of the nucleolus, is also found throughout the nucleoplasm. Treatment with interferon results in a translocation of 41 to the periphery of the nucleolus and it is in this region that the two proteins colocalize. 41 and 75 were found to colocalize with nucleolin but not with B23 or fibrillarin, three nucleolar proteins involved in ribosome synthesis. This colocalization was not affected by low concentrations of actinomycin D. In view of this and since 41 and 75 have homology to proteins involved in regulating gene expression, we suggest that, in association with nucleolin, they play a role in ribosome biogenesis.

Localization of the human interferon-induced, ds-RNA activated p68 kinase gene (PRKR) to chromosome 2p21-p22.

The interferon-induced dsRNA-activated protein kinase (PRKR) belongs to a subclass of serine/threonine kinases, involved in the regulation of protein synthesis by phosphorylation of the alpha subunit of initiation factor eIF2. Somatic cell hybrids segregating human chromosomes were used to assign this kinase to human chromosome 2. Fluorescence in situ hybridization confirmed this assignment and further localized the gene (PRKR) to the boundary region of bands p21 and 22.

Positive effect of the hepatitis C virus nonstructural 5A protein on viral multiplication.

Hepatitis C virus infection (HCV), is a major cause of liver disease worldwide, and are frequently resistant to the interferon alpha treatment. The nonstructural (NS) 5A protein of HCV has been proposed to be involved in this resistance. Additional studies have pointed out a role for NS5A in several other cellular interactions as well as an important role of its adaptative mutations in HCV genome replication. However, no infectious system is available to assess the role of NS5A in the HCV life cycle. Thus, we have constructed a recombinant system directly demonstrating for the first time that the expression of NS5A confers a multiplicative advantage to Sindbis virus, a virus close to HCV. This advantage seemed to be related to an anti-apoptotic effect of the NS5A protein. At a later stage, a possible nuclear localization of NS5A was observed, likely due to apoptotic cleavages of this protein. The NS5A protein was also shown to induce the interleukin-8 (IL-8) mRNA and to activate the NF-kappaB pathway independently of the Sindbis virus. Together, our data suggest that the activation of NF-kappaB could lead to the anti-apoptotic activity of NS5A and explain the viral multiplicative advantage conferred by the expression of the NS5A protein.

Constitutive expression of human double-stranded RNA-activated p68 kinase in murine cells mediates phosphorylation of eukaryotic initiation factor 2 and partial resistance to encephalomyocarditis virus growth.

The cDNA encoding interferon-induced human double-stranded RNA-activated p68 kinase was expressed in murine NIH 3T3 cells by using the pcDNA1/neo vector. Several stable clones were selected which expressed either the wild-type kinase or an inactive mutant possessing a single amino acid substitution in the invariant lysine 296 in the catalytic domain II. The transfected wild-type kinase showed properties similar to those of the natural kinase, such as subcellular ribosomal localization and dependence on double-stranded RNA for autophosphorylation. Upon infection with encephalomyocarditis virus (EMCV), wild-type- but not mutant-expressing clones were found to partially resist virus growth. Such natural antiviral activity was virus specific, since no inhibition was observed in the case of vesicular stomatitis virus infection. In accord with EMCV inhibition, the wild-type p68 kinase was found to be highly phosphorylated during infection. Furthermore, its natural substrate, the small subunit of protein synthesis initiation factor eIF2, was phosphorylated. These results demonstrate that p68 kinase is activated during EMCV infection, leading to reduced virus production.

Expression of hepatitis C virus proteins interferes with the antiviral action of interferon independently of PKR-mediated control of protein synthesis.

Hepatitis C virus (HCV) of genotype 1 is the most resistant to interferon (IFN) therapy. Here, we have analyzed the response to IFN of the human cell line UHCV-11 engineered to inducibly express the entire HCV genotype 1a polyprotein. IFN-treated, induced UHCV cells were found to better support the growth of encephalomyocarditis virus (EMCV) than IFN-treated, uninduced cells. This showed that expression of the HCV proteins allowed the development of a partial resistance to the antiviral action of IFN. The nonstructural 5A (NS5A) protein of HCV has been reported to inhibit PKR, an IFN-induced kinase involved in the antiviral action of IFN, at the level of control of protein synthesis through the phosphorylation of the initiation factor eIF2alpha (M. Gale, Jr., C. M. Blakely, B. Kwieciszewski, S. L. Tan, M. Dossett, N. M. Tang, M. J. Korth, S. J. Polyak, D. R. Gretch, and M. G. Katze, Mol. Cell. Biol. 18:5208-5218, 1998). Accordingly, cell lines inducibly expressing NS5A were found to rescue EMCV growth (S. J. Polyak, D. M. Paschal, S. McArdle, M. J. Gale, Jr., D. Moradpour, and D. R. Gretch, Hepatology 29:1262-1271, 1999). In the present study we analyzed whether the resistance of UHCV-11 cells to IFN could also be attributed to inhibition of PKR. Confocal laser scanning microscopy showed no colocalization of PKR, which is diffuse throughout the cytoplasm, and the induced HCV proteins, which localize around the nucleus within the endoplasmic reticulum. The effect of expression of HCV proteins on PKR activity was assayed in a reporter assay and by direct analysis of the in vivo phosphorylation of eIF2alpha after treatment of cells with poly(I)-poly(C). We found that neither PKR activity nor eIF2alpha phosphorylation was affected by coexpression of the HCV proteins. In conclusion, expression of HCV proteins in their biological context interferes with the development of the antiviral action of IFN. Although the possibility that some inhibition of PKR (by either NS5A or another viral protein) occurs at a very localized level cannot be excluded, the resistance to IFN, resulting from the expression of the HCV proteins, cannot be explained solely by inhibition of the negative control of translation by PKR.

Nuclear localization of the interferon-inducible protein kinase PKR in human cells and transfected mouse cells.

The levels and subcellular distribution of the interferon-inducible double-stranded RNA-dependent protein kinase PKR have been measured in human Daudi cells and stably transfected mouse NIH 3T3 cells expressing the human protein kinase. Immunofluorescence of intact cells and quantitative immunoblotting of cell extracts indicate that PKR occurs in both the cytoplasm and the cell nucleus, with staining specifically in the nucleolus. The ratio of cytoplasmic to nuclear PKR is approximately 5:1 in control cells; in response to interferon treatment the protein kinase is induced severalfold in the cytoplasm whereas the level in the nucleus does not increase significantly. Analysis of individual transfected cells by confocal microscopy reveals a pattern of distribution of PKR similar to that in Daudi cells, with immunostaining of cytoplasm and nucleoli. Similar results are observed whether cells expressing wild-type PKR or a catalytically inactive mutant form of the kinase are analyzed, but untransfected 3T3 cells are not stained by the antibody used. Two-dimensional isoelectric focusing analysis of PKR in whole cell extracts reveals the presence of multiple forms with different pI values whereas similar analysis of the nuclear fraction indicates only one predominant species with a relatively basic pI. These results suggest that PKR may have a role in the cell nucleus as well as the cytoplasm and that the subcellular distribution of the protein kinase may be related to post-translational modifications.

Full peptide synthesis, purification, and characterization of six Tat variants. Differences observed between HIV-1 isolates from Africa and other continents.

AIDS in Africa is characterized by the equal distribution of mortality between the two genders because of highly virulent human immunodeficiency virus type 1 (HIV-1) strains. The viral protein Tat trans-activates viral gene expression and is essential for HIV-1 replication. We chemically synthesized six different Tat proteins, with sizes ranging from 86 to 101 residues, from HIV-1 isolates located in different parts of the world including highly virulent African strains. Protein purification, mass spectroscopy, and amino acid analysis showed that the synthesis was successful in each case but with different yields. We show that all have the ability to bind the HIV long terminal repeat (LTR) RNA trans-activation response element (TAR) region, involved in Tat-mediated trans-activation, but structural heterogeneities are revealed by circular dichroism. These Tat synthetic proteins cross membranes but differ in their ability to trans-activate an HIV LTR-reporter gene in stably transfected HeLa cells. Two Tat proteins from virulent African HIV-1 strains were much more active than those from Europe and the United States. The interferon-induced kinase (PKR), involved in cell antiviral defense, phosphorylates only Tat variants corresponding to less or nonvirulent HIV-1 isolates. Our results indicate that the high virulence of some African HIV-1 strains could be related to Tat activity.

Two dimerization domains in the trans-activation response RNA-binding protein (TRBP) individually reverse the protein kinase R inhibition of HIV-1 long terminal repeat expression.

Trans-activation response (TAR) RNA-binding protein (TRBP) is a cellular protein that binds to the human immunodeficiency virus-1 (HIV-1) TAR element RNA. It has two double-stranded RNA binding domains (dsRBDs), but only one is functional for TAR binding. TRBP interacts with the interferon-induced protein kinase R (PKR) and inhibits its activity. We used the yeast two-hybrid assay to map the interaction sites between the two proteins. We show that TRBP and PKR-N (178 first amino acids of PKR) interact with PKR wild type and inhibit the PKR-induced yeast growth defect in this assay. We characterized two independent PKR-binding sites in TRBP. These sites are located in each dsRBD in TRBP, indicating that PKR-TRBP interaction does not require the RNA binding activity present only in dsRBD2. TRBP and its fragments that interact with PKR reverse the PKR-induced suppression of HIV-1 long terminal repeat expression. In addition, TRBP activates the HIV-1 long terminal repeat expression to a larger extent than the addition of each domain. These data suggest that TRBP activates gene expression in PKR-dependent and PKR-independent manners.